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PROCEEDINGInternational Conference Collaboration Seminar Of Chemistryand Industry (CoSCI 2016)

Development of Sensor Technology to

Support Indonesian Industry

Proceeding TeamCoSCI 2016

(October 5-6th, 2016)

PublisherDepartement of ChemistryFaculty of Science and TechnologyAirlangga University, c campusJalan Mulyorejo, Surabaya 60115, IndonesiaWebsite: http//www.chem.fst.unair.ac.idEmail: [email protected]

The proceeding of International Conference CollaborationSeminar of Chemistry and Industry

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CoSCI 2016October 5-6th, 2016Surabaya Indonesia

VENUE, ORGANIZER, AND COMMITTEE

Conference VenueHotel Santika Premiere SurabayaJalan Raya Gubeng no.54 Surabaya, East java 60281, IndonesiaTelp./Fax. +62 31-5922427Email: [email protected]

Organizer Department of Chemistry, Airlangga University

Patrons: Rector of Airlangga University

Dean of Faculty of Science and Technology

Advisory Board:Chairman Department of Chemistry, Airlangga University

Organizing CommitteeChairperson:Dr. rer. nat. Ganden Supriyanto, M.Sc

Co-chairperson:Dr. Miratul Khasanah, M.Si

Secretary:1. Mochamad Zakki Fahmi, M.Si, Ph.D2. Dr. Purkan, M.Si

Treasurer:1. Dr. Abdulloh, M.Si2. Alfa Akuista Widati, S.Si, M.Si

Scientific CommitteeScientific and Proceeding:1. Dr. Hery Suwito, M.Si (Coordinator)2. Prof. Dr. Afaf Baktir, M.S.3. Dr. Suyanto, M.S.4. Dr. Muji Harsini, M.Si5. Ali Rohman, M.Si, Ph.D6. Mochamad Zakki Fahmi, M.Si., Ph.D7. Yanuardi Rahadjo, S.Si, M.Sc


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Technical CommitteeSecretariate:1. Dr. Sri Sumarsih (Coordinator)2. Siti Wafiroh, S.Si, M.Si3. Alfa Akuista Widati, S.Si, M.Si4. Andriani, S.E

Promotion and Web Registartion:1. Ahmadi Jaya Permana, S.Si, M.Si (Coordinator)2. Harsasi Setyawati, S.Si., M.Si.3. Mochamad Zakki Fahmi, M.Si, Ph.D

Food and Baverages:1. Dra. Usreg Sri Handajani, M.S (Coordinator)2. Dr. Hartati, M.Si3. Aning Purwanti, S.Si, M.Si

Equipments:1. Drs. Sofijan Hadi, M.Si (Coodinator)2. Ahmadi Jaya Permana, S.Si, M.Si

Conference and Poster:1. Dr. Alfinda Novi Kristanti, DEA (Coodinator)2. Dr. Pratiwi Pudjiastuti, M.Si3. Dr. Mulyadi Tanjung, M.Si

Transport and Accommodations:1. Yanuardi Rahardjo, S.Si, M.Sc (Coordinator)2. Drs. Sofijan Hadi, M.Si3. Drs. Handoko Darmokoesoemo, DEA

Sponsorship and Public Relation:1. Prof. Dr. Ni Nyoman Tripusphaningsih, M.Si (Coordinator)2. Tjitjik Srie Tjahjandari, Ph.D.3. Dr. Nanik Siti Aminah, M.Si4. Dr. Abdulloh, M.Si.

Security:1. Rochadi S.Si2. Giman, S.Sos3. Fendik


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Keynote Speakers

Dr. Warsito Purwo TarunoUniversity of Indonesia, Indonesia

Prof. Dr. Jia-Yaw ChangDepartment of Chemical Engineering, National Taiwan University ofScience and Technology, Taiwan

Dr. Yoshiaki TakayaFaculty of Pharmacy, Meijo University, Nagoya

Dr. Didik Sasono Setyadi, S.H.,M.HumIndustrial Speaker oF SKK Migas, Indonesia

Invited Speakers

Prof. Dr. BuchariAnalytical Chemistry Division, Faculty of Mathematics and NaturalSciences, Institute of Technology Bandung, Indonesia

Dr. rer. nat. Ganden Supriyanto, M.ScDepartment of Chemistry, Airlangga University, Indonesia

Prof.Dr.Nor Azah Binti YusofInstitute of Advanced Technology , Universiti Putra Malaysia, Malaysia

Prof. Gao ZhiqiangDepartment of Chemistry, National University of Singapore, Singapore


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FOREWORD BY DEAN FACULTY OF SCIENCE ANDTECHNOLOGY, UNIVERSITAS AIRLANGGA

Assalamualaikum Wr. Wb. Ladies and Gentlemen,

On behalf of all member of The Faculty of Science andTechnology, Universitas Airlangga, It gives me a great pleasure toextend my warm welcome to the participants of the InternationalCollaboration Seminar of Chemistry and Industry, we call CoSCI2016, now is held in Hotel Santika Premiere Gubeng Surabaya. It isan Honor and Joy for us to able to host of this special internationalcollaboration seminar, which partisipant come from variouscountries including Indonesia, Japan, Germany, Taiwan, Australia, Malaysia, Singapore andIrak, where all participants have a chance to present and discuss our knowledge aboutdevelopment of basic and applied science of chemistry. The International CollaborationSeminar of Chemistry and Industry 2016 is organized as implementation of existingcollaboration among Airlangga University with some world-class universities in the word, inorder to promote the development of science and their prospects in industry application.

Ladies and Gentlemen, basic chemical scienceplays an pivotal rolein the development ofadvance chemical kowledge and is become basic for development of chemicalapplied scienceand has grown very rapidly into more specified fields, including biochemistry as basic ofgenetic enginering, material science,medicinal chemistry, computational chemistry andBiotechnology.Hopefully, this seminarprovides a great opportunity for lecturers, researchersand industries to build better communications between university and industry is one of thebest ways to share the development of research in the applied science which will be used inindustries.

Ladies and Gentlemen, finally let me congratulate to all of you once again for organizing andparticipating in this seminar. Also to the committee member, moderator, editor board,sponsors and participants for your kind contributions, I would like to express my gratitude forall the hard work on the succeeding this Seminar.

In this ocassion, I also would like to give my appreciation to all speaker, especially keynotespeaker from Japan, Germany, Taiwan, Australia, Malaysia, Singapore and Indonesia of theseminar for joint to make conference a success. Hopefully the new friendship and newcollaboration will start from this moment, and hopes to become a real cooperation forindustry.

Thank to Allah SWT for His blessing so far. Wassalamu’alaikum Wr. Wb.

Prof. Win Darmanto, M.Si, Ph. D


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FOREWORD BY CHAIRMAN DEPARTMENT OF CHEMISTRY,UNIVERSITAS AIRLANGGA

Distinguished, Ladies and Gentlemen,

The first time I would like to extend the warmest greetings andwelcome to all distinguished speakers and participantsin theinternational conference “ Collaboration Seminar of Chemistry andIndustries” (CoSCI-2016)

It is a pleasure for our institution to be host of the seminar, whichmeans that we can facilitate the realization of the dissemination ofresearch results among researchers in various institutions both academics and industries, aswell as the possibility of the establishment of cooperation with each other.

Through this international seminar, I wish that all the participants get fruitful discussion andit should promote local and international collaboration as an integral aspect of theresearch. I would like also to express my appreciation to all the committees for their supportto this seminar

Thank You very much for participation in the CoSCI 2016

Sincerely Yours,

Dr. Purkan, M.Si


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FOREWORD BY CHAIRMAN OF COSCI 2016

Assalamu’alaikum warohmatullohi wabarokatuh,Distinguished guest, speaker, participants.All welcome to 1st International conference: CollaborationSeminar on Chemistry and Industry (CoSCI) 2016.

I am so pleased to see so many participants from universities andindustries, all in one place. The CoSCI Organizing Committee haveworked very hard to prepare an outstanding conference. Thisconference aims to give researchers the opportunity to share theirresearch achievements with their colleagues from universities and industries and to promotecloser collaboration between universities and industries.

To my knowledge, so far there is no yet a significant collaboration between researchers inuniversities with the industries, especially in related with development of join researchresulting excellent products that can then applied in industries. This is one of the importantreasons why this conference is held.

We hope that this conference will be held every two years and give a significant contributionto the mutual collaboration among researchers and industries. Your strong support and activeparticipation have made the CoSCI a record breaking event. The spectrum of topics is verycurrent and broad.

I would like to express my sincere gratitude to speakers who spend their time to give a lecturein this conference. Also, the organizing committee members who have been working veryhard to prepare this conference.Finally, I would like to thank companies and institutions that have given valuable sponsorshipand support.

The Organizing Committee members is committed to provide maximumhospitality. Please feel free to ask questions to them. We are here to serve you. Enjoy yourparticipation in the 1st CosCI 2016 and memorable time visiting Surabaya. We hopeyou return next two years with even more colleagues for the2nd CosCI 2018.

Thank you. Have a wonderful day.Wassalamu’alaikum warohmatullohi wabarikatuh.

Dr.rer.nat. Ganden Supriyanto, M.Sc


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TABLE OF CONTENT

VENUE, ORGANIZER, AND COMMITTEE............................................................................. II

FOREWORD BY DEAN FACULTY OF SCIENCE AND TECHNOLOGY, UNIVERSITAS AIRLANGGA........................................................................................................................................V

FOREWORD BY CHAIRMAN DEPARTMENT OF CHEMISTRY, UNIVERSITAS AIRLANGGA......VI

FOREWORD BY CHAIRMAN OF COSCI 2016 .....................................................................VII

TABLE OF CONTENT .......................................................................................................VIII

ORAL PAPERS ................................................................................................................. 10LEAD (II) SELECTIVE ELECTRODE BASED ON S-METHYL N-(METHYLCARBAMOYLOXY) THIOACETIMIDATE AS ASENSING MATERIAL .................................................................................................................................1

Qonitah Fardiyah*, Atikah, Linda Noviana, Shilza EkaTITRIMETRIC METHOD OF TOTAL EDTA CONTENT DETERMINATION IN NA2H2EDTA - NAFEEDTA ANDNA2H2EDTA-NA2CAEDTA-NAFEEDTA MIXTURE.......................................................................................5

Kusumaningrum. K Irma, Sanjaya H Eli., Arief,Munzil, Wijaya , R Anugrah., Laily, M Umi, Meyana,E . NurulSTICK CHEMICAL SENSOR USING DITHIZONE TO DETECT MERCURY IN PHARMACHEUTICAL PREPARATIONS .....8

Atina .K.N. 1,2 , Siska A. 1,2 , Ganden S. 1,2, Miratul K. 1,2

CHEMICAL SENSOR FOR MELAMINE BASED ON DIAZOTITATION REACTION USING NAPHTHYLETHYLENEDIAMINE ...13Nia K.Rukman, Syarifah A., Tjijik S. T, Miftakhul J, Ganden Supriyanto

COMPARATIVE COD ADSORPTION KINETIC OF COCONUT SHELL ACTIVATED CARBON....................18Nur Indradewi Oktavitri*, Hery Purnobasuki, Eko Prasetyo Kuncoro, Indah Purnamasari

SYNTHESIS AND CHARACTERIZATION OF COPPER(II) COMPLEX WITH 2,4,5-TRIPHENILIMIDAZOLE .......................24Teguh Hari Sucipto1,2*, Harsasi Setyawati3, Siti Qamariyah Khairunisa1, Fahimah Martak224

INHIBITION OF UV RADIATION BY PIPER COCATUM, PISONIA ALBA SPANOGHE, CHRYSOPHYLLUM CAINITO

L., IPOMEA BATATAS L., MIRABILIS JALAPA L., AND CURCUMA DOMESTICA VAL. EXTRACTS .............27Fredy Kurniawan, Widia Rachmawati, Debora Kartikasari27

ELECTROSPINNING OF CELLULOSE ACETATE NANOFIBER MEMBRANE FOR HEMODIALYSIS ...........32Mohammad Zakki Fahmi, Yanuardi Raharjo, Siti Wafiroh, Iqlima Ayu Prestisya, and Kukuh Ferlanda

BIOACTIVE COMPOUNDS OF PROPOLIS FROM NATURAL BEEHIVES FROM EAST JAVA, INDONESIA 40Zjahra Vianita Nugraheni*1, Anil Kumar Anal2, Agus Wahyudi1, Rhiby Ainur Basit Haryanto140

NANOCOMPOSITE OF SODIUM ALGINATE POLY ACRYLATE-ACRYLAMIDE-BENTONITESUPERABSORBENT: SYNTHESIS AND CHARACTERIZATION...............................................................43

Helmiyati1, Malida Aprilliza243POTENTIAL SYNERGISM OF ASTAXANTHIN FROM HAEMATOCOCCUS PLUVIALIS AS AN ANTIOXIDANT SUPPLEMENTS47

Yuyun Yuniati a, Renny Indrawatia, b, Tatas H. P. Brotosudarmoa, b, Wynona Agatha Nimpoenoa, LeenawatyLimantarab,c ................................................................................................................................................. 47

POSTER PAPERS.............................................................................................................. 51CYCLIC VOLTAMMETRY OF HYDROQUINONE BY CARBON NANOPOROUS PASTE ELECTRODES MODIFIED BYFERROCENE ..........................................................................................................................................52

Untari, Muji Harsini, M. Zakki Fahmi52ISOLATION AND CHARACTERIZATION OF ALKALINE PROTEASE PRODUCING MARINE BACTERIA AND MACRO-ALGAEFROM TANJUNG TIRAM BEACH ................................................................................................................56

Prima E. Susilowati1), Wulan Purnamasari 1), Citrawana B. Ladjamu2), Masdidi3), Desi Kurniawati1)56


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PARTIAL PURIFICATION AND CHARACTERIZATION OF BROMELAIN FROM THE CORE AND FLESH OFPINEAPPLE EXTRACTS (ANANASCOMOSUS) ..........................................................................................60

Siswati Setiasih, Nita Magfirah Ilyas, Sri Handayani and Sumi Hudiyono60SYNTHESIS AND CHARACTERIZATION OF COMPLEX COMPOUND OF ZN(II)-EDTA FOR ANTIALGAECOMPOUNDS IN INDUSTRIAL COOLING WATER ..............................................................................65

Sevia Ayuningtyas., Sri Sumarsih, Harsasi Setyawati65ANALYSIS OF PARTIAL LEACHING IN THE GEOCHEMICAL FRACTION OF FE CONTENT IN SEDIMENT SENDANG BIRUBEACH.................................................................................................................................................69

Anugrah Ricky Wijaya1*, Ulfa Romlah1, Ahmad Fariq Imas1, Irma Kartika Kusumaningrum1, SurjaniWonorahardjo1, Eli Hendrik Sanjaya169

GC-MS ANALYSIS OF N-HEXANE, ETHYL ACETATE, AND METHANOL EXTRACT OF CALLICOSTELLAPRABAKTIANA(C. MῢLL.) BOSCH & SANDE LAC. .........................................................................................73

Junairiaha, Tri Nurhariyatia, Suaibaha, Ni’matuzahroha, Lilis Sulistyorinib73ANTIFUNGAL ACTIVITY OF RAIN TREE (SAMANEA SAMAN JACQ.) LEAF EXTRACT AGAINST FUSARIUM SOLANI, THECAUSE OF STEM ROT DISEASE ON DRAGON FRUIT (HYLOCEREUS SP.) ............................................................77

Wiwik S. Ritaa*, Dewa N. Supraptab, I Made Sudanab, I Made D. Swantaraa77



ORAL PAPERS


1Fardiyah et al.


Lead (II) Selective Electrode Based on S-Methyl N-(Methylcarbamoyloxy) Thioacetimidate as a Sensing Material

Qonitah Fardiyah*, Atikah, Linda Noviana, Shilza EkaDepartment of Chemistry, Faculty of Mathematic and Natural Science, Brawijaya University, Jl. Veteran

Malang 65145*)Email: [email protected]; [email protected]

ABSTRACT

The construction and performance characteristics of a novel lead (II) membrane sensor based on a new S- methyl N-(methyilcarbamoyloxy) thioacetimidate has been developed. S- methyl N-(methyilcarbamoyloxy) thioacetimidate,namely methomyl, was chosen as ligand in membrane electrode. It was selected due to its tendency to form a stablechelates with any heavy metals ions of environmental concern. This methomyl is used as ionopore and mixed withpolyvinylchloride (PVC) and dioctylphtalat (DOP) dissolved in tetrahydrofuran (THF) (1:3 w/v) to form anelectrode membrane sensor. Basic characteristics of lead (II) selective electrode were examined in Nernst factor,limit of detection, linear concentration range, response time, and life time. The immersion time of membrane is 60minutes, apply in methomyl : PVC : DOP = 17 : 17 : 66 (% w/w) membrane composition. The design sensorexhibited a wide linear response with a slope of 29.26 mV per decade over range of 8.054 x 10-6 M to 10-1 Mconcentration. The electrode shows a response time of 60 seconds. The proposed electrode can be used for at leasttwo month or 73 days.

Keywords: lead (II), sensor, potentiometric, PVC membrane, methomyl

INTRODUCTION

Lead is one of chemical elements classified asheavy metals [1]. Lead can accumulates in air,water, soil and in food products, so on. Lead reactwith other compounds to form various lead

compounds such as lead oxide (PbO), lead chloride(PbCl2), and others. Lead may enter to the bodythrough breathing and food. Consumption of leadin large quantities directly causing tissue damage.

Lead exposure in baby and children causing braindamage, growth retardation, kidney damage andhearing loss, whereas an excessive lead exposure inadults causeing increased of blood pressure andimpaired digestion [2]. Lead accumulates in thebody is quite dangerous than not digested in the

excretory system consequently can accumulate inthe body and may cause central nervous system,kidney function, inhibiting the formation of

Figure 1. Determination of membrane immersion time optimization


2Fardiyah et al.


hemoglobin, and can affect the level of intelligence(IQ) in children [3].

One of the popular method of lead analysis isatomic absorption spectrophotometry (AAS)methods. The advantages of this method are bitsample needed, analyze ability at lowconcentrations, high sensitivity and quickrelatively. However, this methods has drawbacksfor sample analysis of which can not be used forfield analysis and require different cathode lampsfor different metals. Furthermore, the AASmethods less practical when used for analysis in thefield. Therefore, these obstacles can be overcomeusing other simple methods such as potentiometry.Potentiometric methods commonly used is usingthe Ion Selective Electrode (ISE). Ion SelectiveElectrode is a working electrode that is able tomeasure selectively against certain ion. Potentialthat will measurably changed reversibly reactivityof ions determined [4]. Ion Selective Electrode is avery important part in the electrochemical sensorsystem, this is because the sensor tip contains anactive material (ionophore) which thethermodynamic and kinetic events [5].

Lead (II) selective electrode have beendeveloped by M. Ghaedi et al, 2011 [6] by usingorganic materials as ionophores. Other membranesupport material used is Polyvinylchloride (PVC),dibutylphthalate (DBP). From the obtained results,the Nernst factor generated price of 25.79 mV perdecade, the concentration range 10-6 - 10-1 M, thedetection limit of 4.0 x 10-7 M with a response timeof 10 seconds. Nernst Factor resulting from thesestudies are still not close to the theoretical price(for the divalent ion is equal to 29.58 mV / decadeof concentration) that need to be modifiedmembrane of Lead (II) selective electrode toproduce Nernstian electrode.

Based on the results of previous studies, needsto be further developed lead (II) selective electrodewith modification of ionophores. In this study,ionophores used are S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate. Thiscompounds was selected as ionophores due tointeract very strongly to metal ions and have a

tendency to form a stable chelate with heavy metalsions of environmental concern [7].

The membrane electrode consist of mixture amethomyl as ionophore, polyvinylchloride (PVC)and dioktilftalat (DOP) dissolved intetrahydrofuran (THF) solvent (1:3 w/v). Variablesof this research consist of immersion time andcomposition of the membrane. Basiccharacterization of lead (II) selective electrodewere examined included: Nernst Factor, limit ofdetection, linear concentration range, responsetime, and life time.

EXPERIMENTALMaterials and Equipment

The materials used in this study are S-Methyl-N-(Methylcarbamoyloxy) ThioacetimidatePb(NO3)2 p.a. (E.Merck), dioktilftalat (DOP)(sigma), polyvinylchloride (PVC) (sigma),tetrahydrofuran (THF) (E.Merck), HNO3 65% (v/v)p.a. (E.Merck), alcohol 96% (v/v) and aquadest.Equipment used in this study are : lead (II)selective electrode based on S- methyl N-(methyilcarbamoyloxy) thioacetimidate have incharacterization, electrode Ag/AgCl type HI5313Hanna, potentiometer (Schoot Geräte CG model820), pH meters (Hanna), alligator clamps,analytical balance (Adventurer AR model 2130),centrifuges, whatman No.40 filter paper, oven,desiccator, the sample bottles, hot plate, and glassequipment

Fabrication lead (II) selective electrode based on S-Methyl-N-(Methylcarbamoyloxy) Thioacetimidate

Manufacture of membranes is done by mixingthe constituents of the membrane which include S-Methyl-N (Methylcarbamoyloxy) Thioacetimidate,PVC and DOP. After it was dissolved with THFwith a ratio of 1: 3 (% w / v). Membrane coated onPt wire with a thickness of 0.3-0.5 mm followed byheating at 50 ° C for 12 hours. Platinum wire thathas been coated membrane is cooled and immersedwith a solution of Pb (NO3)2 1.5 M.

Optimization of membranes composition andmembrane immersion time


3Fardiyah et al.


A lead (II) selective electrode based on S-Methyl-N (Methylcarbamoyloxy) Thioacetimidatemade with certain variations in composition coatedon Pt wire. The electrode with certain compositionused to measure potential of solution Pb(NO3)2 withvariation concentration 1x10-8, 1x10-7, 1x10-6,1x10-5, 1x10-4, 1x10-3, 1x10-2, 1x10-1 M. Theelectrode immersion in a solution of Pb(NO3)2

saturated with a variety of immersion time 10, 30,50, 60, 80 and 120 minutes. Then used to measurethe potential of the solution of Pb (NO3) 2 solutionwith a concentration of 1x 10-1, 1x10-2, 1x10-3,1x10-4, 1x10-5, 1x10-6, 1x10-7, 1x10-8 M.

Basic characterization of lead (II) selective electrodebased on S-Methyl-N-(Methylcarbamoyloxy)Thioacetimidate.

Determination Nernst factor is done bymeasuring the Nernst potential lead (II) selectiveelectrode on the variation of the concentration of

the test solution and using the electrode Ag / AgClelectrode as a comparison. Processed measurementdata and extrapolated into the relationship betweenlog activity of lead graph with the measuredpotential (mV) to obtain the Nernst Factor, linearconcentration range, and the limit of detection.Determination of response time was done bymeasuring potential test solution with an interval of10-180 seconds. Determination of long service lifedone to determine how long the Lead (II) selectiveelectrode based on S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate can beused are shown on the Nernst factor price obtainedon unit time.

RESULT AND DISCUSSIONFabrication lead (II) selective electrode based on S-Methyl-N-(Methylcarbamoyloxy) Thioacetimidate

Active material or ionophore in the fabrication

Figure 2. Determination of the concentration range and limit of detection

Figure 3: Determination of life time Lead (II) selective electrode based on S-Methyl-N-(Methylcarbamoyloxy)Thioacetimidate


4Fardiyah et al.


of membrane lead(II) selective electrode isimportant. Membrane composition have been effectto the Nernst factor produced. In this study whichused methomyl as ionophore membrane and usingother support material of membrane that someplasticizers DOP, and PVC as polymer matrix weredissolved in THF solvent. S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate wasselected as ionophore membrane because thiscompounds can interact very strongly to metal ionsand have a tendency to form a stable chelate withheavy metals ions of environmental concern .

Optimization of membranes composition andmembrane immersion time

In the determination of the optimumcomposition of the building blocks of membranesobtained the optimum composition methomyl:PVC: DOP are 17: 17: 66 (% w / w) where theresulting Nernst factor approaching the theoreticalvalue (29.58 mV / decade concentration of divalentions) is 29.26 mV / decade of concentration.Measurements on 3 variations of membranecomposition showed that approaches the theoreticalNernst factor compositions the ionophoresmembrane has the most, it affects the ability of thecoated wire Lead (II) ion selective electrodebecause the more ionophore in Lead (II) selectiveelectrode then the more lead is bound within themembrane so that when the Nernst factormeasurements produced a more theoreticalapproach. The optimum immertion time is done byimmersing the membrane into the Pb(NO3)2

saturated with the goal of bringing the membranewith a cation lead to be measured potential andproduce Lead (II) selective electrode based on S-Methyl-N (Methylcarbamoyloxy) Thioacetimidate.See Figure 1

The results obtained in 60 minutes to have avalue of Nernst factor 28,2 mV per decade thatapproaches the theoretical Nernst factor. In the 60minutes into the show membrane optimumimmersion time. Determination of optimumimmersion time can be seen from the resultingNernst Factor in 10-20 minutes membrane was notdissociate completely so the resulting Nernst factorhas not approached theoretical Nernst value factor.In the 65-120 minutes to the resulting value of theNernst factor decreases, this caused that membranewas saturated.

Basic characterization of lead (II) selective electrodebased on S-Methyl-N-(Methylcarbamoyloxy)Thioacetimidate

The basic characterization of coated wire lead(II) ion selective electrode with Nernst factorapproaching the theoretical value that is 29.26 mV /decade of concentration. Linear concentration inthis study from the 10-6 to 10-1 M with a limit ofdetection 8.054 x 10-6 M, equivalent to 1,669 ppmof lead. The determination of the concentrationrange and limit of detection can be see in Figure 2.

Response time for lead (II) selective electrodebased on S-Methyl-N (Methylcarbamoyloxy)Thioacetimidate resulted is 60 seconds. It showsthat the electrode has a quick response timerelatively. the resulting graph (Figure 3.) can beseen that in the first 0-10 days resulting NernstFactor was stable approaching the theoretical value(29.58 mV / decade of concentration), but when itgets to 65 days showed a Nernst factor prices beganto fall from the threshold exposure limits. Howeverlead (II) selective electrode based on S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate can still beused because the price of R2 is close to 1. Thismeans that the correlation of data with one anotherdid not find, but on days 65-73 Nernst Factorvalues are no longer Nernstian this can happenbecause the lead (II) selective electrode based onmethomyl prolonged contact with water acting asthe solvent in the test solution. Water is the solventof the test solution will be entered into the poresmethomyl fill gaps that should be filled with lead.Therefore, when the measurement of lead cationinteractions that exist in solution and lead cationsthat exist at the membrane interface can not workoptimally so as to produce the value of the Nernstfactor away from the theoretical value.

CONCLUSIONS

Based on the research that has been done can beconcluded that the optimum composition of theCW-ISE Lead (II)-based on pyrophyllite obtainedby comparison methomyl: PVC: DOP = 17 : 17 :66 (% w/w). The optimum immertion time for Lead(II) selective electrode based on S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate was in 60minutes. As for the basic characterization Lead (II)Selective electrode based on S-Methyl-N(Methylcarbamoyloxy) Thioacetimidate producesNernst Factor of 29.26 mV / decade ofconcentration, with a linear concentration rangefrom 10-6 to 10-1 M lead (II) and it was able todetect cations lead up to 8.054 x 10-6 M orequivalent to 1,669 ppm of lead. It had responsetime of 60 second and could be used for 73 days.

REFERENCES

[1] Wood, O.W. , m.s.c. m.e.d, LL.B,F.R.I.C ,1967., Inorganic Chemistry An IntermediateText Third Edition. Butter Worth & coPublisher limited. London

[2] Standar Nasional Indonesia. 2009. BatasCemaran Logam Berat. Badan StandarisasiNasional Indonesia.

[3] WHO Region Publication European Series,1999, Baku Mutu Kualitas Udara Ambien , PPno 41 Tahun 1999, Serpong

[4] Fardiyah Q., 2003, Aplikasi Elektroda SelektifIon Nitrat Tipe Kawat Terlapis UntukPenentuan Secara Tak Langsung Gas NO,


5Fardiyah et al.


Tesis, Program Studi Kimia, ProgramPascasrjana ITB, Bandung.

[5] Suyanta, 2004, Penentuan Tetapan SelektivitasElektroda Selektif Ion Sistem PotensiometriDengan Metode MPM, Jurusan PendidikanKimia, Universitas Negri Yogyakarta

[6] M. Ghaedi, M. Montazerozohori, Z. Andikaey,A. Shokrollahi, S. Khodadoust, M. Behfar, S.Sharifi, 2011. Fabrication of Pb2+ IonSelective Electrode Based on 1-((3-((2-Hydroxynaphthalen-1-yl)Methyleneamino)-2,2-Dimethylpropylimino) Methyl)Naphthalen-2-ol as New Neutral Ionophore,International Journal of ElectrochemicalScience, Department of Chemistry, Universityof Yasouj, Iran

[7] Guptha, Vinod Kumar, Bhavana Sethi, NirajUpadhyay, Sunita Kumar, Rakesh Singh, LokPratap Singh, 2011, Iron (III) SelectiveElectrode Based on S-Methyl N-(Methylcarbamoyloxy) Thioacetimidate as aSensing Material, Int. J. Electrochem. Sci., 6,pp. 650 – 663.

[8] Patnai. P., 2004, Dean’s Analytical ChemistryHandbook Second Edition, McGraw-HillCompanies Inc, New York.

[9] R.A. Goyer, 1990, Lead Toxicity: FromOvert to Subclinical to Subtle Health Effects,Environmental Health Perspectives, 86, 177-181.


6Kusumaningrum et al.


Titrimetric Method of Total EDTA Content Determination inNa2H2EDTA - NaFeEDTA and Na2H2EDTA-Na2CaEDTA-

NaFeEDTA MixtureKusumaningrum. K Irma, Sanjaya H Eli., Arief,Munzil, Wijaya , RAnugrah., Laily, M Umi, Meyana,E . NurulChemistry Department, Science and Mathematics Faculty of Universitas Negeri Malang

*email: [email protected]

ABSTRACT

EDTA (Etilendiamintetraasetat) is one of the food additives that commonly used in various processed food products.Maximum using of EDTA in foods should be limited. As food additives EDTA is given as Na2H2EDTA, Na2CaEDTA,and NaFeEDTA. The commonly methods for EDTA content determination have been used are spectrophotometricand chromatographic methods with UV-Vis spectrophotometry and HPLC, but those are couldnot analyze the totalcontents of EDTA. Needed an alternative method to determine the contents of EDTA in food ingredients. This paperdescribe a method of total EDTA content determination in Na2H2EDTA-Na2FeEDTA and Na2H2EDTA-Na2CaEDTA-NaFeEDTA mixture. Determination of total EDTA contents has been done with titrimetric method withmurexide as indicator. Based on the results, recovery percentage have reached of total EDTA content determinationin Na2H2EDTA-NaFeEDTA mixture is 91.4% and recovery percentage have reached of total EDTA contentdetermination in Na2H2EDTA- Na2CaEDTA-NaFeEDTA mixture is 85.8%

Keywords: Na2H2EDTA, Na2CaEDTA, NaFeEDTA, titrimetric,

INTRODUCTION

EDTA salt ,Na2H2EDTA, NaFeEDTA andNa2CaEDTA often added to some processed foodproducts as a sequestrant. The maximum EDTAcontent in processed food limited by the NationalAgency of Drug and Food of the Republic ofIndonesia Number 18 Year 2013, the maximumcontent is 33-1000 mg/kg (1). Previously, researchon EDTA content determination in foodstuff hadbeen carried out by using UV-Visspectrophotometer (2), the method could be used todetermine EDTA in the form Na2H2EDTA only,the other methods had been developed in EDTAdetermination method using HPLC equipped withUV-Vis as detector, was developed by ShimadzuFood Product, however, the determination ofEDTA content using HPLC instruments are veryexpensive(3). This research will be described amethod of the total EDTA content determination ina mixture of Na2H2EDTA- NaFeEDTA andNaFeEDTA Na2H2EDTA- Na2CaEDTA mixture bytitrimetric method.

EXPERIMENTALChemical and instrument

Analytical balance, sentrifugator (KOKUSANH-103n), Spectrophotometer UV-Vis(JASCO),

Atomic Absorption Spectrophotometer (AAS),spektronik-20, test tubes, magnetic stirer, watchglass, Erlenmeyer 100 mL, beaker glass, measuredglass, pipette volume, filler, universal indicators.The chemicals used are 5M NaOH solution, 98%HNO3 solution, NaFeEDTA.3H2O,Na2H2EDTA.2H2O powders, Co(NO3) .6H2O,distilled water, aqua demineralization, andmurexide.

Determination of total EDTA content in the mixture ofNa2H2EDTA and NaFeEDTA

The basic solution are Na2H2EDTA,NaFeEDTA. Na2H2EDTA 10,000 ppm solution isprepared by dissolving 1.291 grams in 100 mL ofdemineralized water. NaFeEDTA 4,000 ppmsolution prepared by dissolving 0.584 gramsNaFeEDTA in 100 mL of demineralized water.NaFeEDTA-Na2H2EDTA 400:400 ppm mixtureprepared by mixing and diluting of the basicsolution. Decomposition of NaFeEDTA have doneby following procedure, sample solution mixturewas put in a flask of 50 mL, diluted with aquademineralization up to the mark, then, NaOH 5M isadded until saturated. The precipitate is separatedby centrifugation at 3,000 rpm for 10 minutes.Then, the supernatant is filtered to separate thepellet .Determination of EDTA in the sample




mixture have done by following procedure, 5 ml ofsupernatant put in 25 ml flask, pH of thesupernatant was set at 4, 3 drops of indicatormurexide added to the sample and diluted withaqua demineralization up to 25 mL. Then, 5 ml ofsolution put in Erlenmeyer, titrated with a solutionof Co(NO3) 2 until the color changes from pink topurple.

Determination of total EDTA concentration in themixture Na2H2EDTA- Na2CaEDTA-NaFeEDTA

The samples used is the mixture ofNa2H2EDTA, Na2CaEDTA, and NaFeEDTA, theproportion of the sample Na2H2EDTA:Na2CaEDTA: NaFeEDTA is 400: 400: 400 ppm.Titrant solution used is Co (NO3)2 solution, theCo2+ content of the titrant have standarized withAAS method (Khaira, 2014). To determine EDTAtotal content, 15 mL sample taken anddecomposited to precipitate Fe3+ ions.Decomposition is accomplished by the addition ofNaOH 5 M. Supernatant separated bycentrifugation using sentrifugator. Fe3+ content ofsupernatant was determined with atomic absorptionspectrophotometer (AAS), then 5 mL ofsupernatant was conditioned at pH= 4,3 dropsmurexide indicator was dropped and then dilutedto 25 mL, then 5 ml of dilution product titratedusing Co(NO3)2.

RESULTS AND DISCUSSION

Determination of EDTA content in this studywas conducted in two stages, first, decompositionof NaFeEDTA in the sample solution, this processaccording the following reaction equation:

Scheme 1. Decomposition of NaFeEDTA

NaFeEDTA (aq) + 3NaOH (aq) →Fe(OH)3 (s) + 3Na+ (aq) + EDTA4-(aq) (4)

Decomposition of NaFeEDTA was done byadding NaOH, Fe(OH)3 were separated bycentrifugation method, this process resultingsupernatant and pellet. The Fe3+ content ofsupernatant measured using atomic absorptionspectrophotometry, it’s appears that after thisprocess, the Fe3+ content of supernatant decreasedfrom 1379.7 ppm to 1.5230 ppm. AfterNAFeEDTA decomposition process, supernatantwas separated titrated with Co(NO3)2 as titrant,titration have done at pH= 4, the titration processfollowing the reaction below

Scheme 2. Determination of EDTA as [Co(EDTA)]2-

reaction

Co2+(aq) + H2[EDTA]2-(aq) [Co(EDTA)]2-

(aq) + 2H+(aq)

(5)

Supernatant was-titrated with Co(NO3)2,

recovery percentage of total EDTA contentdetermination of Na2H2EDTA -NaFeEDTAmixture is 91%. Endpoint of the titration markedwith a color changes of murexide as indicator. Thecolor changes at the end point of titration is fromburgundy red color to the red color, indicates thatthe Co2+ ion has reacted with murexide to formingCo-murexide complex. The recovery percentage ofEDTA content determination via H2CoEDTAcomplex formation is not satisfied, this fact iscaused by the difficulty on the end pointobservation. At the beginning of the titration,murexide give a purplish red color and when theendpoint reached, Co –murexide complex formed,the color of this complex is red. The murexide andCo-murexide complex color looks almost similar,so that the color change before and after the endpoint becomes difficult to observe. The clarity ofend point observation analyzed based on the λ maxmeasurement of murexide, Co-murexide complexand H2CoEDTA absorption curve measured byUV-Vis Spectrophotometer, that the λ maxabsorption of H2CoEDTA is 514 nm, Co-murexide513 nm and murexide 521 nm, by this observationit appears there are overlapping between murexide,Co-murexide and H2CoEDTA absorption curve.

Figure 1. Absorption Curve of murexide, Co-murexideand H2CoEDTA

Determination of total EDTA content of themixture of Na2H2EDTA: Na2CaEDTA:NaFeEDTA have done in two step, decompositionof NaFeEDTA and determination of EDTA contentby titrimetric method, the recovery percentage havereached is 85.8%, this recovery percentage is notsatisfied. The low percentage of recovery wasachieved may be due to the incompleteness ofprecipitation and separation process of Fe3+ ions,most of Fe 3+ will precipitate as Fe(OH)3, but asmall portion of Fe3+ remain in the supernatant, thisincompleteness of the process before, reducing theH2EDTA2-, which will be titrated to formH2CoEDTA, was formed. The reducing ofH2EDTA2- in the system reducing the Co2+ ions




needed, as a result EDTA contents were measuredthrough complex formation H2CoEDTA will besmaller than the actual contents. The highercontents of EDTA in the sample, titration errorbecomes larger and the smaller the recoverpercentage achieved. Titration of H2CoEDTAcomplex formation is performed at pH = 4, titrationat this pH can trigger the formation of a stablecomplex compounds NaFeEDTA. Based on thepercent recovery was achieved, the determinationof total EDTA concentration in the Na2H2EDTA-NaFeEDTA mixture is higher than thedetermination of total EDTA concentration inNa2H2EDTA- Na2CaEDTA-NaFeEDTA mixture,this fact may be due to the availability ofNa2CaEDTA in the mix. H2CoEDTA complexformation from NaH2EDTA easier thanH2CoEDTA complex formation from Na2CaEDTA,Na2CaEDTA complex is stable, although theirstability is lower than NaFeEDTA stability, so canbe failure on Ca2+ replacement by Co2+ processthat will be reducing the titrant needed, the titrantneeded not equivalent with the EDTA content

CONCLUSION

The total EDTA content of Na2H2EDTA-NaFeEDTA and Na2H2EDTA- Na2CaEDTANaFeEDTA mixture can be determined withtitrimetric method

Determination of total EDTA content has beendone with several stages, (1) decomposition ofNaFeEDTA (2) the determination of EDTA content

as H2CoEDTA with titrimetric method, therecovery percentage have reached of EDTAcontent determination in Na2H2EDTA-NaFeEDTAmixture is 91.4% and recovery percentage havereached of EDTA content determination inNa2H2EDTA- Na2CaEDTA NaFeEDTA mixture is85.8%

REFERENCES

[1] Peraturan Kepala Badan Pengawas Obat danMakanan Republik Indonesia Nomor 18 TahunTentang Batas Maksimum Penggunaan BahanTambahan Pangan Sekuestran, 2013

[2] A.S. Dwi, P. Anna, S. Hokcu, PengembanganMetode Penetapan Kadar EDTA dalam ProdukPangan Mayonais secara Spektrofotometri UV,Jurnal Sains dan Teknologi Kimia, 5,1, 2014

[3] Carolina E. Cagnasso, Laura B. Lo´pez,Viviana G. Rodrı´guez, Mirta E. Valencia,Development and validation of a method forthe determination of EDTA in non-alcoholicdrinks by HPLC, Journal of Food Compositionand Analysis, 20,2007, 248–251

[4] Svehla, G. Vogel, Setiono,L., &Pudjaatmaka,H (eds), Buku Teks AnalisisAnorganik Kualitatif Makro dan Semimikro,PT. Kalman Media Pusaka, Jakarta, 1985

[5] Paramita.G., Murwani. I. 2012. Atom PusatCo2+ (d7) dengan Konfigurasi Low Spin dalamSenyawaKompleks Co-EDTACo2+ (d7),Prosiding Seminar Nasional Kimia Unesa,2012


9Nisa et al.


Stick Chemical Sensor Using Dithizone To Detect Mercury InPharmacheutical PreparationsAtina .K.N. 1,2 , Siska A. 1,2 , Ganden S. 1,2, Miratul K. 1,2

1Department of Chemistry, Faculty Science and Technology, Airlangga UniversityJl. Mulyorejo Surabaya60115, Indonesia

2Laboratory of Sensor and biosensor, Institute of Tropical Disesase, Airlangga University Jl. MulyorejoSurabaya 60115, Indonesia

ABSTRACT

A stick chemical sensor using dithizone to detect mercury in pharmacheutical preparations has beendeveloped. Dithizone reagent was reacts with mercury(II) to form complex that can be observed from thecolor changing from green to orange. In this research, it is obtained optimum dithizone concentration is 0,01%at pH 4. Sensor selectivity was tested by adding Pb2+. It gives significant interference to this sensor at

concentration ratio 1:1. This sensor has a precision value of 3,6% with the limit of detection 4,6 x 10-3

ppm,and accuracy is 97,2%. Linearity was showed by the correlation coefficient (R2) is 0,997 from standardsolution, while the sensitivity obtained from the slope of the standard solution is 0,1166/ppm.

Keywords : mercury, dithizone, chemical sensor, pharmaceutical

INTRODUCTION

Mercury (Hg) is a metallic element that occursnaturally in the environment. There are threeprimary categories of mercury and its compounds:elemental mercury, which may occur in both liquidand gaseous states; inorganic mercury compounds,including mercurous chloride, mercuric chloride,mercuric acetate, and mercuric sulfide; and organicmercury compounds. [1].

The usage of Hg has been popular in humanand environment aspects nowadays. In some yearsHg has been used in medical, agriculture, andindustrial aspects. In medical aspects, Hg has beenused for the theraphy of sipilis. Many methods foranalysis of mercury have been developing such aresynthesized ionophore [2], synergistic catalyticeffectof gold nanoparticles (AuNPs) and Hg usingan ultrasensitive colorimetric [3], phosphonic acidbased polymeric fluorescent sensor [4],development of a visual optode sensor [5], cold-vapor atomic absorption spec-trometry (CV-AAS),spectrophotometry, anodic stripping vol-tammetry,energy dispersive X-ray fluorescence (EDXRF)andhigh-performance liquid chromatographyhyphenated with induc-tively coupled massspectrometry (HPLC-ICPMS) [6-14] have beenreported for the Hg(II) measurement. Thesemeasurement have a high cost. In this study, asimple chemical sensor that is more practical,inexpensive, sensitive, and selective to detect thepresence of mercury in pharmaceutical preparations(medicines) using dihtizone reagents with dipsticktechnique was made by dipping Whatman paper

into a dithizone reagents and measurement by UV-Vis spectrometry.

Dithizone reagents will react with mercury andgive yellow to orange compound depending on theconcentration of mercury. Sticks chemical sensorcan be used as a form of screening an analyte in aparticular sample both qualitative andsemiquantitative. Qualitative test done by lookingat the color changes on the surface of the stick.While the semiquantitative test done by looking atthe intensity of the resulting color and colorintensity compared to the color series produced byvarying mercury standard solution concentrations.Change the color of the sticks chemical sensorfrom colorless to yellow is caused by reaction ofmercury with dithizone. These color changes occurdue to the formation of complex compounds ofmercury-dithizone.

EXPERIMENTAL

2.1 Material InstrumentationThis research used dithizone (C13H12N4S),

CH3COOH, CH3COONa, NaH2PO4.2H2O, NaOH,CHCl3, Pb(NO3)2, HgCl2, Whatman paper no. 1,destilled water, UV-Vis spectrophotometryShimadzu-1800, pH meter eutech instrument.

2.2 Analytical Parameters Optimization

2.2.1 Determination of the Maximum WavelengthDithizone Reagents

As much 1 mL of dithizone reagents 0.01% wastaken and put in a 10 mL volumetric flask thenadded CHCl3 to mark and then shaken untilhomogeneous. The solution wasmeasured by UV-


10Nisa et al.


Vis spectrophotometer to obtain maximumwavelength of dithizone CHCl3 was used as blank.

2.2.2 Determination of the Optimum pH, MaximumWavelength, Optimum Concentration ofComplex Hg (II) -Dithizone

Into a separating funnel was filled with 5 mLCHCl3, then successively as added with thestandard solution of 100 ppm Hg2+ as much as 1mL, 0.01% dithizone solution in 1 mL and 2 mL ofbuffer solution pH 3, then extracted by meanswhisk for 30 minutes. The organic phase (in thebottom layer) was transferred into a 10 mLvolumetric flask. The solution left in the separatingfunnel was added with 3 mL solvent CHCl3 thenre-extracted by shaking for 30 minutes. Theorganic phase (located in the bottom layer) wastransferred into a flask which already contains theresults of the previous extraction, then addedCHCl3 to mark and shaken until homogeneous. Thesolution absorbance was measured using UV-Visspectrophotometer. The same procedure wascarried out for addition of a buffer at pH 4, 5, 6, 7,8 for determining optimum pH, and then it makescorelation between pH with absorbance. Theoptimum pH was can be used to next procedure todetermine maximum wavelength and optimumconcentration of the complex Hg(II)-dithizone withthe same prosedur based on above. The maximumwavelength was measured from 380 until 780 nm.The optimum absorbance was meant a optimumwavelength from complex compounds Hg (II)-dithizone. The optimum concentration usingdithizone solution with concentration 0.003; 0,004;and 0.01%. Then it makes corelation between pHwith absorbance.

2.2.3 Preparation of Sticks Chemical SensorWhatman size of 5 cm x 10 cm was prepared. A

total of 2 mL dithizone reagents as a results of theabove procedure was filled into petridis thenWhatman was inserted until the entire section ofthe paper submerged for 20 minutes. Whatmanwhich already contain reagents was cut to a size of1 cm x 1 cm and placed on the supporting materialin the form of photo paper, using glue so that theresulting chemical sensors such as universalindicator stick form.

2.2.4 Preparation of Color Intensity Hg-DithizoneInto a 100 mL glass beaker Hg2+ standard

solution with a final concentration of 1 ppm atoptimum pH was added with 3 drops of ethanol,then stirred with a magnetic stirrer and then

chemical sensor was dipped for 20 seconds, Thechange of chemical sensor color was then capturedby camera. The same procedure was done for Hg2+

standard solution of 2,3,4,5 ppm.

2.2.5 Determination of Chemical Sensor SelectivityThe selectivity test was conducted by the

addition of Pb2+ ion. A 1 mL of 10 ppm Hg2+

standard solution was added with 1 mL of 10 ppmPb2+ standard solution in order to obtainconcentration ratio of Hg2+ / Pb2+ at 1: 1. Thenchemical sensor was dipped into a solution. Thecolor intensity obtained was compared with thecolor intensity of Hg2+ concentrations of the samesolution without the addition of Pb 2+, in order toknow the effect of Pb2+ addition. The sameprocedure is performed for Hg 2+ / Pb 2+ 1: 0, 1:10and 1: 100.

2.2.6 Comparison With Spectrophotometry MethodInto 6 pieces separating funnel, each filled with

5 mL of solvent CHCl 3, then into each of theseparating funnel successively added to thestandard solution of 100 ppm Hg 2+ as much as 0.0;1.0; 2.0; 3.0; 4.0 and 5.0 ml, then each added asolution ditizon excess of 0.01% and 2 mL ofbuffer solution pH optimum, then extracted byshaking for 30 minutes. The organic phase (locatedon the bottom layer) was transferred to 10 mLvolumetric flask. The solution left in the separatingfunnel was added with 3 mL solvent CHCl3 thenre-extracted by shaking for 30 minutes. Theorganic phase (located in the bottom layer) wastransferred to a flask already containing theextracted earlier, then added with CHCl3 to markand shaken until homogeneous. The solution wasanalyzed using UV-Vis spectrophotometer. Thedata obtained is used to create a standard curvebetween concentration of Hg 2+ and absorbance andthen used to test the validity.

2.2.7 Sample AnalysisA total of 0.05 grams of the drug sanpicilin was

dissolved with distilled water and added with 1 mLof standard solution of 3 ppm Hg2+, then wetdigested using HNO3 1N and heated to atemperature of 300 °C. After a clear solution, thesample is filtered. Furthermore, the sample wasneutralized using 1N NaOH and transferred into a10 mL volumetric flask. Sensors that have beenprepared, dipped into the sample and the colorformed compared to the sequence of colors.


11Nisa et al.


RESULT AND DISCUSSION

Determination of the dithizone maximumwavelength was done by measuring absorbancedithizone 0.01% solution in chloroform using UV-Vis spectrophotometer. Blank used waschloroform. The results of measurements ofmaximum wavelength of 0.01% dithizonecompounds Fig 1 shows that dithizone hasmaximum wavelength 605 nm. Whiledetermination of optimum pH was conducted todetermine the optimum formation condition ofcomplex Hg(II) -dithizone.

In this study, determination of the optimum pHwas carried out using a 1 mL solution of 100 ppmHg2+ with variations in pH 3, 4, 5, 6, 7, and 8 andreagent dithizone 0.01%. Blank used are CHCl3,dithizone reagent, and a buffer solution withdifferent variations. The color change is from greento orange. Results of the determination of theoptimum pH Fig 2 shows that at pH 4 is anoptimum pH of complex Hg (II) -dithizone becauseit produces the highest absorbance. At pH 5, 6, 7,and 8 decreased absorbance value, becausetheoretically based on the constant solubilityproduct (Ksp) of Hg(OH)2 3,6 x 10-26 possible forHg(II) to form Hg(OH)2, so the concentration of Hg2+ form complexes with dithizone will decrease andcause the absorbance value decreases.

While the determination of the maximumwavelength of complex Hg(II) -dithizone was doneby measuring the maximum absorbance of thereaction between 1 ppm Hg2+ and 0.01% dithizonereagents at optimum pH. Blank used is a mixture ofCHCl3, 0.01% dithizone reagents and buffer

solutions of each variation. Complex Hg(II)-dithizone will yield an orange solution, thenmaximum wavelength of complex Hg(II)-dithizonewas determined UV-Vis spectrophotometry at awavelength of 380-780 nm. Hg(II)-dithizonewavelength is 494.5 nm, and it can be seen atFigure 1. Wavelength of complex compound hasshifted to a lower wavelength when compared withthe maximum wavelength of dithizone.

The optimum concentration of the dithizone cannot be distinguished visually but an increase inabsorbance take place when the dithizoneconcentration of 0.002% to 0.01%. Therefore it canbe concluded that the concentration of the dithizonereagents affect the formation of complex Hg(II)-dithizone. Thus, the optimum concentration ofdithizone was considered 0.01%.

Rows of color intensity is made to be used as areference in the analysis of Hg2+ both qualitativelyand semiquantitatively. Range of Hg2+

concentrations used in the development of colorintensity of this series is the same as thedevelopment of standard curve (1, 2, 3, 4 and 5ppm) Fig 3. Fig 3 shows that the intensity of thecolor complex Hg(II)-dithizone becomeincreasingly apparent with increasingconcentrations of Hg2+. Because of the ability of themetal to form a complex with the dithizone.

0 ppm 1 ppm 2 ppm 3 ppm 4 ppm 5 ppm

Figure 3. The series colour of Hg2+ stick intensitysensor

The sample used in this study is sanpicilin drug.Sanpicilin drug is suspected to contain mercury sothat it becomes a choice in this study to analyze thesamples. Sanpicilin drug sample was weighed 0.05grams and then dissolved in destilled water thenadds across standard with the addition of Hg2+..Once matched with a series of color seen that thesamples that have been added to the standard

-0.1

0

0.1

0.2

0.3

0.4

0.5

380 580 780

Dithizone

ComplexHg(II)-Dithizone

Figure 1. The maximum wavelength of the dithizone 0.01% and complex Hg(II)-dithizone

Figure 2. The effect of pH complex Hg(II)-dithizone

0

0.2

0.4

0.6

3 5 7 9

Abs

orba

nce

pH


12Nisa et al.


solution so that the concentration of Hg 2+ 3 and 5ppm in color similar to the color sequence ofcomplex Hg (II) -ditihzone 3 and 5 ppm. It can beconcluded that the sanpicilin used to test theapplication method does not contain Hg2+ ions.

The result from the research can be validatedusing some parameters such as precision, accuracy,and limit of detection. Based on research , the valueof coefficient of variation (precision) is 3.6531%,the limit of detection (LOD) is 4.6738 x 10-3 ppm,linearity value of 99.7, its value amounted to 0,97accuracy, and sensitivity values obtained from thestandard curve slope is equal to 0.1166 / ppm Table1, 2, and 3.

Table 1 Validation parameters of methodValidation parameter Result

Limit of detection 4.6738 x 10-3 ppmLinearity 0,997sensitivity 0.1166 / ppm

Table 2 Data of the coefficient of variation

Table 3 Data of accuracyThe actual

concentration(ppm)

Measurableconcentration

(ppm)Accuracy (%)

1 1.0823 92.393 3.0274 99.095 4.9944 100.11

Average 97.20

CONCLUSION

Based on this research, we have successfullydeveloped a stick chemical sensor using dithizoneto detect mercury in pharmacheuticalpreparations.The optimum concentration ofdithizone in the development of chemical a sticksensors to detected mercury in pharmaceuticalpreparations is 0.01%, and the optimum pH is 4.

ACKNOWLEDGEMENTS

The authors are grateful to AnalytycalLaboratory of Chemistry Department of AirlanggaUniversity, Surabaya, Indonesia for providing thefacilities to carry out this research work.

REFERENCES

[1] WHO. Elemental Mercury And InorganicMercury Compounds:Human Health Aspects.Published under the joint sponsorship of theUnited Nations Environment Programme, theInternational Labour Organization, and theWorld Health Organization, and producedwithin the framework of the Inter-OrganizationProgramme for the Sound Management ofChemicals. Genewa 2003.

[2] Ali R. Firooz a , Ali A. Ensafi, K. Karimi a,H. Sharghi, Development of a specific andhighly sensitive optical chemical sensor fordetermination of Hg(II) based on a newsynthesized ionophore, J. Materials Scienceand Engineering., C 33, 2013, 4167–4172.

[3] Neha Thakur, Sanjukta A. Kumar, K.S. AjishKumar, Ashok K. Pandey,Sangita D. Kumar,A.V.R. Reddy, Development of a visualoptode sensor for onsite determinationofHg(II), J. Sensors and Actuators., B 211,2015, 346–353.

[4] Soner C¸ubuka, Melike Fırlakb, Nes¸e Tas¸cia, Ece Kök Yetimo˘glua,,Memet VezirKahraman, Phosphonic acid based polymericfluorescent sensor for Hg(II) analysis, J.Sensors and Actuators., B 224, 2016, 640–647.

[5] Lulu Tana, Yimeng Zhanga, Hong Qianga,Yonghui Lic, Jingyan Suna, LiangyuHua,Zhengbo Chena, A sensitive Hg(II)colorimetric sensor based on synergisticcatalyticeffect of gold nanoparticles and Hg, J.Sensors and Actuators., B 229, 2016, 686–691.

[6] Y. Gao, Z. Shi, Z. Long, P. Wu, C. Zheng, X.Hou, Determination and specia-tion ofmercury in environmental and biologicalsamples by analytical atomicspectrometry,Microchem. J., 103 (2012) 1–14.

[7] M. Jose da Silva, A. Paula, S. Paim, M.Fernanda Pimentel, M. Luisa Cervera, M. delaGuardia, Determination of mercury in rice bycold vapor atomic fluorescencespectrometryafter microwave-assisted digestion, Anal.Chim., Acta 667 (2010)43–48.

[8] J. Djedjibegovic, T. Larssen, A. Skrbo, A.Marjanovic, M. Sober, Contents ofcadmium,copper, mercury and lead in fish from Neretvariver (Bosnia andHerzegovina) determined byinductively coupled plasma massspectrometry(ICP-MS), Food Chem. 131(2012) 469–476.

[9] E. Kenduzler, M. Ates, Z. Arslan, M.McHenry, P.B. Tchounwou, Determinationofmercury in fish otoliths by cold vaporgeneration inductively coupled plasmamassspectrometry (CVG-ICP-MS), Talanta 93(2012) 404–410.

Hg2+

concentration(ppm)

StandardDeviation

(SD)

Coefficientof Variation (%)

1 0.0082 6.623 0.0104 2.885 0.0182 3.05

Average 3.65


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[10]10.Y. Zhao, J. Zheng, L. Fang, Q. Lin, Y. Wu,Z. Xue, F. Fu, Speciation analysis ofmercuryin natural water and fish samples by usingcapillary electrophoresis-inductively coupledplasma mass spectrometry, Talanta 89 (2012)280–285.

[11]P.R. Aranda, R.A. Gil, S. Moyana, I.E. Devito,L.D. Martinez, Cloud point extractionofmercury with PONPE 7.5 prior to itsdetermination in biological samplesbyETAAS, Talanta 75 (2008) 307–311.

[12]F. Okcu, H. Ertas, F.N. Ertas, Determination ofmercury in table salt samplesby on-linemedium exchange anodic strippingvoltammetry, Talanta 75 (2008)442–446.

[13]X. Jia, Y. Han, X. Liu, T. Duan, H. Chen,Speciation of mercury in water samplesbydispersive liquid–liquid microextractioncombined with high performanceliquidchromatography-inductively coupled plasmamass spectrometry, Spec-trochim. Acta Part B66 (2011) 88–92.

[14]14 J.L. Rodrigues, D.P. Torres, V.C. deOliveira Souza, B.L. Batista, S.S. deSouza,A.J. Curtius, F. F. Barbosa Jr.,Determination of total and inorganic mercuryinwhole blood by cold vapor inductivelycoupled plasma mass spectrometry (CV-ICP-MS) with alkaline sample preparation, J. Anal.At. Spectrochem. 24 (2009)1414–1420.


14Rukman et al.


Chemical Sensor for Melamine Based on Diazotitation Reaction UsingNaphthylethylenediamine

Nia K.Rukman, Syarifah A., Tjijik S. T, Miftakhul J, GandenSupriyanto1Department of Chemistry, Faculty Science and Technology, Airlangga UniversityJl. Mulyorejo Surabaya

60115, Indonesia2Laboratory of Sensor and biosensor, Institute of Tropical Disesase, Airlangga University Jl. Mulyorejo

Surabaya 60115, Indonesia

ABSTRACT

The development of chemical sensor of melamine with diazotitation reaction using naphthylethylenediamine hasbeen done. The objectives of this research are to know the chemical sensor capability with diazotitation reactionusing reagent naphthylethylenediamine for melamine analysis, to determine optimum reagent of chemical sensor,and also to determine validity method. Melamine is reacted with NaNO2 and hydrochloride acid in acidic solution toform diazonium salt and then added with naphthylethylenediamine reagent to form azo compound that gives coloredcomplex. The best result of optimum reagent for chemical sensor is NaNO2 8.10-3 M, HCl 9 N andnaphthylethylenediamine 8.10-5 M. Respon time of chemical sensor is very quick with reaction condition oftemperature is less than 15°C and pH is in acid condition. But, the stability and durability chemical sensor is low,the color is sharpeless and easily loss. The maximum of wave length is 549 nm. Standard of deviation and precisionare 0,0612 and 16,06% respectively. Linearity of chemical sensor in concentration of 8.10-6 up to 8.10-5 M. Limit ofdetection is 2,21 ppm, sensitivity 640,4 L/mol and recovery is 96,83%.

Keywords : Sensor, Melamine, naphthylethylenediamine, Milk

INTRODUCTION

Milk is a basic need that is very essential. Inbabies, the largest food intake found in dairyproducts. Scandal addition of melamine inimported Chinese powdered milk products hasshocked some countries, such as Asia, Europe,America, including Indonesia. As in the Reutersreports, the victims not only in China, in Taiwanthere are three toddlers and one positive womensuffering from kidney stones. Even a two year oldbaby is showing symptoms of kidney disease. Laterin Hong Kong, has reported five children showedsigns of kidney disease. It was shown afterconsuming milk production China [1].

Melamine is an organic compound with thechemical formula C3H6N6. Melamine has theIUPAC name 1,3,5-triazine-2,4,6-triamine, andcontains 66% nitrogen which is a toxic chemicalthat is commonly used to manufacture plastics,fertilizers and cleaning products. Melamine is awhite solid form with a molar mass of 126.12g/mol and a boiling point of 350°C. Melamine is

soluble in water, glycol, glycerol, pyridine, veryslightly soluble in ethanol, insoluble in ether,benzene, and carbon tetrachloride [2].

Melamine, commonly used as a protectivematerial as plastic resin fire and this, added to themilk during processing as if to increase the proteincontent because during this time the protein contentassessed by analysis of total nitrogen content.Based on the information on the WHO website,mixing melamine to milk is milk adulterationpractices. Manufacturer of adding water to rawmilk so that the milk will be many. After becomingmore dilute, of course, the concentration of proteinin the milk will go down. In order for proteincontent remains high, it is added melamine. Userenterprises generally check raw milk proteincontent of milk by testing the levels of nitrogen.Because melamine is rich in nitrogen compounds(66% melamine is nitrogen), the addition ofmelamine to raw milk will increase the levels ofnitrogen, up to as if the protein content is also high[3].

Analysis of melamine requires an analyticalmethod which has high sensitivity and selectivity.


15Rukman et al.


The measurement technique used to analyzemelamine includes glassy carbon electrode [4],High Performance Liquid Chromatography(HPLC) [5], Gas Chromatography–MassSpectrometry (GC-MS) [6], a variation of liquidchromatography (hydrophilic liquidChromatography, HILIC) combined withUltraviolet (UV) [7]. Advantages of these methodsare that it can produce high levels of melamine inmilk quickly and accurately. But for the generalpublic is difficult to use and requires substantialinvestment and high operating costs. Therefore, thedevelopment of the new analysis method ofmelamine is strongly required which are lowoperational costs, high sensitive, selective, simpleand easy to be used by everyone. Therefore, for theanalysis of melamine need to develop newtechniques and methods that require operationalcosts are quite low but has high sensitivity andselectivity of chemical sensors which method issimple so easy to use everyone.

Melamine having a primary amine groupattached to an aromatic ring diazotized usingHONO (derived from NaNO2 with HCl) whichwould produce a diazonium salt. The salt whenreacted with naftiletilendiamin produce compoundsthat are purple. In this study, created a chemicalsensor in the form of a solution to detect thepresence of melamine. These reagents are expectedto be sensitive to melamine. The analysis ofmelamine in liquid samples can be done by addingreagents Hono and naftiletilendiamin certainvolume into the sample. Fluid samples containingmelamine will react with Hono through diazotizingreaction, then reacts with naftiletilendiamin. Theconcentration of melamine in the sample solutionwill be proportional to the intensity of the colorpurple are formed and compared with the colorintensity of a reaction between melaminenaftiletilendiamin with standard solutions.

MATERIALS AND METHODSMaterials

The chemicals used in this study includedmelamine (C3H6N6), NaNO2, HCl,naphthylethylenediamine solution, distilled water.All materials used were pure analytical grade.

InstrumentationInstruments used was UV-Vis

Spectrophotometer Shimadzu-1700.

Preparation of reagentMelamine (0.1250 g) was weighed

quantitatively and dissolved in water while heatedthen the solution was quantitatively transferred to a250 mL volumetric flask and made up withdistilled water to mark and whipped untilhomogeneous.

Melamine solution 4.10-4 M was prepared bypipetting 10,0 ml of melamine standard solution4.10-3 M and was diluted with distilled water in a100 mL volumetric flask and made up and shakenuntil homogeneous. Then standard solution with aconcentration 0,8.10-5; 1,6.10-5; 2,4.10-5; 4.10-5;5,6.10-5 and 8.10-5 M were created.

NaNO2 (138 mg) was weighed quantitativelyand dissolved in distilled water. The solution wastransferred to a 250 mL volumetric flask and madeup with distilled water to mark and whipped untilhomogeneous. Then standard solution with aconcentration of 8.10-4 M; 8.10-5 M were created.

Concentration optimization ofnaphthylethylenediamine and NaNO2/HCl

A 5.0 mL of standard solution containingmelamine was pipetted and put in a 25 mLvolumetric flask. Distilled water was added to asolution of melamine consecutive NaNO2 and HClat temperatures <15°C then addednaphthylethylenediamine reagent so that theconcentration of melamine, NaNO2, HCl andnaphthylethylenediamine each 8.10-5 M. Thenvariation of the concentration of melamine:NaNO2, HCl, naphthylethylenediamine was createdby 1:1; 1:3 and 1:9. The same procedure was donefor various concentration 8.10-3 and 8.10-4 M.

Data Comparative With UV-Vis spectrophotometryA series of melamine solution; 0,8.10-5; 1,6.10-

5; 2,4.10-5; 4.10-5; 5,6.10-5 and 8.10-5 M was reactedwith NaNO2, HCl, and naphthylethylenediamine atthe optimum concentration. Absorbance of thesolution was measured by UV-Visspectrophotometer at the maximum wavelength.Then standard curve was made where theconcentration as the x-axis and absorbance as they-axis.

Determination of Melamine in Milk with StandardAddition Method

Accurately, it was weighed 1000 mg the milkand put in a centrifuge tube quantitatively, thenadded with 5 ml of standard solution of melaminewith 6 variations of concentration that are 0,8.10-5;1,6.10-5; 2,4.10-5; 4.10-5; 5,6.10-5 and 8.10-5 M.Then milk with the addition of melamine wasadded with 1.5 ml TCA evenly through stirring toprecipitate the protein.

Determination of Validation ParameterThe determination of variation coefficient of

sample was done with three replications. Then theabsorbance of each sample was measured by usingUV-Vis spectrophotometer and the value of thestandard deviation (standard deviation = SD),coefficient of variation (CV) and the detection limitwere determined. Linearity was obtained fromstandard curve of standard solution. The sensitivityis obtained from the slope of the standard curve


16Rukman et al.


equation. Accuracy is obtained by calculating therecovery of known melamine concentration levels.


The maximum wavelength of melamine-naftiletilendiamin complex compound wasdetermined by measuring the absorbance of thecomplex compounds. Measurements were taken atvarious concentrations, while the pH used is atacidic pH because diazotitazion reaction only takesplace at acidic conditions with temperatures around0°C. In this study NaNO2 and HCl were used toform diazonium ions, and then added naphthylethylenediamine to form complex compounds. Themaximum wavelength was obtained from thosemeasurement using UV-Vis spectrophotometer at awavelength of 450-800 nm. The maximumwavelength of melamine-naphthylethylenediaminecomplex solution was used to develope thestandard curve of melamine at a concentration of8.10-3 M NaNO2, HCl 9 M, andnaphthylethylenediamine 8.10-5 M. The formationof a complex between melamine-naphthylethylenediamine produces purplecompound. This complex response time is veryfast, but the stability and durability is low. It isbecause the complex is dependent on thetemperature and acidic conditions. The results ofthe study can be seen in Fig.1. Based on thepictures, the maximum wavelength of melamine-naphthylethylenediamine complex compound is549 nm. Absorbance measurement of the complexof melamine-naphthylethylenediamine the visiblearea will lead to the absorption of radiation by thecomplex of visible light so that it will raise theenergy of the ground state to an excited state,followed by the excitation of electrons of orbitalbonding and non-bonding orbital into the anti-bonding orbital. Electron excitation of bonding andnon-bonding orbital to orbital anti-bonding occursbetween N on melamine with N innaphthylethylenediamine diazonium ion that hasbeen shaped first by the addition of NaNO2 withHCl.

Figure. 1 The maximum wavelength of melamine-naphthylethylenediamine complex.

Determination the optimum reagentconcentration of NaNO2, HCl andnaphthylethylenediamine in this study used avariation of concentration 8.10-5; 8.10-4 and 8.10-3

M. In addition, the optimization was done by theaddition of excess reagent. The most significantchange in chemical sensors was at the time ofaddition of reagents with each concentration 8.10-3

M. Only the addition of this reagent, will form acolor also in the form of water without melamine.In a variation NaNO2 concentration 8.10-3 M, 9 MHCl, and naphthylethylenediamine 8.10-5 Mintensity of the color change is not too high. This isbecause the conditions are not too acidic and it candegrade performance naphthylethylenediaminereagent to form a bond with the azo compoundmelamine. But the difference in the color of theblank water with melamine samples are quitedifferent, so that the chemical sensors with NaNO2

concentration 8.10-3 M, 9 M HCl, andnaphthylethylenediamine 8.10-5 M was selected foruse in the determination of the color intensity of theseries.

This standard curve was made from reagentnaphthylethylenediamine 8.10-5 M. In thisexperiment, a solution of melamine with variousconcentrations 0,8.10-5; 1,6.10-5; 2,4.10-5; 4.10-5;5,6.10-5 and 8.10-5 M were used. The results of thisstudy can be seen in Table 1 which shows theresults of the average absorbance of the threereplications in a solution of melamine-naphthylethylenediamine. The standard curvegraph based on average absorbance of melamineand naphthylethylenediamine complex can be seenin Fig.2. From the calibration curve, the linearregression equation is y = 640,4x + 0.348 with acorrelation coefficient R2 = 0.963. The standardcurve was then used to determine the detectionlimit, linearity and accuracy.

Table 1. Absorbance of melamine complex withnaphthylethylenediamine in waterNo. Concentration (M) Absorbance n=31. 0,8.10-5 0,34822. 1,6.10-5 0,35903. 2,4.10-5 0,36564. 4.10-5 0,37755. 5,6.10-5 0,38526. 8.10-5 0,3961

0

0.5

1

1.5

450 500 550 600 650 700 750 800

Abso

rban

ce

Wavelength (nm)y = 640.48x + 0.348

R² = 0.96380.340.360.38

0.40.42

0 0.00005 0.0001

Abso

rban

ce

Concentration (M)


17Rukman et al.


Figure 2. Standard curve of melamin-naphthylethylenediamine in water

The standard curve was also developed byaddition of standard melamine into milk because itis difficult to find milk containing melamine on themarket. Standard solution of melamine in milk hasa concentration 0,8.10-5; 1,6.10-5; 2,4.10-5; 4.10-5;5,6.10-5 and 8.10-5 M. The results showed theaverage absorbance of the melamine-naphthylethylenediamine in milk. Standard curveof melamine in milk is shown on Fig.3. From thecalibration curve is obtained the linear regressionequation y = 430,4x + 0.303 with a correlationcoefficient R2 = 0.976. The resulting absorbance isalmost the same as the absorbance resulting inmeasurements for pure melamine used in theexperiment.

Table 2. The absorbance of melamine milk-naphthylethylenediamine in milk matrixNo. Concentration (M) Absorbance n=31. 0,8.10-5 0,30532. 1,6.10-5 0,30943. 2,4.10-5 0,31564. 4.10-5 0,32305. 5,6.10-5 0,32886. 8.10-5 0,3362

Figure 3. Standard curves of melamine-naphthylethylenediamine in milk matrix

The result of the calculation of standarddeviation and coefficient of variation based on datagenerated are 0.0612 and 16.06% respectively.Small standard deviation indicates that the figuresobservation is less scattered or nearly uniform.Conversely, when the value of the standarddeviation greater then the numbers fluctuate widelyor further observations are not uniform. Thecoefficient of variation for an experimentconducted with both ranged between 15-20% forbioanalytical and below 5% for non bioanalyticalresearch. If the coefficient of variation is too smallor too large, it is an indicator among others there isan error in the measurement or recording, perhapseven in the analysis of data and if the coefficient ofvariation is too large, it can be due to the samplesize is too small or little.

The detection limit is determined using astandard curve and linear regression equation is y =

640,4x + 0.348 with a correlation coefficient R2 =0.963. The result of the calculation indicates thatdetection limit is 1,75.10-5 M, which equivalent to2.21 ppm. From the result of detection limits, it canbe seen that the sensor which consists of a mixtureof NaNO2, HCl and naphthylethylenediamine isquite sensitive. Because normally the levels ofmelamine found in milk ranged from 0.09 mg/kg ofmilk up to 619 mg/kg of milk, equivalent to 0.09ppm up to 619 ppm levels.

Levels of melamine in the range of 8.10-6 to8.10-5 M provide proportional correlation betweenthe concentration of melamine with an absorbancevalue. The levels of melamine above 8.10-5 Mshows the absorbance values are not linear, then itthen causes the regression coefficient decreases andthe value of linearity reduced. Based on linearregression obtained from the graph, the sensitivityof the developed sensor is 640.4 L/mol. Accuracywas obtained from the standard curve by takingthree concentrations of melamine standard solutionin the small concentrations, middle, and large (ie8.10-6 M, 4.10-5 M and 8.10-5 M). Recoveryobtained of chemical sensors in this study is96.83%. It can be conluded that the results ofmeasurements in this study is accurate, since thevalue of recovery is close to 100%.

CONCLUSION

We have successfully demonstrated thatchemical sensors by usingnaphthylethylenediamine as complexing reagent todetect melamine can be developed. From thepresent study it can be concluded that this methodhas simple methodology, easy work-up, shortreaction times, low cost, and accurate.

ACKNOWLEDGEMENTS

The authors are grateful to AnalyticalLaboratory of Chemistry Department of AirlanggaUniversity, Surabaya, Indonesia for providing thefacilities to carry out this research work.

REFERENCES

[1] Irhama, 2008, Melamin Dalam Produk Pangan,Info POM Bahan Pengawas Obat dan MakananRI, Vol.9, No.6, November 2008

[2] Nshisso, L.D., 2010, Melamine Contaminationof Infant Formula, Case Western University

[3] Lewis, 1993, Hazardous Chemicals DeskReference, Third Edition, New York, 799.

[4] Wanqing Zhang, Guangri. Xu, Runqiang Liu,Jun Chen, Xiaobo Li, Yadong Zhang, YupingZhang, Novel MOFs@XC-72-Nafionnanohybrid modified glassy carbon electrodefor the sensitive determination of melamine,Electrochimica Acta, 211, 2016, 689-696.

y = 430.45x + 0.3036R² = 0.9766

0.30.310.320.330.34

0 0.00005 0.0001

Abso

rban

ce

Concentration (M)


18Rukman et al.


[5] Virginia de L. M. Finete, Marcos M. Gouvea,Flávia F. de C. marques, Annibal D. PareiraNetto, Validation of a method of highperformance liquid chromatography withfluorescence detection for melaminedetermination in UHT whole bovine milk,Food Control, 51, 2015, 402-407.

[6] Jia. Li, Huang-Yang Qi, Yan-Ping Shi,Determination of melamine residues in milkproducts by zirconia hollow fiber sorptivemicroextraction and gas chromatography–massspectrometry, Journal of Chromatography A,1216, 2009, 5467-5471.

[7] Xiao-Lin Zheng, Bing-Sheng. Yu, Ke-Xian Li,Ying-Na Dai, Determination of melamine indairy products by HILIC-UV with NH2

column. Food Control, 23, 2012, 245-250.


Oktavitri et al. 19


COMPARATIVE COD ADSORPTION KINETIC OF COCONUTSHELL ACTIVATED CARBONNur Indradewi Oktavitri*, Hery Purnobasuki, Eko PrasetyoKuncoro, Indah Purnamasari

Department of Biology, Faculty of Science and Technology, Airlangga University, Indonesia, Mulyorejo street,Airlangga University, Surabaya 60115,

*Email: [email protected]

ABSTRACT

Coconut shell activated carbon was used as adsorbent in batch experiment for COD removalfrom dairy waste simulated waste water. This research concerned adsorption kinetics of CODremoval from several activation periods of coconut shell. Classical adsorption kinetic,traditional and innovative Pseudo kinetic was observed in this research. The coconut shell wasphysically activated in two steps. First step was activation in 500oC and the second step was220oC. This research focused on secondary step of physical activation in 1, 2, and 4 hours in220 oC. Coconut shell also activated with chemical activation using H3P04. 20 g of coconutshell activated carbon added in reactor contained 10 l dairy simulated wastewater. The contacttime of coconut shell and dairy simulated waste water was 15 d in batch reactor. Theregression coefficient of the kinetic models of 1, 2, and 4 hours were followed the pseudosecond order kinetic model. The highest coefficient relation value was 1 h activation period,r2=0.989.

Keywords:Activation periods, Adsorption Kinetic, Activated Carbon, COD

INTRODUCTION

Indonesia is one of the largest coconutproducing countries in Asia. Indonesia is able toproduce 16.846 million tons of coconut. All thecomponents in a palm tree can be used. It includeswood, leaves, and fruit. The greatest utilization ison its fruit. In 2008, produced 61 million tons ofcoconuts in the world, and 85% came from Asia,including Indonesia. The large utilization ofcoconuts will produce waste that is great as well,especially waste coconut shells. Coconut shellwaste will accumulate and not worth if it is notprocessed into something more valuable.In fact,Indonesia as well as the largest producer of coconutin Asia [1].

One of ways to reduce coconut shells waste isto utilize coconut shells for the wastewatertreatment process. Waste coconut shells can beused as an adsorbent to remove organic matter inthe wastewater treatment process. Coconut shellsneed to be processed first before being made intoactivated carbon as adsorbent by carbonization andactivation process [2]. .In the carbonizationprocess, the volatile material will be removed andthe activation process was conducted to form thepores and also increase the surface area of activated

carbon from coconut shells [3]. In this research, weused several activation periods for the coconutshells. There are 1, 2, and 4 hour at 220oC. Theproperties of the coconut shells activated carbonare high number of pore and surface area as well ashigh adsorption capacity [4].

Wastewater which has a high organic mattercontent can degrade the quality of water bodies.Organic materials contained in wastewater must beremoved. Organic matter will reduce the oxygendissolved in the water body for the degradationprocess [5]. Organic matter in wastewater can bemeasured by chemical oxidation using potassiumdichromate called Chemical Oxygen Demand(COD).

Coconut shells activated carbon as adsorbentsproved capable of removing organic matter inwaste water. Some studies suggest that activatedcarbon from coconut shells capable to removeorganic material in the form of Chemical OxygenDemand (COD). According to Cruz et al. [6], whoused coconut shells activated carbon in theanaerobic treatment, the effluent reached a removalof 73% of the organic matter in COD terms. Han[7] stated that the wastewater treatment usinganaerobic reactor with activated carbon coconutshells at the OLR of 4-8 g /L.days able to remove


Oktavitri et al. 20


COD by 80%.Meanwhile, according to Akram andStuckey [8] stated that the wastewater treatmentusing activated carbon media by the OrganicLoading rate of 4-16 g / L.hari, capable ofremoving COD by 98%. The results of theOlafadehan, et al. [9] also produces 98% CODremoval using coconut shells activated carbon asadsorbent.

When the adsorption of COD using coconutshells activated carbon is concerned,thermodynamic and kinetic aspects should beinvolved to know more details about itsperformance and mechanisms. Except for adsortioncapacity, kinetic performance of a coconut shellsactivated carbon is also of great significance for thepilot application. From the kinetic analysis, thesolute uptake rate, which determines the residencetime required for completion of adsorptionreaction, may be established. Also, one can knowthe scale of an adsorption apparatus based on thekinetic information.

At present, adorption reaction models have beewidely developed or employed to describe thekinetic process of adsorption [10]. There areseveral adsorption reaction models, such as zeroorder rate equation, pseudo first order rateequation, and pseudo second order rate equation.This research aims to observe the adsorptionkinetics equation of COD removal from severalactivation periods of coconut shells activate carbon.

EXPERIMENTALSPreparation of Adsorbents

The coconut shells charcoal was prepared fromlocally industry. The charcoal was crushed untilthey had a same size at 20 mesh (0,3 cm asdiameter). Then, the charcoal was washed usingdemineralized water to eliminate dirt from its. Thecoconut shells activated carbon were prepared intwo steps. The first step was chemical activationand the second step was physical activation. In thechemical activation process, 50 g of the coconutshells charcoal was agitated with 250 g of aqueoussolution containing 10% H3PO4 by weight. Thechemical activation and coconut shells charcoalwere homogenously mixed at 85oC for 4 h in amixer. After mixing, the coconut shells charcoalslurry was dried under vaccum at 110oC. Thesecond process was physical activation. Thecoconut shells from the first step was heated to200oC for about 1, 2, and 4 hours. After cooling,the activated carbon was washed successivelyseveral times with hot water until the pH becameneutral and finally with cold water to remove theexcess phosphorus compounds.

Simulated WastewaterAbout 2 g NH4Cl, 1 g KH2PO4; 0,05

MgSO4.7H2O; 0,038 g CaCl2; 2 g NaHCO3 were

prepared for 1 litre dairy milk simulatedwastewater [11]. Thel 1 mL inoculum fromslaughter house was used for 100 mL dairy milksimulated wastewater. The Chemical OxygenDemand (COD) concentration of dairy milksimulated wastewater was 4000 mg/l.

The Batch Adsorption ExperimentsThe batch reactors were prepared from LDPE

which had volume 15 L. There were 2 area in theinside of reactors, the 10 L of reactor's volume wasthe dairy milk simulated wastewater and the 5 Lwas a void area. This reactor was operated inanaerobic condition. The outlet point was placed at13 cm of height reactor. The porous bag containedcoconut shell activated carbon was placed at thebottom of reactor. The coconut shells activatedcarbon was contacted with dairy milk simulatedwastewater for 16 days in the anaerobic reactor.The reactors were monitored every 2 days. Theschematic of reactor can be seen at Figure 1.

Figure 1. Illustration of Reactor

15 cm

26 cmOutlet for sampling Simulatedwastewater

Activatedcarbon

Figure 1 Ilustration of reactor

RESULTS AND DISCUSSIONEffect of Activation Time in COD Adsorption

Activation process of coconut shell wasrecommended to improved the adsorption capacityof adsorbent [12, 13]. Increasing the activation timewas assumed can increasing the pores of adsorbent. It caused increasing the ability to removed thepollutant. [14, 15]. Activation time in physicalactivation increased the ability of adsorbent forCOD adsorption after 16 days incubation (Figure2). Adsorption capacity was measured by comparedthe concentration at time-0 and concentration attime-t (equation 1). 1 hour activation time effectedto adsorption capacity. In other side, the adsorptioncapacity of 2 and 4 hours activation time hadsimilar adsorption capacity. It indicated thatincreasing 2 to 4 hours activation time hadstationer performance in activation capacity.= ( )

(1)

where: Co, Ce, qe as TAN concentration at t=0; asTAN concentration after adsorption process; asadsorption capacity at equilibrium, respectively.


Oktavitri et al. 21


050

100150200

1 2 4

qe (m

g/g)

Activation time (Hours)

139.832175.688

0

50

100

150

200

0 2 4 6 8 10 12 14 16

qe (m

g/g)

Contact Time (days)

Figure 2. Effect of activation time to adsorption capacity

Effect of Contact Time in COD AdsorptionContact time was contact duration between

adsorbent and adsorbate. Figure 3 illustrated thatthe early 4 days, the adsorption capacity of 2 hoursactivated time of coconut shell was fluctuated. In 6to 16 days, the adsorption capacity in the range140-175 mg/g. It was similar with stationerposition after 6 days. The stationer position inadsorption capacity represented that adsorbent wassaturated [16]. The degradation of COD inanaerobic reactor contain media was influencedwith adsorption capacity of media andmicroorganism activity inside wastewater. Theslowly process of COD degradation also caused theslowly process of organic compound degradationin anaerobic reactor [17].

Figure 3. Effect of contact time to adsorption capacity

Simple Adsorption KineticThe adsorption kinetic was used to determined

the residence time to complete the adsorptionreaction. Adsorption kinetic also used as scaleinformation for pilot scale. There are severaladsorption kinetic to predict the adsorptionperformance in batch studies. Zero order kineticwas the simple adsorption kinetic. Figure 4represented that the determination coefficient fromzero order kinetic was less than 0.8. It wasindicated that the zero order kinetic was unsuitablefor COD adsorption. Zero order reaction indicatedthat adsorption rate was independent from reactant[18]. It means that the increasing of adsorption timewill not effect to adsorption rate. The adsorptionrate for zero order kinetic in equation (2) [19].

Zero order kinetic model :Ct=-k0.t+A0 (2)

where Ct is the COD concentration (mg/L) at time t(d) and calculated by Ct=C0(1-(φt/100)); φt is theremoval of COD (%) at time t (d); k0, was the zeroorder constants respectively; A0, was constanta.

The another types of simple kinetic reactionswere first and second order reaction (Equation 3and 4). The first order reaction described that therate of change Ct was proportional with reagentwas given in certain contact time. Meanwhile, thesecond order reaction indicated that the change ofCt was propotional to one concentration squared, orto the product of two concentration,First order kinetic model :

lnCt = -k1.t+A1 (3)Second order kinetic mo :

1/Ct = k2.t + A2 (4)

Figure 4. Zero order kinetic models in variation


Oktavitri et al. 22


Where Ct is the COD concentration (mg/L) attime t (d) and calculated by Ct=C0(1-(φt/100)); φt isthe removal of COD (%) at time t (d); k1, and k2 arethe first order and second order reaction rateconstants respectively; A1, A2 are constanta.

Figure 5 represent the low coefficient relation(R2) in first order kinetic reaction, under 0.8 for allactivation time of coconut shell. The coefficientrelation increased when activation time of coconutshell also increased. Nevertheless, the maximum ofcoefficient relation at coconut shell in 4 hoursactivation time only 0.519. Actually, VonSperling [18] recommended first order kinetic forwastewater treatment because first order describedthe removal of organic matter and the decay ofpathogenic microorganism such as research ofWong & Springer [20] used a first order kineticmodel in anaerobic ponds.This research presented that simple kinetic reactionwas unsuitable for the reaction of activated coconutshell to removal organic matter. Von Sperling [18]added that stabilization of organic matter removalcan described with pseudo reaction. Simple kineticreaction has limitation to figured out the removalprocess of high concentration of organic matter.This research treated around 4000 mg/l of organicmatter in which it was represented in COD value.

Pseudo Kinetic ReactionPseudo First and second reaction were the

another kinetic reaction for adsorption. This kinetic

reactions were used to solved the limitation ofclassical kinetic. Lin et al [19] successfully usedpseudo reaction to describe COD removal. Thepseudo reaction based on the hypothesis that thereactions related to substance was occurred insurface of adsorbant. The equation of pseudoreaction was shown at equation (5) and (6).Pseudo first order:

log (Cr,max – Cr,t) = log Cr,max – K1.t (5)Pseudo second order:

t/Cr,t = t/(K2. (Cr,max)2) + t/Cr,max (6)

Where Cr,t (mg/l) and Cr,max (mg/l) are theconcentration of COD removal at t and themaximum concentration of COD removal, k1 andk2 were the pseudo first and second constant,respectively.

Figure 8 Shown that pseudo second orderreaction more fittet to described COD removalrather than Pseudo first reaction. Even, manyresearcher recommended pseudo first order suitablefor wastewater [21, 22]

The R2 values were above 0.9 for all adsorbentactivation time from linear regression.Thisevidence indicated that pseudo second order moresuitable for COD removal. It was similar with Linet al [19] research. It proved that COD removalwas fitted with assumption that the valent forcesinvolving ion exchange between organic compoundand component ions at activated coconut shell[21].

Figure 5. First order kinetic models in variation of activation time

Figure 6. Second order kinetic models in variation of activation time


Oktavitri et al. 23


Actually, regarding on theory of Qiu et al[21],Pseudo second order was not suitable for activatedcarbon from coconut shell such as Wen et al [23]used zeolite as non polar adsorbent also fitted withpseudo second order in ammonium adsorptionprocess. Pseudo second order also had limitationunsuitable described adsorption process for organicpollutant onto non polar polymeric adsorbent.Activated carbon from coconut shell was non polaradsorbent. It adsorbs non polar molecules betterthan polar molecule [22]. Research of Joseph andChinonye [24] using Kola Nut Activated Carbon toadsorb dyestuffs also fitted with pseudo secondorder reaction. It indicated that the wastewater inthis research was dominated with non polarmolecule. It also shown that pseudo secondaryorder kinetic fitted to non polar adsorbent in whichthe adsorbent have valent forces with adsorbate.Organic substance removal usually easily adsorbedwith media from natural adsorbent [24, 25].Polarity of adsorbant and adsorbate wereinfluenced factors of valent forces between both ofthem.

CONCLUSIONS

The simple kinetics and pseudo kinetic werecompared to find the best fitted model of CODadsorption using activated coconut shell in 1, 2,and 4 h activation time. The adsorption process wasobserved in 16 d. Extend the duration of activation

time until 4 h was not increased the adsorptioncapacity of COD. Activated coconut shell easilysaturated when removed COD only 6 d contacttime. The best fitted adsorption kinetic for CODremoval using activated coconut shell was pseudosecond order reaction.

ACKNOWLEDMENT

This research is part of research grant whichsponsored by DIPA Ditlitabmas 2015 and 2016

REFERENCES

[1] Siriphanich, J., Saradhuldhat, P., Romphopak,T., Krisanapook, K., Pathaveerat, S., danTongchitpakdee, S., Woodhead PublishingSeries in Food Science, Technology andNutrition, 2011, 8-28.

[2] Tani, D., Setiaji, B., Trisunaryanti, W., andSyouflan A., Effect of activation time onchemical structure and quality of coconut shellactivated carbon, Asian Journal of Science andTechnology, 5, 9, 2014, 553-556.

[3] Kirk and Othmer, Encyclopedia of chemicaltechnology, Third Edition, New York, WileyInterscience Publication, 1983.

[4] Verma, S., Prasad, W., and Mishra, I. M.,Adsoption kinetics and thermodynamics ofCOD removal of acid pre-treatedpetrochemical wastewater by using granular

Figure 7. Pseudo first order kinetic models in variation of activation time

Figure 8. Pseudo second order kinetic models in variation of activation time


Oktavitri et al. 24


activated carbon, Separation Science andTechnology, 49, 2014, 1067-1075.

[5] Von Sperling, M. 2007. WastewaterCharacteristic, Treatment, and Disposal. IWAPublishing, London. 77-82.

[6] Cruz, L. M. D., Stefanutti, R., Filho, B. C., andTonetti, A. L. Cocomnut Shell as FillingMaterial For Anaerobic Filters, Springerplus2013, 2: 655.

[7] Han, W., Chen, H.,Yao,X., Li, Y., and Yang,C. Biohydrogen production with anaerobicsludge immobilized by granular activatedcarbon in a continous stirred-tank, Journal ofForestry Research, 21, 4, 2010, 509-513.

[8] Akram, A. and Stuckey, D. C. Flux andperformance improvement in a submergedanaerobic membrane bioreactor (SAMBR)using powdered activated carbon (PAC),Journal of Bioresource Technology, 43, 2008,93-102.

[9] Olafadehan, O. A., and Jinadu, O. W.,Treatment of brewery wastewater effluentusing activated carbon prepared from coconutshell, International Journal of Applied Scienceand Technology, 2, 1, 2012, 165-178.

[10] Chen, Y., Cheng, J. J., and Creamer, K. S.Inhibiton of anaerobic digestion process: Areview, Bioresource Technology, 99, 2008,4044-4064

[11] Dawood, A. T., Kumar, A., and Sambi, S. S.,Study on anaerobic treatment of synthetic milkwastewater under variabel experimentalconditions, International Journal ofEnvironmental Science and Development, 2, 1,2011, 17-23.

[12] Meisrilestari, Y., Khomaini, R., Wijayanti, H.Pembuatan Arang Aktif dari Cangkang KelapaSawit Dengan Aktivasi Secara Fisika, Kimiadan Fisika dan Kimia, Jurnal Konversi, 2(1):46-51.

[13] Guo, Y. Rockstraw, D. A.. PhysicochemicalProperties of Carbons Prepared From PecanShell by Phosporic Acid Activation.Bioresources Technology, 98: 1513-1521.

[14] Katesa, J., Junpiromand, S., andTangsathitkulchai, C.. Effect of CarbonizationTemperature On Properties of Char AndActivated Carbon From Coconut Shell.Suranaree J. Sci. Technol., 20(4): 269-278.

[15] Legrouri, K., El Harti, M., Oumam, M.,Khouya, E., Wahbi, R., Hannache, H.,Zarrouk, A. Characterization and EvaluationPerformance of Activated Carbon PreparedFrom Coconut Shell Argan. Journal ofChemical and Pharmaceutical Research, 4(12):5081-5088.

[16] Kučič, D., Markić, M., Briški F. (2012).Ammonium Adsorption On Natural Zeolite(Clipnoptilolite): Adsorption Isotherms and

Kinetics Modeling. The Holistic Approach toEnvironment, 2(4): 145-158.

[17] Chernicharo, C. A. L. (2007). AnaerobicReactors. IWA Publishing, London. 1-38.

[18] Von Sperling, M. 2007. Basic Principle ofWastewater Treatment. IWA Publishing,London. 23-28.

[19] Lin, H., Lin, Y., and Liu, L., Treatment ofdinitrodiazophenol production wastewater byFe/C and Fe/Cu internal electrolysis and theCOD removal kinetics, Journal of The taiwanInstitute of Chemical Engineers, 50, 2015,148-154.

[20] Wong, K. K., A first order kinetic model fordesigning anaerobic ponds in the treatment ofpalm oil mill effluent, Agricultural Waste, 3,1981, 35-42.

[21] Qiu, H., Lv, L., Pan, B., Zhang, Q., Zhang, W.,and Zhang, Q., Critical review in adsorptionkinetic models, Journal of Zhejiang UniversityScience, 10, 5, 2009, 716-724.

[22] Çeçen, F., Aktaş, Ő. (2012). Activated CarbonFor Water and Wastewater Treatment. Wiley-VCH Verlag GmbH dan Co. KgaA. Germany.13-86.

[23] Wen, D., Ho, Y. S., and Tang, X.,Comparative sorption kinetic studies ofammonium onto zeolite, Journal of HazardousMaterials, B133, 2006, 252-256.

[24] Joseph, N. T. and Chinonye, O. E. Isothermand Kinetic Modeling of Adsorption DyestuffsOnto Kola Nut (Cola acuminate) ShellActivated Carbon.

[25] Halim, A. A., Abidin, N. N. Z., Awang, N.,Ithnin, A., Othman, M. S., Wahab, M. I.(2011). Ammonia and COD Removal FromSynthetic Leachate Using Rice HuskComposite Adsorbent. Journal of Urban andEnvironmental, 5(1): 24-31.


Sucipto et al. 25


Synthesis and Characterization of Copper(II) Complex with 2,4,5-TriphenilimidazoleTeguh Hari Sucipto1,2*, Harsasi Setyawati3, Siti QamariyahKhairunisa1, Fahimah Martak2

1Institute of Tropical Disease, Airlangga University, Kampus C Jl. Mulyorejo, Surabaya 60115, East Java,Indonesia

2Department of Chemistry, Faculty of Mathematic and Natural Science, Sepuluh Nopember Institute ofTechnology, Kampus Sukolilo Jl. Raya ITS, Surabaya, 60111, East Java, Indonesia

3Department of Chemistry, Faculty of Science and Technology, Airlangga University, Kampus C Jl. Mulyorejo,Surabaya 60115, East Java, Indonesia

*Corresponding author: Institute of Tropical Disease, Airlangga University, Kampus C Jl. Mulyorejo, Surabaya,East Java 60115, Indonesia.

E-mail: [email protected]

ABSTRACT

This research aims to synthesize and characterize complex compound of copper(II)-2,4,5-triphenylimidazole. Thiscomplex compound synthesized by reacted copper(II) from CuCl2.6H2O and 2,4,5-triphenylimidazole ligand withstoichiometric copper(II):2,4,5-triphenylimidazole = 1:2. The complex was synthesized and characterized by UV-Visspectroscopy, IR spectroscopy, N2 adsorption-desorption, and thermal gravimetric analyzer (TGA). This researchobtained solid yellow green. Characterization of compound are 309 nm and 243 nm, vibration Cu-N band appearsat 493 cm-1 which supports the formation of Cu-N bond. This clearly proves the coordination of imidazole to metalions through this N atom and stability thermal this compound up to 250 °C.

Keywords: copper(II), 2,4,5-triphenylimidazole, complex compound, characterization.

INTRODUCTION

Complex compounds are the one which playsan important role in human life. These compoundsare formed because of the bond between a ligandthat acts as an electron-pair donor (Lewis base)with a central metal ion (metal) that act as electron-pair acceptors (Lewis acids). Nowadays, thedevelopment of science complex compounds morerapidly.

Copper(II) complexes of imidazole are of greatpharmacological interest and importance as severalof them present a wide spectrum of effects,including, anticancer, antibacterial, and antifungal[1,2,3]. The copper(II) complexes of imidazole wasshowed an interesting inhibition of the growth ofall Gram-positive bacteria and fungi tested atconcentrations of 12-50 µg/ml [3]. Low IC50 meansthe compound has high activity, high CC50 meansthe compound has less toxicity, and high selectivityindices (SI) means the compound has highpossibility of the drug. Therefore, in this study thesynthesis and characterization of complexcompounds Copper(II)-imidazole so that later canbe used as a drug.

EXPERIMENTALMaterials

Chemical reagents used in this research is thecopper(II)chloride hexahydrate (CuCl2.6H2O)(Merck 99.0%), N,N-dimethylformamide (DMF)

(Merck 99.8%), 2,4,5-triphenylimidazole ligand(Sigma-Aldrich 90%), and ethanol (Sigma-Aldrich96%).

Synthesis of complex compoundComplex compound synthesis was performed

using a mole ratio. Copper(II)chloride hexahydratereacted with 2,4,5-triphenylimidazole ligand in aDMF [4] as much as 2 ml. The complex compoundsolution was inserted into the bottle autoclave,stirred for 3 hours and heated to a temperature of120° C. The complex was separated from thereaction mixture by filtration, washed with ethanoland dried [5].

Figure 1. UV vis Spectra of complex compound Cu(II)-2,4,5-triphenylimidazole


Sucipto et al. 26


UV-Vis spectroscopyThe complex compound was dissolved in DMF

and determined its spectrum in the visible region(200-700 nm).

Infrared spectroscopyThe complex compound dispersed in KBr with

the ratio of 1: 100. Complex compound and KBrwere mixed then shaped into pellets withcompressed using hydraulic pressure up to athickness of 0.01 mm to 0.05 mm. Pellets thusformed is then placed in the holder and spectra wasrecorded in the area of wave numbers 400-4000cm-1 with spectral separation of 2 cm-1.

Thermal gravimetric analyzer (TGA)TGA analysis was used to determine the

thermal stability of complex compound usingMettler-Toledo TGA, procedures performed with10 mg of sample and included in the holder.Complex compound was heated at a rate of 10°C/min from a temperature of 0 °C to 800 °C undera nitrogen atmosphere.


Wavelength of complex compound

The electronic spectra of the copper(II)complex with 2,4,5-triphenylimidazole ligand arepresented in figure 1. The absorbance was showedat 310 nm. This is not consistent with the theory,the color of compound that absorbs the wavelength

of the complementary color, this compound has agreen yellow color, so that the complex compoundsthat absorb wavelengths in the complementarycolor is red (620-750 nm), this metal complex canbe attributed to intra and inter ligand transitionswhich has merged with the metal-ligand chargetransfer transitions dM π*N-ligand [6]. Transitionsof d-d are not observed due to the very lowconcentration of metal complex in the leachate [7].

Functional groups of complex compoundInfrared spectra of Copper(II) complex with

2,4,5-triphenylimidazole ligand are presented infigure 2. The C=N stretching frequency is observedat 1640 cm-1, N-H whereas two fairly strong bandsat 3443 and 3038 cm-1, C-N stretching frequency in1176 cm-1. The bands at 621 cm-1 are assigned toC-H bending vibrations. The C-H aromatic wasshowed at 697 cm-1, N-H wagging vibration waspresent at 734 cm-1 and peak at 1441 cm-1 isassigned to the symmetric C=N-C=C stretchingvibration. Cu-N band appears at 493 cm-1 whichsupports the formation of Cu-N bond [8]. Thisclearly proves the coordination of imidazole tometal ions through this N atom [7].

Stability thermal of complex compoundThe thermo gram of Cu(II) complex with 2,4,5-

triphenylimidazole showed in figure 3. In thisresearch weight loss corresponds to that of thecomplex which have been found to decompose inthe temperature range of 250-360 °C. The complexcompound has stable up to 250 °C. Decompositionof imidazole occurs only after 200 °C [9].

CONCLUSIONS

This research obtained solid yellow green.Wavelength of compound are 310 nm, vibrationCu-N band appears at 493 cm-1 which supports theformation of Cu-N bond. This clearly proves thecoordination of imidazole to metal ions throughthis N atom and stability thermal this compound upto 250 °C.

ACKNOWLEDGEMENTS

This work was supported by Research GrantMandate Universitas Airlangga (HRMUA);Institute of Tropical Disease (ITD) the Center of

Figure 2. Infrared spectra of complex compound Cu(II)-2,4,5-triphenylimidazole

Figure 3. Thermo gram of complex compound Cu(II)-2,4,5-triphenylimidazole


Sucipto et al. 27


Excellence (COE) program by the Ministry ofResearch and Technology (RISTEK) Indonesia.

REFERENCES

[1] M. Devereux, D.O. Shea, A. Kellett, M.McCann, M. Walsh, D. Egan, C. Deegan, K.Kedziora, G. Rosair, H. Müller-Bunz,Synthesis, X-ray crystal structures andbiometic and anticancer activities of novelcopper(II)benzoate complexes incorporating 2-(4′-thiazolyl)benzimidazole(thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline as chelating nitrogen donorligands, J. Inorg. Biochem., 101, 2007, 881-892

[2] F. Arjmand, B. Mohani, S. Ahmad, Synthesis,antibacterial, antifungal activity and interactionof CT-DNA with a new benzimidazole derivedCu(II) complex, European. J. Med. Chem., 40,2005, 1103-1110

[3] M.C. Rodríguez-Argüelles, E.C. López-Silva,J. Sanmartín, P. Pelagatti, F. Zani, Coppercomplexes of imidazole-2-, pyrrole-2- andindol-3-carbaldehyde thiosemicarbazones:Inhibitory activity against fungi and bacteria, J.Inorg. Biochem., 99, 2005, 2231-2239

[4] S. Han, A.J. Lough, J.C. Kim, Synthesis,crystal structures and properties of macrocycliccopper(II) complexes containing imidazolependants, Bull. Korean Chem. Soc., 33, 7,2012, 2381-2384

[5] S.O. Podunavac-Kuzmanović, L.S. Vojinović,Synthesis and physico-chemicalcharacterization of zinc(II), nickel(II) andcobalt(II) complexes with 2-phenyl-2-imidazole, APTEFF, 34, 2003, 119-124

[6] J.G. Malecki, A. Maron, Synthesis,characterization and molecular structure ofruthenium complexes containing imidazole-2-carboxylic acid derivatives, Polyhedron, 40,2012, 125-133

[7] S.S. Thavamani, T.P. Amaladhas,Encapsuation of Cu(II), Ni(II) and V(IV) –imidazole complexes in fly ash zeolite,characterization and activity towardshydroxylation of phenol, J. Mater. Environ.Sci., 7, 7, 2016, 2314-2327

[8] Sajila, H. Mohabey, IR spectra, magnetic andthermal studies of copper (II) complex of N-hydroxy-N-(4-chloro) phenyl N’(4-fluoro)phenyl benzamidine hydrochloride, Mater. Sci.Res. Ind., 11, 1, 2014, 63-65

[9] Omrani, L.C. Simon, A.A. Rostami, M.Ghaemy, Study on curing mechanism ofDGEBA/nickel-imidazole system,Thermochimica Acta, 468, 2008, 39-48


Kurniawan et al. 28


Inhibition of UV radiation by Piper cocatum, Pisonia alba spanoghe,Chrysophyllum cainito L., Ipomea batatas L., Mirabilis jalapa L.,

and Curcuma domestica Val. extractsFredy Kurniawan, Widia Rachmawati, Debora KartikasariLaboratory of Instrumentation and Analytical Sciences, Chemistry Department, Faculty of Mathematics andNatural Sciences, Institut Teknologi Sepuluh Nopember, Arief Rahman Hakim, Surabaya 60111, Indonesia

Email: [email protected]

ABSTRACT

The extracts of Piper crocatum, Pisonia alba spanoghe, Chrysophyllum cainito L., Ipomea batatas L., Mirabilisjalapa L., and Curcuma domestica Val. have been studied for inhibition of UV radiation. The various solvent wasapplied, i.e. methanol, ethanol and demineralized water using maceration method. The behavior of the extracts oninhibition of UV-Vis radiation have been characterized by fluorescence spectrometry. The fluorescence spectraobtained that the excitation wavelength of water extracts of Piper crocatum, Pisonia alba spanoghe, Chrysophyllumcainito L., Ipomea batatas L., Mirabilis jalapa L., and Curcuma domestica Val. are 200-300 nm, 200-350 nm, 200-400 nm, 325-350 nm, 320-350 nm and 320-350 nm, respectively. The emission wavelength of water extracts of Pipercrocatum, Pisonia alba spanoghe, Chrysophyllum cainito L., Ipomea batatas L., Mirabilis jalapa L., and Curcumadomestica Val. are occur at 300-750 nm, 650-720 nm, 350-750 nm, 650-700 nm, 620-720 nm and 620-700 nm,recpectively. The extracts of Piper crocatum, Pisonia alba spanoghe, Chrysophyllum cainito L., Ipomea batatas L.,Mirabilis jalapa L., and Curcuma domestica Val. are in methanol and ethanol also give similar spectra. Theseresults indicate that combination of the extracts obtained will absorb at all UV radiation area which causes skinaging (i.e. UV-A (320-400 nm), UV-B (290-320 nm) and UV-C (200-290 nm)) then emit the energy at near infra-redregion (>750 nm). Therefore, the combination of the extracts has high possibility to be used as a sunscreen that canprotect the skin completely from harmful UV radiation.

Keywords: sunscreen, UV radiation, plant extract, fluorescence, skin aging.

INTRODUCTION

Skin aging are classified into intrinsic aging(chronology) and extrinsic aging (photoaging) [1].Intrinsic aging caused by the aging process inaccordance with human life and correlated withgenetic factor. Intrinsic aging causes the skinbecame wrinkled and pale. Whereas, extrinsic skinaging caused by external factor such as UVexposure. UV exposure in long term causesdegeneration of skin collagen [2], pathologicaldisorders of skin such as erythema, edema,hyperpigmentation, prematurely aged, and skincancer [3].

UV rays are classified into UV-A (320-400nm), UV-B (290-320 nm) and UV-C (200-290 nm)[3]. The distribution of UV rays on the earth are90% of UV-A, 5% of UV-B and 5% of UV-C. Thepercentage of UV rays which entering on the earthsurface based on the thickness of ozone layer [4].In the recent years, the depletion of ozone layercauses an increasing of UV exposure on the earthsurface. Hung and Chi Feng was reported that UV-A is an important factor on the radiation of UV-Brays [5].

The mechanism of UV radiation on the skininvolves free radical and reactive oxygen species(ROS) [6]. The natural antioxidant can be drainedby them, and causes reactive oxidative stress. ROSis a singlet oxygen [7], superoxide, peroxide,hyperoxide and ICAL RAD hydroxyl [8]. UV-Bwith UV-A produce superoxide, either directly orusing enzyme activation [6]. The effect of UV-B onthe DNA causing the formation of cyclobutanepyrimidine dimers (CPDS) and low pyrimidine (6-4) pyrimidine (6-4 PPs) photoproducts. If thechanging was not repaired, the DNA mutation onthe epidermis cell lead to skin cancer [9]. Itencourages the researcher to explore correlationbetween the application of sunscreen and skincancer [10].

The sunscreen that commonly used only protectthe skin from UV-B rays [11]. It makes the damagefrom UV-A rays cannot be prevented and causingROS will demolish the natural antioxidant from theskin [12]. Based on US Food and DrugAdministration (FDA) on the June 14, 2011,sunscreen should be containing antioxidantcompound which gives protection from UV-A andUV-B rays [13]. The adding of antioxidant on the


Kurniawan et al. 29


sunscreen is recommended, because it can beminimalizing of skin damage which causes ofsunlight exposure. The sunlight exposure make theendogen antioxidant amount loss significantly [14].

-carotene, one of carotenoid compound, isreducing effective singlet oxygen which has highantioxidant activity [15]. The effect of carotenoidprotective can protect (ex-vivo and in vitro) theskin from UV-A, UV-B and infrared rays [16]. But,skin penetration still have problem from thedevelopment of sunscreen which containantioxidant [4]. Some of sunscreen product causestoxicity risk or allergic on the skin. The plasma andurine on human body which uses sunscreen thatcontaining antioxidant was detected containingbenzo-3-phenone. It means that the sunscreen wascontaining the molecule which can be diffused onthe human skin [17]. The diffusion process of themolecule from sunscreen on the skin was occurringbecause the molecular size. The molecular weightand lipophilicity of the compound on sunscreen hasimportant role on the skin penetration andsunscreen production [11].

Phytochemical from plants can be used assunscreen material and protect the skin from UVradiation [2]. Polyphenol plants have ability toclean the strong radical on the human body [3]. Inthe recent years, the ability of polyphenol plantstowards UV rays give a big attention on theresearch area [18]. The utilization of plants extractsuch Punica granatum, Melissa officinalis, L. [3],Anemarrhena asphodeloides rhizome [2], andBlackberry as sunscreen, have been reported.Flavonoid and phenylpropanoid content on theMelissa officinalis, L. makes the ability as anantioxidant. On the 2016, Pérez-Sánchez promotethe extract of Melissa officinalis, L. as a sunscreenwhich can be preventing from UVB-oxidativestress and the damage of DNA on the skin [3].

Piper crocatum, Pisonia alba spanoghe,Chrysophyllum cainito L.[19], Ipomea batatasL.[20], Mirabilis jalapa L. [21], and Curcumadomestica Val. [22] contains flavonoid andpolyphenol compound which can be acted asantioxidant [23]. In this research, the effect of skinprotection towards UV rays from the extract ofPiper crocatum, Pisonia alba spanoghe,Chrysophyllum cainito L., Ipomea batatas L.,Mirabilis jalapa L., and Curcuma domestica Val.will be studying based on fluorescence analysis.

MATERIALS AND METHODExtraction of plants

The leaves of Piper crocatum, Pisonia albaspanoghe, Chrysophyllum cainito L., Ipomeabatatas L., Mirabilis jalapa L. and rhizome ofCurcuma domestica Val. obtained at InstitutTeknologi Sepuluh Nopember. The leaves and

rhizome were dried at 60°C for 48 hours andmashed into powder. 0.4 gram of the powder wasextracted by maceration method using 2 mL ofsolvents for 6 hours. In this study, we use 3solvents, i.e., demineralized water, 98% methanol,and 98% ethanol which purchased from the localmarket. The filtrate from extraction process wasseparated and freeze drying for 24 hours. Theextract of plants are ready to analyze usingfluorescence spectrometry. Coding of the plantextracts conducted to simplify the analysis processand presented at Table 1.Table 1. List of the plants that has been extractedPlant Solvent Code of

samplePipercrocatum

Demineralized waterMethanolEthanol

A1A2A3

Pisonia albaspanoghe


B1B2B3

Chrysophyllumcainito L.


C1C2C3

Ipomeabatatas L


D1D2D3

Mirabilisjalapa L.


E1E2E3

Curcumadomestica Val.


F1F2F3

Characterization of plants extract using fluorescencespectrometry

The extracts of Piper crocatum, Pisonia albaspanoghe, Chrysophyllum cainito L., Ipomeabatatas L., Mirabilis jalapa L., and Curcumadomestica Val. (Table 1) were characterized byfluorescence spectrometry (Perkin Elmer).Excitation of the extract was measured at range ofwavelength of 200-500 nm. While, the emission ofthe extracts was carried out from 200 to 800 nm.

RESULTSCharacterization of water extract using fluorescencespectrometry

The ability of Piper crocatum (A1), Pisoniaalba spanoghe (B1), Chrysophyllum cainito L.(C1), Ipomea batatas L. (D1), Mirabilis jalapa L.(E1), and Curcuma domestica Val. (F1) extract toblock the UV rays was measured by fluorescencespectrometry (Fig 1). Based on the spectra ofexcitation and emission, the extract of A1, B1, C1,D1, E1 and F1 was indicated to prevent theradiation of UV-C, UV-B, UV-A and visible. It isdue to the component on the A1, B1, and C1extracts have ability to absorb at 200-300 nm (UV-


Kurniawan et al. 30


C and UV-B radiation region). Whereas, thecomponent on the D1, E1, and F1 extracts haveability to absorb at 300-350 nm (region of UV-Aradiation). The prevention of visible rays from theextract was caused by emission on > 350 nm.

Fig 1. Fluorescence spectra of excitation (a) andemission (b) of piper cromatum (A1), Pisonia albaspanoghe (B1), Chrysophyllum cainito L. (C1),Ipomea batatas L. (D1), Mirabilis jalapa L. (E1)and Curcuma domestica Val. (F1) extract usingdemineralized water as solvent.

Characterization of methanol extract usingfluorescence spectrometry

Fig 2(a) shows that the A2, B2, and C2 extractshave a good potential to block UV-C radiation. It isdue to the extracts have ability to absorb at morethan 240 nm. Whereas, the extract of F2 has a goodpotential to block UV-B because has ability toabsorb at 280-320 nm. The excitation of D2 and E2extract appear at UV-A region (> 320 nm).

The emission of A2, D2, E2, and F2 extract onFig 2(b) indicate that the extracts have ability toblock visible rays and near infrared rays. Theradiation of UV rays more dangerous than visiblerays. But, the component of sunscreen which hasability to block visible rays will be giving goodeffectively of sunscreen. The effectiveness ofsunscreen not only based on the compound which

can block the rays, but the antioxidant componenton the extract have good role on the sunscreen [11].Antioxidant component on the methanol extractswas obtained from Curcuma domestica Valrhizome (C2). Nakayama et. al., explain that theisolation of curcuma compound from Curcumadomestica Val. rhizome using methanol as solventhas a good stability of antioxidant than usingethanol [24].

Fig 2. Fluorescence spectra of excitation (a) andemission (b) of piper cromatum (A2), Pisonia albaspanoghe (B2), Chrysophyllum cainito L. (C2),Ipomea batatas L. (D2), Mirabilis jalapa L. (E2)and Curcuma domestica Val. (F2) extract usingmethanol as solvent.

Characterization of ethanol extract using fluorescencespectrometry

Ethanol extract of F3 indicate to block UV-B. Itis due to the excitation of F3 extract was at 280-320 nm (Fig 3(a)). Whereas, A3, B3, C3, D3 andE3 were excited at 325-350 nm. It shows that theethanol extracts have a potential as sunscreen, butthe ability of the extracts only to block UV-B andUV-A rays.

Fig 3(b) shows that the extracts have ability toblock visible and near infrared rays. It because ofthe extracts can absorb at 500-725 nm. Therefore,the ethanol extract less effective to be used as

(b)

200 250 300 350 400 4500

100

200

300

400

500

600

Fluor

esce

nce I

ntens

ity (a

.u)

Excitation Wavelength (nm)

A1B1C1D1E1F1

300 400 500 600 700 8000

100

200

300

400

500

600

700

Fluo

resc

ence

Inte

nsity

(a.u

)

Emission Wavelength (nm)

A1B1C1D1E1F1

(a)

(b)

575 600 625 650 675 700 7250

100

200

300

400

500

600

700

800

900

Fluo

resc

ence

Inte

nsity

(a.u

)


A2B2C2D2E2F2

240 280 320 360 4000

100

200

300

400

500

600

700

800

900

Fluo

resc

ence

Inte

nsity

(a.u

)Excitation Wavelength (nm)

A2B2C2D2E2F2

(a)


Kurniawan et al. 31


sunscreen. It is because the extract cannot blockUV-C rays.

Fig 3. Fluorescence spectra of excitation (a) andemission (b) of piper cromatum (A3), Pisonia albaspanoghe (B3), Chrysophyllum cainito L. (C3),Ipomea batatas L. (D3), Mirabilis jalapa L. (E3)and Curcuma domestica Val. (F3) extract usingethanol as solvent.

DISCUSSION

According to the results, all extracts have beenshown to have the potential to be used as asunscreen. However, each extracts have differentregion of absorption and emission. It means that ifwe combine the whole extracts as raw material forsunscreen will be obtained the material that canprotect a longer area. Combination of the extractscan improve protection for UV rays.

In addition, the antioxidant component on thesunscreen can be minimalizing the skin damagewhich caused of sunlight exposure. The sunlightexposure causes the endogen of antioxidant amounton the skin was releases significantly [15]. Thatcompound contained in the water extract of plants.The water extract is rich in flavonoid andpolyphenol compounds which has function asantioxidant [19]. Furthermore, the compound ofZnO in the sunscreen can be replace with thecompound in the methanol extracts.

CONCLUSION

Combination of the water, methanol andethanol extracts of Piper cocatum, Pisonia albaspanoghe, Chrysophyllum cainito L., Ipomeabatatas L., Mirabilis jalapa L.,and Curcumadomestica Val. have a potential to block UV-C (<320 nm), UV-B (290-320 nm), UV-A (> 320 nm),visible (350-700 nm) and near infrared (< 750 nm)rays. Therefore, the extracts can be used as naturalsunscreen.

ACKNOWLEDGMENT

The authors acknowledge the IndonesianGovernment, especially DIKTI (Directorate ofHigher Education of Indonesia) Research Grant forsupporting under the scheme Penelitian UnggulanPerguruan Tinggi (PUPT).

REFERENCES

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[2] H.-S. Kim et al., ‘Inhibition of UVB-inducedwrinkle formation and MMP-9 expression bymangiferin isolated from Anemarrhenaasphodeloides’, Eur. J. Pharmacol., vol. 689,no. 1–3, pp. 38–44, Aug. 2012.

[3] A. Pérez-Sánchez, E. Barrajón-Catalán, M.Herranz-López, J. Castillo, and V. Micol,‘Lemon balm extract (Melissa officinalis, L.)promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage ina skin cell model’, J. Dermatol. Sci.

[4] A. Portantiolo Lettnin et al., ‘Protective effectof infrared-A radiation against damageinduced by UVB radiation in the melan-a cellline’, J. Photochem. Photobiol. B, vol. 163,pp. 125–132, Oct. 2016.

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575 600 625 650 675 700 7250

100

200

300

400

500

600

700

800

900

Fluo

resc

ence

Inte

nsity

(a.u

)


A3B3C3D3E3F3

200 250 300 350 4000

100

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800

Fluo

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Inte

nsity

(a.u

)

Excitation Wavelength (nm)

A3B3C3D3E3F3

(a)

(b)


Kurniawan et al. 32


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Fahmi et al. 33


ELECTROSPINNING OF CELLULOSE ACETATENANOFIBER MEMBRANE FOR HEMODIALYSISMohammad Zakki Fahmi, Yanuardi Raharjo, Siti Wafiroh, IqlimaAyu Prestisya, and Kukuh Ferlanda

Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, Indonesia*Corresponding author Email:[email protected] and [email protected]

ABSTRACT

Nanofiber membrane from cellulose acetate was produced by electrospinning technique using a dope solution withratio of 15% cellulose acetate, 8% formamide, and 77% acetone. The result showed that the optimum conditionswere reached on solution with flow rate at 0.1 μL.h-1, high voltage of 10 kV using a drum or cylinder-shapedcollector with its distance of 10 cm and the optimum time at 5 hours. Optimum characteristic of nanofiber membraneinclude the thickness of membrane was 0.32 mm, hydrophilicity of the membrane was positive hydrophilic with thecontact angle smaller for less than 5 minutes, the results of pore size was less than 1 μm, the mechanical strength ofthe membrane in terms of stress was 0.00245 MPa, strain about 2.1209 and 1.15777 x 10-5 GPa of Young'sModulus. Nanofiber membrane applied in creatinine and urea hemodialysis process resulting from kinetic assayresults of the membrane. The membrane had a flux value of 9,171.974 L.m-2.h-1 and removal of creatinine by98.65% and urea by 87%.

Keywords: Nanofiber membrane, cellulose acetate, electrospinning, hemodialysis, creatinine, urea

.

INTRODUCTIONKidney failure is a case where the kidney

functionally impaired. And the most effective wayto treat on patients is hemodialysis. The principleof hemodialysis is transfer the patient's bloodthrough dialyzer occurring diffusion andultrafiltration, then the blood back into the patient'sbody. In the process in dialyzer, blood and fluiddialyzer separated by a dialysis membrane. Thedialysis membrane must have strong mechanicalproperties, efficiency, and also does not require theaddition of chemicals in the separation process(Edward et al., 2008). The dialysis membrane inthe form of nanofiber can be made from a varietyof organic materials (polymers) such aspolysulfone and cellulose acetate. Cellulose acetateis used as a dialysis membrane because it hashydrophilic properties that hold fouling inhemodialysis and had good permeability andthermostability.

In the previous research that have been done bymaking cellulose acetate nanofiber membraneswith template synthesis method (Zelenski et al.,1998), and phase inversion method (Indarti et al.,2013) but the membrane was shown underperformance, such as pore size of the membrane isbig when used as hemodialysis membranes canallow other molecules to pass through themembrane. So, it would require another method ortechnique such as electrospinning techniques toimprove its perfomance by measuring the thickness

of the membrane, mechanical tests, and testmorphology with SEM instrument then nanofibermembrane was applied to the hemodialysis ureaand creatinine.

MATERIALS AND METHODSReagents and materials

Acetone, cellulose acetate, formamide, NaOH,ethanol, creatinine, picric acid, urease,phenolftalein indicator and urea.

InstrumentErlenmeyer with TS glass stopper, magnetic

stirrer, hot plate, Nachriebe 600 electrospinning,Autograph AG-10 TE Shimadzu, UV-VisSpectrophotometer Mapada UV-6100PCS andScanning Electron Microscopy (SEM).

Dope Solution Preparation ProcedurePreparation of dope was produced with a ratio

of 15% cellulose acetate, 8% formamide, and 77%acetone. Cellulose acetate put into the Erlenmeyerand mixed with acetone, then it placed on a hotplate and stirred using a magnetic stirrer. Then itwas added formamide and stirred again for about 2hours until a homogeneous solution. After ahomogeneous solution was left to stand for onenight to remove air bubbles arising from thedissolution process.

Electrospinning Process ProcedureDope solution was put into syringe and fed with

high-voltage electricity and a collector membranes


Fahmi et al. 34


which made of metal horizontally against thespinneret (Christoforou and Doumanidis, 2010).The cable clamp of power supply clipped onspinneret and other wires clipped to the collector.After passing through spinnerets, dope solutionattracted to the collector and becomes solidnanofiber.

Dope solution was treated with varying flowrate of 0.1, 0.3, 0.5 and 0.7 μL.h-1, varying thevoltage of 5, 10, 15 and 20 kV and also varying theduration of 1, 3, 5, and 7 hours. In addition, it wastreated with varying distance of the needle tocollector of 5, 10 and 15 cm. The collector also bevaried with drum-shaped or cylindrical and flatshape. These obtained the optimum nanofibermembrane.

Characterization

The thickness of the membrane.Membrane thickness measurement using a

screw micrometer with a precision of 0.01 mm.There were 2 scales on micrometer screw whichwere main scale and Nonius scale. Measurementswere clamping membrane and read the scale.

Hydrophobicity and Hydrophilicity test.Dropped off aquadest about 10 µL to membrane

with distance 10 cm. Then observed every minuteuntil the water was completely absorbed into themembrane and water droplets were photographed.This determination was made by determining thecontact angle of water that has dripped onto themembrane.

Figure 1. Membrane on drum collector

Mechanical test of Membranes.Sample dried by oven at 80-90°C temperature

within 2 hours. Then the membrane dripped with0.1 M NaOH and then soaked with ethanol at aratio of 1: 4 overnight. After that, it heated back inthe oven for an hour at a temperature of 80-90°C(Ma et al., 2005). After that tested the tensile wherethe membrane was clamped by autographinstrument. The membrane was then withdrawn ata speed of 1 cm.min-1 until the break.

Morphology of membranes. Determination of cellulose acetate membrane

morphology using Scanning Electron Microscope(SEM). Membrane dried and it was cut to a certainsize and then dipped into liquid nitrogen for a fewseconds. Then the membrane cut to the size of 1x1

cm. Then the membrane attached to the specimenchamber for observation SEM.

Figure 2. The results of the membrane with a variationof 1, 3, 5, and 7 hours (Left to right).

Flux.Determination of flux value can be determined

using a dead end method. The resulting membranecompacted beforehand in order to obtain a constantflux. Then the membrane used for separation ofurea and creatinine separately. The dead-end tubewas closed and pressurized at 0.2 atm. After thecompaction process was complete then the fluxvalues measured with distilled water volumecollected for an interval of 20 minutes.

Rejection.Urea and creatinine solution used as the feed

were 50 mmol.dl-1 or 0.05 M and 2 ppm. Thevolume of feed before passing through themembrane and the permeate volume after passingthrough the membrane would be collected in aseparate container. Then in phase feed andpermeate each were taken 15 ml and testedquantitatively measured with a UV-VisSpectrophotometer to determine the concentrationof urea and creatinine in the sample and permeate.


Fahmi et al. 35


RESULTS AND DISCUSSIONNanofiber Membranes CA

CA nanofiber membrane made usingelectrospinning technique. Dope solution passedthrough a spinneret hole and subsequently drawnusing electrostatic energy with high electric voltagedirect current (DC) then the fibers collected on thecollector (Herdiawan, 2013). To obtain optimumCA nanofiber membranes were optimizedcollector, flow rate, voltage, distance betweencollector and spinneret and duration optimization.

Collector OptimizationThe results can be seen in Figure 1 that the

nanofiber membranes were collected on the drumcollector produced a thicker membrane than the flatcollector and evenly to all parts of the surface ofthe membrane while the yield on flat collectorgathered at the center so just the central part is

thicker than the other side. Therefore, the optimumresults obtained when using the drum or cylinder-shaped collector.Figure 1. Membrane on drum collector

Flow Rate OptimizationBased on the flow rate optimization has been

done, the faster the flow rate, more the dopesolution is wasted. This is because the electricitycurrent supplied unable to pull the dope solution onTaylor cone formed quickly. The force of gravityalso affects the flow rate optimization. As researchconducted Taylor (1969) in which the shape of acone at the end of the spinnerets cannot bemaintained if the flow rate of dope solution is toolarge and also research conducted by Megelski etal. (2002) in which the effect of the flow rate of thepolymer solution shows that the fiber diameter andpore size increase with increasing flow rate.However, the flow rate of the high number ofvisible defects, due to the inability of fiber to becompletely dry before it reaches the collector.

Table 1. CA Membrane for distance of the needle to collector 5, 10 and 15 cm.


Fahmi et al. 36


Inability to dry fibers can also lead to the formationof ribbon-like fiber compared than a cross-sectionfiber. Therefore, the flow rate of 0.1 μL.h-1 as theoptimum rate that is effectively used resultingsolution cause of efficiency of not wasted dopesolution.

Time OptimizationThe results of nanofiber membrane pore size

can be seen in Figure 2 where the membrane wasprocessed by the electrospinning technique at alonger time produce a thicker membrane. As sameas the results of research Gorji et al. (2011) showedthat increasing duration of the process of makingthe membrane, the membrane would get thicker.

Figure 2. The results of the membrane with avariation of 1, 3, 5, and 7 hours (Left to right).

Distance of the Needle to the Collector OptimizationResults of making membranes with these

optimizations can be seen in Table 1.Shown in Table 1 that the optimal membrane

was using a distance of 10 cm. The distance wastoo distant or too close greatly affect the diameterand morphology of the membrane. This is due tothe effect of a long or absence tylor cone formed toreach the collector and forming the membrane sothat the deposition rate and the rate of evaporationof the polymer solution greatly affects themorphology of the membrane (Bhardwaj et al.2010).

Table 2 CA membrane with voltage variations of 5, 10, 15 and 20 kV


Fahmi et al. 37


High Voltage OptimizationThe result of making membranes in

optimization voltage which can be seen in Table 2.Table 2 CA membrane with voltage variations

of 5, 10, 15 and 20 kVIn Table 2 shows that the optimal cellulose

acetate membrane is by a voltage of 10 kV with adistance of 10 cm using a flow rate of 10 µL.h-1 for2.5 hours.

Characterization

The Thickness of MembraneThe thickness of the membrane resulting in an

optimization voltage, duration and needle tocollector distance shown in Figure 3. It shows thatthe longer time spent in making nanofibermembrane CA, it produces a thicker membrane.This is due to the longer time spent in the processof electrospinning, the more the polymer solutioncan be deposited on the membrane so that themembrane produced thicken.Figure 3 the thickness of CA membrane

The voltage and distance optimization’s resultsseen in the visible produces a different thicknesshowever when it measured quantitatively using thetool, it produces the same thickness. It is becausewhen the electrospinning process was only done for2.5 hours so that the thickness of the membrane ateach other is almost the same. The duration couldaffect the thickness of the resulting membrane.

Hydrophobicity and HydrophilicityHydrophilicity of the membrane surface

evaluated from the results of water contact anglemeasurements. Low water contact angle showedhigh hydrophilicity of the membrane surface andvice-versa (Haitao et al. 2009). Results ofdetermination hydrophobicity and hydrophilicitycan be shown in Figure 4.

From Figure 4 shows that the decrease arch ofwater so it can be concluded that the membrane ishydrophilic. This was due to the structure ofcellulose acetate contained hydroxyl groups (OH)that is polar so that membranes can absorb waterand cause the contact angle of water decreased(Amri et al., 2015).

Figure 4 hydrofobicity and hydrofilicity of CAmembrane

Mechanical Test of MembraneNanofiber membrane heated below the melting

point up to dry so that the nanofiber membrane isbonded to one another and retaining themorphology. If using a temperature above themelting point (210°C) can easily destroy thematerial (Ma et al., 2005). After heated to atemperature of 80-90°C, the membrane wasdropped with 0.1 M NaOH and soaked with asolution of ethanol and distilled water in the ratio 1:4 for 24 hours. The function of this treatment is toremove the acetyl group contained in the celluloseacetate through the hydrolysis reaction (Ma et al,2005). Once it rinsed with distilled water and thendried.

A test conducted to determine the mechanicalproperties of the membrane strength of the forceapplied to the membrane broke. The parametersused to determine the mechanical properties of themembrane were tension, strain and Young'smodulus. Rated stress, strain, and young modulusof the membrane at each other was shown in Table3.

Table 3 Value of stress, strain and young’s modulus

Vol

tage

(kV

)

Dis

tanc

e(c

m)

Stre

ss(M

Pa)

Stra

in

You

ng’s

Mod

ulus

(MPa

)

10 100,0204

170,0525

0,388888889

15 100,0163

330,1441

0,113347213

15 50,0126

580,0953

0,132826163

15 150,0134

750,1183

0,113905325

In Table 3 shows that the membrane by voltage10 kV has a higher stress than others. This was dueto the membrane 10 kV has a tight pore structureand has a distance between the molecules of themembrane meeting others and thus required ahigher voltage than other membranes. Membrane

020406080100120140

0 1 2 3 4

Con

tact

Ang

le (°

)

Drop Age (minute)

00.10.20.30.40.50.6

5 10 15

Thic

knes

s (m

m)

Optimization

Duration(hour)

Voltage(Kv)

Distance(cm)

Figure 3 The thickness of CA membrane


Fahmi et al. 38


with 20 kV was not carried out due to tensile testwas formed nanofiber membrane is very thin and

easily broken even by small force and in order tothat it is not possible to do the tensile test.

Furthermore, to stretch the membrane with avoltage 10 kV has a strain value smaller than themembrane with other. This is because themembrane 10 kV has a pore size that is not easilychanged. Membrane good is a membrane that has avalue of strain is relatively small due to the smallelastic properties (Callister, 2010). Then for themembrane with 10 kV has value of Young'smodulus higher than other membranes. This wasdue to the membrane 10 kV has a higher stressvalue and the strain that is lower than the others.However, when seen from the table that theYoung's modulus of cellulose acetate membraneshas a relatively small value because on eachmembrane has a relatively low stress value andstrain value is relatively high so as to produceyoung's modulus values were relatively smallbecause of the value of stress, strain and Young'smodulus of the membranes can be influenced bythe structure, composition and defects in themembrane. So from the results of measurement ofmechanical of cellulose acetate membranesoptimum membrane against the value of stress,strain and Young's modulus is membranes with 10kV.

Seen from the time variation of the membrane,the longer time is pore membrane can produce abig tight so that when tensile tested to breakingproduces a large stress. For the measurement ofstrain of the time variation shown in Figure 5.Increasing the value of the strain caused by theincreasing number of hydrogen bonds so that theviscoelastic response was increased so that it canbe concluded from the results of the stretch, CAnanofiber membranes with a long time is amembrane that is elastic. In Young's modulusmeasurement results depends on the type ofmaterial (material composition) used. The value ofYoung's modulus of the membrane is smaller thana pure CA Young's Modulus value. This decreasewas due to membrane given the influence oftemperature or heat in preparation before the tensiletest so that the lower value of Young`s Modulus.The higher the working temperature of thematerial, the smaller the value of Young's modulus(Dieter, 1986).

Figure 5. Measurement results a) tension b) strain and c)Young's modulus versus time.

Figure 6. Morphology structure of membrane cellulose acetate, (a) Morphology membrane 10 kV, (b) Morphologymembrane optimization (Christoforou et al. 2010), (c) Morphology membrane 5 hours optimization..


Fahmi et al. 39


Morphology of MembraneDetermination the morphology structure of

cellulose acetate membrane can be determined byusing instruments Scanning Electron Microscope(SEM). The results of SEM test aims to determinethe structure of the membrane surface obtained.SEM test results of cellulose acetate membranes inoptimum condition indicated is presented in Figure6.

Seen from Figure 6 shows the uniformity of thenanofiber produced on cellulose acetate membranesby electrospinning technique and the size ofproduced nanofiber membranes for less than 1 µm.Also seen in Figure 5 that the fiber nanofiltrationare formed scattered evenly, tightly andhomogeneous (Cristoforou et al. 2010). Nanofibermembrane with a time of 5 hours and voltage 10kV summed up as membrane optimal than othervariations due to the uniformity of the fibermembrane that allows for the molecule creatinineand urea can be through the membrane.

Kinetic Test of MembraneFlux. Flux used a dead-end method. The flux

value produced from cellulose acetate membranewas at 9171.974 L.m-2.h-1. These big resultsdepended on the feed solution is inserted and getpressurized. Membrane missed all the feedsolution. It shows that the membrane washydrophilic because it could quickly pass all of thefeed solution. Cellulose acetate in its structure hada hydroxyl group (OH), which was polar so that themembrane was hydrophilic. Besides the membranehas a very thin thickness so that the feed solutionwill be faster flow.

Rejection.The results of the rejection at urea and

creatinine hemodialysis were 13% and 1.35%.These results, based on the calculation of theabsorbance concentration permeate solution againstdata regression curve creatinine standard solutions.It means that a reduction in urea after passingthrough cellulose acetate membranes by 87% andcreatinine retained by the membrane at 1.35% sothe reduction in creatinine after passing throughcellulose acetate membranes by 98.65%. Theamount of urea and creatinine that can pass throughthe membrane because the membrane pores are sonumerous and hydrophilic membranes.

CONCLUSIONS

Based on the results of research on themanufacture of nanofiber membrane made ofcellulose acetate can be concluded that:The use of electrospinning technique was effectivein producing of nanofiber membranes for having aparticle size or morphology correspondingapproximately less than 1 μm, the mechanicalstrength of the membrane amounted to 0.00245

MPa of stress, strain at 2.1209, and a Young'smodulus of 1.15777 x 10-5 GPa. Nanofibermembrane was not yet applicable in creatinine andurea hemodialysis process resulting from kineticassay results of the membrane. The membrane hada flux value of 9,171.974 L.m-2.h-1 and removal ofcreatinine by 98.65% and urea by 87%.

The various optimizations performed startingfrom the optimization of flow rate solution,collectors, duration, voltage and distance of needleto collector of nanofiber membrane obtainedoptimum flow rate 0.1μL.h-1 with a drum orcylinder-shaped collector during the optimal timeof 5 hours by a voltage 10 kV and a distance of 10cm.

ACKNOWLEDGEMENT

The authors express gratitude to the Ministry ofResearch and Higher Education, ChemistryDepartment, Faculty of Science and Technology,and Eye Clinic Center Pondok Jati Dr Daddy forelectrospinning equipment facility.

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Nugraheni et al. 41


BIOACTIVE COMPOUNDS OF PROPOLIS FROM NATURALBEEHIVES FROM EAST JAVA, INDONESIAZjahra Vianita Nugraheni*1, Anil Kumar Anal2, Agus Wahyudi1,Rhiby Ainur Basit Haryanto1

1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut TeknologiSepuluh Nopember, Surabaya, Indonesia, 60111

2Food Engineering and Bioprocess Technology, Asian Institut of Technology Pathumthani, Thailand*Corresponding Author : [email protected]

Abstract

Propolis has been considered as antioxidant, antibacterial, antinflammatory substances. These bioactive activitiesdue to rich in polyphenolic compounds, flavonoids, peptides etc. This study mainly uses natural beehives from EastJava, Indonesia to get propolis extracts as primary material. It focused on the composition of propolis. The propolisextracts have phenolic and flavonoid compounds.

Key words : propolis, phenolic, flavonoid, Ultrasonicated Assisted Extraction.

INTRODUCTION

Propolis contains many bioactive compoundssuch as resinous compounds, balms, beeswax,aromatic essential oil and bee pollen. Propolis alsocontains amino acids, trace elements and at least 38bioflavonoids including the presence of benzylcaffeat [1]. Propolis also contains some aminoacids such as alanine, valine, glycin, leucin andothers. It has also been reported that propoliscontains some essential vitamins [2]. Thesebioactive compounds cause the bioactivities ofpropolis such as antioxidant, antibacterial, antiviraletc.

Propolis extract can be obtained by somemethods, for examples, nanofiltration, supercriticalfluid extraction [3] and ultrasonic-assistedextraction [4]. Ultrasounic-assisted extraction is anextraction method which uses the principle ofsound wave. It is one of new extraction method.Ultrasounic-assisted extraction (UAE) can beconsidered as a useful alternative for solid samplepretreatment (propolis extraction) because theenergy imparted facilitates and accelerates somesteps, such as dissolution, fusion, and leaching,among others. In addition, extraction process usingUAE spend less time than other extractionmethods. Operating cost of UAE is low, it canproduce more extracts depend on the characteristicof material, more simple and take a short time ofextraction process [5].

EXPERIMENTALMaterials

Raw materials used in this experiments arenatural beehives. Other reagents used such asethanol 95 %, gallic acid, folin-ciocalteau reagents,Na2CO3, quercetin, NaNO2, H2SO4, and NaOH.

Extraction Process.The crushed beehives (25 g) were mixed with

20 mL of ethanol (95% v/v). Various amplitudesand operation time were studied by Design-Expertsoftware with Response Surface Methodology(RSM). Amplitudes that are used are from 60 to100 % and variation of operation time from 15 to45 minutes at 60°C under continuous stirring (150rpm). The solution was then filtered, collected andstored overnight at 4 °C. The resultant solution wasfurther filtered, and the prectecipitate was washedwith cold 95% (v/v) ethanol. The filtrate wasevaporated at 50°C, giving a resinous brownproduct. The borwn colored filterate was furtherdisintegrated in 10 mL of water, containing 8% w/vof L-lysine by mixing for 30 min at 50°C. Thepropolis extracts were stored at -20 °C in dark forfurther studies [6][7].

Quantifications of Phenolics ContentA mixture of 1.0 mL of the sample, 5.0 mL of

Folin-Ciocalteau reagent diluted in water (1:10)was incubated for 5 min at room temperaturefollowed by addition of 4.0 mL of 0.4 M Na2CO3solution, leaving to rest for 2 h in dark. The resultswere read on a spectrophotometer at 765 nm andgallic acid was used as reference [8].

Quantifications of Flavonoids ContensThis analysis consisted by mixing 0.4 mL of the

sample with 9.6 mL of distilled water, 1.2 mL of0.2 M sulphuric acid, and 1.2 mL of 3 M sodiumnitrite and 1.2 mL of 10 % sodium hydroxide. After15 min of incubation of this mixture at room


Nugraheni et al. 42


temperature (25 °C), the absorbance of the sampleswas measured in a spectrophotometer at 395 nm.Flavonoid quercetin was used as reference.

RESULTS AND DISCUSSIONExtraction Process

The moisture content of dry beehive wasdetermined before the extraction process. Thisextraction process using Ultrasonic assistedextraction (UEA) method in variation of amplitudeand time. The amplitude was in range 60 % to 100% and the time was in the range of 15 to 45minutes, respectively. The optimum condition wasobtained by using Response surface methodology(RSM) design software. The maximum temperatureof extraction was 60 °C because propolis can easilybe degradated in the temperature more than 60 °C[6]. The objective of evaporating extracts is toreduce ethanol content. Evaporating temperaturewas 50°C to prevent the degradation of propolisextract. L-lysine was added to enhance thesolubility of protein contents in propolis extracts.The final results of propolis extracts kept in 4 °Ctemperature and without presence of the light dueto propolis extract can easily be degradated if itplaced in room temperature with the presence ofthe light.

Phenolics ContentThe composition of compounds that containing

in propolis are as key candidate for evaluatingpropolis quality. Phenolic content for each extractfrom different run was determined. Gallic acid wasused as standard. Measurement of absorbance wasreplicate for three times. From this measurements,the highest phenolics content was 10.81 ± 0.10μg/g dry beehive. The extraction amplitude andtime conditions for the propolis extract with highestphenolic content are 100 % and 31.35 minutes. It’smean that high amplitude will produce propolisextract with high phenolics content. according tothis experiments, increasing amplitude will producepropolis extract that contain high phenolic content.However, time does not has affect in phenoliccontent of propolis extract. Another study ofphenolic content of propolis that quantify phenoliccontent of Portugal propolis in methanol solvent[9]. who quantification of. Propolis samples gotfrom two different places, Bornes and Fundao andthe phenolic content were 329 mg/g and 151.mg/g,respectively.

Flavonoids ContentFlavonoids content were determined by using

quercetin as reference. All propolis extracts weredetermined the flavonoids content. The resultsshow that the sample at condition which are 100 %of amplitude and 31.35 minutes of time extractionhas the highest flavonoid content (0.45 ± 0.01 μg/gor 0.45 % of dry beehive). From the previous

results of phenolic content, it can be concluded thatthe sample has high phenolics and flavonoidscontent. The highest amplitude will producepropolis extract with extraction time around 31.35minutes. The flavonoids content of propolis extractwill increase by increasing of amplitude condition.But, time does not has effect in flavonoids content.Flavonoids content of propolis from differentcountries have been studied. For exampleflavonoids content of Romanian propolis [10].They studied flavonoid content using two differentethanolic extracts of propolis and two differentmethods of determination. Other propolis extractswhich had been determined the flavonoids contentare Taiwan, Brazil and China [11]. The results offlavonoids content measurement of propolis in thesame material may vary depend on what solventthat use for extraction and method that use todetermine flavonoids content.

CONCLUSIONS

Propolis ectracts were obtained by extractingthe natural beehives. The extraction method usedwas ultrasonic-assisted extraction with variation ofamplitudes and times of extraction. The highestvalue of phenolics and flavonoids content wereobtain at 100 % of amplitude and 31.35 minutes oftime extraction. The highest phenolics andflavonoids content are 10.81 ± 0.10 μg/g drybeehive and 0.45 ± 0.01 μg/g dry beehives,respectively.

ACKNOWLEDGEMENTS

Laboratorium of Food Engineering andBioprocess Technology (AIT) and ChemistryDepartement (ITS), who have always been helpfuland support this research.

REFERENCES

[1] R. Yamauchi, K. Kato, S. Oida, J. Kanaeda, Y.Ueno, Benzyl caffeate, an antioxidativecompound isolated from propolis, Bioscience,Biotechnology, and Biochemistry, 56, 1992,1321–1322.

[2] S.M. Alencar, T.L.C. Oldoni, M.L. Castro,I.S.R. Cabral, C.M. Costa-Neto, J.A. Cury, P.L.Rosalen, M. Ikegaki, Chemical composition andbiological activity of a new type of Brazilianpropolis: Red propolis, Journal ofEthnopharmacology, 113, 2007, 278-283.

[3] Y.J. Chen, A.C. Huang, H.H. Chang, H.F. Liao,C.M. Jiang, L.Y. Lai, J.T. Chan, Y.Y. Chen, J.Chiang, Caffeic acid phenethyl ester, anantioxidant from propolis, protects peripheralblood mononuclear cells of competitive cyclistsagainst hyperthermal stress, Journal of FoodScience, 74, 2009, 162-167.


Nugraheni et al. 43


[4] J. Zhou, X. Xue, Y. Li, J. Zhang, F. Chen, L.Wu, L. Chen, J. Zhao, Multiresiduedetermination of tetracycline antibiotics inpropolis by using HPLC–UV detection withultrasonic-assisted extraction and two-step solidphase extraction, Food Chemi., 115, 2009,1074-1080.

[5] H. Li, L. Pordesimo, J. Weiss, High intensityultrasound-assisted extraction of oil fromsoyabeans., Food Research International, 37,2004, 731-738.

[6] G.M. Sulaiman, et.al., Chemicalcharacterization of Iraqi propolis samples andassessing their antioxidant potentials, Food andChemical Toxicology, 49, 2011, 2415-2421.

[7] I. Gülçin, et al., Polyphenol contents andantioxidant activity of lyophilized aqueousextract of propolis from Ezurum, Turkey, Foodand Chemical Toxicology, 48, 2010, 2227-

2238.[8] R.A. Laskar, R. Ismail , B. Nayan, A. Naznin,

Antioxidant activity of Indiana propolis and itschemical constituents, Food Chem., 122, 2010,233-237.

[9] L. Moreira, L.G. Dias, J.A. Pereira, Antioxidantproperties, total phenols and ollen analysis ofpropolis sample from Portugal, Food ChemistryToxicology. 46, 11, 2008, 3482-3485.

[10] C.M. Mihai, L. Al Marghitas, O. Bobis, D.Dezmirean, M. Tamas, Estimation of flavonoidcontent in propolis by two different colorimetricmethods, Animal Science and Biotechnologies,43, 1, 2010.

[11] C. Chia-Chi, Y. Ming-Hua, W. Hwei-Mei,Jiing-Chuan, Estimation of total flavonoidcontent in propolis by two complementarycolorimetric methods, Journal of Food andDrug Analysis, 10, 3, 2002, 178-182.


Helmiyati et al. 44


NANOCOMPOSITE OF SODIUM ALGINATE POLYACRYLATE-ACRYLAMIDE-BENTONITE SUPERABSORBENT:

SYNTHESIS AND CHARACTERIZATIONHelmiyati1, Malida Aprilliza21,2 Department of Chemistry, Faculty Mathematic and Natural Science, Universitas Indonesia, Depok, 16424,

Indonesiae-mail : [email protected]

Abstract

The superabsorbent nanocomposite synthesis of sodium alginate with nanocrystal size as the back boneon copolymerization process using acrylic acid, acrylamide monomer and bentonite inorganic particlesas filler. The characterization nanocomposite of sodium alginate -poly (AA-co-AM)/bentonitesuperabsorbent as slow release fertilizer by FTIR show presence of the strong peaks Si-O stretching at1000 cm-1 and Al-O stretching at 500 cm-1 are characteristic peaks of bentonite. The strong peaks at1680 cm-1 due to the influence of N-H group from urea in nanocomposite. XRD patterns ofnanocomposite have semi-crystalline with crystalline index is a 62.12 % and obtained size particle 90.6nm. The surface morphology nanocomposite of sodium alginate-poly (AA-co-AM)/bentonite with SEMlook more rough and pores, due to the addition of bentonite, which are useful in the process ofabsorption of water and urea. The best swelling capacity by addition 15% bentonite in formulationobtained for water 576 g/g and urea 629 g/g. The release capacity obtained for 10 days to water 77.9 %and urea 60.66 %. This result is quite satisfied to applied for slow release fertilizer

Keywords: superabsorbent, nanocomposite, , sodium alginate, slow release fertilizer, release capacity

INTRODUCTION

Fertilizer is one of the important input materials forthe plant. However, most of the the applied amount ofcommon fertilizers can not reach the plant, but it iswashed off by rain and irrigation water [1,2]. The plantscan not absorb all the fertilizers applied, approximately40-70% of nitrogen, phosphorus 80-90%, and 50-70%potassium can not be absorbed by plants. The part of lostfertilizer not only causes large economic losses but alsovery serious environmental pollution. The use of slowrelease fertilizers is a new trend to save on fertilizerconsumption and to minimize environmental pollution[3,4]. Because of slow release fertilizers are designed togradually release fertilizer to the plant at a rate coincideswith the nutritional needs of plants [5,6].

At the sametime, water is one of the main factors thatimportant the production of agriculture, so it is veryimportant to use the water resource efficiently. Theresearch on the use of superabsorbent as watermanagement materials for agricultural applications hasattracted great attention. Superabsorbent polymers arecross linked hydrophilic polymers that can absorb water,or other liquids up to hundreds of times their own weight[7]. The ability of the superabsorbent to absorb waterincreased with increasing hydrophilic groups attached tothe polymer main frame. Based on the properties ofsuperabsorbent are widely used to control the release ofthe absorption and release of water and fertilizer in

agriculture [8]. In agricultural uses especially in aridareas, the use of superabsorbents causes to increase thewater holding capacity and therefore the fertility of thesoil [9]. Therefore, the soluble fertilizer absorbed orfertilizer coated by the superabsorbent formulation willbe a slow-release fertilizer the ideal [10].

The majority of conventional superabsorbents aremade from synthetic hydrophilic polymers such as polyacrylic acid or copolymer of poly Acrylamid. However,the poor degradability in soil, this will cause seriousenvironmental problems. The need for using safeenvironmentally, so that the research on the synthesis ofdegradable superabsorbent is continuously increasing.The composites of superabsorbent eco-friendly andbiodegradability were made from some natural polymermaterials, such as starch, cellulose, chitosan and alginate[11,12]. Alginate based superabsorbents prepared bygraft polymerization with acrylic acid and acryl amidemonomers onto a chain of alginate has been widely usedin agriculture [13,14].

Introduction of inorganic into superabsorbentpolymer is an effective way to develop materials withgood functions. In many previous studies,montmorillonite, kaolin dan attapulgite, have been usedto fabricate superabsorbent composites. Theincorporation inorganic mineral can not only reduceproduction cost, but also to increase swelling ability, andthermal stability of corresponding superabsorbentcomposites [14,15,16]


Helmiyati et al. 45


In this research, the synthesis of nanocomposite usedsodium alginate as backbone, acrylic acid, acrylamide asmonomer, potassium peroxodisulfate as initiator, N'N-methylene bisakrilamida as crosslinking agent andinorganic bentonite as filler material. Characteristics ofpolymer functional groups of nanocompositesuperabsorbent with fourier transform spectroscopyinfrared (FTIR), surface morphology with scanningelectron microscopy (SEM) and diffraction pattern withx- ray diffraction (XRD). Swelling and release capacitynanocomposite of sodium alginate -poly (AA-co-AM)/bentonite superabsorbent expected can be appliedto control slow release of water and fertilizer.

EXPERIMENTALMaterial

Brown algae used in this work was obtained fromBanten Indonesia as sources of sodium alginate (Na-Alg). Acrylic acid, Acrylamide (Nippon Shokubai) usedas a monomer, Potassium persulphate (KPS; Merck) asinitiator, N,N'-methylenebis- acrylamide (MBA; Sigma)as crosslinking, ammonium chloride (Merck), Potassiumdihydrogen phosphate (Merck), Urea (Merck) was usedas the absorbate and Bentonite as filler (Sigma).

Synthesis of Superabsorbent NanocompositeThe synthesis of nanocomposite refers to research

Rashidzadeh et al, 2014 [14]. the sodium alginateextracted from brown algae with the size of thenanocrystal was dissolved in distilled water, then addedthe bentonite suspension was stirred for 6 hours. Thesolution fed into the reactor three neck flasks equippedwith mechanical stirrer, reflux condenser, thermometer,and nitrogen gas. After being purged with nitrogen gasflowed for 30 minutes to remove dissolved oxygen, andheating to 600C in a water bath, then added a solution of5 ml of potassium persulfate (KPS) as the initiator,acrylamide (Aam), acrylic acid (AA) monomer and N,N'metilenabisakrilamida (MBA) into the reactor. Thewater bath was heated slowly to 700C and kept for 4 h tocomplete polymerization. Nanocompositesuperabsorbent product was dried in oven at 500C untilits weight is constant. The composition of the materialsvariation used in synthesis of superabsorbentnanocomposite can be seen in Table 1. Table 1Composition of the superabsorbent nanocomposite

Swelling Capacity of the superabsorbent nanocompositeSuperabsorbent dried (0.5 g) dipped in water and urea

solution at room temperature until get equilibrium.Superabsorbent already absorb adsorbate removed andallowed to constant weight. Swelling capacity (Se) iscalculated by the equation (1):(%) = 100 (1)

Where:

WS (g) is the weight of the swollen sample, and Wd (g) isthe initial weight of the dry sample.

Release Capacity of the superabsorbent nanocompositeThe swelling nanocomposite superabsorbent dipped

into 200 mL of distilled water at room temperature toreach equilibrium. Capacity release is calculated by theequation (2):(%) = 100 (2)

Where:

W0 (g) is the weight of the initial sample, and Wt (g) isthe sample weight of every time.

RESULTS AND DISCUSSIONAnalysis of Functional groups by FTIR

The FTIR spectra of the copolymers of poly- acrylicacid-co-acrylamide are shown in Fig1a, it appears thatthe O-H stretching peak at 3200-3400 cm-1 and the N-Hbands at 1600-1700 cm−1 are related to the overlappedstretching vibration of the carbonyl groups of AA andAAm. Fig 1b is the spectra of nanocomposite of sodiumalginate -poly (AA-co-AM)/ bentonite shown presenceof the strong absorption peaks around 1000 cm-1 (Si-Ostretching) and around 500 cm-1 (Al-O stretching) arecharacteristic peaks of bentonite. The nanocompositewhich have absorbed urea can be seen in Fig 1c showsharper peaks at 1680 cm-1 due to N-H of urea have getinto nanocomposite

Fig 1. Spectra of FTIR, a. Poly (AA-co-AM), b.Nanocomposite of sodium alginate -poly (AA-co-AM)/bentonite and c. Nanocomposite absorb urea.

Analysis of Crystallinity Index by XRD


Helmiyati et al. 46


Fig 2. XRD pattern of a. Sodium alginate isolation, and b.Nanocomposite of sodium alginate -poly (AA-co-AM)/bentonite

Figure 2a is the diffraction pattern of sodium alginatecan be seen two crystalline peaks at 2θ = 13.360, 2θ =22.220 and one amorphous peak at , 2θ = 8.120, thencalculated its crystalline index is 35.62%. This showsthat the sodium alginate isolation has semi-crystallinestructure and

particle size of cellulose calculated by scherrer’s lawobtained 54.6 nm. The sodium alginate isolation with thesize of nanocrystals will be used for the synthesis ofnanocomposite. The XRD patterns of nanocomposite ofsodium alginate -poly (AA-co-AM)/ bentonite (b) can beseen one crystalline peak at 2θ = 9,970, this peak derivedfrom peak of bentonite as inorganic and one amorphouspeak at 2θ = 6.220, crystalline index obtained is 62.12%.This shows that the superabsorbent nanocompositesodium alginate has semi-crystalline structure andparticle size of superabsorbent nanocomposite sodiumalginate obtained 90.6 nm.

Analysis of Surface Morphology by SEMFigure 3a shows that the morphology of the surface

of poly (AA-co-AM) can be seen from the surface of thecopolymer have pores that are small, uniform surfacedistribution and more homogeneous. In Figure 3b can beseen the morphology sodium alginate poly (AA-co-AM)have more pores, rough and heterogeneous than Fig 3a, itdue to the sodium alginate was added as a backbone sothat more porous. In Figure 3c can be seen themorphology nanocomposite of sodium alginate -poly(AA-co-AM)/ bentonite superabsorbent looks more andbig pores than Fig 3b , it due to addition of bentonite willincrease swelling and release process of water and urea.

Fig. 3 Micrograph a. Poly (AA-co-AM), b. Sodium alginatepoly (AA-co-AM), c. Nanocomposite of sodium alginate -poly(AA-co-AM)/ bentonite

Swelling Capacity of Nanocomposite SuperabsorbentExamination of the swelling capacity of the

superabsorbent important to be done for a variety ofapplications such as agriculture. The results of theswelling capacity of the water and urea at equilibrium,variety of samples of superabsorbent nanocomposite canbe seen in Figure 4.

Fig. 4. The swelling capacity of various samples

In Figure 4 can be seen the swelling capacity forwater and urea of a wide variety of compositions that issodium alginate, MBA, bentonite, acrylic acid andacrylamide monomer. The best swelling capacity is SA3,the composition of the SA3 nanocomposite is 3% sodiumalginate, 0.75% MBA, 15% bentonite, 6 gram acrylamide and 7 gram acrylic acid obtained the swellingcapacity to water 576 g/g and urea 629 g/g. The moresodium alginate as the back bone so that the activegroups that can be draw water into superabsorbent is alsoincreasing. The use of bentonite from 10 to 15% showedan increase in the capacity of the swelling water andurea, it is caused repulsive forces between the surface ofthe nanocomposite COO- ions is negatively chargedwith the surface bentonite negatively charged led toincrease the distance between the network so that thewater and urea absorption increase[12].

Release Capacity of Nanocomposite SuperabsorbentIn application as the slow release fertilizer,

nanocomposite which has swelling water and urea, thenperformed testing of release capacity in water, theexperiment was measured until reach equilibrium. Thecapacity of the release of water and urea to SA3nanocomposite can be seen in Figure 5.

Fig. 5. The Release capacity to water and urea


Helmiyati et al. 47


In Figure 5, it can be seen the release of water andurea the trend increases with increasing time, until thesecond day of a high release rate and increase until 10days, obtained release capasity 77.9 % for water and60.66 % for urea. This result is quite satisfied to appliedfor slow release superabsorbent.

CONCLUSION

Superabsorbent nanocomposite was succesfullysynthesized by free radical polymerization method. Theaddition of bentonite on the superabsorbent can increasethe swelling capacity and good release capasity. Thisresult is supported from FTIR spectra showed that theincorporation of bentonite superabsorbent matrix. It canalso be observed by XRD diffraction pattern. The resultsof surface morphology by SEM seen with the addition ofbentonite, the structure superabsorbent nanocompositewill be more porous. It was due to increase theabsorption of water and urea in the superabsorbentnanocomposite. The capacity of release quite well up today 10 th for urea 60.88% and water 77.9 %, whichmeans fertilizer and water are still in nanocomposite.Finally, the superabsorbent nanocomposite prepared inthis work have a good release capacity, thus reducing theloss of fertilizer and can withstand water, can be appliedin agricultural.

ACKNOWLEDGEMENTS

This research was supported by Research Fund ofGrant Research Universities, Directorate General forHigher Education, for fiscal year 2016

REFERENCES

[1] A. Bajpai, A. Giri, Swelling dynamics of amacromolecular hydrophilicnetwork and evaluationof its potential for controlled release ofagrochemicals, Reactive and Functional. Polymers.,53, 2, 2002, 125–141.

[2] M. Guo, M. Liu, Z. Hu, F. Zhan, L. Wu,Preparation and properties of a slowrelease NPcompound fertilizer with superabsorbent andmoisture preservation.Journal of Applied PolymerScience., 96, 6, 2005, 2132–2138.

[3] A. Jarosiewicz, M. Tomaszewska, Controlled-release NPK fertilizer encapsulated by polymericmembranes. Journal of Agricultural and FoodChemistry., 51, 2003, 413–417.

[4] Wu. L, Liu. M, Preparation and properties ofchitosan-coated NPK compound fertilizer withcontrolled elease and water retention, CarbohydratePolymers., 72, 2008, 240-247

[5] M. Teodorescu, A. Lungu, P.O. Stanescu,Preparation and properties ofnovel slow-releaseNPK agrochemical formulations based onpoly(acrylic acid)hydrogels and liquid fertilizers,Industrial and Engineering Chemistry Research.,48,14, 2009, 6527–6534.

[6] R. Liang, M. Liu, Preparation and properties ofcoated nitrogen fertil-izer with slow release andwater retention, Industrial and EngineeringChemistryResearch., 45, 25, 2006, 8610–8616.

[7] N. Seetapan, J. Wongsawaeng, S. Kiatkamjornwong,Gel strength and swelling of acrylamide–protic acidsuperabsorbent copolymers, Polymers for AdvancedTechnologies., 22, 12, 2011,1685–1695.

[8] M. Zohuriaan M. J and Kabiri. K, Superabsorbentpolymer Materials: A Review. Iran. Polym. J., 17 6,2008, 451.

[9] M. Zohuriaan, H. Omidian, S. Doroudiani, K.Kabiri, Advancesin non-hygienic applications ofsuperabsorbent hydrogel materials., Journal ofMaterials Science, 45, 21, 2010, 5711–5735.

[10]A. Rashidzadeh, A. Olad, A. Reyhanitabar,Hydrogel/clinoptilolite nanocomposite-coatedfertilizer: swelling, water-retention and slow-releasefertilizer properties, Polym. Bulletin., 72, 10, 2015,2667–2684.

[11]J. Zhang, A. Li, A. Wang, Synthesis andcharacterization of multifunctional poly(acrylic acid-co-acrylamide)/sodium humate superabsorbentcomposite, Reactive and Functional Polymers., 66,7, 2006, 747–756.

[12] J. Zhang, Q. Wang, A. Wang, Synthesis andcharacterization of chitosang poly (acrylicacid)/attapulgite superabsorbent composites,Carbohydrate Polymers., 68, 2, 2007, 367–374.

[13] A. El-Rehim, A. Hassan, Characterization andpossible agricultural application ofpolyacrylamide/sodium alginate crosslinkedhydrogels prepared byionizing radiation, Journal ofApplied Polymer Science., 101, 6, 2006, 3572–3580.

[14]A. Rashidzadeh, A. Olad, D. Salari, A.Reyhanitabar, On the preparation and swellingproperties of hydrogel nanocomposite based onsodiumalginate-g-poly(acrylic acid-co-acrylamide)/clinoptilolite and its application asslowrelease fertilizer, Journal of Polymer Research., 21,2, 2014, 1–15.

[15]A. Bortolin, F, A. Aouada, L.H. Mattoso, C.Ribeiro, Nanocomposite PAAm/methylcellulose/montmorillonite hydrogel: Evidence ofsynergisticeffects for the slow release of fertilizers,Journal of Agricultural and Food Chemistry., 61, 31,2013, 7431–7439.

[16]Y. Bao, J. Ma, Y. Sun, Swelling behaviour oforganic/inorganic composites based on variouscellulose derivates and inorganic particles,Carbohydrate Polymers., 88, 2012, 589-595.


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Potential Synergism of Astaxanthin from Haematococcus pluvialis asan Antioxidant SupplementsYuyun Yuniati a, Renny Indrawatia, b, Tatas H. P. Brotosudarmoa, b,Wynona Agatha Nimpoenoa, Leenawaty Limantarab,c

aChemistry Department, Faculty of Science and Technology, Universitas Ma Chung, Jl. Villa Puncak Tidar N-1, Malang, East Java

bMa Chung Research Center for Photosynthetic Pigments, Universitas Ma Chung, Jl. Villa Puncak Tidar N-1,Malang, East Java

cUniversitas Pembangunan Jaya, Jl. Cendrawasih, South Tangerang, Banten, West JavaCorresponding author: [email protected]

ABSTRACT

Nowadays the epidemiology of degenerative diseases have reached a point of particular concern, sincedegenerative desease becoming a major mortality cause in almost all over the world. The imbalance offree radicals and antioxidants in the body whether due to normal bodily process or modern lifestylepreferences leads to degenerative cell alterations, which then disrupts the function of tissues or organs.Dietary intake of antioxidants is in someways of importance to prevent degenerative diseases.Haematococcus pluvialis, a rich source of astaxanthin, which is known to have good antioxidantproperties, is already applied in most antioxidant supplements. This research aims to discover thepotential synergism of astaxanthin. As a such, the research is to encourage future researchers ondeveloping the most effective formula of astaxanthin with other pigments.

Keywords: antioxidant, astaxanthin, Haematococcus pluvialis, supplement, synergism

DEGENERATIVE DISEASES AS AMAJOR MORTALITY CAUSE

Degenerative disease is the most common deathcause, even more so in modern countries. Someexamples of degenerative diseases are heartdisease, stroke, diabetes mellitus, and hypertension.Degenerative diseases are resulted from acontinuous process based on degenerative cellchanges, affecting tissues or organs, whichincreasingly deteriorates over time, whether due tonormal bodily wear or lifestyle choices such as lackof exercise or bad eating habits. Modern lifestylescan be a trigger for degenerative diseases. Despitebeing caused by normal occurrences such asnormal bodily wear, exercise, and eating habits,degenerative diseases have become a majormortality cause. Human death percentage causedby these diseases are reported to become more andmore significant each year.

Figure 1 shown the most apparent death causeby around 13% is ischaemic heart disease, followedby stroke at 12% and both chronic obstructivepulmonary disease and lower respiratory infectionsfollowing at 6% [1]. In that figure, it is shown thatmajor death causes are causes are caused bydegenerative diseases, which are heart disease,stroke, obstructive pulmonary disease, cancer,diabetes mellitus, and hypertension, whichaltogether makes 39% of world death cause in2012.

These diseases are caused by imbalance ofradicals and antioxidants in the body. Free radicalscome from normal essential metabolic processes inthe human body or from external sources such asexposure to X-rays, ozone, cigarette smoking, airpollutants, and industrial chemicals. Free radicalsare formed continuously in cells as a consequenceof enzymatic process such as those involved in therespiratory chain, in phagocytosis, or inprostaglandin synthesis and non-enzymaticreactions of oxygen with organic compounds aswell ionizing reactions [2]. Industrial chemicalsand air pollutants are two things almost impossibleto erase from our life these days. They exist in fastfoods, packaged foods, even room freshener,instant beverages, snacks, everything. Airpollutants come from motorized vehicle residues,industrial waste, cooking, etc.


Yuniati et al. 49


Fig 1. Percentages of Death Causes in 2012 basedon GHO report [1]

Human body usually has a natural process inscavenging the free radicals we retrieve from ourdaily life, but since the amount of pollution, addedby other lifestyles, is much higher, theneutralization process becomes slower, and thus,this ends up damaging our body more than itshould have.

The desire to have a higher life quality and tokeep our body healthy has significantly promptedto society to consume supplements to balancebodies’ nutritional needs. To ward off free radicals,antioxidant is needed. Antioxidant will scavengefree radicals by inhibiting them from attackingbody tissues and cells. Some of well-known dietaryantioxidants are ascorbates, tocopherols andcarotenoids. There are also a lot of naturalantioxidants sources such as fruits and vegetables,seeds, cereals, berries, wine, tea, onion bulbs, oliveoil and aromatic plants [3].

HAEMATOCOCCUS PLUVIALIS ASNATURAL SOURCE OFASTAXANTHIN

Haematococcus pluvialis is the richest source ofnatural astaxanthin and it has already beencultivated at industrial scale [4]. Astaxanthinbelongs to xanthophyll group which has the mainfunction as antioxidants. The antioxidant propertiesof astaxanthin is ten times stronger than those of β-carotene. Astaxanthin consists of 40 carbons joinedtogether with both single and double bonds (3,3'-dihydroxy- β, β -carotene-4,4'-dione) [5]. Fig 2shows the chemical structure of astaxanthin.

Fig 2. Chemical structure of astaxanthin

Astaxanthin has been widely used in foodindustry, medicine, health supplements, andaquaculture [6]. Most dietary supplements employH.pluvialis as the source of astaxanthin whereasaquaculture industries prefer to cultivate P.rhodozyma [7].This pigment has a bright red colorfrom the long, conjugated double chains at thecenter of the compound. This structure allowsastaxanthin to have stronger antioxidant propertiesthan other carotenoids. [5] The mechanism of itsantioxidant properties is by protecting cells fromoxidation by scavenging singlet oxygens, thenreleasing the excess energy in the form of heat.Astaxanthin can stop oxidation since it has free

radical neutralizing properties. With this structure,electron decentralization – that can reduce theamount of reactive oxygens – happens [6]. Due tothese properties, it is highly potential to use H.pluvialis as a natural source of antioxidant. Theselection of H.pluvialis as the astaxanthin source isfrom the high astaxanthin concentration comparingto the other sources i.e. Salmonidae with theconcentration ranging from 0-37 mg/kg, and theCrustacean family ranging from 10-1160 while theconcentration of astaxanthin in H. pluvialis rangesaround 10,000-30,000 mg/kg. [6]

TRACING THE ANTIOXIDANT-RICH WITH ASTAXANTHINPIGMENT

To trace the distribution of product containingastaxanthin that is sold in market, a survey wascarried out by browsing through 5 online shops thatsell supplement.

Fig 3. Product types containing astaxanthin

In the market, there hasn’t been much use ofastaxanthin, especially in the form of processedfoods or beverages. The main usage is as anantioxidant supplement (79%) to tackle freeradicals due to UV radiation and as bioactivecompound in anti-aging cream, with one productusing astaxanthin to stimulate hair growth. All theproducers are found to be originated in USA. Fig 3.shows the percentages of each products containingasthaxanthin found in online market.

bodycream16%

shampoo5%

dietary. supp79%

O

CH3 CH3 CH3

CH3 CH3

CH3

OHCH3

CH3

O

OH

CH3 CH3


Yuniati et al. 50


Fig 4. Supplements composition in market

Fig 4. shows the composition of commercialproducts containing astaxanthin. The supplementscontaining pure astaxanthin compose as much as83% of the total products, leaving only 17% forsupplements with combinations. The combinationsare krill (9%) as the highest number followed byspirulina, carotenoid blend (containing zeaxanthin,lutein, cyanidin 3-glucoside, and meso-zeaxanthin)taking 4% of the total products count.

SYNERGISTIC POTENTIAL OFPIGMENT COMBINATION

The antioxidant activity of astaxanthin is tentimes stronger compared to β-carotene. Based onmarket data, we observe that the use of astaxanthinis not only applied as a lone antioxidant, but itcombined with spirulina, carotenoid, and krill. Thesynergistic effect of pigments has been researchedon and reported by scientists. A research shows thatmixtures of carotenoids were more effective thanthe single compounds, with the synergism effectmost pronounced when lycopene or lutein waspresent. The superior protection of mixtures maybe related to specific positioning of differentcarotenoids in membranes [9]. In another research,binary and ternary combination of quercetin, lutein,caffeic acid, chlorogenic acid, gallic acid androsmarinic acid was found to able to influence theantioxidant ability [10]. It is also proven that acombination of β-carotene and α-tocopherol resultsin an inhibition of lipid peroxidation significantlygreater than the sum of the individual inhibitions[11].

ASTAXANTHIN EXTRACTION FROMH. PLUVIALIS

The equipment used in astaxanthin extraction isbeaker glass for extraction, Whatman 0,2 μmNYLON filter membrane, Rotary Evaporator IKA

RV 10 Basic D, Shimadzu (Japan)’s, UV-VISSpectrophotometer UV-1700 PharmaSpec, HPLC-20 AD (Shimadzu, Japan), and ultrasonic fromMosonic USA. The main materials used wereHaematococcus pluvialis Flotow microalgae. Thebiomass of H. pluvialis was obtained from PT.Setia Kawan Abadi in the form of dried cells.Ethanol, acetone, methanol, and acetonitrile,KH2PO4, K2HPO4, nitrogen gas, and purified waterwere used for extraction procedure. The extractionwas accomplised in a dark room to minimalize thedegradation of pigment. Extraction method bysonication at the frequency of 32 kHz and bulktemperature range of 30-40°C is safe enough forthe pigment. Variables set are the variation ofsolvents: 100% ethanol, ethanol-water (50:50 v/v),and ethanol-water (25:75) v/v

First, 1 gram of H. pluvalis powder wasweighed in watch glasses, then poured into abeaker glass and is added with 5 ml of solvent. Thesonication process was done for 1 minute. Thesonication method results in the formation of microhot spots in the solution, so every one minute, thesonication process was interspersed withcooling for one minute. This process wasrepeated for three times. After that, the supernatantwas separated from the pellets and was strained byusing Whatman 0,2 µm filter membrane. Thepellets were then saluted again in 5 ml of solventsand the process is repeated. The strainedsupernatant was then kept in a flask to beevaporated with a Rotary Evaporator (IKA RV 10Basic D). The astaxanthin pigment produced wasthen dried with nitrogen gas and kept in a freezer of-20°C in temperature. The crude pigment extractwas then subjected to antioxidant assay. Freeradical scavenging activity of different solvent inextraction were measured by 1,1-diphenyl-2picrylhydrazyl (DPPH).


Astaxanthin identification usingSpectrophotometry produces an absorption spectrawith its main peak corresponding to astaxanthin at482 nm and one small peak at 670 nm. The peak at670 nm wavelength shows the presence ofchlorophyll in the pigment extract [12]. The spectraprofile in Fig. 5 shows that the absorbance valueincreases in the presence of pure ethanol, ethanol-water (50:50, v/v), and ethanol-water (25:75, v/v)as solvent proportional to the amount of extractedastaxanthin. The value of absorbance in differenttypes of solvent were 0.1523, 0.1031 and 0.1001respective to pure ethanol, ethanol-water (50:50,v/v), and ethanol-water (25:75, v/v).

83%

4%4%9%

pure spirulina carotenoid blend krill


Yuniati et al. 51


Fig. 5. Spectra profile extract of H. pluvialis in varioussolvent

The antioxidant activity of astaxanthinextracted from H. pluvialis microalgae in ethanolicsolution was determined using DPPH method. Thisassay provides an easy method to rapidly determinethe antioxidant activity of astaxanthin. The colorchange in the astaxanthin-DPPH solution frompurple to yellow causes a decrease in absorbancedue to the reduction of the antioxidant compoundsin astaxanthin. The percentage of antioxidantactivity (%AA) also gives different results based onsolvents used. The value of astaxanthin’santioxidant activity in different types of solventwere 21.51%, 18.8%, and 15.65% respective topure ethanol, ethanol-water (50:50, v/v), andethanol-water (25:75, v/v). Studies has alsoreported that xanthopylles (astaxanthin, lutein, andxeazanthin) have antioxidant effect to preventoxidative stress [13,14].

CONCLUSION

Astaxanthin is a potential antioxidativecompound that can be isolated from H. pluvialis.The highest value of antioxidant activity wasobtained in pure ethanol solvent. It has been knownthat astaxanthin show antioxidant activity and hasgood effect on human health. Astaxanthin itselfalso has a synergistic effect with spirulina,carotenoid, and krill. The innovation of astaxanthinuse as a natural source of antioxidant still needs tobe researched further, especially related to themost effective formula of astaxanthin incombination with other antioxidative pigments.Finding the combination and formula that has theoptimal synergistic effect is extremely important inorder to obtain the highest antioxidant activity

ACKNOWLEDGMENT

This research was supported by Ministry ofResearch, Technology and Higher Education of theRepublic of Indonesia, PUPT Research Grant 2016.

REFERENCES

[1] Global Health Observatory (GHO). 2012.Mortality and global health estimates.(http://www.who.int/gho/mortality_burden_disease/en/)

[2] Lobo, V., Patil, A., Phatak, A., and Chandra, N.2010. Free radicals, antioxidants and functionalfoods: Impact on human health. PharmacognRev. 4(8): 118–126.

[3] Dimitrios, B. 2006. Sources of natural phenolicantioxidants. Trends in Food Science &Technology 9:505-12.

[4] Guerin, M., Huntley, ME., and Olaizola, M.2003. Haematococcus astaxanthin: applicationsfor human health and nutrition. Trends inBiotechnology, 21(5): 210–216.

[5] Pratiwi, R and Limantara, L. 2008. Astaxantindan Kesehatan Manusia. Prosiding Sains danTeknologi Pigmen Alami, 322-333.

[6] Pratiwi, R and Limantara, L. 2008. PotensiAstaxantin sebagai Senyawa Antikanker.Indonesian Journal of Cancer, 4:149-154.

[7] Lorenz, RT and Cysewski, GR. 2000.Commercial Potential for HaematococcusMicroalgae as a Natural Source of Astaxanthin.Trends in Biotechnology, 18:1-8.

[8] Kurashige, M., Okimasu, E., Inoue, M., andUtsumi, K. 1990. Inhibition of Oxidative Injuryof Biological Membranes by Astaxanthin.Physlol. Chem. Phys. & Med. NMR, 22:27-38.

[9] Stahl, W.,Junghans, A., Boer, BD., Driomina,ES., Briviba, K., and Sies, H. 1998. Carotenoidmixtures protect multilamellar liposomesagainst oxidative damage: synergistic effects oflycopene and lutein. FEBS Letters 427: 305-308.

[10] Hajimhdipoor, H., Shahrestani, R., andShekarchi, M. 2014. Investigating thesynergistic antioxidant effects of someflavonoid and phenolic compounds. ResearchJournal of Pharmacognosy (RJP) 1(3): 35-40.

[11] Palozza and Krinsky, NI. 1992. β-Caroteneand α-Tocopherol Are Synergistic Antioxidants.Archives of biochemistry and biophysics,297(1): 184-187.

[12]Smith, J.H.C and Benitez, A. 1955.Chlorophylls : Analysis in plant material. InModern Methods of Plant Analysis, 142-196.Springer, Berlin.

[13] Miller, NJ., Sampson, J., Candeias, LP.,Bramley, PM., and Rice-Evans, CA. 1996.Antioxidant activies of carotenes andxanthophylls. FEBS Letters 384: 240-242

[14] Guerin, M., Huntley, ME., and Olaizola, M.2003. Haematococcus astaxanthin: applicationsfor human health and nutrition. TRENDS inBiotechnology 21(5): 210-216.



POSTER PAPERS


Untari et al. 53


Cyclic Voltammetry of Hydroquinone by Carbon Nanoporous PasteElectrodes Modified by FerroceneUntari, Muji Harsini, M. Zakki Fahmi

Department of Chemistry, Universitas Airlangga, Jalan Mulyorejo, Surabaya 61115, IndonesiaEmail: [email protected]

Abstract

A cyclic voltammetry of carbon nanoporous paste electrodes modified by ferrocene (Fc) in hydroquinone solutionhave been studied. The object of this research to predict of reaction mechanisms hydroquinone at carbonnanoporous paste electrodes modified surface. The modified carbon nanoporous paste electrode shows excellentelectrocatalytic activity with higher conductivity toward the oxidation of hydroquinone (HQ) in phosphate buffersolution (pH=7.0) between -0.3 to 1.0 V. The separation of oxidation and reduction peak (ΔE) is decreased from 547to 353 mV and peak the anodic currents for the oxidation of hydroquinone are greatly increased at modified carbonnanoporous paste electrode. The peak current obtained on carbon nanoporous paste electrodes modified was twotime higher than obtained on bare electrode. A anodic oxidation of HQ (in pH=7.0) at the Fc-modified carbonnanoporous paste electrode occurred at low overpotensial and treatment of the voltammetric data showed that itwas a purely diffusion-controlled reaction with the involvement of two electron in the rate-determination step.Conclusion of the research show that followed quasi-reversible chemical reaction mechanism.

Keywords: cyclic voltammetry, hydroquinone, ferrocene, carbon nanoporous electrode.

INTRODUCTION

Hydroquinone (HQ) is an important organiccompound that is widely used in many fieldssuch as pharmaceutical, antioxidant, dye,photography and cosmetic industries. Also HQis considered as an environmental pollutant bythe US Environmental Protection Agency (EPA)and the European Union (EU) for the hightoxicity and low degradability in the ecologicalenvironment [Hu et al, 2012].

Cyclic voltammetry (CV) is perhaps the mostversatile electroanalytical technique for the studyof electroactive species. . Cyclic voltammetry isoften the first experiment performed in anelectrochemical studv of a compound, a biologicalmaterial, or an electrode surface. The effectivenessof CV results from its capability for rapidlyobserving the redox behavior over a wide potentialrange. The resulting voltammogram is analogous toa conventional spectrum in that it conveysinformation as a function of an energy scan. Acyclic voltammogram is obtained by measuring thecurrent at the working electrode during thepotential scan. The current can he considered theresponse signal to the potential excitation sienal.The voltammoaram is a disolav of current (verticalaxis) versus potential (horizontal axis). Because thepotential varies linearly with time, the horizontalaxis can also be thought of as a time axis. This ishelpful in understanding the fundamentals of thetechnique (Kissinger & Heineman, 1983).

The relationship to concentration is particularlyimportant in analytical applications and in studiesof electrode mechanisms. The values of Ipa and Ipcshould be identical for a simple reversihle (fast)couple. Electrochemical irreversibility is caused byslow electron exchange of the redox species withthe working electrode. Electrochemicalirreversibility is characterized by a separation ofpeak potentials greater than indicated by eqn(Kissinger & Heineman, 1983).

To improve the performance of carbonnanoporous paste electrodes, it can be modified.Modifications of working electrode by ferosen.Ferrocene (Fc) and its derivatives are widely usedin electrochemistry because of their good stabilityin solution and rapid response to manyelectroactive substances (Kamyabi & Aghjanloo(2009). This research to predict of reactionmechanisms hydroquinone at carbon nanoporouspaste modified electrodes surface with optimationof working electrode composition and variation ofthe scan rate.

EXPERIMENTALInstruments and Reagen

Cylic voltammetry (CV) were carried out on apotensiostat edaq e-corder 201 (model ed201) withsoftware Echem versi 1.5. A three-electrode systemwas used with an carbon nanoporous pasteelectrodes modified by ferrocene as workingelectrode, Ag/AgCl reference electrode andplatinum wire counter electrode. A digital pHmeter was applied for the preparation of the buffer


Untari et al. 54


solution, which was used as supporting electrolytein the voltammetric experiments.

Ferrocene and hydroquinone were from sigmaand were used as received. All the other chemicalswere of analytical-reagent grade used directlywithout further purification. Double distilled waterwas used to prepared buffer and reagent solution.The supporting electrolyte used in all experimentswas 0.1 M phosphate buffer solution.

Carbon nanoporous paste electrode can beprepared by mixing carbon nanoporous powderwith as appropriate amount of parafin pastilles. Aportion of the composite mixture was packed into atube that connected to the end of a carbonnanoporous paste electrode. The tip of the electrodewas polished with a weighing paper. The modifiedelectrode was prepared by mixing unmodifiedcomposite with Fc (modifier mass fraction, w (Fc)= 1.0%) and then homogenized by spatula. Themodified composite was then used in the same wayas the unmodified electrode for analysishydroquinone 1mM in phosphate buffer solutionbetween -0.3 to 1.0 V at variation scan rate 10, 20,40, 50, 80, 100,200 and 250 mV/s.


Figure 1 shows the typical cyclicvoltammogram of hydroquinone in a pH = 7.0phosphate buffer solution at bare CPE and aferrocene (w(Fc) = 1.0%) modified CPE between -0.3 to 1.0 V at scan rate 100 mV/s in 1 mMhydroquinone solution. This figure illustrates thecyclic voltammetric responses of a bare carbonpaste electrode (curve B and D) and Fc-modifiedCPE (curves A and C) without and withhydroquinone solution 1 mM respectively.

Figure 1. Cyclic voltammogram: at bare carbonnanoporous paste electrode (CPE) (D) in the absence, (B)in the presence of hydroquinone 1 mM; and at 1% Fc-modified carbon paste electrode (C) in the absence, (A)in the presence of hydroquinone 1mM; supportingelectrolyte, 0.1 M phosphate buffer (pH= 7.0); scan rate100 mV/s.

At the surface of the unmodified electrode thedirect oxidation of hydroquinone produces a pair ofredox peaks were observed for hydroquinone 1mM.The oxidation and reduction peak potentialsoccurred at 410 and -137 mV, respectively. Underthe identical conditions, the ferrocen modified CPEgives increased peak currents to hydroquinoe. Awell-defined redox wave of hydroquinone wasobserved with the anodic peak potential at 297 Vand the corresponding cathodic peak potential at -63 mV. So, the peak separation was smaller thanthat at the bare CPE, and further, substantialincreases in peak current were also observed due tothe improvements in the reversibility of theelectron transfer processes. This suggests anefficient oxidation reaction of hydroquinone at theFc modified CPE. By using Fc as an electronmediator in the matrix of the modified electrode,the overpotential for the anodic oxidation ofhydroquinone becomes considerably lower.

The separation between the peak potentials (fora reversible couple) is given by:

ΔEp = Epa - Epk =. ……(1)

The peak separation can be used to determinethe number of electronstransferred, and as acriterion for a Nernstian behavior. Accordingly, afast one-electron process exhibits a ∆Ep of about59 mV (Wang, 2006). From the equation 1 Thepeak separation (∆Ep )for this research 353 dan547 for unmodified an modified respectively.

Current generated on the electrodes modifiedtwo times higher than the electrodes withoutmodification. this indicates the presence of ferosenaccelerate the electrochemical reaction themodified electrode has a good stability in aqueoussolutions. The results show that the modifiedelectrode has a good stability in aqueous solutions.(Kamyabi & Aghajanloo, 2009).

The amount of ferrocene in the carbonnanoporous paste has a significant influence on thevoltammetric response of the modified electrode.This is shown more distinctly in Figure 2A, whichcyclic voltammogram dan a plot of peak currentvs. the modifier mass fraction, w(Fc) at figure 2B.As this figure illustrates, the oxidation current forhydroquinone 1 mM increases gradually withmodifier, and at w(Fc) = 0.8% the oxidation currentachieves a maximum and then decreases with afurther increase of the modifier mass fraction. Thisoccurs may be, due to a decrease in the carbonnanoporous content in the paste and, consequentreduction of the conductive electrode area. Theselected electrode composition is the compositionof the electrode 1%, carbon nanopori 0.6 grams and0.3 grams of paraffin pastilles.


Untari et al. 55


Figure 2. cyclic voltammogram on hyroquinone 1mMsolution (A) and effect modifier fraction w/%, on thepeak current for hydroquinone 1 mM in phosphate buffersolution (pH = 7.0) (B); scan rate 100 mV/s.

Catalytical mechanism that is compatible withthe observed behavior is:

Fc Fc+ + e-

Fc+ + HQ

Fc + HQ (quinone)0Fc + HQoks + H+

Fc + HQ(quinone)0 + H+

The overall chemical reaction is as follows:2Fe 2Fe+ + 2e-

2Fe+ + DA 2Fe + HQox

In other words, the rate-determining step is aone electron transfer step followed by a fast oneelectron process to give HQox as a final product.

Figure 3. Dependence of Ipa/Ipk (A) and ΔE (B) ofhydroquinone 1mM in phosphate buffer solution ondifferent composition of electrode at scan rate 100 mV/s.

Carbon paste electrode modified with ferosen1% shows the separation of cathodic and anodicpeak potential is the smallest as well as thecomparison Ipa /Ipk approach to 1 as compared toother compositions. This suggests that electrodeproduces the best electron transfer mechanism asshown in figure 3 (A) and 3 (B).

Kinetic aspects and mechanistic ofelectrocatalytic oxidation and reduction ofhydroquinone at the surface of a modified electrodestudied from variation of scan rate. Scan rate caninfluence the current responses of hydroquinoneand corresponding electrochemical parameterscould be deduced from the relationship betweenscan rate of potential sweep and current responsesof hydroquinone. The dependence of oxidation andreduction peak current of hydroquinone 1 mM onscan rate at the Fc-modified CPE in 0,1 Mphosphate buffer (pH = 7), was illustrated in Figure5 (A) and (B). As the scan rate increased; theoxidation and reduction peak current increasedwith coefisien correlation (R2) 0,9133 dan 0,931respectively. The Ipa was directly proportional tothe square root of potential scan rate, ν1/2, over therange of 10–250 mV/s. This result indicates that theoverall electrochemical reaction of hydroquinone atthe modified electrode might be controlled by thediffusion of hydroquinone as well as by a kineticprocess.

Figure 4. Dependence of the cyclic voltametric responseat modified carbon nanoporous paste electrode on sweeprate in 0.1 M phosphate buffer (pH = 7.0) containinghydroquinone 1 mM. Scan rate: over the range of 10 –250 mV/s.

In addition, with increasing scan rate, thecatalytic oxidation peak potential (Epa) shifts tomore positive values and there is a linearcorrelation between the peak potential and the scanrate, as is illustrated in Figure 6.


Untari et al. 56


Figure 5. Dependence of the catodic peak current (Ipc)(A) and anodic peak current (B) of hydroquinone 1 mMon variation of scan rate range 10 – 250 mV/s.

Figure 6. Dependence ΔE of hydroquinone 1mM inphosphate buffer solution on different scan rate.

CONCLUSIONS

In this paper, a simple methode was used tostudied reaction mechanisms hydroquinone atcarbon nanoporous paste electrode modified byferrocene (Fc) which showed good electrocatalyticeffect to the redox reaction of hydroquinone. Theelectrochemical response of hydoquinone oncarbon nanoporous paste electrodes modifiedsurface was a diffusion-controlled quasi-reversibleprocess with two electrons and two protonsinvolved.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude toanalytical chemistry laboratory AirlanggaUniversity for support of this work.

REFERENCES.

[1] Hu, S., Wang, Y., Wang, X., Xu, L., Xiang, J.and Sun, W., 2012, Electrochemical detectionof hydroquinone with a gold nanoparticle andgraphene modified carbon ionic liquidelectrode, Sens. Actuators B, 168, 27-33

[2] Kamyabi, M.A. dan Aghajanloo, F., 2009,Electrocatalytic Response of Dopamine at aCarbon Paste Electrode Modified withFerrocne. Croatica Chemica Acta, 82 (3), 599-606.

[3] Kissinger, P.T. dan Heineman, W.T., 1983,Cyclic Voltammetry. Journal of ChemicalEducation, Vol. 60, No. 9

[4] Wang, J., 2006, Analytical Electrochemistry,3rd ed, John Wiley & Sons, Inc.,USA


Susilowati et al. 57


Isolation and Characterization of Alkaline Protease Producing MarineBacteria and Macro-algae from Tanjung Tiram BeachPrima E. Susilowati1), Wulan Purnamasari 1), Citrawana B.Ladjamu2), Masdidi3), Desi Kurniawati1)

1)Department of Chemistry, Faculty of Mathematics and Science2)Department of Pharmacy, Faculty of Pharmacy3)Department of Aquaculture, Faculty of Fisheries and Marine Science, University Halu Oleo,

Kendari, IndonesiaE-mail : [email protected]

1

ABSTRACT

Protease enzyme performs proteolysis, which is catabolism of protein by hydrolysis of the peptide bonds. Alkalineprotease has various applications in industrial products and processes, such as detergent, food, pharmaceuticalsand leather. The objective of our work is to isolates Bacteria and macro-algae from Tanjung Tiram beach. Enzymeprotease extracellular produced from bacteria, when cultured with 1% milk non-fat, and enzyme proteaseintracellular produced from macro-algae. The enzyme was activated and stabilized by relatively high saltconcentrations (> 0.2 M). Enzyme activity was temperature optimum 500C, pH optimum 8, and salt concentration 1M NaCl. The activity was stable up to 300C, from pH 8 to pH 9.

Keywords: bacteria, enzyme, macro-algae, marine, protease

INTRODUCTION

Proteases (EC.3.4) are a distinct subgroup ofhydrolytic enzymes which catalyze the cleavage ofpeptide bonds in protein substrates. Depending ontheir mode of action and catalytic mechanism,proteases are divided into four major groupsincluding: serine, cysteine, metallo- aspartate andthreonine. They are widely distributed in plants,animals and microorganisms.

Protease have importance in both commercialand physiological fields. For industrial productionof the proteases micro-organisms are preferred sothat large scale production of enzymes can beachieved. Bacillus, Aspergillus, Pseudomonas etc.are the organisms that produce protease. Proteasesare widespread in nature, microbes serve as apreferred source of these enzymes because of theirrapid growth, the limited space required for theircultivation and the ease with which they can begenetically manipulated to generate new enzymeswith altered properties that are desirable for theirvarious applications [1]. Screening of proteasesproducing bacteria symbion algae from differentecological environments can result in isolation ofnew alkaline proteases with unique physiochemicalcharacteristics [2].

Since the majority of industrial processes areaccomplished under harsh conditions, it would beof great importance to enjoy microbial enzymesthat demonstrate optimal activities at wide ranges

of pH, temperature and salt concentration.Microorganisms inhabiting in hypersalineenvironments are a remarkable source forproducing such enzymes. They are expected tohave specific proteins presenting characters whichare different from proteins produced by organismsfrom non-saline environments.

Few interesting studies have been presented onfungi, microalgae and protozoa as associates ofmacroalgae [3, 4, 5, 6]. Detailed knowledge of theinteraction of algae with their associated microbesand among microbes on algal surfaces and tissuesis still lacking [7, 8].

It was suggested that the proteolytic activitymay play a significant role in the symbioticrelationship between the bacterium and the algae.Furthermore, proteases are utilized in severalcommercial processes, including brewing, cheesemaking, and as detergent additives. In this paperwe aimed to isolate newer source of extracellularprotease from the local marine sample at TanjungTiram Beach Southeast Sulawesi to potentialapplication of the proteases for industrialapplications.

EXPERIMENTALIsolation and identification

Algae samples were collected from differentregions of Tanjung Tiram Beach, SoutheastSulawesi, Indonesia and screened for protease




producing microbes. The collection was performedin plastic containers and transferred to laboratory atroom temperature and the process of isolation wasinitiated immediately. The selected strain wasidentified using morphological tests.

Isolation and preparation of protease enzymeThe algae, approximately fifty grams (50g) in

amounts, were washed separately and then soakedin distilled water at room temperature. After that,all algae were finely powdered in a pre-chilledmortar and mixed with chilled 10mMTris-HClbuffer at pH 7.0 for 3 hours. The extracted mixtureswere filtered were centrifuged at 6000 rpm for 10minutes. The collected supernatant was used forpurifications.

The algae were soaked in saline water. Serialdilution was done for isolation of organisms.Halophilic medium consisting of Zobell agarplates. For bacteria the Petriplates were incubatedat room temperature for 24 hours. The obtainedcolonies were checked for cultural characteristics,morphology. Both the isolates were checked for theproduction of protease on skim milk agar plate [9].

The collected supernatants were saturated withsolid (NH4)2SO4 for overnight precipitation. Afterprecipitation, they were centrifuged at 7000 rpmfor 30 min. The collected precipitated weredissolved in 10 nm Tris-HCl buffer (pH 7) anddialyzed against the same buffer and finallycentrifuged at 5000 rpm for 10 min. Thesupernatant was used as crude enzyme for the assayof activity of enzyme and characterization.

Screening for Protease EnzymeThe crude enzyme was screened for protease

production using skim milk agar medium. Plateswere incubated for 24-48 h at room temperature.The clear zone around the streak was evaluated asprotease producers.

Parameters for Protease EnzymeThe influence of different temperature and pH

on proteolytic activity of the crude enzyme wasdetermined by medium Zobell, at various pH for 2days.


In the present study, algae were collected fromTanjung Tiram beach. Bacteria from macro-algaewere serially diluted and spread plated. About fourdominant morphologically distinct colonies wereselected and pure cultured by repeated streaking onthe Zobell agar plates. The isolated bacterial strainswere screened for protease producing ability onskim milk agar. The zone formation around thestreak of bacterial growth was identified as thepositive protease, which may be due to hydrolysisof casein (Table 1).

Table 1. Characterization Enzyme from Bacteriaand Algae

Microorganisms/Algae

Purificationenzyme

Clearzone(cm)

Isolate 1Crude extract 0.20(NH4)2SO4 0-30%

0.57

(NH4)2SO4 30-60%

0.50

(NH4)2SO4 60-90%

0.53

Algae 1Crude extract 1.20(NH4)2SO4 0-30%

1.20

(NH4)2SO4 30-60%

1.40

(NH4)2SO4 60-90%

1.09

The salt tolerance of the isolate was studied atvarious NaCl and KCl concentrations. The resultswere shown in (Figure 1). It was found that thetested strain has the ability to tolerate up to 0.5 MNaCl and 0.5 M KCl concentration.

Fig.1 Effect of salt on protease activity on KCl andNaCl




One would expect the extremely halophilicbacteria to be proteolytic because they occur inprotein-rich salt brines and they cause salted fish tospoil. Our results show that some Halobacteriumstrains do form a true extracellular proteinase, butthat different strains vary in proteolytic activity.

The effect of a change in pH on enzymeactivity is shown in Fig 2. As with temperature,each enzyme has an optimum pH. If pH increasesor decreases much beyond this optimum, theionisation of groups at the active site and on thesubstrate may change, effectively slowing orpreventing the formation of the enzyme substratecomplex. At extreme pH, the bonds which maintainthe tertiary structure, hence the active site, aredisrupted and the enzyme is irreversibly denatured.

pH played an important role in the enzymeproduction. In the present study the pH optimal ofprotease activity was studied at different pHranging from 6 to 9. The highest proteaseproduction was observed in pH 8 for algae. Belowand above that pH the enzyme activity was foundto be decreased. The obtained results werepresented in (Figure 2).

Figure 2 Effect of pH on protease activity

Enzymes have an optimum temperature. Thisis the temperature at which they work mostrapidly. Below the optimum temperature,increasing temperature will increase the rate ofthe reaction. This is because temperature increasesthe kinetic energy of the system, effectivelyincreasing the number of collisions between thesubstrate and the enzyme’s active site.Temperatures above the optimum will lead todenaturation. This occurs because the hydrogenbonds and disulphide bridges which maintain theshape of the active site are broken. Thus, enzymesubstrate complexes can no longer be formed.

Temperature also played an important role inactivating and inactivation of enzymes. Eachenzyme has an optimum temperature for maximumenzyme activity. In the present study, the effect oftemperature on protease production was studiedwith various temperatures ranging from 30 - 60℃.

The obtained results were noted in (Figure 3). Theprotease activity is relatively stable in thetemperature range 40℃ for Bacillus subtilis (0.232U/ml).).The enzyme activity was found to bedecreased above and below those temperatures forboth strains. The present investigation showed thatBacillus subtilis produced maximum protease at50ºC.

Figure.3 Effect of temperature on protease activity

Proteases are important enzymes obtainedfrom marine microorganisms and algae whichsynthesis bioactive compounds. Screening wasperformed and maximum proteolytic activity wasrevealed. Enzyme from isolate 1 has tolerated up to0.5 M NaCl and 0.5 M KCl concentration. Whereasoptimization of pH revealed that enzyme proteasefrom isolate 1 and algae, exhibited to increase atpH 8. Temperature optimum enzyme protease at500C.

CONCLUSIONS

Various bacterial isolates and algae fromTanjung Tiram Beach were studied for proteaseproducing activity. Proteolytic activity wasmeasured for high enzyme producing algae andmarine bacteria.

ACKNOWLEDGMENT

Support by the Ristek-Dikti (PKMP 2016).

REFERENCES

[1] G.S. Kocher, S. Mishra, Immobilization ofBacillus circulans MTCC 7906 for enhancedproduction of alkaline protease under batch andpacked bed fermentation conditions, Internet J.Microbiol., 7, p. 2.

[2] J. Singh, R. Vohra, D. Sahoo, Alkaline proteasefrom a new obligate alkalophilic isolate ofBacillus sphaericus. Biotech. Lett., 21, 1999,921-924.

[3] C. Hellio, J.P. Berge, C. Beaupoil, Y. Le Gal,N. Bourgougnon, Screening of marine algal




extracts for anti-settlement activities againstmicroalgae and macroalgae, Biofouling, 18,2001, 205–215

[4] S. Raghukumar, Ecology of the marine protists,the Labyrinthulomycetes (Thraustochytrids andLabyrinthulids), Eur J Protistol, 38, 2002, 127–145

[5] J. Kohlmeyer, B. Volkmann-Kohlmeyer,Marine Ascomycetes from algae and animalhosts. Bot Mar, 46, 2003, 285–306

[6] C. Lam, A. Grage, D. Schulz, A. Schulte, T.Harder, Extracts of North Sea macroalgaereveal specific activity patterns againstattachment and proliferation of benthic diatoms:a laboratory study, Biofouling, 24, 2008, 59–66

[7] P.D. Steinberg, R. de Nys, S. Kjelleberg,Chemical cues for surface colonization. J ChemEcol 28, 2002, 1935–1951

[8] J. Kubanek, P.R. Jensen, P.A. Keifer, M.C.Sullards, D.O. Collins, W. Fenical, Seaweedresistance to microbial attack: a targetedchemical defense against marine fungi, ProcNatl Acad Sci USA, 100, 2003, 6916–6921

[9] K.R. Aneja, Experiments in Microbiology,Plant Pathology and Biotechnology (4th ed.),New Delhi, India, New Age. ISBN 13 : 978-81-224-1494, 2009


Setiasih et al. 61


PARTIAL PURIFICATION AND CHARACTERIZATION OFBROMELAIN FROM THE CORE AND FLESH OF PINEAPPLE

EXTRACTS (Ananascomosus)Siswati Setiasih, Nita Magfirah Ilyas, Sri Handayani and SumiHudiyonoDepartement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424,

IndonesiaEmail: [email protected]

ABSTRACTThis research was initiated by comparing the specific activity of bromelain from core and flesh extract of pineapple(Ananascomosus) after fractionation using ammonium sulfate followed by dialysis. In the ammonium sulfatefractionation, precipitation of the crude enzyme was conducted by using different range of ammonium sulfateconcentration. Fraction 3 (50-80%) showed the highest specific activity both for the core and flesh of pineapple. Thepineapple core has a specific activity (0.30 U/mg protein) higher than the flesh (0.21 U/mg protein). After dialysis,the core fraction (0.33 U/ mg protein) also showed higher specific activity than flesh fraction (0.24 U/mg protein).The core fraction has the purify level 141.58 fold compared to crude extract. Fraction of the core has higher thermalstability than the fraction of the flesh. The optimum temperature and pH of this enzyme was 37oC and 7.0.Proteolytic activity of this enzyme was inhibited by EDTA, Hg2+, Cu2+ with the inhibition value of 29.33%, 13.88%and 6.43% respectively and was activated by Ca2+ and Na+ with activitation value of 1.62% and 1.95% respectively.

Keywords: bromelain, pineapple, proteolytic activity, specific activity.

INTRODUCTION

Indonesia is the fifth largest pineapple producer inthe world. Pineapples contain a group of proteolyticenzyme, namely called bromelain. This enzyme is usedin many therapeutic applications and was known hasantithrombic effect that can reduce clumping of platelets(antiplatelet), formation of plaques in the arteries andblood clots. All these effects are useful in the treatmentof cardiovascular diseases. (Bhattacharya, 2008).

These enzyme break down a protein by breaking apeptide bond and produce a more simple peptide.Bromelain enzyme was found in all plant tissues ofpineapple. The enzyme found in pineapple stem (EC3.4.22.32) is sulphydrylic and the sulphydryl group isessential to the proteolytic activity (Arshad, et al, 2014).About half of the protein in the pineapple containsbromelain protease. The benefits of bromelain is quiteextensive in the field of pharmacology and the foodindustry, stimulate many researchers to learn more. Theresearch about bromelain in pineapple plants have beenstarted since 1894 until today. Various ways of isolationhas been done to get bromelain from pineapple with thebest enzyme activity (Neta, et al, 2012).

In this experiment the crude bromelain was extractedfrom pineapple core and flesh. This enzyme was thenisolated and purified through several stages.The initialstage of this research was separation of crude enzyme byfractionation using ammonium sulfate followed bydialysis. The next step was determination the proteolyticand specific activity. The effects of inhibitor andactivator were also determined.

EXPERIMENTALEnzyme Extract

The pineapples that were used in this research wereobtained from Mount Cisalak, Cijeruk, Bogor. Crudeenzyme was prepared by making pineapple core or fleshpuree using 0.2 M phosphate buffer, pH 7.0 at 4°C andthen filtered. The filtrate had been centrifuged at 6000rpm for 15 min at 4°C. The supernatant obtained fromeach sample was crude bromelain extract. The crudebromelain was stored in a refrigerator to be used as asource of enzyme.

Fractionation of Enzyme with Ammonium Sulfate


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The crude bromelain was fractionated by usingammonium sulfate at different range of concentration (0-20%, 20-50%, 50-80%). About 150 ml of crude enzymeextract was put in a beaker glass which was placed in asalt ice bath. The crude enzyme was slowly added byammonium sulfate (0-20%) with constant stirring using amagnetic stirrer. After the addition of salt wascompleted, the stirring process had been continued for 20minutes. The solution was then allowed to settleovernight in the refrigerator. The solution wascentrifuged (6000 rpm) for 15 minutes at 4oC and theprecipitate was dissolved in 0.2 M phosphate buffer, pH7.0.

DialysisThe enzyme solution was put into a cellophane bag.

The cellophane bag was then immersed in a solution of0.05 M phosphate buffer, pH 7.0. The dialysis was takenplace at 4°C using ice salt bath by constant stirring. Thebuffer was changed for every 2 hour.

Protein and Enzyme Activity AssayThe protein concentration was determined by Lowry

Method. The enzymatic activity assay was perfomed byKunitz method using casein as substrate at 37°C for 30minutes. The enzyme was inactivated by adding 3 ml of5% TCA. The solution then had been incubated in icewater bath for 30 minutes.

Determination of Optimum pH and TemperatureFor determination of optimum temperature, reactions

were conducted at 30°C, 35°C, 37°C, 40°C and 45°C.Meanwhile the pH variations used were 6.0, 6.5, 7.0, 7.5and 8.0 using 0.2 M phosphate buffer (pH 6.0-8.0).Sample solution had been incubated at 37°C for 30minutes. The enzyme was inactivated by adding 3 ml of5% TCA.

The Effect of Various Compounds on Bromelain Activity

This experiment was conducted to determine theeffect of various compound on the proteolytic activity ofbromelain. The compounds used in this study were Hg2+

(HgCl2), Cu2+ (CuSO4), Ca2+ (CaCl2), NaCl and EDTA0.01 M. The enzyme solutions containing the compoundswere incubated at 37oC for 10 minutes, incubation wascontinued for 30 minutes after the addition of substrate.The enzyme was inactivated by adding 3 ml of 5% TCA.

Determination of Thermal StabilityDetermination of thermal stability of bromelain was

examined by incubating the enzyme and substrate for 15minutes at various temperature. The temperaturevariations used were at 40oC, 45oC, 50oC, 55oC, 60oC,65oC, 70oC, 75oC and 80oC. The enzyme was inactivatedby adding 3 ml of 5% TCA.

FractionVolume

(ml)

Total SpecificActivity(U/mg)

PurificationFactorActivity

(U)Protein

(mg)Pineapple Core

Fraction 1(0-20%)

1.5 11.76 212.25 0.04 23.87

Fraction 2(20-50%)

4.5 28.83 684.25 0.05 18.14

Fraction 3(50-80%)

1.5 16.86 54.75 0.30 132.65

Pineapple FleshFraction 1(0-20%)

6 34 589 0.05 29.51

Fraction 2(20-50%)

5 31.6 743.61 0.06 21.73

Fraction 3(50-80%)

2 16.66 78.55 0.21 108.47

Table 1.Purification of The Crude Enzyme by using Ammonium Sulfate


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Crude bromelain extract from pineapple core andflesh has been purified by fractionation using ammoniumsulfate, followed by dialysis. Crude enzyme obtainedfrom the core and flesh pineapple have a specific activityof 0.0023 U/mg and 0.0019 U/mg protein respectively.The results of crude enzyme purification usingammonium sulfate with different of range concentrationsproduced enzyme fractions that have different proteolyticactivity and protein content (Table 1). Fraction 2 ofpineapple core has higher proteolytic activity thanfraction 1 and 3, while fraction 2 and 1 has higherprotein content than fraction 3. In the pineapple fleshfraction 1 shows higher proteolytic activity than fraction2 and 3. Based on these data, it was known that fraction3 obtained from pineapple core or flesh have the highestspecific activity with the value of 0.30 U/mg and 0.21U/mg respectively and the purity level of 132.65 and108.47 fold compared to the crude enzyme. In this study,fraction 3 of pineapple core has higher purity level thanpineapple flesh and then further purified by dialysis.

Table 2 shows that the specific activity before andafter dialysis. The fraction 3 from the pineapple coreafter dialysis has higher specific activity than pineappleflesh. This is because in dialysis some proteins that hasmolecular weight less than bromelain can pass throughthe cellophane membrane. The specific activity ofbromelain from pineapple core is 0.33 U/mg protein witha purity level of 141.58 fold to the crude enzyme extract.While the specific activity of bromelain from pineappleflesh is 0.24 U/mg with a purity level of 122.21 foldcompared to extract of crude enzyme.

Determination of the Optimum pHDetermination of optimum pH was conducted only

for bromelain from pineapple core after dialysis, becauseit has the highest specific activity. Figure 2 shows that

the effect of pH against enzyme activity of bromelain.The enzyme activity of bromelain increase with theincreasing of pH and above pH 7.0 the enzyme activitydecrease. The optimum pH obtained in this research is7.0 with the enzyme activity value of the bromelain is6.51 U/ml. In this research, Bresolin (2013) found thatthe optimum pH of bromelain isolated from pineapplepeel was 7.0. Wuryanti (2004) also reported on herresearch that the optimum pH of bromelain obtainedfrom pineapple core is between pH 6 until 8.

Determination of Optimum Temperature

FractionVolume

(ml)

Total SpecificActivity(U/mg)

PurificationFactorActivity

(U)Protein

(mg)PineappleCore

Fraction 3Before Dialysis

2 16.86 54.75 0.30 132.65

Fraction 3After Dialysis

3 32.15 97.8 0.33 141.58

PineappleFleshFraction 3

Before Dialysis2 16.67 78.56 0.21 108.47

Fraction 3After Dialysis

3.5 28.86 120.75 0.24 122.21

Table 2.The Result of Dialysis on Bromelain Enzyme

Figure 1. Comparison of Specific Activity of Each Stage on thePineapple Core and Flesh


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Enzyme activity is affected by temperature. In thisresearch, determination of optimum temperature wasalso conducted only for bromelain from pineapple coreafter dialysis, because it has the highest specific activity.Figure 3 shows that the effect of temperature on enzymeactivity of bromelain. The enzyme activity of bromelain

increase with the increasing of temperature and above37oC the enzyme activity decrease. The optimumtemperature obtained in this research is 37oC with theenzyme activity value of the bromelain is 12.52 U/ml. Inthis study bromelain has the highest activity at 37°C anddecreased at temperatures around 40°C, but still has

activity at a temperature of around 45°C (6,47 U/ml).

The Effect of Inhibitors and Activators against BromelainActivity

The effect of Ca2+ and Na+ showed an increase ofproteolyticbromelain activity. It could be assumed thatthe calcium ion is a positive modulator which canfacilitate the interaction between substrate and enzyme(Table 3). Hg2+ and Cu2+ that a heavy metal ion act asinhibitor. This enzyme activity is also inhibited byEDTA.

This result shows that bromelain from pineapple corehas a higher thermal stability than the flesh.If thetemperature is higher than 37oC, the enzyme will bedenatured. Denaturation caused by changes of theenzyme active site that can decrease or eliminate theactivity of the enzyme. (Zusfahair et al, 2014).

Test of Thermal Stability on Enzyme SolutionDetermination of thermal stability was needed to

determine the effect of temperature changes on structurestability of bromelain. Bromelain from pineapple corewas more stable than bromelain from pineapple flesh atthe range temperature of 40-55ºC (Figure 4). Thebromelain from pineapple core completely inactivated at80ºC, while bromelain from pineapple flesh completelyinactivated at 70ºC.

This result shows that bromelain from pineapple corehas a higher thermal stability than the flesh.If thetemperature is higher than 37oC, the enzyme will bedenatured. Denaturation caused by changes of theenzyme active site that can decrease or eliminate theactivity of the enzyme. (Zusfahair et al, 2014).

Compound(0,01 M)

Activation(%)

Inhibition(%)

HgCl2 - 13.88

CuSO4 - 6.43

CaCl2 1.62 -

NaCl 1.95 -

EDTA - 29.33

Figure 2.The Effect of pH on BromelainActivity Figure 3. The Effect of Temperature on Bromelain Activity

Figure 4.Termal Stability of Bromelain on the Pineapple Core and Flesh

Table 3.Effect of compound on Enzyme Activity


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CONCLUSIONS

Partial purification of bromelain has been quitesuccessful and indicated by the increasing value ofbromelain activity. The pineapple core has higherspecific activity than pineapple flesh in eachprocess. Bromelain from pineapple core wasinhibited by EDTA, Hg2+ and Cu2+, while activatedby Ca2+ and Na+. Bromelain can be classified intocysteine protease and it was indicated by increasingits activity in the presence of calcium. Theoptimum pH and temperature obtained in thisresearch were 7.0 and 37oC. Core-bromelain wasmore on a higher temperature than flesh-bromelain.

ACKNOWLEDGEMENT

This project was funded by Biaya OperasionalPerguruan Tinggi Negeri (BOPTN) DIKTI 2016.

REFERENCES

[1] Arshad, Z.I.M., et al. Bromelain: an overviewof industrial application and purificationstrategies. Journal of ApplMicrobiolBiotechnol(2014) 98:7283–7297.

[2] Bhattacharyya, B.K., Bromelain, J. NaturalProduct Radiance, Vol 7(4), 2008, 359-363.

[3] Bresolin, et al., Isolation and purification ofbromelain from waste peel of pineapple fortherapeutic application, J. Biological andApplied Science, 2013.

[4] Burgess, R.R. 2008. Protein Purification,ISBN: 978-3-527-31716-5.

[5] Gautam, S.S., et al., Comparative Study ofExtraction, Purification, and Estimation ofBromelain from Stem and Fruit of PineapplePlant, 2010.

[6] GE Healthcare, Strategies for ProteinPurification Handbook, 28-9833-31 AA.

[7] Ji, Xuebin.,Hou, Ming, Novel agents for anti-platelet therapy, Journal of Hematology andOncology, 2011.

[8] K.Z. Nadzirah, S. Zainal, A. Noriham, and I.Normah, Efficacy of selected purificationtechniques for bromelain, International FoodResearch Journal 20(1): 43-46 (2013).

[9] Lakshminarasimaiah, N., Vibhuti, R.B.,Ghosh, B. Extraction of Bromelain frompineapple waste, Internatioanl Journal ofScientific & Engineering Research, Volume 5,Issue 6, June-2014.

[10]Mulyono, Noryawati., et al. Quantity andQuality of Bromelain in Some IndonesianPineapple Fruits, International Journal ofApplied Biology and PharmaceuticalTechnology, Volume 4, Issue -2, April-June-2013.

[11]Maurer, H.R., Bromelain:biochemistry,pharmacology and medical use,J. Cell. Mol. Life Sci. 58 (2001) 1234–1245.

[12]Neta, J.L.V., et al., Bromelain Enzyme fromPineapple : In-vitro Activity Study underDifferent Micropropagation Conditions,ApplBiochemBiotechnol (2012) 168:234–246.

[13]Ramalingam, C., Srinath, R., Islam, N.N.,Isolation and Characterization of Bromelainfrom Pineapple (AnanasComosus) andComparing its anti-browning activity on applejuice with anti-browning agents, J. Elixir FoodScience, 45 (2012) 78


Ayuningtyas et al. 66


SYNTHESIS AND CHARACTERIZATION OF COMPLEXCOMPOUND OF Zn(II)-EDTA FOR ANTIALGAE

COMPOUNDS IN INDUSTRIAL COOLING WATERSevia Ayuningtyas., Sri Sumarsih, Harsasi SetyawatiDepartement of Chemistry, Fakulty of Science and Technology

Universitas AirlanggaEmail : [email protected]

ABSTRACT

A research on the synthesis and characterization of complex compounds of Zn (II)-EDTA as antialgae compound isapplied to the cooling water industry. This research aims to determine the activity of complex compounds of Zn (II)-EDTA against algae that live in the water cooling water. The activity antialgae assay of comple compound of Zn(II)-EDTA with UV-Vis spectrophotometer method and dry cell weight method. Complex compound of Zn (II)-EDTAmade with mole ratio of ZnCl2: Na2EDTA is 1:1. Complex compound of Zn (II)-EDTA analyzed using UV-Visspectrophotometer and FTIR spectrophotometer. The results of UV-Vis spectrophotometer analysis showed that thecomplex compounds of Zn (II)-EDTA has a maximum wavelength at 752 nm. While the results of FTIR analysisshowed Zn-O vibration absorption at wave number 478.35 cm-1 and Zn-N vibration absorption at wave number516.92 cm-1. In the activity antialgae assay of complex compound of Zn (II)-EDTA made with a concentration of 5ppm, 10 ppm, 50 ppm and 100 ppm. The test results showed that the activity of complex compounds of Zn (II) -EDTAcan kill green algae and brown algae. Of the four concentrations of complex compounds of Zn (II)-EDTA, greenalgae and brown algae can be killed optimally at a concentration of 50 ppm.

Keywords: complex Zn(II)-EDTA, cooling water, antialgae, green algae, brown algae

INTRODUCTION

One of the main needs of the industry that isoften used during the production process is thatwater is used as cooling water (cooling water).Water used as cooling water should have a lowdissolved solids content, free of microorganisms,especially fungi and moss, and not corrosive(Widiasa et al, 2005). In industrial cooling systemsare typically used to use an open circulatorysystem. The heat contained in the cooling water isreleased into the air by direct contact. As a result,cooling water contaminated by microorganismsfrom the air, such as algae. If this is allowed, thenthe cooling machine will have corrosive andlumutan, so that the cooling process becomes lessthan the maximum and the production process willbe disrupted. The presence of microorganisms incooling water must be addressed immediately, soas not to cause further problems. In this study, toaddress the problem of the growth ofmicroorganisms in cooling water used complexcompounds of Zn (II) -EDTA. According PatentNo. 2002098231 AI, there is a water treatmentagent and water treatment methods to inhibit,reduce and prevent the formation of algae andbacteria growing in the water. One of these is Na 2EDTA complexed with the metal to preventbacteria and algae growing in the water. EDTA

compound selected because previous research hasdescribed that the ligand has the ability EDTAchelating agent capable of forming complexcompounds are very stable with divalent metal ions(Satroutdinov et al, 2000) and can be dissolved inwater (Tarasov, 2011). Oligodinamik Zn metal hasproperties that power on the metal which at smallconcentrations can kill microbes such as bacteriaand algae. Synthesized in this research instrumentis characterized by UV-Vis spectrophotometer andFTIR spectrophotometer. And a complexcompound of Zn (II) -EDTA tested their activitywith perpendaran method and the method of drycell weight.

EXPERIMENTAL SECTIONS

This study begins with the synthesis of complexcompounds of Zn (II) -EDTA. Zn mass thatweighed as much as 1.3637 grams and the mass ofEDTA that weighed as much as 3.3820 gram. Thenthe metal Zn and EDTA ligands each dissolved indistilled water. Furthermore, the two solutions weremixed and stirrer for 1 hour while heated with alow temperature. Stirrer and heating process wasstopped when the volume of the solution up to athird of the initial volume. Then the solution wascooled and then filtered using a filter paper toobtain sediment and filtat. The filtrate obtained ismuted and kept then recrystallized. Crystals formed




analyzed characteristics of the instrument UV-Visspectrophotometer and FTIR spectrophotometer.

Complex compound Zn (II) -EDTA tested itsactivity against cooling water samples containingalgae. Previously tested samples of cooling waterhas been added commercial compounds that seyton10 ppm and complex compounds Zn (II) -EDTAwith a concentration of 5 ppm, 10 ppm, 50 ppm,100 ppm, and there is a cooling water samples thatdid not get any additions. Then all samples in ashaker for 24 hours. After 24 hours, test the activityof compounds in complex samples. First, thecomplex compounds tested by the UV-Visspectrophotometer method. UV-Visspectrophotometer method to determine theconcentration of algae are still alive in the sample.After 24 hours, all the samples was measured itsabsorbance at a wavelength of 590 nm to 550 nmgreen algae and brown algae for. The secondactivity test was conducted using dry cell weight.This method is used to determine the dry weight ofalgae cells whether alive or dead in the sample.After the shaker for 24 hours, the sample wasfiltered using filter paper. The filter paper is thendried by in oven at low temperature for 1 hour.After 1 hour, the filter paper is cooled and thenweighed to determine the weight of the dry cell.

RESULT AND DISCUSSIONMaximum Wavelength Characteristics and FunctionalGroups Complex Compounds of Zn (II) -EDTA

The wavelength maximum and functionalgroups of complex compounds of Zn (II) -EDTApresented in Table 3.1 and Figure 3.1.

Tabel 3.1 The wavelength maximum compound ofZnCl2, Na2EDTA and complex compounds of Zn(II)-EDTA

CompoundsThe wavelengthmaximum (nm)

ZnCl2 652,00

Na2EDTA 472,50

Zn(II)-EDTA 752,00

Complex compound of Zn (II) -EDTA showedabsorption in visible region is at 752.00 nm. Thisshows that the new compounds are formed, which

is a complex compound of Zn (II)-EDTA. Theemergence of a wavelength at 752.00 nm showedbathochromic shift, namely the absorption peakshifts towards larger wavelengths. Theirbathochromic shift caused by the structure ofcomplex compounds of Zn (II) -EDTA have achromophore group (C = O bond) and auxochromegroup (OH group) derived from the ligand EDTA.Clusters that causes a complex compound of Zn(II) -EDTA can absorb light in the visible region /visible (Hendayana, 1994).

Figure 3.1. FTIR result of Na2EDTA dan complexcompounds of Zn(II)-EDTA

Figure 3.2a Absorbance Green Algae with AdditionComplex Compounds Zn (II) -EDTA and WithoutAddition

Figure 3.2b Comparison Green Algae with AdditionComplex Compounds Zn (II) -EDTA 50 ppm andCompound Seyton 10 ppm.




Based on the results of the spectra in Figure 3.1can be seen the inception of the formation of acomplex compound of Zn (II) -EDTA can be seenfrom the emergence of cluster vibration Zn and Zn-O-N. Where in the spectra, vibration Zn-O groupappeared at a wave bilangam 478.35 cm-1.According to the literature vibration Zn-O groupwill appear at wave number 486 cm-1 (Palomino,2006). And vibration groups Zn-N in spectraappears at wave number 516.92 cm-1. According tothe literature vibration Zn-N group will appear atwave number 570.93 cm-1 (Setiyani R, et al,2015).

The Anti Algae Activity Assay Complex Compounds Zn(II) -EDTA with UV-Vis Spectrophotometer Method

The result of the activity of complexcompounds of Zn (II) -EDTA against green algaeand brown algae with UV-Vis spectrophotometermethod can be seen in Figure 3.2a, 3.2b, 3.2c and3.2d.

Figure 3.2d Comparison Brown Algae with AdditionComplex Compounds Zn (II) -EDTA 50 ppm andCompound Seyton 10 ppm

Based on Figure 3.2a, 3.2b, 3.2c and 3.2d canbe seen that for 14 days, a complex compound ofZn (II)-EDTA showed a decrease in theconcentration of algae that are still alive. And whencompared with seyton compound, a complex

compound of Zn (II)-EDTA more effectively killalgae, both green algae and brown algae.

The Anti Algae Activity Assay Complex Compounds Zn(II) -EDTA with Dry Cell Weight Method

The result of Anti-algae activity of complexcompounds Zn (II) -EDTA with dry cell weightmethod are presented in Figure 3.3.

Based on Figure 3.3, for cooling water samplesby addition of complex compound Zn (II) -EDTAcan be seen that on the first day to the second dayof the algae growth has increased slightly. Then onthe third day until the seventh day there was anincrease significant growth. In these conditions, thealgae undergo cell division to the maximum so thegrowth is fast. While on the eighth day until thefourteenth day of the growth of algae began todecline slowly. In this condition, the algae began todecline cell division until eventually all cells diebecause they can not perform cell division again.For a sample by adding Seyton and without theaddition of any given growth chart continues toincrease the number of cells. This means that witha given sample without adding anything then algaewill grow continuously without being hamperedgrowth. While the sample is given Seytoncompound also experienced continued growthdespite being inhibited growth. This is because thecompound Seyton added ability to kill algae lessleverage than with the ability of the algae to growevery day.

CONCLUSION

The complex compounds of Zn (II)-EDTA hasthe characteristics of long. Maximum wave 752 nmand Zn-O functional groups on the uptake vibration478.35 cm-1 as well as the functional groups Zn-Non the absorption of vibration 516.92 cm-1.Complex compounds of Zn (II) -EDTA activitykilling algae green and brown algae atconcentrations of 50 ppm to percentage inhibitionof 62.4% in green algae and 76.2% on brown algae.

Figure 3.2c Absorbance Brown Algae with AdditionComplex Compounds Zn (II) -EDTA and WithoutAddition

Figure 3.3 Grafik Berat Sel Kering Alga




REFERENCES

[1] Hendayana, Sumar, dkk. 1994. InorganicChemistry. New Jersay: Prentice- Hall Inc.

[2] Palomino, A.G. 2006. Room TemperatureSybthesis and Characterization of HighlyMonodisperse Transition Metal-Doped ZnONanocrystals. Physics. University of PuertoRico.

[3] Paten No. 2002098231 A1, Method andApparatus for Preventing Bacteria and AlgaeGrowth in Water.

[4] Satroutdinov, A.D., Aidar, D., Emiliya, G.D.,Tat’yana, I.C., Margarete, W., Igor, G.M.,Valery, K.E., Thomas, E., 2000, Degradationof Metal-EDTA Complexes by Resting Cellsof the Bacterial Starin DSM 9103, Environ.Sci. Technol., 34 : 1715-1720.

[5] Setiyani, R. & Kartika, M.D. 2015.Pemanfaatan Komposit Kitosan Zn-O-SiO2sebagai Agen Antibakteri Terhadap BakteriStaphylococcus aureus Pada Kain Katun.Jurnal of Chemistry, Vol 4. No.2

[6] Tarasov, Konstantin, Patricia B., Michel C.,Eric M., Ynling L., 2011, Genesis ofSupported Crabon-coated Co Nanoparticleswith Controlled Magnetic Properties, Preparedby Decomposition of Chelate Complexes, JJNanopart Res., 13: 1873-1887.

[7] Widiasa, N.I., dkk. 2005. Rekayasa KontaktorHollow Fiber Longitudinal sebagai ClosedRecirulation Modular Cooling Water untukOptimalisasi Penggunaan Air dan Energi diIndustri . Semarang : Fakultas TeknikUniversitas Diponegoro.


Wijaya et al. 70


Analysis of partial leaching in the geochemical fraction of Fe content insediment Sendang Biru BeachAnugrah Ricky Wijaya1*, Ulfa Romlah1, Ahmad Fariq Imas1, IrmaKartika Kusumaningrum1, Surjani Wonorahardjo1, Eli HendrikSanjaya1

1State University of Malang, Department of Chemistry, Jl. Semarang No. 5 Postal Code 65145, Malang, EastJava, Indonesia

*Corresponding author: [email protected], [email protected]

Abstract

In order to get provide insight how to develop methodology in the partial leaching for assessment of metal contents,we investigated of Fe content in the geochemical fraction sediment in Sendang Biru Beach. Samples were leachedusing HNO3 (2-10 M), time contact (15-180 minutes), and varied volume of optimum condition in the leached HNO3

(10-100 mL) using reflux method to dissolve Fe in carbonate and sulfide phase due to anthropogenic effects. Afterleaching, filtrate samples were measured by Atomic Absorbance Spectroscopy (AAS). The optimum condition partialleached of Fe content showed 50 mL of HNO3 8M in time contacted of 180 minute were higher comparing with aquaregia (standard partial leaching). This optimum condition of leached Fe in sediment can be used as alternativemethod for assessment of Fe content in Sendang Biru Beach in the future.

Keywords: Partial Leaching, Fe, Sediment, Sendang Biru Beach

INTRODUCTION

Sediment in the geochemical fractions is achemical fossil consisting of anthropogenic andnatural sources (Wijaya et al. 2012, Wijaya et al.2013). Sediment has the ability to adsorb heavymetal. Heavy metal in sediment is warmly topicdue to its adverse effects on biological systems,causing a variety of diseases and disorders. One ofthe most common heavy metals is iron (Fe) whichis an essential heavy metal. According to Yoon etal. 2006, essential heavy metals are those which arerequired by the organism for biological processes,but excessive quantities can be toxic. Healthproblems caused by excess levels of Fe in humansinclude vomiting, diarrhea, and cardiovasculardisease.

Partial leaching is very important in theanalytical chemistry method to determine andanalyze of surface sediment in the beach accuratelyand precision. Akan, et al (2012) has conductedresearch on the assessment of water pollutants andsediment samples in Lake Chad, Baga, North EastNigeria. One of the parameters analyzed is Feconcentration in sediments be destructed usingaqua regia (a mixture of HNO3 : HCl ratio of 1: 3)and 30% H2O2. The addition of 30% H2O2

potentially cause the explosion in highlytemperature and reduce of Fe conten measurement.The use of aqua regia as a solvent cannot leachmetals in the overall analysis (Kisser, 2005).Furthermore, the presence of HCl solution can alsoprecipitate other metals that cause deviations in

analytical results. In the research that has beendone, there is no information on the effectivenessanalysis of the sediment samples. Effectivenessanalysis methods are expressed by validationleaching method with standard partial leaching.The accuracy and precision of the determination ofFe content is influenced by leaching process. It isnecessary to optimize the leaching process throughthe selection of the solvent and the leachingconditions.

As a solvent, HNO3 is a strong oxidizingreagent which can leach metals contained inenvironmental media (Ahn et al., 2011). Fe can beleached in a solution of HNO3 due to its highsolubility. HNO3 is also used as a standard blank tomeasure Fe in samples using AAS. Soluble Femetal is a form of metallic Fe located in sedimentin the mobile and exchangeable fractions (Wijayaet al. 2013). Here, a series of tests were made todetermine the optimum conditions for the leachingof sediments with HNO3 as the solvent. Thisalternative method of leaching of Fe in optimumconditions for HNO3 will be compared with aquaregia without the addition of a solution of H2O2 inorder to prevent explosive.

EKSPERIMENTALMaterials

The sediments used in this study were obtainedfrom Sendang Biru Beach, East Java, Indonesia islocated at point 8o43’ S and 112o68’E. Sedimentsamples were obtained via sediment grabber. Thechemicals used in this study are: Powder


Wijaya et al. 71


Fe(NO3)3.9H2O grade, HNO3 (pa), HNO3 solutions(2M; 4M; 6M; 8M, and 10M), HCl (pa), anddistilled water.

MethodsThe partial leaching process was conducted

using reflux method at temperature of 85oC asfollows: (1) Preparation of sediment samples, (2)Testing leaching solution HNO3 in variousconcentrations (2; 4; 6; 8; and 10M), (3) Testingthe leaching using a solution of HNO3 with variousdecomposition times (15; 30; 60; 120; 150; and 180minutes), (4) Testing variations of the volumeHNO3 at the optimum concentration (10; 25; 50;75; and 100 mL), (5) Comparing Fe leached usingthe optimum condition with aqua regia. All offiltrates in the leached solution in 0.5 g sedimentwere measured using AAS Shimadzu AA-6200under standard operating conditions havingr>0.999. Instrumental parameters of Fe was usedthe acetylene (2.3 L/min), air (17 L/min),wavelength (288.3 nm), slit width (0.2 nm), lampcurrent (30 Ma) and detection limit of Fe (5 µg/L).The reproducibility in the measuring in the leachedsolution was found to be at 95% confidence level.

RESULTS AND DISCUSSIONEffect of varied concentration HNO3

Partial leaching was tested from the smallestconcentration to higher concentration for maximalleaching Fe ions in the matrix surface sediment.HNO3 was chosen due to its high solubility todissolve Fe ions. The effect of differingconcentrations of HNO3 solvent on the leaching ofFe metal in sediment was tested. The optimumconcentration of HNO3 is chosen to consider thesmallest concentration of HNO3 to leach amaximum concentration of Fe in sediment. Asshown in Figure 1, the concentration of Fe isdirectly proportional to the increase in theconcentration of HNO3 at various concentrationsfrom 2M to 8M. The ion of H+ exchanged ion ofFe3+ in the leached sediment at optimum at HNO3

8M. In other hand, the leached of Fe decreased at10M due to the higher competitive of matrix ions,such as Mg and Ca ions contained in the sedimentsamples.The chemical reaction of HNO3 with Fecontaminants in the sediment can be written asfollows:3Fe2+(aq) + HNO3(aq) + 3H+(aq)

3Fe3+(aq) + NO(g) + 2H2O(l)

Fe(s) + HNO3(aq) + H+(aq)

Fe3+(aq) + 2H2O(l) + NO(g)

Fe3+(aq) + HNO3(aq) + 3H+(aq)

Fe3+(aq) + NO(g) + 2H2O(l)

Using solvent of HNO3 can leach Fe releasingFe in acid soluble fraction. The source of Fe in thegeochemical fraction in sediment was caused theprecipitation or co-precipitation as Fe-carbonateand Fe-dolomite or carbonate phase. Other sourceof Fe came from sulfide minerals including pyrite,sphalerite, and chalcopyrite due to the weatheringwaste piles. HNO3 8M accurately leached maximalion of Fe in the carbonate and sulfide fraction insediment Sendang Biru Beach.

Effect of time contactThis study investigated at variations time

contact to determine the effect on the leached Fefrom the sediment. HNO3 possibly could release Feions becoming passive in the several times (Svehla,1979). Leaching was performed with the timecontact variations in order to choose the accuratelytime for leaching Fe and prevent over time causedthe passive Fe ions.

The optimum leaching time is the shortest timeof decomposition that can leach Fe to provide amaximum concentration of Fe. The concentrationof Fe is directly proportional to the longer leachingtime contact with variations of 15, 30, 60, 120, and150 minutes using HNO3 8M, respectively. The

0.00.10.20.30.40.50.60.7

0 5 10 15

Fe (p

pm)

HNO3 (M)

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200

Fe (p

pm)

Time (minutes)

Figure 1. Relationship between of HNO3 concentration(M) with leached Fe

Figure 2. Relationship between time contact(minutes) with leached of Fe (ppm) in the sediment


Wijaya et al. 72


research data of the influence of variations ofleaching times on the concentration of leached Fein the sediment can be seen in Fig. 2.

In the relatively short time of leaching from 15to 120 minutes, the HNO3 possibly leached of Fe insediment in the carbonate phase and then from 120to 150 minutes the HNO3 attacked Fe in the sulfidephase. The leaching of HNO3 revealed to contact ofion active Fe in sediment from 15 to 150 minutesand then after 150 minutes ion of Fe undergoingpassive. The condition optimum of time contact at150 minute, the HNO3 can leach maximal ion of Fein the geochemical fraction of sediment SendangBiru Beach.

Effect of volume HNO3 8MVariations on the volume of an 8 M HNO3

solution is measured to see how the number ofmole of H+ ions that are exchanged with mole of Feions in the fraction of sediment. The optimumvolume of 8 M HNO3 solution is chosen toinvestigate the minimum volume that is used todissolve Fe with a maximum output. The researchdata on the effect of variations of volume of 8MHNO3 solution on the leaching of Fe in thesediments can be seen in Fig. 3.

Mole of Fe in leached solution is directlyproportional to mole of H+ from HNO3 8M in thevolume of 10, 25, 50, 75 and 100 mL, respectively.As shown in Figure 3, the volume from 10 to 50 mlof HNO3 8M increases the leached Fe in sediment.At higher volumes of the 75 and 100 mL, leachedFe concentration decreased. The optimum volumeidentified is 50 mL HNO3 8 M.

Comparison of Decomposition of Fe in Sediment UsingAqua Regia with HNO3

Aqua regia is a mixture volume of solute HNO3

and solute HCl in the ratio of 1:3. Aqua regia isstandard partial leaching of Fe in geochemicalfraction sediment. Aqua regia was compared to the

optimum values for concentration, time, andvolume of a HNO3 solution obtained from the this

experiments to determine which is the moreoptimum solvent for leaching Fe in sediment. Thereaction equation of the dissolution of Fe in aquaregia is as follows:

The research data can be shown in Figure 4 andTable 1. The experiment shows a higherconcentration of Fe was obtained by using 50 mLof 8M HNO3 solution at 150 minute (optimizedcondition) compared to their optimized conditionof aqua regia. In order to compare the effectiveleaching solution to leach Fe in sediment, wecalculated the leached Fe using optimizedcondition of HNO3 with aqua regia. As shown inTable 1, Leaching solution of HNO3 could leach 72ppm comparing with aqua regia (60 ppm) in 0.5gram in sediment. This optimum condition ofleached Fe in sediment can be used as alternativemethod for assessment of Fe content in SendangBiru Beach in the future.

CONCLUSIONS

Based on this research can be concluded thatthe partial leaching in optimum conditions wasobtained at 50 mL of 8 M HNO3 and the leachingtime contacted for 150 minute. The content of Fe insediment in optimized condition leaching of HNO3

solution was more efficient than using aqua regia.This method of partial leaching in optimizedcondition can be used for assessment of Fe contentin the sediment Sendang Biru Beach in the futureassociated with human health

ACKNOWLEDGMENTS

0

0.2

0.4

0.6

0.8

0 50 100 150

Fe (p

pm)

Volume of HNO3 8M (mL)

2Fe2+(s) + HNO3(aq) + 3HCl(aq) 2Fe3+(aq) + NOCl(g) + Cl-(aq) + 2H2O(l)

0.500.550.600.650.700.75

Asam Nitrat Akuaregia

Fe

(ppm

)

Leaching Solution

Optimized HNO3 Aqua regia

Figure 4. Comparison of optimized leaching HNO3 withaqua regia

Figure 3. Relationships of volume HNO3 8M withleached of Fe (ppm) in sediment


Wijaya et al. 73


The authors would like to thank the chemistrydepartment, State University of Malang who hasgiven the place and equipment in completing thisresearch, and chemical research laboratoriesFMIPA UM. The authors would also like to thankthe Ministry of Research, Technology and HigherEducation of Indonesia who has funded thisresearch through the Fund for FundamentalResearch Grant in 2016.

REFERENCE

[1] J. W. Ahn, D. W. Chung, K. W. Lee, J. G.Ahn, H. Y. Sohn, Nitric Acid Leaching ofBase Metals from Waste PDP Electrode Scrapand Recovery of Ruthenium Content fromLeached Residues, Mater. Trans, 52, 5, 2011,1063–1069.http://doi.org/10.2320/matertrans.M2010417

[2] J. C. Akan, M. T. Abbagambo, Z. M. Chellube,Assessment of Pollutants in Water andSediment Samples, J. Environ. Protect,

3(November 2012), 2012, 1428–1441.http://doi.org/10.4236/jep.2012.311161

[3] M. I. Kisser, Digestion of solid matrices Part1 : Digestion with Aqua Regia, Report ofevaluation study, (November), 2005, 1–38.

[4] A. R. Wijaya, A. J. Ouchi, K. Tanaka, R.Shino, S. Ohde, Metal contents and Pbisotopes in road-side dust and sediment ofJapan. J. Geo. Exp, 118, 2012, 68–76.

[5] A. R. Wijaya, A. J. Ouchi, K. Tanaka, D. K.Cohen, S. Sirirattanachai, R. Shinjo, S. Ohde,Evaluation of heavy metal contents and Pbisotopic compositions in the Chao PhrayaRiver sediments: Implication foranthropogenic inputs from urbanized areas,Bangkok. J. Geo. Exp, 126-127, 2013, 45–54.

[6] A. I. Vogel, Textbook of Macro and Semimicro Qualitative Inorganic Analysis. London:Logman Group Limited, 1979.

[7] J. C. Yoon, Z. Xinde, Qixing, L.Q. Ma,Accumulation of Pb, Cu, and Zn in NativePlants Gowing on a Contaminated Florida Site,Sci. Total. Environ, 52, 2006, 456-464.

No. Leaching

Solution

Optimized

Condition

Fe in solution

(ppm)

Fe in sediment

(ppm)

1. HNO3 8 M 50 mL, 150 minutes, 85oC 0.72±0.05 72 ppm

2. Aqua regia 50 mL, 150 minutes, 85oC 0.60±0.07 60 ppm

Table 1. The calculation of leached Fe in sediment


Junairiah et al. 74


GC-MS ANALYSIS OF N-HEXANE, ETHYL ACETATE, ANDMETHANOL EXTRACT OF Callicostella prabaktiana(C. Mῢll.)

Bosch & Sande Lac.Junairiaha, Tri Nurhariyatia, Suaibaha, Ni’matuzahroha, LilisSulistyoriniba Department of Biology Faculty of Science and Technology, Airlangga University,

Surabaya, Indonesiab Faculty of Public Health Airlangga University, Surabaya, Indonesia

*Corresponding Author’s E-mail: [email protected]

ABSTRACT

Callicostella prabaktiana is one species of the moss , orders Hookeriales, family Hookeriaceae. So farthere has been no research on bioactive compounds C. prabaktiana. The objective of this research toknow the bioactive compounds the moss C. prabaktiana. Gametophyte of the moss are cleaned, dried,and made into a powder, then extracted with hexane, ethyl acetate, and methanol by maceration method.The identification of bioactive compounds by using Gass Chromatography Mass Spectra (GC-MS). Theresults showed that the extract of n-hexane, ethyl acetate, and methanol C prabaktiana each containing21, 26, and 17 compounds. The dominant component is hexadecanoic acid and neophytadiene

Key words: Callicostella prabaktiana, bioactive compounds

INTRODUCTION

Recently the use of plants as herbal remedies toincrease significantly. This is due to plantcontaining the bioactive compounds [1,2,3]. Theplant is used as an antimicrobial, antiinflammatoy,analgesic, antiseptic, anti-tumor, antioxidant,fungicide, nematicide, pesticides,hypercholesterolemic, antihistaminic,antiandrogenic,etc[4]

Callicostella prabaktiana is one species of themoss , orders Hookeriales, family Hookeriaceae.Based on literature review, there has been noresearch on bioactive compounds of Callicostellaprabaktiana. The objective of this research todetermine the bioactive compounds of the mossCallicostella prabaktiana by Gass ChromatographyMass Spectra (GC-MS). GC-MS is the besttechnique to identify bioactive compoundsalkaloids, terpenoids, long-chain hydrocarbons,alcohols, esters, amino acids and nitrogencompounds, and others [5].

EXPERIMENTALCollection of Plant Material

C. prabaktiana wass collected from Cangarforest, Batu, East Java, Indonesia, and the mossidentified and voucher specimen was deposited in

the Department of Biology, Faculty of Science andTechnology, Airlangga University.

Preparation of ExractThe moss of C. prabaktiana was washed with

running water and dried. Dry moss plants is madein the form of a powder by cutting. Powdered plantmaterial was extracted using maceration method.Solvents used are n-hexane, ethyl acetate andmethanol. Each volume of solvent used was 400

Figure 1. Chromatogram profile of n-hexaneextract of C. prabaktiana


Junairiah et al. 75


ml. Maceration repeated three times. Identificationof bioactive compounds using GC-MS analysis.


Based on the chromatogram profile of extract n-hexane, ethyl acetate and methanol C. prabaktianaconsists of 21, 26, and 17 peaks (Figure 1, 2, 3).This is showed that the extract of n-hexane, ethylacetate and methanol consist of 21, 26, and 17compounds.

Figure 2. Chromatogram profile of ethyl acetate extractof C. prabaktiana

Figure 3. Chromatogram profile of ethyl acetate extractof C. prabaktiana

Identification of compounds each extract of n-hexane, ethyl acetate, and methanol can be seen ontables 1,2, and 3. Based on the identification, thedominant component is hexadecanoic acid andneophytadiene.

Table 1.Phytocomponents identified in the n-hexaneextract of the moss Callicostella prabaktiana

peak RetentionTime

Compound Area(%)

1 4.15 Naphthalene 2.52

2 7.03 Butylated

Hydroxytoluene

3.16

3 7.16 14-Beta-H-Pregna 0.52

4 7.48 14-Beta-H-Pregna 2.72

5 7.52 Nonahexacontano

ic acid

0.73

6 7.57 14-Beta-H-Pregna 1.11

7 7.64 14-Beta-H-Pregna 1.43

8 7.73 14-Beta-H-Pregna 2.58

9 8.00 14-Beta-H-Pregna 6.72

10 8.03 Tetrahydroxycycl

opentadienone

2.97

11 8.11 3,6-Epoxy-

2H,8H-pyrimido

3.30

12 8.61 Cyclohexyl 2-

Methyelenebutan

yl

9.87

13 9.31 Phenol 2.27

14 9.92 2,5-furandone 7.66

15 10.89 Hexadecanoic

acid

12.78

16 11.16 1,2Benzenediol 2.92

17 11.76 1,2-

Benzisothiazole

11.88

18 11.90 Cyclooctacosane 10.69

19 15.29 1-Dotriacontanol 7.29

20 15.59 Heptadecene-(8)-

Carbonic acid-(1)

3.95

21 16.57 Bicyclo(2,2,1)hep

t-2-en-7’-yli

2.93


Junairiah et al. 76


Table 2. Phytocomponents identified in the ethyl acetateextract of the moss Callicostella prabaktianapeak Retention

TimeCompound Area

(%)1 2.42 3,4-furandiol 0.41

2 3.89 Hexane, 1-

isopropylidenecyclop

0.78

3 4.15 Naphthalene 0.56

4 6.42 Decane, 2 methyl- 0.87

5 6.67 Vulgarol B 0.62

6 6.81 Methanesulfonamide 0.67

7 6.95 7-Hydroxy-3-(1,1-

dimethylprop-2

0.87

8 7.65 Camphane hydrate 0.55

9 8.47 Oxiranepentanoic

acid

0.60

10 8.61 Delta 8p menthen 3,4

diol

1.98

11 8.71 Trans2 nonadecene 0.77

12 8.93 Cis 8-(N-pyrrolidyl)-

2,2,5,5-t

0.74

13 9.31 1-Hexadecene 4.57

14 9.83 Neophytadiene 24.01

15 10.11 4-Tetradecyne 3.57


17 10.90 8-Acetyl-3,3-

epoxymethanol

4.01

18 11.16 7,9-Di tert-butyl-1-

oxaspiro

1.55

19 11.30 Benzenepropanoic

acid

1.91

20 11.73 n-Hexadecnoic acid 14.31

21 11.90 Cycloeicosane 8.17

22 12.75 Heptenoic acid 7.35

23 13.81 9-Octadecanoic acid 1.55

24 14.47 1-Ditriacontanol 4.23

25 15.30 Cis-2-nonadecene 4.86

26 15.50 Oleic acid 3.43

Table 3. Phytocomponents identified in themethanol extract of the moss Callicostellaprabaktiana

peak Retention

Time

Compound Area(%)

1 2.47 1,2-Propandiol, 3 chloro 5.58

2 3.15 Delta 2 tetrazaboroline 2.99

3 6.30 Cyclopropanecarboxylic

acid

4.44

4 6.84 Cytidine 16.9

4

5 7.81 Thiosulfuric acid 4.78

6 8.43 Tetrahydroxycyclopentadie

none

1.82

7 8.73 Cyclotriadecane 4.12


9 9.92 2-docosanol 6.63

10 10.34 1-docosanol 3.79

11 10.90 4-dimethylhydrazonometh 2.93

12 11.73 n-hexadecanoic acid 22.6

1

13 12.17 Oleic acid 2.85

14 14.40 Cyclopentadecanone 3.78

15 15.30 Cis, trans-farnesal 4.09

16 15.59 Heptadecene-8-carbonic

acid

3.12

17 19.52 8-acetyl-3,3-epoxymethano,

6,6,7

1.98

The ethyl acetate extract of Guiera senegalensiscontaining 9-hexadecanoic acid (20.93%). Thiscompound has potential as pesticides, andantibiotics [6]. The n-hexane extract of Epaltesdivaricata containing n-hexadecanoic acid andneophytadiene [7]. The extracts metanol ofWattakaka volubilis containing hexadecanoic acid[1]. Neophytadiene is reported to be anantibacterial activity, headache, reumatism, andsome skin diseases [8,9].

CONCLUSIONS

The extract of n-hexane, ethyl acetate, andmethanol C prabaktiana each containing 21, 26,and 17 compounds. The dominant component ishexadecanoic acid and neophytadiene


Junairiah et al. 77


REFERENCES

[1] S. Usharani and M. Chitra, GC-MS Analysis ofmethanol extract of leaf of Wattakaka volubilis(L.F), International Research Journal ofPharmaceutical and applied Sciences.,3,4,2013,161-165.

[2] SH.Lahigi, K. Amini, P. Moradi, K. Assadi,Investigating the chemical composition ofdifferent parts extracts of bipod nettle Urticadioica L, In Tonakabon Region, Iranian Journalof Plant Physiology, 2,1, 2001,337-340.

[3] E. Menghani, A. PareekRS. Negi, CK. Ojha,Search for antimicrobial potential from certainIndian medicinal plants, Res J Med Plants,5,2011, 295-301.

[4] S. Gopalakrishnan, E. Vadiel, GC-MS Analysisof some bioactive constituents of Mussaendafrondosa Linn, International Journal of Pharmaand Bio Sciences, 2,1, 2011,313-320.

[5] A. Muthulaksmi, R. Jothibai Margret, VR.Moham, Analysis of bioactive components ofFeronia ephantum correa (Rutaceae), Journal ofApplied Pharmaceutical Science,02,02,2012,69-74.

[6] A.Y.Sheetima, Y. Karuni, O.A. Sodipo, H.Usman, M.A. Tijjani, Gass Chromatography.Mass Spectrometry (GC-MS) analysis ofbioactive components of ethyl acetate rootextract of Guiera senegalensis J.F. Gmel, 3,03,2013,146-150.

[7] K. Amala, A. Saraswathy, S. Amerjothy, GC-MS Analysis of n-hexane extract of Epaltesdivaricata (L.) Cass, Journal of Pharmacognosyand Phytochemistry, 2,1, 2013, 33-35.

[8] R.G. Palicee, G. Stojanovic, S. Alagic, M.Nikolic, Z. Lepojevic, Chemical compositionand antimicrobial activity of the essential oiland CO2 extracts of the oriental tobbacco,Prilep Flavour Fragr J, 17, 2002, 323-326.

[9] L. Suresh, R.M. Veerabah, S.R. Gnanasingh,GC-MS analysis of ethanolic extract ofZanthoxylum rhetsa (roxb).dc spines, J. HerbalMed Toxicol, 4, 2010, 191-192.


Rita et al. 78


Antifungal Activity of Rain Tree (Samanea saman Jacq.) Leaf ExtractAgainst Fusarium solani, The Cause of Stem Rot Disease on Dragon

Fruit (Hylocereus sp.)Wiwik S. Ritaa*, Dewa N. Supraptab, I Made Sudanab, I Made D.Swantaraaa Department of Chemistry, Faculty of Math and Sciences

Udayana University, Kampus Bukit Jimbaran, Badung, Bali, Indonesiab Laboratory of Biopesticide Faculty of Agriculture

Udayana University, Jl. PB. Sudirman Denpasar Bali Indonesia*Corresponding Author’s E-mail: [email protected]

ABSTRACT

Fusarium solani is one of the pathogenic fungi which causes stem rot disease on dragon fruit. Synthetic fungicidesare commonly applied to control the pathogen. However, it may cause environmental and health problems. Theextract of several higher tropical plants were proven to possess antifungal activity, one of which was rain tree(Samanea saman Jacq.). This study aims to determine the effectiveness of ethanol and water extracts of the leaf ofrain tree in inhibiting the growth of F. solani and analyze the compounds in the ethanol extract. The effectiveness ofethanol extract of the leaf of rain tree against F. solani was performed in vitro by a diffusion well method and that ofwater extract was done in vivo by spraying rain tree leaf extract into the stem of dragon fruit inoculated the sporesof F. solani. Analysis of compounds in the ethanol extract of rain tree was performed by phytochemical analysis.Ethanol extract of the leaf of rain tree could inhibit the growth of F. solani with an inhibition diameter of 30.0 mmon day 5 of incubation and MIC of 0.9 % with an inhibition of 8.12 mm in diameter on day 3 of incubation. Theethanol leaf extract significantly inhibited the fungal radial growth, sporulation, and the total biomass. Compared tocontrol, water extract of rain tree at a concentration of 2.5 % (w/v) was able to inhibit the growth of F. solani invivo. The effectiveness of the extract was comparable to synthetic fungicide (antracol ) at concentraction of 0.2 %).According to phytochemical analysis, the compounds in the ethanol extract of the leaf of rain tree were alkaloid,flavonoid, phenolic compounds, tannin, triterpenoid, and steroid.

Keywords: Rain Tree, Samanea saman Jacq., Antifungal Activity, Fusarium solani

INTRODUCTION

In recent years, dragon fruits have beendeveloped in Indonesia, especially Bali, becausethey have high commercial valuable fruits. Dragonfruit is commonly consumed as fresh fruit assatiation, because the water level is high enoughthat approximately 90.20% of the weight of thefruit with the sugar content reaches 13-18 briks.The content of other nutrients: carbohydrates (11.5g), acid (0.139 g), protein (0.53 g), fiber (0.71 g),calcium (134.5 mg), phosphorus (8.7 mg),magnesium (60.4 mg) and vitamin C (9.4 mg). Theexotic fruits also contain vitamins B1, B2 and B3.Dragon fruit was classified as horticulturalproducts that is beneficial to health. It contains alot of fiber. The fiber can bind carcinogens causingcancer and facilitate the digestive process. Dragonfruit is also used for balancing sugar levels in theblood [1].

Dragon fruit orchard developed in Sobanganvillage, Badung Regency, Bali has an area of 2hectares consisting of 9,600 plants of white dragonfruit, 2,000 plants of super red dragon fruit, and

1,200 plants of red dragon fruit. Dragon fruit plantsin the garden can not produce fruit to themaximum, but only produced about 20 tons peryear, while the market demand quite a lot of, sosupply of dragon fruit in supermarkets in Balibrought in from outside Bali. This is due to thepresence of pathogens that attack these plants,particularly fungal pathogen that resulting in stemrot disease.

There were various diseases on dragon fruitsthat have been reported. Hawa et al. [2] reportedthat stem rot disease on dragon fruit was caused byFusarium semitectum, Fusarium moniliforme, andFusarium oxysporium. Meanwhile anthracnosedisease was caused by Colletotrichumgloeosporioides [3]. Rita et al. [4] reported thatpathogenic fungus causing stem rot diseases ondragon fruit in Bali was Fusarium solani. Inaddition, black spot caused by Alternaria sp., wiltdisease caused by Fusarium oxysporium, stemblight caused by Diplodia sp., Ascochyta sp., andPhoma sp., speck blight caused by Nectriella sp.have reported by Wang et al. [5].


Rita et al. 79


One of the alternative measures to control thedisease is the use of botanical pesticides thatcontaining plant extracts as an active ingredient.This botanical pesticide is considered to be anenvironmentally friendly measure. Botanicalpesticides do not cause resistance to pests becausethe active ingredient is composed of severalcompounds and most of them are biodegradableunder natural condition [6].

Biodiversity of plants provide opportunities forthe development of botanical fungicides incontrolling fungal pathogens. Some scientiststudied the effect of different plant extracts tofungal growth. Aba-Alkhail [7] used the extracts ofAllium sativum (garlic), Cymogopogon proximus(lemongrass), Carum carvi (caraway), Azadirachtaindica (neem), and Eugenia caryophyllus (clove) toinhibit the growth of Fusarium oxysporum f. sp.lycopersici. El-Assiuty et al. [8] utilizedCymbopogon proximus to control toxigenic fungiAspergillus flavus and Fusarium vertiillioides.Fawzi et al.[9] utilized cinnamon, bay leaves,avocado and ginger in controlling the fungusAlternaria alternata and Fusarium oxysporum.

Many extracts of higher plants have beenstudied and showed remarkable antifungalactivities against plant fungal pathogens. In thisstudy, 30 plant extracts were tested for theirantifungal activity against the causal fungus ofstem rot disease on dragon fruit grown in Bali. Theresult showed that the extract of rain tree (Samaneasaman (Jacq.) was the most active in inhibiting thefungal growth on PDA medium. Prasad et al. [10]reported that rain tree leaf extract could inhibit thegrowth of Candida albicans at a concentration of10 mg/mL. Phytochemical screening showed thepresence of tannins, flavonoids, saponins, steroids,cardiac glycosides, and terpenoids in the leaves ofrain tree. Ukoha et al. [11] reported the tannins inthe ethyl acetate fraction (TEA) rain tree podextract showed the highest activity in inhibitingCandida albicans. Rita et al. [12] reported thattotal flavonoid and phenolic contents of rain treeleaf n-butanol extract have a positive correlation toits activity in inhibiting the growth of Escherichiacoli and Staphylococcus. Meanwhile, Suteja et al.[13] stated that isoflavon with a hydroxy groups atC-5 and C-7 in rain tree leaf butanol extract wasable to inhibit the growth of Escherichia coli.

Although the use of plant extracts in the controlof pathogenic fungi have been carried out, butspecifically for the control of fungal pathogens ondragon fruit has not been widely studied. Thisstudy aims to determine the effectiveness ofethanol and water extracts of the leaf of rain tree ininhibiting the growth of the causal fungus of stemrot disease and to reduce the incidence of stem rotdisease on dragon fruit. In addition, secondary

metabolites in ethanol extract of rain tree leaf werevery worth to identify.

EXPERIMENTALPlant Material

Rain tree leaves were collected aroundDenpasar Bali and dried at room temperature. Thedried samples subsequently were milled to apowder.

Fungal IsolatePure culture of Fusarium solani was obtained

from the Laboratory of Biopesticide, University ofUdayana that was maintained at 4°C. The fungalisolate was then grown on potato dextrose agar(PDA) on a Petri dish and incubated at roomtemperature (27 + 2oC) before use for further test.

ExtractionAround 1 kg of rain tree leaf powder was

extracted with 10 L of ethanol 70% for 24 hours atroom temperature (27 + 2°C). The same procedurewas applied to get the water extract, but it usedwater as solvent. The extracts were kept for 24 h at4°C, filtered through Whatman No. 1 filter paper,evaporated to dryness under vacuum, and stored at4°C until use for antifungal test and phytochemicalanalysis. Water extract was done in the sameprocedure.

Antifungal Activity TestAntifungal activity of ethanol extract of rain

tree against the main fungal pathogens of stem rotdisease was done by diffusion well methods.According to Ardiansyah [14], if the diameter ofzone of inhibition 20 mm, the inhibition was verystrong; 10-20 mm: strong inhibition; 5-10 mm:moderate inhibition; and <5 mm: weak inhibition.Several tests were performed in this experimentsuch as determination of minimum inhibitoryconcentration (MIC) of the crude extract; theeffects of extract on the fungal colony growth onPDA, sporulation and biomass formation in theliquid medium.

Determination of Minimum Inhibitory ConcentrationThe suspension of Fusarium solani (200 L)

was spread on melted PDA medium in a laminarflow. After the medium become solid, diffusionwell was made using cork borer (5 mm indiameter). Into the well, 20 l ethanol extract ofrain tree leaf was applied at concentrations of0,2%, 0,4%, 0,8%, 1,0%, 2,0%, 3,0%, 4,0%, 5,0%,10,0% (w/v). For control, 20 l sterile distilledwater containing 0.2% Tween-80 was used. ThreePetri dishes were prepared for each concentration.The cultures were then incubated for 48 h in thedark under room temperature. The formation ofinhibition zone around the diffusion well wasobserved and was used to determine the antifungal


Rita et al. 80


activity. The lowest concentration in which theethanol extract of rain tree leaf produced inhibitionzone is known as minimum inhibitoryconcentration (MIC).

Effect of Extract on Colony GrowthThe ethanol leaf extract of rain tree at various

concentrations (0.5%, 1%, 2% and 4%, w/v) wereput on Petri dishes and mixed with 10 mL meltedPDA medium. The sterile distilled water containing0.2% Tween-80 was used as control. The Petridishes were shaken gently to allow the extract todistribute evenly. After the medium solidified, amycelia plug (5 mm in diameter) of Fusariumsolani taken from the edge of a 3-day old culturewas put in the center of the PDA. Three Petridishes were prepared for each concentration. Thecultures were incubated for 7 days in the darkunder room temperature. The diameter of fungalcolony was measured daily. The inhibitory activityto the radial growth (IR) was determined accordingto the following formula: [15](%) = 100

……………….. (1)

where: IC = inhibitory activity to the colonygrowth

dc = average increase in mycelia growth incontrol plates

dt = average increase in mycelia growth intreated plates.

Effect of Extract on SporulationSpores were harvested in sterile distilled water

from a culture maintained in slant PDA. Thesuspension was passed through a filter paper(Whatman No.2) to separate the spore and myceliaor hypae. As much as 200 L spore suspension(2x105 spores mL-1) was added into 10 mL potatodextrose broth in a test tube containing variousconcentrations of rain tree leaf extract, i.e. 0%,0.5%, 1%, 2% and 4% (w/v). The cultures wereincubated in the dark under room temperature forfive days. The number of spores were countedusing haemocytometer under light microscope. Theinhibitory activity to the spore formation (IS) wascalculated according to the following formula: [16](%) = 100

……………….. (2)

where: IS = inhibitory activity to the sporulationdc = spore’s density on control (without

extract treatment)dt = spore’s density with extract treatment.

Effect of Extract on Fungal BiomassThe determination of the effect of rain tree

extract on fungal biomass was done in 100 mLpotato-dextrose broth (PDB) medium that was

placed in a 200-ml Erlenmeyer flask. The extractwas added into the flask at concentration variedfrom 0%, 0.5%, 1%, 2% and 4% (w/v). Themedium was then inoculated with 1 ml of sporesuspension (the spore density was 2x105 sporesmL-1). The final volume of the culture was 100 mLwith five flasks for each concentration. Thecultures were incubated in the dark for 8 daysunder room temperature. The biomass washarvested through centrifugation at 5,000 rpm for 5minutes. The pellet (biomass) was taken and placedon glass filter paper and dried in an oven at 60oCuntil constant weight. The inhibitory activityagainst the fungal biomass (IB) was calculatedaccording to the formula: [16](%) = 100 ……………..

(3)

Where: IB = inhibitory activity to the fungalbiomass

wc = dry weight of biomass on control(without extract treatment)

wt = dry weight of biomass with extracttreatment.

Phytochemical AnalysisPhytochemical analysis was performed on

ethanol extract of leaf of rain tree. The extract wastested for the presence of alkaloids, triterpenoids,steroids, flavonoids, phenols, glycosides, saponins,and tannins using detection reagent compounds[17].

Effectiveness of Water Extract of the Leaf Rain Tree toControl Stem Rot Disease on Dragon Fruit

Rain tree leaf water extract with variousconcentrations of 0.0, 0.5; 1.0; 1.5; 2.0; and 2.5%(w/v) were tested in this experiment to control stemrot disease. The extract treatment was allocatedaccording to the randomized block design (RBD)with 4 replications for each concentration. Onemonth-old dragon fruit plants grown inpolyethylene bags filled with cultural medium(fertile soil: compost, 3:1) under green house wereartificially inoculated with the spore’s suspensionof Fusarium solani (0.5 mL per plant). Theinoculation sites were wounded with 5 needlesprior to the inoculation. Treatment with plantextract was done a day after inoculation. Theextract of respective concentration was evenlysprayed onto the surface of the plant using sprayer.Observation was done every day to determine thedisease incidence. The disease incidence wasdetermined according to the following formula:(%) = 100 …….. (4)

Where: DI = Disease incidencedp = The number of diseased plantsdi = The number of inoculated plants


Rita et al. 81



The minimum inhibitory concentration (MIC)of ethanol extract of the leaf of rain tree againstFusarium solani on PDA was 0.9% with diameterof inhibition by 8.12 mm on day 3 of incubation.The extract significantly (P<0.05) suppressed thegrowth of colony of F. solani on PDA medium.Treatment with extract at concentration of 1%inhibited the colony growth by 48.35%, while thetreatment with extract at concentration of 4%inhibited the colony growth by 93.95%. Results ofthis study showed that the higher the concentrationof extract, the higher the inhibitory activity (Table1).

Table 1. Inhibitory activity of ethanol extract of rain treeleaves against the colony growth of Fusarium solani

Extractconcentration

(%, w/v)

Diameter offungal

colony (mm)

Percent ofinhibition

0 71.67 a* -0.5 71.00 a 0.911 37.00 b 48.352 14.67 c 79.544 4.33 d 93.95

*Values followed by the same letters in the samecolumn are not significantly different according tothe Duncan’s Multiple Range Test at P<5%.

The treatment with ethanol extract of rain treeleaf effectively inhibited the formation of spore ofF. solani. Treatment of the extract at aconcentration of 0.5% significantly (P <0.05)inhibited the formation of spores by 19.05%, whileno spore formation occurred when the fungus wastreated with extract at concentration of 4% (Table2). The ethanol extract at concentration of 0.5 to4.0% significantly (P<0.05) inhibited the biomassof F. solani in PDB medium. Treatment withextract at a concentration of 0.5% was able toinhibit the biomass by 30.41%, whereas treatmentat a concentration of 4% was able to inhibit thebiomass by 97.68% (Table 3).

Al-Reza et al. [18] evaluated the antifungalactivity of essential oils (1000 ppm) and flowerextracts of Cestrum nocturnum L. (1500 mg / disc)against Botrytis cinerea, Colletotrichum capsici,Fusarium oxysporum, Fusarium solani,Phytophthora capsici, Rhizoctonia solani, andSclerotinia sclerotiorum. The inhibitionconcentration of the essential oils was ranging from59.2 to 80.6%, while the inhibition of flowerextracts was from 46.6 to 78.9%. The MIC of theessential oils and the flower extracts was 62.5-500and 125-1,000 mg mL-1.

Table 2. Inhibitory activity of ethanol extract of rain treeleaves against spore’s formation of Fusarium solani

Extract Spore’s Percent of

concentration( %, w/v )

density ml-1

(x 105 spores)inhibition

0 69.33 a* -0.5 55.67 b 19.051 29.67 c 56.992 2.33 d 96.674 0 d 100.00


Table 3. Inhibitory activity of ethanol extract of rain treeleaves against biomass of Fusarium solani

Extractconcentration

(%, w/v)

Dry weightof biomass

(mg)

Percent ofinhibition

0 360 a* -0.5 250 b 30.411 183.33 c 48.972 116.67 d 67.504 8.33 e 97.68


Bajpai and Kang [19] reported that the essentialoil of Magnolia liliflora DESR. with aconcentration of 62.5 mg/mL could inhibitcompletely the formation of spores of Fusariumsolani. Hadi and Kashefi (2013) conducted a studyon the effects of some medicinal plants againstFusarium oxysporum Schlecht spore formation invitro. Cinnamomum zeylanicum, Mentha piperita,Allium hirtifolium, and Allium sativum extractsshowed maximum inhibition of spore formation ineach 1,000, 1000, 1000, and 500 ppm after 8 days,while the formation of spores at least have occurredin each of 1000, 100, 100 and 25 ppm after 10 dayson PDB medium. Siripornvisal [20] reported thatajowan oil with concentrations of 120-480 mg mL-1

had a significant inhibitory effect on the biomass of3 strains Fusarium oxysporum, including F.oxysporum f.sp. lycopersici, F. oxysporum f.sp.cubense, and F. oxysporum f.sp. capsici, pathogensof tomato, banana, and chili wilt diseases.

The inhibition of ethanol extract of rain tree leafon the growth of F. solani was caused byantifungal active compounds contained in theleaves of rain tree. According Pelczar et al. [21],the mechanism of antimicrobial substances inkilling or inhibiting the growth of microbes was to:(1) damage the microbial cell wall, resulting inlysis or inhibit the formation of the cell wall ingrowing cells, (2) changing the permeability of thecytoplasmic membrane which causes the leakage ofnutrients from within the cell, for example by


Rita et al. 82


phenolic compounds, (3) cause denaturation ofcells, and (4) inhibit the enzyme action in the cell.

Phytochemical test of the extract showed thatthe ethanol extract of rain tree leaves containalkaloids, flavonoids, phenolics, tannins,triterpenoids, steroids, and saponins. Prasad et al.(2008) [10] reported that rain tree leaves containtannins, flavonoids, saponins, steroids, cardiacglycosides, and terpenoids. Raghavendra et al. [22]reported the presence of alkaloids in the leaves ofrain tree. While Ferdous et al. [23] reported thepresence of lupeol and epilupeol (triterpenoidsgroup) obtained from n-hexane fraction.

The activity of rain tree leaf to controlFusarium solani was probably caused by flavonoidand tannins. The activity of flavonoid as antifungalwas probably caused by its ability to bind thefungal proteins by forming hydrogen bondsbetween the OH group on the C atom number 3 orcarbonyl group (C number 4) with the aminogroups of the fungal protein, so the composition ofthe fungal cell wall was disrupted. This wasconfirmed by Amjad et al. [24] which states thatthe antifungal activity of flavonoid was due to theirability to form complexes with proteins ofextracelluler and soluble protein, whereas thelipophilic flavonoid may probably interfere fungalmembranes. According to Cushnie and Lamb [25],there were different mechanisms for differentflavonoid, such mechanisms included inhibiting thesynthesis of nucleic acids, the cytoplasmicmembrane function, and energy metabolism. Themechanism involved the interaction between thefungal enzymes with flavonoid. Enzyme was anactive protein that consists of various amino acids,resulting in the formation of hydrogen bondsbetween the amino acids, it caused the flavonoidenzymes activity to be disturbed.

Mailoa et al. [26] reported the antimicrobialactivity of tannin extract of guava leaves againstmicrobial pathogens. The results showed that theinhibitory activity of tannins on five microbialpathogens was different. This was because thecomposition of the microbial cell wall fivemicrobes was different. The results showed that thetannin extract can inhibit the growth of Escherichiacoli, Pseudomonas aureginosa, Staphilococcusaureus, Aspergillus niger and Candida albicans.

Treatment with various concentrations of thewater extract showed significant effect (P <0.05) tothe incidence of stem rot disease on dragon fruit aspresented in Table 4 and Figure1. The diseaseincidence of control (K) was 91.25%, Treatmentwith concentration of 0.5; 1.0; 1.5; and 2.0 showedthe incidence of the disease respectively for 62.50;36.25; 21.25; and 15.00%, the highest among othertreatments, while no disease incidence wasobserved on dragon fruit plant treated with extract

at concentration of 2.5%. This treatment wascomparable to the treatment with syntheticfungicide (Propineb) at concentration of 0.2%(recommended concentration).

Table 4. Effectiveness of water extract of rain treeleaf to suppress stem rot disease on Dragon Fruit

Treatment Disease incidence (%)K (control) 91.25 a*A (0.5%) 62.50 bB (1.0%) 36.25 cC (1.5%) 21.25 dD (2.0%) 15.00 dE (2.5%) 0 e

F (synthetic fungicide,0.2%)

2.50 e


Figure 1. Bar chart of stem rot disease incidence (%) ondragon fruit with and without treatment of water extractof rain tree leaves

This result suggested that water extract of theleaf of rain tree potentially can be used to controlthe stem rot disease on dragon tree caused by F.solani. This measure can be considered as a safeand environmentally friendly control method inorder to minimize the use of synthetic fungicides.

CONCLUSIONS

Based on the results of research and discussion,it can be concluded that the leaf extract of rain treeeffectively inhibited the growth of Fusarium solaniand reduce the incidence of stem rot disease ondragon fruit. The ethanol extract of rain tree leavescontain alkaloids, flavonoids, phenolics, tannins,triterpenoids, steroids, and saponins.

ACKNOWLEDGEMENTS

Authors would like to express theirappreciation to The Udayana University, BaliIndonesia for providing research grant to supportthis study. Appreciation is also sent to the FloraBali Desa Sobangan Bali for providing dragon fruitseedlings.

020406080

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(%)

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Rita et al. 83


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