ISSN 2320 -5083 Journal of International · Er.Chandramohan, M.S Syste m Specialist - OGP ABB...

Journal of International Academic Research for Multidisciplinary

ISSN 2320 -5083

A Scholarly, Peer Reviewed, Monthly, Open Access, Online Research Journal

Impact Factor – 1.393

VOLUME 1 ISSUE 10 NOVEMBER 2013

A GLOBAL SOCIETY FOR MULTIDISCIPLINARY RESEARCH

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A GREEN PUBLISHING HOUSE

Editorial Board

Dr. Kari Jabbour, Ph.D Curriculum Developer, American College of Technology, Missouri, USA.

Er.Chandramohan, M.S System Specialist - OGP ABB Australia Pvt. Ltd., Australia.

Dr. S.K. Singh Chief Scientist Advanced Materials Technology Department Institute of Minerals & Materials Technology Bhubaneswar, India

Dr. Jake M. Laguador Director, Research and Statistics Center, Lyceum of the Philippines University, Philippines.

Prof. Dr. Sharath Babu, LLM Ph.D Dean. Faculty of Law, Karnatak University Dharwad, Karnataka, India

Dr.S.M Kadri, MBBS, MPH/ICHD, FFP Fellow, Public Health Foundation of India Epidemiologist Division of Epidemiology and Public Health, Kashmir, India

Dr.Bhumika Talwar, BDS Research Officer State Institute of Health & Family Welfare Jaipur, India

Dr. Tej Pratap Mall Ph.D Head, Postgraduate Department of Botany, Kisan P.G. College, Bahraich, India.

Dr. Arup Kanti Konar, Ph.D Associate Professor of Economics Achhruram, Memorial College, SKB University, Jhalda,Purulia, West Bengal. India

Dr. S.Raja Ph.D Research Associate, Madras Research Center of CMFR , Indian Council of Agricultural Research, Chennai, India

Dr. Vijay Pithadia, Ph.D, Director - Sri Aurobindo Institute of Management Rajkot, India.

Er. R. Bhuvanewari Devi M. Tech, MCIHT Highway Engineer, Infrastructure, Ramboll, Abu Dhabi, UAE Sanda Maican, Ph.D. Senior Researcher, Department of Ecology, Taxonomy and Nature Conservation Institute of Biology of the Romanian Academy, Bucharest, Romania Dr. Reynalda B. Garcia Professor, Graduate School & College of Education, Arts and Sciences Lyceum of the Philippines University Philippines Dr.Damarla Bala Venkata Ramana Senior Scientist Central Research Institute for Dryland Agriculture (CRIDA) Hyderabad, A.P, India PROF. Dr.S.V.Kshirsagar, M.B.B.S,M.S Head - Department of Anatomy, Bidar Institute of Medical Sciences, Karnataka, India. Dr Asifa Nazir, M.B.B.S, MD, Assistant Professor, Dept of Microbiology Government Medical College, Srinagar, India. Dr.AmitaPuri, Ph.D Officiating Principal Army Inst. Of Education New Delhi, India Dr. Shobana Nelasco Ph.D Associate Professor, Fellow of Indian Council of Social Science Research (On Deputation}, Department of Economics, Bharathidasan University, Trichirappalli. India M. Suresh Kumar, PHD Assistant Manager, Godrej Security Solution, India. Dr.T.Chandrasekarayya,Ph.D Assistant Professor, Dept Of Population Studies & Social Work, S.V.University, Tirupati, India.

JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: 2320-5083, Volume 1, Issue 10, November 2013

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EXTRACTION, MODELING AND PURIFICATION OF FLAVONOIDS FROM ZEPHYRANTHES CANDIDA

MANOJ KUMAR SINDIRI*

MANASA MACHAVARAPU** DR. MEENA VANGALAPATI***

*M.Tech, Dept. of Chemical Engineering, Center For Biotechnology, AUCE(A), Andhra University, Visakhapatnam, India ** M.Tech, Dept. of Chemical Engineering, Center For Biotechnology, AUCE(A), Andhra University, Visakhapatnam, India

***Associate Professor, Dept. of Chemical Engineering, Center For Biotechnology, AUCE(A), Andhra University, Visakhapatnam, India

ABSTRACT

Zephyranthes candida is a perennial herb has many pharmacological activities

because of flavonoid and other biological components. The objective of present work is to

develop a modelling equation and purification of flavonoids by solvent-solvent extraction and

Column chromatography. Extraction was carried out by 80% methanol as a solvent Soxhlet

extractor. Soxhlet extraction with methanol was carried out with varying time intervals, to

evaluate modelling equation. The proposed modelling equation was E= 7.831 (t) 0.0037. High

yield was obtained for 4hrs of Soxhlet extraction with 80% methanol. Yield of flavonoids

after solvent-solvent extraction and column chromatography was 265.67µg/ml and 270.4

µg/ml respectively. The proposed model showed good agreement with the experimental data.

KEYWORDS: Zephyranthes Candida, Flavonoids, Soxhlet Extraction, Solvent-Solvent

Extraction, Column Chromatography.

INTRODUCTION

Zephyranthes candida is a perennial herb, native to the South America regions. It

belongs to the genus Zephyranthes of family Amaryllidaceae [Meerowet al, 1999]. The genus

Zephyranthes Herb is composed of approximately60 species distributed in the American

continent. A number of species are cultivated due to their gorgeous flowers, and are known

by plant breeders as “rain lilies”owing this name to their tendency to flower after a rain

period [Fernández et al, 2004]. Plants this genius are well known for ornamental plants in

China, besides these are also used in the treatment of diseases because of their

pharmacological activities [Hostettmann et al, 2006]. Because of the presence of various

biological constituents, this plant has many has biological activities like antimalarial,

antiviral, antiproliferative antibacterial, antidiabetic etc [Campbell et al, 1988; Hohmann et

al, 2002; Szlávik et al, 2004].Other species this genus were named popular because of their

various therapeutic uses [Zaida et al, 1989].


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Z. candida leaves are the moderate reserves for flavonoids[Manoj et al, 2013], the

present research work is to extract and purify flavonoids from the leaves of Z. candida.

MATERIALS AND METHODS:

Collection of plants:

Z. candida plants were procured from local nursery, Visakhapatnam, Andhra Pradesh.

Leaves were air dried and grated for further use.

Soxhlet extraction:

300 ml of 80% methanol was taken in the round bottom flask of the soxhlet extractor,

since it was the best solvent for the extraction flavonoids: quercetin, rutin. 3 gm of leaf

powder packed in filter paper then placed in the thimble and it is connected to the condenser.

Now the entire was placed in the heater. Extraction was done continuously for 8hrs. Then the

extract was filtered and methanol was evaporated on vacuum rotary evaporator

[Suryaprakash et al, 2012].

Purification:

Crude extract obtained after the Soxhlet extraction, was purified by two different

methods: solvent-solvent extraction and column chromatography.

Solvent – solvent extraction:

Solvent – Solvent extraction was done with extract (i.e., obtained from Soxhlet

extractor) andhexane of different proportions by varying extract: hexane proportion in the

ratio of 1:0.5 to 1:2.0 used as solvent. Extraction was done in separating funnel for 1hr to

2hrs and then the two phases: raffinate and extract phases were separated. The sample is

collected from both the phases for the estimation of flavonoids: quercetin, rutin

concentrations and also the partition coefficient was estimated.

Partition coefficient =

Column chromatography:

In Column Chromatography, 100 – 200 mesh size silica gel was used as stationary

phase. Before starting the experiment, first insert a piece of cotton down to the column, then

wash with solvent. Fix the column to the clamp tightly. Pour the sea sand of 1cm bed in the

column followed by washing with solvent. Add silica gel powder in the column up to 10cm

length from the sand bed of the column. Run the solvent hexane in the column up to the bed

was entirely wet.Add excess solvent on the top of the silica gel bed. Gently tap the column

with hand or soft materials. After tapping, gentle pressure can be applied. Before loading the


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sample in the column, little silica gel was added to the sample. Pour the sample and rinse the

wall. Add sand on the top of the sample [sreesatya et al, 2012].

Modelling of Extraction of Flavonoids using Soxhlet Extractor Apparatus:

In order to describe the flavonoids transfer from leaf powder of Zephyranthes candidato the

bulk of the solvent the following hypothesis was used. The mass transfer coefficient is

constant. The solvent in the extractor is perfectly mixed, while the transfer resistance in the

liquid phase is negligible and flavonoid concentration in the solvent depends only on time.

The transfer of the flavonoids was a diffusion phenomenon and independent of time. The

final form of the modelling equation was obtained from the extraction of flavonoids by using

Soxhlet extractor.

Where

E = micro grams of flavonoids per ml of solvent

t = extraction time (min)

b & d are constants.

Estimation of Flavonoids:

Concentrations of flavonoids were expressed in terms of quercetin equivalents, here

the procedure is as follows.

0.5 mL of a sample (methanolic extract of leaves of Z. candida) was mixed with 1.5

mL 95 % methanol (V/V), 0.1 mL 10 % aluminum chloride (m/V), 0.2 mL of 1 mol L–1

potassium acetate and 2.8 mL water. A volume of 10 % (m/V) aluminum chloride was

substituted by the same volume of distilled water in blank. After incubation at room

temperature for 30 minutes, the absorbance of the reaction mixture was measured at 415 nm

[Suhadet al, 2008] .

Results and Discussions:

Soxhlet extraction:

Flavonoid were extracted from the leaves of Z. candida with 80% methanol in the

Soxhlet extractor for 8hrs. Polar solvents shows high yield of extraction when compared to

non-polar solvents. Non- polar solvents were almost for the extraction of flavonoids.

Methanol was found to be the best solvent for the extraction of flavonoids. The highest

amount of flavonoids were extracted from 80% of methanol. The final form of proposed


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model equation for flavonoids was E= 7.831 (t) 0.0037. Results for the extraction of flavonoids

with 80% methanol were tabulated in Tab 1. From the obtained data it was observed that

maximum extraction of flavonoids: Quercetin and Rutin has occurred for 4 hrs, further

continuing the extraction for 4 hrs and then it shows depletion in flavonoid concentration.

Table 1: Extraction of flavonoids w.r.t time using Soxhlet extractor.

S.no Time (hrs) Yield Extract (µg/ml)

1 1 75.854

2 2 81.854

3 3 85.30

4 4 96.218

5 5 89.309

6 6 90.218

7 7 90.218

8 8 89.672

Figure 1: Extraction yield of flavonoid with extraction time

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9

Flav

onoi

d Co

ncet

ratio

n (µ

g/nl

)

Time (hrs)


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Figure 2: Soxhlet extractor for the extraction of flavonoids

Solvent-Solvent Extraction:

Methanolic extract of leaves of Z. candida obtained from Soxhlet extractor was

purified by solvent – solvent extraction method, using hexane as a solvent. Purification was

done with different hexane proportion ranging from 0.5 to 2 times of extract. 1: 0.5, 1:1,

1:1.5, and 1:2 of extract: hexane mixture was taken in four different conical flasks and

allowed to shake continuously in rotary shaker for 2hrs and then separated by using

separating funnel. After 1hr, among the different ratios of extract and hexane, 1:0.5 has

showed optimum purification Tab 2and the partition coefficient was found to be 4.07.

Table 2: purification of flavonoids by solvent-solvent extraction.

Extraction time(hrs) Extract : Hexane

Flavonoid concentration (µg/ml)

Quercetin Rutin

Extract Raffinate Extract Raffinate

1

1: 0.5 265.67 65.12 167.40 65.12

1 : 1.0 241.85 75.30 151.23 75.30

1 : 1.5 200.03 81.12 122.83 81.12

1: 2.0 168.58 105.12 101.48 105.12

2

1: 0.5 247.49 64.94 155.06 64.94

1 : 1.0 224.76 66.4 139.62 66.40

1 : 1.5 156.21 68.03 93.08 68.03

1: 2.0 126.94 73.49 73.20 73.49


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Partition coefficient = = 4.07

Column Chromatography:

Extract obtained from soxhlet extractor is subjected to column chromatography. 100

ml of extract was taken in column for purification, finally 80 ml of purified extract was

collected. Quercetin and rutin concentrations before the purification were 106.76 µg/ml and

59.50 µg/ml, after column chromatography their concentrations have been increased to 270.4

µg/ml and 170.61 µg/ml respectively.

Figure 3: Column Chromatography

Conclusion

Methanol was the best solvent for extraction of flavonoids from the leaves of Z.

candida. 80%methanol shows highest yield of flavonoids. The final proposed model equation

was E= 7.831 (t) 0.0037. Among the two purification methods, column chromatography was

concluded as the best purification process, since it shows the highest yield of flavonoids

compared to solvent-solvent extraction. From the experimental observation the proposed

model shows the best match.


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REFERENCES 1. CAMPBELL, W.E., NAIR, J.J., GAMMON, D.W., BASTIDA, J., CODINA, C., VILADOMAT,

F., SMITH, P.J., ALBRECHT, C.F. 1988. Cytotoxic and antimalarial alkaloids from Brunsvigia littoralis, PlantaMed, 64, 91-93.

2. FERNÁNDEZ-ALONSO, J.L., GROENENDIJK, J.P. 2004.A new species of Zephyranthes Herb. S. L. (Amaryllidaceae Hipperastreae), with notes on the Genus in Colombia. Rev. Acad. Colomb. Cienc, 28, 177-186.

3. HOHMANN, J., FORGO, P., MOLNÁR, J., WOLFARD, K., MOLNÁR, A., THALHAMMER, T., MÁTHÉ, I., SHARPLES, D. 2002. Antiproliferative Amaryllidaceae alkaloids isolated from the bulbs of Sprekelia formosissima and Hymenocalis x festalis, Planta Med, 68, 454-457.

4. HOSTETTMANN, K., BORLOZ, A., URBAIN, A., MARSTON, A. 2006. Natural product inhibitors of acetylcholinesterase, Curr Org Chem,10, 825-847.

5. MANOJ KUMAR SINDIRI., MANASA MACHAVARAPU., MEENA VANGALAPATI. 2013. Antibacterial Activity of Methanolic Extracts of Zephyranthes candida. Asian Academic Research Journal of Multidisciplinary. (In press).

6. MEEROW, A.W., Fay, M.F., GUY, C.L., LI, Q.B., ZAMAN, F.Q., CHASE, M.W. 1999. Systematics of Amaryllidaceae based on cladistic analysis of plastid RBCL and TML-F sequences of data. American Journal of Botany, 86, 1325-1345.

7. Nandam Sree satya., Surya Prakash, D. V., Meena Vangalapati. 2012. Purification of Cinnamaldehyde from Cinnamon Species by Column Chromatography. International Research Journal of Biological Sciences, 1(7), 49-51.

8. SUHAD S HUMADI., VIORICA ISTUDOR. 2008. Quantitative analysis of bio-active compound in hibiscus sabdariffal. Extracts. Note i quantitative analysis of flavonoids. Farmacia, 4(6), 699-707.

9. Surya Prakash, D. V., Sree satya N., Meena Vangalapati. 2012. Extraction of Chebulinic acid from Terminalia chebula sp by Soxhlet Extractor – An Experimental & Modelling studies. Asian Journal of Biochemical and Pharmaceutical Research, 3(2), 170-176.

10. SZLÁVIK, L., GYURIS, Á., MINÁROVITS, J., FORGO, P., MOLNÁR, J., HOHMANN, J. 2004. Alkaloids from Leucojum vernum and antiretroviral activity of Amaryllidaceae alkaloids, Planta Med 70, 871-873.

11. Zaida, T.A., Myrna, C.R., Iraida, S.S.1989. Preliminary study of Zephyranthes eggersiana Urban. Revista Cubana de Farmacia, 23, 147–150

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