. ~ . \

ISSN 0975 -1491

Impact (Cites per doc)- 0.91 (SCimago, SJR 2013)

www.scimagojr.com and see option

Journal Search ICV (2011)- 5.00

Co Ri htForm

!I ELSEVIER

CURRENT ISSUE

International Journal of Pharmacy and Pharmaceutical Sciences (Int J Phann Phann Sci) is peer reviewed, Monthly (Onward April 2014) open access Journal. IJPPS publishes original research work that contributes significantly to further th1 scientific knowledge in pharmacy and pharmaceutical sciences (Pharmaceutical Technology, Pharmacognosy, Natural Product Research, Pharmaceutics, Novel Drug Delivery, Biopharmaceutics, Pharrnacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics Bioinformatics, Phannacoeconomics). Research outcomes from medical sciences/case study and biotechnology of pharmaceutical interest are also considered. IJPPS publishes original research work either as an Original Article or as a Short Communication. Review articles on current topic under mentioned scopes are also considered for ublication. l • It ~ 1 ~ ,: • l 0

1 j ;.

' . Abstracting and Indexing

1 Indicator 2006- 2013 Val~

Google Scholar, Scopus, Elsevier, EBSCO, SJR ~ 0•

48 EMBASE, SCI mago (SJR), CAS, CASSI (American Chemical Society), Directory of Open Access Journal (DOAJ), Index Copernicus, ICAAP Scientific corumnnc::, PSO~ Open-J-Gate, Indian Citation Index (ICI), Index Medtcus 10..: '.VH0

-.. .sci111a~jr.~ South-East Asia (IMSEAR), OAI, LOCK.KS, OCLC (World Digital Collection Gateway), UIUC

Under process: Chemical Abstracts, Medline, Pubmed, Pubmed Central

ARCHIVES S UBMIT MANU SCRIPT TRACK ARTICL( S Tl\TlJ~

.. '

... ~:;._Cite Factor DOAJ g~~~l~~~~l •·.~ -- JOURNAlS ·:~ ...

INDEX~COPERNICUS u:e; IMSEAJl f>... OCLC. ·--

11/S ~ ... --'"-.... -- 'QI - · I M l I I W A T I 0 • A L > ~ey,,;_,-t ~

.. .

'ol 4, Suppl 1, 2012

~eview Articles

~URRENT TREATMENT OF IDV INFECTION A REVIEW SIMPLIFIED: AN 1-14 JNDERST ANDING ABOUT HIV INFECTION AND ANTI IllV DRUGS MECHANISAM OF lCTION

'ISHAL MODI, TARA SHANKAR BASURI, ISHV ARCHANDRA PARMAR, VIRAG SHAH

:HE ROLE OF SEX HORMONES IN RHEUMATOID ARTHRITIS 15-21 ' ISHAL BABUSHETTY, CHANDRASHEKHAR M SULTANPUR

JOVEL COLON SPECIFIC DRUG DELIVERY SYSTEM: A REVIEW 22-29

:.PRADEEP KUMAR, D.PRATIDBHA, R.P ARTHIBARAJAN, C.RUBINA REICHAL

MMUNOMODULA TORS: A PHARMACOLOGICAL REVIEW 30-36

J.S. PATIL, A.V. JAYDEOKAR, D.D. BANDA WANE

MPROVEMENT IN BIOA VAILABILITY OF CLASS-III DRUG: PHYTOLIPID DELIVERY 37-42 :YSTEM.

' IPIN K AGRAWAL, AMRESH GUPTA, SHASHANK CHATURVEDI

:URRENT CLINICAL STRATEGIES IN RHEUMATOID ARTHRITIS: A REVIEW 43-46 7.SUBHASHINI, A.M.MAHALAKSHMI AND B.SURESH

:HANGING LANDSCAPE OF HERBAL MEDICINE: TECHNOLOGY AITRIBUTING 47-52 tENAISSANCE

:AT ARUP A BANERJEE AND ANALA VA MITRA

JNRESECTABLE COLORECT AL CANCER CASES RECIEVING NO-DOSE-~DUCTION 53-55 'OLFIRI THERAPY WITH THE REGULAR ADMINISTRATION OF GRANULOCYTE :OLONY-STIMULATING FACTOR SUPPORT: CASE REPORT WITH REVIEW OF ,JTERATURE

:HOICIDRO OHTA, YUKIKO CHO, HIROSID KOJIMA

1ARK1NSON'S DISEASE AND NEUROTOXIC ANIMAL MODELS: A MECHANISTIC VIEW 55-60

'IVEK SHARMA

~ABA RECEPTOR -A WELL ESTABLISHED OLD TARGET 61-66

IEMANT U. CHIKHALE, ELAHE K.HAMISADEH, AMIT G. NERKAR, AND SANJA Y D. :A WANT

~esearch Articles

N VITRO EVALUATION OF ANTIBACTERIAL ACTIVITY OF PTEROCARPUS MARSUPIUM 67-68 tOXB

CACHHA WA J.B.S., SHARMA N., TY AGI S., GUPTA R.S., SHARMA K.K.

ilICROENCAPSULATION FOR PREP ARING SUSTAINED RELEASE DRUGS

•.SHASHIKALA, A. LAV ANY A, M.BHAGA V ANTH RAO

HAGNOSTIC CONSIDERATIONS FOR NOVEL INFLUENZA A <HlNl)

if.ARWAN SHEIKH-TAHA, EDWARD ff. EILAND, III., JIAN HAN, WILLIAM LINDGREN, 'HOMAS MACANDREW ENGLISH, ALI HASSOUN.

69-72

73-75

'ORMULATION AND EVALUATION OF SUSTAINED RELEASE MULTIPLE EMULSION OF 76-80 IYDROXYPROGESTERONE

UMBEKAR T.P., WANJARI B.E., SANGID D.K., GAIKWAD N.J.

N VIVO AND IN VITRO BIOCHEMICAL ESTIMATION OF PRIMARY METABOLITES FROM 81-84 ATROPHA CURCAS: AN IMPORTANT BIODIESEL PLANT

t.SETH, R. SARIN

:v ALUATION OF A NOVEL, NATURAL BADAM GUM AS A SUSTAINED RELEASE AND 85-91 ,flJCOADHESIVE COMPONENT OF ATENOLOL BUCCAL TABLETS

:HELLANVIJAYARAGHAVAN,KRISHNAMOORTHYBALAKUMAR,NATARAJANTAMIL :EL VAN, JUDITH JUSTIN 1HARMACOKINETICS OF INJECTABLE BETA-CYCLODETRIN INCLUSION COMPLEX IN 92-95 VISTARRATS

SAO-QIU LI, NA LI, SHU-JUAN WANG JI-YOU GAO, SID-HONG FANG 1HYTOCONSTITUENTS AND HPTLC ANALYSIS IN SARACA ASOCA CROXB.)WILDE 96-99

A VITA SAHA, T ANIY A MITRA, KAMALA GUPTA, SUMONA MUKHERJEE >EVELOPMENT AND VALIDATION OF A DENSITOMETRIC HPTLC METHOD FOR )UANTITATIVE ANALYSIS OF CEFPODOXIME PROXETIL IN HUMAN PLASMA

:mT AL BHANDARI, NIKHIL KBISTI

tECLINICAL PHARMACOKINETIC EVALUATION OF PIOGLITAZONE FLOATING ~ABLETS FORMULATED EMPLOYING CROSS-LINKED STARCH-UREA

;. CHINNA DEVIAND K.P.R.CHOWDARY

100-10:

104-lOr

IEPATOPROTECTIVE ACTIVITY OF NELSON/A CANESCENS (LAM.) SPRENG ON ACUTE 107-11: IEPATOTOXICITY INDUCED BY PARACETAMOL

SEDABATI DASGUPTA, JOGEN CHANDRA KALITA, ARUDYUTI CHOWDHURY AND JIBON ~OTOKY

~NTIMICROBIAL EFFICACY OF SOME NATURAL COSMECEUTICALS, 113-120 JEUTRACEUTICALS AND MEDICINAL PLANT EXTRACTS AND ULTRASTRUCTURAL ~LTERATIONS IN FOOD BORNE PATHOGENS

~ VITHA SAGAR, REETID B, AK.SHA THA G AND SHY AM PRASAD

~OMPARITIVE STUDIES OF GLUTATHIONE-S-TRANSFERASE KINETICS IN CONTROL. 121-123 ]SPLATIN AND ETOPOSIDE TREATED HEPATIC AND RENAL TISSUE OF MALE RAT

'RA TIBHA R.KAMBLE, SAMEER KULKARNI, DAY ANAND A. BHIWGADE

>AVONIA ALNIFOLIA A. ST. IDL.: IN VIVO HYPOTENSIVE EFFECT AND IN VITRO ACE 124-126 NIDBITORY ACTIVITY

~ADEU UGGERE DE ANDRADE, BRUNELL Y TSCHAEN EWALD, PAULA DA ROS FREITAS, >OMINIK LENZ, DENISE COUTINHO ENDRINGER 1ROPHYLACTIC EFFECTS OF FLA VERIA TRINERVIA EXTRACT AGAINST ETHANOL 127-133 NDUCED HEPATOTOXICITY USING RATS

OY HOSKERI H., KRISHNA V. RAMESH BABU K., BADARINATH D. K.

:NZYMATIC ALTERATION IN THE VITAL ORGANS OF STREPTOZOTOCIN DIABETIC 134-14i tATS TREATED WITH AQUEOUS EXTRACT OF ERYTHRINA V ARIEGATA BARK

\.NUP AMA V, NARMADHA R, GOP ALAKRISHNAN VK AND DEV AKI K

NVITRO ANTI-INFLAMMATORY ACTMTY OF MOMORDICA CHARANTIA BY NIDBITION OF LIPOXYGENASE ENZVME

;.LEELAPRAKASH, J .CAROLINE ROSE , S.MOHAN DASS

148-152

'ORMULATION AND EVALUATION OF SUBMICRONIC EMULSIONS OF AMPHOTERICIN 153-159 l

' ANITHA KONDI, SINGH KAMALINDER, MOHAN VARMA, RAMESH ALL URI, SUNIL mMAR KARUMURI

)EVELOPMENT OF A VALIDATED STABILITY-INDICATING HPTLC METHOD FOR 160-16~

)ETERMINATION OF MELOXICAM IN BULK AND PHARMACEUTICAL FORMULATIONS: 1ERTINENCE TO ICB GUIDELINES

ESSY SHAJI, DHANILA VARKEY

lNTIULCEROGENIC AND FREE RADICAL SCAVENGING ACTMTY OF FLAVONOID 170-174 'RACTION OF PSIDIUM GUAJAVA LINN LEAVES

:. JAY AKUMARI, J. ANBU,V. RA VICHANDIRAN, ASHWINI ANJANA, G.M. SIVA KUMAR, lfAHARAJ SINGH.

'ORMULATION AND EVALUATION OF METHOTREXATE PRONIOSOMAL POWDER 175-17f t.P ARTHIBARAJAN, C.RUBINAREICHAL, S.LOGANATHAN

iOVEL SPECTROPHOTOMETRIC ESTIMATION OF OXCARBAZEPINE USING MIXED 179-18' IYDROTROPIC TECHNIQUE

'RAMEELA RANI A, HEMA VEESAM

~OPICAL ANTIINFLAMMATORY ACTIVITY OF TRIPODANTHUS ACUTIFOLIUS 183-18C LOWERS GEL FORMULATION

>AUD ADRIANA, REYNOSO MARCOS, ARISTIMUNO EUGENIA, SANCHEZ RIERA ALICIA

;c-MS, HPLC AND AAS ANALYSIS OF FATTY ACIDS, AMINO ACIDS AND MINERALS IN 187-19( t.ED ALGAEAMPHEROA ANCEPS

A Y ASREE.N.B, ANEESH.T.P, VISAKH PRABHAKAR, R.ANANDAN

: .

:OMPARATIVE STUDY ON THE TEACHING EFFECTIVENESS OF CHALK & TALK AND 191-193 l[ICROSOFT POWERPOINT PRESENTATION FROM THE STUDENT PERSPECTIVE

' AMSID KRISHNA T., M. VISHNU DATTA, Y. S.S. KISHAN, V. ADITYA, G. lHANUPRAKASH

ifETHOD DEVELOPMENT AND VALIDATION OF OSELTAMIVIR PHOSPHATE IN BULK 194-196 >RUG BY UV SPECTROSCOPY

:ANDEEP SAHU, SHRUTI ACHARYA, AMRITA CHOURASIA, ANUJ ASATI.

~HE PHYSICOCHEMICAL CHARACTERISTIC OF MICROCRYSTALLINE CELLULOSE, 197-200 >ERIVED FROM SAWDUST, AGRICULTURAL WASTE PRODUCTS

>YENIYI, Y.J AND ITIOLA, O.A

l NOVEL VALIDATED IDGH PRESSURE LIQUID CHROMATOGRAPHY METHOD FOR 201-209 :EP ARA TION OF PIOGLITAZONE DEGRADENT IN DRUG PRODUCT

:URENDRA SINGH RAO, NITISH SHARMA, A. MALLESW ARA REDDY

:v ALUATION OF MOR/NGA OLE/FERA GUM AST ABLET DISINTEGRANT

llNIT V PATEL, NEELESH CHOBEY

.fiCROSPHERES OF 5-FLUOROURACIL FOR COLON TARGETING

lMIT KUMAR PANIGRAHI, M. MATHRUSRI ANNAPURNA, K. HIMASHANKAR

'HARMACOGNOSTICAL AND HYPOGLYCEMIC ACTIVITY OF DIFFERENT PARTS OF 'OLANUM NIGRUMLTNN PLANT

:.r. SATHYA MEONAH, M. PALANISWAMY,S.T. IMMANUEL MOSES KEERTHY, L.A. 1RADEEP RAJKUMAR, R. USHA NANDHINI

210-214

215-220

221-224

~!OXIDANT ACTIVITIES OF EXTRACTS FROMACACL4 MELANOXYLON. ACACL4 225-231 >EALBATA AND OLEA EUROPAEA AND ALKALOIDS ESTIMATION

~NGELO Luis, NUNO GIL, MARIA EMILIA AMARAL,ANA PAULA DUARTE

•HYTOCHEMICAL SCREENING AND ANTHELMINTIC ACTIVITY OF METHANOLIC 232-234 :XTRACT OF IMPERATA CYLINDRICA

'ARVATHYN.G, PADMA.R,RENJITH.V,KALPANA. P. RAHATE, SARANYA T.S.

:FFECT OF METHANOLIC EXTRACTS OF BLUMEA ERIANTHA DC LEAVES ON PROTEIN 235-238 tfETABOLISM AND MARKER ENZYMES IN STREPTOZOTOCIN- INDUCED IYPERGLYCEMIC ANIMALS

JMESH PRAT AP SINGH, ARVIND KUMAR SINGH, DR. R. PARTHA SARATHY

~TIDIABETIC ACTIVITY OF AQUEOUS EXTRACT OF CORIANDRUM SATIVUML. FRUITS 239-24f1 N STREPTOZOTOCIN INDUCED RATS

:AMR.ANJA VED NAQUVI, MOHD. ALI, JAVED AHAMAD :XCIPIENT SCREENING AND' DEVELOPMENT OF FORMULATION DESIGN SPACE FOR 241-248 >ICLOFENAC SODIUM FAST DISSOLVING TABLETS

AGADEESHINDURUANDPADMAJABOOKYA

HO ANALYTICAL METHOD DEVELOPMENT AND VALIDA TI ON OF LEV ALBUTEROL AB2- 249-253 lDRENERGIC AGONIST BY RP-HPLC METHOD

l. S. GHEMUD, B. SANTHAKlJMARI, A. B. PHARNE, M. M. JADHA V, K. S. JAIN, M. J. \:ULKARNl

. . .

)EVELOPMENT AND IN-VITRO EVALUATION OF TASTE MASKED ONDANSETRON HCI 254-26( >RAL DISPERSIBLE TABLETS BY DIRECT COMPRESSION METHOD BY USING HFFERENT DILUENTS

1 AMSHIDHAR REDDY D, DODDA YY AH, SAISIRISHA A, BHARATHI T

1ROWTH CHARACTERIZATION OF CALCIUM OXALATE MONOHYDRATE CRYSTALS 261-27( NFLUENCED BY COSTUS IGNEUS AQUEOUS STEM EXTRACT

~. MANJULA, K. PAZHANICHAMY, S.KUMARAN, T. EEVERA, C. DALE KEEFE AND K. ~JENDRAND

lNTIOXIDANT ACTIVITY OF METHANOLIC EXTRACT OF PHASEOLUS TRILOBUS ROOT 271-27~ 10WDER

iA VPREET KAUR, LALIT KISHORE

,EVELOPMENT AND VALIDATION OF REVERSED-PHASE HIGH PERFORMANCE LIQUID 276-27i :HROMATOGRAPIDC METHOD FOR SIMULTANIOUS ESTIMATION OF SUMATRIPTAN :ucCINATE AND NAPROXEN SODIUM IN PHARMACEUTICAL DOSAGE FORM

:AGARD. SOLANKI, DR. PARESH U. PATEL

~CID IC METHOD FOR THE LOW MOLECULAR PECTIN PREPARATION 279-28~

lfAXIM KHOTIMCHENKO, V ALERIKOVALEV, ELENA KOLENCHENKO, ~OTIMCHENKO

,ESIGN AND EVALUATION OF COLON SPECIFIC DRUG DELIVERY OF NAPROXEN 284-28t :ODIUM USING GUAR GUM AND CROSSLINKED GUAR GUM

C.RAJY A LAKSHMI, Y.INDIRA MUZIB & VIJAYA KUMAR.VOLETI

'ORMULATION AND EVALUATION OF FINGOLIMOD CAPSULES 289-29:

C. KA THIRESAN, M. BHAGATH KUMAR REDDY, C. MOORTHI, N. AHAMED DA WOOD :HA, KIRAN KRISHNAN, R. MANAV ALAN

lNTIDEPRESSIVE-LIKE EFFECT OF MICROCYSTIN-FR IN SWISS ALBINO MICE TESTED 293-29~ SY A BATTERY OF BEHAVIOURAL DEPRESSION MODELS

t ASHWIN KUMAR, SUSHIL KUMAR Y ADA V, SANJAY KUMAR VERMA

'ALIDATION OF HPLC METHOD FOR DETERMINATION OF ANTIOXIDANT VITAMIN C 300-30· ~VITAMIN B6 IN FOOD SUPPLEMENTS AND DRUGS

&OGDAN KIRILOV, DANKA OBRESHKOV A, DOBRINA TSVETKOV A

:FFECT OF ETHANOLIC EXTRACT OF ACALYPHA IND/CA LINN. ON ETHYLENE GLYCOL 305-30l INDUCED KIDNEY CALCULI IN RA TS

.f.SA THY A, DR. R.KOKILA V ANI

IPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF PROTON-PUMP NHIBITORS WITH DOMPERIDONE IN HUMAN PLASMA EMPLOYING RESPONSE :URFACE DESIGN 7• SREE JANARDHANAN,R. MANA V ALAN AND K. V ALLIAPP AN

>ETECTION OF MYCOBACTERIAL ANTIBODIES IN SERUM SAMPLES USING BCG 'ACCINE BASED ELISA

:ooRAJ. s. NATH, NATHIYA. K , ANGAYARKANNI. J, PALANISWAMY. M

309-31'

318-321

>EVELOPMENT OF AV ALIDA TED ST ABILITY-INDICATING HPLC ASSAY METHOD FOR 321-321 )EXKETOPROFEN TROMET AMOL

'IDHY A KISHORE BHUSARI, SUNIL RAJARAM DHANESHW AR

:FFECT OF GROWTH REGULATORS IN CALLUS INDUCTION, PLUMBAGIN CONTENT lND INDIRECT ORGANOGENESIS OF PLUMBAGO ZEYLANICA

,UBAJNA A.S, MURUGAN K

tfANJUNATH SANGAPPA, PADMA TIDAGARAJAN

ffiWER DRUGS IN ANAESTHESIA

'EENA BANSAL, SARLA HOODA

Ubitech Solutions Website Design, Website Development Copyright © 2009 IJPPS. All rights reserved

& Website Maintenance

327-333

334-33(

658-667

668-670

·: AcCloteV\A.Lc scteV\,ce.s International Journal oi Pharmacy and Pharmaceutical Sciences

ISSN· 0975-1491 VOl4, SnpplJ,2012

Research Article

ALPHA·GLUCOSIDASE INHIBITOR ACTIVITY AND CHARACTERIZATION OF ENDOPHYTIC ACTINOMYCETES ISOLATED FROM SOME INDONESIAN DIABETIC MEDICINAL PLANTS

SRI PUJIYANTQt.l, YULIN LESTARJl", ANTONIUS SUWANTOl, SRI BUDIARTP, LATIFAH K. DARUSMAN2

'Department or Biology, ?Department or Chemistry, Faculty or Mathematics and Natural Sciences, Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia, >Department ofBiology, Faculty or Mathematics and Natural Sciences, Diponegoro University

Tembalang Campus, Semarang 50275, Indonesia. 'Email: yulinlestari@yahoo.com

Received: 13 Oct 2011. Revised and Accepted: 10 Nov 2011

ABSTRACT

An alpha glucosidase inhibitor is one of the compounds for the treatment or diabetes. This inhibitor can retard the liberation of glucose from dietary complex carbohydrates and delay glucose absorption. resulting in reduced postprandial plasma glucose levels and suppress postprandial hyperglycaemia. The purpose of this study was to isolate and select alpha glucosidase inhibitor-producing endophytic actinomycetes from various diabetic medicinal plants. Endophytlc actinomycetes were isolated from the roots, leaves and stems of diabetic medicinal plants: A//oe vera, Tinosporo crispa, Phalerio mocrocorpa. Curcumo oeru9inoso, Centela aslatica, Xoncus orvensis, Andro9rophis ponicu/ata, Caesa/plnia sappan, Curcumo xanthoriza, Parcia speciosa. Gynura procumbens, Physolis peruviana and Hibiscus sabdariffa. Sterilized plant sample were inoculated on the HV Agar medium containing 50 ppm cycloheximide and 30 ppm nalid1x1c acid and were incubated for 2-3 weeks at room temperature. Sixty-five isolates were obtained and tested for their ability to inhibit the alpha·glucosidase. Identification for the selected isolates was based on 16S rDNA sequences. The inhibitor activity to alpha glucosidase was determined spectrophotometrically at 400 nm usmg p·N1trophenyl-alpha-D-glucopyranoside as a substrate, and acarbose as a positive c;ontroL The results showed that endophytic actinomycetes isolated from selected antidiabetic plants produced various inhibition activities. The highest inhibition activity to alpha·glucosidase was shown by BWA65 found from Tmospora cnspa. Production of alpha·glucosidase inhibitor compounds in this plant largely related with the contribution of its actinomycetes endophytes. The molecular Identification based on 16S rONA sequence revealed that the potential BWA65 isolate showed 92% similarity to Streptomyces olivochromogenes.

Keywords: Alpha·glucosidase inhibitor, Endophytic actinomycete, Diabetes mellitus, Indonesian medicinal plants.

INTRODUCTION

Diabetes mellitus (OM) is the highest cause of death among other chronic diseases. This disease can cause complications such as cardiovascular dJsease, kidney failure, blindness. impotence and gangrene. More than 95% of diabetes is type 2 diabetes or often called non-insulin dependent diabetes•. OM cannot be cured, but can be controlled. Treatment of OM In principle is to maintain blood glucose levels in normal conditions (80·120 mg/di). Both modern and traditional antidlabetlc drugs have commonly use by Indonesian community experiencing DM. One of antidiabetic drugs mechanism Is by inhibiting digestion of complex carbohydrates (starch) into glucose in the small intestine, resulting of reducing the intake of glucose from the intestine into the blood. One of active compounds that have this activity is an alpha glucosidase inhibitor. The alpha glucosidase inhibitor can be produced by some organisms, including microbes. An example is acarbose, a commercial alpha glucosidase inhibitor produced by Actinoplones sp., an actinomycetes isolated from Kenya2.

Traditionally, OM treatments utilize various types of medicinal plants which contain active ingredients that can decrease blood sugar levels. Empirically, some medicinal plants are known to have capability to cure diabetes. The plant active ingredients are commonly used to decrease blood sugar levels. Various medicinal plants have been reported as anti hyperglicaemic e.g. Terminalio arjuna, Tinospora crispo, Pho/eria macrocorpo, Andrographis paniculatu, Momordica charantia, Tribulus terestris, and Berberis aristata 3.10.

Exploration of endophyt1c microbes 1s expected to produce important secondary metabolites that have properties similar to that produced by the host plant metabolites. Endophytic microbes that live in plants can produce secondary metabolites similar to those produced by its host as a result of genetic exchange and evolution of a long relationship• •.12. Medicinal plants for diabetes are a potential source of microbial producers of alpha glucosidase inhibitors. With potential isolates obtained from medicinal plants. we will be able to produce an alpha glucosidase inhibitor compounds for diabetes drug microbiologically, with greater numbers and better quality. The purpose of this study was to obtain

isolates of endophytic actinomycetes from some Indonesian medicmal plants that have been known to have antldiabetic properties. potentially as an alpha glucosidase inhibitor producer.

MATERIALS AND METHODS

Medicinal plants samples

Thirteen medicinal plants were collected from the Collection of Medicinal Plants Garden of Blopharmaca Research Center, Bogor Agricultural University, Bogor, Indonesia. The sample of medicinal plants namely: Alloe vera, Tinosporo crispa, Phaleria mocrocarpa, Curcuma aeruginosa. Centela asiatica, Xoncus orvensis, Andro9raphis poniculata, Curcumo xanthonza, Physa/is peruviana, Gynura procumbens, Hibiscus sabdorif!o, Coesalpinia sappan and Parcia speciosa.

Isolation of endophytlc actlnomycetes

Isolation of endophytic actmomycetes was base on previous researcher13• Surface sterilization of the plant samples were done by soaking in alcohol 70% (I minute), sodium hypochlorite 1% (S minutes). alcohol 70% (1 minute) and finally rinsed with sterile distilled water. The sterilized samples were then aseptically grounded and added by 4 ml of 12.5 mM sterile phosphate buffer. Amount of 100 µi of sample suspension was platted on Humic Acid Vitamin (HV) agar medium contain SO ppm of cycloheximide and 30 ppm of nalidixlc acid, and incubated for 2·3 weeks at room temperature (25·2BoC). Actinomycetes colonies that grow from agar medium were purified on Yeast Malt Extract Agar (YMA) medium and stored in the refrigerator for further examination.

Selection of an alpha glucosldase inhibitor-producer

All isolates obtained were grown in a liquid medium containing 0.1% soluble starch, 0.5% peptone, and 0.1% yeast extract (pli 7) for 14 days with agitation (120 rpm) at room temperature. The cell biomass were separated by centrifugation at 1432 x g for 20 minutes and the supernatant were tested for alpha glucosidase Inhibitor activity according to previous researcher••· Isolates with the greatest inhibitory activity was selected for further investigation.

Pujiyanto et al.

Assay of alpha glucosidase inhibition

Inhibition of alpha glucosidase activity was examined according to previous researcher14• Enzyme inhibition assay was measured based on solving the substrate to produce colored products. Enzyme alpha­glucosidase (Sigma) with a concentration 0.75 units / ml was dissolved in 0.1 M phosphate buffer pH 7. As a substrate. we used p­nitrophenyl-alpha-0-gluco pyranoside 20 mM dissolved in 0.1 M phosphate buffer pH 7. The mixture of reaction contains 125 µJ substrate, 240 µl 0.1 M phospate buffer pH 7 and 10 µI sample. After the reaction mixture was incubated at 37oc for 5 minutes, amount of 125 µJ of enzyme were added and incubated for 15 minutes at 37°C. The reaction was stopped by adding 500 µI sodium carbonate and p­nitrofenol produced was measured its absorbance at 400 nm. As a comparison, we used 1 mg/ml solution of acarbose (Sigma). Inhibition of alpha glucosidase activity was determined by the formula:

Inhibition(%): (Ac-(As-Ab) /Ac x 100 %

(Ac: absorbance of control, Ab: absorbance of background, As: absorbance of sample)

Role of endophytic actinomycetes in the production of alpha­glucosidase inhibitor

To determine the role of endophytic actinomycetes in producing alpha·glucosidase inhibitors, we used free endophytic of selected medicinal plant tissue culture, Tinospora crispa. The 0.5 g of samples plant tissue culture was grounded and added 0.5 ml phosphate buffer, and then centrifuged 1432 x g for 5 minutes. The supernatant obtained was tested against the alpha glucosidase inhibitory activity. In the same way, the inhibition activity of the naturally grown plant was also examined. The ability of plant inhibitors were compared with the inhibitor from endophytic actinomycete culture

Molecular identification and morphological characterization

DNA extraction and amplification of 16S rDNA gene

DNA extraction of BWA65 isolate was carried out using GES methods1s followed 16S rDNA gene amplification using Primer 20F (5'-GATITIGATCCTGGCTCAG-3') and 1500R (5-GTTACCTTGTTACGACTT-3'). PCR reactions were done using Thermakycler (Trootsa Shuzo Co, Ltd., Shiga. Japan) for 30 cycles. Amplification results was visualized by electrophoresis using Mupid Mini Cell in 1% agarose gel in TAE buffer (Tris-EDTA Acetate) for 25 minutes at 100 V. Purification of PCR products was done with PEG precipitation method. 16s rDNA pure samples was stored at -zooc.

Cycle sequencin.g

The cycle sequencing was carried out by using primers 520 F (5'· GTGCCAGCAGCCGCGG-3'), 920R (5'-CCGTCAATTCATTTGAGTTT-3'), 520R (5'-ACCGCGGCTGCTGGC-3'), 920F (5'· AAACTCAAATGAATTGACGG-3'), 20 F (5'-GATITIGATCCTGGCTCAG· 3') and 1500 R (5·GTTACCTTGTTACGACTT-3'). The composition used for each tube was 0.5 ml 10 pmol primer, 1 µI purified DNA, 0.5 ml of Big Dye Terminator sequence premix kit (Applied Biosystems Inc., Warington. UK), 1.5 ml Sx sequence buffer and deionized water until a volume of 10 ml. Subsequently the mixture reaction was performed by PCR amplification of 40 cycles.

Preparation and sequencing

Preparation was performed by mixing 10 ml of the product cycle sequencing with 1 ml of 3M Na-acetate, 1 ml of 125 mM EDTA (pH 8) and 25 ml absolute ethanol. The next stage was centrifugation at 8586 x g for 25 minutes at 4oc. Supernatant discarded and the pellet was washed with 70% ethanol and then re-centrifuged 8586 x g for 10 minutes. The supernatant was discarded followed by drying pellets for 10 minutes. Dried DNA pellets were added to 10 ml HiDi· formamide (Applied Biosystems Inc., Warington, UK). Samples were then heated at 95°C for 2 minutes and immediately cooled in lee. The next step, sample was injected to ABI 3130 sequencer (Applied Biosystems Inc., Foster. Calif.).

Analysis of molecular data

DNA sequences were analysed using the BioEdit program and data blasting at the NCBI Gene Bank data library. Phylogenetic analysis

Int) Plrnrm Ph arm Sci, Vol 4, Suppl 1, 327-333

was conducted using multiple alignment programs Clustal X version 1.83. Construction of phylogenetic trees as based on genetic distance with the Neighbor Joining method. Construction of evolutionary distance in degrees of confidence was done using the bootstrap values in NJ plot program.

Observation of morphology

Morphological observations were carried out by microscopic observation with light microscope at magnification of lOOx, 400x and scanning electron microscope QSM-5310LV) with magnification of 10,000x.

RESULTS AND DISCUSSION

Isolation of endophytic Actinomycetes

Endophytic microbes are microbes that live inside plant tissues at specific periods and are able to live by forming colonies in plant tissue without harming their host.

In this study, 13 samples of medicinal plants used as sources of endophytic actinomycetes isolates, namely: Alloe vera, Tinospora crispa, Phaleria macrocarpa, Curcuma aeru9inosa, Centela asiatica, Xoncus orvensis. Andrographis paniculata, Caesalpinia sappan, Curcuma xanthoriza, Parcia speciosa, Gynura procumbens, Physalis peruviana and Hibiscus sobdariffa.

In this study, 65 endophytic actinomycetes isolates were successfully isolated from different species of medicinal plants using HV agar medium. All of the isolated endophytic actinomycett:s obtained can be regarded as the culturable isolates, although they may not represent all the endophytic microbial populations living on these plants. This is due largely that microbes cannot all grow on synthetic medium.

The result of endophytic actinomycetes isolates from each medicinal plant examined was shown in Figure land Table 1. Around 69.2% of tested plants contained endophytic actinomycetes. Tinospora crispa contained the highest number of endophytic actinomycetes (32 isolates). Curcuma aeruginosa (9 isolates). Gynura procumbens (6 isolates), Curcuma xanthoryza (5 isolates) and other plants (1·4 isolates). The results of investigation showed that most endophytic actinomycetes obtained from the roots (45 isolates), followed by the rhizome (14 isolates), the stems (3 isolates) and the leaves (3 isolates).

Each plant generally contains several endophytic actinomycetes that live in the plants. Each higher plants may contain some endophytic actinomycetes that can produce biological compounds or secondary metabolites that allegedly as a result co-evolution or genetic transfer (genetic recombination) from the host plant into endophytic microbes' 1•

The results indicate that several endopbytic actinomycetes capable of producing active compounds similar to their host. This phenomena support the findings from other workers. Strobel! and Daisy16 reported that endophytic Taxomyces andreanae produced paclitaxel in Taxus plants. Anticancer compound paclitaxel was also produced by the plant Taxus brevivolia. Taechowisan et al" found that Streptomyces aureofaciens, endophytic in ginger plant produced arylcoumarin compound which has antitumor activity, where the ginger plant also has anti-tumor compounds such as reported by previous researcherl8. Meanwhile Castillo et a/19 showed that Streptomyces NRRL 30562, the endophytic in Kennedia nigriscans plant capable of producing broad-spectrum of antibiotics. This plant is traditionally used to prevent infection of microbes in the wound by Aboriginal tribes.

Screening of endophytic actinomycetes producln~ a­glucosidase inhibitor

Examination for all endophytic actinomycetes revealed that 12 isolates which comprised of 10 isolates from Tinospora crispa, 1 isolate from Caesa/pinia sappans and 1 isolate from Curcuma aeruginosa respectively, generate positive alpha-glucosidase inhibitors, whereas 53 other isolates were having negative results (Table 1).

328

I• Pujiyanto et al.

Int] Pharm Pharm Sci, Vol 4, Suppl 1, 327-333

35 - ·· ~··- .. ----·-·---

30 "' QI .... 25 "' 0 .~ 20 _._ -- -- -· .... 0 ... 15 QI .Q

E :s 10 .-..... -- -- - ·--·-- --z

t1_··~~--5

0 ···-- --·

___ ............... __ ------.. ·-·----------" Fig. 1: The number of endophytic actinomycetes Isolates from each plant

Table 1: The endophytlc actinomycetes isolates from various diabetic medicinal plants and their properties of alpha glucosidase inhibitor

Medicinalplants

Gynura procumbens

Alloe vera

Curcuma xanthoriza

Centela asiatica

Physalis peruviana

Tinospora crispa

Part of plants roots

Stems

Leaves

Roots Leaves

Roots Stems Leaves Rhizome

Roots Stems Leaves

Roots Stems Leaves

Roots

Number of isolates 4

2

1

s

1

32

Code of isolates SNA 11 SNA 12 SNA 2 SNA21 SNB 1 SNB lA SND 22

TLR 1 TLR2 TLR21 TLR3 TLR4

CPl

BWA 14

BWA14A BWAlS BWA lSA BWA 16 BWA 2 BWA3 BWA33 BWA34 BWA35 BWA36 BWA3A BWA4 BWA4A BWASl

Characteristic onYMA medium

no spores, pink colony no spores, red colony no spores, brown colony no spores, dark brown colony no spores, brown colony no spores, pink colony no spores, red colony

brown spores white spores gray spores white spores gray spores

white spores

no spores, brown colonie~. producing a brown pigment no spores, brown colonies brownish white spores gray spores gray spores white-gray spores white spores, produce reddish pigments white spores white spores, black colony no spores, brown colony no spores, brown colony white spores white spores. brown colony gray spores white spores

Inhibitory activity (%)

0.684

2.66 4.853 0.478 1.162 0.273

329

Pujlyonto et al. Int} Phorm Pharm Sci, Vol 4, Suppl 1, 327-333

8WA54 no spores, dark brown colonies, dark brown 1.162 pigment

BWA61 no spores, dark brown colonies. brown pigment

BWA62 no spores. copper brown colonies BWA63 white-brown spores BWA64 white spores BWA65 no spores, dark brown colony 4.511 BWA66 white spores BWA71 no spore. brown colony 0.752 BWA 72 no spores, yellow-brown colony BWA73 white spores, brown colony 0.889 BWA74 no spores, brown colony BWA75 white spores BWA 76 white spores, brown colony, black pigmen BWA82 no spores, brown colony BWA84 no spores, yellow colony BWA85 white spores BWA86 no spores, brown colony BWA93 no spores, black colony

Stems Leaves

Hibiscus sobdor1ffo Roots Stems 1 ROB 12 no spore, pink colony Leaves Flower

Phaleria macrocarpa Roots 3 MDA2 brown spores MDA22 brown spores MDA52 no spore, orange colony

Stems Leaves Fruits

Andrographis Roots 2 SBLAl no spore, black colony panlcu/ata SBLAZ white spores, no pigment

Stems Leaves 1 SBLD 3 no spore, brown colony

Xoncus arvensis Roots Stems Leaves

Caesa/pinia sappan Roots 4 SC A 13 white spores 0.547 SCA 11 white-brown spore SCA 14 white spores SCA 1 white spores

Stems Leaves

Parcia speciosa Roots Stems Leaves

Curcuma aeruginosa Roots Stems Leaves Rhizomes 9 TIRll white spores, brown colony ,. TIR 12 white spores - brown

TIR 13 gold spores, brown colony 3.623 TIR14 no spore TIRlA white spores, brown colony TIR 18 white spores TIR 182 white spores, brown colony TIRZ gray spores, dark brown colony TIR3 gray-white spores

Number of Isolates 65

On further testing quantitatively assay using nitrophenyl-alpha- extract of 8WA65 isolates from Tinospora crispa produce the D-gluco pyranoside as a substrate showed that the crude extract highest inhibition {11.01%) to alpha glucosidase, that was equal (supernatant) from four isolates of endophytic actinomycetes of to 80% if compared to lmg/ml of acarbose (13.61%) as control Tinospora er/spa had alpha glucosidase inhibition activity. Crude (Figure 2).

330

Pujiyanto et al. Int/ Pharm Pharm Sci, Vol 4, Suppl 1, 327-333

16.00

14.00

12.00

~ 10.00 . c .2 8.00 ... :s :a 6.00 .E

4.00

2.00

0.00

BWA36 BWA65 BWA35 BWA54 Ac

Isolates

Fig. 2: Inhibitory activity of the a lpha glucosidase by endophytic actinomycetes isolated from Tlnospora crlspa

The discovery of endopbytlc actlnomycetes isolates from Tinospora crispa which produce the alpha glucosidase inhibitor in this research, strengthen the opinions that any plant can contain several endophytic microbes that can produce biological compounds or secondary metabolites that allegedly as a result genetic transfer (genetic recombination) from the host plant into endophytic microbes11• Information about the presence of hypoglycemic agents in Tinospora had been reported by previous researchersW·Z3. There were reported that the daily administration of alcoholic or aqueous extract of Tinospora cordifol1a decreased the blood sugar in alloxan­induced hyperglycemia in rats and rabbits in the dose of 400 mg/kg.

In this study alpha glucosldase inhibitor activity was assayed by comparing the activity produced by host plants Tinospora crispa,

free endophytic Tinospora crispo plant tissue culture and endophytic actinomycetes isolates. The result can illustrate the role of endophytic actinomycetes in contributing to the production of the inhibitor compounds. The results showed that free of endophytes plant tissue culture has only a very low capability to produce inhibitor compounds.

Moreover. the native plants part of Tinospora crispo capable of producing much larger inhibitor compounds (Figure 3). However, the capability of endophytic actinomycetes BWA65 was more than twice the activity of the host plants. The data clearly Indicates that the endophytic actinomycetes in this plant contribute significantly to the production of alpha-glucosidase inhibitor compounds.

12.00 ~~~~~~~---,-1

10.00 I

~ 8.UU

c=-.s G.00 f

:s :a .5 4.0U

2.00

0 .00

~·I _._J_a -PTC Callus Stem Root Loaves BWASll BWAGS

Sample

Fig. 3: The activity ofalpha·glucosidase inhibitor produced by plant tissue culture (PTC, callus), native plants parts (stem, root, leaves) and eodophytic actinomycetes isolates (BWA54, BWA65)

Morphologycal and molecular identification of isolate BWA65

In this study we used three types of media to see the culture characteristics of isolates BWA65 ie: Yeast Extract Malt Extract Agar (YMA), Yeast Extract Soluble Starch Agar (YSA) and Oatmeal Agar (OA). The results of investigation showed that BWA65 has good growth on YSA and OA media, and moderate growth on YMA media.

In all media, the BWA65 isolate produced white aerial mycelium. The substrate mycelium of isolate BWA65 was brown on OA media and dark brown on both YSA and YMA media.

This isolate also produce soluble pigment dark brown on YSA media and pink pigment on OA media. The culture characteristics of BWA65 isolate on various media are listed in Table 2.

331

... Pujlyanto et al.

Int} Phann Pharm Sci, Vol 4, Suppl 1, 327-333

Tabel 2: Cultural characterizatlcs or potential isolate BWA65 on various media (7 days Incubation at r oom temperature)

Cultural characteristic Media YMA YSA OA

Growth moderate good good Aerial mycellum white white white Substrate mycehum dark brown dark brown brown Soluble pigment no pigment brown pink

The morphological observation under a hght microscope with 400x magnification showed the spiral spore chains. The spiral spore chains are typical characteristics for Streptomyces. Further

testing by SEM showed that BWA65 isolates has unbranched aerial hyphae and spores cyhndrical spiral chains with smooth surface (Figure 4).

Fig. 4 : The morphology of the endophytlc actinomycetes BWA65 observed on Oaoneal Agar, ligh microscope (400x) and SEM (10,000x)

Phylogenetic tree of endophytlc actinomycete of isolate BWA65 based on the 16s rDNA sequences ls showed at Figure 5. The results or molecular identification using partial sequences of 16S rDNA showed that BWA65 has a 92% similanty with Streptomyces o/lvochromogenes. Base on other previous studies, Sceptomyces olivochromogenes was known to produce many active compounds

such as: glucose isomerase. xilose isomerase and phospolipase2Hs. Until now, there has been no report of alpha-glucosidase Inhibitor produced by Steptomyces o/ivochromogenes. Accordingly, we believe that the isolate BWA65 was a new type of alpha·glucosidase inhibitors producer. The results of this investigation also Indicate that the BWA65 may be a novel species.

~eptor.tycesafbo.,fm:>~e••s .----S't?•ptowjvcas lfqnJha7t:tC'l~$l:..

Kirasat~pora lclll.1n.inn.s 5),oeptomyc~ oburo111 cn:.i;

~cptomyce• ••urcofaci~H• ~~plo•11;fC'eS ""!Yl2mlNUSU

~op1c,,,.,cc. n11tt.:Mu~ St><1pto"l}'cc; :wcllan<u~ ~•ptomyc~>P""'"'"°"'"' -Sttcp~""1>1'-·eJ psa11m10hcus.

Streptomyc4• cod1c<>io1 .$"rr~FJ.:>P'rt)IC4S l i».;O/,Ht1.1>: ~•plomyc,;s gti:o1eruber

St>-epfomyc1;•charf>cus1J S,.cpl<'~l)'C">Or~llaceuJ 5,.,c.r>romy,;cJ.corchoriuu $~Q}'tt':'~,f)'ct1:. ramcu..1

tStt-c..oto1•~ycc:; r«b> ;:>'>>V;oh~lvolu l-~ept0Ytt~V'Ctt,~t<ild1n Streptt!,ny .. ~J. t"'J.~g;tlv1:." ~cptO»IJ'C<il:> u1:ula "t1;.

$h-q.1tcmy:.;.s :.;"-=>7ov1,.&tli1s !;tri<ptc·»V'i.·fl.z n-ic.o/.<:>r i ('l'r,~tomy~<C!l to'1n;m-1ns1:,

.'\h "p!C"Yl'i_VCPS .:o/omb! vnSI l

.:)'.).,."}"'IC-mf:C.,~ ..... ,.o~U!"UC-0,.,,,IJ

QS'f-.CFt.ON'l)' ..... C.;.pn . .oue.c-lcr SIY~pf.jtt:ycc:. o/~...,c-chTc-.mogent?.5 SIY~p1c·niy{'V.':i gnJ•oc"tr-..-.mogcn.e.s

Stt.tFIC·rn,fCll s. can us. Srreptom)'c~;._l)mbn1?1'1'1~

.'S"/1--&p1omye4: ,.::i:,!o-mycif~.:u.)

IS?epl.:;,'!'11)'"~:. 6''l1ia.:i.:;, ~C-p!omy~'"~.· SVJCE'UJ. ~~pt.-:om)'c..:.:. .. ·,nnamo>t~1~!•.l

S't1'1;ptt:•tn)•t."'e i at.:?tJt.:~ !;'t., .. .epromy"·a~ kun11un<!c11n~ .sb-<ptO?!YN$ .:n:<1..rm>h /u

'---+-+--------------8\\'A.65 SJrcpte>.'n)'.:~; bob1,,

•pft()m)'C<l u1tKJnnul ~cplcmycc:. um&,.u·"'~

S'/7•pfo"')'CCJ f!dU.tn:.1:. Strep!Om)-"t:¢' fm,,;,1cu,;

OD2

Fig. S: Phylogenetic tree of endophytlc actinomycete of isolate BWA6S based on the 16S rDNA sequences

332

. . .. • Pujlyanto et al .

CONCLUSION In this study, 65 endophytic actinomycetes have been isolated from various diabetic medicinal plants. Endophytic actinomycete isolate BWA65 from Tinospora crispa has high ability to inhibit the alpha glucosidase activity. Production of alpha·glucosidase inhibitor compounds in this plants largely due to the contribution of its actinomycetes endophytes. Isolate BWA65 has 92% similarity with Streptomyces olivochromogenes.

ACKNOWLEDGEMENT This research was supported by a Graduate Research Grant from the Directorate General of Higher Education of Republic of Indonesia granted to Dr. Yulin Lesrari. Authors are grateful to the Laboratory Conseivatlon of Forest Resources, Forestry Faculty. Bogor Agricultural University for Tinospora crispa plant tissue culture. We also thank Arif Nurkanto from Biological Research Center, Indonesian Institute of Sciences (LIPI) for 16$ rDNA analysis assistance of selected isolates

REFERENCES

1. Bailey CJ, Day C. Antidiabetlc drugs. Br J Cardiol 2003;10 :128· 36.

2. McGown J. Out of Africa: Mysteries of Access and Benefit Sharing. Washington (USA): The Edmonds Institute; 2006.

3. Morshed MA. Haque A, Rokeya B. Ali L. Anti·hyperglycemic and lipid lowering effect of Terminalia arjuna bark extract on streptozotocin induced type 2 diabetic model rats. Int J Pharm Pharm Sci 2011;3(4) :449-453.

4. Subroto A. Herbs for Diabetes Mellitus. Jakarta (INA): Penebar Swadaya; 2006.

5. Klein G, Kim J. Himmeldirk K. Cao Y, Chen X. Antidiabetes and anti-obesity activity of Lagerstroemia speciosa. eCAM 2007;4 :401-407.

6. Bnouham M, Z1yyat A. Mekhfi H. Tahn A, Medicinal plants with potential antidiabetic activity-a review of ten years of herbal medicine research. Int J Diabet and Metabolism 2006; 14:1·25.

7. Subramanian R. Asmawi MZ. Sadikun A. Effect of ethanohc extract of Andro9raphis paniculata nees on a combination of fat.fed diet and low dose streptozotocin induced chronic insulin resistance in rats. Diabetologia Croatica 2008;37 :13-22.

8. Yulinah E, Sukrasno. Fitri MA. Antidiabetic activity of ethanol extract of Andrographis paniculata Nees (Acanthaceae). J Matematika dan Sains 2001;6 :13-20.

9. Lamba HS, Bhargava CS, Thakur M, Bhargava S. Alpha glucosldase and aldolase reductase inhibitory activity in vitro and anti diabetic activity in vivo of Tribulus terrestris I... (Dunal). Int J Pharm Pharm Sci 2011;3(3) :270-272.

10. Upwar N. Patel R. Waseem N, Mahobia NK. Hypoglycemic effect of methanolic extract of Berberis aristata DC stem on normal

Int} Phann PharmSci, Vol 4, Suppl 1, 327-333

and streptozotocin induced diabetic rats. Int J Pharm Pharm Sci 2011;3(1) ·222·224

11 Tan RX. Zou WX. Endophytes: a rich source of functional metabolites. Nat Prod Rep 2001;18 :448·459.

12. Radji M. The role of biotechnology and endophytic microorganisms in herbal drug development Majalah llmu Kefarmasian 2005;2 :113-126.

13. Coombs JT. Franco CM. Isolation and identification of Actinobacteria from surface-sterilized wheat roots. Appl Environ Mlcrobiol 2003;69 :5603-5608.

14 Anam K. Widharna RM. Kusrini D. Alpha glucosidase inhibitor activity of Terminalia species. Int J Pbarmacol 2009;5 :277-280.

15. Pitcher DG. Saunder NA and Own RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Appl Microbiol 1989;8 :151-156.

16. Strobel GA and Daisy B. B1oprospecting for microbial endophytes and their natural products. Microbiol and Molecular Biology Rev 2003;67 :491·502.

17. Taechowisan T. Lu C, Shen Y, Lumyong S. Antitumor activity of 4-arylcoumarins from endophytic Streptomyces aureofaciens CMU Acl30. J Cancer Res Ther 2007;3 :86·91.

18. Katiyar SK. Arwal R, Muhtar H. Inhibition of tumor promotion in SENCAR mouse skin by etanol extract of Zfngiber olficinale rhizome.Cancer Research 1996;56 :1023-1030.

19. Castillo UF, Strobel GA. Ford EJ. Hess WM, Porter H, Jensen JB et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nlgriscans. Microbiol 2002;148 :2675-2685.

20. Singh SS. Pandey SC. Srivastava S, Gupta VS. Patro B, Ghosh AC. Chemistry and medical properties of Tinospora cordifolia. Ind J Pharmacol 2003;35 :83-91.

21. Rajalakshmi M, Eliza J, Priya CE, Nirmala A. Daisy P. Anti· diabetic properties of Tinospora cardifo/ia stem extracts on streptozotocin-induced diabetic rats. Afr J Pharm Pharmacol 2009;3 :171-180.

22. Chougale AD, Ghadyale VA, Panaskar SN. Aivindekar AU. Alpha glucosidase inhibition by stem extract of Tinospora cordifolia. J Enzyme Inhibition and Med Chem 2009;24 :998-1001.

23. Sriyapal C, Rawadee D, Sangwatanaroj S, Kongkathip N, Krittiyanunt S. Hypoglycemic effect of Tinospora crlspa dry powder in outpatients with metabolic syndrome. J Healt Res. 2009;23 :125·133.

24. Azin M. Maozaml N, Bogan MN. Production, purification and immobilization of glucose isomerase from Streptomyces olivochromogenes PTCC 1547. World J Microbiol Biotechnol 1997;13 :597-598.

25. Simkada JR. Lee HJ, Jang SY. Cho SS, Park EJ, Sohng JK. Yoo JC. A novel alkalo- and thennostable phospholipase D from Streptomyces olivochromogenes. Biotechnol Letters 2009;31 :429-435.

333

. ... •

0 ~ -· 1c1llll9C."'cm

I SClmago .ot.rnal & Cour l~ Rank

lbne Journal Search

~~ ~chqueiy

ES~ MODUS 11\ REBUS

CdrtSerdl International Journal of Pharmacy and Pharmaceutical Sciences

Wby:nii

~jectAIM: ~. TaO:liff rdltimam