Abdul Mun’im et al. Int. Res. J. Pharm. 2013, 4 (5)

Page 128

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

SCREENING OF ENDOPHYTIC FUNGI FROM CASSIA SIAMEA LAMK LEAVES

AS α-GLUCOSIDASE INHIBITOR Abdul Mun’im*, M Gama Ramadhan, Atiek Soemiati

Faculty of Pharmacy, University of Indonesia, West Java, Indonesia *Corresponding Author Email: munim@farmasi.ui.ac.id

Article Received on: 10/03/13 Revised on: 09/04/13 Approved for publication: 11/05/13

DOI: 10.7897/2230-8407.04526 IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com © All rights reserved. ABSTRACT Alpha glucosidase inhibitor is one of therapeutic approaches for diabetes mellitus which is known for its safety compare to other oral anti diabetic drugs. Therefore searching of α-glucosidase inhibitor from natural compounds was recently done by many researchers to find the new active compounds. Endophytic fungi have great potential as a source of α-glucosidase inhibitory compounds. This research aims to isolate the endophytic fungi from Cassia siamea Lamk. leaves and then to evaluate their α-glucosidase inhibitory activity. We successfully isolated five endophytic fungi colonies, and then each isolate was fermented and extracted with ethyl acetate and methanol. Each extract was assayed for its α-glucosidase inhibitory activity using spectrophotometry method. Nine extracts showed better α-glucosidase inhibitory than acarbose with the smallest IC50 value was 28.40 ppm. Keywords: α-glucosidase inhibitory activity, anti diabetic, Cassia siamea Lamk., endophytic fungi. INTRODUCTION Diabetes mellitus is a metabolic syndrome characterized by hyperglycemia, due to insulin secretion, insulin sensitivity or both. It can be caused by bad life style, genetics, virus, hormonal disturbance and others1. Diabetes mellitus becomes one of major health problems in Indonesia because of the increasing of diabetes prevalence in all age. A study from WHO shows that there are 8.4 million people with diabetes in Indonesia in 2000 and estimates it will be 21.3 million people with diabetes in Indonesia in 20302. This health problem challenges researchers to find an alternative therapy for diabetes mellitus. Therapy of diabetes, such as oral anti diabetes from α-glucosidase inhibitor group, promises to decrease glucose intake with low hypoglycemic effect3. Recently, the searching of natural α-glucosidase inhibitors were increasing regards it provides huge possibilities to find lead compounds4. Various medicinal plants had been investigated their hypoglycemic effect. Some of them showed good activity in vivo or in vitro experiments5. One of promising candidate is Cassia siamea Lamk belongs to Leguminosae. This plant has antihyperglycemia effect in streptozotocin induced diabetic rats6. However, the bioactive compounds haven’t been isolated. In the other hand, exploration medicinal plants as raw material to produce mass natural drug substance would decrease natural diversity if we exploited too much7. Finding another source that can answer this question is needed. Therefore, microorganism can be an alternative source of α-glucosidase inhibitor substances. Aspergillus aculeatus is proved to have good α-glucosidase inhibitory activity8. Koji and Nojirimycin isolated from some species of fungi and bacteria are well known as potential α-glucosidase inhibitors4,9. Another microorganism is endophytes which have potency to produce α-glucosidase inhibitory substance. Tan and Zou (2001) found endophytes that are able to produce similar natural substances as their hosts10. This is the main benefit of endophytes that they can substitute their host as producer of natural substances. Moreover, endophytes do not need large area as plants do to grow up and need less time in order to produce natural

substances11. In this study we report the isolation of endophytic fungi from C. siamea Lamk and the evaluation of α-glucosidase inhibitory activity from the extracts of its fermentation culture. MATERIALS AND METHODS Materials Cassia siamea Lamk. leaves were collected from University of Indonesia area, and were identified by Herbarium Bogoriensis Indonesia Institute of Science. Corn Meal Malt Agar, Potato Dextrose Agar, Water Agar, Potato Dextrose Yeast broth, Lactophenol Cotton Blue, methanol (Merck), recombinant S. cerevisae α- glucosidase (Sigma), p-Nitrophenyl α-D-glucopyranoside (Wako Pure Chemical Industry), and Acarbose (Actavis Pharmaceutical Industry, Indonesia). Endophytic Fungi Isolation The leaves of Cassia siamea Lamk were thoroughly surface sterilized by immersing them in 70% v/v ethanol solution for 3 minutes, 5.25% v/v NaOCl solution for 5 minutes, and then 70% v/v ethanol for 30 seconds. After sterilization, samples were air-dried on sterile tissue paper inside the laminar air flow chamber. Samples were inoculated on isolation media which had been added with 0.05% w/v chloramphenicol and incubated for 5-21 days in 27°C. The growing endophytic fungi were purified on PDA media and incubated for 5-7 days. The isolated endophytic fungi were transferred to PDA slant agar as working culture and stock culture. Endophytic Fungi Colonies Examination The colonies were identified based on their morphological characteristics by macroscopic and microscopic methods. Macroscopic examination method was made by observing colony morphology, diameter, color and reverse colony color. Microscopic examination was made by staining with lactophenol cotton blue at object glass and observing it under microscope.

Abdul Mun’im et al. Int. Res. J. Pharm. 2013, 4 (5)

Page 129

Table 1: Enzymatic Reaction System Scheme

Control C0 (μL) Control C1 (μL) S1 (μL) S0 (μL) Sample - - 20 20 Buffer 980 980 980 980 DMSO 20 20 - -

Substrate 500 500 500 500 Incubate 5 minutes at 37°C Enzyme - 500 500 - Na2CO3 2000 - - 2000 Incubate 15 minutes at 37°C Enzyme 500 - - 500 Na2CO3 - 2000 2000 -

Table 2: Results of isolated fungi

No Isolate code Morphology and color of colony in PDA medium 1 5.PDA.5a white undulate, reverse white milk, roseleaf-like surface, rough periphery 2 3.CMM.2b greenish brown, reverse brown, rough surface like pumice-stone, rough periphery 3 5.PDA.1b blackish green, reverse blackish green, aerial concentric 4 4.WA.3a light-yellow, reverse light-yellow, rough periphery 5 3.WA.5b white, reverse light-yellow, cotton-like surface, aerial concentric

Table 3: IC50 value of crude extract of fermentation culture

No. Isolat Extract IC50 (ppm) 1 5.PDA.5a Water 9126.20

Methanol 397.06 Ethyl acetate 28.40

2 3.CMM.2b Water 1132.87 Methanol 227.03

Ethyl acetate 262.46 3 5.PDA.1b Water 277.07

Methanol 1335.18 Ethyl acetate 243.34

4 4.WA.3a Water 1115.93 Methanol 620.18

Ethyl acetate 157.94 5 3.WA.5b Water 627.74

Methanol 416.67 Ethyl acetate 127.43

6 Acarbose 503.91

Table 4: Results of TLC pattern assessment of ethyl acetate extract from 5.PDA.5a isolate under UV 366 nm

No Solvent system Spots number Rf value 1 Hexane-ethyl acetate (7:3) 2 0.18; 0.61 2 Hexane-ethyl acetate (4:6) 3 0.14; 0.55; 0.71 3 Hexane-ethyl acetate-acetone (1:4:2) 4 0.18; 0.48; 0.80; 0.89 4 Hexane-ethyl acetate (2:8) 5 0.05; 0.18; 0.36; 0.63; 0.9

Table 5: Km value at each concentration of ethyl acetate extract of 5.PDA.5a isolate

Extract concentration Km value

No Inhibitor 1.4419 6.25 ppm 0.5112 12.5 ppm 1,0957 25 ppm 112.28

Figure 1: Isolate 5.PDA.5a from leaves of Cassia siamea Lamk at PDA medium

Figure 2: Isolate 5.PDA.1b from leaves of Cassia siamea Lamk at PDA medium

Figure 3: Isolate 3.WA.5b from leaves of Cassia siamea Lamk at PDA medium

Abdul Mun’im et al. Int. Res. J. Pharm. 2013, 4 (5)

Page 130

Figure 4: Lineweaver-Burk plot of α-glucosidase inhibition of ethyl acetate extract of 5.PDA5a isolate at 25ppm, 12.5ppm, 6.25ppm Extraction of Fermentation Culture Isolated fungi that had sporulated (aged 7-10 days) were transferred to 500 mL PDY broth in 1000 mL Erlenmeyer flask, and then shaken at 120 rpm for 7 days at 27°C. Fermentation culture was divided into two parts; first part was centrifuged at 3000 rpm for 15 minutes and the supernatant was used for α-glucosidase inhibition assay. Its biomass was mixed with methanol using vortex mixer for 5 minutes then centrifuged at 3000 rpm for 15 minutes, and the supernatant was used for α-glucosidase inhibition assay. The second part was mixed with ethyl acetate using vortex mixer for 5 min then centrifuged at 3000 rpm for 15 minutes and the supernatant was used for α-glucosidase inhibition assay. Each extract was freeze-dried for water extract and dried in vacuum oven (30º-40ºC) for methanol and ethyl acetate extract. Thin Layer Chromatography Each extract (1 mg) was dissolved in 1mL appropriate solvent. Extract solution was spotted on TLC plate (Silica F254) and eluted with hexane-ethyl acetate in various combination ratio. After elution, the plates were sprayed by10% H2SO4 solution. α-Glucosidase Inhibitory Activity Assay Enzyme solution (750 units) was diluted in phosphate buffer (pH 7) containing 0.2% bovine serum albumin until final concentration 0.05 unit/mL. Extracts were dissolved in DMSO, and then diluted in the phosphate buffer12. The assay mixture containing 20 μL sample solution was added with 980 µL phosphate buffer pH 7 and 500 µL 20 mM p-Nitrophenyl α-D-glucopyranoside (PNP) as substrate, then incubated for 5 minutes. Enzyme solutions (500 µL) which has diluted was added to mixture and incubated for 15 minutes. Enzymatic reaction is stopped by adding 2000 µL Na2CO3 (200mM). The absorbance of assay mixture was measured at 400 nm. The complete enzymatic reaction scheme can be seen as in Table 1. Samples were water extract, methanol extract, and ethyl acetate extract. Each extract was assayed with triple repetition. Inhibition percentage was calculated with equation;

In which S is absorbance of S1-S0 and C is absorbance of C1-C0. IC50 can be calculated by using linier regression, with formula y = a + bx, where Y is inhibition percentage and X is sample concentration, then the equation;

Enzyme Kinetic Assay Substrates PNP with increasing concentrations were added to assay solutions at three different extract concentrations, and then measured at 400 nm9. Inhibition mode was determined by using Lineweaver-Burk plot calculated from Michaelis-Menten kinetic equations. RESULTS AND DISCUSSION In this study 5 fungi colonies were isolated from leaves of Cassia siamea Lamk. The endophytic fungi had characteristic: (1) growing time within 5-21 days; (2) growing around the leaves; and (3) it has different morphologies from the fungi in control Petri plate (Figure 1-3). Results of isolated fungi can be seen in Table 2. Endophytic fungi is defined as fungal microorganism which spent the whole or part of their life cycle colonizing inter- and/or intra-cellularly inside the tissue of host plant10. The symbiosis between endophytic fungi and their host is commonly mutualism or commensalism, even though in unbalance plant-endophytic fungi relationship, the symbiosis possibly turns to parasitism13. Endophytic fungi can be isolated from tissue of host plant and cultivated on appropriate agar medium. Endophytic fungi are slow-growing micro organism, therefore they need appropriate media and optimum incubation condition15. Malt extract agar is commonly used as media with or without antibiotic supplement to prevent bacteria growth. To reduce contamination, in the experiment weak medium, such as water agar can be used14. Fermentation of endophytic fungi was made in PDY media which supply source of carbon and nitrogen. Using culture broth as fermentation medium provides benefit which is multiplication of cell number which can lead to optimize production of fungi metabolites. Some metabolites are excreted into the broth culture whilst some others are found in mycelium15. Both culture broth and mycelium need to be extracted with appropriate solvent to

Abdul Mun’im et al. Int. Res. J. Pharm. 2013, 4 (5)

Page 131

obtain the metabolites. This study is a preliminary screening of α-glucosidase inhibitor from crude extract from fermentation culture of endophytic fungi of Cassia siamea Lamk. Leaves we used α-glucosidase from recombinant S. cerevisae and acarbose as α-glucosidase inhibitor standard. The principle reaction of this assay is hydrolysis of p-Nitrophenyl α-D-glucopyranoside as substrate by α-glucosidase to produce glucose and p-nitrophenol with yellow color12. The absorbance of reaction mixture was measured by spectrophotometer UV-Vis. In this assay, we found 9 extract with IC50 value less than that acarbose (Table 3). Crude extract of ethyl acetate from 5.PDA.5a isolate showed the strongest α-glucosidase inhibitory activity with IC50 of 28.4 ppm. All ethyl acetate fractions had IC50 lower than that acarbose, methanolic extracts mostly showed similar phenomenon. Otherwise, only one of water extracts stronger α -glusidase inhibitory than acarbose. It was probably because most active compounds were semi polar compounds. So they were more soluble in ethylacetate. These had the semilar result as Sugiwati, et al (2009) and Dewi, et al (2007) studies. They isolated α -glucosidase inhibitors from ethyl acetate fraction of A. terreus9, 12. In this study, ethyl acetate fraction showed stronger α -glucosidase inhibitory activity than that the commercial α-glucosidase, acarbose. It’s probably because acarbose had low activity against α-glucosidase from microorganism rather than from mammals15. Moreover, synergic effects from compounds in the extracts could have a role in their potential activity15. Thin layer chromatography was performed on extract which showed the strongest α -glucosidase inhibitory activity. TLC chromatogram of ethyl ethyl acetate extract of 5. PDA. 5a showed some spots with hexane-ethyl acetate as developing solvent system. Based on the TLC profile hexane-ethyl acetate (2:8) is optimum mobile phase. In this system were detected 5 spots (Table 4). Chemical identification did not performed in this study due to limited amount of samples. This study also investigated inhibition mode of sample by using Lineweaver-Burk plot. We investigated only the most potent extracts in three concentrations which were 25ppm, 12.5ppm and 6.25ppm, also in absence of the extract (NI). Lineweaver-Burk plot showed mixed inhibition type which is characterized by combination of competitive and non competitive inhibitor (Figure 4). Constanta of Michaels (Km) also showed mixed inhibition type. If we compare Km values from three different sample concentrations with Km values of NI, we will find that sample at concentration 12.5ppm had the same Km value as NI which is nature of noncompetitive inhibitor (Table 5). Otherwise, sample at concentration 25ppm had higher Km value than NI which is nature of competitive inhibitor 15. This phenomenon may be due to the extract contained many compounds with different inhibition mode. Endophytic fungi from the leaves of Cassia siamea have great potency as a source of α-glucosidase inhibitory compounds. Further study is needed to isolate the α-glucosidase inhibitors and elucidate their structures.

CONCLUSION We succeeded in isolating five endophytic fungi colonies from leaves of Cassia siamea. In the α-glucosidase inhibitory activity assay, ethyl acetate extract exhibited stronger activity than acarbose. The most potent α-glucosidase inhibitor was demonstrated by ethyl acetate extract of 5.PDA.5a fermentation culture with IC50 value of 28.40ppm. Mode of inhibition of 5.PDA.5a ethyl acetate was mix inhibition. ACKNOWLEDGEMENTS The authors thank Directorate General of Higher Education, Ministry of National Education, and Republic of Indonesia for financial support of this study via Riset Madya UI 2012 Scheme. REFERENCES 1. Corwin EJ. Handbook of pathophysiology. Philadelphia: Lippincott

Williams & Wilkins; 2008. 2. Wild S, G Roglid, A Green, R Sicree and H King. Global prevalence of

diabetes. Diabetes Care 2004; 27(5): 1047-1053. http://dx.doi.org /10.2337/diacare.27.5.1047 PMid:15111519

3. Hanefeld M. Alpha-glucosidase inhibitors. In B. J. Goldstein dan D. Muller-Wieland (Ed.). Type 2 diabetes principles and practice second edition. New York: Informa Healthcare USA Inc; 2008. p. 27-43

4. Borges de Melo E, AS Gomes and I Carvalho. α- and β-glucosidase inhibitors: chemical structure and biological activity. Tetrahedron. 2006; 62: 10277-10302. http://dx.doi.org/10.1016/j.tet.2006.08.055

5. Bnouham M, A Ziyyat, H Mekhfi, A Tahri and A Leggsyer. Medicinal plants with potential antidiabetic activity – a review of ten years of herbal medicine research (1999-2000). International Journal of Diabetes Metabolism. 2006; 14: 1-25.

6. Kumar S, V Kumar and O Prakash. Antidiabetic and anti-lipemic effects of Cassia siamea leaves extract in streptozotocin induced diabetic rats. Asian Pasific Journal of Tropical Medicine, 2010; 871-873. PMCid:2916039

7. Strobel G, B Daisy, U Castillo and J Harper. Natural products from endophytic microorganism. Journal of Natural Products 2004; 67: 257-268. http://dx.doi.org/10.1021/np030397v PMid:14987067

8. Ingavat N, J Dobereiner, S Wiyakrutta, C Mahidol, S Ruchirawat and P Kittakoop. Aspergillusol A, an α-glucosidase inhibitor from marine-derived fungus Aspergillusaculeatus. Journal of Natural Products 2009; 72: 2049-2052. http://dx.doi.org/10.1021/np9003883 PMid:19824618

9. Dewi RT, YM Iskandar, M Hanafi, LBS Kardono, M Angelina, ID Dewijanti and SDS Banjarnahor. Inhibitory effect of koji Aspergillus terreus on α-glucosidase activity and postprandial hyperglycemia. Pakistan Journal of Biolological Sciences. 2007; 10(8): 3131-3135. PMid:19090111

10. Tan RX and WX Zou. Endophytes: a rich source of functional metabolite. National Product Reports 2001; 18: 448-459. http:/ dx.doi.org/10.1039/b100918o PMid:11548053

11. Strobel G and B Daisy. Bioprospecting for microbial endophytes and their natural products. Microbiology and Molecular Biology Review 2003; 67(4): 491-502. http://dx.doi.org/10.1128/MMBR.67.4.491-502.2003 PMid:14665674 PMCid:309047

12. Sugiwati S, S Setiasih and E Afifah. Antihyperglicemic activity of the mahkota dewa [Phaleriamacrocarpa (Scheff.) Boerl.] leaf extracts as an alpha glucosidase inhibitor. Makara Kesehatan 2009; 13(2): 74-78.

13. Kogel K, P Franken, R Huckelhoven. Endophyte or parasite – what decides? Current Opinion Plant Biology. 2006; 9: 358-363. http:// dx.doi.org/10.1016/j.pbi.2006.05.001 PMid:16713330

14. Stone JK, JD Polishook, JF Whitte. Endophytic fungi. In MS Foster, GF Bills, GM Mueller. Biodiversity of fungi inventory and monitoring methods. Burlintong: Elsevier Academic Press; 2004. http://dx.doi.org/ 10.1016/B978-012509551-8/50015-5

15. Hanson JR. The Chemistry of Fungi. Cambridge: RCS Publishing; 2008. Cite this article as: Abdul Mun’im, M Gama Ramadhan, Atiek Soemiati. Screening of endophytic fungi from Cassia siamea Lamk leaves as α-glucosidase inhibitor. Int. Res. J. Pharm. 2013; 4(5):128-131

Source of support: Nil, Conflict of interest: None Declared