Potential Endophytic Microbes Selection for Antidiabetic Bioactive Compounds Production

MATERIALS AND METHODS

Research material: Research materials are 6 species of well known Indonesian medicinal plants for antidiabetic, i.e., Mahoni (Swietenia mahagoni Jacq.), Sambiloto (Andrographis paniculata Ness.), Kumis kucing (Orthosiphon spicatus BBS), Mengkudu (Morinda citrifolia L.), Sirih merah (Piper crocatum L.) and Sirih hitam (Piper ornatum sp.) collected from Bogor and Bandung areas. This research was conducted during February-October 2010.

Isolation and purification of endophytic microbes: Plant’s stem in 2 cm for length and 1 cm for diameter were washed by water followed by sterilization using ethanol 70% for 1 min and ethanol 75% for 30 sec. Stems were cut longitudinally into 2 pieces then were placed on petri disc filled with Corn Meal Malt Agar (CMMA) mixed with 0.05 mg mL^-1 chloramphenicol (Theantana et al., 2007). Others were placed on Potato Dextrose Agar (PDA) mixed with 0.05 mg mL^-1 chloramphenicol (Croizer et al., 2006). Those agars were incubated at 25°C for 3 days. Purification were done by transferring colony to fresh PDA followed by incubation at 25°C for few times until we got pure colony. Pure colony are kept on slanted PDA in the refrigerator (-80°C) for further treatment.

Production of endophytic microbes bioactive compounds: Production and fermentation of endophytic microbes chemical compounds were done by cutting 1 week old endophytic microbe colony in medium agar using cork borer tobe inoculum disc with diameter of 1.8 cm. Inoculum was inoculated into 250 Potato Dextrose Broth (PDB) medium in Erlenmeyer flask size 1000 mL and fermented for 14 days with agitation 150 rpm at room temperature (Onifade, 2007). Fermented fungi were extracted using ethylacetate and filtrated using Whatman filter paper number 41 until we got filtrate and biomass of fungi. Filtrate collected was evaporated using evaporator and were dried using oven at 40°C. Dried biomass was extracted again using ethylacetate to get the extract (Sunil et al., 2009).

In vitro antidiabetic activity test of active compounds: Screening was done to get isolates which can produce a chemical compound with inhibitory activity to α-glucosidase enzyme. In this screening, we were having test to all ethylacetate extracts of endophytic fungi (both filtrate and biomass extracts). As control we used quercetin, a well known flavonoid compound which have inhibitory activity to α-glucosidase enzyme. All samples and standard solution were measured at concentration of 50 ppm (Suarsana et al., 2008).

In vitro antidiabetic activity test were done using α-glucosidase method (Saijyo et al., 2008). Inhibition capability were calculated using formula:

where, S showed sample absorbance and C showed blank absorbance with quercetin solution as standard.

Thin layer chromatography test: Both filtrate and biomass ethylacetate extract of endophyte isolates were tested by thin layer chromatography analysis using eluent chloroform-methanol (5:1) and silica gel GF254 as solid phase. Spray reagent used is Serium sulphate.

RESULTS AND DISCUSSION

Isolation of endophytic fungi: Isolation and purification of endophytic fungi from Mahoni (Swietenia mahagoni Jacq.), Sambiloto (Andrographis paniculata Ness.), Kumis kucing (Orthosiphon spicatus BBS), Mengkudu (Morinda citrifolia L.), Sirih merah (Piper crocatum L.) and Sirih hitam (Piper ornatum sp.) gave results 45 fungi isolates. Complete results of isolation can be seen in Table 1.

These endophytic isolates were extracted with ethylacetate and produced 45 sample of ethylacetate filtrate and 45 sample of ethylacetate biomass.

Screening of endophytic fungi isolates using α-glucosidase test: α-glucosidase is an enzyme in intestine which can hydrolyze carbohydrate into simple sugar (glucose). Compound which can inhibit this enzyme activity will be very potential to be an antidiabetic drug since it can decrease blood sugar level by slowing the absorption of carbohydrate post-prandial. The results of the screening were summarized in Table 2.

In this test, we compared absorbancy of standard with samples. DMSO solution was used as blank solution while quercetin as standard. Absorbancy value of DMSO solution 0.1183 and absorbancy value of quercetin solution 0.1007. So, Inhibition percentage of standard 14.88%.

Table 1:	Endophytic fungi isolated from some indonesian antidiabetic plants
Notation A is refers to endophytic fungi isolates planted on CMM (Corn Meal Medium), Notation B is refers to endophytic fungi isolates planted on PDA (Potato Dextrose Agar)

Table 2:	The results of the screening of endophytic fungi isolates using α-glucosidase test
Absorbancy of DMSO solution 0.118, absorbancy of quercetin solution 0.1007:

Highest inhibition percentage against α-glucosidase enzyme was showed by biomass extract of fungus B.Sm.1F from Mahoni (Swietenia mahagoni Jacq.) plant which gave 98.84% inhibition while its filtrate extract only gave moderate inhibition percentage at 63.40%. On the contrary, fungus B.Sm.3F gave 93.91% inhibition percentage from its filtrate extract while its biomass extract only gave 51.48% inhibition. The chosen potential fungus is expected to have high inhibition percentage from both biomass and filtrate extracts.

From 9 endophytic fungi obtained from Sambiloto (Andrographis paniculata Ness.) there are 3 fungi which have high inhibition percentage both from biomass and filtrate extracts. They are A.Ap.3F (98.84 and 81.40%), A.Ap.4F (96.87 and 81.40%) and B.Ap.1F (98.48 and 87.49%).

Endophytic fungus B.Os.1F from Kumis kucing (Orthosiphon spicatus BBS) gave highest inhibition percentage compared to 7 other fungi obtained from this plant. It gave 93.91% from filtrate extract and 89.01% from biomass extract.

Most endophytic fungi obtained from Mengkudu (Morinda citrifolia L.) have moderate inhibition percentage. From 5 fungi, there are 2 potential fungi, i.e., A.Mc.2F with 89.77% inhibition for its filtrate extract and 30.94% for biomass extract and B.Mc.3F (89.77 and 72.78%).

From 7 endophytic fungi obtained from Sirih Merah (Piper crocatum L.) there are 3 fungi which have high inhibition percentage both from biomass and filtrate extracts. They are A.Pc.1F (87.66 and 82.08%), B.Pc.1F (85.63 and 89.35%) and B.Pc.2F (83.51 and 87.57%).

Most endophytic fungi obtained from Sirih Hitam (Piper ornatum L.) have low to moderate inhibition percentage (28-80%). From 9 fungi, only fungus A.Ps.1F which gave quite high inhibition percentage. Its biomass extract gave 80.22% inhibition while filtrate extract gave 55.45%.

Suarsana et al. (2008) obtained antidiabetic activity of quercetin standard using acarbose at concentration 50 ppm at 15.33%. In this research, antidiabetic activity of quercetin standard was 14.88%.

Screening to endophytic fungi from 6 Indonesian medicinal plants using α-glucosidase method resulted in 7 potential endophytic fungi with high inhibition percentage both from their filtrate and biomass extracts. They are A.Ap.3F, A.Ap.4F and B.Ap.1F from Sambiloto (Andrographis paniculata Ness.), B.Os.1F from Kumis Kucing (Orthosiphon spicatus BBS), A.Pc.1F, B.Pc.1F and B.Pc.2F from Sirih Merah (Piper crocatum L). Among those 7 potential fungi, A.Ap.1F has highest inhibition percentage. In a study conducted by Suarsana and his friends obtained value of tempeh extract on the inhibition of α-glucosidase enzyme of 11.89% (Suarsana et al., 2008). Several compounds that can inhibit the enzyme α-glucosidase has been obtained by Saijyo et al. (2008) in the research by using ethanol from plant extracts bergenia ligulata, such as compound 1 inhibited 57.5% of α-glucosidase activity at a concentration of 0.25 mM and the ID₅₀ (50% inhibition dose) value was 0.1 mM and Compound 5 inhibited 90.4% of α-glucosidase activity at a concentration of 0.25 mM and the ID₅₀ (50% inhibition dose) value was 0.05 mM. In this study, 7 potential endophytic fungi provides inhibition values ranging between 80 to 98%.

CONCLUSION

Isolation and purification of endophytic fungi from 6 Indonesian medicinal plants obtained 45 fungi isolates. Mahoni (Swietenia mahagoni Jacq.) gave 7 isolates, Sambiloto (Andrographis paniculata Ness.) 9 isolates, Kumis kucing (Orthosiphon spicatus BBS) 8 isolates, Mengkudu (Morinda citrifolia L.) 5 isolates, Sirih merah (Piper crocatum L.) 7 isolates and Sirih hitam (Piper ornatum sp.) gave 9 isolates.

Screening procedure using Thin Layer Chromatography (TLC) test and α-glucosidase test resulted in 7 potential endophytic fungi with antidiabetic activity both from their filtrate and biomass extracts. They are A.Ap.3F, A.Ap.4F and B.Ap.1F from Sambiloto (Andrographis paniculata Ness.), B.Os.1F from Kumis Kucing (Orthosiphon spicatus BBS), A.Pc.1F, B.Pc.1F and B.Pc.2F from Sirih Merah (Piper crocatum L.).

ACKNOWLEDGMENT

The writer would like to thanks Research Center for Biotechnology Indonesian Institute of Sciences (LIPI) for their help in this research.