Ambika Vasantakumar, G. M. Vidyasagar*
Medicinal Plants and Microbiology Research Laboratory, Department of P G Studies and Research in Botany, Gulbarga University, Kalaburagi-585106, Karnataka, India.
*Address for Corresponding Author
G. M. Vidyasagar
Medicinal Plants and Microbiology Research Laboratory,Department of P G Studies and Research in Botany, Gulbarga University, Kalaburagi-585106, Karnataka, India.
Abstract
Objective: Endophytes act as major reservoir of biologically active compounds for health care applications and an increasing number of novel bioactive compounds are being extracted from fungal endophytes.so the present study is isolation and identification of endophytic fungi from Gymnema sylvestre. Material and methods: Fresh leaves and stem were washed thoroughly in running water and with distilled water to remove dust and debris. The material was surface sterilized by mercury chloride. The leaf segments were placed in petri dishes (2–3 segments/petri dish) containing Potato Dextrose Agar (PDA) medium. The petri dishes were sealed using parafilm and incubated at 26±2 °C in the light chamber for 3 weeks. The fungi grown were transferred to PDA plate and identified based on their morphology and other features. Results: A total of 60 endophytic isolates were isolated from Gymnema sylvestre and among them 10 endophytes belonging to seven genera were identified. The other fungal isolates need further efforts to identify. Conclusion: A total of 10 endophytic fungi belongs to seven genera were isolated and identified from Gymnema sylvestre and they may be very much useful in the extraction of potent active compounds that the plant is known far. The study may be useful in protecting the highly useful medicinal plant by giving the alternate source for drug.
Keywords: Gymnema sylvestre , endophytes
Introduction
Gymnema sylvestre R.Br is a medicinal herb native to central and western India, tropical Africa and Australia. It is often called “gurmar” (destroyer of sugar), as chewing the leaves causes a loss of sweet taste (Yeh et al., 2003). G. sylvestre containing the triterpenoid gymnemagenin and Gymnemic acid, extracted from leaves and roots is mainly used in India and parts of Asia as a natural treatment for diabetes as it helps to lower the blood sugar(Duke et al., 1997). In addition, it possesses antimicrobial, antihyper cholesterolemic (Bishayee et al., 1994), sweet suppressing (Kurihara et al., 1992) and hepatoprotective (Rana et al., 1992) activities. It also acts as feeding deterrents to caterpillar, prodenia eridania (Granich et al., 1974), prevent dental caries caused by Streptococcus mutans (Hiji et al., 1990) and skin cosmetics (Komalavalli et al., 2000). Besides, it is also used in the treatment of asthma, eye complaints, inflammations, snake bite (Kini et al., 1982). Gymnemagenin, the wonder drug for diabetes (Liu et al., 1992,) and obesity (Kanetkar et al 2007), also possess antiviral properties (Sinsheimer et al., 1971). They are being isolated from the leaves and roots of Gymnema sylvestre, which leads to the destruction of natural resources. Alternatively, gymnemagenin-producing endophytic fungi Penicillium oxalicum is isolated from Gymnema sylvestre, so the fungus can serve as a potential material for the production of gymnemagenin. As G. sylvestre has been used in the treatment of several ailments, it is assumed to have several active compounds such as, Betaine, Beta-Carotene, Choline, Gymnemic-Acid, Niacin, Ascorbic-Acid. Hence the present study was carried out to identify the fungal endophytes capable of producing the active compounds reported from G. sylvestre.
Figure 1. Gymnema sylvestre
Materials and methods
Collection of the plant material
G. sylvestre collected from Botanical garden of Gulbarga University, Kalaburagi, Karnataka and was authenticated with the help of flora of Gulbarga district (Seetharam et al., 2000). A specimen is deposited in the herbarium, Dept. of Botany, Gulbarga University Kalaburagi (Voucher speciemen No HGUG-58).
Isolation and identification of endophytic fungi
Fresh leaves and stem were washed thoroughly in running water and with distilled water to remove dust and debris. The material was surface sterilized by mercury chloride. Leaves and stem samples were cut into 2–3 cm segments under aseptic conditions, The leaf segments were placed in petri dishes (2–3 segments/petri dish) containing Potato Dextrose Agar (PDA) medium. The petri dishes were sealed using parafilm and incubated at 26±2 °C in the light chamber for 3 weeks. The plates were monitored every day to check the growth of endophytic fungal colonies on leaf segments. The fungi grown were transferred to PDA plate and identified based on their morphology and other features.
Results and discussion
Isolation and identification of endophytic fungi
In the present study, a total of 60 isolates were isolated from the leaf and stem materials of G. sylvestre. Of which, ten fungal species belonging to seven genera were isolated and identified. Cylindrocladium parvum, Cladosporium variabile, cochliobolus geniculatus were recorded during September 2015, Alternaria brassicicola, Colletotrichum capsici, colletotrichum dematium were recovered during August and Aspergillus fumigates, Aspergillus niger, Fusarium oxysporum and Alternaria alternate were recorded during February month of 2015 (Figure 2).Aspergillus niger and Alternaria alternata were also reported from Calotropis gigantean (Srimathi et al., 2011) Calotropis procera (Rezwana et al., 2007), Withania somnifera (Rezwana et al., 2010), Azadirachta indica (Tenguria et al., 2011) and Melia azadirachta (Kaushal et al., 2010). Ascomycota forms have been reported as endophytes (Blodgett et al., 2000; Suryanarayanan et al., 1998) and natural products from endophytes have a broad spectrum of biological activity (Zhang et al., 2006).
Identification of endophytes
Endophytic fungi were identified according to their microscopic and macroscopic characteristics such as the morphology of fruiting structures and spore morphology under a bright-field microscope (10X and 40X) using Motic image microscope. Fungal features have been recorded with the help of a hand book of soil fungi by Nagamani (Nagamani et al., 2006) and illustrated genera of imperfect fungi by Barnet HL and Hunter BB (Barnet et al., 1972).
Table 1. Fungal endophytes isolated from leaf and stem parts of Gymnema sylvestre
|
S. No. |
Name of the endophyte |
Plant part used |
Growth in diameter |
|
1 |
Cylindrocladium parvum |
Stem |
2.5 |
|
2 |
cladosporium variabile |
Stem |
2 |
|
3 |
cochliobolus geniculatus |
Leaf |
4.32 |
|
4 |
Alternaria brassicicola |
Leaf |
3.72 |
|
5 |
Colletotrichum capsici |
Leaf |
3.9 |
|
6 |
colletotrichum dematium |
Stem |
2.38 |
|
7 |
Aspergillus fumigates |
Stem |
4.16 |
|
8 |
Aspergillus niger |
Stem |
2.03 |
|
9 |
Fusarium oxysporum |
Stem |
2.06 |
|
10 |
Alternaria alternata |
Stem |
3.27 |
Figgure 2. Endophytic fungi isolated from Gymnema sylvestre: (a) Cylindrocladium parvum, (b) Cladosporium variabile, (c) Cochliobolus geniculatus, (d) Alternaria brassicicola, (e) Colletotrichum capsici, (f) Colletotrichum dematium, (g) Aspergillus fumigates, (h) Aspergillus niger, (i) Fusarium oxysporum, (j) Alternaria alternate
Conclusion
A total of 10 endophytic fungi belongs to seven genera were isolated and identified from Gymnema sylvestre and they may be very much useful in the extraction of potent active compounds that the plant is known far. The study may be useful in protecting the highly useful medicinal plant by giving the alternate source for drug.
Conflicts of interest: Nil
References
Barnet HL, Hunter BB. 1972. Illustrated genera of imperfect fungi 3rd edn. Burgess publishing company, USA.
Bishayee A, Malay C. 1994. Hypolipidaemic and antiatherosclerotic effects of oral Gymnema sylvestre R.Br. Leaf extract in albino Rats fedon high fat diet. Journal of Phytotherapy Research, 8(2):118-120.
Blodgett JT, Swart WJ, Louw SV, Weeks WJ. 2000. Species composition of endophytic fungi in Amaranthus hybrid leaves, petioles, stems, and roots. Journal of Mycologia, 92(5):853-859.
Duke, James A, Japnes PM, Danny B, Jony D, Bully P. 1997. The Green Pharmacy. Emmaus, Pennsylvania:Roale Press, Inc. 1997.
Gloria Yeh, David M, Eisenberg, Ted J, Kaptchuk,Russell S. Phillips. 2003.Systematic Review of Hers and Dietary Supplements supplements for Glycemic Contrl in Diabetes. Diabetes Care, 26(4):1277-1294.
GranichMark S, Halpern P, EisnerThomas. 1974. Gymnemic acids: Secondary plant substances of dual defensive action. Journal of Insect Physiology, 20(2):435-439.
Hiji Yasutake. 1990. Cariostatic materials and foods, and method for Preventing dental caries.United States Patent, Patent No.4:912089.
Kanetkar PV, Singhal RS, Kamat MY. 2007. Gymnema sylvestre: a memoir. Recent advances in Indian herbal drug research. Journal of Clinical Biochemistry and Nutrition, 41:77–81.
Kaushal KS, Rao DV, Batra A. 2010. Morphological study of endophytic fungi inhabiting leaves of MeliaazedarachL. International Journal of Pharmaceutical Sciences Review and Research, 5(3):177-180.
Kini RM, Gowda TV. 1982. Studies on sanke venom enzymes: Part I purification of ATPase, a toxic component of Naja naja venom and its inhibition by potassium gymnemate. Indian Journal of Biochemistry & Biophysics, 22(2):152-154.
Kini RM, Gowda TV. 1982. Studies on sanke venom enzymes: Part II Partial characterization of ATPase from Russell’s viper (Vipera russelli) venom and their interaction with potassium gymnemate. Indian Journal of Biochemistry & Biophysics, 19(5):342-46.
Komalavalli N, Rao MV. 2000. In vitro micropropagation of Gymnema sylvestre -A multipurpose medicinal plant. Plant Cell, Tissue and Organ Culture, 6:97-105.
Kurihara Y. 1992. Characteristics of antisweet substances, sweet proteins, and sweetness inducing proteins. Critical Reviews in Food Science and Nutrition, 32(3):231-52.
Liu HM, Kiuchi F, Tsuda Y. 1992. Isolation and structure elucidation of gymnemic acids, anti-sweet principles of Gymnema sylvestre. Chemical and Pharmaceutical Bulletin, 40:1366–1375.
Nagamani K, Unwar IK, Manoharachary CA. 2006. A hand book of soil fungi I K International Pvt. Ltd.
Rana AC, Avadhoot Y. 1992. Experimental evaluation of hepatoprotective activity of Gymnema sylvestre and Curcum Zedoaria. Fitoterapia, 63:60.
Rezwana K, Saleem S, Choudhary MI, Shakeel AK, Aqeel A. 2007. Biodiversity of the endophytic fungi isolated from Calotropisprocera. Pakistan journal of Botany, 39(6):2233- 2239.
Rezwana K, Saleem S, Choudhary MI, Shakeel AK, Aqeel A. 2010.Communities of endophytic fungi In Medicinal plant Withania somnifera. Pakistan Journal of Botany, 42(2): 1281-1287.
Seetharam YN, Kotresha K, Uplaonkar SB. 2000. Flora of Gulbarga District, Gulbarga university Kalburgi First edition.
Sinsheimer JE, Subbarao G. 1971. Constituents from Gymnema sylvestre VIII: Isolation, Chemistry and Derivatives of gymnemagenimn and gymnemastrogenin. Journal of Pharmaceutical Sciences, 60:190-193.
Srimathi S, Indrakumar I, Johnpaul M. 2011. Biodiversity of the endophytic fungi isolated from Calotropisgigantea (L.) R. BR. Recent Research in Science and Technology, 3(4):94-100.
Suryanarayanan TS, Kumaresan V, Johnson JA. 1998. Foliar endophytes from two spies of mangrove Rhizophora. Canadian journal of Microbiology, 44:1003-1006.
Tenguria RK, Firoz NK. 2011. Distribution Of Endophytic Fungi In Leaves Of Azadirachta indica A. JUSS. (Neem) of Panchmarhi Biosphere Reserve. Current Botany, 2(2):27-29.
Zhang HW, Song YC, Tan RX. 2006. Biology and chemistry of endophytes Journal of Natural product Reports, 23:753-771.
