INT J CURR SCI 2013, 7: E 118-127
RESEARCH ARTICLE
ISSN 2250-1770
Studies on endophytic fungi of ayurvedic medicinal plant Gymnema sylvestre
*Nithyananda Sastry Darbhaa and Sucheta Shivaji Tikoleb
a
Department of Pharmaceutical Biotechnology, KLE University’s College of Pharmacy, Belgaum, Karnataka, India
b
Department of Pharmaceutics, MSS’S College of pharmacy, Medha, Satara, Maharastra, India
*Corresponding author: [email protected]; Phone: 0091-831-247139; Fax: 0091-831-2472777
Abstract
Antioxidants are considered to be important dietary supplements and promising therapeutic agents investigated and
shown to have beneficial effects for treatment of reactive oxygen species linked diseases. Metabolites isolated from
medicinal plants and their endophytes have been reported to be a potential source of natural antioxidants. Plant extract of a
well known medicinal plant used in ayurvedic and homeopathic system Gymnema sylvestre belonging to family
Asclepediacea was shown to exhibit high antioxidant activity. In the present study, a total 18 endophytic fungi were isolated
from leaves, stem, and roots of this plant which were identified on the basis of morphological, microscopical and
biochemical characteristics. Fermentation studies were carried out. Cell-free broths were extracted with ethyl acetate after
fermentation. TLC profiles of ethyl acetate extract of fermented extract were carried out in comparison with the native plant
extract.TLC profile of 3 fungal extracts of Cladosporium sp. (GSEF-06), Mycelia sterilia sp. (GSEF-08), Aspergillus sp.
(GSEF-14) indicated spots of comparable Rf value with standard drug and native plant extract indicating possible presence
of similar secondary metabolites of plant into these endophytic fungal extracts. Extracts were also tested for antimicrobial,
antioxidant and siderophore activity. Cladosporium sp., Mycelia sterilia and Acremonium sp (GSEF-09) were found to
possess relatively higher antibacterial activity while Cladosporium sp., Mycelia sterilia and Curvularia sp. (GSEF-15) were
found to possess antifungal activities. Mycelia sterilia and Aspergillus sp. were found to possess relatively higher antioxidant
activity and three strains GSEF-01, GSEF-09, and GSEF-11 were found to secrete pigments under solid state and submerged
conditions.
Keywords: Gymnema sylvestre, endophytic fungi, antimicrobial activity, antioxidant activity
Received: 18th April; Revised: 06thMay; Accepted: 14thJune; © IJCS New Liberty Group 2013
Introduction
considered to be good sources of natural antioxidants.
Antioxidants are considered to be important dietary
However metabolites from endophytes, microorganisms
supplements and potential therapeutic agents which are
living within plant tissues have also been reported to be a
shown to have beneficial effects for treatment of reactive
potential source of novel natural antioxidants (Huang et al.,
oxygen species linked diseases such as cancer, diabetes
2007; Mariana et al., 2011; Sadananda et al., 2011)
mellitus, rheumatoid arthritis, ischemia, cardiovascular
Endophytes have symbiotic association with their hosts.
diseases and neurodegenerative diseases (Halliwell, 1994;
Because of their association and possible inter-generic
Packer and Ong, 1997; Valko et al., 2007). A variety of
exchange of genetic information, they may have the ability
fruits, vegetables and medicinal plants in general are
to produce the same or similar bioactive compounds as
Nithyananda et al., 2013
those originated from their host plants (Strobel, 2002).
tomentosa was investigated by Srinivasan and coworkers.
Isolation of bioactive metabolites from endophytes known
They claim ethanolic extract of Phyllosticta sp. Contains
to
anticancer
significant amount of phenols and flavanoid (Srinivasan
compounds is reported in literature (Strobel et al., 1996;
et al., 2010). Sadananda and coworkers investigated
Wiyakrutta et al., 2004; Ramasamy et al., 2010).
antimicrobial and antioxidant activities of endophytes
possess
antimicrobial,
antiviral
and
A significant number of interesting bioactive
isolated from Tabebuia argentea. They claim that phenolic
molecules have been found and reported to be produced by
acids, flavonoids and Phenolic terpenoids and certain
endophytes which include antibiotics, alkaloids, steroids,
volatile and aliphatic constituents identified in their study
terpenoids,
present study might be responsible to total antioxidant
isocoumarins,
phenylpropanoids,
lignans,
quinones,
peptides,
flavonoids,
phenolics,
and
properties of the host plant and the isolated endophytic
volatile organic compounds (Tan and Zou, 2001; Strobel
fungi
(Sadananda,
2011).
and Daisy, 2003). This triggers the expectation some
Graphislactone A, was isolated from the endophytic fungus
metabolites of the endophytes isolated from medicinal
Cephalosporium
plants known to possess antioxidant activity may yield
Trachelospermum jasminoides was shown to have free
known and novel natural antioxidants (Harper et al., 2003;
radical-scavenging and antioxidant activities in vitro than
Huang et al., 2007). Liu and coworkers claim methanolic
butylated hydroxytoluene (BHT) and ascorbic acid (Song
extract of an endophytic Xylaria sp. isolated from the
et al., 2005).
sp.
A
phenolic
IFB-E001
metabolite
obtained
from
medicinal plant Ginkgo biloba to have strong antioxidant
Gymnema sylvestre, a well known medicinal plant
activity due to the presence of phenolic and flavonoid
belonging to family Asclepiadaceae is used for over 3000
compounds (Liu et al., 2007). Similarly Huang and
years in ayurvedic, folk and homeopathic and other
coworkers isolated endophytic fungal cultures from
indigenous medical systems. It is also referred to as gur-
Chinese medicinal plants and have investigated the
mar, Madhunashini or mesasrngi. It is a slow growing
antioxidant activity of their metabolites.They claim that the
perennial, woody, climbing plant native to central and
phenolic metabolites as the major antioxidant constituents
peninsular India, well distributed throughout Asia,
(Huang et al., 2007).
Australia, Tropical Africa, Malaysia and Srilanka (The
Antioxidant activity, antimycotic and antifungal
activities
of
metabolites
pestacin
and
Wealth of India, 2005; The Ayurvedic Pharmacopoeia of
isopestacin
India, 2006). Both the dried leaf and dried root are used
structurally similar to the flavonoids obtained from the
therapeutically. The leaves of this plant have sweet taste
endophytic fungus Pestalotiopsis microspora isolated from
suppressing activity were shown to have an excellent effect
a plant growing in the Papua New Guinea, Terminalia
in controlling hyperglycemia of both types 1 and 2 diabetic
morobensis have been investigated by (Harper et al., 2003)
patients (Shanmugasundaram et al., 1990; Baskaran
they believe Pestacin is a more powerful antioxidant a
et al.,1990). Extracts of this plant are used in treatment of
vitamin-E derivative Trolox. Antioxidant activity of
obesity, eye complaints, snake bites, allergies, dental
endophytic fungus Phyllosticta sp isolated from Guazuma
carries and in controlling cholesterol level. It is also used
www.currentsciencejournal.info
Nithyananda et al., 2013
as a blood purifier, antibiotic in urinary and viral
thorough wash with tap and autoclaved distilled water. All
infections, and in rheumatism. Gymnemic acids, triterpene
samples were surface sterilized by dipping in 70% ethanol
saponins are the major constituents of leaves are shown
for 2 min, followed by 5% sodium hypochlorite for 10
inhibit of intestinal glucose absorption and lowering of
mins, and finally 70% ethanol for 1 min.
plasma glucose levels. Other plant constituents include
Conduritol A, flavones, hentri-acontane, pentatriacontane,
Lupeol,
β-amyrin
related
glycosides,
anthraquinone
derivatives, alkaloids, betain, choline and trimethylamine,
The Surface sterilized samples were washed thrice
with sterile distilled water. The surface sterilized samples
leaf and stem were cut into small segments and were
placed on potato dextrose agar petriplates containing
phytin, resins, dquercitol, tartaric acid, formic acid, butyric
streptomycin sulphate solution (150 mgl-1). The petridishes
acid, and stigmasterol as other minor constituents.
were incubated for one week at 27˚C. The emerging fungal
Gymnemic acids are reported to have antidiabetic, antiinflammatory activities and anti-oxidant activities (Evans,
2002; Porchezhian and Dobriyel, 2003; Kokate et al., 2006;
Kanetkar et al., 2007). Although the antioxidant and other
medicinal values of the plant and its extracts have been
investigated (Fujimoto et al., 1991; Murakami et al., 1996;
Rachh et al., 2009) to our knowledge endophytes of this
plant have not been investigated fully more so with
antioxidant activity. Secondary metabolites of this plant are
unique. Therefore, Study on the production of secondary
metabolites from the endophytic fungi of Gymnema
sylvestre and study for its biological activity is undertaken.
hyphae after inoculation were picked and transferred onto
Potato dextrose agar (PDA) and sabouraud dextrose agar
(SDA)
plates
Appropriate
for
positive
identification
and
negative
and
preservation.
agar
petriplates
containing non sterilized plant tissues and only the
impression
of
sterilized
tissues
were
incubated
simultaneously to check the efficiency of surface
sterilization and ensure obtained fungal cultures were
possibly endophytic. Isolated fungi were identified on the
basis of their morphological and microscopic studies using
relevant taxonomic features mentioned in various standard
manuals and with the help of local fungal taxonomists.
Materials and Methods
Fungal cultivation and extraction of metabolites
Isolation of Endophytic fungi
The plant materials of Gymnema sylvestre was
Isolated fungi were grown in 250 ml Erlenmeyer
collected from campus of Regional Medical Research
flasks containing 100 ml Potato Dextrose Broth. The flasks
Centre, Belgaum, Karnataka, and was authenticated by Dr.
were incubated at 28 ± 2°C for 2 weeks, with periodical
Harsha Hegde, Scientist B, Regional Medical Research
shaking at 150 rpm. The fungal culture was filtered to
Centre, Belgaum, Karnataka. Endophytes from leaves stem
remove mycelium. The filtered broth was then extracted
and roots of authenticated plant material were isolated
with ethyl acetate three times. The fungal mycelia were
from following the protocol (Fisher et al., 1993), with
soaked in acetone for overnight and filtered to remove
some minor modifications. In brief, plant specimens were
mycelium. The filtrate was then extracted with ethyl
thoroughly washed in running tap water for 2-3 times.
acetate three times. All the ethyl acetate extracts were
,treated with 5% tween-20 solution for 3 min followed by a
mixed. The organic phase was evaporated to dryness under
www.currentsciencejournal.info
Nithyananda et al., 2013
reduced pressure using a rotary evaporator to constitute the
solution of 1 mg/ml of the crude fungal broth
crude broth extract.
extract/drug/plant extract is prepared in distilled water and
Phytochemical investigation of Gymnema sylvestre:
diluted to get various concentrations (10-100 µg/ml) in a
The shade dried leaves of authenticated plant
final volume of 1 ml. 1 ml of the working solution was
Gymnema sylvestre were reduced to coarse powder (# 40
mixed
with
1ml
various
crude
fungal
broth
size mesh) and around 200 gm of powder was subjected to
extract/drug/plant extract concentrations (10-100 µg/ml)
hot successive soxhlet extraction with ethyl acetate and
prepared, the reaction mixture was incubated for 20 min at
then with alcohol.
room temperature in dark conditions. Absorbance of
Qualitative Chemical Investigation
reaction mixtures was recorded at 517 nm. In all the
Ethyl acetate extracts of plant and endophytic fungal
experiments, distilled water served as blank and reaction
extracts and ethanolic extract of plant were subjected to
mixtures without plant extract/drug dilutions (1 ml of
qualitative chemical investigations. Phytochemical tests for
working solution) served as control samples. The changes
the presence of chemical constituents sterols, tritepenoids,
in the absorbance of the reaction mixtures were measured
glycosides,
carbohydrates,
using a spectrophotometer and the percent scavenging or
alkaloids, flavonoids, tannins, amino acids, phenolic
inhibition was calculated according to the following
content and xanthoproteins in the extracts were done.
formula. Percentage scavenging inhibition or % inhibition=
Determination of antimicrobial activity
[(Absorbance of control - Absorbance of test)/ Absorbance
anthraquinones,
Endophytic
extracts
saponins,
were
screened
for
their
of control] x 100,Where Abs control is the absorbance of
antifungal and antibacterial activity by cup plate method.
the DPPH radical + ethyl acetate, Abs sample is the
All the test strains were obtained from NCIM, NCL
absorbance of DPPH radical + sample extract/standard.
(National Chemical Laboratories), Pune (India) were sub-
ABTS Radical Decolourization Assay
cultured and maintained in the media specified. Antifungal
activity
was
compared
with
nystatin
100
g/ml,
ABTS
decolourization
diammonium
test
was
salt
radical
cation
performed
using
antibacterial is compared with streptomycin sulphate at
spectrophotometric method of (Pellegrini et al., 1999).
concentration of 100 g/ml.
Stock solution of ABTS reagent was freshly prepared by
In-vitro antioxidant studies: DPPH radical scavenging
dissolving 4 mg of ABTS to 100 ml. of distilled water, 38
activity
mg of potassium persulphate was dissolved in 1ml.
The effect of the plant extract on the DPPH radical
distilled water, 88 µl of potassium persulfate solution as
was estimated according to the method of (Hou et al.,
added to 100 ml of ABTS solution and keep it for
2001) with minor modifications. Stock solution of DPPH
overnight incubation at 40C. The working solution of
solution was freshly prepared by dissolving 9.8mg of
ABTnt was prepared by diluting the stock solution with
D.P.P.H in 50 ml of methanol and was stored at -20°C
ethanol to give an absorbance of 0.7 ± 0.05 at 734 nm.
before use. Working solution was prepared by dissolving
Stock solution of 1 mg/ml of the crude fungal broth
10ml of the stock solution in 40 ml. of methanol. Stock
extract/drug/plant extract is prepared in distilled water and
www.currentsciencejournal.info
Nithyananda et al., 2013
diluted to get various concentrations (10-100 µg/ml) in a
characteristics; microscopic studies and biochemical tests.
final volume of 1 ml. 1 ml of the working solution was
These strains were selected for their production and
mixed
broth
characterization of secondary metabolites by fermentation
extract/drug/plant extract concentrations (10-100 µg/ml)
method (Fig. 1). All the strains were producing some kind
prepared, the reaction mixture was incubated for 20 min at
of organic acid as evident from significant reduction in pH
room temperature in dark conditions. Absorbance of
of fermentation broths. All the fungal extracts of these
reaction mixtures was recorded at 734 nm. In all the
strains demonstrated antioxidant activity. Four of them
experiments, distilled water served as blank and reaction
produced pigments. 12 of them demonstrated siderophore
mixtures without plant extract/drug dilutions (1 ml of
activity under iron-limited conditions. These fermented cell
working solution) served as control samples. The changes
free broth extracts were studied for antimicrobial and
in the absorbance of the reaction mixtures were measured
invitro antioxidant activity (Fig. 2). Out of 18 isolates, 12
using a spectrophotometer and the percent scavenging or
strains showed anti-microbial activity. Cladosporium sp.,
inhibition was calculated according to the following
M. sterilia and Acremonium sp. were found to possess
formula. Percentage scavenging inhibition or % inhibition=
relatively higher antibacterial activity while Cladosporium
[(Absorbance of control - Absorbance of test)/Absorbance
sp., M. sterilia and Curvularia sp. were found to possess
of control] x 100, Where Abs control is the absorbance of
antifungal activities.
with
1ml
various
crude
fungal
the ABTS radical + ethyl acetate, Abs sample is the
Native plant extract of G. sylvestre was done by
absorbance of ABTS radical+ sample extract/standard.
using ethyl acetate and ethanol. Percentage yield of ethyl
Fe2+ ion chelating activity
acetate and ethanol were found to be 1.95 and 1.6%w/w
The Chrome Azurol S (CAS) chemical assay was
resp. Preliminary qualitative tests for presence of
used to detect the iron chelating siderophore activity
triterpenoid,
saponins,
flavonoids,
anthraquinones,
(Schwyn et al., 1987). TLC for Triterpene saponins was
carbohydrates, xantho protein and phenol were carried out
carried out using Silica gel GF-254 Stationary phase,
for endophytic ethyl acetate fungal broth extracts, ethanol
CHCl3: Methanol: Water (64:50:10); CHCl3: Methanol:
and ethyl acetate fractions of native plant extracts of
Acetic acid (5:1:1) mobile phase. Vanillin sulphuric acid
Gymnema sylvestre. Above tests indicate the presence of
was used as visualising agent. Ethyl acetate extract of
triterpenoid saponins in GSEF-06, GSEF-08, GSEF-14 and
leaves of G. sylvestre and endophytic fungal extracts were
anthraquinones in strain GSEF-01, GSEF-09, and GSEF-
compared with standard.
11. TLC study was carried out using ethyl acetate fraction
Results
of fungal extract, native plant extract and commercial
A total of 18 promising (12 from the leaves, 2 from
Gymnema powder samples as control. Result indicates, Rf
stems and 4 from roots) endophytes of medicinally
value of single spot observed for endophytic fungal
important G. sylveste were isolated in the present study
extracts of GSEF-06, GSEF-08, GSEF-14 was comparable
belonging to wide range of fungal groups. Identification of
to that of natural extract and control indicating the
fungal strains was carried out on the basis of culture
possibility of presence of similar secondary metabolites.
www.currentsciencejournal.info
Fig. 1. Isolated endophytes of Gymnema sylvestre
The DPPH and ABTS radical scavenging activity was
GSEF-4, GSEF-7 GSEF-8, GSEF-11, GSEF-13, and
carried out using 18 fungal extracts and compared with
GSEF-14 showed good DPPH radical scavenging activity.
standard Ascorbic acid. Result indicates the scavenging
The fungal extracts of GSEF -1, GSEF -4, GSEF -6, and
activity of endophytic fungal extracts in a concentration
GSEF -9 GSEF -13 GSEF -14, GSEF-11, GSSF-15
dependant manner. Several concentrations ranging from
showed
10-1000 µg/ml of fungal extracts were tested for
Scavenging effects of all these extracts were comparable
antioxidant activity. The scavenging effects of different
with that of ascorbic acid used as control (Fig. 2). Extracts
fungal extracts on Ascorbic acid were taken as a standard
also demonstrated siderophore activity based on the
compound. The fungal extracts of GSEF-1, GSEF-3,
universal CAS assay.
good
ABTS
radical
scavenging
activity.
Fig. 2. Antimicrobial and in-vitro antioxidant activity of endophytic fungal extracts of Gymnema sylvestre
Antimicrobial activity(AA) of endophytic fungal extracts
AA of endophytic fungal extracts
against Bacillus subtilis
against Staphylococcus aureus
AA of fungal extracts
AA of endophytic fungal
Antimicrobial activity of endophytic fungal extracts
against P. aeurginosa
against N. crassa
against Aspergillus niger
Antimicrobial activity of endophytic fungal extracts
TLC of Triterpene saponines
against candida albicans
TLC of Triterpene
saponines
Stationary phase: Silica gel GF254
Mobile Phase: Chloroform:
Methanol: Acetic acid (5:1:1);
Visualising agent: Vanillin
sulphuric acid
Native extract,GSEF 06,08,14
Fig. 3. In vitro freeradical scavenging activity of endophytic fungal extracts
will provide the necessary tools to identify and culture
Discussion
These results indicate endophytes of Gymnema
endophytes that produce novel potent antioxidants, organic
sylvestre can be potential sources for antimicrobial,
acids, pharmaceutically active lead metabolites and other
siderophore, biopigments, secondary metabolites and for
bioactive secondary metabolites, on an economically viable
production of organic acids. It is assumed that endophytes
scale. With the available techniques of fermentation, it is
because of their symbiotic association and possible
possible to harvest the antioxidant metabolites in large
intergeneric exchange of genetic information with host
quantities on an economically viable scale to prepare
plants may have the ability to produce the same or similar
suitable cost-effective nutraceutical formulations.
bioactive compounds as host plants. Native plant is known
Acknowledgements
to possess anthraquinone pigments. Few of our endophytes
The authors are grateful to Dr. Ch. Ramesh, Dr. J.
are secreting biopigments under solid state and submerged
Santosh Kumar, Dr. S. S. Metgud for their help in
conditions. Native plant leaf extract was shown to possess
identifying the fungal isolates. The authors also thank Dr.
invitro antioxidant activity and iron chelating activity
Harsha
(Rachh et al., 2009).Some of our endophytic extracts are
authenticating the plant and The Principal, K.L.E.
possessing invitro antioxidant activity and and iron
University's College of Pharmacy, Belgaum, India, for
chelating activity (siderophore activity). Many of our
providing the necessary facilities to carry out the work.
endophytes are secreting some kind of organic acids. Our
References
chromatographic studies and preliminary qualitative tests
Packer L, Ong OSH (1997). Biological oxidants and
indicate the possibility of presence of similar secondary
antioxidants: Molecular mechanisms and health
metabolites as that of the native plant Gymnema sylveste
effects. Champaign, USA: AOCS Press.
such
as
organic
acids,
triterpenoids,
saponin
Hegde,
RMRC,
ICMR
for
his
help
in
and
Halliwell B (1994). Free-radicals, antioxidants and human
anthraquinones into the fungal extracts. However precise
diseases: curiosity, cause and consequences. Lancet
characterization of both plant and fungal metabolites has to
322: 721-724.
be carried out which are in progress.
Valko M, Leibfritz D, Moncol J, Cronin M T D, Mazur M,
A larger number of samples, sources in different
Telser J (2007). Free radicals and antioxidants in
seasons would give a better and wider picture of
normal physiological functions and human disease.
endophytic biodiversity of this plant. It is likely that more
International Journal of Biochemistry and Cell
promising strains may be found. Results seen in this study
Biology 39(1): 44-84.
shows and re-emphasize that endophytic fungi are potential
Mariana Recco Pimentel, Gustavo Molina, Ana Paula
sources of secondary metabolites and worthy of further
Dionisio, Mario Roberto Marostica Junior, Glaucia
investigation regardless whether such metabolites are
Maria Pastore (2011). The Use of Endophytes to
present in the native plant or not. We expect that the
Obtain Bioactive Compounds and Their Application
combined knowledge of microbial biodiversity, rational
in Biotransformation Process. Chem. Rev 111
and random mutagenesis, along with bioprocess knowledge
(1):117-51.
Nithyananda et al., 2013
Sadananda TS, Nirupama R, Chaithra K, Govindappa M,
Liu X, Dong M, Chen X, Jiang M, Lv X, Yan G (2007).
Chandrappa CP, Vinay Raghavendra B (2011).
Antioxidant activity and phenolics of an endophytic
Antimicrobial
Xylaria sp. from Ginkgo biloba. Food Chemistry
endophytes
and
from
antioxidant
Tabebuia
activities
argentea
of
and
identification of anticancer agent (lapachol) Journal
of Medicinal Plants Research. 5(16): 3643-3652.
fungi
from
Nerium
oleander
Huang WY, Cai YZ, Xing J, Corke, Corke H, Mei Sun M
(2007). A potential; antioxidant resource: endophytic
Huang WY, Cai YZ, Hyde KD, Corke H, Sun M (2007).
Endophytic
105(2): 548–554.
L
fungi from medicinal plants. Econ. Bot 61(1):14-30.
Harper JK, Arif AM, Ford EJ (2003). Pestacin: a 1,3-
(Apocynaceae): main constituents and antioxidant
dihydro
activity. World J. Microbiol. Biotechnol 23: 1253-
microspora possessing antioxidant and antimycotic
1263.
activities. Tetrahedron 59(14): 2471-2476.
Strobel GA (2002). Rainforest Endophytes and Bioactive
Products. Crit Rev. Biotechnol, 22: 315.
Srinivasan
isobenzofuran
KLK,
Jagadish
from
R,
Pestalotiopsis
Shenbhagaraman,
Muthumary J (2010). Antioxidant activity of
Wiyakrutta S, Sriubolmas N, Panphut W, Thongon N,
Danwiset-Kanjana K, Ruangrungsi N, Meevootisom
endophytic fungus Phyllosticta sp. isolated from
Guazuma tomentosa. J. Phytol 2(6): 37-41.
V (2004). Endophytic fungi with anti-microbial,
Song YC, Huang WY, Sun C, Wang FW, Tan RX (2005).
anti-cancer and anti-malarial activities isolated from
Characterization of graphis lactone a as the
Thai
antioxidant and free radical-scavenging substance
medicinal
plants.
World
J.
Microbiol.
Biotechnol 20: 265-272.
from the culture of Cephalosporium sp. IFB-E001,
Ramasamy K, Lim SM, Bakar AB, Ismail N, Ismail MS,
Ali MF, Weber JFF, Cole ALJ (2010). Antimicrobial
and cytotoxic activities of Malaysian endophytes.
Phytothear. Res 24(5): 640-643.
an
endophytic
fungus
in
Trachelospermum
jasminoides. Biol. Pharmaceut. Bull 28: 506-509.
The Wealth of India Vol. IV (2005). A Dictionary of
Indian Raw Materials and Industrial Research,
Strobel GA, Yang XS, Sears J, Kramer R, Sidhu RS, Hess
NISCAIR Press, New Delhi, India pp. 276-277.
WM (1996). Taxol from Pestalotiopsis microspora,
The Ayurvedic Pharmacopoeia of India Volume-V ( 2006),
an endophytic fungus of Taxus wallachiana.
Government of India, Ministry of Health and Family
Microbiol. 142: 435-440.
Welfare Department, New Delhi, India pp. 110-114.
Strobel GA, Daisy B (2003). Bioprospecting for Microbial
Endophytes
and
Their
Natural
Products.
Microbiol.Mol. Biol. Rev 67: 491-502.
Shanmugasundaram ERB, Rajeswari G, Baskaran K,
Kumar BR Rajesh, Shanmugasundaram, K Radha,
Ahmath B Kizar (1990). Use of Gymnema sylvestre
Tan RX, Zou WX (2001). Endophytes: A rich source of
leaf extract in the control of blood glucose in insulin
functional metabolites. Nat. Prod. Reports 18: 448-
dependent diabetes mellitus. J Ethnopharmacol 30:
459.
281-294.
www.currentsciencejournal.info
Nithyananda et al., 2013
Baskaran K, Ahamath B Kizar, Shanmugasundaram K
Radha,
Shanmugasundarama
ERB
Fisher PJ, Petrini O, and Sutton BE. (1993). A comparative
(1990).
study of fungal endophytes in leaves, xylem and bark
Antidiabetic effect of a leaf extract from Gymnema
of Eucalyptus in Australia and England. Sydowia 45:
sylvestre in non-insulin-dependent diabetes mellitus
338-345.
patients. J Ethnopharmacol 30: 295-305.
Hou WY, Chen H, Chen Y, Lin L, Yang, Lee M (2001).
Evans WC (2002). Trease and Evans 15th edition,
Antioxidant activities of trypsin inhibitor, a 33kDa
Pharmacognosy, WB Saunders, an imprint of
root storage protein of sweet potato (Ipomoea
Elsevier Limited, pp. 471.
batatas (L.) Lam cv. J. Agric. Food Chem 49: 2978-
Kokate
CK,
AP
Purohit,
SB
Gokhale
(2006).
Pharmacognosy. 36th Edition, Nirali Prakashan,
Pune, India, pp.252.
of
Gymnema
Pellegrini N, Re R, Yang M, Rice-Evans CA (1999)
Screening of dietary carotenoids and carotenoid rich
Porchezhian E, Dobriyal RM (2003). An overview on the
advances
298.
sylvestre:
chemistry,
pharmacology and patents. Pharmazie 58: 5-9.
fruit extracts for antioxidant activities applying the 2,
2′-azobis
(3-ethylenebenzothiazoline-6-sulfonic)
acid radical cation decolorization assay. Methods
Khanna VG, Kannabiran K (2008). Antimicrobial activity
Enzymol. 299: 379-389.
of saponin fractions of the leaves of Gymnema
Schwyn B, JB Neilands (1987). Universal chemical assay
sylvestre and Eclipta prostrata. World J. Microbiol.
for the detection and determination of siderophores.
Biotechnol 24(11): 2737-2740.
Analytical Biochem. 160: 47-56.
Kanetkar P, Singhal R, Kamat M (2007). Gymnema
sylvestre: A Memoir. J Clin Biochem Nutrition
41:77-81.
Murakami N, Murakami T, Kadoya M, Matsuda H,
Yamahara
J,
Yoshikawa
M
(1996).
New
hypoglycemic constituents in gymnemic acid from
Gymnema sylvestre. Chem Pharm Bull 44: 469-471.
Fujimoto T, Nagai T, Yamashita F, Kensho G, Nakano Y.
(1991) Physiological activating substances isolated
from Gymnema Sylvestre (Part 2). Seitogijutsu
Kenkyukaishi 39: 71-75.
Rachh PR, Patel SR, Hirpara HV, Ruparelia MR, Rachh M
R, Bhargava AS, Patel NM, Modi DC (2009).In vitro
Evaluation of antioxidant activity of Gymnema
sylvestre R. BR. leaf extract Rom. J. Biol. Plant Biol
54(2):141-148.
www.currentsciencejournal.info