Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,

Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358

Impact Factor: 6.057

Isolation and Screening of Resveratrol

Producing Endophytes from Wild
Grape Cayratia Trifolia
Chetana Roat*, Meenu Saraf
Department of Microbiology & Biotechnology, University School of Sciences,
Gujarat University, Ahmedabad-380 009, India
* Corresponding author. Tel.: +91 79 2630 3225; fax: +91 79 26303225.
E-mail address: chetana.roat@gmail.com (Chetana Roat)

Abstract: The aim of the present study was to obtain endophytes strains with effective resveratrol
production from wild grapevive Cayratai trifolia in Gujarat, India. 30 strains of endophytes were
isolated, including 20 fungus and 10 bacteria. The distribution of endophytes were found in different
tissues and organ. The presence of the resveratrol produced by endophytes were confirmed by
biochemical test like ferric chloride-potassium ferricynaide colour reaction and quantitative
confirmation confirmed was done by Thin layer chromatagraphy, however some endophytes-free
samples from the same species also contained resveratrol, usually in reduced concentration. 21 fungal
and 6 bacterial isolates showed positive potential for the production of resveratrol. This observation
represents the first report of the resveratrol from the Cayratia trifolia

Keywords: “Endophytes”; “Cayratia trifolia”; “Resveretrol”; “FeCl3”; “Thin layer chromatography”.

Introduction:
The distribution of endophytes like fungal and bacterial is ubiquitous and almost without exception; the
endophytes have been reported from all tissues, including roots, leaves, stems, fruits and flowers ( Kumara P.M.
et al. 2014). Endophytes were distributed in each and every plant species and were investigated for endophytic
microbial components (Carroll 2004). Endophytic, colonizing both inter and intracellular spaces of tissues of higher
plants without causing any damage on the plants in which they live. Endophytes have proven to be rich sources of
bioactive natural products (Li et al. 2008; Molina et al. 2012). Endophytes have been intensively studied in several
unexplored environments around the world (Dar et al. 2015). Endophytes have proven to be rich sources of novel

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 Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358
Impact Factor: 6.057

natural compounds with a wide-spectrum of biological activities and a high level of structural diversity. Bioactive
natural compounds produced by endophytes have been promising potential usefulness in safety and human health
concerns, although there is still a significant demand of drug industry for synthetic products due to economic and
time-consuming reasons (Strobel et al., 2004).Resveratrol, a stilbene polyphenol substances from natural secondary
metabolites of plants (Burns et al. 2002; Poltronieri et al. 2013), often found in grapes, peanuts, Polygonum
cuspidatum, veratrum, or cassia seed, had a large number of beneficial medical functions to human body, such as
anti-inflammatory, antiallergic, antitumor, regulating blood fat and anti-pathogenic microorganism.
Although many fungal endophytes isolated from healthy plants of Vitaceae family, there are no reports on fungal
and bacterial endophytes in Cayratia trifolia. Cayratia trifolia endophytes are worth investigating because of their
potential to produce bioactive molecule resveratrol.
In this manuscript, fungal and bacterial endophytes were isolated from Cayratia trifolia and screened for resveratrol
production. Endophytes producing resveratrol may provide a new valuable medicinal resource in addition to plant
resources. 21 fungal and 6 bacterial isolates were then identified having the potential to produce resveratrol.

Material and Methods:

Sampling
Wild grapre, Cayratia trifolia was collected from the Gandhinagar, Gujarat, in the month of June, July and August
2017, respectively. The root, stem, leaf and fruit of C.trifolia were transferred to the laboratory with the sterile bag
at 4 °C, respectively. The plant materials were immediately rinsed with aseptic water on a sterile operating table to
remove the dust, dirt and some microorganisms on the sample surface. After drying at room temperature, the root,
stem, leaf and fruits collected were preserved at 4 °C, respectively.

Surface sterilization and pure culture of endophytes

The samples above were soaked by 80 % alcohol for 30 seconds before washing with sterile water and drying using
filter paper, respectively, and then treated by 8 % of sodium hypochlorite for 7-8 minutes prior to drying employing
filter paper again, and finally rinsed by aseptic water for 5 times. Following the pices of 7–9 mm long segment of
leaves, roots, stem, fruits etc. The samples placed on the middle of each Petri dish, and cultured under the 28-30° C
temperature. The three parallel Petri dishes were prepared for each tissue. The colonies cultivated were picked out
and transferred carefully to a new sterile plate for re-culturing when the new hyphae or fungi culture appeared. After
repeating the procedure five-six times, the endophytes of pure culture in different wild grapevine organs were just
obtained.

Chemical agents and Medium

Standard resveratrol was purchased from Sigma. While the chemical agents as analytical reagents, such as methanol,
acetonitrile, beef extract, peptone, agar, soluble starch, sodium hypochlorite, ethanol, glucose, sodium chloride,

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 Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358
Impact Factor: 6.057

KNO3, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, ethyl acetate, ferric chloride, potassium ferricyanide, toluene, acetic
acid, and ethyl acetate, were purchased by Sigma and Merk.

The potato dextrose agar (PDA), the separation and purification culture media of endophytic fungi, was prepared as
follows: the 200 g of fresh potato filtrate through four layers of gauze, after boiling for 30 min, was added to
1000 mL with supple water, and supplemented 20 g of the filtrate agar and 20 g of glucose, respectively. Then the
mixture, adjusted to pH 7.0, was subjected to 121 °C high pressure steam sterilization for 20 min. Agar was not
added in the fermentation liquid of PDA. Gaus No. l medium acting as the isolation and purification culture of
endophytic actinomycete was prepared according to the method described by Cao et al. (2004), and nutrient broth–
yeast extract medium employed for the isolation and purification culture of the endophytic bacteria was also made
up according to the method of Zinniel et al. (2002).

Biochemical test for screening of Resveratrol producing endophytes

Endophytic fungi purified were inoculated to 250 mL of Erlenmeyer flask including 100 mL liquid medium,
respectively, and incubated at 100 rpm for 5 days at 28 °C simultaneously. Then the fermentation liquid was
centrifuged at 4000 rpm for 15 min. The screening of chromogenic reaction: the endophytic fungus producing
resveratrol could be screened according to the resveratrol property with ferric chloride–potassium ferricyanide color
reaction. While the color reaction of the fermentation liquor was as follows: the mixture, 0.1 % FeCl3: 0.1 %
K3[Fe(CN)6] = 1:1 (v/v), was served as chromogenic agent. 2 mL of coarse extraction liquid per sample
concentrated above was mixed with the same volume of methanol, and then added two drops of chromogenic agent,
meanwhile the color changes were recorded, respectively. The polyphenols containing resveratrol would be
indicated by the indication of the blue color.

Quantitative Test thin Layer Chromatography

The fermentation liquid culture of the strains of ten microliter after the process of centrifugation and
−1
5 μg mL resveratrol standard solution was spotted into the same silica gel plate (20 × 20 cm) in chromatography
cylinder, then expanded vertically upward with the function of the developing solvent (Chloroform: ethyl acetate:
water = 8.5:0.3:0.1, v/v), respectively. When the developing solvent reached to 1 cm of location close to the top of
the thin layer plate, it was immediately removed and blowed dry by blower.

After the silica gel plate above was sprayed uniformly by the color developer, 0.1 % FeCl3: 0.1 % K3[Fe(CN)6] = 1:
1 (v/v) and dried, the distance was measured immediately between the blue spot center and origin as well as between
the origin and solvent. Finally, the Rf value (rate of flow or retention factor) was calculated. The Rf value is a
constant for a given component under the same experimental conditions. The Rf value may be calculated from the
following equation.
Rf=ab

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 Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358
Impact Factor: 6.057

The Rf value itself is unit less.

a = distance of the center of the sample spot from the origin

b= is distance of the solvent front from the origin.

Result and Discussion

Isolation and screening of wild grape endophytes

Table: The endophytes distribution from different tissue of Cayratia trifolia

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 Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358
Impact Factor: 6.057

As could be seen in Table 1, 25 strains of endophytes were obtained from different tissue of wild grapevines
collected from gandhinagar, Gujarat. While Zeng et al. (2012) obtained only 30 endophytes from skin, spike stalk
and stalk of mature wine grape ―Melot‖ . In the trial, two types of endophytes were found , including the 20 strains
of endophytic fungi, 5 strains of endophytic bacteria were isolated from Cayratia trifolia. Therefore, the value of
the endophytic microbes varied with the different tissues and which reflected an abundant biodiversity of
endophytes in the same plant.

In contrast to the research of other plants, the number of the endophytes obtained in the present work were
relatively small, which was explicate by the many reasons: like the careful disinfection to the samples surface killed
some useful endophyttes, some endophytic fungi could not remain active after continuous subculture.

Screening of resveratrol producing endophytes

The studies had shown that the resveratrol was a kind of the secondary metabolite of polyphenols group of stilbenes
class, and appeared the blue under the potassium ferricyanide solution and ferric chloride. This is the preliminary
qualitative analyses of the resveratrol (Bavaresco et al. 2012; Díaz et al. 2012).The fermentation liquids of 25 strains
of endophytic fungi were ensured with color reaction, respectively. The color range, from the green, yellow, light
blue and dark blue, was notice during the whole color process reaction. As a result, the fermentation liquid of a-
total-18-strains of fungus and 4 strains of bacteria revealed the blue reaction, The fungal strains including
CTF1,CTF2,CTF3,CTF5,CTF6,CTF7,CTF8,CTF10,CTF11,CTF12,CTF13,CTF15,CTF17,CTF19,CTF20,CTF21,C
TF22,CTF24 and bacterial strains including CTB1,CTB3,CTB4,CTB5 promoted the liquid of the deep blue.
Therefore, the above fungal and bacterial strains were deduced to have the capability of producing polyphenol
substance—resveratrol (Table-1).

Thin layer chromatography analysis

The twenty strains of the endophytic fungi showing blue color reaction were tested by thin layer chromatography for
qualitative detection of resveratrol. Four kinds of the strains, C2J6, C2Y4, XP2-03 and C2Y6, presented obviously
blue spots in the experiment. TLC was often used for the separation and identification of organic compounds and it
is qualitative test for bioactive molecules like resveratrol (Roat et al 2008).Therefore, the composition of the organic
matter could be identified by comparing with the compound of the known structure. When there were the same
conditions, such as expansion agent, adsorbent, the thickness of the thin layer plate and temperature, for a
compound, the Rfvalue, as a constant, could be used as the basis of qualitative analysis. In this trial, the Rf of the
selected strains which showed positive result in the FeCl3 test showed Rf value 0.334, same to the value of the
resveratrol standard, which could conclude preliminarily that the C2J6 could promote the synthesis of the
resveratrol. Nevertheless, the Rf values of other strains, such as CTF4, CTF9, CTF14, CTF16, CTF18, CTF23,
CTF25 and CTB2 were different from that of the standard sample, which excluded the possibility that these strains

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 Chetana Roat et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.11, November- 2017, pg. 27-33 ISSN: 2348-1358
Impact Factor: 6.057

produced resveratrol. Therefore, the strains which gave positive FeCl3 test and showed same Rf with the standard
resveratrol were selected as the target strains for the production of resveratrol, which was isolated from the stems,
skin of fruit, leaf, tendril, seed of fruit,cob, root of wild grapevine Cayratia trifolia collected in June, July and
August.

Ultraviolet wavelength scanning

The result that the resveratrol sample analyzed was scanned in the range of 200–500 nm was that the maximum
absorption wavelength of the resveratrol sample was at 306 nm, which was the same as the maximum wavelength of
resveratrol standard. Based on the result, the product recovered from liquid fermented was just the resveratrol.

Conclusion:
From this trial the 25 fungal strains and 5 bacterial strain were isolated from the different tissue of the Cayratia
trifolia plant. 21 fungal strain and 4 bacterial strain isolates showed resverarol producing capability confirmed by
biochemical test, thin layer chromatography and Ultraviolrt test.

Acknowledgments
This work was supported by the University grant Commission, Government of India, New Delhi, India for providing
Dr. D.S Kothari Post Doctoral Fellowship with Ref. No.F.4-2/2006(BSR)/BL/16-17/0021&01-09- 2063.

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Impact Factor: 6.057

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