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Buddhan Sundaram Vijayakumar
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Italian Journal of Mycology
alpha-amylase inhibitors
<em>In-vitro</em> anti-oxidant and anti-diabetic potential of endophytic fungi associated with <em>Ficus religiosa</em>
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Articles
2021-03-31
2020-12-29
2021-02-08
2021-06-25
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Ital J Mycol
Buddhan Sundaram Vijayakumar
2021/03/31
10.6092/issn.2531-7342/12104
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Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Puttaparthi- 515134, Anantapur District, Andhra Pradesh, India
2531-7342
alpha-amylase inhibitors
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Zhang YJ, Zhang S, Liu XZ, Wen HA,Wang M (2010) A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains. Letters in applied microbiology 51(1):114–118. https://doi.org/10.1111/j.1472-765X.2010.02867.x
<em>In-vitro</em> anti-oxidant and anti-diabetic potential of endophytic fungi associated with <em>Ficus religiosa</em>
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                 IJM - Italian Journal of Mycology
                 ISSN 2531-7342 - Vol. 50 (2021): 10-20
                 Journal homepage: https://italianmycology.unibo.it/

Original paper

     In-vitro anti-oxidant and anti-diabetic potential of endophytic
                  fungi associated with Ficus religiosa

              Kottapalli Kameswara Jayant*, Buddhan Sundaram Vijayakumar

Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam, Puttaparthi- 515134,
Anantapur District, Andhra Pradesh, India

Corresponding author e-mail: kottapallikjayant@sssihl.edu.in


Article info
Received 29/12/2020; accepted 19/3/2021
https://doi.org/10.6092/issn.2531-7342/12104

Abstract
Endophytic fungi are capable of producing plethora of important secondary metabolites, previously known from medicinal
plants, thereby raising the potential of exploring endophytes as alternate sources for these metabolites. In the present
investigation, ten endophytic fungi were isolated from Ficus religiosa L. (Moraceae) and the extracts obtained from fungi
were evaluated for their potential as anti-oxidant and anti-diabetic agents. Phytochemical screening of extracts revealed
presence of phenols, flavonoids, alkaloids, steroids, terpenes and terpenoids. Anti-oxidant and anti- diabetic activities
of the extracts were tested by DPPH free radical scavenging assay and alpha amylase inhibitory assay, respectively. In
general, all the extracts showed moderate inhibition of DPPH thereby providing an insight that extracts have a good
proton-donating ability and could be potential free radical inhibitors. Similarly, moderate inhibition of alpha amylase was
shown by all the extracts suggesting their possible role in reducing the breakdown and flow of glucose from complex
dietary carbohydrates into the bloodstream. Among the ten fungi tested, Curvularia lunata displayed the highest anti-
oxidant and anti-diabetic activities.

Keywords
Endophytes, medicinal plant, secondary metabolites, phytochemicals, anti-oxidants, alpha-amylase inhibitors



Introduction
Endophytic fungi inhabit the host plant by living in their tissues and exhibit a mutual relationship
with the plant without causing any disease symptom (Bacon and White, 2000). According to the
plant-endophyte coevolution hypothesis suggested by Ji et al. (2009), it is believed that endophytes
assist the plants in chemical defense by producing bioactive secondary metabolites. Since endophytes
reside within plants and are in continuous interaction with their hosts, it is fairly relatable that plants
would have a substantial influence in the metabolic processes of the endophytes. Occasionally, the
compounds produced by endophytic fungi are similar as those produced by the plants (Kusari et
al., 2012). Several metabolites produced by endophytic microorganisms have varied pharmaceutical
applications and are used in production of anti-microbial, anti-fungal, anti-oxidant, anti-diabetic,
anti-inflammatory and many such other potential lead drug compounds (Manganyi et al., 2020).
    Ficus religiosa L. (Moraceae) is a large, perennial tree commonly planted in the temple areas
and is considered sacred, especially in Southeast Asia (Government of India, 2016). Various parts
of F. religiosa were assessed for their therapeutic qualities and found to have significant medicinal
properties (Singh et al., 2011).


Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
Jayant, Vijayakumar                                             Italian Journal of Mycology 50 (2021): 10-20

https://doi.org/10.6092/issn.2531-7342/12104



   It is a universal understanding that oxygen is major component of human existence. However in
certain cases, this oxygen may be killer of cells when it generates reactive species that causes necrosis
and untimely cell death. Reactive oxygen species (ROS) along with Reactive nitrogen species (RNS)
causes oxidation by disrupting the physiological processes in the cell (Weseler and Bast, 2010). Free
radicals such as ROS and others are generated as a result of biological and metabolic reactions in
the body and also exogenous factors. Many human degenerative disorders such as diabetes mellitus,
Alzheimer’s, Parkinson’s disease, atherosclerosis and inflammatory diseases are as a result of
oxidative stress and membrane damage (Uttara et al., 2009). An ‘antioxidant’ can be labeled for any
molecule/substance which inhibits or delays the oxidation of a substrate. There are several molecules
either synthesised endogenously or exogenously consumed which play a role in antioxidant defense
(Somogyi et al., 2007). There are many antioxidant compounds produced by endophytic fungi, for
example, pestacin and isopestacin was obtained from Pestalotiopsis microspora (Speg.) G.C. Zhao &
Nan Li, an endophytic fungus isolated from medicinal plant Terminalia morobensis Coode (Strobel
and Daisy, 2003). Endophytes from Passiflora incarnata L. have been reported to produce phenolic
compounds, methoxymethylphenol, orcinol and sorbicillin which have notable antioxidant activities
in vitro and can be regarded as promising sources of new drugs (da Silva et al., 2020).
   Diabetes mellitus and in specific type-II diabetes is emerging as a grave medical consideration
in global scenario. It is estimated that about 422 million people worldwide have diabetes and about
1.6 million deaths in the world are directly attributed to diabetes each year (Roglic, 2016). This
alarming situation demands research on novel therapeutic agents to treat diabetes. One of the major
challenge, among many others, in diabetes treatment is normalising the blood glucose levels in
patients. A potential preventive or control measure to achieve this is by controlling monosaccharide
absorption and regulating the action and network of carbohydrate degrading/hydrolysing enzymes.
Two principle digestive enzymes α-amylase and α-glucosidase are responsible for the degradation
of complex dietary carbohydrates into sugar in the digestive tract. Regulation of these enzymes
provides scope for lowering post-prandial rise in blood glucose levels and thereby bringing the insulin
action/regulation under control (Alqahtani et al., 2019). Previous work on anti-diabetic potential of
endophytic fungi isolated from F. religiosa showed that Aspergillus species among five other fungal
isolates exhibited highest percentage of inhibition for α-amylase inhibition assay, α-glucosidase
inhibition assay and showed good results in glucose diffusion assay. These results gave an indication
about the hypoglycemic activity of the endophytic fungal crude extract (Tiwari et al., 2017).
   With this background, this study attempts to report more endophytic fungi associated with F.
religiosa and to evaluate their phytochemical and pharmacological potential in the context of oxidative
stress and diabetes.

Materials and Methods
Collection of plant samples and culturing endophytic fungi
Fresh and healthy leaf samples of F. religiosa (identified by Botanical Survey of India, Regional
Centre, Hyderabad) were collected from Puttaparthi, Andhra Pradesh, India during the year 2018.
Composite sampling technique was used: multiple temporally or spatially discrete leaf samples were
collected and combined, thoroughly homogenised, and treated as a single sample. The plant specimens
were processed by thoroughly rinsing with tap water (Hallmann et al., 2006). Samples were cut into
3-4 cm pieces prior to surface sterilization (75% ethanol for 2-5 minutes followed by 4% sodium
hypochlorite for 1-2 minutes and again 75% ethanol for 2-5 minutes). Surface sterilized tissues
were rinsed three times with sterile water, blot dried, cut into small pieces (1–1.5 cm), and plated on
Potato Dextrose Agar (PDA, Hi Media Laboratories) plates supplemented with streptomycin (250 mg
L-1). The efficacy of surface sterilization was confirmed by inoculating the surface sterilized water
collected from last wash of the sample in a nutrient medium. The absence of growth of any fungi

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on the media confirms the efficient surface sterilization of the segments. The inoculated petri plates
were sealed and incubated at 25 ᴼC in an incubation chamber. After 3 days of inoculation, the plates
were observed daily for growth of the fungi from cultured segments up to two weeks. The fungus that
emerged from tip of the segment was picked up, sub-culturedand the pure culture was maintained on
PDA for the extraction of fungal metabolites.

Phenotypic and genotypic identification of the endophytic fungi
The isolated endophytic fungi were initially identified by microscopic examination (Biological
binocular microscope, Dewinter Optical Inc, New Delhi, India) of colony morphology and reproductive
characteristics using slide cultures. Morphological identification of the endophytic fungi was carried
out at Indian Type Culture Collection Centre, Division of Plant Pathology, IARI, New Delhi.
   For genotypic identification of the fungus showing high bioactivity, total genomic DNA of the
endophytic fungus was isolated directly from actively growing mycelium growing in potato dextrose
broth (PDB). Isolation of DNA and the subsequent steps of Polymerase chain reaction (PCR) and
DNA sequencing were done by the Pentavalent Biotechnology Pvt. Ltd., Bengaluru, India. Fungal
DNA was extracted using the protocol of Zhang et al. (2010) and the extracted DNA was amplified
using ITS1 – ITS4 primers (White et al., 1990). Amplified DNAs were sequenced and the obtained
sequences were compared with already existing DNA sequences in NCBI Gen Bank (http://www.
ncbi.nlm.nih.gov.blast) to identify the respective fungi.

Extraction of fungal metabolites
The mycelia from growing colony edge were inoculated into PDB, for production of fungal metabolites
by fermentation method. Mycelia were inoculated into 1000 mL
   Erlenmeyer flask containing 500 mL of PDB medium and kept on a rotary shaker (Orbital shaker,
Scigenics, Chennai, India) for 14 days at 25 ᴼC. Each culture was filtered using Whatman filter paper.
Solvent extraction procedure using ethyl acetate as organic solvent was employed to obtain fungal
crude extracts. To the culture filtrate equal volume of ethyl acetate (AR grade, 99% pure, Merck) was
added, mixed well for 10 min and kept aside for 5 min till the two clear immiscible layers formed.
The upper layer of solvent containing the extracted compounds was separated using separating funnel
(Bhardwaj et al., 2015), and evaporated using rotary vacuum evaporator (Heidolph, model: 571-
01300-06-0 Hei-VAP Core HL G3) to yield the crude dry extracts (yield obtained ranged from 1.2
mg to 2 mg for ten fungal cultures). The crude extracts were then dissolved in ethyl acetate at various
concentrations (0.1-0.5 mg mL-1) and kept at 4 °C.

Qualitative phytochemical analysis
The endophytic fungal extracts were tested for the presence of the secondary metabolites such as
alkaloids, phenols, flavonoids, steroids and terpenoids by various qualitative techniques (Harborne,
1998).

Alkaloids
The endophytic fungal crude extract (0.5 mg mL-1) was evaporated and the residues were dissolved in
2 N HCl. The mixture was filtered and the filtrate was used for the following test:

• Wagner’s test: reddish brown precipitate formation indicated presence of alkaloids on addition of
Wagner’s reagent (Tiwari and Gupta, 2020).
• Hager’s test: presence of alkaloid was confirmed with yellow colour precipitate on addition of
Hager’s reagent (Tiwari and Gupta, 2020).


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Flavonoids
• Alkaline reagent test:fungal extract was treated with 10% ammonium hydroxide solution. Presence
of flavonoids was detected by a yellow fluorescence (Tiwari and Gupta, 2020).
• Shinoda test: to 0.5 ml of crude extract, 5-10 drops of dil. HCl and a small piece of zinc was added
followed by boiling solution for few minutes. In the presence of flavonoids, pink or dirty brown
colour was observed (Tiwari and Gupta, 2020).

Phenols
• Ferric chloride test: extract was treated with 5% ferric chloride solution. Dark green colour indicated
the presence of phenol (Devi et al., 2012).
• Lead acetate test: 10% lead acetate solution was treated with fungal crude extract and a bulky white
colour indicated phenolic presence (Vimalkumar et al., 2014).

Steroid
• Salkowski test: to 2 mL of chloroform, 0.5 mL of extract was added and mixed. Sulphuric acid was
added along walls and allowed to stand for some time after shaking. A reddish brown colour at the
interface indicated the presence of the steroidal ring (Tiwari and Gupta, 2020).

Terpenoids
• The Libermann–Burchard test: a deep red layer at the junction of two layers indicated the presence
of terpenoids, when 1 mL of the fungal crude extract was treated with few drops of acetic acid and 1
mL concentrated sulphuric acid (Tiwari and Gupta, 2020).

Total phenolic content
Total phenolic content of ethyl acetate extracts of endophytic fungi was estimated by Folin-Ciocalteau
reagent based assay using gallic acid as standard (Halliwel and Guttridge, 1989). Total phenolic value
was obtained from the regression equation: y = 0.0009X +0.0686 with R2 = 0.999 and expressed as
mg of gallic acid equivalents (GAE)/g of extract (dw).

Anti - oxidant activity of fungal extracts

DPPH free radical scavenging assay
DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable, nitrogen-centred free radical which produces
violet colour in methanol solution. Change in absorbance of DPPH was monitored for anti-oxidant
activity through free radical scavenging. DPPH free radical scavenging assay is considered to be
one of the basic, accurate, and widely used methods to find out the antioxidant potential of various
natural products (Brand-Williams et al., 1995). Endophytic fungal extracts at a concentration range
of 0.1 – 0.5 mg mL-1 were used. DPPH solution (0.5 mM L-1) was prepared in 95% methanol and a
total of 2 mL of DPPH solution was added to the test sample (200 µL) and incubated for 30 min at
room temperature in darkness. After incubation, the absorbance was measured at 517 nm (Manzocco
et al., 1998). Ascorbic acid was taken as standard. Free radical scavenging activity was expressed as
percentage inhibition which was calculated as:

                Acontrol - Asample
Inhibition (%) =		                 X 100
                     A control



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Inhibition of carbohydrate digestive enzyme (Anti-diabetic activity)

Alpha amylase Inhibitory (AAI) Assay
Reaction mixture was prepared by adding 40 μL of fungal extract (0.1 – 0.5 mg mL-1), 200 μL of
phosphate buffer (pH 6.9), and 40 μL (24 U mL-1) porcine pancreatic amylase. It was incubated
at 37 °C for 10 min followed by addition of 50 μL of starch (1%) and incubation at 37 °C for 20
min. Reaction was terminated by adding 0.5 mL DNS (3,5-dinitrosalicylic acid) reagent followed by
incubation in boiling water for 5 min. The reaction mixture was diluted with 5 mL of distilled water
and absorbance was read at 540 nm. For each concentration, blank tubes were prepared by replacing
the enzyme solution with 40 μL of buffer. Control representing enzyme activity was prepared in a
similar manner without extract. Acarbose was used as standard and compared with the test samples.
The experiments were repeated thrice using the same protocol (Nair et al., 2013).

The AAI activity was calculated using the formula:
                              A control - A sample
% Alpha amylase Inhibition =                       X 100
                                   A control
Results
Morphological and genetic identification of endophytic fungi
Ten endophytic fungi were isolated from F. religiosa and identified by morphological features and ITS
gene sequencing. The cultures obtained were Aspergillus aculeatus Iizuka, Penicillium sp., Aspergillus
sydowii (Bainier & Sartory) Thom & Church, Curvularia lunata (Wakker) Boedijn (NCBI accession
number: MT825246), Cephaliophora irregularis Thaxt., Diaporthe sp., Aspergillus quadrilineatus
Thom & Raper, Aspergillus flavus Link, Aspergillus versicolor (Vuill.) Tirab. and Aspergillus sp.
(Taxonomic citation source: http://www.indexfungorum.org/names/names.asp).

Phytochemical analysis and determination of total phenolic content
The ethyl acetate extracts of endophytic fungi revealed presence of alkaloids, phenols, flavonoids,
saponins, terpenes, terpenoids and steroids as depicted in Table 1. Total phenolic content was estimated
and the highest concentration of phenolics was observed in the extract of C. lunata, equal to of 21.7
± 0.19 mg GAE/g of extract (dw).

Anti-oxidant activity

DPPH free radical scavenging assay
The reaction was visible as a color change from purple to yellow. Ethyl acetate extract of C. lunata
at a concentration of 0.5 mg mL-1 showed the best anti-oxidant capacity (inhibitory % of 90.7 ±
1.0%). Ascorbic acid asstandard exhibited 95.95 ± 1.45% inhibitory activity (Fig. 1). The effect of
antioxidants on DPPH is thought to be due to their hydrogen donating ability. The result showed that
the extract has a good proton-donating ability and could be a potential free radical inhibitor. The IC50
values of the standard and C. lunata extract (showing high inhibitory %) were calculated (https://
www.aatbio.com/tools/ic50-calculator) and are as depicted in Tables 2 and 3, respectively.




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Table 1 - Phytochemicals present in the fungal extracts and total phenolic content (mg GAE/g of extract [dw]).
                                                                                                  Total
     Fungal
                                                                    Phytochemicals detected       Phenolic
   endophyte
                                                                                                  Content
                                                                                                  [mg GAE/g
                               Alkaloids Phenols Flavonoids Steroids Terpenes Terpenoids Saponins of extract
                                                                                                  (dw)]
Aspergillus
                                       +               +                          +                                                        13.78 ± 0.13
aculeatus
Penicillium sp.                        +               +            +                                                                      13.14 ± 0.06
Aspergillus
                                       +               +                                          +                                        12.4 ± 0.05
sydowii
Curvularia
                                       +               +            +             +               +             +               +          21.7 ± 0.19
lunata
Cephaliophora
                                       +               +                                                                        +          0.80 ± 0.10
irregularis
Diaporthe sp.                          +               +            +                                                           +          0.78 ± 0.09
Aspergillus
                                       +               +                                                        +                          1.12 ± 0.19
quadrilineatus
Aspergillus
                                       +               +                                          +                                        1.39 ± 0.22
flavus
Aspergillus
                                       +               +                                                                        +          1.62 ± 0.08
versicolor
Aspergillus sp.                        +               +                          +                                                        2.42 ± 0.04



                             120

                             100
         % DPPH Inhibition




                             80

                             60

                             40

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                                              Concentration of endophytic fungal extracts (0.5-1mg
                                                                                                ) mL
          Fig. 1 - DPPH Inhibition by fungal extracts. Each bar represents particular fungus and the last bar is
          % inhibition of ascorbic acid (standard).




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Table 2 - DPPH % Inhibition and IC50value of Ascorbic Acid (Standard).
 AscorbicAcid (Standard)      Concentration (mg mL-1)               % Inhibition                     IC50 value

                                                                                                     (mg mL-1)
                                          0.1                        35.67 ± 0.94                   0.217± 0.75
                                          0.2                        58.32 ± 0.77
                                          0.3                        87.16 ± 0.16
                                          0.4                        91.77 ± 0.43
                                          0.5                        95.95 ± 1.45

Table 3 - DPPH% Inhibition and IC50value of C. lunata extract (showing high % inhibition).
Ethyl acetate extract of C.         Concentration                   % Inhibition                     IC50 value
          lunata
                                      (mg mL-1)                                                       (mg mL-1)
                                         0.1                          7.88 ± 0.03                    0.42 ± 0.54
                                         0.2                         14.90 ± 0.23
                                         0.3                         22.29 ± 0.65
                                         0.4                         57.27 ± 0.79
                                         0.5                          90.77 ± 1.0

Anti-diabetic Activity of fungi

α-Amylase inhibition (AAI) assay
Antidiabetic activity of fungal extracts was determined by inhibition of α-amylase. All the extracts
showed enzyme inhibitory activity and the C. lunata extract was the most effective in this assay also
(Fig. 2). Ethyl acetate extract of C. lunata at a concentration of 0.5 mg mL-1 exhibited an inhibition
of 80.4%. Standard α-amylase inhibitor - Acarbose showed an inhibitory activity of 98.8%. The IC50
values of the standard and C. lunata extract (showing high inhibitory %) were calculated (https://
www.aatbio.com/tools/ic50-calculator) and are as depicted in Tables 4 and 5 respectively.




                  Fig. 2 - Alpha amylase inhibition by fungal extracts. Each bar represents particular
                  fungus and the last bar is % inhibition of acarbose (standard).


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Table 4 - Alpha amylase% Inhibition and IC50 value of Acarbose (Standard).
         Acarbose              Concentration (mg mL-1)               % Inhibition                       IC50 value

        (Standard)                                                                                    (mg mL-1)
                                           0.1                         10.8 ± 0.08                  0.3187 ± 0.24
                                           0.2                         21.6 ± 0.05
                                           0.3                         56.8 ± 0.20
                                           0.4                         80.4 ± 0.36
                                           0.5                         98.8 ± 0.55



Table 5 - Alpha amylase % Inhibition and IC50 value of C. lunata extract (showing high % inhibition).
   Ethyl acetate extract       Concentration (mg mL-1)               % Inhibition                       IC50 value

        of C. lunata                                                                                 (mg mL-1)
                                           0.1                          5.6 ± 0.44                  0.3563± 0.46
                                           0.2                         14.4 ± 0.24
                                           0.3                           28 ± 0.4
                                           0.4                          64 ± 0.67
                                           0.5                         80.4 ± 0.56

Statistical analysis
All the experiments were carried out in triplicate and the data was analyzed by t-test using Microsoft
excel 2010. The error bars in the graphs represent mean of triplicate data ± standard error with P ≤
0.05. The data represents average of three independent experiments carried out with three replicates
from an experiment.

Discussion and Conclusions

Ten endophytic fungal strains were isolated from leaves of F. religiosa. Strains of the genus Aspergillus
were found predominantly. Other endophytic fungi from the genus Penicillium, Curvularia,
Cephaliophora were found in F. religiosa.
   Fungal extracts were obtained using ethyl acetate as solvent, asethyl acetate extraction is reported
to be most efficient method of isolating secondary metabolites from fungal species (Yadav et al.,
2014). The qualitative phytochemical analysis confirmed the presence of phenols, flavonoids,
alkaloids, terpenoids and steroids, varyingamong the different fungal extracts. The extracts were
further evaluated for antioxidant and anti-diabetic activities.
   The α-amylase inhibition assay performed as part of this study demonstrated that all the fungal
extracts showed activity and the ethyl acetate extract of C. lunata had significant inhibitory potential.
The IC50 value (0.35 ± 0.46 mg mL-1) of C. lunata extractis comparable to that of Acarbose (0.31 ±
0.24 mg mL-1), a widely used and marketed anti-diabetic drug. Previously, Tiwari et al. (2017) had
isolated twenty-one endophytic fungi from leaf explants of F. religiosa and these were evaluated for
anti-diabetic or hypoglycemic activities. Among these nine pure endophytic cultures were isolated
and five tested for alpha amylase inhibition and alpha glucosidase inhibition. Petroleum ether extract
of Aspergillus sp. showed highest inhibitory action against alpha amylase and alpha glucosidase
enzymes. Lower concentration (100 µg) of crude extract showed maximum inhibitory activity with
91 ± 0.06% for alpha amylase and 43 ± 0.01% for alpha glucosidase enzymes. The endophytic fungus
Dendryphion nanum (Nees) S. Hughes isolated from leaves of F. religiosa yielded a naphtoquinone

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compound which induced glucose uptake in rat skeletal muscles in the presence of insulin with an
EC50 of 0.80 ± 0.090 μM. Rosiglitazone, a known glucose uptake activator (EC50 = 3.0 ± 0.040 μM)
was used as a standard in the assay. The ability of compound to induce glucose uptake at such low
concentration indicated that it could be a good scaffold as a starting point for drug development
(Mishra et al., 2013).
   It is predicted that diabetic complications occur as a result of the oxidative stress due to the
formation of free radicals with the glucose oxidation (Mehta et al., 2006). Therefore, the use of
antioxidants along with anti-diabetic drugs is frequently recommended to avoid such complications.
The antioxidant activities measured by DPPH scavenging assay and the total phenolic content of the
fungal extracts indicate that all extracts possess antioxidant properties with C. lunata exhibiting the
best DPPH scavenging ability (IC50 of 0.42 ± 0.54), comparable to that of standard Ascorbic acid (IC50
of 0.21 ± 0.75 mg mL-1), and the highest total phenolic content (21.7 ± 0.19 mg GAE/g extract). Our
findings support some of the previous studies, such as that of Yadav et al. (2014) which have reported
about the relationship between total phenolic content and antioxidant activity of ethyl acetate extracts
of endophytic fungi.
   Literature on Curvularia sp. isolated from various medicinal plants suggests that about 143
secondary metabolites were purified from their crude extracts. These metabolites belonged to several
chemical classes such as alkaloids, anthraquinones, polyketides, quinones, terpenes and peptides.
Several isolated compounds showed diverse biological activities including antimalarial, antifouling,
antilarval, anti-inflammatory, anti-oxidant, anti-bacterial, anti-fungal, anti-cancer, leishmanicidal
properties and phytotoxicity (Khiralla et al., 2019). The new endophytic fungus Curvularia sp. T12
was isolated from the medicinal plant Rauwolfia macrophylla Stapf. Its large scale fermentation and
working up of the crude extract, led to the isolation of 2’-deoxyribolactone, hexylitaconic acid and
ergosterol. These compounds displayed antimicrobial, antioxidant and acetylcholinesterase inhibition
activities (Kaaniche et al., 2019).
   In conclusion the study provides an overview of the role of endophytes in providing new compounds
or leads for treating certain medical disorders. Curvularia lunata isolated from F. religiosa showed
greater anti-oxidant and anti-diabetic potential and this can be attributed to various phytochemicals
detected in the ethyl acetate extract of the fungus. However, it is pertinent to mention that results
obtained are performed as in vitro experiments and need to be confirmed by in vivo tests. Future
direction for this study would be elucidation of the mode of inhibition and structural characterization
of the bio-active fractions and QSAR studies. There is a further need to evaluate the toxicological
properties of these extracts/compounds obtained from extracts and test for ADME properties.

Acknowledgements
Authors gratefully acknowledge the benediction of the Founder Chancellor of the Institute, Bhagawan
Sri Sathya Sai Baba. Authors are grateful to University Grants Commission (UGC) for providing
BSR Fellowship in Sciences and to UGC SAP DRS level II funding for infrastructural support.



References
Alqahtani AS, Hidayathulla S, Rehman MT, El Gamal AA, Al-Massarani S, Razmovski-Naumovski
     V, Alqahtani MS, El Dib RA, AlAjmi MF (2019) Alpha-amylase and alpha-glucosidase enzyme
     inhibition and antioxidant potential of 3-oxolupenal and katononic acid isolated from Nuxia
     oppositifolia. Biomolecules 10(1):61. https://doi.org/10.3390/biom10010061
Bacon CW, White J (2000) Microbial endophytes. CRC press, Boca Raton.


                                                  18
Jayant, Vijayakumar                                         Italian Journal of Mycology 50 (2021): 10-20

https://doi.org/10.6092/issn.2531-7342/12104



Bhardwaj A, Sharma D, Jadon N, Agrawal PK (2015) Antimicrobial and phytochemical screening of
      endophytic fungi isolated from spikes of Pinusroxburghii. Archives of Clinical Microbiology
      6 (3):19.
Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free radical method to evaluate
      antioxidant activity. LWT Food Science Technology 28:25-30. https://doi.org/10.1016/S0023-
      6438(95)80008-5
da Silva M, Cueva-Yesquén LG, Júnior SB, Garcia VL, Sartoratto A, de Angelis DF, de Angelis DA
      (2020) Endophytic fungi from Passiflora incarnata: an antioxidant compound source. Archives
      of microbiology 202(10):2779–2789. https://doi.org/10.1007/s00203-020-02001-y
Devi NN, Prabakaran JJ, Wahab F (2012). Phytochemical analysis and enzyme analysis of endophytic
      fungi from Centella asiatica. Asian Pacific Journal of Tropical Biomedicine 2:S1280–S1284.
      https://doi.org/10.1016/S2221-1691(12)60400-6
Government of India, Ministry of Ayush (2016) The ayurvedic pharmacopoeia of India. Pharmacopoeia
      Commission for Indian Medicine & Homoeopathy, pp. 17-20.
Halliwel B, Guttridge JM (1989) Free radicals in biology and medicine, 2nd ed. Clarendon Press,
      London.
Hallmann J, Berg G, Schulz B (2006) Isolation procedures for endophytic microorganisms. In: Schulz
      BJE, Boyle CJC, Sieber TN (eds) Microbial Root Endophytes. Soil Biology, vol 9. Springer,
      Berlin, Heidelberg. https://doi.org/10.1007/3-540-33526-9_17
Harborne JB (1998) Phytochemical methods A guide to modern techniques of plant analysis. Chapman
      and Hall Limited, 3rd ed. London, pp 142-175.
Ji HF, Li XJ, Zhang HY (2009) Natural products and drug discovery. EMBO Reports 10:194-200.
      https://doi.org/10.1038/embor.2009.12
Kaaniche F, Hamed A, Abdel-Razek AS, Wibberg D, Abdissa N, El Euch IZ, et al. (2019) Bioactive
      secondary metabolites from new endophytic fungus Curvularia. sp isolated from Rauwolfia
      macrophylla. PLoS ONE 14(6), e0217627. https://doi.org/10.1371/journal.pone.0217627
Khiralla A, Spina R, Saliba S, Laurain-Mattar D (2019) Diversity of natural products of the genera
      Curvularia and Bipolaris. Fungal Biology Reviews 33:101-122. https://doi.org/10.1016/j.
      fbr.2018.09.002
Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of
      secondary metabolites. Chemistry & Biology 19(7):792–798. https://doi.org/10.1016/j.
      chembiol.2012.06.004
Manganyi MC, Ateba CN (2020) Untapped potentials of endophytic fungi: A review of novel bioactive
      compounds with biological applications. Microorganisms 8(12):1934. https://doi.org/10.3390/
      microorganisms8121934
Manzocco L, Anese M, Nicoli MC (1998) Antioxidant properties of tea extracts as affected by
      processing. LWT - Food Science and Technology 31(7-8):694-698. https://doi.org/10.1006/
      fstl.1998.0491
Mehta JL, Rasouli N, Sinha AK, Molavi B (2006) Oxidative stress in diabetes: a mechanistic
      overview of its effects on atherogenesis and myocardial dysfunction. International Journal of
      Biochemistry and Cell Biology 38(5-6):794–803. https://doi.org/10.1016/j.biocel.2005.12.008
Mishra PD, Verekar SA, Kulkarni-Almeida A, Roy S K, Jain S, Balakrishnan A, Vishwakarma R,
      Deshmuk SK (2013) Anti-inflammatory and anti-diabetic naphthoquinones from an endophytic
      fungus Dendryphion nanum (Nees) S. Hughes. Indian Journal of Chemistry Section B 52:565-
      567.
Nair SS, Kavrekar V, Mishra A (2013) In-vitro studies on alpha amylase and alpha glucosidase
      inhibitory activities of selected plant extracts. European Journal of Experimental Biology
      3(1):128-132.

                                                19
Jayant, Vijayakumar                                          Italian Journal of Mycology 50 (2021): 10-20

https://doi.org/10.6092/issn.2531-7342/12104



Roglic G (2016) WHO Global report on diabetes: a summary. International Journal of
      Noncommunicable Diseases 1:3-8.
Singh D, Singh B, Goel R (2011) Traditional uses, phytochemistry and pharmacology of Ficus
      religiosa: a review. Journal of ethnopharmacology 134:565-83. https://doi.org/10.1016/j.
      jep.2011.01.046
Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G (2007) Antioxidant measurements. Physiological
      measurement 28(4):R41–55. https://doi.org/10.1088/0967-3334/28/4/R01
Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products.
      Microbiology and Molecular Biology Reviews 67(4):491–502. https://doi.org/10.1128/
      MMBR.67.4.491-502.2003
Tiwari P, Gupta R (2020) Preliminary phytochemical screening of bark (powder) extracts of Ficus
      religiosa (peepal) plant. International Journal of Research and Development in Pharmacy &
      Life Sciences 9(1):1-6. https://doi.org/10.47128/IJRDPL.2278-0238.2020.9(1).1-9
Tiwari P, Nathiya R, Mahalingam G (2017) Antidiabetic activity of endophytic fungi isolated from
      Ficus Religiosa. Asian Journal of Pharmaceutical and Clinical Research 10(4):59-61. https://
      doi.org/10.22159/ajpcr.2017.v10i4.14718
Uttara B, Singh AV, Zamboni P, Mahajan R (2009) Oxidative stress and neurodegenerative
      diseases: A review of upstream and downstream antioxidant therapeutic options. Current
      Neuropharmacology 7(1):65–74. https://doi.org/10.2174/157015909787602823
Vimalkumar CS, Hosagaudar VB, Suja SR, Vilash V, Krishnakumar NM, Latha PG (2014)
      Comparative preliminary phytochemical analysis of ethanolic extracts of leaves of Olea dioica
      Roxb., infected with the rust fungus Zaghouania oleae (E.J. Butler) Cummins and non-infected
      plants. Journal of Pharmacognosy and Phytochemistry 3(4):69-72.
Weseler AR, Bast A (2010) Oxidative stress and vascular function: implications for pharmacologic
      treatments. Current hypertension reports 12(3):154–161. https://doi.org/10.1007/s11906-010-
      0103-9
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal
      ribosomal RNA enes for phylogenetics. In: Innis, MA, Gelfand DH, Sninsky JJ, White TJ
      (eds.), PCR Protocols: A guide to methods and applications. Academic Press, New York, pp
      315-322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1
Yadav M, Yadav A, Yadav J P (2014) In vitro antioxidant activity and total phenolic content of
      endophytic fungi isolated from Eugenia jambolana Lam. Asian Pacific journal of tropical
      medicine 7(S1):S256–S261. https://doi.org/10.1016/S1995-7645(14)60242-X
Zhang YJ, Zhang S, Liu XZ, Wen HA,Wang M (2010) A simple method of genomic DNA extraction
      suitable for analysis of bulk fungal strains. Letters in applied microbiology 51(1):114–118.
      https://doi.org/10.1111/j.1472-765X.2010.02867.x




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