Antimicrobial and Enzyme Activity of Endophytic Fungi Isolated from Tulsi

Abstract

A study was carried out to isolate endophytic fungi from Ocimum species (Tulsi) and to assess their antimicrobial activity against Pseudomonas aeroginosa, Mycobacterium smegmatis, Salmonella typhimurium, Candida albicans and Penicillium chrysogenum and production of enzymes such as amylase, protease and tyrosinase. In this study forty endophytic fungi were isolated from leaves and branches of Tulsi. Out of forty endophytic fungal isolates, six isolates showed the inhibition activity against test microorganisms done by dual culture method. The isolate P14T1 exhibited a high antimicrobial activity against Candida albicans (22mm). The crude extract of P13T5 isolate showed highest zone of inhibition against Pseudomonas aeroginosa (21mm) by well and disc diffusion method. 50% of fungal isolates found positive for amylase and protease enzymes production and 27.5% showed positive for tyrosinase. The results of the study suggest that endophytic fungi associated with Tulsi are potential agents for antimicrobial activity and production of enzyme.

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Ananda K. et al. / JPBMS, 2012, 16 (12)
1 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 16, Issue 16
Available online at www.jpbms.info
JPBMS
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES
Antimicrobial and Enzyme Activity of Endophytic Fungi Isolated from Tulsi
N. Pavithra1, L. Sathish 2, K. Ananda*3
1,2 Research Scholar, Department of Biological Sciences, Poornaprajna Institute of Scientific Research, Devanahalli,
Bangalore- 562110, Karnataka, India.
3 Assistant Professor, Department of Biological Sciences, Poornaprajna Institute of Scientific Research,
Devanahalli, Bangalore- 562110, Karnataka, India.
Abstract:
A study was carried out to isolate endophytic fungi from Ocimum species (Tulsi) and to assess their antimicrobial activity
against Pseudomonas aeroginosa, Mycobacterium smegmatis, Salmonella typhimurium, Candida albicans and Penicillium
chrysogenum and production of enzymes such as amylase, protease and tyrosinase. In this study forty endophytic fungi
were isolated from leaves and branches of Tulsi. Out of forty endophytic fungal isolates, six isolates showed the inhibition
activity against test microorganisms done by dual culture method. The isolate P14T1 exhibited a high antimicrobial
activity against Candida albicans (22mm). The crude extract of P13T5 isolate showed highest zone of inhibition against
Pseudomonas aeroginosa (21mm) by well and disc diffusion method. 50% of fungal isolates found positive for amylase and
protease enzymes production and 27.5% showed positive for tyrosinase. The results of the study suggest that endophytic
fungi associated with Tulsi are potential agents for antimicrobial activity and production of enzyme.
Key Words: antimicrobial activity, enzymes, endophytic fungi, Tulsi.
Introduction:
Endophytic fungi are the microorganisms that are present
in living tissues of various plants, establishing mutual
relationship without causing any symptom of diseases.
Endophytes are rich sources of bioactive metabolites,
which have important potentials in medicine, agriculture
and industries [1-3]. The production of secondary
metabolites from endophytes is associated with
environmental factors. The endophytic interaction with its
host may favor the synthesis of secondary metabolites [4
_-6]. Endophytes are known to produce metabolites such as
alkaloids, terpenoids, steroids, quinones, isocoumarin
derivatives, flavanoids, phenols, phenolic acids, and
peptides. Some species produce novel antimicrobial agents
(cryptocandin from Cryptosporiopsis quercina), other
produce potent anti-cancer compounds (taxol from
Taxomyces andreanae) and yet others produce compounds
that can be utilized industrially, such as enzymes and
solvents [7].
The search of bioactive compound from isolated
endophytic fungi and higher plants are considered for
developing the therapeutic drugs. Thus there is a need to
isolate and synthesize the antibiotics, therapeutic agents
and agrochemicals from endophytic fungi, which are highly
effective, low toxic and having minor environmental
impact [8].
Ocimum (Tulsi) plants are considered as sacred plants in
India and known for its antimicrobial, immunomodulatory,
anti-stress, anti-inflammatory, antipyretic, anti-asthmatic,
hypoglycemic, hypotensive and analgesic activities [9-11]. It
is also reported that Ocimum Sanctum L exhibited
antibacterial activity against Klebisella, Escherichia coli,
Proteus, Staphylococcus aureus and Vibrio cholerae.
Maximum zone of inhibition was noticed against
Staphylococcus aureus [12]. Endophytic bacteria isolated
from leaves of Ocimum sanctum screened in dual culture
was found to be active against various phytopathogenic
fungi viz. Rhizoctonia solani, Sclerotium rolfsii, Fusarium
solani, Alternaria solani, and Colletotrichum
lindemuthianum [13]. Eleven endophytes have been isolated
from leaves of O. sanctum and their biodiversity has been
reported by Banerjee at al [14]. The endophytic fungal
extract from leaf of Ocimum basilicum was found to have
antimicrobial activity against Bacillus cereus and
Staphylococcus aureus [15]. Many of the drugs available
commercially are derived from plant-based chemicals.
Recent years the isolation of endophytic fungi and
screening of antimicrobial activity has gained more
attention [2]. The goal of this study was to isolate
endophytic fungi from Tulsi (Ocimum Sanctum) and screen
them for anti-microbial activities against Pseudomonas
aeroginosa, Mycobacterium smegmatis, Salmonella
typhimurium, Candida albicans and Penicillium
chrysogenum and the production of enzymes such as
amylase, protease and tyrosinase.
Materials and Methods:
Isolation of endophytic fungi
Isolation of endophytic fungi from Ocimum sanctum was
carried out using the protocol described by Strobel et al
with slight modifications [16]. Fresh plant materials
(Branches and leaves) were collected from the field located
at Devanahalli, Bangalore, India. The leaves and small
branches were washed under running tap water for 10
minutes. Before surface sterilization, leaves and branches
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2 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 16, Issue 16
were cut into small pieces with 1 cm long and sterilized in
series with 70% ethanol for 1 min, 1.0 % sodium
hypochlorite (NaOCl) (v/v) for 1 min and further cleaned
by passing through two sets of sterile distilled water. The
sterile samples were placed on plate containing potato
dextrose agar (PDA) media with 200 mg/L concentration
of streptomycin to suppress the bacterial contamination.
The parafilm wrapped petridishes were incubated at 25± 2
°C till the fungal mycelia starts growing from the samples.
The endophytic fungi was transferred into a new agar
slants and stored at 4°C for the further studies.
Test microorganisms
Lyophilized three test bacteria and two test fungi cultures
were purchased from Microbial Type Culture Collection
(MTCC) of Institute of Microbial Technology, Chandigarh.
Cultures of Pseudomonas aeroginosa (MTCC No. 4676),
Mycobacterium smegmatis (MTCC No. 943), Salmonella
typhimurium (MTCC No. 3232) were grown on nutrient
agar media and used for antibacterial activity. Candida
albicans (MTCC No. 183) were grown on Yeast extract
Peptone Dextrose Agar (YEPD) and Penicillium
chrysogenum (MTCC No. 6795) were grown on Czapek
yeast extract agar (CYA) and used for antifungal activity.
0.5 McFarland standard bacterial and fungal suspension
was used for antimicrobial assay.
Preliminary-Antimicrobial assay
Antimicrobial activity of isolated endophytic fungi was
tested based on the protocol of Zhang et al with slight
modifications [17]. The petri dishes containing respective
media for the growth of bacteria and fungi were prepared
and 100 µL of test organism was spread over the surface of
the agar media using sterile cotton swab. Nine millimeter
diameter of actively growing fungal culture discs from PDA
plates were cut using a sterile cork borer and placed on the
surface of the respective agar media seeded with test
bacteria or fungi. These plates were sealed with parafilm
and kept in refrigerator at 4°C for 12 hours for complete
diffusion of antimicrobial compounds if any, thereafter
they were incubated at room temperature for next 12
hours for organisms such as Pseudomonas aeroginosa,
Mycobacterium smegmatis, Salmonella typhimurium and
Candida albicans and 48-60 hours for Penicillium
chrysogenum. After incubation the diameter of the
inhibition zone was measured in millimeter by using scale.
The experiment was carried out in triplicates.
Secondary metabolites extraction
Secondary metabolites extraction was carried out by using
Radji et al method [18]. Positive endophytic fungal isolates
were inoculated into 250 mL Erlenmeyer flasks containing
100 mL potato dextrose broth and incubated at room
temperature for 21 days under stationary conditions with
intermittent shaking. The broth culture was filtered to
separate the mycelia and filtrate. To the filtrate equal
volume of ethyl acetate was added, mixed well for 10
minutes and kept for 5 minutes till the two clear
immiscible layers formed. The upper layer of ethyl acetate
containing the extracted compounds was separated using
separating funnel. The mycelium was grinded properly in a
pestle and mortar using ethyl acetate as solvent and then it
was filtered using cheese cloth. Both mycelia and culture
filtrate extracts were pooled together and evaporated to
dryness in hot air oven. The extract residue was dissolved
in dimethyl sulfoxide (DMSO) and stored at 4°C to be used
as stock solution for antimicrobial assay.
Secondary antimicrobial assay
Antimicrobial activity of secondary metabolites was tested
by disc and well diffusion method [19-20]. The sterile growth
media plates specific for test organisms were prepared and
inoculated with the test organism. Twenty micro liter of
crude extract was added on to a sterile 6 mm paper disc
(Hi-Media) using a micropipette and allowed to dry. Discs
containing compounds were placed on the surface of the
medium. In the same plate 6 mm diameter wells were
made using a sterile cork borer and 20 µL of sample was
added to each well. The experiment was carried out in
triplicates. Ciprofloxacin (5 mcg; Hi-Media) was used as
positive control for bacteria. Flucanozole (5 mg/ mL;
Mankind pharma Ltd) was used as a positive control for
fungi. Similarly, 20 µl of DMSO as well as ethyl acetate were
used as negative controls. The plates were incubated at 35
± 2°C for 12-24 hours for bacteria of Pseudomonas
aeroginosa, Mycobacterium smegmatis and Salmonella
typhimurium, 30 ±2 °C for 12-24 hours for Candida albicans
and 28-30 °C for 48-60 hrs for fungi Penicillium
chrysogenum. The diameter of inhibition zone around the
disc and well was measured by using scale.
Enzyme assay
The production of enzyme by fungal endopytes was
qualitatively determined by using Hankin and
Anagnostakis method [21]. Amylase enzyme activity was
assessed by growing the fungi on glucose yeast extract
peptone (GYP) agar medium (glucose-1g, yeast extract
0.1g, peptone 0.5g, agar 16 g, distilled water 1000mL and
pH 6) containing 1% soluble starch. After 5 days
incubation, the plates with fungal colony were flooded with
1% iodine in 2% potassium iodide. The appearance of clear
zone surrounding the colony was considered positive for
amylase enzyme.
Protease assay was performed by growing the fungi on
GYP agar medium amended with 0.4 % gelatin and
adjusted the pH to 6. After 5 days of incubation, plates
were flooded with saturated aqueous ammonium sulphate.
The undigested gelatin will precipitate with ammonium
sulphate and digested area around the fungal colony would
appear as clear zone.
Similarly, Tyrosinase activity of fungal isolates was
determined by growing them on GYP agar medium. After 5
days of fungal growth a mixture of 0.11% p-cresol and 0.05
% glycine was overlaid on the surface of the fungal colony.
Culture plates were observed after 24 hours for the
appearance of reddish brown color around the colony
which indicates the presence of tyrosinase enzyme.
Results:
A total of 40 endophytic fungi were isolated from leaves
and branches of Ocimum sanctum. Each isolate were sub-
cultured into a PDA agar plates to remove the adherent
plant metabolite from the mycelia and stored at 4°C for the
further studies. The anti-microbial activity of isolated
endopytic fungi tested against representative Gram
positive and Gram negative bacteria, yeast and a
filamentous fungi by dual culture, well diffusion and disc
diffusion method which have been reported in the Table 1.
Six endophytic fungal isolates inhibited three test
microorganisms (Pseudomonas aeroginosa, Mycobacterium
smegmatis & Candida albicans) in all tested methods and
the zone of inhibition was in the range of 10 to 22mm
diameter. All the six isolates inhibited Candida albicans

Ananda K. et al. / JPBMS, 2012, 16 (12)
3 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 16, Issue 16
significantly high compared to other tested microbes and even it is nearly equivalent to the standard antibiotic tested.
Further the isolate P14T1 exhibited a highest antimicrobial activity against Candida albicans (22mm) compared to other
isolates. The isolate P13T5 showed highest zone of inhibition against Pseudomonas aeroginosa (21mm) by well diffusion
and disc diffusion method. Out of six bioactive fungal isolates one isolate (P13T5) showed inhibition of Salmonella
typhimurium up to 16mm diameter inhibition zone. None of the isolates could inhibit the filamentous fungi Penicillium
chrysogenum. Ciprofloxacin used as positive control for bacteria showed a zone of inhibition of 20 mm and Flucanozole
used as a positive control inhibited the fungi with a zone of inhibition of 20 mm diameter.
Table 1: Antimicrobial activity of endophytic fungi isolated from Tulsi
Fungal isolates were named based on the parent source. Abbreviations: P; plant, T; branches, L; leaf. PA-Pseudomonas aeruginosa, MS-Mycobacterium
smegmatis, ST-Salmonella typhimurium, CA-Candida albicans, PC-Penicillium chrysogenum
Figure 1 shows representative petri plates having
antimicrobial activity of endophytic fungal isolate P14L3
against Candida albicans (plate A) in dual culture method,
the crude ethyl acetate extracts of P14L3 fungal isolate
showed inhibition against Mycobacterium smegmatis by
disc diffusion (D) and well diffusion (W) method (Plate B)
and Plate (C) shows the positive (C1) and negative (C2)
controls of DMSO and ethyl acetate respectively.
The results of qualitative estimation of endophytic fungal
enzymes (amylase, protease and tyrosinase) in solid state
media are represented in the Table 2.
Figure 1: Antimicrobial zone of inhibition by endophytic fungi from Tulsi.
Representative petriplates showing inhibition of Candida albicans in Dual culture method (Plate A) and inhibition of Mycobacterium smegmatis by Disc
Diffusion (D) & Well diffusion (W) method by P14L3 isolate (Plate B). A Positive (C1) & Negative (C2) control is represented in (Plate C)
Endophytic
fungal
isolates
Zone of inhibition by the test microorganisms (mm)
(Mean±SD,n=3)
PA
MS
ST
CA
PC
Dual
Culture
method
P14T1
14.3±0.6
12.3±0.6
-
22.0±0.0
-
P14T2
12.6±0.6
12.0±0.0
-
13.6±0.6
-
P13T5
13.3±0.
6
11.3±0.6
16.3±0.6
16.0±1.0
-
P14L3
12.0±0.0
13.3±0.6
-
21.0±0.0
-
P14L2
10.0±0.0
12.0±0.0
-
12.6±0.6
-
P14T4
10.3±0.6
13.3±0.6
-
21.6±0.6
-
Well Diffusion method
P14T1
14.3±0.6
13.0±0.0
-
17.3±1.2
-
P14T2
17.0±0.0
14.0±0.0
-
17.6±1.2
-
P13T5
22.0±
0.0
20.0±0.0
09.0±0.0
20.0±0.0
-
P14L3
16.0±0.0
15.0±0.0
-
19.3±0.6
-
P14L2
17.0±0.0
15.0±0.0
-
20.6±0.6
-
P14T4
16.0±0.0
14.0±0.0
-
17.6±1.2
-
Disc Diffusion method
P14T1
12.6±0.6
13.0±0.0
-
15.3±1.6
-
P14T2
16.0±0.0
14.0±0.0
-
18.0±0.0
-
P13T5
21
.0±0.0
19.0±0.0
09.0±0.0
20.0±0.0
-
P14L3
15.0±0.0
13.6±0.6
-
18.0±0.0
-
P14L2
16.0±0.0
15.0±0.0
-
20.0±0.0
-
P14T4
14.0±0.0
13.0±0.0
-
17.0±0.0
-
Ciprofloxacin
standard
20.0±0.0
23.0±2.3
22.0±0.0
-
-
Flucanozole standard
-
-
-
22.0±0.0
20.0±0.0

Ananda K. et al. / JPBMS, 2012, 16 (12)
4 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 16, Issue 16
Table 2: List of endophytic fungal isolates producing enzymes
Test enzymes
Sl No.
Fungal isolates
Amylase
Protease
Tyrosinase
1
P02T2
+
+
+
2
P03T1
+
+
+
3
P11T3
+
+
-
4
P12L2
+
+
-
5
P13L2
+
+
-
6
P13T1
+
+
-
7
P13T12
+
+
+
8
P13T14
+
+
+
9
P13T2
+
+
-
10
P13T3
+
+
-
11
P1
3T5
+
+
+
12
P13T7
+
+
+
13
P14L1
+
+
+
14
P14L2
+
+
+
15
P14L3
+
+
+
16
P14L4
+
+
-
17
P14T1
+
+
-
18
P14T2
+
+
+
19
P14T4
+
+
-
20
P14T6
+
+
+
Qualitative analysis for the presence of enzymes tested for all the isolates and only positive isolates were listed. Fungal
isolates were named based on parent source. Abbreviations: P; plant, T; branches, L; leaf.
‘+’ indicates positive for enzyme test.
‘-’ indicates negative for enzyme test.
Fifty percent of the isolates showed positive for amylase
and protease activity and 27.5% of the isolates showed
positive for tyrosinase enzyme activity. Out of 40, only
seven isolates are producing tyrosinase enzyme. Figure 2
shows representative petri plates indicating the presence
of enzymes from isolated endopytic fungi by qualitative
test. In the figure 2, plate (A) showing a clear zone around
the fungal colony indicating the degradation of starch by
the amylase enzyme produced by the fungi. Plate (B in
figure 2) represents the production of protease enzyme by
endopytic fungi. The clear zone around the fungal colony
indicates degradation of gelatin due to protease activity.
Plate (C in figure 2) represents the production of
tyrosinase enzyme from isolated endophytic fungi.
Presence of reddish brown color around the fungal colony
indicates the tyrosinase activity.
Figure 2: Production of fungal enzymes by endophytic fungi from Tulsi.
Representative petriplates showing
A; Clear zone indicating degradation of starch by amylase enzyme,
B; Clear zone showing degradation of gelatin by the protease enzyme &
C; Reddish brown color under and around fungal colony indicating production of tyrosinase
Discussion:
The need for new antimicrobial agents, in general, comes
from the increasing rates of resistance to existing
antibiotics. This problem extends beyond the clinical
application of antimicrobial drugs, such as agricultural
microorganisms are also known to have acquired
resistance to commonly used antimicrobial chemicals [22].
Six of the endophytic fungi isolated from Tulsi have
significantly inhibited representative Gram positive, Gram
negative and yeast like fungi. The zones of inhibition by
these fungi are very much comparable to the standard
antibiotics. It has been reported that Pseudomonas
aeroginosa have developed drug resistance towards many
antibiotics [23]. In this study all the six isolates has inhibited
Pseudomonas aeroginosa in dual culture, well and disc
diffusion method. The crude extract of ethyl acetate was
dried in oven, dissolved in DMSO and used for well and disc
diffusion method gave better inhibition than dual culture
which leads to the conclusion that bioactive compounds
are easily extracted by the medium polar solvent. There
are also evidences that these compounds are consistently
produced by these isolates even after several generations
grown in lab using fungal media. Among the tested
organisms Candida albicans is most

Ananda K. et al. / JPBMS, 2012, 16 (12)
5 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 16, Issue 16
susceptible for the compounds produced by all six
endophytic fungal isolates. The zone of inhibition by the
endophytic fungal isolates for Candida albicans is up to 22
mm diameter, which is comparable to the inhibition by the
antifungal standard Fluconazole (22mm). The only
endophytic fungal isolate P13T5 has shown inhibition
against Salmonella typhimurium a representative of
typhoid causing bacteria. Inhibition of this bacterium
indicates that fungal isolate P13T5 might lead to a path for
the production of novel antibiotic against typhoid.
Inhibition of S.typhimurium is about 17 mm in dual culture
method but it has been reduced almost 50 % with the
crude ethyl acetate extract. This may be due to partial
extraction of bioactive compound by ethyl acetate, there is
need for a suitable extracting solvent for the bioactive
compounds extraction in this case.
The other important Gram positive bacteria
Mycobacterium smegmatis is also inhibited by the
endophytic fungi from Tulsi plant. This bacterium has lot of
similarity for the deadly organism M. tuberculosis. The
results of this study show that there are potential
endophytic fungi from tulsi which can produce compounds
which can destroy M. smegmantis, therefore possibly can
also act on M. tuberculosis. Detailed study of these
endophytic fungi for the production of bioactive
compounds against tuberculosis is worth doing. There is a
growing need for new, environmentally-friendly
antimicrobial agents that may be used safely to control
plant pathogens [24]. These active endophytic fungi might
be the future antibiotic producing microorganisms. Though
these six endophytic fungal isolates can inhibit yeast like
fungi, none of them are able to inhibit filamentous fungi
Penicillium chrysogenum which is interesting.
While much of the interest in endophyte bioactive
compound is for medicinal use, compounds that may have
industrial or agricultural applications are also gaining
attention. In particular, amylase is an important enzyme
that is used in numerous applications in a variety of
industries and there is growing interest in amylases with a
wider spectrum of biological properties that can function
at diverse pH and temperature ranges [25]. There are
reports on out of 17 endophytes isolated from Ocimum
sanctum only 3 endophytic bacteria were capable of
producing L-asparaginase enzyme [26]. In this study 40
endohpytic fungi isolated and twenty of them were able to
degrade starch by amylase an enzyme, which has wide
range of industrial applications. The rate of enzyme
production by these endophytic fungi would be estimated
after growing them in large batch culture for purification.
Present study indicates that some of these might produce
good amount (high activity) of amylase enzyme. Some of
the isolates have shown significant area of clear zone
where as some of them showed less area indicating
different rate of enzyme production by different isolates.
The same 20 fungal isolates that gave positive for amylase
enzyme were also found to produce proteases. Proteases
are used in clinical applications especially in the
treatments like diabetes. It is known from the history that
extracts of Tulsi plant being used for the diabetic control.
Proteases are one of the enzymes involved in controlling
diabetes as reported by Wiest-Ladenburger et al [27] whose
administration delays Insulin-dependent diabetes mellitus
(IDDM) onset in an animal model for autoimmune
diabetes, in the non obese diabetic mice.
We try to conjecture that the protease enzymes from the
endophytic fungi isolated from the Tulsi plant will have
similar properties. This can be confirmed only after
purification, characterization and testing of these
compounds in animal models.
Tyrosinase is known for lignin degradation and also has a
role in melanin synthesis. In the present study we have
screened at preliminary level and do not know the rate of
production of this enzyme by these endophytic fungi.
Further detailed study on this enzyme from the available
endophytic fungi is essential to get a clear picture. The
current study carried out paves way for wide openings in
endophytic fungal research tending towards development
of resistance in infectious microorganisms by determining
bioactive metabolites of various importance in medicine,
agriculture and industries.
Conclusion:
The present study leads to the need of further in depth
studies on these isolated bioactive endophytic fungal
isolates. Many are able to produce quite a good amount of
antimicrobial compounds tested in preliminary test.
Further growing those in large scale, modifying culture
conditions like changing pH, changing growth media and
supplying some stimulants might help in getting better
production of the particular bioactive compound and
enzyme. Further, the best proved active isolates should be
identified using available methods to place these fungi in
the fungal kingdom. There are no reported studies on
bioactive compounds and amylase, protease and tyrosinase
enzymes to the present knowledge.
Acknowledgement:
The authors of this research work would like to thank
Poornaprajna Institute of Scientific Research (PPISR) of
Admar Mutt Education Foundation (AMEF) for over all
support; mainly the financial support for the Research
Fellows, Faculty and providing research facilities. Authors
would like to thank Dr. A.B.Halgeri, Director, PPISR for his
valuable suggestions and Dr. K. Ravi Babu, ROHC(S),NIOH-
ICMR for his help in writing of this manuscript.
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Conflict of Interest: - None
Source of funding: - Admar Mutt Education Foundation, Udupi.
*Corresponding author:
Dr K. Ananda
Assistant Professor
Department of Biological Sciences , Poornaprajna Institute of Scientific Research
No.4, 16th Cross, Sadashivnagar, Bangalore-560080 ,Karnataka, India.
Phone: 080-23611836, M: 9945900336
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