The document summarizes a study on isolating and characterizing endophytic fungi from Ocimum tenuiflorum (holy basil) leaves. Two fungi, Aspergillus sp. and Fusarium sp., were isolated. Phytochemical analysis found similar compounds in both fungi. Aspergillus sp. showed higher total phenolic content and antioxidant activity. Both fungi were able to synthesize silver nanoparticles when added to silver nitrate solution. In conclusion, the endophytic fungi of O. tenuiflorum contain beneficial compounds and show potential for applications in treating diseases.

1. M.Sc. 4th Semester
Examination
BIOTECHNOLOGY
Roll: PG/ VUOGP57/BIT IV S
No. 044
Reg. No. : 02195 of 2020-21
Evaluation Of Secondary
Metabolites Of
Endophytic Fungi Isolated From
Ocimum
Tenuiflorum
GUIDED BY: Dr. Debjani Roychowdhury (Asst. Professor, OIST, Burdwan)
ORIENTAL INSTITUTE OF SCIENCE AND TECHNOLOGY,BURDWAN

2. Items to
cover
OBJECTIVE OF THE STUDY
INTRODUCTION
MATERIALS & METHODS
RESULT
CONCLUSION

3. INTRODUCTION
Ocimum tenuifloram Known as "Queen of Herbs",
"The Incomparable One", 'Mother Medicine of Nature".
 Called "Elixir of life”.
Commonly known as basil, tulasi or tulsi.
Belongs to family Lamiaceae.
Holy basil is an erect, many-branched subshrub.
30—60 cm tall with hairy stems.
Leaves are green or purple.
The three main morphotypes cultivated in India and Nepal are Ram tulsi,
Krishna tulsi, vana tulsi.
Benefits : Protection and detoxification, Toxicant stress, Anti bacterial,
Metabolic stress, Anti cancerous, Antioxidant and many others.

4. Endophytes
2. Discussion – Virus, a mortal enemy
"microbes that colonize living internal tissues of plants
without causing any immediate, overt negative effects"
• Found in virtually every plant on earth.
• Reside in the living tissues of the host plant and do so in a
variety of relationships ranging from symbiotic to
pathogenic.
• "Plant host is able to supply the necessary nutrients and the
compounds required for the endophyte to complete its life
cycle
• Endophytes produce a wide range of bioactive natural
compounds.
• Plants are directly used as medicines by a majority of
cultures around the world, for example Chinese
medicine and Indian medicine (Ayurveda).
• Medicinal plants are known to harbour Endophytic fungi.

5. OBJECTIVE OF THE STUDY
 Isolation of different endophytes from the leaves of Ocimum tenuiflorum
 Identification and characterization of the different endophytic fungi
 Detection of phytochemicals of the isolated fungi
 Study of antioxidant activity of isolated fungi
 Study of Nanoparticle formation of the fungal isolates

6. MATERIALS
 Leaves of Ocimum tenuiflorum
 PDA (Potato dextrose agar)  For staining:
 Lactophenol Cotton Blue (Used
as fungal Stain)
 PDB (Potato dextrose broth)
 For surface sterilization:
 70% ethanol
 Sodium hypochlorite (2%).
 Double distilled Water
 Qualitative Identification:
 Hydrochloric acid
 Hager’s reagent
 Benedict’s reagent
 Fehling’s reagent
 Conc. Nitric acid
 Sodium hydroxide solution
 Ferric chloride solution
 Glacial acetic acid
 Quantitative estimation:
 DPPH Solution (2,2-diphenyl-1-
picryl-hydrazyl-hydrate)
 1mM Silver Nitrate
 Glasswares:
 Petriplates
 Beaker
 Conical flask
 Test tubes

7. Methods
Sample Collection
(Plant material
collection)
Medicinal Plant Ocimum
tenuiflorum
Surface sterilization
Using 2% sodium
hypochloride,75% ethanol and
distilled water
Isolation of
Endophytic Fungi
sterilized samples were placed
in potato dextrose agar for 10-
12 days incubation at room
temp
Identification of
Endophytic Fungi
using
Lactophenol cotton blue stain
and observed under low and
high power of microscope.
Preparation of fungal
extract
endophytic fungi were inoculated into
250 ml.
flasks containing 100 ml potato
dextrose broth and incubated at room
temperature for 12 days
Qualitative
Identification of
Phytochemicals from
Endophytic
Fungal Extracts
Quantitative estimation of
phenolics:
1. Study of antioxidant
activity
2. Characterization of
Silver Nanoparticles
Result Conclusion

8. Result
Name of the plant Segment of the plant Name of the fungus isolated
Ocimum tenuiflorum Leaves
Aspergillus Sp.
Fusarium Sp.
Fig(a) & Fig(b), both inoculation of sample in Potato Dextrose
Agar (PDA) media
Fig (c ) & Fig (d) ,Endophytic fungi grown from sterilized
inoculated segment of Ocimum tenuiflorum on PDA

9. Fig (g) 1 A in
100X
Fig (h) 2B in
100X
Fig (g ), (h ), Microscopic image of endophytic fungi

10. Qualitative Estimation:
Comparative analysis of phytochemical constituents of two different species of Fusarium Sp.&
Aspergillus Sp.
Phytochemical
constituents
Chemical tests Aspergillus Sp. Fusarium Sp.
Alkaloids Hager’s test - -
Carbohydrates
(Reducing sugar)
Benedict’s test
Fehling’s test
+
+
+
+
Proteins Xanthoproteic test + +
Flavonoids Alkaline reagent test - +
Phenolic
compounds
Lead acetate test + +
Saponin Froth test - +
Cardiac
glycosides
Keller-killiani test + +
Key: + = Present and – = Absent

11. Quantitative estimation of phenolics:
Total phenolics was calculated using the standard curve of Galic acid in various concentrations and the value were
expressed as mg of gallic acid equivalent (GAE) per gram of sample extract. All data in the experimental results were
expressed as mean ± standard deviation of the measurements.
Table: OD values & conc of different sample extracts
Sample OD at 720nm Concentration Conc. (Mean ± SD)
1A
0.49 11.33
11.4±0.12
0.5 11.54
0.49 11.33
2B
0.25 6.17
6.24±0.12
0.26 6.39
0.29 6.17
0
2
4
6
8
10
12
14
Sample 1A Sample 2B
TOTAL PHENOLIC CONTENT
FIG : Graphical representation of Total phenolic contents of the extracts

12. Study of antioxidant activity
Among the dominant isolates of Ocimum tenuiflorum highest free radical scavenging activity
showed by Aspergillus sp (42.71±0.73) followed by Fusarium sp (34.89±0.73). Thus, it
is concluded that Aspergillus sp have the higher antioxidant activity among these two.
Table-: OD values(520nm) and % of antioxidant of different sample extract
Samples OD at 520nm % Of antioxidant % Of antioxidant (Mean±SD)
1A
0.42 34.37
34.89±0.73
0.41 35.93
0.42 34.37
2B
0.37 42.19
42.71±0.73
0.37 42.19
0.36 43.75
0
5
10
15
20
25
30
35
40
45
50
Sample 1A Sample 2B
ANTI-OXIDANT ESTIMATION
Fig : Sample 1A and 2B showing antioxidant activity

13. Characterization of Silver Nanoparticles
It was observed that solution of silver nitrate turned dark brown on addition of fungal
extract; it indicates the formation of AgNPs, while no color change was observed in the
absence of fungal extract.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 100 200 300 400 500 600 700
Absorbance
Wavelength(nm)
UV-Vis Spectra of Photosynthesized AgNps

14. Conclusion:
The phytochemical analysis of the fungal extracts of Ocimum tenuiflorum shows the presence of almost similar groups of
compounds. This present study can help in choosing superior race of the plant. It has also been found that the fungal
extracts of these (Ocimum tenuiflorum) contain a good quantity of phenolic compounds, good antioxidant activity and
synthesis of nanoparticles. These plants can be studied further to know their biological effects which could be a beneficial
in the treatment and controlling of various diseases.

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