This document summarizes the evaluation of anti-HIV potential in selected medicinal plants. Cell culture assays were conducted to test crude extracts of several plants for anti-HIV activity. Methanolic extracts of Ocimum gratissimum, Tinospora sp., and Swertia bimaculata showed anti-HIV activity, reducing green foci in the cell culture. Further fractionation of the active O. gratissimum extract did not identify any fractions with improved anti-HIV activity compared to the crude extract. The study found preliminary evidence of anti-HIV activity in some plant extracts warranting further investigation.

1. Evaluation of anti-HIV potential
in selected medicinal plants
Titas Mallick. Sem IV. Dept. Of Botany. M.Sc. CU.
Reg No. 621-1121-1230-14
Roll No. 91/BOT/1710008
Under the guidance of
Prof. Binay Chaubey
Department of Botany
UNIVERSITY OF CALCUTTA

2. HIV as a global pandemic
36.9 Million
3%
14%
24%
2%
HIV Statistics, Report: UNAIDS, 2017
Total infected
children
unaware of HIV
No drug avilable
Death
Fig 1. Chart Showing UNAIDS 2017 statistic of HIV-AIDS Fig 2. Global distribution of AIDS
0.502% of the global population suffers from HIV, still no cure is known. And
no vaccines have been produced. HIV is truly a global pandemic.

3. HIV as a global pandemic
36.9 Million
3%
14%
24%
2%
HIV Statistics, Report: UNAIDS, 2017
Total infected
children
unaware of HIV
No drug avilable
Death
Fig 1. Chart Showing UNAIDS 2017 statistic of HIV-AIDS Fig 2. Global distribution of AIDS
0.502% of the global population suffers from HIV, still no cure is known. And
no vaccines have been produced. HIV is truly a global pandemic.

4. HIV and AIDS: Overview, causes, symptoms,
and treatments
• HIV is a Group VI ssRNA-RT virus belong to the genus Lentivirus.
• It infects the Human CD4 T cells. As a result of reduction of the number of T cells
in the immune system, the body becomes progressively more susceptible to
opportunistic infections, leading to the development of AIDS.
• The treatment includes HAART (highly active anti retroviral therapy) or PrEP (Pre-
exposure prophylaxis).
• HAART or highly active anti retroviral therapy includes a combination of 7 classes
of HIV drugs [non-nucleoside reverse transcriptase inhibitors (NNRTIs),
nucleoside reverse transcriptase inhibitors (NRTIs), post-attachment inhibitors,
protease inhibitors (PIs), CCR5 antagonists, integrase strand transfer inhibitors
(INSTIs), fusion inhibitors] that prevents the virus in different points.
• Constantly developing drug-resistant strain is the major challenge. So new drug
development is required.

5. HIV and AIDS: Overview, causes, symptoms,
and treatments
• HIV is a Group VI ssRNA-RT virus belong to the genus Lentivirus.
• It infects the Human CD4 T cells. As a result of reduction of the number of T cells
in the immune system, the body becomes progressively more susceptible to
opportunistic infections, leading to the development of AIDS.
• The treatment includes HAART (highly active anti retroviral therapy) or PrEP (Pre-
exposure prophylaxis).
• HAART or highly active anti retroviral therapy includes a combination of 7 classes
of HIV drugs [non-nucleoside reverse transcriptase inhibitors (NNRTIs),
nucleoside reverse transcriptase inhibitors (NRTIs), post-attachment inhibitors,
protease inhibitors (PIs), CCR5 antagonists, integrase strand transfer inhibitors
(INSTIs), fusion inhibitors] that prevents the virus in different points.
• Constantly developing drug-resistant strain is the major challenge. So new drug
development is required.

6. Plant could be a potent source of new drug research
• Plants naturally synthesize thousands of compounds. Many of the
naturally occurring phytochemicals show anti-viral properties.
• This huge numbers of phytochemicals could potentially be HIV
inhibitors.
• This natural compounds can be screened for their anti-HIV activity.
• Anti-HIV natural compounds could be used in new drug development.
• New HAART drug could be developed from these naturally occurring
phytochemicals.

7. Objectives of the project
• Objective 1: Bioactivity guided fractionations of selected plants.
• Selection of Plants
• Collection and Drying
• Crude extract preparation
• Fractionation through column chromatography.
• Objective 2: Evaluation of anti-HIV activity in cell culture and in in-vitro.
• Preparation of crudes and other sub-fractions
• Virus production and infection
• Cell culture assay
• In-silico analysis
• RT production and purification
• In-vitro assay.

8. Bioactivity guided fractionations of selected plants.
Collection of
plants
Drying and
grinding
Crude
extraction by
cold extraction
method
Bioactive
assays
Further
fractionations

9. Bioactivity guided fractionations of selected plants.
Collection of
plants
Drying and
grinding
Crude
extraction by
cold extraction
method
Bioactive
assays
Further
fractionations
1. Some plants are selected based on their specific selection criteria
2. Plants are dried and ground
3. Cold extraction performed in non-polar to polar solvents
4. Bioactive assays performed to evaluate crude extracts
5. Crudes showing anti-HIV activity were again fractioned through chromatographic methods and were
evaluated

11. Evaluation of anti-HIV activity in cell culture
Isolation of transfection
grade plasmid: pNL4-3-
EGFP (env-)
Isolation of transfection
grade plasmid: pVSV-G
Co transfection into 293
T cells
Production of pseudo
virus
Infecting Huh 7.5 cells
Application of crude at
24hr with controls
Microscopy: Count of
green foci
Positive Negative
Further
Fractionation
Plant Crude prepared MTT Assay
Drug dissolved below
its cytotoxic level

12. 293 T cells after 60 hours treated with Catharanthus crude extract in Ethyl Acetate
CC50 value 426.81 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Catharanthus crude extract in Methanol
CC50 value 449.64 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Catharanthus crude extract in Petroleum Benzene
CC50 value 451.95 µg/ml Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofCatharanthusroseus

13. 293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Ethyl Acetate
CC50 value 411.1 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy,
Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Methanol
CC50 value 448.5 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
Significant reduce in green foci is observed.
293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Petroleum Benzene
CC50 value 441.24 µg/ml Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
ResultsofCellCulturebasedAssay
ANTI-HIV ACTIVITY WAS DETECTED IN METHANOLIC CRUDE EXTRACT
EvaluationofCrudeextractsofOcimumgratissimum

14. 293 T cells after 60 hours treated with Tinospora crude extract in Petroleum benzene
CC50 value 411.1 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy,
Bottom row: Bright field Microscopy
Significant reduce in green foci is observed.
293 T cells after 60 hours treated with Tinospora crude extract in Ethyl Acetate
CC50 value 384.5 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
CC50 value 441.24 µg/ml 293 T cells after 60 hours treated with Tinospora crude extract in Methanol
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy, Bottom
row: Bright field Microscopy
ResultsofCellCulturebasedAssay
EvaluationofCrudeextractsofTinosporasp.
ANTI-HIV ACTIVITY WAS DETECTED IN PETROLEUM BENZENE METHANOLIC CRUDE EXTRACT

15. 293 T cells after 60 hours treated with Mangifera indica Hexane extract
CC50 value 418.5 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy,
Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Mangifera indica Chloroform extract
CC50 value 435.25 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Mangifera indica Methanolic extract
CC50 value 389.56 µg/ml Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofMangiferaindica

16. 293 T cells after 60 hours treated with Swertia bimaculata Hexane extract
CC50 value 411.62 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy,
Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Swertia bimaculata Chloroform extract
CC50 value 348.55 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Swertia bimaculata Methanolic extract
CC50 value 441.39 µg/ml Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
ResultsofCellCulturebasedAssay
ANTI-HIV ACTIVITY WAS DETECTED IN METHANOLIC CRUDE EXTRACT
EvaluationofCrudeextractsofSwertiabimaculata

17. 293 T cells after 60 hours treated with Solanum sp. Hexane extract
CC50 value 390.5 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy,
Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Solanum sp. Chloroform extract
CC50 value 357.3 µg/ml
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence
microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with Solanum sp. Methanolic extract
CC50 value 398 µg/ml Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofSolanumsp.

18. Further Fractionations of methanolic crude extract of
Ocimum gratissimum
• Methanolic crude extract of Ocimum gratissimum showed positive result in cell
culture assay, Further fractionation of the crude performed.
• Each Fractions were evaluated
Petroleum benzene fraction of Tinospora sp. and Methanolic fraction of Swertia
bimaculata also showed positive result but no further fractionations were done
due to lack of time and lack of sufficient amount of the samples
SOLVENT NUMBER OF FRACTION CC50 VALUES
E (Ethyl Acetate 100%) 3 392.5, 398, 384 µg/ml
E7M3 (Ethyl Acetate: Methanol 70:30) 4 395.6, 393, 440.25, 385.6 µg/ml
E5M5 (Ethyl Acetate: Methanol 50:50) 0 -
E3M7 (Ethyl Acetate: Methanol 30:70) 1 411.2 µg/ml
M (Methanol 100%) 4 393, 421.25, 389.6, 396.25 µg/ml

19. 293 T cells after 60 hours treated with E1
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with E2A
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with E2B
Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy, Bottom
row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofFractionsofOcimum
gratissimumMethanoliccrudeextract

20. 293 T cells after 60 hours treated with E7M3-1
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with E7M3-2
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with E7M3-3
Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy, Bottom
row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofFractionsofOcimum
gratissimumMethanoliccrudeextract

21. 293 T cells after 60 hours treated with E7M3-4
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with E3M7
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
Significant reduce in green foci is observed.
293 T cells after 60 hours treated with M1
Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy, Bottom
row: Bright field Microscopy
ResultsofCellCulturebasedAssay
ANTI-HIV ACTIVITY WAS DETECTED IN E3M7 FRACTION
EvaluationofCrudeextractsofFractionsofOcimum
gratissimumMethanoliccrudeextract

22. 293 T cells after 60 hours treated with M2
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with M3
Observed under a fluroscent microscope
Blank: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row:
Fluorescence microscopy, Bottom row: Bright field Microscopy
No significant reduce in green foci is observed.
293 T cells after 60 hours treated with M4
Control: No drug treated, DMSO: Solvent control, EFV: Efavirenz. Top row: Fluorescence microscopy, Bottom
row: Bright field Microscopy
ResultsofCellCulturebasedAssay
NO PROMINENT ANTI-HIV ACTIVITY WAS DETECTED IN ANY OF THEM
EvaluationofCrudeextractsofFractionsofOcimum
gratissimumMethanoliccrudeextract

23. Thin Layer Chromatography for further fractionation of crude
extract showing positive anti-HIV activity
• Thin Layer chromatography was performed to identify components of E3M7 (Ethyl Acetate 30%+ Methanol
70% - Methanolic Extract of Ocimum gratissimum) that showed anti-HIV activity in cell culture assay. Three
different bands were observed under UV. As solvent 100% Methanol was used.
Thin Layer chromatography to identify components of E3M7 (Ethyl Acetate 30%+ Methanol 70% -
Methanolic Extract of Ocimum gratissimum) showing three bands.
BAND Y X 𝑿
𝒀
Rf Value
Band 1 9.8 9.5 9.5
9.8
0.96
Band 2 9.8 8.4 8.4
9.8
0.857
Band 3 9.8 7.6 7.6
9.8
0.775

24. After cell culture assay, in-silico analysis was performed before
evaluating natural compounds in in-vitro analysis
• Semi-purified crude extract of Ocimum gratissimum showed positive anti-
HIV-1 activity in cell culture assay.
• But their target is unknown, i.e. it could be a RT inhibitor or PR inhibitor or
may be INT inhibitor.
• To check whether it have RT inhibitory property or not in-vitro studies are
required.
• Before in-vitro studies, in-silico analysis of natural compounds from O.
gratissimum was performed.

25. In-silico molecular docking of HIV-1 RT with different
natural compounds
3D PDB of HIV RT
downloaded
>3HVT|PDBID|
Literature studied:
GC MS studies [1], [2]
on O. gratissimum
List of Molecules
prepared
Molecules
downloaded from
PubChem as *.sdf
Converted to
.mol2 using
OpenBabel
Used as binding site
Used as ligands
Docked using igemDock,
Population size: 1000,
Generation: 40, Solution: 1
Default scoring Matrix
Results Analyzed
Standard Drugs
[1] Joshi, Dr. R. K.. (2016). GC-MS Analysis of the Essential Oil of Ocimum gratissimum L. Growing Desolately in South India. Acta Chromatographica. 29. 1-9.
10.1556/1326.2017.29.1.10.
[2] C.R. Unnithan and Undrala Sushen. Chemical composition of ocimum gratissimum l by gc-ms analysis. ejpmr, 2017,4(06), 410-412. ISSN 2394-3211

26. Results of in-silico docking of different drugs and compounds
from GC-MS analysis of Ocimum gratissimum with HIV-1 RT
114.393
94.2916
88.94
86.5418
84.6878
83.798
82.1133
79.6954
78.8038
78.062
73.2777
73.0664
72.8843
68.111
67.8104
67.1006
66.824
66.5471
65.4708
64.9085
64.8692
64.7053
64.0577
63.9537
63.512
63.4689
63.2099
63.0086
62.9203
62.8871
62.5295
62.4625
61.915
61.7671
61.3877
60.3479
60.2459
58.6813
58.6808
58.5824
56.3893
56.3679
55.9886
55.7536
55.2911
53.3258
52.6158
51.8232
51.6938
51.4951
50.9915
50.7968
50.4048
50.2654
50.1984
50.0245
48.8194
48.8129
47.3481
46.7882
46.0505
45.8804
45.7896
0
20
40
60
80
100
120
140
CID_2117
CID_2153
CID_12409
CID_6744
zidovudine
Rilpivirine
CID_3314
abacavirsulfate
CID_1752
Etravirine
lamivudine
CID_12302222
stavudine
CID_3327
CID_12389
CID_23274265
CID_5281516
CID_62566
CID_441005
CID_91457
CID_6918391
CID_519857
CID_64139
CID_15560276
CID_12302131
CID_3314
CID_7127
CID_19725
CID_440967
CID_5317570
CID_1742210
CID_442393
Efavirenz
CID_92138
CID_2237
CID_6448
CID_440666
CID_86609
CID_18815
CID_3314
CID_9018
CID_6549
CID_5281515
CID_12302844
CID_1930
CID_17100
CID_31253
CID_5281553
CID_62387
CID_10363
CID_101629835
CID_64685
CID_11230
CID_2537
CID_17900
CID_7461
CID_2758
CID_14745
CID_520384
CID_11463
CID_1105
CID_18818
CID_6616
AFFINITY->
Different Ligands

27. Results of in-silico docking of different drugs and compounds
from GC-MS analysis of Ocimum gratissimum with HIV-1 RT
114.393
94.2916
88.94
86.5418
84.6878
83.798
82.1133
79.6954
78.8038
78.062
73.2777
73.0664
72.8843
68.111
67.8104
67.1006
66.824
66.5471
65.4708
64.9085
64.8692
64.7053
64.0577
63.9537
63.512
63.4689
63.2099
63.0086
62.9203
62.8871
62.5295
62.4625
61.915
61.7671
61.3877
60.3479
60.2459
58.6813
58.6808
58.5824
56.3893
56.3679
55.9886
55.7536
55.2911
53.3258
52.6158
51.8232
51.6938
51.4951
50.9915
50.7968
50.4048
50.2654
50.1984
50.0245
48.8194
48.8129
47.3481
46.7882
46.0505
45.8804
45.7896
0
20
40
60
80
100
120
140
CID_2117
CID_2153
CID_12409
CID_6744
zidovudine
Rilpivirine
CID_3314
abacavirsulfate
CID_1752
Etravirine
lamivudine
CID_12302222
stavudine
CID_3327
CID_12389
CID_23274265
CID_5281516
CID_62566
CID_441005
CID_91457
CID_6918391
CID_519857
CID_64139
CID_15560276
CID_12302131
CID_3314
CID_7127
CID_19725
CID_440967
CID_5317570
CID_1742210
CID_442393
Efavirenz
CID_92138
CID_2237
CID_6448
CID_440666
CID_86609
CID_18815
CID_3314
CID_9018
CID_6549
CID_5281515
CID_12302844
CID_1930
CID_17100
CID_31253
CID_5281553
CID_62387
CID_10363
CID_101629835
CID_64685
CID_11230
CID_2537
CID_17900
CID_7461
CID_2758
CID_14745
CID_520384
CID_11463
CID_1105
CID_18818
CID_6616
Natural Compounds with potential activity
Natural Compounds without significant activity
Anti HIV Drugs (NRTIs, NNRTIs)
AFFINITY->
Different Ligands

28. Natural compounds showing comparable binding affinity with established
anti-HIV-1 medicines in in-silico docking with HIV-1 RT
Compound ID Compound Name Formula Affinity
(igemdock arbitrary unit)
CID_2117 DL-alpha-Tocopherol acetate C31H52O3 114.393
CID_2153 Theophylline C7H8N4O2 94.2916
CID_12409 Nonacosane C29H60 88.94
CID_35370 Zidovudine C10H13N5O4 84.6867
CID_3314 Eugenol C10H12O2 82.1133
CID_1752 4-Nonylphenol C15H24O 78.8038
CID_12302222 Isopropyl-dimethyl-
octahydronaphthalen-ol
C15H26O 73.0664
CID_3327 Farnesol C15H26O 68.111
CID_64139 Efavarinez C14H9ClF3NO2 61.915
Results Suggests some molecules significantly binds with HIV RT at its active binding site. Some of the molecules
even binds with RT with more stability than some established drugs. So in-vitro studies are needed.

29. Evaluation of anti-HIV activity in in-vitro assay
Primary Culture
of E. coli BL21DE3
containing
PET28a-RT51,66
Secondary
Culture
Induction
Cell Pellet Sonication
Ni-iMAC
Dialysis
Quantification RNA isolation
RT PCR
RT activity
tested
Pico-green Assay
Evaluation of natural
compounds

30. Result of Protein production and purification
FIG1. Gel showing induction with IPTG. Lane 1: 1x dye, Lane 2: RT66 (uninduced), Lane 3: RT66 (induced), Lane 4: BSA, Lane 5: RT51
(Uninduced), Lane 6: RT51 (Induced), Lane 7: BSA, Lane 8 & 9: RT51+RT66
FIG2. Gel showing Flow through in Lane 1, 2 and 3. Lane 4: BSA, Lane 5: Last elution
FIG3. Gel showing Elution. Lane 1: 1x dye, Lane 2: BSA, Lane 3-10: Every Odd elution.
FIG 4. Gel showing RT51 after second dialysis. Lane 2, 8: BSA, Lane 4, 6: RT51
Protein Quantification Result: The concentration of RT51 is 3.7 µg/ml and the concentration of RT66 is 4.2 µg/ml.
Further work was not continued due to lack of time.
FIG 1 FIG 2 FIG 3 FIG 4

31. Limitations and future prospects
• In-vitro assay was not performed due to lack of time, in-vitro assay needed to be done
• Further evaluation of TLC derived compounds
• Fractionation and evaluation of other crudes showing positive activity
• Extraction of natural compounds that showed promising results in in-silico analysis using
different solvents and their evaluation
• Evaluation of natural compounds in in-vitro analysis that showed promising result in in-
silico experiment.

32. Conclusions
• Plants have immense potential in anti-HIV drug research
• More and more traditional and medicinal plants are needed to be
screened
• Identification of molecular mechanism of anti-HIV activity by the
phytochemicals are required.
• Systematic studies of phytochemicals having anti-HIV activity in drug
development is needed

33. Thank You