Evaluation of anti-HIV potential in selected medicinal plants
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Acquired immunodeficiency syndrome (AIDS) is a global pandemic. Since its discovery >36.9 million people have lost their lives. Although highly active antiretroviral therapy (HAART) has been successful in significantly lowering viral titers as HIV-1 integrates into host genome it is not targeted by HAART and hence not eliminated. Therefore, the virus rebounds on discontinuation of drugs. Hence, patients are likely to take therapy for a lifetime and long-term drug treatment leads to severe drug-induced toxicity and also the emergence of multidrug-resistant viruses. It is pertinent to mention that the rate of emergence of resistant strains of HIV-1 is much greater than the rate of new drug development. Hence there is an urgent need to develop new strategies to tackle HIV-1 and emerging resistant strains. Compounds from plant origin have enormous potential in terms of their incomparable structural diversity and plant kingdom needs to be explored for anti-HIV-1 molecules. In the present study medicinal plants namely Catharanthusroseus, Ocimumgratissimum, Mangifera sp., Tinospora sp., Solanum sp., Swertia bimaculata were evaluated for their anti-HIV-1 activity in cell culture. Crude extracts of the dried plants were prepared using solvents viz. hexane, chloroform, and methanol or Ethyl Acetate. Extracts were dissolved in DMSO and filter sterilized and screened for anti-HIV-1 activity using pseudotyped HIV-1 virus with GFP (Green Fluorescent Protein) reporter system. Prior cytotoxic concentrations 50 (CC50) of each extract was determined and all the extracts used in the present study were below their respective CC50 concentrations. The significant anti-HIV-1 activity was observed in methanolic extracts of Ocimum gratissimum, ethyl Acetate extracts of Tinospora sp. Further fractionation of Ocimum gratissimum showed anti-HIV-1 activity in- E3M7 1 fraction.
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Evaluation of anti-HIV potential in selected medicinal plants
- 1. Evaluation of anti-HIV potential in selected medicinal plants Titas Mallick. Sem IIII. Dept. Of Botany. M.sc. CU.
- 2. 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 symptom ranges from fever and sore throat in early stages to diseases like Toxoplasmosis, HIV-related encephalopathy and many more that ultimately causes the death of the patient. • The treatment includes HAART (highly active anti retroviral therapy) or PrEP (Pre-exposure prophylaxis).
- 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 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.
- 5. HAART and necessity of new drug development • 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. • HAART is a potent solution but no sterilizing cure of the disease is possible. • The drugs doesn’t work for long because of the error prone replication machinery of the virus. The drug target sites of the enzymes alters and the drugs stop to work. 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. • Objective 2: Evaluation of anti-HIV activity in cell culture and in in-vitro.
- 8. 1. Bioactivity guided fractionations of selected plants. Collection of plants Drying and grinding Crude extraction by cold extraction method Bioactive assays Further fractionations
- 9. 1. 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. Further Fractinations • Methanolic crude extract of Ocimum gratissimum showed positive result in cell culture assay, Further fractionation of the crude performed • Each Fractions should be evaluated SOLVENT NUMBER OF FRACTION E (Ethyl Acetate 100%) 3 E7M3 (Ethyl Acetate: Methanol 70:30) 4 E5M5 (Ethyl Acetate: Methanol 50:50) 0 E3M7 (Ethyl Acetate: Methanol 30:70) 1 M (Methanol 100%) 4
- 12. 2. Evaluation of anti-HIV activity in cell culture and in in-vitro Isolation of transfection grade plasmid: pNL4-3- GFP (env-) Isolation of transfection grade plasmid: pVSV-G Co transfection into 293 T ells 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
- 13. 293 T cells after 60 hours treated with Catharanthus crude extract in Ethyl Acetate 293 T cells after 60 hours treated with Catharanthus crude extract in Methanol 293 T cells after 60 hours treated with Catharanthus crude extract in Petrolium Benzene 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
- 14. 293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Ethyl Acetate 293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Methanol 293 T cells after 60 hours treated with Ocimum gratissimum crude extract in Petroleum Benzene 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
- 15. 293 T cells after 60 hours treated with Tinospora crude extract in Petroleum benzene 293 T cells after 60 hours treated with Tinospora crude extract in Ethyl Acetate 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
- 16. 293 T cells after 60 hours treated with Mangifera indica Hexane extract 293 T cells after 60 hours treated with Mangifera indica Chloroform extract 293 T cells after 60 hours treated with Mangifera indica Methanolic extract 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
- 17. 293 T cells after 60 hours treated with Swertia bimaculata Hexane extract 293 T cells after 60 hours treated with Swertia bimaculata Chloroform extract 293 T cells after 60 hours treated with Swertia bimaculata Methanolic extract 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
- 18. 293 T cells after 60 hours treated with Solanum sp. Hexane extract 293 T cells after 60 hours treated with Solanum sp. Chloroform extract 293 T cells after 60 hours treated with Solanum sp. Methanolic extract 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.
- 19. 293 T cells after 60 hours treated with E1 293 T cells after 60 hours treated with E2A 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 E7M3-1 293 T cells after 60 hours treated with E7M3-2 293 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 E7M3-4 293 T cells after 60 hours treated with E3M7 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 293 T cells after 60 hours treated with M3 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 • Thin Layer chromatography was performed to identify components of E3M7 (Ethyl Acetate 30%+ Methanol 70% - Methanolic Extract of Ocimum gratissimum). Three different bands were observed under UV. 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. Cell culture assay to in vitro study • Cell culture assays shown positive anti HIV activity of some extracts. • Methanolic fraction of Ocimum gratissimum and Petroleum Benzene fraction of Tinospora sp. Have shown positive result. • 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-silico molecular docking was done.
- 25. In-silico molecular docking 3D PDB of HIV RT downloaded >1REV:A|PDBID| Literature studied: GC MS studies 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
- 26. Results of in-silico docking 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 Docking of HIV-1 RT with Different Drugs and Plant Molecules
- 27. Results of in-silico docking 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 Docking of HIV-1 RT with Different Drugs and Plant Molecules Natural Compounds with potential activity Natural Compounds without significant activity Anti HIV Drugs (NRTIs, NNRTIs)
- 28. Results of in-silico docking • 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. • Molecules with significant binding are: DL-Alpha-Tocopherol Acetate C31H52O3, CID 2117 and Theophylline, C7H8N4O2, CID 2153 and others.
- 29. Evaluation of anti-HIV activity in cell culture and in in-vitro 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 Radioactive Primer Extension Assay Evaluation of natural compounds
- 30. Radioactive primer extension assay Poly RA Oligo DT Primer Primer/template dimer Radio labeled 3HdTTP HIV RT as enzyme incubation Treatment with different Drugs Stop the Reaction Filtration Reading in scintillation counter PROCESS IS UNDER OPTIMISATION
- 31. Thank You