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1 SUPPLEMENTARY MATERIAL Acetone and methanol fruit extracts of Terminalia paniculata inhibit HIV-1 infection in vitro Ankita Durge a, Pratiksha Jadaun a, Ashish Wadhwani a, Ashish A. Chinchansure b, Madhukar Said b, H.V Thulasiram b, Swati P. Joshi b and Smita S. Kulkarni a * a Department of Virology, National AIDS Research Institute, Pune , India b Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, , India *Corresponding author. skulkarni@nariindia.org Abstract In this study, we report the in vitro anti-hiv1 activity of acetone and methanol extracts of fruit of Terminalia paniculata. Cytotoxicity tests were conducted on TZM-bl cells and peripheral blood mononuclear cells (PBMC), the CC 50 values of both the extracts were 260 µg/ml. Using TZM-bl cells, the extracts were tested for their ability to inhibit replication of two primary isolates HIV-1 (X4, Subtype D), HIV-1 (R5, Subtype C). The activity against HIV-1 primary isolate (R5, Subtype C) was confirmed using activated PBMC and by quantification of HIV-1 p24 antigen. Both the extracts showed anti-hiv1 activity in a dose dependent manner. The EC 50 values of the acetone and methanol extracts of T. paniculata were 10.3 µg/ml. The enzymatic assays were performed to determine the mechanism of action which indicated that the anti-hiv1 activity might be due to inhibition of reverse transcriptase ( 77.7% inhibition) and protease ( 69.9% inhibition) enzymes. Keywords: Terminalia paniculata, anti-hiv1 activity, acetone extract, methanol extract, protease, reverse transcriptase
2 Experimental Plant collection and processing Terminalia paniculata fruits were collected from Varandha Ghat, Dist. Pune in December A herbarium sample was deposited in the Botanical Survey of India, Western Circle, Pune (Voucher No. SPTP2N). Fruits were shade dried and pulverized. Preparation of T. paniculata acetone (TPA) extract Pulverized fruits (3000 g) were extracted with acetone (4L 3 14h) at room temperature. The acetone-soluble fractions were filtered and concentrated under reduced pressure to yield a greenish acetone extract (150 g, 5% yield based on dry fruits). Preparation of T. paniculata methanol (TPM) extract TPM extract was prepared from the residue remained after acetone extraction. Residue was extracted with methanol (4L 3 14h) at room temperature. The methanol-soluble fractions were filtered and concentrated under reduced pressure to yield a brownish methanol extract (190 g, 6.33% yield). Cell line TZM-bl cell line was used for primary screening of extracts. The TZM-bl cell line is derived from recombinant HeLa cells containing β-galactosidase and luciferase genes under HIV-1 LTR promoter. The cells were obtained from National Institutes of Health AIDS Research and Reference Reagent Program (NIH ARRRP) and maintained in DMEM (Gibco- Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat inactivated FBS (Moregate, Australia), HEPES (Gibco, USA), penicillin and streptomycin (Gibco, USA). Cultures were incubated at 37 o C in a humidified 5% CO 2 atmosphere and used when 80% cell confluency was achieved. Phytochemical analysis The phytochemical analysis of TPA and TPM extracts was carried out as per the standard methodology (Kokate 1994; Harborne 1998).
3 Virus Stock The primary isolate HIV-1 UG070 (X4, Subtype D from Uganda) was obtained from NIH ARRRP, whereas Indian primary isolates HIV-1 VB59 and HIV-1 VB51 (R5, Subtype C) were isolated and propagated in our laboratory. The viruses were grown in PHA-p (5 µg/ml) activated peripheral blood mononuclear cells (PBMC) derived from healthy donors and quantified by HIV-1 p24 antigen detection assay (Advanced Bioscience Lab [ABL] Inc, USA). These virus stocks were titrated in the TZM-bl cell line and TCID50 (50% Tissue culture infective dose) of each virus stock was determined in the TZM-bl cells and PHA-p PBMC using Spearman Karber formula. Cytotoxicity assays The cytotoxicity of the extracts for TZM-bl cells and activated PBMC was determined using MTT [3-(4,5 dimethyl thiazole-2 yl) - 2,5-diphenyl tetrazolium bromide] (Sigma Aldrich, Inc., USA) assay. Briefly, two-fold dilutions of the working stock (1mg/ml) of each extract were prepared and added on overnight cultured TZM-bl cells ( cells/well). After incubation for 48 h, the cell viability was determined using MTT, the percent viability was determined and the results were expressed as CC 50 (concentration showing 50% cell viability). Assays for anti-hiv1 activity In vitro anti-hiv1 assay was carried out using published protocol (Geonnotti et al. 2014; Singh et al. 2015). Based on the CC 50 value, the anti-hiv1 activity was evaluated using a range of non cytotoxic concentrations of the extracts by following assays. In cell associated assay (CA), the TZM-bl cells ( cells/well) were first infected with the viral stocks (400 TCID 50 ) for 2 h at 37 o C in 5% CO 2 atmosphere before exposing the infected cells to the serial dilutions of extracts 0.78µg/mL to 100µg/mL in duplicate. After 48 h, luciferase activity was measured using Britelite plus reagent (Perkin Elmer, USA) (Singh et al. 2015). While in cell free assay (CF), the viral stocks (400 TCID 50 ) were first treated with the serial dilutions of extracts (0.78µg/mL to 100µg/mL) in duplicate for 1 h, at 37 o C in 5% CO 2 atmosphere preceding to its addition on the TZM-bl cells ( cells/well) and after 48 h, luciferase activity was measured. Azidothymidine (AZT) and Dextran sulfate (DS) were used as positive controls in cell associated and cell free anti-hiv1 assays, respectively. The EC 50
4 (effective concentration showing 50% virus inhibition) was calculated and compared with positive control. Confirmatory Assays using PBMC Briefly, in cell associated assay, PHA-p activated PBMC were infected with HIV-1 VB51 (400 TCID 50 ) and seeded ( cells/well). This was followed by the addition of sub toxic concentrations of extracts (0.78µg/mL to 100µg/mL). After 120 h, the virus supernatant was analyzed for p24 detection as described above. The EC 50 value was calculated and compared with positive control. For cell free assay, the viral stock of HIV-1 VB51 (400 TCID 50 ) was treated with the serial dilutions (0.78µg/mL to 100µg/mL) of extracts and incubated for 1 h. The pretreated virus was added onto activated PBMC ( cells/well) and incubated for 24 h. The next day, cells were washed with PBS and incubated for 120 h at 37 C after adding fresh media. After 120 h, virus supernatant was analyzed for p24 antigen detection as described above. Azidothymidine (AZT) and Dextran sulfate (DS) were used as positive controls in cell associated and cell free anti-hiv1 assays, respectively. HIV reverse transcriptase (RT) and protease assay (PR) The inhibitory action of TPA and TPM extracts on the HIV-1 reverse transcriptase (RT) and protease enzymes was evaluated using commercially available kits (Roche Applied Sciences Mannheim, Germany and Anaspec, CA, USA respectively) by following instructions of the manufacturers.
5 Table S1: In vitro cytotoxicity and anti-hiv1 activity of TPA and TPM extracts using TZM-bl cells and PBMC Details of the extract (*CC 5 0, µg/m L) Anti-HIV activity (**EC 50, µg/ml) TZM-bl PBMC HIV-1 UG070 HIV-1 VB59 HIV-1 VB51 *CC 50 HIV-1 VB51 CA CF CA CF CA CF, CA CF µg/m L) Acetone- Extract Methanol - Extract *CC 50 : The cytotoxic concentration of the extracts that caused the reduction of viable cells by 50%. All data presented are averages of three independent experiments. **EC 50 : The effective concentration of the extracts that resulted in 50% inhibition in HIV-1 infection. All data presented are averages of three independent experiments.
6 Table S2: Phytochemical analysis of TPA and TPM extracts T. paniculata Extracts Code Tannins Glycosid es Flavonoid s Saponins Phytosterol s Triterpen es Alkaloids Acetone Extract Methanol Fraction TPA TPM Key: +: Present in low quantity; ++: Present in moderate quantity; +++: Present in high quantity; - : Absent.
7 Figure S1: Anti-HIV1 activity of TPA and TPM extracts on TZM-bl reporter cell Legend: TZM-bl cell infected with (a, b) HIV-1 UG070 (X4, Subtype D) (c, d) HIV-1 VB59 (R5, Subtype C) and (e, f) HIV-1 VB51 (R5, Subtype C) through cell associated (CA) and cell free (CF) assays. AZT and DS were used as positive controls in CA and CF assays respectively. Data is represented as mean ± SE of three independent experiments
8 Figure S2: Anti-HIV1 activity of TPA and TPM extracts with HIV-1 VB51 infected PBMC (a) cell associated (CA) and (b) cell free (CF) assays Legend: Quantification of HIV-1 p24 antigen following treatment of extracts at various concentrations. AZT and DS were used as positive controls in CA and CF assays respectively. Data is represented as mean ± SE of three independent experiments.
9 Figure S3: Determination of mechanism of action Legend: Enzymatic assays (a) HIV1 RT inhibition assay (b) Protease inhibition assay. HIV-1 RT with AZT treatment and HIV-1 protease with pepstatin treatment were taken as positive controls in the respective experiment. Data is represented as mean ± SE of duplicates experiments. Referances Geonnotti A. R., Bilska M., Yuan X., Ochsenbauer C., Edmonds T. G., Kappes J. C., Liao H. X., Haynes B. F., Montefiori D. C Differential inhibition of human immunodeficiency virus type1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin mediated chemokine and gamma interferon production. AIDS Research Human Retroviruses, 26: Harborne, A. J Phytochemical methods a guide to modern techniques of plant analysis. Springer Science & Business Media. Kokate C. K Practical pharmacognosy. In Vallabh Prakashan, New Delhi 4: Singh A., Yadav D., Yadav M., Dhamanage A., Kulkarni S., Singh R. K Molecular modeling, synthesis and biological evaluation of N-Heteroaryl Compounds as reverse transcriptase inhibitors against HIV-1. Chemical biology & drug design, 85:
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