In vitro anti-HIV activity of some Indian medicinal plant extracts

Background Human Immunodeficiency Virus (HIV) persists to be a significant public health issue worldwide. The current strategy for the treatment of HIV infection, Highly Active Antiretroviral Therapy (HAART), has reduced deaths from AIDS related disease, but it can be an expensive regime for the underdeveloped and developing countries where the supply of drugs is scarce and often not well tolerated, especially in persons undergoing long term treatment. The present therapy also has limitations of development of multidrug resistance, thus there is a need for the discovery of novel anti-HIV compounds from plants as a potential alternative in combating HIV disease. Methods Ten Indian medicinal plants were tested for entry and replication inhibition against laboratory adapted strains HIV-1IIIB, HIV-1Ada5 and primary isolates HIV-1UG070, HIV-1VB59 in TZM-bl cell lines and primary isolates HIV-1UG070, HIV-1VB59 in PM1 cell lines. The plant extracts were further evaluated for toxicity in HEC-1A epithelial cell lines by transwell epithelial model. Results The methanolic extracts of Achyranthes aspera, Rosa centifolia and aqueous extract of Ficus benghalensis inhibited laboratory adapted HIV-1 strains (IC80 3.6–118 μg/ml) and primary isolates (IC80 4.8–156 μg/ml) in TZM-bl cells. Methanolic extract of Strychnos potatorum, aqueous extract of Ficus infectoria and hydroalcoholic extract of Annona squamosa inhibited laboratory adapted HIV-1 strains (IC80 4.24–125 μg/ml) and primary isolates (IC80 18–156 μg/ml) in TZM-bl cells. Methanolic extracts of Achyranthes aspera and Rosa centifolia, (IC801-9 μg/ml) further significantly inhibited HIV-1 primary isolates in PM1cells. Methanolic extracts of Tridax procumbens, Mallotus philippinensis, Annona reticulate, aqueous extract of Ficus benghalensis and hydroalcoholic extract of Albizzia lebbeck did not exhibit anti-HIV activity in all the tested strains. Methanolic extract of Rosa centifolia also demonstrated to be non-toxic to HEC-1A epithelial cells and maintained epithelial integrity (at 500 μg/ml) when tested in transwell dual-chamber. Conclusion These active methanolic extracts of Achyranthes aspera and Rosa centifolia, could be further subjected to chemical analysis to investigate the active moiety responsible for the anti-HIV activity. Methanolic extract of Rosa centifolia was found to be well tolerated maintaining the epithelial integrity of HEC-1A cells in vitro and thus has potential for investigating it further as candidate microbicide.


Background
Human Immunodeficiency Virus (HIV) persists to be a significant public health issue worldwide. In 2018, 37.9 million people are living with HIV globally; out of which 36.2 million are adults and 1.7 million are children less than 15 years old. There were 1.7 million new infections and 770,000 people died from AIDS related illness worldwide [1]. The current strategy for the treatment of HIV infection is Highly Active Antiretroviral Therapy (HAART), based on combination of inhibitors of reverse transcriptase and protease. Although HAART has considerably reduced deaths from AIDS related disease, it often has side effects and not well tolerated especially in persons undergoing long term treatment and maintains the risk of developing multidrug resistance [2]. Moreover, HAART is an expensive regime for underdeveloped and developing countries where the drugs are inaccessible to the HIV infected patients. Thus, there is a need for the discovery of novel therapeutic strategies, which identify new anti-HIV compounds from natural sources particularly from medicinal plants.
Natural sources provide a large reservoir for screening of anti-HIV agents with novel structure and antiviral mechanism due to their structural diversity. For the purpose of this study, ten Indian traditional medicinal plants, Albizzia lebbeck, Tridax procumbens, Achyranthes aspera, Ficus benghalensis, Mallotus philippinensis, Rosa centifolia, Strychnos potatorum, Annona reticulate, Ficus infectoria and Annona squamosa were selected to investigate their in vitro inhibitory activity against entry inhibition/ replication of HIV-1 as first step towards identification of potential anti-HIV microbicide. The microbicides provide protection by directly inactivating HIV or preventing HIV from attaching, entering or replicating in susceptible target cells as well as dissemination from target cells present in semen or the host cells that line the vaginal/rectal wall [3]. These plants were selected on the basis of detailed patent survey and scientific articles on the ethnomedicinal usages of the plant genera directly in HIV/AIDS or for symptoms/conditions closely associated with this disease (Table 1).
Plants such as R. centifolia, S. potatorum, F. infectoria, F. benghalensis and M. philippinensis were selected because other species of the same genera have exhibited anti-HIV activity [56][57][58][59][60]. Its traditional use in gonorrhoea and leukeorrhea [61] and suppressive effects on sperm motility [39] further made S. potatorum, a plant of choice for this study. Fruit pulp of A. squamosa has been reported to inhibit HIV replication significantly in H9 lymphocytes [49] therefore the seeds of A. squamosa which have also shown spermicidal property, an additional desirable attribute for a vaginal microbicide [62] was selected for the study. In addition the leaves of other species A. reticulate were also selected for assessing the anti-HIV activity.
Taylor et al., [63,64] reported methanolic extract of T. procumbens to exhibit in-vitro anti-Herpes Simplex Virus activity in Vero cells; hence it was selected for investigating its anti-HIV activity. Anticipating the potential of spermicide-based vaginal contraceptives in the reproductive health of women such as Nonoxynol (N-9) and Praneem polyherbal (Azadirachta indica leaves, Sapindus mukerossi pericarp of fruit and Mentha citrate oil) [65]; two plant extracts, methanolic leaf extract of A. aspera that has exhibited safety as well as good antifertility property [66] and methanolic pod extract of A. lebbeck which has been shown to suppress spermatogenesis and alter the structure and activity of the Sertoli and Leydig cells [4] were considered worthwhile to explore for anti-HIV activity.

Plant materials and extraction
10 plant materials were collected from various parts of India in different seasons. A plant taxonomist at publicly available herbarium, Botanical Survey of India, Pune, India, validated scientific names and classification of these plants. The specimens were also deposited in the herbarium. Table 2 presents ethno-botanical information and solvents used for extraction of the selected plants.
The collected plant materials were cleaned, freed of foreign contaminates and washed with water, first air dried and then dried in an electric oven at 40°C. The dried plant materials were pulverized in an electric mixer. The plant materials were extracted with various solvents individually by hot continuous Soxhlet extraction method for 18-24 h. After extraction, the extract obtained was filtered through 0.2-μm syringe filter and then concentrated on a rotary evaporator by distilling off the solvent under vacuum at 40°C. The concentrated extracts was finally lyophilized to obtain free flowing powder and stored in airtight bottles in the refrigerator at 4-8°C. The extractive yields of the individual extracts are recorded in Table 2. Powder was reconstituted in DMSO for final concentration of extract 10 mg/ml and stored at -20°C until tested for anti-HIV1 activity.

Preliminary phytochemical investigation
Qualitative tests were carried out to ascertain the presence of various phytochemicals in the plants extract of the selected plants using the methods described by Harbourne [67] (Table 3). It involved the appropriate addition of chemicals and reagents to the concentrated extract of the plant material in a test tube. The changes in the appearance of the colour, as the case may be, confirmed the presence of alkaloids, flavanoids, tannins, steroids and saponins.
Cells, viral strain and culture conditions TZM-bl (recombinant HeLa cells expressing high levels CD4 receptor, CXCR4 and CCR5 co-receptors) and PM1 cells (Clonal derivative of HUT 78) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (NIH ARRRP). The HEC-1A (human endometrial adenocarcinoma) cell line was kindly provided by Dr. R. Fichorova (Associate Professor, Brigham and Women's Hospital, Boston, USA) and National Institute of Virology, Pune, respectively.
The TZM-bl cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, USA) and PM1 and HEC-1A cells in RPMI-1640 (Sigma-Aldrich, USA), supplemented with 10% heat inactivated fetal bovine serum (FBS, Moregate Biotech, Australia) and standard antibiotic-antimycotic cocktail. The laboratory adapted HIV-1 strains [HIV-1IIIB (X4, subtype B), HIV-1Ada5 (R5, subtype B)] and the primary isolate HIV-1UG070 (X4, Subtype D) were procured from National Institutes of Health-AIDS Research and Reference Reagent Program, while the Indian isolate HIV-1VB59 (R5, subtype C) was obtained from the National AIDS Research Institute (NARI), Pune. Phytohemagglutinin-P (5 μg/ml, Sigma Aldrich, USA) activated peripheral blood mononuclear cells (PBMC) derived from healthy donors were used for the growth of all the viral strains. HIV-1 p24 antigen detection kit (Vironostika HIV-1 Antigen, Netherlands) was used to determine the virus production in cell culture supernatants. Samples of viral culture supernatants free form cells were obtained by centrifugation and further filtered and finally stored at -70oC for further use. Spearman Karber formula was used to ascertain the 50% tissue culture infectivity dose (TCID50) of each virus stock in both TZM-bl and PM1 cells [68].

Anti HIV1 assays Determination of cytotoxicity in the uninfected TZM-bl and PM1 cell lines
The cytotoxicity of the extracts was determined in uninfected TZM-bl cells using colorimetric assay that measures the reduction of a yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase (Sigma Aldrich, USA) [69]. Briefly, two-fold dilutions of the extracts were prepared, added to 96 well plates pre-seeded with TZM-bl cells (10,000 cells/well) in quadruplicate and incubated for 48 h at 37°C. The MTT (20 μl, 5 mg/ml) solution was added and the plates were incubated  further for 4 h. The supernatant was removed, 200 μl of DMSO was added, the plates were incubated for 1 h and the absorbance was read at 550 nm and 630 nm. The percent viability was calculated by comparing cell viability in the absence of extract using following formula and the results were expressed as CC 50 (50% cytotoxic concentration).
The cytotoxicity in uninfected PM1 cells of all the extracts was determined in a similar manner by using a similar dilution scheme and procedure as mentioned above for TZM-bl cells. The cell viability was determined by the trypan blue dye exclusion assay (Sigma Aldrich, USA) and the results were expressed as CC 50 [70].
Preliminary screening for anti-HIV1 activity against laboratory adapted strains in TZM-bl cell lines The anti-HIV1 activity was tested against Cell-free (CF) and Cell-associated (CA) X4 tropic (HIV-1 IIIB ) and R5 tropic (HIV-1 Ada5 ) laboratory adapted strains in TZM-b1 cell lines.
In cell free assay, the viral stocks (400 TCID 50 ) were pre-treated in duplicate with sub toxic concentrations of the extracts/fractions for 1 h, at 37°C prior to addition onto the TZM-bl cells (10,000 cells/well). While in cell-associated assay, the cells (10,000 cells/ well) were pre-infected with the viral stocks (400 TCID 50 ) for 2 h at 37°C before exposure to the extracts/fractions [71]. After 48 h, the supernatant was collected and luciferase activity was determined using Britelite plus (Perkin Elmer, USA). Dextran Sulphate (Sigma Aldrich, USA) and Azidothymidine (AZT, CIPLA, India) were used as positive controls for cell free and cell associated assays respectively. The results were expressed as IC 50 (50% inhibitory concentration), IC 80 (80% inhibitory concentration) and Therapeutic Index (TI=CC 50 /IC 50 ).
The procedure for anti-HIV1 activity against primary isolates in TZM-bl cell lines followed was same as mentioned above for anti-HIV1 activity against laboratory adapted strains. The results were expressed as percentage inhibition calculated using following equation The results were expressed as IC 80 (80% inhibitory concentration).
The anti-HIV activity against primary isolates was also evaluated in PM1 cell lines using 24-well plate (Corning, USA). In the cell free assay, 20 TCID 50 the viral stock (HIV-1 UG070 and HIV-1 VB59 ) was pre-treated with sub toxic concentrations of the extracts/fractions, before addition onto the cells (5x10 4 cells/well). Whereas, in the cell associated assay, the PM1 cells (5x10 4 cells/well) were pre-infected with 20 TCID 50 of the viral stock and then exposed to the extracts/fractions [72]. The virus growth was monitored by Vironostika®p24 antigen ELISA (Biomerieux, France). Dextran sulphate and AZT were used as positive controls for cell free and cell associated assays respectively. The percent inhibition was calculated by comparing activity in absence of the extracts/fractions/control drug using the formula mentioned above and the results were expressed as IC 80. Toxicity testing using Transwell epithelial model Cytotoxicity assay The toxicity of selected plant extract was determined in HEC-1A using similar protocol as described for TZM-bl cells, only with a difference of the read out system, i.e. LDH cytotoxicity detection kit (Roche Diagnostics, Germany).

Determination of epithelial integrity in Transwell dualchamber system
The epithelial integrity was determined as described by Gali et al., [73]. Briefly, HEC-1A cells (1 × 10 5 /100 μl) were cultured for 7 days on the apical chamber of a Laminin coated dual-chamber Transwell® system (growth area: 0.3cm 2 , pore size: 3.0 μm) (Corning Costar Corp, USA). After 7 days incubation, two-fold serial dilutions of test preparations (100 μl) were added on to the HEC-1A cells and incubated for 24 h (37°C, 5% CO 2 ). The test preparations were removed and 100 μl of a 1/20 dilution of yellow-green fluorescent microspheres (FluoSpheres® sulphate microspheres, Molecular Probes Europe NV, Netherlands) were added in the apical chamber. After 24 h, 100 μl of medium was harvested from the basal chamber and the fluorescence was measured using a fluorometer (Perkin Elmer, USA). Untreated HEC-1A cells and 1% Nonoxynol-9 were used as controls for measuring percent transmission.

Preliminary phytochemical investigation
The preliminary phytochemical evaluation of plant extracts for the presence of steriods, flavanoids, alkaloids,

%Inhibition ¼ 1−½ðluminescence in presence of the test extract or fraction
Àluminescence of uninfected control cells=luminescence of cells infected with virus Àluminescence of uninfected control cellsÞ Â 100 saponins, tannins and phenolic acids was done for 10 plants extracts from 8 different families. Steroids were not present in T. procumbens, M. philippinensis, R. centifolia and A. squamosa extracts and the Saponins in T. procumbens and A. reticulate extract. Only flavanoids was present in T. procumbens extract while it was not present in S. potaotorum extract ( Table 3).

Determination of cytotoxicity in TZM-bl and PM1 cell lines
Six methanolic extracts, two aqueous extracts and two hydroalcoholic extracts of 10 medicinal plants were examined for their ability to inhibit HIV-1 entry and replication. The in vitro toxicity of these extracts to TZM-bl cells was investigated by MTT assay. Methanolic extracts of, A. aspera, hydroalcoholic extract of A. squamosa and water extract of F. benghalensis tested were relatively non-toxic to TZM-bl cells at a CC 50 value between 51 and 72 μg/ml. The CC 50 values of other extracts such as methanolic extract of, R. centifolia, S. potatorum and aqueous extract of F. infectoria were found to be comparatively higher ranging between 118 and 147 μg/ml. However, methanolic extract of A. reticulate was found to be toxic at a very low concentration (CC 50 = 11 μg/ml) as compared to the other extracts (Tables 4 and 5). Cytotoxicity of plant extracts, A. aspera, F. benghalensis, R. centifolia, S. potatorum, F. infectoria and A. squamosa showing activity in preliminary anti-HIV-1 assay was carried out in PM1 cells using trypan blue dye exclusion assay. The 50% cytotoxicity was observed at a concentrations ranging from 2.9-46 μg/ml. Aqueous extract of F. benghalensis was toxic at a very low concentration as compared to other extracts (Table 5).

Preliminary screening for anti-HIV1 activity against laboratory adapted strains in TZM-bl cell lines
Plant extracts of A. aspera, F. benghalensis, R. centifolia, S. potatorum, F. infectoria and A. squamosa showed inhibition of HIV-1 IIIB and HIV-1 Ada5 laboratory adapted strains in both cell free and cell associated assays. Aqueous extract of F. infectoria revealed significant activity against the laboratory adapted strains with estimated IC 80 in the range of 4.24-125 μg/ml giving TI of 189, 49 and 27 in cell free HIV-1 IIIB , HIV-1 Ada5 and cell associated HIV-1 IIIB respectively. This was followed by methanolic extract of A. aspera which showed activity with preliminary IC 80

Confirmation of anti-HIV activity against primary isolates in TZM-bl and PM1 cell lines
The plant extracts showing activity in preliminary screening against laboratory adapted strains were further screened both cell free and cell associated assays against primary isolates HIV-1 UG070 and HIV-1 VB59 in TZM-b1 In TZM-b1 cell lines methanolic extract of A. aspera and hydroalcoholic extract of A. squamosa exhibited a very good activity with lowest estimated IC 80 of 4.8-53 μg/ml and 25-27 μg/ml respectively against primary isolates of HIV-1 strains. This was followed by aqueous extract of F. infectoria, methanolic extract of S. potatorum and R. centifolia and aqueous extract of F. benghalensis with preliminary IC 80 in the range of 18-73 μg/ml, < 31.25-105 μg/ml, 17-> 125 μg/ml, and < 78-< 156 μg/ml respectively (Table 5).
In PM1 cell lines, methanolic extract of A. aspera and R. centifolia showed activity with estimated IC 80 ranging 1-9 μg/ml. Aqueous extract of F. benghalensis exhibited activity at preliminary IC 80 of 2.9-3.5 μg/ml in cell free assay. Methanolic extract of S. potatorum exhibited activity at IC 80 of 6-29 μg/ml except for HIV-1 VB59 cell free assay. The hydroalcoholic extract of A. squamosa showed very good activity at lower concentration 0.8 μg/ ml in cell associated assay. Aqueous extract of F. infectoria showed activity in cell free assay at a concentration ranging 2-29 μg/ml ( Table 5). The representative dose response bar graphs for A. aspera and R. centifolia, in cell free and cell associated assays for TZMb1 and PM1 are given in Additional file 1: (Figures S1 to S5).

Toxicity testing using Transwell epithelial model
Amongst the three extracts which showed highest activity, methanolic extract of R. centifolia exhibited least toxicity both inTZM-b1 and PM1 cell lines, therefore it was further tested for in vitro activity against HEC-1A cells and demonstrated minimal cytotoxicity with CC 50 of 1443 μg/ ml. Epithelial integrity of HEC-1A in Transwell dual-chamber system was maintained with only 1% relative fluorescence (percent of positive control) detected after treatment with 500 μg/ml of methanolic extract of R. centifolia. At higher concentration of 1000 μg/ml integrity was affected with 24% leaked fluorescence relative to positive control (Fig. 1).

Discussion
Natural products continue to be major sources of innovative therapeutic agents for treatment of infectious diseases, and their exploration has been one of the most successful strategies for the discovery of medicines. The development of new microbicides as preventive interventions is a promising area in AIDS research [3]. They could be valuable addition in prevention of sexual transmission of HIV-1 and could be an important way to reduce the number of cases of HIV infection globally [74,75]. Currently available anti-HIV drugs are chemically synthesized and are often limited by side effects and emergence of drug resistance [76].
In order to find such potential anti-HIV agents from natural sources, ten traditional medicinal plants from India were studied for their inhibitory effects against laboratory adapted strains HIV-1 IIIB, HIV-1Ada5 and primary isolates HIV-1UGO70, HIV-1VB59 in TZM-b1 and primary isolates HIV-1UGO70, HIV-1VB59 in PM1 cell lines. HIV viruses can spread in the body via either a cell-free (virus floating free in plasma) mode or a cell associated (virus particles that remain attached to or within the host cell after replication) mode involving direct cell-cell contact. Hence all the selected plant extracts were evaluated to depict their mechanism of action, whether they will act as an entry inhibitor or at the HIV replication stage [77]. The selected plant extracts were subjected to high throughput (cost-effective, quick and reproducible) TZM-bl assay model which is useful for preliminary screening allowing screening of large number of products against HIV [70,78]. The results presented here indicate that the methanolic extracts of aerial parts of A. aspera, leaves of R. centifolia and seeds of S. potatorum and aqueous extract of leaves of F. benghalensis and F. infectoria possess anti-HIV properties of therapeutic interest inhibiting HIV-1 virus at an estimated TI of 3-35, 1-6, 7-24,12-32, and 2-189 respectively against laboratory adapted strains and at a very low preliminary IC 80 ranging from 4.8-26, 17-125, 31-105, 78-156 and 18-73 μg/ml respectively against primary isolates using TZM-b1 assay.
The lead extracts were also confirmed for anti-HIV activity in PM1 cell line which supports persistent HIV-1 infection [72]. The PM1 cell line have been reported to be comparable to peripheral blood mononuclear cells (PBMCs) for culturing of any of the HIV-1 strains and subtypes and thus provide a valuable research tool for studying new anti-HIV therapies [79]. This cell line has been previously used for studying the anti-HIV1 properties of the polyherbal cream Basant [80]. Hence PM-1 was used for confirming the anti-HIV activity of the methanolic extracts of aerial parts of A. aspera and leaves of R. centifolia which showed anti-HIV activity (IC 80 ) ranging between 1 and 8.4 and 2.2-6.8 μg/ml respectively. These extracts may potentially inhibit the entry and also inhibit HIV-1 replication if the virus enters the vaginal cells. However further work on more replicates and wider concentration range studies are required for confirmation. Future studies on PBMCs for qualifying the results should also be considered.
Our earlier work has shown that methanolic extract of R. centifolia has also shown activity against four strains of N. gonorrhoeae [81]. It substantially lacks cytotoxicity even at high concentrations (CC 50 greater than 1 mg/ml) when tested in vitro on HEC-1A cell line (endometrial origin) and maintained its epithelial integrity when studied in Transwell model at concentrations up to 500 μg/ ml thus showing potential for investigating it further as candidate anti-HIV microbicide.
As per the literature these extracts have not been further analyzed chemically, although the active components such as oleanolic acid and pomolic acid isolated from Rosa wodsii leaves the other species of Rosa, have been reported to inhibit HIV replication in acutely infected H9 cell growth at IC 50 of 40 μg/ml [56,57]. The literature indicates the phytosteroids, polyphenols and saponins present in the methanolic leaf extract of A. aspera are responsible for its anti-fertility effect [66] and methanolic root extract possess anti-herpes virus activity at EC 50 of 64.4 μg/ml for HSV-1 and 72.8 μg/ml for HSV-2 [82] The other selected medicinal plants extract showed anti-HIV activity against at least any one of the assay model except for hydroalcoholic extract of whole pods of A. lebbeck, methanolic extract of aerial parts of T. procumbens, methanolic extract of leaves of M. philippinenis and methanolic extract of leaves of A. reticulate, they were incapable of showing anti-HIV1 activity against cell free and cell associated HIV-1 IIIB , HIV-1 Ada5 laboratory adapted strains and HIV-1 UG070 , HIV-1 VB59 primary isolates in TZM-b1 and PM1 cell lines. It's worth mentioning that these plants were selected on basis of their sub species showing activity against other strains and primary isolates of HIV and the same species having contraceptive and activity related to this infectious disease. The inactivity of these plants against our test strains and primary isolates of HIV does not prove that they do not possess anti-HIV1 activity. These plants can be taken further for the activity against other strains and primary isolates of HIV virus using other anti-HIV assays. Some plants extract such as F. benghalensis, S. potatorum and F. infectoria showed moderate to mild anti-HIV1 activity. These plants extracts had variable activities across the assays presented in this study where the extract exhibited inhibition of one strain of the primary isolates in one assay but did not inhibit the same primary isolates in another assay model. Aqueous extract of leaves of F. benghalensis showed anti-HIV1 activity against all HIV-1 laboratory adapted strains and primary isolates using TZM-b1 assay (TI: 12-32, IC 80 : 78-156 μg/ml) but did not inhibit cell associated primary isolates in PM1 assay. Methanolic extract of seeds of S. potatorum showed anti-HIV1 activity against all HIV-1 laboratory adapted strains and primary isolates using TZM-b1 assay (TI: 7-24, IC 80 : 31.25-105 μg/ml) but was not capable of inhibiting cell free primary isolate HIV-1 VB59 in PM1 assay. Aqueous extract of leaves of F. infectoria showed anti-HIV1 activity against all HIV-1 laboratory adapted strains and primary isolates using TZM-b1 assay (TI: 2-189, IC 80 : 18-73 μg/ml) but did not inhibit cell associated primary isolates in PM1 assay. Hence these extracts may not altogether be classified as extracts not having anti-HIV1 inhibitory potential.
Hydroalcoholic extract of seeds of A. squamosa exhibited activity against all HIV-1 laboratory adapted strains and primary isolates using TZM-b1 assay (TI: 2-4, IC 80 : 26-27 μg/ml) but was not capable of inhibiting cell free primary isolates in PM1 assay. This plant extract has a some potential to be explored further and may be used supplementary as a replication inhibitor.

Conclusion
To conclude the study, out of 10 plants screened for anti-HIV activity using TZM-b1 and PM1 assays, methanolic extracts of aerial parts of A. aspera and leaves of R. centifolia has prospective anti-HIV1 potential as an entry and replication inhibitors. Hence these experimental moieties may have favourable implications on the prevention or management of HIV/AIDS. Additionally methanolic extract of leaves of R. centifolia have shown good safety and maintained the epithelial integrity on HEC-1A cells. Plant extracts are complex mixtures of many compounds. Some compounds may mask the anti-HIV1 potential of plant extract due to their cytotoxicity. Therefore our next step would be isolating the phytoconstituents and increasing the chances to find active anti HIV1 compounds with low cytotoxicity.
Additional file 1: Figure S1. Plot of % HIV inhibition Vs Concentration (μg/ml) in TZMb1 (primary isolates) Cell free (CF) and Cell associated (CA) assay for R. centifolia. Figure S2. Plot of % HIV inhibition Vs Concentration (μg/ml) in TZMb1 (primary isolates) Cell free assay for A. aspera. Figure S3. Plot of % HIV inhibition Vs Concentration (μg/ml) in TZMb1 (primary isolates) Cell associated assay for A. aspera. Figure S4. Plot of % HIV inhibition Vs Concentration (μg/ml) in PM1 Cell free assay for R. centifolia and A. aspera. Figure S5. Plot of % HIV inhibition Vs Concentration (μg/ml) in PM1 Cell associated assay for R. centifolia and A. aspera.