Potential Anti-HIV Activity of Jatropha curcas Linn. Leaf Extracts

Objectives: The presence of drug-resistant HIV is a major global concern and warrants the development of novel anti-virals as alternative and inexpensive therapy. In the current study, potentially drug-resistant HIV was isolated and previously unreported anti-viral activity of Jatropha curcas Linn. leaf extracts was assessed. Methods: HIV isolation was done using in vitro micro co-culture methods followed by drug susceptibility assays to determine resistance to Zidovudine (AZT), Lamivudine (3TC) and Stavudine (d4T). Soxhlet apparatus was used for extraction of metabolites from leaves of Jatropha curcas Linn. and Methanolic and Aqueous Extracts were chosen for further study. Secondary metabolites were detected by High-Performance Thin Layer Chromatography and in vitro cytotoxicity established by MTT assay. The extracts were then used in postand pre-infection studies by measuring inhibition of HIV replication to determine anti-viral activity. Results: Seven HIV isolates were obtained (isolation rate: 23.33%) with drug IC50 values ranging from 0.00141882.73 μM AZT, 2.645-15.35 μM 3TC and 18.55-66.23 μM d4T. Tannins, Flavonoids, Saponins were detected in Aqueous Extract and Flavonoids, Saponins in Methanolic Extract. The CC50 values were 32.07 mg/mL and 35.5 mg/ mL for Aqueous and Methanolic Extracts respectively. Anti-viral activity was evaluated by inhibition of HIV replication as determined by HIV p24 antigen ELISA. Post-infection (4 isolates) interaction studies showed IC50 values ranging from 0.0255-0.4137 mg/mL and 0.00073-0.1278 mg/mL for Aqueous and Methanolic Extracts respectively and preinfection (1 isolate) interaction studies showed 100% inhibition by Methanolic and 97.19% inhibition by Aqueous Extract at 25 mg/mL each. Conclusions: HIV isolates potentially resistant to AZT/3TC/d4T were obtained and Jatropha curcas Linn. leaf extracts showed effective anti-viral and probable entry inhibition activity against potentially drug-resistant HIV, which has not been reported earlier. The study indicates that Jatropha curcas Linn. is a good candidate for anti-HIV therapy with further research. Journal of Antivirals & Antiretrovirals J o u r n al o f A nti virals & Antitrovira l s


Introduction
People living with HIV/AIDS often choose traditional or complementary and alternative medicine to complement or replace conventional treatment. The presence of multi-drug or even multiclass resistance in HIV also warrants the need to explore additional means to combat HIV and provide further justifications for the need of alternative and complementary medicines in the treatment of HIV/ AIDS.
Most of the traditional systems of medicine in India include some form of 'medicinal plant', herbs or natural plant products. It is therefore not surprising that the activity of these traditional medicines against HIV can be scientifically analysed to deduce the role of natural plant products in their anti-HIV activities. A number of medicinal plants have been reported to have anti-HIV properties [1]. Over the past two decades, substantial progress has been made in research on the natural products possessing anti-HIV activity. A variety of secondary metabolites obtained from natural origin showed moderate to good anti-HIV activity [2]. Jatropha curcas Linn., (colloquially known as errand/danti/katri in Marathi) is a multipurpose, drought resistant, perennial plant that belongs to Euphorbiaceae family, widely distributed in the wild or semi-cultivated areas in Central and South America, Africa, India and South East Asia. The genus name 'Jatropha' derives from the Greek word 'jatr´os' (doctor) and 'troph´e' (food), which implies medicinal uses [3]. Practically all parts of Jatropha have some use including green manure, soil erosion control, soap production from oils, leaves as animal feed, biocidal activity from natural products, leaves and/or stems for sericulture and vermiculture and the more famously known biodiesel production from oil apart from its numerous medicinal uses from various parts [4,5]. All parts of Jatropha (seeds, leaves and bark) have also been used in traditional medicine and for veterinary purposes for several centuries [6]. Some of the known medicinal properties of Jatropha curcas Linn. include antitumor activities, molluscicidal, insecticidal and fungicidal properties. The seed oil (Jamalgota) can be applied to treat eczema and skin diseases and to soothe rheumatic pain [6]. The latex from branches have been found to be strong inhibitors to watermelon mosaic virus and the leaves and latex are used in healing of wounds, refractory ulcers, and septic gums and as a styptic in cuts and bruises [7]. Topical application of Jatropha root powder in paste form is a common ethnobotanical practice for the treatment of inflammation being followed by Bhil tribes from Rajasthan area in India and the methanol extract of these roots exhibited systemic and significant anti-inflammatory activity in acute carrageenan-induced paw oedema in albino mice [8]. Four antitumor compounds, including jatropham and jatrophone, are reported from other species of Jatropha [8]. The plant has also been homoeopathically used for cold sweats, colic, collapse, cramps, cyanosis, diarrhoea, leg cramps [7,8]. Some other potential antimicrobial activity of Jatropha curcas Linn. have also been found [9,10].
Although several studies have shown the antimicrobial activity, there are a very limited number of previous reports on the specific anti-HIV activity of Jatropha curcas Linn. One such study showed that Jatropha curcas Linn. had specific in vitro anti-RT enzyme activity and that the water extract of the branches also strongly inhibited HIV induced cytopathic effects with low cytotoxicity [9]. Several unknown active components of plants belonging to the family Euphorbiaceae (Jatropha curcas, J. multifida, Spirostachys africana and Trigonostema xyphophylloides) were found to possess anti-HIV activities by inhibiting HIV-1 cell entry [11][12][13]. Another study that looked at traditional medicines in the management of HIV/AIDS in Tanzania found that Jatropha curcas Linn. leaves were being used for treatment in HIV related conditions such as skin rash and oral candidiasis [14].
With this background, the current study was designed to study drug-resistant HIV-1 in the patient cohort in Mumbai. A small sample size was chosen to evaluate the presence of drug-resistant HIV-1 by phenotypic drug resistance assays. Alternative strategies such extracts from the leaves of medicinal plant Jatropha curcas Linn. were evaluated for the potential antiviral and virucidal and/or entry inhibitory activity of these plant extracts.

Study population
A total of 30 HIV-seropositive patients (both on Antiretroviral therapy (ART) and drug naïve) attending the ART centre at AIDS Research and Control Centre (ARCON), Sir JJ Hospital Campus, were included in the study. Institutional Ethics Committee approval and written informed consent was acquired prior to blood collection.

HIV isolation
Isolation of HIV from blood samples was done using a PBMC micro-co-culture assay [15]. Briefly, healthy, HIV-seronegative donor PBMCs were stimulated with the mitogen phytohemagglutinin-P (PHA-P; at a final concentration of 5.0 μg/mL; Sigma-Aldrich Corporation, Bengaluru, India), in the presence of human interleukin 2 (IL-2; Sigma-Aldrich) for 24-72 hours before use to promote blast formation and replication of T-cells. The cells were counted and 1×10 6 PHA-stimulated donor cells and 1×10 6 patient PBMCs (samples) were added in duplicate wells of a 24-well tissue culture plate. The final volume was adjusted to 2.0 mL with growth media containing RPMI-1640 (GIBCO ® , Invitrogen BioServices India Pvt. Ltd, Bengaluru, India) with 20% FBS (Sigma-Aldrich) and 10.0 U/mL IL-2. The plate was then incubated at 37°C with 5% CO 2 in a CO 2 incubator (New Brunswick Scientific, Connecticut, USA). On day 7 and 14, the cultures were replenished with fresh growth media containing 5×10 5 PHAstimulated donor cells (feeder cells). The culture was continued until day 21 and then terminated. Supernatant fractions from duplicate wells from day 14 and 21 were saved separately, and stored at -70°C until analysis for HIV p24 antigen by ELISA using a HIV-1 p24CA Antigen Capture Assay procured from the AIDS & Cancer Virus Program, National Cancer Institute, Frederick, Maryland, USA as per the manufacturer's instructions. Cultures were considered positive only when day 21 supernatants showed an increase in p24 antigen concentrations measured in pg/mL from day 14 supernatants.

Drug susceptibility assay using zidovudine (AZT), lamivudine (3TC) and stavudine (d4T)
Drug susceptibility assay was carried out by using a standardized PBMC susceptibility assay [15,16] with slight modifications. Since we were working with small volumes of virus (micro-culture), an additional step of 50% Tissue Culture Infectious Dose (TCID 50 ) calculation was not required. This modification results in a faster and less expensive assay [17]. Dilutions of 2X concentrations (20.0 μM, 2.0 μM, 0.2 μM, 0.02 μM and 0.002 μM) of the drugs of interest (AZT, 3TC and d4T) were prepared by in Growth Medium. The 2X drug working solutions were then added to their respective wells in a 96-well microtitre plate with PBS in the corner wells to help maintain humidity. A single row of uninfected cells and drug was included to serve as a drug toxicity control. Next, 1-to 3-day old PHA-stimulated normal donor PBMC were re-suspended in Growth Medium to a concentration of 4×10 6 PBMC/mL. A volume of 100.0 μL of virus stock was added to 1.0 mL of these donor cells. After mixing gently, the suspension was incubated for 1-3 hours at 37°C, 5% CO 2 with humidity to allow the virus to infect cells. After incubation, the cells were washed to remove any uninfected HIV and 100.0 μL of infected cells was dispensed into the well of the microtitre plate containing the appropriate 2X drug concentrations. The final volume in each well was 200.0 μL. The plates were then incubated at 37°C, 5% CO 2 with humidity. On day 7, 20.0 μL of the supernatant from each of the drug susceptibility assay wells was added to the lysing solution (10% Triton X-100™; Sigma-Aldrich) and used for determining the p24 antigen levels. The 50% Inhibitory Concentration (IC 50 ) values were calculated by the median effect equation using GraphPad Prism software (GraphPad Software, Inc., California, USA). The potential phenotypic drug resistance was determined by calculating the fold increase in IC 50 values of the drugs against the HIV isolates compared with IC 50 values against a standard strain of HIV (HIV-1 IIIB ).

Collection, identification and authentication of Jatropha curcas Linn.
The plant material was collected from Rajaramnagar (Islampur), Sangli District, Maharashtra. Since the plant material was collected from wild, it needed to be authenticated prior to further use. The identification and authentication of the plant was done at Blatter Herbarium, Department of Botany, St. Xavier's College, Mumbai and a voucher specimen was deposited in the herbarium (Specimen no. K.V.S. 2298).

Solvent extraction of plant material and detection of secondary metabolites
Extraction of leaves from Jatropha curcas Linn. was carried out using a Soxhlet apparatus and various solvents in increasing order of polarity. The solvents used for extraction were hexane, dichloromethane (DCM), methanol (Sigma-Aldrich) and water (Distilled). Briefly, Jatropha curcas Linn. leaves were thoroughly washed with water, shade dried on a clean filter paper and ground into fine powder. A total of 30.0 g of leaf powder was wrapped in a thimble made of Whattman filter paper no. 1, and was placed into the main extraction unit of the Soxhlet apparatus. A round bottom flask containing solvent was placed on a heating mantle and temperature adjusted to the boiling point of each solvent. Once the solvent in the side arm became colourless, the extraction process was stopped. The desired extract containing small amount of solvent was taken in a clean and dry pre-weighed petriplate and the solvent was evaporation completely. The extracts were then stored in a cool and dry conditions until further in vitro assays.
Detection of the secondary metabolites of the plant extracts was carried out by High Performance Thin Layer Chromatography (HPTLC) on a third-party commercial basis (M/s Anchrom Test Lab (I) Pvt. Ltd., Mumbai) using a Linomat 5 Semi-automatic Sampler and densitometric evaluation done using Camag TLC Scanner and Visualizer. The documentation was done using winCATS Planar Chromatography Manager software.

Cytotoxicity assay
Before using the Jatropha curcas Linn. extracts for antiviral assay it was necessary to assess their in vitro toxicity against human cells. The cytotoxicity of experimental moieties (ranging from 200 mg/mL to 6.25 mg/mL) was determined using both Vero cell lines as well as PBMCs by MTT assay [18]. The 50% cytotoxic concentration (CC 50 ) values were then calculated using GraphPad Prism software.

Assays to assess potential antiviral activity
Potential antiviral and entry inhibitory activity of the experimental moieties was assessed using standardized drug susceptibility assays using PBMCs and measured by inhibition of HIV p24 antigen. Two types of assays were carried out: Post-infection interaction (cellassociated virus) to determine antiviral activity and a preliminary Preinfection interaction (cell-free virus) to assess probable inactivation of virus (virucidal activity) or entry inhibitory activity.

Post-infection interaction (cell-associated HIV)
The HIV isolate/s were first allowed to infect PBMCs and the plant extracts (ten-fold dilutions ranging from 25.0 mg/mL to 0.025 mg/mL, based on the CC 50 values of the extracts) were added to the suspension afterwards. Briefly, a volume of 100.0 μL of viral isolates were individually added to 1.0 mL of PHA-stimulated PBMCs in a 1.5 mL microcentrifuge tube and incubated for 1 hr in a CO 2 incubator to allow the virus to infect the cells. After incubation, the cells were washed carefully to remove any uninfected HIV and 100.0 μL of infected cells were plated into wells of 96-well tissue culture plate. Next, 100.0 μL of 2X concentrations of different plant extracts were added. The plates were then incubated for 7 days at 37°C in the CO 2 incubator. The contents of each well was transferred to microfuge tubes, cells were pelleted and the supernatants used for determining the p24 antigen levels. The results obtained were then analysed to calculate IC 50 values and compared with the IC 50 values of standard drugs AZT, 3TC and d4T against the respective isolates.

Pre-infection interaction (cell-free HIV)
The HIV isolate/s were first interacted with the plant extracts (25 mg/mL) for 1 h and then allowed to infect PBMCs. Briefly, a volume of 10.0 μL of viral isolates were individually added to 10.0 μL of 2X concentrations of the plant extracts in a 1.5 mL microcentrifuge tube and incubated for 1 hr at 37°C in a CO 2 incubator. After 1 h of interaction the experimental moiety-virus suspension was added to 96well tissue culture plate containing 100.0 µL of PHA stimulated PBMCs and incubated for 1 h in the CO 2 incubator to allow the virus to infect the cells. After incubation, the cells were washed carefully to remove any uninfected HIV and the plate was incubated for 7 days in the CO 2 incubator. The contents of each well was transferred to microfuge tubes, cells were pelleted and the supernatants used for determining the p24 antigen levels. The results obtained were then analysed to calculate p24 antigen inhibition.

HIV isolation by PBMC co-cultivation and determination of phenotypic drug resistance
Thirty samples were processed for Peripheral Blood Mononuclear Cells (PBMC) co-cultivation from which seven HIV isolates were obtained giving an isolation rate of 23.33%. The phenotypic drug resistance was determined by calculating the fold increase in IC 50 values of the drugs against the HIV isolates compared with reported IC 50 values against a standard strain of HIV (HIV-1IIIB) [19]. The fold increases in IC 50 values are given in Table 1.

Solvent extraction and detection of secondary metabolites
Solvent extraction using Soxhlet apparatus yielded in 2.2 g of Hexane extract, 1.5 g of Dichloromethane (DCM) extract, 1.3 g of Methanolic extract and 1.8 g of Aqueous extract from leaves of Jatropha curcas Linn. Since the Hexane and DCM extracts were extremely sticky and insoluble in water or RPMI-1640 medium, only Methanolic (ME) and Aqueous extracts (AE) were used for all the further assays.
Secondary metabolites of Jatropha curcas Linn. leaf extracts were detected by HPTLC analysis. The plant extracts showed the presence of Tannins, Flavonoids and Saponins in the AE and Flavonoids and Saponins in the ME (Table 2). Figure 1 shows the HPTLC fingerprint of the plant extracts.

In vitro cytotoxicity of plant extracts
The in vitro cytotoxicity was carried out using MTT assay and CC 50 calculated. The CC 50 values of Jatropha curcas Linn. plant extracts were 35.49 mg/mL for Methanolic and 32.07 mg/mL for Aqueous extract as shown in Figure 2.

Anti-HIV activity of Jatropha curcas Linn. methanolic and aqueous extracts
Anti-HIV activity of Methanolic and Aqueous extracts of Jatropha curcas Linn. was studied using the in vitro drug susceptibility assay with an endpoint determination of p24 antigen. Percent inhibition of p24

2° Metabolite Methanolic Extract Aqueous Extract
Alkaloids --  antigen was determined against 4 isolates, and it was observed that the IC 50 values of the Methanolic leaf extracts ranged from 0.00073-0.1278 mg/mL (Figure 3; Selective index = 19095.34 ± 17709.64) while those of the Aqueous leaf extracts ranged from 0.0255-0.4137 mg/mL (Figure 4; Selective index = 705.86 ± 438.33) as shown in Table 3. Since this was a preliminary study, the pre-exposure interaction to assess the potential entry inhibitory activity was carried out only against 1 isolate (15C), and percent p24 antigen inhibition was determined. It was seen that the methanolic extract showed 100.0% inhibition while the aqueous extract showed 97.19% inhibition.

Discussion
The need for novel antivirals for managing HIV/AIDS is indisputable. The nature of the virus to evolve faster than the antivirals being made available has made it imperative that newer approaches to combating the virus are considered.
Our laboratory has been actively involved in the research of novel experimental moieties for their potential anti-HIV activity. Previous studies carried out in our laboratory with Mangrove plants such as Ocimum sanctum, Withania somnifera, Tinospora cordifolia, Avicennia officinalis, Rhizophora mucronata and 'Shilajit', a herbomineral [20] and other medicinal herbs such as Phyllanthus amarus [21] have shown tremendous potential in their anti-HIV activities. The leaf extracts of medicinal plant Jatropha curcas Linn. have not been studied extensively in the past. A preliminary study carried out by our laboratory for assessing the anti-HIV activity of Jatropha curcas Linn. extracts showed promising results [22]; hence, in this study an attempt was made to determine the potential anti-HIV activity using potentially drug-resistant clinical isolates of HIV-1.     The plant extracts showed effective antiviral activity as compared to standard drug AZT, 3TC and d4T ( Figure 5) in HIV p24 antigen inhibition assays. The aqueous leaf extracts showed IC 50 values ranging from 0.0255-0.4137 mg/mL and the methanolic leaf extracts showed IC 50 values ranging from 0.00073-0.1278 mg/mL; while in the pre-infection studies, the aqueous leaf extracts showed 97.19% p24 inhibition and the methanolic leaf extract showed 100.0% p24 inhibition at 25 mg/mL each indicating potential denaturation of virus (virucidal activity) or entry inhibitory activity.
These findings are quite significant since the extracts seem to work in both pre-and post-infection regimes and hence suggest that the effect may be mediated by a direct action on the virus particle. Perhaps it blocks binding or uptake to cells or causes immediate particle lysis. While we have planned experiments to elucidate a preliminary mode of action to evaluate the future potential of the extracts, it would be unreasonable to expect a precise mechanism at this stage.
Previous studies using Jatropha curcas Linn. stem bark extracts have shown inhibition of HIV-induced cytopathic effects [9,23]. Recently, the anti-HIV effect of 12-deoxyphorbol-13-phenylacetate, a compound synthesised from Jatropha phorbol esters, has been demonstrated; it inhibits HIV entry into target cells [24,25]. The anti-HIV activity of Jatropha curcas Linn. may be attributed to the secondary metabolites present in the leaf extracts. The aqueous leaf extract showed the presence of Tannins, Flavonoids, and Saponins as detected by HPTLC, while Flavonoids, Saponins were detected in the methanolic leaf extract. Recent reports by Kamal et al. [26] have given the chemical composition of leaves as containing Cyclic triterpenes: stigmasterol, stigmast-5-en-3, 7 diol, stigmast-5-en-3,7 diol, cholest-5-en-3,7 diol, campesterol, sitosterol, 7-keto-sitosterol as well as the d-glucoside of sitosterol and Flavonoids: apigenin, vitexin, isovitexin. Leaves also contain the dimer of a triterpene alcohol (C63H117O9) and two flavonoidal glycosides. Further fractionation and purification of these secondary metabolites for active antiviral and entry inhibitory activity screening is required to determine the exact mode of action.
While our laboratory has also shown similar potential anti-influenza (inhibition of hemagglutinin protein of Influenza virus) activity of the Jatropha curcas Linn. plant extracts [27], to the best of our knowledge, this is the primary report of anti-HIV activity of Jatropha curcas Linn. methanolic and aqueous leaf extracts against drug-resistant HIV.
The study also showed that the Jatropha curcas Linn. methanolic and aqueous leaf extracts were equally effective against isolates that were sensitive and potentially resistant to standard anti-retroviral drugs AZT, 3TC and d4T.

Conclusion
To conclude, the study has evaluated that Jatropha curcas Linn. has prospective antiviral activity against potentially drug-resistant HIV-1 and that these experimental moieties have favourable implications on the prevention or management of HIV/AIDS. The plant extracts may be used in the formulation of microbicides or surface disinfectants. It can be therefore deduced from that study that Jatropha curcas Linn. is a good candidate for anti-HIV therapy with further research.