WO2018142428A1 - Herbal microbicide formulation for preventing hiv - Google Patents

Abstract

The present invention relates to an aqueous gel formulation and its method of preparation using combination of four or five medicinal plants for preventing sexually transmitted HIV-1. The gel formulation comprises 50% aqueous ethanolic extracts of medicinal plants namely Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa, Acacia catechu and/or Aegle marmelos. The invention provides an herbal formulation useful as a vaginal/rectal microbicide gel. These herbal formulations showed potent in vitro anti-HIV-1 activity using reporter-gene based cell lines (TZM-bl) as well as human peripheral blood lymphocytes. These formulations also inhibited HIV-1 reverse transcriptase, integrase and protease activities suggesting that these may be working at multiple steps/stages of HIV life cycle and thus may be a valuable tool for alternative therapy.

Description

HERBAL MICROBICIDE FORMULATION FOR PREVENTING HIV

FIELD OF THE INVENTION

The present invention relates to formulations for preventing sexually transmitted HIV-1 infection. More particularly, it relates to a vaginal/rectal microbicide gel formulation comprising plant extracts for preventing HIV-1 infection. The present invention also relates to a method of preparing such gel formulation.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV), a lentivirus belonging to the Retroviridae family, is of two types, HIV-1 and HIV-2. HIV-1 is more virulent and the most common cause of Acquired Immunodeficiency Syndrome (AIDS) worldwide except in West Africa where HIV-2 is relatively common. However, HIV-2 is also slowly diffusing into various parts of the world other than West Africa. Total number of deaths due to HIV- AIDS in the year 2015 was 1.1 million and 2.1 million people were newly infected and enrolled for anti-retroviral treatment. At the end of year 2015, more than 36.7 million people were living with HIV infection. According to UNAIDS 2016 update, although the overall growth of the global AIDS epidemic appears to have stabilized and the number of new infections has been falling, overall level of new infections is still high, and with significant reduction in mortality, the number of people living with HIV infection worldwide has increased.

HIV virus attacks the immune system and in particular T-helper cells. It can also infect macrophages and dendritic cells. HIV entry into the host cells is initiated by the binding of the viral envelope glycoprotein gpl20 to the primary receptor (CD4) and a co-receptor, usually either CCR5 or CXCR4. T cell-tropic HIV strains tend to use the CXCR4 chemokine receptor, while macrophage-tropic strains tend to use the CCR5 chemokine receptor. After the initial binding of gpl20 with the CD4 receptor present on the target cells, it is further stabilized by the heparan sulphate proteoglycans present on the host cell surface. This binding induces a conformational change in the gpl20, exposing sites that interact with the chemokine receptor (CCR5 or CXCR4). The virus fusion protein (gp41) then gets uncovered and undergoes a conformational change. Glycoprotein gp41 inserts into the membrane of the host cells to initiate the fusion of the two bilayers. Viral RNA released into the cytoplasm undergoes reverse transcription with the reverse transcriptase (RT) enzyme and is converted into DNA. This viral DNA enters into host cell nucleus where it integrates in the host genome by integrase enzyme.

HIV-1/AIDS has been a major public health priority for the past three decades. In majority of the cases, the HIV infection occurs through heterosexual route; however, the transmission of HIV from the infected mother to child, through blood transfusion, use of contaminated needles by drug addicts and through homosexual route also contributes to its spread. To prevent HIV transmission through sexual route, 'safe sex' has been proposed as one of the major approaches. Male and female condoms provide an effective means for preventing sexually transmitted HIV infection; however, women fail to negotiate their use. The infection rates of HIV are higher in women than in men, and that could be because of more permissive environment in the female reproductive tract. Because of gender inequality, women (particularly in developing countries) have limited power to implement HIV-1 prevention options like condoms. Use of condoms by men is poor based on religious ground and the perceived notion that it reduces pleasure during sex. The reliance on the male sexual partner in HIV prevention practices often make women more susceptible to acquire the virus infection eight fold higher than men. Finding an effective method to reduce high infection rate among women is vital to control HIV-1 epidemic. Recent studies suggest that circumcision in men may also reduce HIV transmission. The concerted efforts to make vaccine that can effectively prevent HIV transmission have not resulted in practical options and most of these are at different stages of development and clinical trials. Treatment options for HIV infection with highly active anti-retroviral (HAART) drugs have expanded during the past years. These drugs fall into four general categories: nucleoside/nucleotide viral RT inhibitors (NRTIs), non-nucleoside RT inhibitors (NNRTIs), protease inhibitors (Pis), and fusion (or entry) inhibitors. The patients treated with HAART triple-drug cocktail of two nucleoside inhibitors and one protease inhibitor, reduced blood levels of virus below the detectable level (< 50 copies of viral RNA per ml of plasma). However, it is likely that long-term use of chemical drugs either singly or in combinations may lead to toxicity, especially to the bone marrow and suppression of CD8⁺ T cells along with different unwanted secondary effects. Another major concern in long-term chemical anti-retroviral therapy is the development of mutations in HIV genome leading to either partial or complete resistance to these therapies. Keeping in view of the above, it is imperative to discover novel anti-HIV agents from natural 75 sources that may have lesser side-effects. For the management of HIV/ AIDS, in 1989 the World Health Organization (WHO) had declared the necessity to assess ethnomedicines and other natural products. Various studies have shown anti-HIV properties of the extract prepared from variety of plants. The plant extracts or purified phytochemicals may exhibit anti-HIV activity by inhibiting virus entry/fusion, HIV-1 RT, protease or its integrase 80 activity. To prevent hetero- or homo-sexual transmission of HIV, microbicides with anti-HIV properties have been proposed that can be applied topically before sexual act.

A few natural therapies relating to treatment of sexually transmitted HIV are known in the existing art.

85

United States Patent Application 20150190450 to Alice Chang entitled "Ingredient for consumption and application" relates to an ingredient for consumption and application which comprises at least one plant ingredient and one Chinese medicine. The ingredient comprising the medicine can be applied directly to sex organ in the form of gel for treating sexually 90 transmitted diseases.

Patent and Publication Numbers US20060074108, WO2002096440, US7344738, IN243944, IN240422 and US20020182272 relate to herbal composition that may be used for treating or preventing AIDS and/or HIV.

95

United States Patent Application 20050025847 to Florence Camus-Bablon et al. entitled "Microbicidal compositions and method of use" relates to microbicidal compositions comprising bisabolol for preventing the transmission of or treating sexually transmitted infections and/or common vaginal infections, while minimizing disruptions to vaginal 00 ecology.

United States Patent Application 20050037033 to Florence Camus-Bablon et al. entitled "Microbicidal compositions and method of use" relates to microbicidal compositions containing a microbicidal formulation or agent for preventing the transmission of or treating 05 sexually transmitted infections and/or common vaginal infections, while minimizing disruptions to vaginal ecology and epithelium. Here, the microbicidal agent or formulation may include ciclopirox olamine or natural active ingredient.

European Patent 0867115 to Andreas Arndt et al. entitled "Microbicidal composition and its use" relates to an application solution containing the microbicidal agent in an aqueous or aqueous/alcoholic solution.

Still, there is need for an effective herbal formulation that is particularly useful in the prevention of sexually transmitted HIV without any side effects.

Thus, the present invention is aimed to provide a novel vaginal/rectal gel formulation comprising a combination of four and five plant extracts that prevent HIV-1 infection and a method of preparing such formulation. The formulations are having good activity against HIV-1 with no side effects to the normal cell lines.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an effective gel based herbal microbicide formulation to prevent sexually transmitted HIV-1 infection having no cytotoxic and other adverse effect to the normal cells.

The present invention relates to vaginal/rectal gel based herbal formulation, comprising four and five medicinal plants for preventing sexually transmitted HIV infection. The present herbal microbicide formulation is effective against HIV-1 infection with no side effects.

According to the present invention, the microbicide herbal formulation comprises aqueous alcoholic extracts obtained from medicinal plants namely Terminalia chebula, Phyllanthus embilica, Lagersromia speciose and Acacia catechu and/or Aegle marmelos. The herbal formulations showed potent in vitro anti-HIV-1 activity using reporter-gene based cell lines (TZM-bl) as well as human peripheral blood lymphocytes. These formulations also inhibited HIV-1 reverse transcriptase, integrase and protease activities suggesting that these may be acting at multiple steps of HIV life cycle. Further, preclinical safety evaluation revealed that these formulations have no adverse effect on the growth of lactobacilli associated with female reproductive tract, do not possess haemolytic activity, have no adverse effect on the integrity of monolayer formed by Caco-2 cells, and do not affect the viability of human cervico-vaginal cells. In addition, treatment of Salmonella typhimurium Strain 100 with both the herbal formulations do not led to an increase in the Mutagenic Index. Interestingly, treatment of human cervico-vaginal cells with these herbal formulations does not lead to increase in the production of pro-inflammatory cytokines, further suggesting there safety.

The foregoing and other objects and features of the present invention will become more evident from the following detailed description and appended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS:

The present invention will now be described in more detail with reference to the accompanying drawings, in which:

FIG. 1 illustrates a table showing the yield of 50% aqueous ethanolic extract from the respective plants by accelerated solvent extraction method;

FIG. 2 illustrates a table showing the physicochemical characteristics of plant extracts;

FIG. 3 illustrates a table showing the cytotoxicity and anti-HIV-1 activity of the extracts from different plants using HIV_NL4_.3 in TZM-bl cells based assay;

FIG. 4 illustrates a table showing the result of pro-inflammatory cytokines secretion by vaginal keratinocytes cells (Vk2/E6E7) after treatment for 24 hours with herbal formulations; FIG. 5 illustrates a table showing the mutagenic index of herbal formulations using Salmonella typhimurium Strain TA100;

FIG. 6 illustrates a graph showing the HPLC profiles of 50% ethanolic extract prepared from the fruits of T. chebula;

FIG. 7 illustrates a graph showing the HPLC profiles of 50% ethanolic extract prepared from the fruits of Phyllanthus emblica;

FIG. 8 illustrates a graph showing the HPLC profiles of 50% ethanolic extract prepared from the leaves of Lagerstroemia speciose; FIG. 9 illustrates a graph showing the HPLC profiles of 50% ethanolic extract prepared from the fruits of Aegle marmelos;

FIG. 10 illustrates a graph showing the HPLC profiles of 50% aqueous ethanolic extract prepared from the heart wood of Acacia catechu;

FIG. 11 illustrates HPTLC profiles of the methanol extract of the five plants extracts;

FIG. 12 illustrates a graph showing the HPLC profile of Formulation- 1 according to the present invention;

FIG. 13 illustrates a graph showing the HPLC profile of Formulation-2 according to the present invention;

FIG. 14 illustrates a graph showing the cytotoxicity evaluation of herbal formulations;

FIG. 15 illustrates a graph showing theanti-HIV-1 activity of herbal formulations;

FIG. 16 illustrates a graph showing theanti-HIV-1 activity of herbal formulations using human peripheral blood lymphocytes (PBLs);

FIG. 17 illustrates a graph showing the effect of herbal formulations on HIV-1 RT activity;

FIG. 18 illustrates a graph showing the effect of herbal formulations on HIV-1 protease activity;

FIG. 19 illustrates a graph showing the effect of herbal formulation- 1 and -2 on HIV-1 integrase;

FIG. 20 illustrates a graph showing the effect of herbal formulation- 1 and -2 on the viability of lactobacilli associated with female reproductive tract as normal flora;

FIG. 21 illustrates a graph showing the haemolytic activity of herbal formulation- 1 and -2;

FIG. 22 illustrates a graph showing the effect of herbal formulations on the integrity of epithelial monolayer; and

FIG. 23 illustrates a graph showing the effect of herbal formulations on primary human cervico- vaginal keratinocytes (Vk2/E6E7) viability.

DETAILED DESCRIPTION OF THE INVENTION: The present invention relates to a microbicide herbal formulation comprising a combination of four and five plant extracts for preventing HIV-1 transmission and a method of preparing such formulation. The formulation comprises aqueous alcoholic extracts of medicinal plants namely Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa, Acacia catechu and/or Aegle marmelos. Each plant extract is present in an amount of 1 to 5 milligram per 1 gram of the formulation. The invention provides an herbal formulation useful as a vaginal/rectal gel and a women/men adaptive method to prevent sexually transmitted HIV-1 infection.

The herbal formulations of the present invention showed potent in vitro anti-HIV-1 activity using reporter-gene based assay (cell lines TZM-bl) as well as human peripheral blood lymphocytes (PBLs). The herbal formulations inhibit HIV-1 infection at non-cytotoxic concentrations. These formulations also inhibited HIV-1 reverse transcriptase (RT), integrase and protease activities suggesting that these may be acting at multiple steps of HIV life cycle. Further, preclinical safety evaluation revealed that these formulations have no adverse effect on the growth of lactobacilli associated with female reproductive tract. These do not show any significant increase in the haemolytic activity of human red blood cells (RBCs). The herbal formulations do not have any adverse effect on the integrity of monolayer formed by Caco-2 cells and do not affect the viability of human cervico-vaginal cells at the concentrations which are much above their effective concentration to inhibit HIV-1 infection. Further, treatment of human cervico-vaginal cells with these herbal formulations does not lead to increase in the production of pro-inflammatory cytokines such as IL-Ιβ, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor (TNF), further suggesting their safety. In addition, treatment of Salmonella typhimurium Strain TA100 with both the herbal formulations do not led to an increase in the Mutagenic Index.

In an embodiment of the present invention, the microbicide herbal formulation comprises aqueous alcoholic extracts of four medicinal plants (herein referred as Formulation- 1) namely Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa and Acacia catechu. In another embodiment of the present invention, the microbicide herbal formulation comprises aqueous alcoholic extracts of five medicinal plants (herein referred as Formulation-2) namely Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciose, Acacia catechu and Aegle marmelos. The invention is further demonstrated with the following illustrated examples.

Example 1 Preparation of medicinal plants extracts and there characterization:

240 To make herbalgel formulation, 50% aqueous ethanolic extracts of medicinal plants namely Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa, Aegle marmelos and Acacia catechu were prepared, characterized by Reverse Phase High Performance Liquid Chromatography (HPLC), and High Performance Thin Layer Chromatography (HPTLC).

245 Preparation of plant extracts (using accelerated solvent extraction)

Collection of plant material: The fruits of Terminelia chebula, Phyllanthus embilica, Aegle marmelos; leaves of Lagerstroemia speciosa; and heart wood from Acacia catechu were collected from the botanical garden of Ayurveda Research Institute, Department of Ayush, Thiruvananthapuram, Kerala (under administrative control of Ministry of Ayush,

250 Government of India) and the identity of all the five plants was confirmed by the taxonomist from the same institute. The voucher specimens for fruits of Terminelia chebula [Accession Number, HLL/04/2013], fruits of Phyllanthus embilica [Accession Number, HLL/02/2013], fruits of Aegle marmelos [Accession Number, HLL/01/2013], leaves of Lagerstroemia speciosa [Accession Number, HLL/12/2015] and heart wood from Acacia catechu

255 [Accession Number, HLL/11/2015] have been kept at the Natural Products Division of HLL Lifecare Limited, Thiruvananthapuram, Kerala, India. The fruits were selected according to the uniformity of the shape.

Preparation of 50% aqueous ethanolic extract: The air and shade dried fruits of Terminelia 260 chebula, Phyllanthus embilica, Aegle marmelos; leaves of Lagerstroemia speciosa; and heart wood from Acacia catechu were powdered in an electric grinder. The grinded powder (30 gm) was subjected to pressurized sequential extraction using Accelerated Solvent Extractor (ASE 150, Dionex Inc., Sunnyvale, CA, USA; Alonso-Salces et al, 2001; Saha et al, 2015). The powdered material was mixed with diatomaceous earth at 4: 1 ratio. The mixture was 265 placed into a sample cell (100 ml) and loaded onto the ASE 150 system. The extraction was performed using 50% aqueous ethanol under pressure (1500 psi) at 60 C with a rinse volume of 90% using 3 static cycles. The solvent was then evaporated in a rotary evaporator (Biichi Labortechnik AG, 9230 Flawil, Switzerland). The dried extract was weighed and used for further studies. The yield of dried extract obtained by this procedure from all the five plants is 270 shown in table as illustrated in FIG. 1. It ranged from 3 to 64% of the dried powder weight taken to prepare the extract. The 50% aqueous ethanolic extracts can also be prepared by using various other methods such as Ultrasound Extraction (Sonication), Hot Continuous Extraction (Soxhlet), Maceration, Super Critical Fluid Extraction, and Microwave-assisted Extraction etc.

275

Characterization of 50% aqueous ethanolic extracts prepared from medicinal plants by reverse phase HPLC

Terminalia chebula: The Reverse Phase High Performance Liquid Chromatography (HPLC;

280 LC Agilent, Agilent Technologies, Boblingen, Germany) of the plant extract was performed using a Reverse phase XTerra RP 18 column (4.6 X 250 mm, 5 μιη; Waters Corporation, Milford, USA) and pure compounds (chebulagic and chebulinic acids) were used as reference standards. HPLC grade solvents were purchased from Merck, Mumbai, India. The solvent system used was 0.01% orthophosphoric acid in water : acetonitrile (80 : 20) with a flow rate

285 of 1 ml/min. The peaks were detected at 272 nm. The extract was prepared at a concentration of 5 mg/ml and the standards at 1 mg/ml in the mobile phase solvent system and 20 μΐ of each was injected for analysis. The data was processed using open lab software (Agilent Technologies). Chebulagic acid and chebulinic acid (Sigma-Aldrich Inc., St. Lois, MO, USA) were used as standards. Analysis of T. chebula extract by reverse phase HPLC, showed the

290 presence of chebulagic acid, chebulinic acid, gallic acid and ellagic acid in the extract. (FIG.

6).

Phyllanthus emblica: The 50% aqueous ethanolic extract prepared from the fruits of Phyllanthus emblica(100 μg) was also resolved as described for Terminalia chebula with the 295 exception that gallic acid and ellagic acid (Sigma-Aldrich Inc.) were used as reference standard. Reverse Phase HPLC revealed peaks of gallic acid and ellagic acid in the extract (FIG. 7).

Lagerstroemia speciosa: The 50% aqueous ethanolic extract prepared from the leaves of 300 Lagerstroemia speciosa (100 μg) was also resolved as described for Terminalia chebula with the exception that ellagic acid was used as reference standard. Reverse Phase HPLC revealed presence of ellagic acid in the extract (FIG. 8).

Aegle marmelos: The 50% aqueous ethanolic extract prepared from the fruits of Aegle 305 marmelos(l00 μg) was also resolved using same column but with isocratic solvent system comprising of 70% methanol and 30% water and imperatorin was used as reference standard. Reverse Phase HPLC revealed the presence of imperatorin in the extract (FIG. 9).

Acacia catechu: The 50% aqueous ethanolic extract prepared from the heart wood of Acacia catechu(l00 μg)was resolved using a reverse phase XTerra RP 18 column with a acetonitrile: methanol: orthophosphoric acid gradient in water. The acetonitrile:methanol: orthophosphoric acid was 5% at 0 min, 15% at 15 min, 25% at 35 min, 35% at 40 min, 50% at 45 min, 15% at 50 min, 5% at 60-70 min. Catechin hydrate was used as reference standard. Reverse Phase HPLC revealed the presence of catechins (FIG. 10).

C. HPTLC and physiochemical characterization of each extracts

HPTLC was carried out in CAMAG Linomat 5 with CAMAG TLC Scanner3 and Camag Reprostar 3 equipment. The stationary phase was Silica gel 60 (Merck 1.05554.0007) F₂5₄ 10x10 aluminium sheet and developed in CAMAG lOxlOcm Twin trough chamber. 1 gm of each plants extract (Terminalia chebula, Phyllanthus embilica, Lagerstroemia speciosa, Aegle marmelos, and Acacia catechu) sample is weighed and extracted with 10 ml methanol and spotted as 5 microlitre. To run these samples on stationary phase, Toluene: Ethyl acetate: Formic acid: Methanol (7:5: 1:0.5) was used as mobile phase. The plates were viewed under 254nm and 366nmas shown in FIG. 11.

The observed physicochemical characteristics of Terminalia chebula, Phyllanthus embilica, Lagerstroemia speciosa, Aegle marmelos, and Acacia catechu extract are mentioned in table as shown in FIG. 2 and HPTLC profiles of different plant extracts shown in FIG. 11.

Example 2

In vitro anti-HIV-1 activity of the respective plant extracts

The 50% aqueous ethanolic extracts prepared from the above five plants were evaluated for their anti-HIV-1 activity using reporter gene based cell assay employing TZM-bl cells [recombinant HeLa cell line expressing high levels of CD4, HIV-1 co-receptors CCR5 & CXCR4 with β-galactosidase and luciferase reporter genes under HrV-1 long terminal repeat (LTR) promoter]. TZM-bl cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma- Aldrich Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco, 340 Grand Island, NY, USA) and an antibiotic-antimycotic cocktail [Penicillin (100 units/ml), Streptomycin (100 g/ml) and Amphotericin B (250 ng/ml); Pen-Strep-Ampho sol, Biological Industries, Kibbutz beitHaemek, Israel]. In brief, TZM-bl cells (5.0 x 10⁴/well) were seeded in 24-well cell culture plate (Greiner Bio-One, GmbH, Frickenhausen, Germany) and cultured overnight at 37°C in a humidified atmosphere of 5% C0₂. In separate vials,

345 UW-l NL4.3 (CXCR4 using virus; NIH AIDS Research & Reference Reagent Program, Division of AIDS, NIAID, NIH, provided molecular clone of HIV-1_NL4_.3) at a multiplicity of infection (MOI) of 0.05 was treated with varying concentrations of plant extracts for 1 hour at 37°C. Subsequently, pre-treated viruses with the respective plant extracts were added in duplicates to TZM-bl cells and incubated for 4 hours. Nevirapine (2.0 μg/ml) was used as a

350 positive reference control whereas negative control comprised of cells without HIV infection.

After incubation, the cells were washed once with cold 50 mM PBS, pH-7.4 to remove the cell-free virus followed by addition of fresh culture medium with or without the gel formulation/gel base as per layout of the experiment. Cells were further incubated for 48 hours, washed twice with PBS and lysed with IX lysis buffer (Promega Corporation,

355 Madison, USA) by freeze-thaw. The supernatant was analyzed for luciferase activity by BrightGlo Luciferase Assay kit (Promega Corporation) in white opti-plate and luminescence was read using Fluorimeter (BMG Labtech GmbH, Offenberg, Germany) at a spectral range of 240-740 nm. The results were expressed as percent of viral inhibition compared to the untreated control and calculated by taking the luminescence in experimental group divided by

360 the untreated control multiplied by hundred. Percent inhibition was calculated by subtracting the above value from hundred. Next, non-linear regression analysis was used to calculate ICso.

Results: To confirm the anti-HIV-1 efficacy of 50% aqueous ethanolic extracts isolated from five different plants source, reporter gene based cell line (TZM-bl) assay was used. All

365 extracts from A. catechu, A. marmelos, L. speciosa, P. emblica, and T. chebula showed dose dependent inhibition of HIV-1 infection in TZM-bl cells with their IC₅₀ value of 2.68 + 0.3, 1.66 + 0.1, 0.61 + 0.1, 1.15 + 0.6, and 0.37 + 0.2 μg/ml respectively (as per table shown in FIG. 3). These values were calculated by estimated luminescence from the supernatant of lysed infected TZM-bl cells. Nevirapine (2μg/ml) used aspositive control. These plant

370 extracts were also evaluated for their impact on cellular viability of TZM-bl cells by MTT [3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; Sigma-Aldrich Inc.] assay which will be described in Example 4. CC₅₀ values of the extracts are given in FIG. 3. All the extracts showed potent anti-HIV-1 activity without significant cytotoxic effect on the viability of TZM-bl cells.

375

Example 3

A. Preparation of aqueous gel formulations comprising of the extracts from either four or five plants

380 To prepare aqueous gel formulation, first 2.0 gm of carbopol 974P NF polymer (Lubrizol, Belgium) was mixed in 78.60 ml of deionised water. Simultaneously, in a separate tube 0.18 gm of methylparaben and 0.02 gm of propylparaben were dissolved in 4.00 ml of deionized water. The solution containing methylparaben and propylparaben were added into the carbopol 974P NF polymer gel. To prepare the gel base, in the above mixture 0.2 gm

385 triethanolmine and 15.00 ml of glycerine were also added and thoroughly mixed. The formulation was prepared asper QBD (Quality by design) and based on these 8 combinations for four plants and 16 combinations for five plants were prepared. Out of these 1: 1: 1: 1 of four plants and 1: 1: 1: 1: 1 of five plant combinations shows good biological activity and was finalized. Concentration of each extract was finalized as 4 milligram/gram of the gel base. To

390 prepare Formulation- 1 comprising of four plants extracts, 400 mg each of the 50% aqueous ethanolic extracts (4 mg/gm gel base) prepared from the fruits of Terminalia chebula, fruits of Phyllanthus embilica, leaves of Lagerstroemia speciosa and heart wood of Acacia catechu were added. To prepare aqueous gel Formulation-2 comprising of five plants extracts, to the gel base, 400 mg each of the 50% aqueous ethanolic extracts prepared from the fruits of

395 Terminalia chebula, fruits of Phyllanthus embilica, fruits of Aegle marmelos, leaves of Lagerstroemia speciosa and heart wood of Acacia catechu were added.

B. Characterization of formulation

i) HPLC characterization of formulation

400 The Formulation- 1 was subjected to HPLC analysis using the solvent acetonitrile: methanol: orthophosphoric acid gradient in water. The acetonitrile: methanol: orthophosphoric acid was 5% at 0 min, 15% at 15 min, 25% at 35 min, 35% at 40 min, 50% at 45 min, 15% at 50 min, and 5% at 60-70 min. A mixture of chebulagic acid, chebulinic acid, ellagic acid, gallic acid, catechin hydrate were used as reference standards for Formulation- 1 (shown in FIG.12).

405 The Formulation-2 was subjected to HPLC analysis using the solvent acetonitrile: methanol: orthophosphoric acid gradient in water. The acetonitrile: methanol: orthophosphoric acid was 5% at 0 min, 15% at 15 min, 25% at 35 min, 35% at 40 min, 50% at 45 min, 15% at 50 min, and 5% at 60-70 min. A mixture of chebulagic acid, chebulinic acid, ellagic acid, gallic acid, catechin hydrate and imperatonin were used as reference standard for Formulation-2 (shown in FIG. 13). ii) Spreadability of formulation

1 gm of sample at room temperature was loaded on a glass plate placed over a squared paper (in millimeter).A second glass plate was placed over the sample and a weight of 100 gm was allowed to rest on the upper glass plate for 1 minute. The diameter after spreading of the gel was measured.

S = M*L / T

Spreadability = weight applied (M) * length or diameter (L) / time (T)

Spreadability value of formulation- 1 is 4.6 gm.cm/sec

Spreadability value of formulation-2 is 4.5 gm.cm/sec

iii) Extrudability

Extrudability was determined by applying 500gm weight was placed above the tube after removing the cap. The amount of gel extruded was collected and weighed

Extrudability = Applied weight to extrude gel from the tube (in gm)

Area (in cm²) Extrudability of formulation- 1 is 1.5gm/cm

Extrudability of formulation-2 is 1.2gm/cm iv) pH

pH of the formulation was checked by dipping the pH probe directly into the gel formulation pH of the gel formulation- 1 was 3.69

pH of the gel formulation-2 was 3.73 v) Viscosity Viscosity of gel formulation was checked using Anton paar Rheometre at 25 C and 37° C 440 using spindle no: pp25.

Viscosity of formulation- 1 is 1800 mpa.s

Viscosity of formulation-2 is 1750 mpa.s

Example 4

445

In vitro cyotoxicity and anti-HIV-1 activity of herbal Formulation- l/Formulation-2 using TZM-bl cells i) In vitro cytotoxicity

450 The inhibitory activity of the herbal extracts against HIV infection is sometimes a result of their toxic effects. Hence to exclude the non-specific antiviral effect, the toxicity of the herbal formulations on TZM-bl cells was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; Sigma-Aldrich Inc., St. Louis, MO, USA] assay. In brief, TZM-bl cells (8 x 107well/100 μΐ) were seeded in 96-well cell culture plates (Greiner Bio-

455 One, GmbH, Frickenhausen, Germany) and grown overnight at 37°C in a humidified atmosphere of 5% C0₂. Next day, herbal formulations were added in varying concentrations followed by further incubation for 48 hours. Negative control included cells treated with gel base. After incubation, cell viability was assessed by adding 20 μΐ MTT (5 mg/ml in PBS) per well and incubated at 37°C for 3 hours followed by addition of MTT solvent (100 μΐ/well;

460 absolute isopropanol, 0.04 N HC1) for 2 hours. The absorbance (OD) was read at 540 nm with reference filter at 630 nm. Experiments were repeated 3 times and each experiment was performed in duplicates. Percent viability was calculated by dividing the OD obtained in treatment group by OD of untreated cell control multiplied by hundred.

Results: Herbal formulations were first screened for their impact on cellular viability of 465 TZM-bl cells. The result of cellular viability evaluation was exhibited in percent viability and CC₅₀ value. CC₅₀ is the concentration of the formulations that reduced the cell viability by 50%. On the basis of gel amount, herbal formulation- 1 (F-l) showed CC₅₀ = 3.65 + 0.19 mg/ml and Formulation-2 (F-2) CC₅₀ was 1.93 + 0.48 mg/ml (shown in FIG. 14). Under similar experimental conditions, gel base showed CC₅₀ = 3.61 + 0.61 mg/ml.

470

ii) In vitro anti-HIV-1 activity using TZM-bl cells Further, these formulations were evaluated for their anti-HIV-1 activity using reporter gene- based cell assay employing TZM-bl cells as described above. In brief, TZM-bl cells (5.0 x 10⁴/well) were seeded in 24-well cell culture plate (Greiner Bio-One) and cultured overnight

475 at 37°C in a humidified atmosphere of 5% C0₂. In separate vials, HIV-1 NL4.3 (CXCR4 using virus) at a multiplicity of infection (MOI) of 0.05 was treated with varying concentrations of gel formulations as well as gel base for 1 hour at 37°C. Subsequently, pre- treated viruses with herbal formulations were added in duplicates to TZM-bl cells and incubated for 4 hours. Nevirapine (2.0 μg/ml) was used as a positive reference control

480 whereas negative control comprised of cells without HIV infection. Rest of the procedure is as described in Example 2.

Results: Formulation- 1 and -2 showed dose dependent inhibition of HIV infection with IC₅₀ values of 0.047 + 0.004 and 0.047 + 0.006 mg/ml respectively (shown in FIG. 15). Both the Formulations reduced the viral infection in TZM-bl cells as estimated by lucif erase assay

485 from the supernatant of lysed infected cells. Gel base when used at 31.5 mg/ml showed < 20% inhibition in HIV-1 infection. Nevirapine used as positive control showed almost 100% inhibition of HIV-1 infection at 2.0 μg/ml. The therapeutic index (TI; ratio of CC₅₀ by IC₅₀) values of the Formulation- 1 and -2 as compared with MTT assay was 76.8 and 41.06, respectively. Observed TI value suggests the therapeutic potential of these herbal

490 formulations.

Example 5

In vitro anti-HIV-1 activity of herbal formulations using human peripheral blood 495 lymphocytes (PBL)

Further, to confirm the efficacy of the herbal formulation- 1 and -2 in primary cells, phytohemagglutinin (PHA-P)-activated human peripheral blood lymphocytes (PBLs) based assay was used. This experiment was carried out under informed consent of the blood donors and following the clearance from the Institutional Bio-safety and Human Ethical Committee. 500 Blood (5 ml) was taken from healthy HIV sero-negative donors and PBLs were isolated using Ficoll density gradient method. Cells (2 x 10⁶ cells/ml) were stimulated for 3 days with PHA- P (3 μg/ml; Sigma-Aldrich Inc.) and after stimulation washed twice to remove PHA-P. Stimulated cells were infected by HIV-1(NL4.3) at an MOI of 0.05, in presence of IL-2 (10 U/ml) for 4 h. Infected cells were washed twice with plain medium to remove the unbound virus and seeded in 96-well plate (5 x 10⁴ cells/well/ 100 μΐ), in RPMI medium supplemented with 10% FBS and IL-2 (10 U/ml). The Formulations (100 μΐ/well) at varying 2x concentrations, diluted in culture medium, were added in duplicate as per layout of the experiment. Plates were incubated at 37°C, 5% C0₂ and culture supernatant was collected on 5^th day for p24 estimation. The viral load in the supernatant (diluted 1: 10 for virus control and 1: 1 for treatments) of treated human PBLs was measured using ELISA kit (SAIC-Frederick Inc., NCI-Frederick, USA; XpressBio, Life Science Products, MD, USA) by estimating p24, following the instructions of the manufacturer. Result was expressed as p24 concentration (pg/ml) taking into account the dilution of the used culture medium.

Results: With an aim to discover new gel-based herbal formulations, apart from the reporter- gene based assay, the anti-HIV-1 activity of the formulations was also assessed using activated human PBLs (biological targets of HIV including CD4+ T cells, monocytes, dendritic cells, etc.) from blood of HIV seronegative donors. The formulation- 1 and -2 were non-toxic to PBLs up to a concentration of 2.0 mg gel/ml (data not shown). There was a dose-dependent inhibition in p24 secretion by infected PBLs that were treated with different concentrations of the Formulations. The herbal formulation- 1 and -2 showed inhibition in p24 concentration to 25.59 + 0.07 pg/ml and 29.21 + 1.25 pg/ml respectively at 2 mg gel/ml as compared to 617.26 + 73.30 pg/ml with gel base (used as vehicle control) respectively (shown in FIG. 16) i.e. > 90% reduction in HIV-1 infection. Anti-HIV-1 activity of the Formulations in human PBLs was not due to non-specific cytotoxicity as more than 80% cells were viable. The culture medium of AZT (2 μΜ) treated HIV-1 infected human PBLs revealed p24 concentration of 54.5 + 14.96 pg/ml (Fig. 16).

Example 6 Formulation- 1 and -2 inhibit HIV-1 reverse transcriptase, protease and integrase activities i) HIV-1 reverse transcriptase (RT) inhibition activity

Since HIV-1 is a retrovirus, virally encoded enzyme reverse transcriptase (RT) that catalyses the conversion of viral RNA to proviral DNA is an important target where the formulation may act to inhibit HIV infection. For this, the herbal formulation- 1 and -2 were evaluated to know at what stage of HIV-1 virus cycle it inhibits the HIV-1 infectivity. Their activity to inhibit HIV-1 reverse transcriptase was evaluated using commercial ELISA kit (Roche Diagnostics, Mannheim, Germany). In brief, HIV-RT (10 mlU/reaction) was incubated with varying concentrations of herbal gel formulations as well as gel base in a reaction vial containing the template, digoxigenin- and biotin-labeled dUTP in a total reaction volume of 60 ul. The reaction mix was incubated for 60 min at 37°C. Subsequently, the reaction mix was incubated with streptavidin-coated microplate modules followed by incubation with anti- DIG-POD solution and development of color by 2, 2'-azinobis [3-ethylbenzothiozoline-6- sulfonic acid]-diammonium salt (ABTS) as per the instructions of the manufacturer. Nevirapine was used as reference standard. Absorbance was measured at 405 nm with reference wavelength of 490 nm using microplate reader. The resulting signal intensity is directly proportional to the actual RT activity. To analyze the inhibitory activity of the herbal formulations, percent inhibition was calculated as compared to sample that does not contain an inhibitor and IC₅₀ was calculated by non-linear regression.

Results: The in vitro inhibitory activity of the formulation- 1 and -2 on HIV-1 RT was determined as per the manual's instructions of the kit (Roche Diagnostics). Nevirapine (0.002 mg/ml) used as a positive control showed -70% inhibition in RT activity (shown in FIG. 17). Both Formulation- 1 and -2 showed dose dependent inhibition in the RT activity with an IC₅₀ value of 1.78 + 0.4 and 1.64 + 0.1 mg gel/ml respectively, while Gel base used as vehicle control exhibited -4% inhibition at 2 mg/ml (FIG. 17). These results suggest that some of the phyto constituents present in the herbal formulations inhibit HIV-1 RT activity. ii) HIV-1 protease inhibition activity

The ability of the herbal formulation- 1 and -2 to inhibit HIV-1 protease activity was determined by commercial kit (Anaspec, CA, USA). Protease inhibitors block the activity of the protease enzyme, which HIV uses to break up large polyproteins into the smaller pieces required for assembly of new viral particles. Various concentrations of the formulatios, gel base and HIV-1 protease diluent were added in 96-well plate in a total volume of 50 μΐ followed by addition of substrate solution (50 μΐ/well) by shaking plates gently for 30-60 sec. PepstatinA (PenA) was used as a positive control at 2 μΜ. The reaction mix was incubated at room temperature for 60 min followed by addition of Stop Solution (50 μΐ/well) provided in the kit. The fluorescence intensity was measured at 490 nm. Protease activity in presence of formulations was calculated by dividing the absorbance observed in its presence with absorbance observed in its absence multiplied by 100. Values obtained were subtracted from 100 to obtain percent inhibition in the protease activity.

Results: The in vitro inhibitory activity of the formulation- 1 and -2 on HIV-1 protease was determined as per the manual's instructions of the kit (Anaspec, CA, USA). Pepstatin (2 μΜ) was used as a positive control and showed -90% inhibition in HIV-1 protease activity (Fig.

575 13). Both formulation- 1 and -2 showed dose dependent inhibition in HIV-1 protease activity with an IC₅o value of 0.04 + 0.0.003 and 0.035 + 0.008 mg gel/ml, respectively. Gel base used as vehicle control exhibited -60% inhibition at 0.5 mg/ml (Fig. 18) suggesting that some of the components of gel base inhibit non- specifically HIV-1 protease activity. However, there is a significant difference in the anti-HIV- 1 protease activity of both the herbal

580 formulations as compared to gel base. iii) HIV-1 integrase inhibition activity

HIV-1 integrase inhibition activity of the respective herbal formulations was determined by a commercially available kit (XpressBio, Life Science Products, MD, USA). Briefly, double-

585 stranded HIV-1 LTR U5 donor substrate (DS) DNA containing an end-labelled biotin was added to the streptavidin-coated 96-well plates. Full-length recombinant HIV-1 integrase protein was loaded onto the well. Varying concentrations of the herbal formulations were added to the wells followed by the addition of a different double stranded target substrate (TS) DNA containing a 3 '-end modification. The HIV-1 integrase cleaves the terminal two

590 bases from the exposed 3 '-end of the HIV-1 LTR DS DNA and then catalyses a strand- transfer recombination reaction to integrate the DS DNA into the TS DNA. The products of the reaction were detected calorimetrically at 450 nm using an HRP labelled antibody directed against the TS 3 '-end modification. Raltegravir at 2 μΜ was used as positive control. Integrase inhibition activity of the formulations was calculated by dividing the absorbance

595 observed in presence of formulation with absorbance in its absence multiplied by 100. Values obtained were subtracted from 100 to obtain percent inhibition in the integrase activity.

Results: Formulaton-1 inhibited integrase activity with IC₅₀ = 0.51 + 0.05 mg gel/ml in a dose dependent manner, while Formulation-2 inhibited with IC₅₀ = 0.72 + 0.08 mg gel/ml

600 (shown in FIG. 19). However, gel base at 2.0 mg/ml did not show any significant anti- integrase activity, suggesting that the observed anti-integrase activity by both the gel-based herbal formulations may not be due to the components of gel base. Raltegravir showed -90% inhibition at 2 μΜ(ΡΙΰ. 14). The inhibition of HIV-1 integrase by both formulations suggests that impediment of viral DNA integration may be a potential target in the anti-HIV activity of

605 the herbal constituents of the formulations. Example 7

Effect of herbal formulations on the viability of lactobacilli associated with female reproductive tract

Lactobacillus sp. are the dominant members of the human vaginal microflora, where they play a protective role against urogenital infection, as well as prevent attachment of HIV virus, so it is essential that the formulation should be non-toxic to their growth. Various lactobacilli strains such as Lactobacillus casie (MTCC 1423), L. fermentum (MTCC 903), L. plantarum (MTCC 4462) and L. rhamnosus (MTCC 1408) were obtained from Institute of Microbial Technology, Chandigarh, India and cultured in MRS broth (HiMedia, Mumbai, India). The cytotoxicity of herbal formulations on lactobacilli was assessed by MTT assay. In brief, bacterial density was adjusted to an OD of 0.06 at a wavelength of 600 nm i.e. approximately 10 CFU/ml. Seventy microliter of herbal formulation/gel base diluted in MRS broth was added at varying concentrations into 96-well-round bottom plates along with 30 μΐ of bacterial suspension. Final volume was made up to 200 μΐ by adding MRS broth. Positive control included cells treated with ampicillin at 100 μg/ml. After incubation for 24 hours at 37°C, 10 μΐ of MTT (5 mg/ml in 50 mM PBS; Sigma- Aldrich Inc.) was added to each well containing microbial inoculums and herbal formulation/gel base. Plates were incubated for 3 hours at 37°C, followed by centrifugation at 2500 g for 10 min. Supematants were aspirated and 100 μΐ of acid-isopropanol (5 ml of 1 N HC1 in 95 ml of isopropanol) was added to each well. Optical density was measured using microplate spectrophotometer (EL_X 800MS;BioTek Instrument Inc., Vermont, USA) at 540 nm using reference filter at 630 nm. Percent viability was calculated by dividing the absorbance of treated cells to untreated cells multiplied by hundred.

Results: Incubation of Lactobacillus casei, L. fermentum, L. plantarum and L. rhamnosus with herbal formulation- 1 up to 62.5 mg gel/ml and formulation-2 up to 50 mg gel/ml revealed no adverse effect on the viability of the lactobacilli (shown in FIG. 20) respectively. The gel base up to 62.5 mg/ml, used as vehicle control, also had no adverse effect on the viability of lactobacilli (FIG. 20). Under similar experimental conditions, ampicillin (100 μg/ml) used as positive control, showed significant decrease in the viability of lactobacilli (FIG. 20). These results suggest that herbal formulations as described in this present invention do not have any adverse effect on the viability of the lactobacilli at the tested concentrations. Example 8

In vitro haemol tic activity of herbal formulations on human red blood cells (RBCs)

645 Toward pre-clinical safety evaluation of the proposed herbal formulations as candidate microbicides, it is pertinent to investigate, if these have any haemolytic effect on human RBC. In brief, human RBCs were isolated from the 5 ml blood of healthy individual; blood was mixed with equal volume of 50 mM PBS (pH-7.4). The diluted blood was centrifuged at 3000 rpm for 15 min and packed cells were washed two times with PBS. Human RBCs

650 (10⁶/tube/ml) were incubated with varying concentrations of formulation- 1 and -2 at 37°C for 30 to 45 min and haemolysis of RBC was measured spectrophotometrically at 450 nm. Triton X-100 (0.1%) was used as positive control whereas gel base was used as vehicle control. Percent haemolysis was estimated by dividing the optical density (OD) obtained in presence of the test compound by OD obtained by 0.1% Triton X-100 treated RBCs multiplied by 100.

655 Results: Under the experimental conditions as described above, Formulation- 1 revealed less than 5% haemolysis of RBCs even at the highest concentration of 31.2 mg gel/ml used (FIG. 21A). Similarly, Formulation-2 even at 25 mg gel/ml showed less than 8% haemolysis as compared to RBCs treated with Triton X-100 (FIG. 21B). The haemolysis of human RBCs by Formulation- 1 and -2 was not statistically significant as compared to gel base used as vehicle

660 control.

Example 9

Effect of herbal formulations on the integrity of epithelial monolayer

665 Preclinical safety investigations should also comprise evaluation of herbal formulations for their interference with epithelial lining integrity. Maintenance of an intact as well as polarized monolayer in presence of a potential microbicide candidate is an important factor to be considered as any damage in epithelial layer may allow infectious virus to reach the host target cells. Keeping this in view, deleterious effect of Formulation- 1 and -2 was evaluated on

670 the monolayer formed by Caco-2 cells. Transepithelial Resistance (TER), a measure of epithelial integrity was performed on monolayer formed by Caco-2 cells. The cells (5.0 x 10⁵/well) were grown in the apical chamber of transwell plates and culture medium (1.5 ml) was dispensed in the basolateral compartment of each well. The cells were allowed to grow for 36-48 hours in 5% C0₂ at 37°C and assessed for formation of monolayer by measuring 675 TER. Resistance was measured using Millicell-ERS voltmeter (EMD Millipore Corporation, Billerica, MA, USA) each day until resistance reached plateau. After formation of monolayer, the herbal Formulation- 1 and -2 (5 mg/ml) were added in the culture medium and cells were further incubated in humidified atmosphere of 5% C0₂ at 37°C. Gel base at 5 mg/ml and Triton-X at 0.01% were used as vehicle and positive controls respectively. Resistance was

680 measured at 1, 2, 4, 8 and 24 hours after addition of herbal formulations, vehicle control and positive control.

Results: Herbal formulations were also evaluated for integrity of monolayer cells, because preclinical safety investigations should also comprise evaluation of interference with

685 epithelial integrity. Potential microbicide candidate/herbal formulations should avoid any damage in epithelial layer, which may allow infectious virus to reach the host target cells. Formulation- 1 and -2 when used at 5 mg gel/ml did not reduce the TER significantly (p > 0.05) as compared to the untreated cells (shown in FIG. 22). After 24 hours of application, the TER values were same as of untreated cells. Same observations were made in presence of

690 gel base (5 mg gel/ml). However, Triton X-100 (0.1%) led to a non-reversible reduction in TER even after 1 h of its application (Fig. 17). Overall herbal Formulation-1 and -2 did not disturb monolayer formed by Caco-2 cells and hence may be suitable candidate for topical application.

695 Example 10

Cytotoxicity evaluation of herbal formulations on human cervico- vaginal keratinocytes and secretion of proinflammatory cytokines

700 i) In vitro cytotoxicity evaluation

To study, if herbal formulations on local application may induce cytotoxicity to human vaginal epithelial cells and may also induce the secretion of pro-inflammatory cytokines, human cervico-vaginal keratinocyte cell line (Vk2/E6E7) was used as an experimental model. Vk2/E6E7 cells (6.0xl07well) were seeded in 96-well culture plate and incubated in 705 humidified atmosphere of 5% C0₂ at 37°C for overnight. Next day, cells were treated with varying concentrations of herbal Formulation-1 and -2 or gel base for 24 hours. Cell viability was assessed by adding 20 μΐ MTT (5 mg/ml in PBS) per well and incubated at 37°C for 3 hours followed by addition of MTT solvent (100 μΐ/well; absolute isopropanol, 0.04 N HC1). The absorbance (OD) was read at 540 nm with reference filter at 630 nm. The percent 71 0 viability was calculated by dividing the OD obtained in treatment group by OD of untreated cell control multiplied by hundred.

Results: While considering herbal formulations as candidate microbicides to be used for prevention of sexually transmitted HIV-1 infection, their effect on primary vaginal epithelial

71 5 cells viability is highly relevant, as vaginal epithelial cells form a part of the physical barrier that may impede the passage of cell-free or cell- associated HIV-1 into sub-epithelial tissues. Therefore, ruling out any adverse effect of the herbal formulations on vaginal cells is relevant. The viability assay was performed on human cervico-vaginal keratinocytes (Vk2/E6E7) cells using MTT assay as described above. The CC₅₀ observed with

720 Formulation- 1 was 1.6 + 0.04 mg gel/ml whereas CC₅₀ with Formulation-2 was 2.1 + 0.17 mg gel/ml (FIG. 20). The gel base showed CC₅₀ of 2.95 + 0.42 mg gel/ml (FIG. 23). ii) In vitro evaluation of pro-inflammatory cytokines secretion by human cervico-vaginal keratinocytes after application of the herbal formulations

725 Clinical trials based on different microbicides have raised concern that rise in proinflammatory cytokines secretion by their application may increase the susceptibility to HIV- 1 infection. Thus, evaluation of pro-inflammatory cytokines secretion levels after application of the herbal Formulation- 1 and -2 are relevant. To study the inflammatory responses of the herbal formulations, a human cervico-vaginal keratinocyte cell line (Vk2/E6E7) was used.

730 Vk2/E6E7 cells are immortalized human vaginal epithelial cells that proved to be an adequate model for studying the vaginal responses to topical agents. Cells (6.0 x 10 cells/well) were seeded in 96-well culture plate and incubated in humidified atmosphere of 5% C0₂ at 37°C for 24 h. After incubation, cells were treated with Formulation- 1 and -2 (1 mg gel/ml) for 24 hours and culture supernatant was collected for estimation of various cytokines using BD™

735 Cytometric Bead Array Human Inflammatory Cytokines kit (BD Biosciences, San Jose, USA). The kit provides a method of capturing a soluble analytes with beads of known size and fluorescence to detect interleukin IL-Ιβ, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor (TNF) using flow cytometry (BD FACSCanto Flow Cytometer; BD Biosciences Pharmigen, San Diego, CA, USA). This assay was performed according to the manufacturer's

740 specifications and data analysed using BD FACS Diva software. Results: Proinflammatory cytokines play a critical role in HIV-1 pathogenesis. Some of them, such as IL-1, IL-8, and IL-6, have been studied in genital fluids. Proinflammatory cytokines such as IL-1, IL-6, and TNF- stimulate viral replication in latently infected cells.

745 The resulting table as illustrated in FIG. 4shows no increase in pro-inflammatory cytokines as compared to cell control. On the contrary, a decrease in the concentration of IL-Ιβ, IL-6, IL-8 and TNF was observed subsequent to treatment of Vk2/E6E7 cells with both Formulation- 1 (F-l) and -2 (F-2). Human IL10 and IL-12p70 were below the detection limit of the assay both in the culture supematants of untreated cells as well as those treated with the herbal

750 formulations. Thus, the herbal formulations of both four plants (Formulation- 1) and five plants (Formulation-2) appear to be safe and may be useful candidates to be developed as microbicides.

Example 11

755

Mutagenicity of the herbal formulations on Salmonella typhimurium

Herbal formulation to be used as topical microbicide against prevention of sexually transmitted HIV-1 should also be evaluated for any deleterious effect on the genetic material of the organism. Salmonella typhimurium strain TA100, which detects base pair mutations

760 was used to test the mutagenic behavior of herbal Formulations- 1 and -2. In brief, the agar plates (1.5% agar, 2.0% glucose) in Vogel-Bonner medium E (40 mM MgS0₄, 520 mM citric acid, 2.87 M K₂HP0₄, 0.87 M NaHNH₄) were prepared as per the standard procedure. Salmonella typhimurium viz. TA100 (obtained from Microbial Type Culture Collection &Gene Bank, Institute of Microbial Technology, Chandigarh) was grown in Nutrient Broth at

765 37°C overnight with shaking. Herbal Formulation-1 and -2 (1 mg gel/ml) or gel base (1 mg/ml) used as vehicle control were mixed with Salmonella culture (1 x 10⁶ cells/ml) in a total volume of 2.0 ml and incubated at 37°C for 20 min without shaking. Sodium azide (5 μg in two ml) was used as positive control. Subsequently, 2.0 ml of molten top agar (0.8% agar, 0.5% NaCl) supplemented with histidine (0.05 mM) and biotin (0.05 mM) was added into

770 each tube containing a mixture of Salmonella typhimurium cells and test compounds and quickly mixed and poured on top of the agar plate. Plates were subsequently incubated at 37°C for 48-72 hours and colonies are then counted. The resulting table is expressed as the number of revertant colonies per plate (as shown in FIG. 4). The mutagenic index (MI) was also calculated for each formulation tested, this being the average number of revertants per

775 plate with the test formulation divided by the average number of revertants per plate with the negative control (cell control). Formulation was considered mutagenic when a two-fold increase in the number of mutants (MI > 2) was observed.

Results: Salmonella typhimurium (strain TA100) was treated with the herbal Formulation- 1 780 and -2 as well as gel base at 1 mg/ml. The colonies were counted to determine the mutagenic activities of Formulation- 1 and 2. The Formulation- 1 and -2 showed non-mutagenic effects at the concentration of 1 mg/ml/plate by the bacterial reverse mutation assay against chemical mutagens in S. typhimurium strain as shown in FIG. 5.

785 In-vitro studies have been performed for extracts as well as formulation. The advantage of the herbal formulation of the present invention is that the combination of medicinal plants which intervene at different points of lifecycle of HIV virus will exhibit greater potency and a broader spectrum of activity than single-agent microbicides.

790 The prepared herbal gel formulation is effective against HIV-1 and it has no toxicity at the doses tested. Thus, the present invention provides an effective microbicide herbal formulation useful as vaginal/rectal gel for the prevention of sexually transmitted HIV-1.

Although the foregoing invention has been described in some detail by way of examples for 795 purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

800

805 REFERENCES:

Abdool Karim Q, Sibeko S, Baxter C. Preventing HIV infection in women - a global health imperative. Clin Infect Dis. 2010; 50 Suppl 3: S 122-S 129.

81 0 Agwu AL, Fairlie L. Antiretroviral treatment, management challenges and outcomes in perinatally HIV infected adolescents. J Int AIDS Soc. 2013; 16: 18579.

Alonso-Salces RM, Korta E, Barranco A, Berrueta LA, Gallo B, Vicente F. Determination of polyphenolic profiles of Basque cider apple varieties using accelerated solvent extraction. 81 5 Agric Food Chem. 2001; 49: 3761-3767.

Avi R, Pauskar M, Karki T, Kallas E, Jogeda EL, Margus T, Huik K, Lutsar I. Prevalence of drug resistance mutations in HAART patients infected with HIV-1 CRF06_cpx in Estonia. Med Virol. 2016; 88: 448-454.

820

Bessong PO, Obi CL, Andreola ML, Rojas LB, Pouysegu L, Igumbor E, Meyer JJ, Quideau S, Litvak S. Evaluation of selected South African medicinal plants for inhibitory properties against human immunodeficiency virus type 1 reverse transcriptase and integrase. Ethnopharmacol. 2005; 99: 83-91.

825

Bulteel N, Bansi-Matharu L, Churchill D, Dunn D, Bibby D, Hill T, Sabin C, Nelson M. The emergence of drug resistant HIV variants at virological failure of HAART combinations containing efavirenz, tenofovir and lamivudine or emtricitabine within the UK Collaborative HIV Cohort. J Infect. 2014; 68: 77-84.

830

Carlson JM, Schaefer M, Monaco DC, Batorsky R, Claiborne DT, Prince J, Deymier MJ, Ende ZS, Klatt NR, DeZiel CE, Lin TH, Peng J, Seese AM, Shapiro R, Frater J, Ndung'u T, Tang J, Goepfert P, Gilmour J, Price MA, Kilembe W, Heckerman D, Goulder PJ, Allen TM, Allen S, Hunter E. Selection bias at the heterosexual HIV-1 transmission bottleneck. Science 835 2014; 345: 1254031.

De Clercq E. Antiretroviral drugs. Curr Opin Pharmacol. 2010; 10: 507-515. De Clercq E. Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of 840 HW.Int JAntimicrob Agents. 2009; 33: 307-320.

Dumas F, Preira P, Salome L. Membrane organization of virus and target cell plays a role in ΗΓ entry. Biochimie. 2014; 107 Pt A: 22-27.

845 Eberle J, Giirtler L. HIV Types, groups, subtypes and recombinant forms: errors in replication, selection pressure and quasispecies. Intervirology . 2012; 55: 79-83.

Ekstrand ML, Shet A, Chandy S, Singh G, Shamsundar R, Madhavan V, Saravanan S, Heylen E, Kumarasamy N. Suboptimal adherence associated with virological failure and 850 resistance mutations to first-line highly active antiretroviral therapy (HAART) in Bangalore, India. Int Health. 2011; 3: 27-34.

Este JA, Cihlar T. current status and challenges of antiretroviral research and therapy .Antiviral Res. 2010; 85: 25-33.

855

Gandhi RT, Zheng L, Bosch RJ, Chan ES, Margolis DM, Read S, Kallungal B, Palmer S, Medvik K, Lederman MM, Alatrakchi N, Jacobson JM, Wiegand A, Kearney M, Coffin JM, Mellors JW, Eron JJ. The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial. PLoS Med. 860 2010; 7. pii: el000321.

Gupta SK, Nutan. Clinical use of vaginal or rectally applied microbicides in patients suffering from HIV/ AIDS .HIV AIDS (Auckl). 2013; 5: 295-307.

865 Haacker M, Fraser-Hurt N, Gorgens M. Effectiveness of and financial returns to voluntary medical male circumcision for HIV prevention in South Africa: An incremental cost- effectiveness analysis. PLoS Med. 2016; 13: el002012.

Han H, He W, Wang W, Gao B. Inhibitory effect of aqueous Dandelion extract on HIV-1 870 replication and reverse transcriptase activity. BMC Complement Altern Med. 2011; 11: 112. Hocqueloux L, Avettand-Fenoel V, Jacquot S, Prazuck T, Legac E, Melard A, Niang M, Mille C, Le Moal G, Viard JP, Rouzioux C. Long-term antiretroviral therapy initiated during primary HIV-1 infection is key to achieving both low HIV reservoirs and normal T cell 875 counts Antimicrob Chemother. 2013; 68: 1169-1178.

Kapewangolo P, Knott M, Shithigona RE, Uusiku SL, Kandawa-Schulz M. In vitro anti-HIV and antioxidant activity of Hoodia gordonii (Apocynaceae), a commercial plant product. BMC Complement Altern Med. 2016; 16: 411.

880

Klos M, van de Venter M, Milne PJ, Traore HN, Meyer D, Oosthuizen V. In vitro anti- HIV activity of five selected South African medicinal plant extracts. Ethnopharmacol. 2009; 124: 182-188.

885 Kripke K, Njeuhmeli E, Samuelson J, Schnure M, Ncube B, Dalai S, Farley T, Hankins C, Thomas AG, Reed J, Stegman P, Bock N. Assessing progress, impact, and next steps in rolling out voluntary medical male circumcision for HIV prevention in 14 priority countries in eastern and southern Africa through 2014. PLoS One. 2016; 11: e0158767.

890 Kurapati KR, Atluri VS, Samikkannu T, Garcia G, Nair MP. Natural products as anti-HIV agents and role in HIV-associated neurocognitive disorders (HAND): A Brief Overview. Front Microbiol. 2016; 6: 1444.

Montagnier L. 25 years after HIV discovery: prospects for cure and vaccine (Nobel 895 lecture) Angew Chem Int Ed Engl. 2009; 48: 5815-5826.

Ndagije H, Nambasa V, Namagala E, Nassali H, Kajungu D, Sematiko G, Olsson S, Pal S. Targeted spontaneous reporting of suspected renal toxicity in patients undergoing highly active anti-retroviral therapy in two public health facilities in Uganda. Drug Saf. 2015; 38: 900 395-408.

Nunes R, Sarmento B, das Neves J. Formulation and delivery of anti-HIV rectal microbicides: advances and challenges. Control Release. 2014; 194: 278-294. 905 Nutan, Modi M, Dezzutti CS, Kulshreshtha S, Rawat AK, Srivastava SK, Malhotra S, Verma A, Ranga U, Gupta SK. Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat. Virol J. 2013; 10: 309.

Saha S, Walia S, Kundu A, Sharma K, Paul RK. Optimal extraction and fingerprinting of 91 0 carotenoids by accelerated solvent extraction and liquid chromatography with tandem mass spectrometry. Food Chem. 2015; 177: 369-375.

Subbaraman R, Chaguturu SK, Mayer KH, Flanigan TP, Kumarasamy N. Adverse effects of highly active antiretroviral therapy in developing countries. Clin Infect Dis. 2007; 91 5 45: 1093-1101.

Tewtrakul S, Itharat A, Rattanasuwan P. Anti-HIV-1 protease- and HIV-1 integrase activities of Thai medicinal plants known as Hua-Khao-Yen.J Ethnopharmacol. 2006; 105: 312-315.

920 Tshikalange TE, Meyer JJ, Lall N, Munoz E, Sancho R, Van de Venter M, Oosthuizen V. In vitro anti-HIV-1 properties of ethnobotanically selected South African plants used in the treatment of sexually transmitted diseases. J Ethnopharmacol. 2008; 119: 478-481.

Wilen CB, Tilton JC, Doms RW. HIV: cell binding and entry. Cold Spring Harb Perspect 925 Med. 2012; 2. pii: a006866.

Zhan P, Pannecouque C, De Clercq E, Liu X. Anti-HIV drug discovery and development: current innovations and future trends. J Med Chem. 2016; 59: 2849-2878.

930

935

Claims

WE CLAIM:

940 1. A herbal microbicide formulation for preventing the transmission and/or infection of

HIV-1 during sexual activity, comprising: 50% by weight of said formulation comprising an aqueous alcoholic extract of Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa, Acacia catechu and Aegle marmelos.

945 2. A herbal microbicide formulation for preventing the transmission and/or infection of

HIV-1 during sexual activity, comprising: 50% by weight of said formulation comprising an aqueous alcoholic extract of Terminalia chebula, Phyllanthus emblica, Lagerstroemia speciosa and Acacia catechu.

950 3. The formulation according to claim 1 or 2, is in the form of gel.

4. The formulation according to claim 1 or 2, is for vaginal application.

5. The formulations according to claim 1 or 2, is for rectal application.

955

6. The formulation according to claims 1 or 2, wherein said extract of each herb or medicinal plant is present in an amount of 1 to 5 milligram per 1 gram of said formulation.

960 7. A method for preventing the transmission and/or infection of HIV-1 during sexual activity, the method comprising the step of vaginally administering the herbal microbicide formulation of claim 1 or claim 2 to a female subject in need thereof.

8. A method for preventing the transmission and/or infection of HIV-1 during sexual

965 activity, the method comprising the step of rectal application of the herbal microbicide formulation of claim 1 or claim 2 to a male subject in need thereof.

9. The method according to claim 7 or 8, wherein said formulation is administered in the form of a gel.