Field of the invention
The present invention relates to an extract from the plant Rubia cordifolia useful for the treatment Sexually transmitted infections and a process for its preparation . Particularly, the invention relates to a novel extract from the plant Ruhia cordifolia useful for the treatment Sexually transmitted infections caused by Human Immunodeficiency virus, Herpes simplex virus-2, hepatitis B virus, Neisseria gonorrhoea and Trichomonas vaginalis and a process for its preparation . The extract of the present invention is isolated from the various parts of the plant Rubia cordifolia particularly the roots of the said plant.
Background of the invention
Sexually transmitted diseases or sexually transmitted infections, as it is termed today, are among the most common infectious diseases and pose a major public health concern globally (Sahyoun & Shukri, 2004). Sexually Transmitted infections are a major source of morbidity and mortality and represent a major socioeconomic cost in both the developing and industrialized nations (Wu et al.9 2004). The World Health Organization estimates 333 million cases of curable STDs worldwide. These diseases not only affect sexually active individuals but also new borns, who may become infected perinatally (Herold et al., 1999). Women and their infants primarily suffer the consequences of untreated and improperly treated infections, which include the PID, infertility, spontaneous abortion, still birth and cervical cancer. According to the WHO, approximately 125 million new cases of major bacterial and viral STDs occur per year worldwide. STDs, also known as venereal diseases, are infections caused by a variety of pathogens including bacteria (Neisseria gonorrhoea, Treponema pallidum, Haemophillus ducreyi, Gardnerella vaginalis), viruses (human immunodeficiency virus, Herpes simplex virus-2, Human papilloma virus, and Hepatitis B and C virus) and parasites (Trichomonas vaginalis, Giardia lamblia). STIs and HIV are gaining significant importance at present due to rapid spread of these diseases and high cost of treatment.

Human Immunodeficiency Virus
In 1983 the causative agent of AIDS was identified as a human retrovirus, first isolated in France from a patient with multiple lymphadenopathies, a condition linked to AIDS and subsequently in 1984, from AIDS patients. Initially three different names were given to the virus isolated from AIDS patients: human T lymphotropic virus III (HTLV-III), lymphadenopathy-associated virus (LAV) and AIDS-associated retrovirus (ARV). Eventually in 1986, the AIDS -causing virus was given an alternative name, Human Immunodeficieny virus (HIV).
Targets for Anti-HIV Chemotherapy: Every step in the HIV replication is considered as potential target for anti-viral chemotherapy. But, the number of targets for drug interventions is reduced due to the fact that the virus is an intracellular parasite, which relies on the metabolic pathway of the host cell. Thus, most agents that block the replication of the virus are also lethal to the host cell.
The gained knowledge about the replicative cycle of HIV-virus has led to the extraction of virus-specific processes. Scientists have focused their attention on the following processes, (a) viral binding to target cells, (b) virus cell fusion, ( c) virus uncoating, (d) reverse transcription of genomic RNA, (e) viral integration, ( f) gene expression, and (g) protease activity. So far, the strategies involving the inhibition of reverse transcription of genomic RNA and protease activity have been proven to be the most successful in the search for drugs that can be used for the treatment of AIDS.
Reverse Transcriptase Inhibitors; HIV reverse transcriptase (RT) catalyses the replication of single- stranded viral RNA to a double -stranded DNA. Inhibition of RT prevents the formation of this double-stranded DNA that can be integrated in the host DNA. Reverse transcriptase inhibitors can be divided into two categories , nucleoside (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI).Today there are around six NRTI approved; Zidovudine, Zalcitabine, Didanosine, Stavudine, Lamivudine and Abacavir. But, all these substances are associated with associated with side effects, like bone marrow suppression, peripheral neuropathy and acute pancreatitis. The NNRTIs, like

V.;
Nevirapine, Efavirenz and Delaviridine, bind in the highly hydrophobic pockets of the enzyme and exhibit greater affinity for the enzyme-substrate complex .Though NNRTIs have a high selectivity index and a low toxicity, the virus is eliciting rapid resistance to these group of drugs.
HIV Protease Inhibitors: The HIV encodes an aspartyl protease made of 99amino acids that is essential for the cleabvage of the Gag polypreotein precursor, Blockage of theis enzyme leads to the formation of immature, non- infectious virions.Some of the FDA approved protease inhibitors are Saquinavirs, Ritonavir, Indinavir, Nelfinavir, Amprenavir and Lopinavir.Though these drugs are highly selective, they induce side effects such as lipodystrophy, hyperlipidaemia, insulin resistance and emergence of resisitant mutants upon prolonged use. (Alterman, 2001).
Herpes Simplex Virus-2
The herpes simplex virus belonged to the Family Herpesviridae, subfamily Alphaherpesviridae and the genus Simplexvirus. They are large enveloped icosahedral virions.Herpes simplex virus -2 causes genital herpes; a typical lesion being a vesicle, a ballooning degeneration of intra-epithelial cells. In females the virus infects the labia, vagina (or the cervix, while in males, the most frequent sites of infection are the glans and shaft of penis. This virus has a tendency to latently infect the sacral ganglia and may get reactiviated.
The choice of treatment for herpes infection is acyclovir (ACV), a nucleoside analogue of guanosine that has to be phosphorylated three times. The first phosphorylation is completed by the viral encoded thymidine kinase (TK) protein which allows ACV to become active only in virus infected cells. The second and third phosphorylation are achieved by cellular thymidine kinases. ACV triphosphate acts by competitive inhibition of viralDNA polymerase and it is a DNA chain terminator. The other drugs used for herpetic infections are penciclovir, valacyclovir and famciclovir (guanosine analogues ), cidofovir ( a phosphonate molecu;e derived from cytidine) and Foscarnet ( a pyrophosphate analogue that acts directly on viral polymerase). The virus

is gaining resistance to these drugs due to one of these mechanisms: a loss of TK activity ( TK deficient virus), an alteration in the TK substrate specificity (TK altered virus) and /or an alteration of the DNA polymerase activity. These mutations leading to resistance occur spontaneously during viral replication and resistant viruses are then selected by antiviral treatment, treatment (Morfin & Thouvenot, 2003). The frequency at which these drug resistant viruses are encountered ranges from 2-14 % (Sarisky et aL, 2002.).
Hepatitis B Virus
Hepatitis B virus belongs to the family Hepadnaviruses. The viral genome of HBV is partially double stranded circular DNA of approximately 3200 base pairs that encodes four overlapping open reading frames.
The clinical manifestations of Hepatitis B virus are highly variable. Generally newborns do not develop any clinical symptoms and infection produces typical illness in only 5-15% of children of Ito 5 years of age. In 33-50% adults infections are symptomatic. The clinical signs and symptoms of acute hepatitis include fever, anorexia, nausea, malaise, vomiting, jaundice, dark urine, and abdominal pain. Sometimes extra-hepatic manifestations like skin rashes, arthralgia and arthritis also occur. Fulminant hepatitis occurs in about 1-2 % cases and has a fatality of 63-93%. ( McMahon et al9 1985).
The initiative for hepatitis B treatment has been extensively reviewed. The only licensed antiviral drugs effective against HBV are the nucleoside analogues lamivudine and Adefovir. Lamivudine has been used for several years with worthy successes but recently mutative escape of viruses has started occurring. One well-characterized mutation is in the highly conserved YMDD motif of the polymerase enzyme. In addition, protection which follows successful application of lamivudine is often lost when use of the drug is stopped. (Hilleman, 2003). Adefovir apparently has a greater effectiveness; however, toxicity for the kidney by this drug may be a limiting factor. Interferon alpha have been used for the treatment of chronic hepatitis B. but its side effects include fever, malaise, neutropenia and thrombocytopenia. . (Lee, 2003), (Feld & Locarnini, 2002).

Neisseria gonorrhea
These organisms are gram-negative cocci with the adjacent sides concave, being kidney shaped. They possess pili on their surface. This facilitates their adhesion to the mucosal surfaces. Gonorrhorea is an STD known from ancient times. This name Gonorrhorea was given to it by Galen in 130 AD which meant flow of seed. This disease remains one of the most common sexually transmitted diseases worldwide that is associated with serious female genital tract complications such as pelvic inflammatory disease (PID(Rice & Knapp, 1994). Gonococci cause urethritis, proctitis and epididymitis in men, while in female it causes cervicitis, vulvovaginitis, PID and salpingitis that may lead to sterility.
The ability of Neisseria gonorrhea to develop resistance to antimicrobial agents has become a significant problem in the treatment and control of the communicable sexually transmitted disease, gonorrhea. The resistance is both plasmid or chromosomally encoded. The emergence of penicillinase -producing N. gonorrhea (PPNG) in 1976 led to widespread high-level penicillin resistance. Tetracycline resistance was reported for gonococci in 1985 by centers for disease control and prevention (CDC), while spectinomycin resistance has also been reported for many strains of N. gonorrhea (Castillo et al, 1998). Thus treatment failures against gonococcal infections prompt the need to search for newer drugs.
Trichomonas vaginalis
Trichomonas vaginalis, the causative agent of trichomoniasis is a parasitic protozoan. This urogenital pathogen measures approximately 10nm and 7 nm in length and breadth respectively. In axenic culture, the protozoan tends is pear shaped while it is amoeboid in appearance when attached to vaginal epithelial cells. It is a flagellated protozoan possessing five flagella, four of which are located at its anterior portion and the fifth is incorporated within the undulating membrane of the parasite. The flagella and the undulating membrane gives it the characteristic quivering motility.

Trichomonas vaginalis are the causative agents of the most common, non-viral STD. It causes vaginitis, cervicitis and urethritis in women, while in men it is related to prostitis. The protozoan is also associated with adverse pregnancy outcome manifested by preterm rupture of membranes, preterm labour and low birth-weight infants, as well as being associated with upper reproductive tract post - surgical infections. It has also been related to very serious disease like the cervical neoplasia(Fiori et al, 1999), (Barrio,et ah 2002).
The 5-nitroimidazole family drugs, specifically metronidazole and tinidazole, are the only class of drugs approved for the treatment of trichomoniasis. But today up to 10 % cases are not responding to these drugs. Further the broad spectrum of these drugs which extends across many organisms including Entamoeba histolytica, Giardia duodenalis, Blastocysts hominis, Helicobacter pylori, Bacteroids spp and Clostridium spp have necessitated a limited use of these drugs(Dunne et al, 2003).
Herbal medicines for sexually transmitted infections
Finding healing powers in plants is an ancient idea. There are evidences that Neanderthals living 60,000 years ago used plants like hollyhoch for curing illnesses(Cowan, 1999). During the past decade there has been increasing global interest regarding the use of medicinal plants to treat diseases. These traditional systems of medicines are indispensable health care systems in rural communities where modern medicine is not readily available. Plants have long provided mankind with herbal remedies for many infectious diseases and even today, they continue to play a major role in primary health care as therapeutic remedies in developing countries. The search for biologically active extracts based on traditionally used plants is still relevant due to the appearance of microbial resistance and the occurrence of fatal opportunistic infections. (Tshikalange et al, 2005).
Today many plants have been studied for their activity against the organisms associated with sexually transmitted infections and many lead compounds have been

isolated. Coriandrin and Cornusin, tannins from Coriandrum sativum and Hyssops officinalis are reported to be HIV reverse transcriptase inhibitors. So were glycyrrhizin from Licorice {Glycyrrhiza glabra) and Thuja polysaccharide from Thuja occidentalis. Phyllanthus amarus was found to inhibit HIV-1 and HIV-2 in MT-4 lympjoid cell lines(Notka et al, 2003). This was attributed to their inhibitory activity on HIV RT. Many plants have been shown to have anti- HIV protease activity. The prominent among them being Rosmarinus offwialis. The terpenoid from this extract was found to be the active principle. The other compound with anti- protease activity was Ursolic acid from Geum japonicum. (Cowan, 1999).
Herpes virus was found to be inhibited by some herbal formulations. Ginkgetin, a biflavone from Ginko biloba was found be be virucidal on HeLa and Vero cell lines (Hayashi et al, 1992). Viral yield on Vero cell lines were reduced to more than 1,100-fold in the presence of Geum japonicum and Syzygium aromaticum extracts (Kurokawa el al, 1998). The anti-viral activity was also observed with ethanolic extracts of Pachyrrhizus erosus (Apongkul et al, 2002) and Rhusjavanica (Kurokawa et al, 1999).
There are more than 300 preparations in the Indian system of medicine for the treatment of liver diseases. In India, there are more than 87 medicinal plants used in different combinations as herbal drugs for liver diseases. There is a wide range of data analyzing the hepatoprotective activity of Phyllanthus amarus, Picrorhiza kurroa. Silyhum marianum and Glycyrrhiza glabra (Thyagarajan et al, 2002). Some other plants like Andrographis paniculata, (Senthil Kumaran et al 2003) Camellia sinensis, Eclipta alba, Sida cordifolia and Trichopus zeylanicns have also shown activity against viruses causing hepatitis (Subramoniam & Pushpangadan, 1999).
Herbal remedy for gonorrhea, though, have been passed on from one generation to another by word of mouth, it has been scarcely studied in the light of modern medicine. Very few researchers have ventured into the field of herbal remedies and gonorrhea. A study in Guatemala reported some plants like Bixa orellana, Piper aduncum and Physalis angulata (Caceres et al, 1995) to have inhibitory activity against Neisseria

gonorrhoea. Terminalia macoptera was found to be anti-gonococcal with MIC values between 100-200 ugiml (Silva et al, 2002).
Anti- Trichomonas activity of herbal drugs has been studied very rarely. The wide search for the same yielded in a report from Spain, where activity against Trichomonas vaginalis was found in the leaf extracts of Mikania cordifolia and the bark extract of Scuta buxifolia (Muelas-Serrano, 2000)
Medicinal plants which are used in the present invention
In the light of the above cited reports on herbal remedies against organisms associated with sexually transmitted infections, the present study has been planned and conducted to analyze the activity of 20 Indian Medicinal Plants, on five organisms associated with sexually transmitted infections; human immunodeficiency virus, Herpes simplex virus-2, Hepatitis B virus, Neisseria gonorrhea and Trichomonas vaginalis. The plants selected for the study are tabulated in Table 1.
To the best of our knowledge, till date, no body has done any work on the extracts of the plants given in the Table 1 for the treatment of diseases caused by the five organisms associated with sexually transmitted infections; human immunodeficiency virus, Herpes simplex virus, Hepatitis B virus, Neisseria gonorrhea and Trichomonas vaginalis.



Accordingly, we took up R & D studies on the above plants concentrating our attention owards finding out their in-vitro anti-Human Immunodeficiency virus activity by HIV-everse transcriptase inhibition assay, the anti-Herpes simplex virus-2 activity by complete HSV-2 cytopathic effect (CPE) neutralization assay, anti-Hepatitis B virus ictivity by Hepatitis B surface antigen (HBsAg) binding assay and Hepatitis B DNA polymerase inhibition assay, anti-gonococcal activity by screening the extract using the iisc diffusion assay and the anti-Trichornonas vaginalis activity by broth dilution method.
Preparation of extracts
The plant parts were collected and subsequently shade dried, powdered and subjected to the extraction process. In brief, to the plant powder 10% (i.e. to lg of the plant powder, 10 ml of the solvent) of the different solvents (water, methanol, chloroform, hexane and petroleum ether) was added and incubated in a shaker for 48 hours. The extracts were filtered using Whatmann filter paper no.l, evaporated to dryness using a lyophiliser to get a dry powder. The dried powder was stored at -20°C till further assay was performed.

ACTIVITY OF PLANT EXTRACTS AGAINST ORGANISMS ASSOCIATED WITH SEXUALLY TRANSMITTED INFECTIONS
HIV- reverse transcriptase inhibition assay
The reverse transcriptase synthesizes double stranded DNA from the single stranded RNA genome of the virus. The function of this enzyme is to enable the elongation of RNA-DNA primer complex by incorporation of the deoxy nucleotide triphosphates (DNPs) into a newly synthesized DNA strand complementary to the existing RNA strand. The test involves a synthetic RNA template-Poly r(A) ,a short DNA primer oligo (dT), and a radio labeled nucleotide H-thymidine triphosphate ( H-TTP) along with Tris buffer (at optimal pH), metal salts (to provide" ionic strength and required Mg), and dithiothritol (to stabilize the enzyme-free sulfhydryl groups). The RNA-3H-DNA hybrid produced is precipitated with trichloro acetic acid, isolated by filtration, and quantitated by measuring the radioactivity caused by the tritiated thymidine which gets incorporated into the DNA strand complimentary to the adenine in the Poly (A). The level of radioactivity is proportional to the amount of active RT enzyme present.
By employing the above methodology, the extracts of the plants listed in the Table 1 were tested for their activity against HIV reverse transcriptase enzyme. The extracts of the said plants that inhibited the activity of HIV reverse transcriptase enzyme are tabulated in Table 2.


From the Table 2 it can be inferred that the extracts of only three plants are found to have such activity. None of the extracts of the other plants listed in Table 1 have such activity.
Anti-herpes simplex virus-2 studies
The assay was performed on the green monkey kidney cell lines, Vero cell lines. Cells that had formed a monolayer were trypsinised and added into 96 well plate at a concentration of 5 x 104 cells /well. They were then incubated till 80% monolayer was formed and were subsequently infected with a viral suspension with a concentration of 103 TCIDso/ml, The extract (sterilized using syringe filter) was added to the wells and incubated at 37 °C in the presence of 5% CO2 for 3-6 days. The plates were observed for complete CPE inhibition from day 2 to 5 using an inverted phase contract microscope. Complete inhibition of HSV-CPE was taken as positive.
By employing the above methodology, the extracts of the plants listed in Table 2 were tested for their activity against HSV complete HSV-2 CPE neutralization. The extracts of the said plants that inhibited the activity of HSV complete HSV-2 CPE neutralization are tabulated in Table 3.


Anti-hepatitis B virus studies HBsAg Binding Assay
The presence of HBsAg binding activity in the extract was tested using an enzyme immunoassay based on the sandwich principle. HBsAg in the serum is neutralized or bound by pre-incubation within anti-HBs-like substance and hence no longer reacts with the antibody coated in the wells. The extract was tested by incubating it with HBsAg positive serum. An ELISA was done the next day and a reduction of colour or a negative Elisa result was taken as positive result for Hepatitis B surface antigen binding activity. The presence of unbound HBsAg in a test sample is demonstrated by an increase in the colour or a positive Elisa result.
By employing the above methodology, the above-mentioned extracts of the plants were tested for their HBsAg binding activity. The extracts of the said plants having HBsAg binding activity are tabulated in Table 4
HBV DNA polymerase Inhibition Assay
Replication of Hepadna viruses involves a viral DNA polymerase, which is a potential target for chemotherapy against HBV. In the presence of HBV-DNA polymerase, complimentary bases are added to the template (i.e. HBV-DNA), the addition of which is quantitated with the help of tritiated thymidine triphosphate. Reduction in the count of 50% or more in the test is noted as inhibitory activity. Hepatitis B viral DNA and the viral polymerase were isolated from HBV DNA positive serum by centrifuging and treating it with 1/8 volume of NP-40 and 2% mercaptoethanol. The viral preparation was incubated with the reaction mixture consisting of 1M Tris HC1, 400mm mgcl2 ,2M KC1, 10mm dNTPs, 2M 3HTTP and 25u1 of DNAse and RNAse free water and NIM-76. The inhibitory activity of the compound on the functioning of the viral DNA polymerase was measured using Liquid Scintillation Unit.

By employing the above methodology, the above mentioned extracts of the plants were tested for their HBV DNA polymerase inhibitory activities which are tabulated in Table 4.

From the Table 4 it can be inferred that anti-hepatitis B virus activity was found in the extracts of only four plants where in the HBsAg binding activity was exhibited by only the extracts from three plants and HBV DNA Polymerase Inhibition activity was found in the extracts of two plants. None of the extracts of the other have such activity.
Anti- neisseria gonorrhoea studies Disc Diffusion assay
Preparation of discs: The discs for disc diffusion assay were prepared from whatmann filter paper no-1. The filter paper was cut to discs and sterilized by hot air oven. To the sterilized discs the extracts (sterilized by membrane filtrations) were introduced and kept for drying at 40 °C.
Disc diffusion assay: Disc diffusion assay was employed to screen the antibacterial activity of all the extracts. The fastidious bacteria, Neisseria gonorrhea was grown on trypticase soya broth to obtain a turbidity equal to Mcfarland's 0.5 standard and the assay was performed on chocolate agar (using horse blood).

By employing the above methodology, the above mentioned extracts of the plants were tested for their anti-Neisseria gonorrhea activity which are tabulated in Table 5.

From the Table 5 it can be inferred that anti- Neisseria gonorrhoea activity was found in the extracts of only three plants. None of the extracts of the other have such activity.
Anti- Trichomonas vaginalis studies
Trichomonas vaginalis was inoculated in a 12 well plate with Modified Diamond's Media containing the plant extracts and incubated at 37 °C. 5u of the mixture was taken from each well and observed for motile protozoa from day 2 to day 7. Absence of motile protozoan indicated inhibitory activity of the extract.
By employing the above methodology, the above mentioned extracts of the plants were tested for their anti-trichomonas vaginalis activity which are tabulated in Table 6


From the Table 6 it can be inferred that ax&i-trichomonas vaginalis activity was found in the extracts of only three plants. None of the extracts of the other have such activity.
Based on the results shown in the Tables 2, 3, 4, 5 and 6, it was observed that the methanolic extract of the plant Rubia cordifolia , especially the roots only exhibited all the activity of HIV-Reverse transcriptase inhibitory activity, HBsAg Binding activity, HBV DNA polymerase inhibition activity, HSV-2 CPE inhibitory activity, Anti-Neisseria gonorrhoea activity and Anti-Trichomonas vaginalis activity. Based on the above observations this plant was taken up for further detailed studies.
Therefore the main objective of the present invention is to provide a a novel extract of the plant Rubia cordifolia containing a flavanoid useful for the treatment of sexually transmitted infections
Another objective of the present invention is to provide a a novel extract of the plant Rubia cordifolia containing a flavanoid having the following activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f. Trichomonas vaginalis inhibitory activity

Yet objective of the present invention is to provide a process for the preparation of novel extract of the plant Rubia cordifolia containing a flavanoid useful for the treatment of sexually transmitted infections
Accordingly, the Present invention there is provided a novel extract of the plant Rubia cordifolia containing a flavanoid having the following activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f. Trichomonas vaginalis inhibitory activity
According to another embodiment of the present invention there is provided a process for the preparation of Novel extract of the plant Rubia cordifolia containing a flavanoid having the following activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f. Trichomonas vaginalis inhibitory activity
which comprises (i) fractionating the powdered part of the plant Rubia cordifolia with methanol and
(ii) subjecting the 7th faction to HPLC to get the fraction. Containing only a single component, namely a flavanoid.
BIOLOGICALLY-GUIDED FRACTIONATION
Biologically guided fractionation method, the details of which are explained below, was done to isolate the different fractions of the methanolic extract of the plant Rubia cordifolia and to identify the exact fraction responsible for the bioactivity.

Step (i) Silica gel Column chromatography
The column was prepared using silica gel having a mesh size in the range of 60-120. The silica gel fractionation of the methanolic extract of Rubia cordifolia yielded a
total of fourteen different fractions. Out of which only the 7 fraction was found to have all the above said activities as shown in the Table 7


Step (ii) Thin Layer Chromatography-Monitoring of the 7 fraction
The 7th fraction obtained by silica gel column chromatography as explained above was analyzed using thin layer chromatography to ascertain its purity. The plates used for the study consisted of silica gel incorporated with fluorescent F 254. The solvent system used for the assay was hexane: ethyl acetate: petroleum ether preferably in the proportion 3:2:1. The plates were observed in UV and then stained with 5% H2SO4 to visualize the different bands. It was found that this fraction contains only a single component.

Step (iii) Preparative High performance liquid chromatography
The above said purified 7th faction was subjected to HPLC to further purify the fraction. Preparative HPLC was performed using a chromatographic system consisting of a Solvent module pump LCI OAT, Detector module SPD 10A, Column LCI 8 Supercoil with dimensions of 25cm x 4.6cm, a Mobile Phase of Methanol: Water with a Flow rate 1.5ml/min and detected at 210nm. The 7th fraction so purified consist of only a single component namely a flavanoid.
Characterisation of the 7 fraction
Chemical Characterization of the 7th fraction
The fraction -7 (RC-7) was analyzed for the presence of alkaloids, glycosides, flavonoids, phenols, tannins, carbohydrates, saponins, sterols, oils, proteins and amino acids, by very well known and standard chemical assays. The studies resulted in our finding that the active component of this fraction is a flavanoid.
Physical characterization of the 7th fraction
Mass of the fraction, RC-7 was determined by mass spectroscopic analysis. The spectroscopic reading suggests that the mass was in the range of 284g/mol for RC-7, of the plant Rubia cordifolia. Thus, the seventh fraction of Rubia cordifolia was found to be a flavonoid with the mass in the range of 284 g/mol.
Evaluation of activity of the 7th fraction consisting of a flavanoid against organisms associated with sexually transmitted infection
The 7th fraction containing the flavanoid was analysed for its activity against HIV, HSV-2, HBV, Neisseria gonorrhea and Trichomonas vaginalis by methods explained

above namely HIV-RT inhibition studies, HSV CPE inhibition studies, HBsAg binding studies, HBV DNA polymerase inhibition studies, anti- Neisseria gonorrhea studies and anti- Trichomonas vaginalis studies.
The Table 7 given above represents the summary of the activities of the 7l fraction of methanolic extract of Rubia cordifolia.
In vitro cytotoxicity studies
The seventh fraction of the plant Rubia cordifolia (RC-7) containing the flavanoid was subjected to in vitro cytotoxicity analysis on Vero cell lines. The said fraction was found to be non-toxic to the cells till a concentration of 3000 jag/ml.
Advantages of the invention
1. The invention provides an extract containing a flavanoid having the following
activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f. Trichomonas vaginalis inhibitory activity
2. The extract of the invention is non-toxic.
3. The extract containing the flavanoid can be prepared from the plant Rubia cordifolia, especially the roots, which is an abundantly available medicinal plant throughout India

We Claim
1. Novel extract of the plant Rubia cordifolia containing a flavanoid having the
following activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f. Trichomonas vaginalis inhibitory activity
2. A process for the preparation of Novel extract of the plant Rubia cordifolia
containing a flavanoid having the following activities
a. Anti- HIV -RT activity
b. HSV-2 Cytopathic effect neutralization activity
c. HBsAg binding activity
d. HBV DNA polymerase inhibition activity
e. Neisseria gonorrhea inhibitory activity
f Trichomonas vaginalis inhibitory activity which comprises (i) fractionating the powdered part of the plant Rubia cordifolia with methanol
(ii) subjecting the 7l faction to HPLC to get the fraction. Containing only a single component, namely a flavanoid.
4. A process as claimed in claim2 wherein the parts of the plant is the roots
5. A process as claimed in claims 2 & 3 wherein the silica gel used has a mesh size in the range of 60-120.
6. A process as claimed in claims 2 to 5 wherein the HPLC is performed using a chromatographic system consisting of a Solvent module pump LC10AT, Detector module SPD 10A, Column LC I8 Supercoil with dimensions of 25cm x 4.6cm, a

Mobile Phase of Methanol: Water with a Flow rate 1.5ml/min and detected at 210nm.
Novel extract of the plant Rubia cordifolia containing a flavanoid useful for the treatment of sexually transmitted infections for substantially as herein described
A process for the preparation of Novel extract of the plant Rubia cordifolia containing a flavanoid useful for the treatment of sexually transmitted infections for substantially as herein described