Bioactive water fraction from Gomphostemma niveum

#The present invention relates to a novel bioactive water fraction obtained from the leaves of an herb name Gomphostemma Niveum commonly found in North Eastern part of India, and which is useful for inhibiting the growth of malarial parasite Plasmodium falciparum. The present invention also relates to a method for the extraction of said bioactive fraction. The present invention also provides methods for treatment of malaria using such bioactive water fractions and use of such bioactive water fractions for the treatment of malaria.

#Malaria is the most prevalent among the insect borne disease accounting for more than two million deaths out of 400 million cases reported each year (Greenwood et. al., Nature 9415), 670(2002)) caused by Plasmodium falciparum. (Depote et al. Trends in parasitology (20), 4, 165-198, (2004)). More than half of the world's population lives in areas where they remain at risk of malarial infection (Sachs et at Nature, (415), 686 (2002). The derivation of important anti-malarial compound started with the discovery of cinchona bark powder (quinine) (Brooking, G B 106430, (1917)) in 1820. Subsequently post World War-I was a period of intense work in maintaining such ethnobotanical records in which the use of quinine has remained the drug of choice of malaria. In 1940 another anti-malarial drug chloroquine (Ringwald et al, Bulletin of the world health organization, 77(1), 34, (1999)) was synthesized and until recently this was the only drug used for the treatment of malaria. Unfortunately, after an early success, the malarial parasite especially P. falciparum became resistant to chloroquine. Treatment of chloroquine resistant malaria (White et al. Delaying anti-malarial drug resistance with combination chemotherapy Parasitologia, (41), 301-308, (1999)) was done with alternative drug or drug combination (Tulp et. al, Drug discovery today (9)450, (2004)) which are rather expensive and sometimes toxic. Furthermore, these combinations are not always dependent upon pharmacokinetic principle due to inadequate knowledge of metabolism and mechanism of action of most anti-malarial drugs. In the last few years a plant based anti-malarial drug isolated from the Chinese plant Artimisinin annua (Klayman, Science (228), 1049, (1985)), Bharel et al. Fitoterapia, (67), 387-399, (1996)) proved to be very active against P. falciparum. However, the use of Artemisinin, an endoperoxide sesquiterpene lactone is somewhat limited because of its relatively high cost, limited production of GMP standards and reports of toxicity (Haynes et al. Curr. Opin. Infect. Dis, (14), 719-726, (2001)). The current route for the total synthesis of Artemisinin (Qinghaosu) (U.S. Pat. No. 6,710,074 (2004) is too complex for commercial production (Schmid et al J. Am. Chem. Soc. (105), 624-625, (1983). It is currently prepared by large scale extraction from Artemisia annua (sweet warwood) and derivative such as artemether, artesunate, arteether and dihydroartemisinin are prepared semi synthetically from the purified extract. Moreover, difficulties have also been encountered in the production of high quality material (Haynes et al. Curr. Opin. Infect. Dis., (14), 719-726, (2001)).

#Thus, the widespread resistance of malarial parasite to the majority of drugs, necessitates the development of new drug (Wiesner et al. Angew. Chem. Int: Ed. (42), 5274-5293, (2003)) which should be structurally different from classical compounds with novel mechanism of action to overcome the resistance problem. Recently Ihara et. al disclosed about compounds having anti-malarial activity (U.S. Pat. No. 6,710,074 (2002)) from synthesis. In recent past different classes of compound are also reported having anti-malarial activity like substituted 1,2,4 trioxane (U.S. Pat. No. 6,737,438 (2002)), flavonoids (WO2004000306 (2003)), naphthylisoquinoline (U.S. Pat. No. 6,627,641 (2003)), indoloquinazoles (U.S. Pat. No. 6,531,487 (2003)), trioxolanes (U.S. Pat. No. 6,486,199 (2002)), betacarboline alkaloids (U.S. Pat. No. 6,143,756 (2000)), vocamine (WO 9948501(1999)), acetyl glucosamine derivatives. (DE3220426(1983)) and so on. Recently many patents like U.S. Pat. No. 6,710,074, WO2004000319, U.S. Pat. No. 5,264,726, US2003212098, WO2004000306, EP1076057, WO9948501, U.S. Pat. No. 5,290,553, U.S. Pat. No. 6,143,756 and U.S. Pat. No. 6,627,641 disclosed compounds having anti Plasmodium falciparum activity from natural origin mainly plants. Hence the ethnopharmacologial approach for the search of new anti-malarial has proved to be more predictive.

#FIG. 1 is the HPLC profile of the crude extract of the invention.

#FIG. 2 is the total ion chromatogram of the crude extract after further fractionation.

#The increasing resistance of Plasmodium falciparum to currently available drug such as choloroquine, present a challenge in the treatment of malaria, which still remains one of the treatment for malaria, which still remains one of the leading causes of death in the world. Consequently new agents are urgently needed to treat the organism.

#The present invention provides a bioactive water fraction named as GN-W obtained from the leaves of plant Gomphostemma Niveum having inhibitory activity against chloroquine resistant and chloroquine sensitive malarial parasite. According to another embodiment of the present invention it provides the simple and efficient method of extraction, isolation and identification for the bioactive compound from the sun/air dried leaves of Gomphostemma Niveum is provided.

#Further in accordance with another embodiment of the present invention is the development of fingerprinting high performance liquid chromatographic (HPLC) method for the routine monitoring of the crude extract. Additionally this invention provides a suitable technique for in vitro and in vivo bio-evaluation of crude extract.