Title of Invention	A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF CANCER AND RELATED DISORDERS
Abstract	The present invention provides a bio-active composition obtained from Boswellia species, exhibiting in vivo and in vitro anti-cancer activity. The bio-active composition comprises an isomeric mixture of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives. The pharmaceutically effective dose of the said composition is in the range of 250 -lOOOmg/Kg body weight of a subject. The composition is isolated after repeated column chromatography of the neutral extract of the gum resin of Boswellia serrata and related natural sources and can be alternatively prepared from semi-synthetic source. The said composition exhibits anti-cancer activities for breast, leukemia, liver, and colon and brain cancer.

Title of Invention

A PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF CANCER AND RELATED DISORDERS

Abstract

The present invention provides a bio-active composition obtained from Boswellia species, exhibiting in vivo and in vitro anti-cancer activity. The bio-active composition comprises an isomeric mixture of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives. The pharmaceutically effective dose of the said composition is in the range of 250 -lOOOmg/Kg body weight of a subject. The composition is isolated after repeated column chromatography of the neutral extract of the gum resin of Boswellia serrata and related natural sources and can be alternatively prepared from semi-synthetic source. The said composition exhibits anti-cancer activities for breast, leukemia, liver, and colon and brain cancer.

Full Text	Field of the invention; The present invention relates to a bioactive composition useful for induction of apoptosis thereof cytotoxicity and anti-cancer activity, obtained by natural isolate from Boswellia species, alternatively prepared semi-synthetically, that comprises an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1, alone or in combination with pharmaceutically acceptable or other carriers. More particularly the present invention relates to bioactive composition useful for cancer of colon, prostrate, liver, breast, central nervous system (CNS), leukemia and malignancy of other tissues, including ascites and solid tumors wherein the cancer cell death is mediated by induction of apoptosis and inhibition of cell proliferation at specific doses of the composition. The active mixture may be isolated from Boswellia serrata, Boswellia ovalifoliolata and related species or may be prepared semi-synthetically from the natural isolates i.e. boswellic acids, which comprise an isomeric mixture of a and p-boswellic acids or their carboxylic esters by lithium aluminium hydride reduction. The present invention further relates to the bioactive composition that kills cancer cells through induction of programmed cell death (apoptosis) and can be used for the treatment of cancer. The herbal product comprising an isomeric mixture of 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 are isolated from Boswellia species and alternatively prepared semi-synthetically from (a+p) boswellic acids. Background and prior art of the invention; The gum exudates from Boswellia serrata, the source of these products has been used since long in the traditional Ayurvedic system of medicine for the treatment of various inflammatory diseases in Indian subcontinent besides being used in preparations of commercial importance such as in fragrance and cosmetic industry. Gum resin of another species of the same genus i.e. Boswellia ovalifoliolata Roxb (Buseraceae) is also the source of 3oc, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 also known as olibanum was used traditionally in the perfumery industry only. Boswellia serrata is a rich source of pentacyclic triterpenoids also known as boswellic acids while Boswellia ovalifoliolata is rich in tetracyclic triterpenoids. One of the isomers of the bioactive constituents i.e. 3a, 24-dihydroxyurs-12-ene has earlier been isolated and reported as new natural triterpenoid from the gum resin of Boswellia serrata [(B. Mahajan, S. C. Taneja, V. K.Sethi and K. L. Dhar, Phytochemistry 39(2), (1995)453-55], though it was semisynthetically known earlier [Allan, G.G., Phytochemistry (1968), 7, 963]. However, there has been no report of its occurrence in two isomeric forms in plants including their bioactivity. Second to coronary heart diseases, cancer has emerged as the most common cause of death in many countries. Unlike normal cells, cancer cell appears after having undergone accumulation of several genes mutations within it, which account for independent and uncontrolled growth of this cell into a clonal expansion amounting to invasion of adjoining tissues, and some times metastasing to other tissues. Dysregulation of programmed cell death (apoptosis) is the hall mark of all cancer cells and hence provides important therapeutic target to intervene in the development of anti-cancer agents, because activation of apoptosis is a common mechanism of malignant cell killing by anti-cancer agents [(K.M.Deabatin, Activation of apoptosis pathways by anti-cancer treatment, Toxicol Lett., 112/113 (2000) 41-48)]. Several pathways therefore, are proposed to be involved in the death of cancer cells by anti-cancer drugs. The drug may kill cells by activation of death receptors mediated pathways or via mitochondrial dependent pathways involving reactive oxygen and nitrogen species culminating in activation of certain genes in the apoptotic signal transduction pathways. Several agents are known to kill malignant cells by inducing apoptosis via free radicals generation and one such example is doxorubicin [(G. Minotti, P. Mena, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines: Molecular advances and pharmacologic developments in anti-tumor activity and cardiotoxicity, Pharmacol. Rev. 56 (2004), 185-229)]. Most of these agents activate various caspases, genes, translocate cytochrome c that upregulate downstream events leading to DNA fragmentation (DNA laddering) typical of apoptosis. In another two decades world is expected to peak in all kinds of cancer and brain cancer in particular appears difficult to manage. Recently there has been a report of inhibitory activity of boswellic acids against human leukemia HL-60 cells in culture. Out of the four natural boswellic acids tested in vitro against the cell line HL-60 it was reported that acetyl -11- keto-(3-boswellic acid induced the most pronounced inhibition in a dose dependent manner on DNA, RNA and protein synthesis (1C 50 values 0.6, 0.5, and 4.1 jo, mol) while for other boswellic acids of the same group recorded 1C 50 value in the range of 0.6- 7.1 \|n mol. The effect of acetyl -11-keto-p-boswellic acid on DNA synthesis was found to be irreversible. The composition also significantly inhibited the cell growth of HL-60 [Yu Shao,, Chi-Tang Ho, Chee-Kok Chin, Vladimir Badmaev, Wei Ma and Mou-Tan Huang ; Planta Medico. 64 (1998) 328-31]. A patent has claimed the DNA topoisimerse I and II inhibitory effect of pentacyclic triterpenic acids of Boswellia serrate [Y-W. Lee, Q-C. Fang, Z-G. Wang, D-H. Li and C.E. Cook, (1991) USP 5,064,823]. There have also been IPR's related to the use of boswellic acid in the treatment of brain tumors [Simmet Thomas (De) (1994) USP 5919821 and USP 6174876] and other anti-cancer related activities of boswellic acids, [Subbiah, Ven, Natural product based apoptosis inducers (plant extracts), USP, 20050084547. Ammon, Hermann P.T, et al. use of boswellic acid and its derivatives for inhibiting normal and increased leucocytic elastase or plasmin activity (USP, 20020010168). Therefore, it is apparent from the literature search that in the present invention, the isolation and use of isomeric mixture of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 is novel as a cytotoxic agent and inducer of apoptosis in cancer cells and can be used for the treatment of cancer derived from various tissues. Besides, process of semi-synthetic preparation of bioactive mixture from a an (3-boswellic acids by reduction is also novel. Objects of the invention: The main object of the present invention is to provide a bioactive composition comprising of an isomeric mixture of triterpenoids 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 isolated from Boswellia serrata, or alternatively obtained from semi-synthetically. Another object of the present invention is to provide a process of preparation of the bioactive composition comprising of an isomeric mixture of triterpenoids 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 having pharmaceutical effective dose being in the range 250 - lOOOmg/kg body weight of a subject. Also another object of the present invention is to provide the usefulness of bioactive composition of formula 1, comprising of an isomeric mixture of triterpenoids 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1:1 for the treatment of cancer of prostrate, colon, breast, leukemia, liver, CNS and of other tissues. Yet another object of the present invention is to provide the chemically synthesizable, economically viable, commercially high yield of the composition from Boswellia sp, intended to provide analgesic and sedative effects to the patients suffering from advanced malignancy, in sharp contrast to others where prophylactic use of psychotherapeutics and anti-emetics are often recommended during chemo-cum-radiotherapy. Summary of the invention; The present invention deals with the isolation of a natural isolate, alternatively semi-synthetic preparation of a bioactive composition of formula 1 , comprising of an isomeric mixture of triterpenoids 3 a, 24-dihydroxyurs- 1 2-ene and 3cc, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1 : 1 and its usefulness for the treatment of cancer of prostrate, colon, breast, leukemia, liver, CNS and of other tissues. (FigureRemoved) Accordingly, the present invention provides a bio-active composition useful for the treatment of cancer wherein the said composition comprising an isomeric mixture of 3oc, 24 -dihydroxyurs-12-ene and 3ot, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject and the said isomeric mixture having following formula 1, wherein the Rl = methyl when R2= H or R2=methyl when Rl = H. In an embodiment of the present invention, the said composition exhibiting cytotoxicity against human cancer cell lines selected from the group of prostrate, colon, breast, liver, CNS and leukemia. In another embodiment of the present invention, the said composition exhibits anti-cancer activity against Ehrlich Ascites carcinoma mouse tumor model when given orally in 10% alcohol. In still another embodiment of the present invention, the said composition exhibits anti-cancer activity against Ehrlich Ascites Tumor mouse model when given orally in 10% alcohol. In yet another embodiment of the present invention, the said composition is useful for exhibiting anti-cancer activity against mouse sarcoma 180 tumor models when given intra-peritoneally with 10% alcohol or in normal saline with 0.5% Tween-20. In still another embodiment of the present invention, the said composition is useful for inhibiting leukemia cell proliferation with IC50 value of 12-14f4,g/ml. In still anther embodiment of the present invention, the said composition is useful for inhibiting human breast MCF-7 cancer cell proliferation with IC50 value of 5 jig/ml. In also another embodiment of the present invention, the said composition is useful for inducing programmed cell death (apoptosis) in lymphoblastic human leukemia Molt-4 and HL-60 cells. Further in an embodiment of the present invention, the said composition is useful for producing early reactive oxygen species (peroxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells. In still another embodiment of the present invention, the said composition is useful for producing early reactive nitrogen species (nitric oxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells. In yet another embodiment of the present invention, the said composition is useful for inducing mitochondrial depolarization consequent to reactive oxygen or nitrogen species as the mechanism into the apoptotic death of cancer cells. In also another embodiment of the present invention, the said composition is useful for exhibiting anti-cancer activity during the growth of solid and suspension tumors in experimental mouse tumor models. Further in another embodiment of the present invention, the said composition does not show any mortality of BALB/c mice over a period of two weeks after administering single oral dose in the range of 250- lOOOmg/Kg body weight or single intra-peritoneal dose of 500mg/Kg body weight. Breif description of the figures & tables; Figure 1 shows the structural formula 1, comprising an isomeric mixture of 3a, 24 -dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1. Figure 2 shows the inhibition of cancer cell proliferation by composition of formula 1 for determination of IC50. Figure 3 shows the induction of DNA laddering (DNA damage), typical of apoptotic cell death induced by composition of formula 1 in human lymphoblastic Molt-4 and myeloblastic HL-60 leukemia cells. Figure 4 shows flow cytometric measurement for induction of apoptosis by composition of formula 1. Figure 5 shows flow cytometric measurement for early generation of reactive oxygen species by composition of formula 1, in HL 60 cells. Figure 6 shows flow cytometric measurement for copious early endogenous formation of nitric oxide by composition with formula 1, in HL-60 cells. Figure 7 shows depolarization of mitochondrial membrane functions by composition of formula 1. Table 1 represents the influence of composition of formula 1, on human cancer cells growth in terms of percentage inhibition. Table 2 represents the anticancer activity of composition of formula 1 in various mosue tumor models of cancer in vivo. Table 3 represents in vitro cytotoxic activity of natural boswellic acids on human cancer cell lines. Table 4 represents in vitro cytotoxic activity of composition of formula 1 on human cancer cell lines. Detailed description of the invention; Boswellia serrata is well known for its anti-inflammatory activity in Indian system of medicine and the major constituents boswellic acids are also reported for their anti-cancer activity. The composition of formula 1 having isomeric mixture of triterpenoids which are present in Boswellia species extract in minor amounts (0.1-7.0%) comprising of 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1:1 and is isolated after repeated column chromatography of the neutral extract of the gum resin of Boswellia serrata, Boswellia ovalifoliolata and related natural sources. The pharmaceutical preparation comprising triterpenoids display biological activities, particularly cytotoxicity in human cancer cell lines which is useful for its application as anti-cancer agent. The bioactive composition of formula 1, comprising an isomeric mixture of triterpenoids 3a, 24-dihydroxyurs-12-ene and 3oc, 24-dihydroxyolean-12 in the ratio 1:20 to 1:1, also can be obtained by semi-synthetic process that involves chemical reduction of the natural mixture of a and (3-boswellec 11 acids, by converting them to their alkyl esters such as methyl or ethyl esters thereafter subjecting them to reduction by lithium aluminium hydride (LAH) in an inert organic solvent such as diethyl ether, tetrahydrofuran and the like. The semi-synthetic product obtained by above process displays the same level of bioactivity as shown by the natural isolate. The purity of the product is established by physico-chemical characteristics as well as by reverse phase HPLC method. The ratio of the two isomers 3 a, 24-dihydroxyurs-12-ene and 3ot, 24-dihydroxyolean-12-ene has range between 1: 20 to 1: 1. In a feature of the present invention provides isolation as well as semi-synthetic preparation of composition of formula 1, isomeric mixture of triterpenoid compounds comprising of 3 a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in a ratio 1: 20 to 1: 1, that exhibits anti-cancer activities for breast, leukemia, liver, colon and brain cancer. In another feature of the present invention, the bioactive composition of formula 1 also inhibits human breast MCF-7 cancer cells growth by 50% at about 15 \|ig/ml concentration. In also another feature of the present invention, the bioactive composition of formula 1 comprising triterpenoids kills human cancer cells by induction of apoptosis. In still another feature of the present invention, the bioactive composition of formula 1 kills more than 92% of prostrate DU-145 and PC-3 cells at 5x10" 5M concentration, wherein the prostrate cell lines are selected from DU-145 and PC-3 cells. In yet another feature of the present invention, the bioactive composition of formula 1 kills colon cancer cells up to 96% at 5xlO"5M concentration, wherein the colon cancer cell lines are selected from HT-29, SW-620 and Colo205 cells. Also in another feature of the present invention, the bioactive composition of formula 1 inhibits growth of cancer cells of liver up to 92% at 5xlO"5M concentration, wherein the cancer cell line of liver Hep2 is selected. In still a feature of the present invention, the bioactive composition of formula 1 inhibits growth of neuroblastoma cell line SK-N-SH up to 86% at 5xlO"5M, wherein the cell line is selected from neuroblastoma (CNS) cancer cell lines SK-N-SH. The following examples are given by the way of illustration of the present invention and should not be construed to limit the scope of the present invention. Example-1 Isolation of bioactive triterpenoids 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 from the gum resin Boswellia serrata gum resin (IKg) was cleaned and made free of extraneous impurities. The gum is extracted with 3x3L of ethyl alcohol at room temperature. The combined extract after filtration was concentrated under reduced pressure. The concentrated extract (530 g) was diluted with 3% potassium hydroxide solution (3L) and the mixture stirred for an hour. The aqueous extracted thus obtained was extracted with (2x 2L) hexane and then with 10% ethyl acetate in hexane (lx2L). The combined organic solvents were washed with water, dried over anhydrous sodium sulphate and concentrated under reduced pressure to give 130 g of viscous liquid comprising the neutral components of the gum resin. A portion of the concentrated extract (20g) was subjected to repeated column chromatography over silica gel. Gradient elution with hexane/ethyl acetate furnished besides other compounds, a compound (400 mg) mp 1 82°C, [a]D + 63. It was identified as a mixture of two isomeric compounds by 13CNMR, . The ratio of the isomeric mixture was determined by reverse phase HPLC on C-18 column (acetonitrile and water in the ratio of 95:5) and detection at 200 nm. Example-2 Preparation of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyoIean-12-ene of formula 1 by semi-synthetic method a. An isomeric mixture of (a+P) of boswellic acids (R=H) (230 mg) was dissolved in dry solvent ether at room temperature and to the stirring solution under nitrogen atmosphere was added lithium aluminum hydride 20 mg (LiAlH4) while maintaining the temperature between 20-25°C. After the completion of the addition the reaction mixture is further stirred for one hour. The excess of LAH was destroyed by adding ethyl acetate and the organic layer washed with water. Concentration of the solvent gave desired product 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in almost quantitative yield (a:(3 :: 1:4). It was crystallized from hexane-ethyl acetate to furnish white crystal of mixture of formula 1 and confirmed by physicochemical and spectral data. The ratio of isomers (ot:p: 1:4) was estimated by HPLC on C-18 column using acetonitrile : water : acetic acid (95:5 : 0.1) as the mobile phase . b. An isomeric mixture of (a+P) of boswellic acids was converted to methyl ester by diazomethane generated in situ by the method known in the art of synthesis. A mixture of (ct+p) of boswellic acids (230 mg) was dissolved in dry solvent ether and diazomethane solution in diethyl ether was slowly added till completion of reaction as monitored by TLC. The mixture comprising the methyl esters of compound of structure formula 1 (R=Me) without purification was further reduced by LAH by the method described as in part a example 2.(Figure Removed) Example-3 Cytotoxicity of 3d, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyoIean-12-ene of formula 1 against human cancer cell lines in vitro Human cancer cells after trypsinization into single cell suspension were grown in 96-well culture plate for 24 hr. Cells were treated with indicated doses of composition of formula 1 and incubated in CO2 incubator for 48 hr. After 48h cells were stained with sulforhodamine B dye, and the bound dye was eluted to measure the optical density indicating cell growth in Elisa Reader at 540nm [(Monks, D. Seudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd (1991), Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines, J. Natl. Cancer Inst. 83 757-766)]. The OD of untreated cells is considered as 100% while of oil treated group is subtracted from the control group to determine % inhibition as a measure of cell cytotoxicity. The composition inhibited various human cancer cells growth by more than 88% at lOuM.(Table Removed) Example-4 Composition of formula 1 comprising 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene, induced inhibition of cell proliferation of leukemia cells using MTT assay. Human leukemia cells are grown in suspension in 96-well culture plate and were incubated with different concentrations of the composition for 48 hr. The cells were then incubated with MTT and the MTT-formazon formed is eluted with DMSO, and OD measured in ELISA Reader [(Shashi B., SinghJ., Rao, J.M., Saxena, A. K. and Qazi, G. N.(2006), A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells. Nitric Oxide, 14, 72-88)]. The intensity of the color formed in the untreated control wells relates to 100% cell growth. The growth of cells is recorded with different concentrations of the bioactive composition of formula 1, and the concentration that inhibits 50% cell growth is taken as 1C so value. Human breast cancer cells MCF-7 grown in adherent culture are trypsinised to obtain cell suspension. Cells are then distributed to 96-well culture plate to grow as monolayer culture. Cells are treated with indicated concentration of composition of formula 1 and assayed for cell viability using MTT assay as described above. The IC50 values in leukemia cells was in the range of 12-14 ug/ml while for MCF7 cells was about 5 jug/ml. Fig.2. Inhibition of cancer cell proliferation by composition of formula 1: Determination of IC50 (Figure Removed) 50: Example-5 Composition of formula 1 induced DNA fragmentation typical of apoptosis in Molt-4 and HL-60 cells. The composition of formula 1 induces programmed cell death (apoptosis), a desired therapeutic anticancer drug target, in lymphoblastic human leukaemia MOLT-4 and human myeloma leukaemia HL-60 cells. Composition of formula 1 produced dose dependent effect in both the cell lines and the DNA laddering produced is comparable to the one produced by standard anti-cancer drug camptothecin. Human leukemia cell lines grown in culture were exposed to 3-100 (J,g/ml of composition of formula 1 for 18hr. The control group received only the vehicle. Cells were similarly treated with camptothecin, 4uM as positive control. Increasing bioactive product concentration enhanced DNA ladder of about 180bp fragments as indicated by increased ethidium bromide fluorescence intensity of the fragments indicative of apoptosis. Fig. 3. Induction of DNA laddering (DNA damage), typical of apoptotic cell death induced by composition of formula 1 in human lymphoblastic Molt-4 and myeloblastic HL-60 leukemia cells.(FigureRemoved) Example 6 Concentration related influence of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 on apoptosis: Flow cytometric analysis in HL-60 leukemia cells. During the early events of apoptosis, phospholipid phosphatidyl serine of plasma membrane is exteranalized, which has very high affinity for annexinV antibody. HL-60 cells were treated with different concentrations of composition of formula 1 and incubated for a brief period of 6 hr. There after, cells were washed and stained with FITC conjugated annexinV antibody and propidium iodide. The cells (10,000) were analyzed by flow cytometery (BD, LSR) using ProQuest software. The fraction of cell population in the lower right quadrant indicates apoptotic cells, upper right post apoptotic and upper left as necrotic populations. Composition of formula 1 produced concentration dependent increase in apoptotic and post apoptotic cell population. Composition of formula 1 at 1 jag/ml produced almost 18% apoptotic / post-apoptotic population, which increased to 74% at 50ug/ml concentration. The studies indicated that cell killing is mainly mediated via apoptosis and that composition of formula 1 is an interesting lead to be developed into anti-cancer agent. Fig.4. Induction of apoptosis by composition of formula 1: Measurement by flow cytometry. Example 7 Influence of bioactive composition formula 1 on the early generation of reactive oxygen species (peroxides): analysis by flow cytometery The endogenous generation of peroxides was measured using a non-fluorescent probe DCFH-DA which upon entering the cell is deesterified to DCFH which is oxidized by the reactive oxygen species to a fluorescent product DCF that remains entrapped within the cells offering analysis by flow cytometery [(Shashi et al, 2006, cited in Example 4 )]. HL-60 cells were incubated with different concentrations of the composition of formula 1 for a brief period of 6hr. Thereafter cells were washed and stained for 1 hr with DCFH-DA and analysed on flow cytometer. The composition of formula 1 produced dose dependent increase in peroxide positive cell population, being 79% at 50 jug/ml. The pro-oxidant effect was completely impaired in the presence of anti-oxidant, ascorbate. Formation of reactive oxygen and nitrogen species are indicated in the induction of apoptosis. Fig.5. Early generation of reactive oxygen species by composition of formula 1 in HL-60 cells: Measurement by Flowcytometry(Figure Removed) Example 8 Composition of formula 1 comprising 3 a, 24-Dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene mediated early generation of endogenous nitric oxide measured by flow cytometery. Measurement of in situ NO generation involved the use of a fluorescent probe diaminofluoresceine-2-diacetate, which is permeated easily into the cells. Once inside the cell it binds NO soon it is formed and emits fluorescence [H.Kojima et, Development of a fluorescent indicator for the nitric oxide based on the fluoresceine chromophore, Chem. Pharm. Bull., 46 (1998) 373-375]. For this purpose, the HL-60 cells were incubated with DAF-2-DA for 30 min before being incubated with the composition of formula 1 for 6hr. Cells were washed and stained with anti-annexinV-PE. Cells were analyzed on flow cytometer in FL-1 channel for NO and FL-2 channel for apoptosis FL-1. It appears that all the cells formed NO within 6 hr irrespective of the concentration of bioactive composition used, because there were no cells in the lower left quadrant as indicated in control cells. However, the extent of apoptosis was much higher in bioactive composition of formula 1 when the cell populations from the upper two quadrants (apoptotic and early apoptotic) are taken into consideration. Interaction of NO with superoxide, while both are generated simultaneously, may affect the mitochondria! membrane potential that may be responsible for the activation of apoptosis Fig.6. Copious early endogenous formation of nitric oxide by composition of formula 1 in HL-60 cells: Measurement of apoptosis vis-a- vis NO by flowcytometry. (FigureRemoved) Example 9 Mitochondrial membrane depolarization by 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1: measurement by flow cytometery Rhodamine-123 uptake into the mitochondria is driven by mitochondrial transmembrane potential ( \j/mt ) that allows the determination of cell population with active integrated mitochondrial functions. Loss of \j/mt would lead to depolarization of mitochondria consequent to ROS and NO generation leading to cell death. HL-60 cells were exposed to the composition of formula 1 for 12 hr and incubated with Rh-123. Cells, 10000, were acquired for analysis by flow cytometery. The percentage of cells with low Rh-123 fluorescence was calculated from the dot plot stastics. In untreated control cells more than 92% cells showed Rh-123 fluorescence, which decreased with treatment of composition of formula 1 .This shows that reactive oxygen/nitrogen species generated by 1 bring about oxidative damage to mitochondria ensuing apoptosis. Fig.7. Depolarization of mitochondrial membrane functions by composition of formula 1 CM (Figure Removed) ExamplelO Anti-cancer activity of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the mouse model of Sarcoma 180 solid tumor and Ehrlich ascitic carcinoma (suspension) Swiss albino mice, 6-8 weeks old (18-22 gm by weight) were bred and maintained at institute's animal house. Approval of the Institute's ethics committee was taken for use of laboratory animals and the procedures employed therein. The mice were housed in well individually ventilated cage (IVC) system and maintained on pelleted diet and water ad libitum. For the initiation of experiment, 1X107 cells of sarcoma -180 tumors were transplanted intramuscularly in the thigh of mice for the development of solid tumor. The day of tumor transplantation was assigned as day 0, next day (day-1) animals were randomly selected and divided into three groups of seven mice in each group of similar body wt. Group-1 was treated with composition of formula 1 100 mg/kg i.p prepared in 10 % alcohol of the total volume 0.2ml. Group-2 received 0.2 ml of vehicle and served as normal control while group-3 serving as positive control was treated with 5-FU (22 mg/ kg b.wt.) intraperitoneally. The total duration of treatment was of 9 days. The evaluation of effect of treatment on tumor was done on 13th day by measuring the tumor weight. Similarly Ehrlich Ascitic carcinoma (suspension tumor) was produced by transplanting IxlO7 cells of Ehrlich Ascitic Carcinoma intraperitoneally. In case of Ehrlich Ascites tumor model 1x107 cells of Ehrlich Ascitic tumor cells were transplanted intramuscularly in the right hind thigh of the mice for the development of solid tumor. Composition of formula 1 produced significant regression of tumor when given both orally and intraperitoneally in 10 % alcohol or in 0.5% Tween-20 (weight/vol, normal saline) in a total volume of 0.2 ml per mouse. The composition of formula 1 thus offers a novel innovation for its development as anticancer therapeutic. (Figure Removed) Table3. In vitro cytotoxic activity of natural boswellic acids on human cancer cell lines (Table Removed) (Table Removed) Advantages of the invention; The main advantages of the present invention are: Present composition is about 5 times better than the earlier reported natural derivatives of boswellic acid. Present composition is useful for induction of apoptosis of cancer cells, thus preventing the growth of tumour and malignancy of tissues. We claim l. A bio-active composition useful for the treatment of cancer, wherein the said composition comprising an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject and the said isomeric mixture having following formula l, Formula l REMOVED wherein R l = methyl when R2 = H or R2 = methyl when R l = H 2. A bioactive composition of formula l as claimed in claim l, wherein ion is obtained from gum resin of Boswellîa sp. and alternatively by semi synthetic method. 3. A bioactive composition as claimed in claiml, wherein the said subject is a mammal, including human. 4. A bioactive composition as claimed in claiml, wherein the said composition exhibits cytotoxicity against various human cancer cell lines up to 88% at a preferable concentration of at least lOuM. 5. A bioactive composition as claimed in claiml, wherein the said composition exhibiting anti-cancer activity up to 57% against Ehrlich Ascites carcinoma mouse tumor model, when given orally with 10%alcohol. 6. A bioactive composition as claimed in claiml, wherein the said composition is exhibiting anti-cancer activity up to 41% against Ehrlich Ascites Tumor mouse model, when given orally with 10% alcohol. 7. A bioactive composition as claimed in claiml, wherein the said composition exhibiting anti-cancer activity up to 56% against mouse sarcoma 180 tumor model, when given intra peritoneally with 10% alcohol or in normal saline with 0.5% Tween-20. 8. A bioactive composition as claimed in claiml, wherein the said composition is usefial for inhibiting cell proliferation with IC50 value of 12-14\|ug/ml, for leukemia cells. 32 9. A bioactive composition as claimed in claiml, wherein the said composition is useftil for inhibiting cell proliferation with IC50 value of 5 jig/ml, for human breast MCF-7 cancer cells. 10. A bioactive composition as claimed in claiml, wherein the said composition is useful for inducing mitochondrial depolarization consequent to reactive oxygen or nitrogen species as the mechanism into the apoptotic death of cancer cells. 11. A bioactive composition as claimed in claiml, wherein the said composition is useful for inducing programmed cell death (apoptosis) in lymphoblastic human leukemia Molt-4 and HL-60 cells. 12. A bioactive composition as claimed in claiml, wherein the said composition produces 18% - 74% apoptotic / post-apoptotic population at concentration ranging between l ug/ml - 50(j,g/ml. 13. A bioactive composition as claimed in claiml, wherein the said composition is useful for producing early reactive oxygen species (peroxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells. 14. A bioactive composition as claimed in claiml, wherein the said composition is useful for producing early reactive nitrogen species (nitric oxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells. 15. A bioactive composition as claimed in claiml, wherein the said composition is useful for exhibiting anti-cancer activity during the growth of solid and suspension tumors in experimental mouse tumor models. 16. A bioactive composition useful for treatment of cancer as claimed in claim l, wherein the cancer type is selected from group comprising of prostrate, colon, breast, liver, CNS and leukemia. 17. A process for preparation of a bio-active composition comprising an isomeric mixture of 3 a, 24 -dihydroxyurs-12-ene and 3 a, 24- dihydroxyolean-12-ene in the ratio of l :20 to 1:1 as claimed in claim l, wherein the said process of obtaining the composition from Boswellia sp comprising the following steps: (a) extracting the purified gum resin of Boswellia serrata with ethyl alcohol at room temperature; (b) concentrating the filtered extract obtained from step (a) under reduced pressure; (c) diluting the extract obtained from step (b) with potassium hydroxide with continuous stirring; (d)further extracting the aqueous extract obtained from step (c) with 10% ethyl acetate in hexane; (e) washing of the extract with water to remove the organic solvents and drying over anhydrous sodium sulphate. (f) performing column chromatography of the viscous liquid neutral components over silica gel; (g)eluting the components obtained from step (f) with hexane/ethyl acetate separating the desired composition comprising an isomeric mixture of 3ot, 24 -dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1 18. A process as claimed in claim 17, wherein the ratio of the components in the isomeric mixture is identified by reverse phase HPLC. 19. A process of preparation for the composition as claimed in claim 17, alternately through a semi synthetic process, wherein the said process comprising the following steps- (a) dissolving an isomeric mixture of (a+(3) of boswellic acids in dried ether at room temperature; (b)adding lithium aluminum hydride (LiAlH4) to the stirring solution as obtained from step (a), under nitrogen atmosphere at 20°C -25°C; (c) removing the excess of LiAlH4 from the solution as obtained from step (b) by adding of ethyl acetate and washing the organic layer with water; (d) concentrating the solution as obtained from step (c) gives desired composition comprising an isomeric mixture of 3a, 24 -dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio of l :20 to 1:1; (e) crystallizing the solution as obtained from step (d) to furnish white crystal of the desired composition. 20. A bio-active composition useful for the treatment of cancer, wherein the said composition comprising an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject substantially as herein described with reference to the figures, tables and examples accompanying this specification.

Full Text

Field of the invention;
The present invention relates to a bioactive composition useful for induction of apoptosis thereof cytotoxicity and anti-cancer activity, obtained by natural isolate from Boswellia species, alternatively prepared semi-synthetically, that comprises an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1, alone or in combination with pharmaceutically acceptable or other carriers.
More particularly the present invention relates to bioactive composition useful for cancer of colon, prostrate, liver, breast, central nervous system (CNS), leukemia and malignancy of other tissues, including ascites and solid tumors wherein the cancer cell death is mediated by induction of apoptosis and inhibition of cell proliferation at specific doses of the composition. The active mixture may be isolated from Boswellia serrata, Boswellia ovalifoliolata and related species or may be prepared semi-synthetically from the natural isolates i.e. boswellic acids, which comprise an isomeric mixture of a and p-boswellic acids or their carboxylic esters by lithium aluminium hydride reduction.
The present invention further relates to the bioactive composition that kills cancer cells through induction of programmed cell death (apoptosis) and can be used for the treatment of cancer. The herbal product comprising an isomeric mixture of 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 are isolated from

Boswellia species and alternatively prepared semi-synthetically from (a+p) boswellic acids.
Background and prior art of the invention;
The gum exudates from Boswellia serrata, the source of these products has been used since long in the traditional Ayurvedic system of medicine for the treatment of various inflammatory diseases in Indian subcontinent besides being used in preparations of commercial importance such as in fragrance and cosmetic industry.
Gum resin of another species of the same genus i.e. Boswellia ovalifoliolata Roxb (Buseraceae) is also the source of 3oc, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 also known as olibanum was used traditionally in the perfumery industry only. Boswellia serrata is a rich source of pentacyclic triterpenoids also known as boswellic acids while Boswellia ovalifoliolata is rich in tetracyclic triterpenoids. One of the isomers of the bioactive constituents i.e. 3a, 24-dihydroxyurs-12-ene has earlier been isolated and reported as new natural triterpenoid from the gum resin of Boswellia serrata [(B. Mahajan, S. C. Taneja, V. K.Sethi and K. L. Dhar, Phytochemistry 39(2), (1995)453-55], though it was semisynthetically known earlier [Allan, G.G., Phytochemistry (1968), 7, 963]. However, there has been no report of its occurrence in two isomeric forms in plants including their bioactivity.
Second to coronary heart diseases, cancer has emerged as the most common cause of death in many countries. Unlike normal cells, cancer cell appears

after having undergone accumulation of several genes mutations within it, which account for independent and uncontrolled growth of this cell into a clonal expansion amounting to invasion of adjoining tissues, and some times metastasing to other tissues. Dysregulation of programmed cell death (apoptosis) is the hall mark of all cancer cells and hence provides important therapeutic target to intervene in the development of anti-cancer agents, because activation of apoptosis is a common mechanism of malignant cell killing by anti-cancer agents [(K.M.Deabatin, Activation of apoptosis pathways by anti-cancer treatment, Toxicol Lett., 112/113 (2000) 41-48)]. Several pathways therefore, are proposed to be involved in the death of cancer cells by anti-cancer drugs. The drug may kill cells by activation of death receptors mediated pathways or via mitochondrial dependent pathways involving reactive oxygen and nitrogen species culminating in activation of certain genes in the apoptotic signal transduction pathways. Several agents are known to kill malignant cells by inducing apoptosis via free radicals generation and one such example is doxorubicin [(G. Minotti, P. Mena, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines: Molecular advances and pharmacologic developments in anti-tumor activity and cardiotoxicity, Pharmacol. Rev. 56 (2004), 185-229)]. Most of these agents activate various caspases, genes, translocate cytochrome c that upregulate downstream events leading to DNA fragmentation (DNA laddering) typical of apoptosis.
In another two decades world is expected to peak in all kinds of cancer and brain cancer in particular appears difficult to manage. Recently there has been a report of inhibitory activity of boswellic acids against human leukemia HL-60 cells in culture. Out of the four natural boswellic acids tested in vitro against the cell line HL-60 it was reported that acetyl -11-

keto-(3-boswellic acid induced the most pronounced inhibition in a dose dependent manner on DNA, RNA and protein synthesis (1C 50 values 0.6, 0.5, and 4.1 jo, mol) while for other boswellic acids of the same group recorded 1C 50 value in the range of 0.6- 7.1 |n mol. The effect of acetyl -11-keto-p-boswellic acid on DNA synthesis was found to be irreversible. The composition also significantly inhibited the cell growth of HL-60 [Yu Shao,, Chi-Tang Ho, Chee-Kok Chin, Vladimir Badmaev, Wei Ma and Mou-Tan Huang ; Planta Medico. 64 (1998) 328-31]. A patent has claimed the DNA topoisimerse I and II inhibitory effect of pentacyclic triterpenic acids of Boswellia serrate [Y-W. Lee, Q-C. Fang, Z-G. Wang, D-H. Li and C.E. Cook, (1991) USP 5,064,823]. There have also been IPR's related to the use of boswellic acid in the treatment of brain tumors [Simmet Thomas (De) (1994) USP 5919821 and USP 6174876] and other anti-cancer related activities of boswellic acids, [Subbiah, Ven, Natural product based apoptosis inducers (plant extracts), USP, 20050084547. Ammon, Hermann P.T, et al. use of boswellic acid and its derivatives for inhibiting normal and increased leucocytic elastase or plasmin activity (USP, 20020010168).
Therefore, it is apparent from the literature search that in the present invention, the isolation and use of isomeric mixture of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 is novel as a cytotoxic agent and inducer of apoptosis in cancer cells and can be used for the treatment of cancer derived from various tissues. Besides, process of semi-synthetic preparation of bioactive mixture from a an (3-boswellic acids by reduction is also novel.

Objects of the invention:
The main object of the present invention is to provide a bioactive composition comprising of an isomeric mixture of triterpenoids 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 isolated from Boswellia serrata, or alternatively obtained from semi-synthetically.
Another object of the present invention is to provide a process of preparation of the bioactive composition comprising of an isomeric mixture of triterpenoids 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene of formula 1 in the ratio 1:20 to 1:1 having pharmaceutical effective dose being in the range 250 - lOOOmg/kg body weight of a subject.
Also another object of the present invention is to provide the usefulness of bioactive composition of formula 1, comprising of an isomeric mixture of triterpenoids 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1:1 for the treatment of cancer of prostrate, colon, breast, leukemia, liver, CNS and of other tissues.
Yet another object of the present invention is to provide the chemically synthesizable, economically viable, commercially high yield of the composition from Boswellia sp, intended to provide analgesic and sedative effects to the patients suffering from advanced malignancy, in sharp contrast

to others where prophylactic use of psychotherapeutics and anti-emetics are often recommended during chemo-cum-radiotherapy.
Summary of the invention;
The present invention deals with the isolation of a natural isolate, alternatively semi-synthetic preparation of a bioactive composition of formula 1 , comprising of an isomeric mixture of triterpenoids 3 a, 24-dihydroxyurs- 1 2-ene and 3cc, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1 : 1 and its usefulness for the treatment of cancer of prostrate, colon, breast, leukemia, liver, CNS and of other tissues.
(FigureRemoved)

Accordingly, the present invention provides a bio-active composition useful for the treatment of cancer wherein the said composition comprising an isomeric mixture of 3oc, 24 -dihydroxyurs-12-ene and 3ot, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject and the said isomeric mixture having following

formula 1, wherein the Rl = methyl when R2= H or R2=methyl when Rl = H.
In an embodiment of the present invention, the said composition exhibiting cytotoxicity against human cancer cell lines selected from the group of prostrate, colon, breast, liver, CNS and leukemia.
In another embodiment of the present invention, the said composition exhibits anti-cancer activity against Ehrlich Ascites carcinoma mouse tumor model when given orally in 10% alcohol.
In still another embodiment of the present invention, the said composition exhibits anti-cancer activity against Ehrlich Ascites Tumor mouse model when given orally in 10% alcohol.
In yet another embodiment of the present invention, the said composition is useful for exhibiting anti-cancer activity against mouse sarcoma 180 tumor models when given intra-peritoneally with 10% alcohol or in normal saline with 0.5% Tween-20.
In still another embodiment of the present invention, the said composition is useful for inhibiting leukemia cell proliferation with IC50 value of 12-14f4,g/ml.
In still anther embodiment of the present invention, the said composition is useful for inhibiting human breast MCF-7 cancer cell proliferation with IC50 value of 5 jig/ml.

In also another embodiment of the present invention, the said composition is useful for inducing programmed cell death (apoptosis) in lymphoblastic human leukemia Molt-4 and HL-60 cells.
Further in an embodiment of the present invention, the said composition is useful for producing early reactive oxygen species (peroxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells.
In still another embodiment of the present invention, the said composition is useful for producing early reactive nitrogen species (nitric oxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells.
In yet another embodiment of the present invention, the said composition is useful for inducing mitochondrial depolarization consequent to reactive oxygen or nitrogen species as the mechanism into the apoptotic death of cancer cells.
In also another embodiment of the present invention, the said composition is useful for exhibiting anti-cancer activity during the growth of solid and suspension tumors in experimental mouse tumor models.
Further in another embodiment of the present invention, the said composition does not show any mortality of BALB/c mice over a period of two weeks after administering single oral dose in the range of 250-

lOOOmg/Kg body weight or single intra-peritoneal dose of 500mg/Kg body weight.
Breif description of the figures & tables;
Figure 1 shows the structural formula 1, comprising an isomeric mixture of 3a, 24 -dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1.
Figure 2 shows the inhibition of cancer cell proliferation by composition of formula 1 for determination of IC50.
Figure 3 shows the induction of DNA laddering (DNA damage), typical of apoptotic cell death induced by composition of formula 1 in human lymphoblastic Molt-4 and myeloblastic HL-60 leukemia cells. Figure 4 shows flow cytometric measurement for induction of apoptosis by composition of formula 1.
Figure 5 shows flow cytometric measurement for early generation of reactive oxygen species by composition of formula 1, in HL 60 cells. Figure 6 shows flow cytometric measurement for copious early endogenous formation of nitric oxide by composition with formula 1, in HL-60 cells. Figure 7 shows depolarization of mitochondrial membrane functions by composition of formula 1.
Table 1 represents the influence of composition of formula 1, on human cancer cells growth in terms of percentage inhibition.
Table 2 represents the anticancer activity of composition of formula 1 in various mosue tumor models of cancer in vivo.

Table 3 represents in vitro cytotoxic activity of natural boswellic acids on human cancer cell lines.
Table 4 represents in vitro cytotoxic activity of composition of formula 1 on human cancer cell lines.
Detailed description of the invention;
Boswellia serrata is well known for its anti-inflammatory activity in Indian system of medicine and the major constituents boswellic acids are also reported for their anti-cancer activity. The composition of formula 1 having isomeric mixture of
triterpenoids which are present in Boswellia species extract in minor amounts (0.1-7.0%) comprising of 3ot, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the ratio 1:20 to 1:1 and is isolated after repeated column chromatography of the neutral extract of the gum resin of Boswellia serrata, Boswellia ovalifoliolata and related natural sources. The pharmaceutical preparation comprising triterpenoids display biological activities, particularly cytotoxicity in human cancer cell lines which is useful for its application as anti-cancer agent.
The bioactive composition of formula 1, comprising an isomeric mixture of triterpenoids 3a, 24-dihydroxyurs-12-ene and 3oc, 24-dihydroxyolean-12 in the ratio 1:20 to 1:1, also can be obtained by semi-synthetic process that involves chemical reduction of the natural mixture of a and (3-boswellec
11

acids, by converting them to their alkyl esters such as methyl or ethyl esters thereafter subjecting them to reduction by lithium aluminium hydride (LAH) in an inert organic solvent such as diethyl ether, tetrahydrofuran and the like. The semi-synthetic product obtained by above process displays the same level of bioactivity as shown by the natural isolate. The purity of the product is established by physico-chemical characteristics as well as by reverse phase HPLC method. The ratio of the two isomers 3 a, 24-dihydroxyurs-12-ene and 3ot, 24-dihydroxyolean-12-ene has range between 1: 20 to 1: 1.
In a feature of the present invention provides isolation as well as semi-synthetic preparation of composition of formula 1, isomeric mixture of triterpenoid compounds comprising of 3 a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in a ratio 1: 20 to 1: 1, that exhibits anti-cancer activities for breast, leukemia, liver, colon and brain cancer.
In another feature of the present invention, the bioactive composition of formula 1 also inhibits human breast MCF-7 cancer cells growth by 50% at about 15 |ig/ml concentration.
In also another feature of the present invention, the bioactive composition of formula 1 comprising triterpenoids kills human cancer cells by induction of apoptosis.
In still another feature of the present invention, the bioactive composition of formula 1 kills more than 92% of prostrate DU-145 and PC-3 cells at 5x10"

5M concentration, wherein the prostrate cell lines are selected from DU-145 and PC-3 cells.
In yet another feature of the present invention, the bioactive composition of formula 1 kills colon cancer cells up to 96% at 5xlO"5M concentration, wherein the colon cancer cell lines are selected from HT-29, SW-620 and Colo205 cells.
Also in another feature of the present invention, the bioactive composition of formula 1 inhibits growth of cancer cells of liver up to 92% at 5xlO"5M concentration, wherein the cancer cell line of liver Hep2 is selected.
In still a feature of the present invention, the bioactive composition of formula 1 inhibits growth of neuroblastoma cell line SK-N-SH up to 86% at 5xlO"5M, wherein the cell line is selected from neuroblastoma (CNS) cancer cell lines SK-N-SH.

The following examples are given by the way of illustration of the present invention and should not be construed to limit the scope of the present invention.
Example-1
Isolation of bioactive triterpenoids 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 from the gum resin
Boswellia serrata gum resin (IKg) was cleaned and made free of extraneous impurities. The gum is extracted with 3x3L of ethyl alcohol at room temperature. The combined extract after filtration was concentrated under reduced pressure. The concentrated extract (530 g) was diluted with 3% potassium hydroxide solution (3L) and the mixture stirred for an hour. The aqueous extracted thus obtained was extracted with (2x 2L) hexane and then with 10% ethyl acetate in hexane (lx2L). The combined organic solvents were washed with water, dried over anhydrous sodium sulphate and concentrated under reduced pressure to give 130 g of viscous liquid comprising the neutral components of the gum resin. A portion of the concentrated extract (20g) was subjected to repeated column chromatography over silica gel. Gradient elution with hexane/ethyl acetate furnished besides other compounds, a compound (400 mg) mp 1 82°C, [a]D + 63. It was identified as a mixture of two isomeric compounds by 13CNMR,
. The ratio of the isomeric mixture was determined by reverse phase HPLC on C-18 column (acetonitrile and water in the ratio of 95:5) and detection at 200 nm.

Example-2
Preparation of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyoIean-12-ene of formula 1 by semi-synthetic method
a. An isomeric mixture of (a+P) of boswellic acids (R=H) (230 mg) was
dissolved in dry solvent ether at room temperature and to the stirring
solution under nitrogen atmosphere was added lithium aluminum hydride 20
mg (LiAlH4) while maintaining the temperature between 20-25°C. After the
completion of the addition the reaction mixture is further stirred for one
hour. The excess of LAH was destroyed by adding ethyl acetate and the
organic layer washed with water. Concentration of the solvent gave desired
product 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in
almost quantitative yield (a:(3 :: 1:4). It was crystallized from hexane-ethyl
acetate to furnish white crystal of mixture of formula 1 and confirmed by
physicochemical and spectral data. The ratio of isomers (ot:p: 1:4) was
estimated by HPLC on C-18 column using acetonitrile : water : acetic acid
(95:5 : 0.1) as the mobile phase .
b. An isomeric mixture of (a+P) of boswellic acids was converted to methyl
ester by diazomethane generated in situ by the method known in the art of
synthesis. A mixture of (ct+p) of boswellic acids (230 mg) was dissolved in
dry solvent ether and diazomethane solution in diethyl ether was slowly
added till completion of reaction as monitored by TLC. The mixture
comprising the methyl esters of compound of structure formula 1 (R=Me)
without purification was further reduced by LAH by the method described as in part a example 2.(Figure Removed)

Example-3
Cytotoxicity of 3d, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyoIean-12-ene of formula 1 against human cancer cell lines in vitro
Human cancer cells after trypsinization into single cell suspension were grown in 96-well culture plate for 24 hr. Cells were treated with indicated doses of composition of formula 1 and incubated in CO2 incubator for 48 hr. After 48h cells were stained with sulforhodamine B dye, and the bound dye was eluted to measure the optical density indicating cell growth in Elisa Reader at 540nm [(Monks, D. Seudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo, M. Boyd (1991), Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines, J. Natl. Cancer Inst. 83 757-766)]. The OD of untreated cells is
considered as 100% while of oil treated group is subtracted from the control group to determine % inhibition as a measure of cell cytotoxicity. The composition inhibited various human cancer cells growth by more than 88% at lOuM.(Table Removed)
Example-4
Composition of formula 1 comprising 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene, induced inhibition of cell proliferation of leukemia cells using MTT assay.
Human leukemia cells are grown in suspension in 96-well culture plate and were incubated with different concentrations of the composition for 48 hr. The cells were then incubated with MTT and the MTT-formazon formed is
eluted with DMSO, and OD measured in ELISA Reader [(Shashi B., SinghJ., Rao, J.M., Saxena, A. K. and Qazi, G. N.(2006), A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells. Nitric Oxide, 14, 72-88)]. The intensity of the color formed in the untreated control wells relates to 100% cell growth. The growth of
cells is recorded with different concentrations of the bioactive composition of formula 1, and the concentration that inhibits 50% cell growth is taken as 1C so value.
Human breast cancer cells MCF-7 grown in adherent culture are trypsinised to obtain cell suspension. Cells are then distributed to 96-well culture plate to grow as monolayer culture. Cells are treated with indicated concentration of composition of formula 1 and assayed for cell viability using MTT assay as described above. The IC50 values in leukemia cells was in the range of 12-14 ug/ml while for MCF7 cells was about 5 jug/ml.
Fig.2. Inhibition of cancer cell proliferation by composition of formula 1: Determination of IC50
(Figure Removed)

50:

Example-5
Composition of formula 1 induced DNA fragmentation typical of apoptosis in Molt-4 and HL-60 cells.
The composition of formula 1 induces programmed cell death (apoptosis), a desired therapeutic anticancer drug target, in lymphoblastic human leukaemia MOLT-4 and human myeloma leukaemia HL-60 cells. Composition of formula 1 produced dose dependent effect in both the cell lines and the DNA laddering produced is comparable to the one produced by standard anti-cancer drug camptothecin. Human leukemia cell lines grown in culture were exposed to 3-100 (J,g/ml of composition of formula 1 for 18hr. The control group received only the vehicle. Cells were similarly treated with camptothecin, 4uM as positive control. Increasing bioactive product concentration enhanced DNA ladder of about 180bp fragments as indicated by increased ethidium bromide fluorescence intensity of the fragments indicative of apoptosis.
Fig. 3. Induction of DNA laddering (DNA damage), typical of apoptotic cell death induced by composition of formula 1 in human lymphoblastic Molt-4 and myeloblastic HL-60 leukemia cells.(FigureRemoved)

Example 6
Concentration related influence of 3a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 on apoptosis: Flow cytometric analysis in HL-60 leukemia cells.
During the early events of apoptosis, phospholipid phosphatidyl serine of plasma membrane is exteranalized, which has very high affinity for annexinV antibody. HL-60 cells were treated with different concentrations of composition of formula 1 and incubated for a brief period of 6 hr. There after, cells were washed and stained with FITC conjugated annexinV antibody and propidium iodide. The cells (10,000) were analyzed by flow cytometery (BD, LSR) using ProQuest software. The fraction of cell

population in the lower right quadrant indicates apoptotic cells, upper right post apoptotic and upper left as necrotic populations. Composition of formula 1 produced concentration dependent increase in apoptotic and post apoptotic cell population. Composition of formula 1 at 1 jag/ml produced almost 18% apoptotic / post-apoptotic population, which increased to 74% at 50ug/ml concentration. The studies indicated that cell killing is mainly mediated via apoptosis and that composition of formula 1 is an interesting lead to be developed into anti-cancer agent.
Fig.4. Induction of apoptosis by composition of formula 1: Measurement by flow cytometry.
Example 7
Influence of bioactive composition formula 1 on the early generation of reactive oxygen species (peroxides): analysis by flow cytometery
The endogenous generation of peroxides was measured using a non-fluorescent probe DCFH-DA which upon entering the cell is deesterified to DCFH which is oxidized by the reactive oxygen species to a fluorescent product DCF that remains entrapped within the cells offering analysis by flow cytometery [(Shashi et al, 2006, cited in Example 4 )]. HL-60 cells were incubated with different concentrations of the composition of formula 1 for a brief period of 6hr. Thereafter cells were washed and stained for 1 hr with DCFH-DA and analysed on flow cytometer. The composition of formula 1 produced dose dependent increase in peroxide positive cell population, being 79% at 50 jug/ml. The pro-oxidant effect was completely impaired in the presence of anti-oxidant, ascorbate. Formation of reactive oxygen and nitrogen species are indicated in the induction of apoptosis.
Fig.5. Early generation of reactive oxygen species by composition of formula 1 in HL-60 cells: Measurement by Flowcytometry(Figure Removed)

Example 8
Composition of formula 1 comprising 3 a, 24-Dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene mediated early generation of endogenous nitric oxide measured by flow cytometery.
Measurement of in situ NO generation involved the use of a fluorescent probe diaminofluoresceine-2-diacetate, which is permeated easily into the cells. Once inside the cell it binds NO soon it is formed and emits fluorescence [H.Kojima et, Development of a fluorescent indicator for the nitric oxide based on the fluoresceine chromophore, Chem. Pharm. Bull., 46 (1998) 373-375]. For this purpose, the HL-60 cells were incubated with DAF-2-DA for 30 min before being incubated with the composition of formula 1 for 6hr. Cells were washed and stained with anti-annexinV-PE. Cells were analyzed on flow cytometer in FL-1 channel for NO and FL-2 channel for apoptosis FL-1. It appears that all the cells formed NO within 6
hr irrespective of the concentration of bioactive composition used, because there were no cells in the lower left quadrant as indicated in control cells. However, the extent of apoptosis was much higher in bioactive composition of formula 1 when the cell populations from the upper two quadrants (apoptotic and early apoptotic) are taken into consideration. Interaction of NO with superoxide, while both are generated simultaneously, may affect the mitochondria! membrane potential that may be responsible for the activation of apoptosis
Fig.6. Copious early endogenous formation of nitric oxide by composition of formula 1 in HL-60 cells: Measurement of apoptosis vis-a- vis NO by flowcytometry.
(FigureRemoved)
Example 9
Mitochondrial membrane depolarization by 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1: measurement by flow cytometery
Rhodamine-123 uptake into the mitochondria is driven by mitochondrial transmembrane potential ( \j/mt ) that allows the determination of cell population with active integrated mitochondrial functions. Loss of \j/mt would lead to depolarization of mitochondria consequent to ROS and NO generation leading to cell death. HL-60 cells were exposed to the composition of formula 1 for 12 hr and incubated with Rh-123. Cells, 10000, were acquired for analysis by flow cytometery. The percentage of cells with low Rh-123 fluorescence was calculated from the dot plot stastics. In untreated control cells more than 92% cells showed Rh-123 fluorescence, which decreased with treatment of composition of formula 1 .This shows that reactive oxygen/nitrogen species generated by 1 bring about oxidative damage to mitochondria ensuing apoptosis.
Fig.7. Depolarization of mitochondrial membrane functions by composition of formula 1

CM

(Figure Removed)
ExamplelO
Anti-cancer activity of 3 a, 24-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene of formula 1 in the mouse model of Sarcoma 180 solid tumor and Ehrlich ascitic carcinoma (suspension)
Swiss albino mice, 6-8 weeks old (18-22 gm by weight) were bred and maintained at institute's animal house. Approval of the Institute's ethics committee was taken for use of laboratory animals and the procedures employed therein. The mice were housed in well individually ventilated cage (IVC) system and maintained on pelleted diet and water ad libitum. For the initiation of experiment, 1X107 cells of sarcoma -180 tumors were transplanted intramuscularly in the thigh of mice for the development of solid tumor. The day of tumor transplantation was assigned as day 0, next day (day-1) animals were randomly selected and divided into three groups of

seven mice in each group of similar body wt. Group-1 was treated with composition of formula 1 100 mg/kg i.p prepared in 10 % alcohol of the total volume 0.2ml. Group-2 received 0.2 ml of vehicle and served as normal control while group-3 serving as positive control was treated with 5-FU (22 mg/ kg b.wt.) intraperitoneally. The total duration of treatment was of 9 days. The evaluation of effect of treatment on tumor was done on 13th day by measuring the tumor weight.
Similarly Ehrlich Ascitic carcinoma (suspension tumor) was produced by transplanting IxlO7 cells of Ehrlich Ascitic Carcinoma intraperitoneally. In case of Ehrlich Ascites tumor model 1x107 cells of Ehrlich Ascitic tumor cells were transplanted intramuscularly in the right hind thigh of the mice for the development of solid tumor. Composition of formula 1 produced significant regression of tumor when given both orally and intraperitoneally in 10 % alcohol or in 0.5% Tween-20 (weight/vol, normal saline) in a total volume of 0.2 ml per mouse. The composition of formula 1 thus offers a novel innovation for its development as anticancer therapeutic.
(Figure Removed)
Table3. In vitro cytotoxic activity of natural boswellic acids on human cancer cell lines
(Table Removed)

(Table Removed)

Advantages of the invention;
The main advantages of the present invention are:
Present composition is about 5 times better than the earlier reported
natural derivatives of boswellic acid.
Present composition is useful for induction of apoptosis of cancer cells,
thus preventing the growth of tumour and malignancy of tissues.

We claim
l. A bio-active composition useful for the treatment of cancer, wherein the said composition comprising an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, optionally along with pharmaceutically acceptable carriers or additives and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject and the said isomeric mixture having following formula l,

Formula l REMOVED
wherein R l = methyl when R2 = H or R2 = methyl when R l = H
2. A bioactive composition of formula l as claimed in claim l, wherein ion is obtained from gum resin of Boswellîa sp. and alternatively by semi synthetic method.

3. A bioactive composition as claimed in claiml, wherein the said
subject is a mammal, including human.
4. A bioactive composition as claimed in claiml, wherein the said
composition exhibits cytotoxicity against various human cancer cell
lines up to 88% at a preferable concentration of at least lOuM.

5. A bioactive composition as claimed in claiml, wherein the said
composition exhibiting anti-cancer activity up to 57% against
Ehrlich Ascites carcinoma mouse tumor model, when given orally
with 10%alcohol.
6. A bioactive composition as claimed in claiml, wherein the said
composition is exhibiting anti-cancer activity up to 41% against
Ehrlich Ascites Tumor mouse model, when given orally with 10%
alcohol.

7. A bioactive composition as claimed in claiml, wherein the said
composition exhibiting anti-cancer activity up to 56% against mouse
sarcoma 180 tumor model, when given intra peritoneally with 10%
alcohol or in normal saline with 0.5% Tween-20.
8. A bioactive composition as claimed in claiml, wherein the said
composition is usefial for inhibiting cell proliferation with IC50
value of 12-14|ug/ml, for leukemia cells.
32

9. A bioactive composition as claimed in claiml, wherein the said
composition is useftil for inhibiting cell proliferation with IC50
value of 5 jig/ml, for human breast MCF-7 cancer cells.
10. A bioactive composition as claimed in claiml, wherein the said
composition is useful for inducing mitochondrial depolarization
consequent to reactive oxygen or nitrogen species as the mechanism
into the apoptotic death of cancer cells.

11. A bioactive composition as claimed in claiml, wherein the said
composition is useful for inducing programmed cell death
(apoptosis) in lymphoblastic human leukemia Molt-4 and HL-60
cells.
12. A bioactive composition as claimed in claiml, wherein the said
composition produces 18% - 74% apoptotic / post-apoptotic
population at concentration ranging between l ug/ml - 50(j,g/ml.
13. A bioactive composition as claimed in claiml, wherein the said composition is useful for producing early reactive oxygen species (peroxide) as the causative agent ensuing DNA laddering and apoptotic death of cancer cells.

14. A bioactive composition as claimed in claiml, wherein the said
composition is useful for producing early reactive nitrogen species
(nitric oxide) as the causative agent ensuing DNA laddering and
apoptotic death of cancer cells.
15. A bioactive composition as claimed in claiml, wherein the said
composition is useful for exhibiting anti-cancer activity during the
growth of solid and suspension tumors in experimental mouse tumor
models.

16. A bioactive composition useful for treatment of cancer as claimed in
claim l, wherein the cancer type is selected from group comprising
of prostrate, colon, breast, liver, CNS and leukemia.
17. A process for preparation of a bio-active composition comprising an
isomeric mixture of 3 a, 24 -dihydroxyurs-12-ene and 3 a, 24-
dihydroxyolean-12-ene in the ratio of l :20 to 1:1 as claimed in claim
l, wherein the said process of obtaining the composition from
Boswellia sp comprising the following steps:

(a) extracting the purified gum resin of Boswellia serrata with
ethyl alcohol at room temperature;
(b) concentrating the filtered extract obtained from step (a) under
reduced pressure;

(c) diluting the extract obtained from step (b) with potassium
hydroxide with continuous stirring; (d)further extracting the aqueous extract obtained from step (c)
with 10% ethyl acetate in hexane;
(e) washing of the extract with water to remove the organic
solvents and drying over anhydrous sodium sulphate.
(f) performing column chromatography of the viscous liquid
neutral components over silica gel;
(g)eluting the components obtained from step (f) with hexane/ethyl acetate separating the desired composition comprising an isomeric mixture of 3ot, 24 -dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1
18. A process as claimed in claim 17, wherein the ratio of the
components in the isomeric mixture is identified by reverse phase
HPLC.
19. A process of preparation for the composition as claimed in claim 17,
alternately through a semi synthetic process, wherein the said
process comprising the following steps-
(a) dissolving an isomeric mixture of (a+(3) of boswellic acids in
dried ether at room temperature; (b)adding lithium aluminum hydride (LiAlH4) to the stirring
solution as obtained from step (a), under nitrogen atmosphere at
20°C -25°C;

(c) removing the excess of LiAlH4 from the solution as obtained
from step (b) by adding of ethyl acetate and washing the
organic layer with water;
(d) concentrating the solution as obtained from step (c) gives
desired composition comprising an isomeric mixture of 3a, 24
-dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene in the
ratio of l :20 to 1:1;
(e) crystallizing the solution as obtained from step (d) to furnish
white crystal of the desired composition.
20. A bio-active composition useful for the treatment of cancer, wherein the said composition comprising an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and 3 a, 24-dihydroxyolean-12-ene in the ratio of 1:20 to 1:1, and the pharmaceutically effective dose of the said composition being in the range of 250 -lOOOmg/Kg body weight of a subject substantially as herein described with reference to the figures, tables and examples accompanying this specification.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=EAftb0b/2ScQSV9Lh4b23Q==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

268507

Indian Patent Application Number

570/DEL/2007

PG Journal Number

36/2015

Publication Date

04-Sep-2015

Grant Date

31-Aug-2015

Date of Filing

16-Mar-2007

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	SETHI VIJAY KUMAR	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
2	MONDHE DILIP MANIKRAO	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
3	KAPAHI BAL KRISHAN	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
4	BHUSHAN SHASHI	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
5	ANDOTRA SAMAR SINGH	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
6	BHAHWAL ALI SHAH	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
7	SINGH SURJEET	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
8	HARISH CHANDRA PAL	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
9	MALIK MAYAZ	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
10	KUMAR AJAY	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
11	MADHUNIKA SHARMA	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
12	TANEJA SUBHASH CHANDRA	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
13	SINGH JASWANT	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
14	SAXENA AJIT KUMAR	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI
15	QAZI GHULAM NABI	REGIONAL RESEARCH LABORATORY, JAMMU-TAWI

PCT International Classification Number

A61K31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA