FIELD OF INVENTION

The present invention refers to a use of an effective amount of a composition comprising- stamineus leaf extract. ^: _·. _. .;· ^' .V.

BACKGROUND OF THE INVENTION

Orthosiphon stamineus Benth. is a traditional medicinal herb belongs to the family Lamiaceae grown in Southeast Asia, is also called java tea, or Kidney Tea plant as common and folk names. This plant is locally known as Misai Kucing.

Some of the principle bioactive phytpconstituents of O. stamineus leaf extract are pentacyclic- triterpenes like betulinic acid, oleanolic acid, ursolic acid and β-sitosterol, highly-oxygenated isopimarane and staminane - type diterpenes together with monoterpenes, triterpenes, flavonoids, benzochromene, saponins, hexoses, organic acids, rosmarinic acid, chromene, and myo-inositol; some of polymethoxylated flavones such as sinensetin, . tetramethylscutellarein and 3'-hydroxy-5,6,7,4'- tetramethoxyflavone. The plant extract is ^: rich in flavonoids (phenolics) and about twenty . pherioiic ⁵ compounds were reported from this plant including nine lipophilic flavones, two flavonol glycosides and nine caffeic acid derivatives such as rosmarinic acid and 2,3-dicaffeoyltartaric acid.

Rosmarinic acid, a derivative of caffeic acid, is the most abundant component (about 12.8%) of the O. stamineus leaf extract, which is known to have strong antioxidant and anti-inflammatory activity. Other major antioxidants present in the O. stamineus leaf extract are betulinc acid, quercetin, myricetiri and luteolin.

O. stamineus leaves used as traditional medicine mainly as diuretic and an edible stuff as well/This plant is used commonly in Southeast Asia and European countries as herbal tea. Dried or fresh leaves of the plant can be used to make cool refreshing drinks or warm relaxing teas/decoctions, which is good for rheumatism, edema, abdominal pain, kidney and bladder inflammation, hypertension and gout.

As an alternative medicine, the paste or aqueous extract of the leaves was used as abdominal related abnormalities, to remove kidney stones and post pregnancy problems. From the scientific studies, it is . found that leaves have dynamic pharmacological properties such as, antioxidant, anti-inflammatory, cytotoxic, diuretic, antihypertensive and vasodialative properties.

Angiogenesis is the formation of new blood vessels from pre-existing, vessels that occurs in many physiological and pathological conditions such as embryonic development, chronic inflammation, wound hea|ing, and tumor progression and metastasis. Normal angiogenesis occurs during fetal growth and development, while tumor tissues release angiogenic, factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), .angiogenin and transforming growth factor-beta 1 (TGF- beta 1 ) to supply oxygen and nutrients to themselves during tumor growth[Gargett and Rogers, 2001.].: Tumors can remain avascular and latent for years and activation of angiogenesis is a necessary condition for tumor development. It is known that without blood supply the dimensions of a tumor nodule cannot exceed 2-3 mm ³ due to hypoxia leading to death of tumor cells (Folkman 1995). Since a close relationship between tumor growth and angiogenesis had been clarified, various angiogenic inhibitors for use in cancer treatment have been studied. Therefore, the inhibition of angiogenesis emerges as a potent target for prevention and treatment of cancer. Angiogenesis plays a pivotal role In many life threatening pathological conditions. For 'instant; cardiovascular diseases such as angioma, atherosclerosis, vascular deformity, angiofibroma, synechia; and edemic sclerosis; and opthalmological diseases such as neovascularization after cornea, implantation, neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease, macular degeneration, retinal degeneration, retrolental fibroplasias, and granular conjunctivitis are related to angiogenesis. Chronic inflammatory diseases such as arthritis, dermatological disease such, as psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis and acne are also angiogenesis-depehdent. · ^'' . ^' ■ ^' _. · VEGF is a potent angiogenic stimulator produced, by various types of tumor cells, which plays an important role in the growth and metastasis of tumors by stimulating the proliferation of endothelial cells and promoting neovascularization. VEGFs are endothelial cell mitogens in vitro and also stimulate angjpgenesis in vivo. VEGF is a dimeric glycoprotein that binds two distinct receptors on. vascular- endothelial cells, VEGF receptor- 1 (flt-1 ) and VEGFR-2 (fik-1/KDR). VEGF receptor is a tyrosine kinase receptor and has been strongly implicated in tumor angiogenesis. Blockade of the angiogenic process, · through inhibition of VEGF signaling, has been shown to result in significant tumor growth delay in a wide range of preclinical models. The clinical benefit of this approach has also been confirmed. -

Breast cancer remains the most common cancer among women worldwide, with more than 1 million new cases and more than 370,000 deaths each year. Most breast cancer deaths are due to metastatic disease, not primary tumor burden. Metastasis is a muitistep process that involves the spread of malignant cells from the primary tumor and the subsequent growth of secondary tumors in distant tissues and organs of the body. Angiogenesis is a prerequisite for tumor growth and metastasis. Angiogenesis and the development of metastases are intrinsically connected. Arthritis, a well-known inflammatory disease, is initiated as ah autoimmune disease. However, t e growth _: of vascular endothelial cell in the synovial cavity is activated by the inflammatory cytokines, which finall destroy cartilage in the articulation.

Approximately more than half of the population in the developed and developing countries are overweight or obese. The prevalence in children in all developed countries is about 15-25% and this number, is.■ expected to increase significantly in the near future. Obesity is a complex metabolic disorder that is commonly associated with type 2 diabetes mellitus, hypertension, coronary heart disease, stroke, dyslipidemia, gallbladder disease, hepatic steatosis, sleep apnea, stroke, endometrial disorder, and cancer. Interestingly, most of these obesity-related disorders are closely associated with vascular dysfunctions. For example, hypervascularization could result in onset and progression of diabetic ocular . and kidney complications, macular diseases and cancer. Many people are losing their eyesight all over the world because of various ocular diseases. Many patients become blind due to the infiltration of the capillary blood cells into the vitreous humor.

Psoriasis is caused by extremely active proliferation, of ^* skin cells. Fast-growing cells require sufficient blood supply, and angiogenesis is abnormally induced in psoriasis. From the above mentioned literature, it is now convinced that angiogenesis is strongly related to initiation : and development of many lethal diseases. Therefore, inhibition of angiogenesis can be the excellent: target for the treatment of those diseases. Many efforts have been made toward the search of ahti- angiogenic agents with the intention to prevent and/or cure those diseases.

VEGF is a potent angiogenic stimulator produced by various types of tumor cells, which plays an important role in the growth and metastasis of tumors by stimulating the proliferation of endothelial cells and promoting neovascularization. VEGFs are endothelial cell mitogens in vitro and also stimulate angiogenesis in vivo. VEGF is a dimeric glycoprotein that binds two distinct receptors on vascular endothelial cells, VEGF receptor-1 (flt-1 ) and VEGFR-2 (flk-1/KDR). VEGF receptor is a tyrosine kinase receptor and has been strongly implicated in tumor angiogenesis. Blockade of the angiogenic process, through inhibition of VEGF signaling, has been shown to result in significant tumor growth delay in a wide ^" range of preclinical models. The clinical benefit of this approach has also been confirmed.

Since inhibitors for angiogenesis and VEGF signaling pathway can be used for the treatment of many diseases, development of angiogenesis inhibitor as new drugs is expected. Desirable inhibitors should not have toxic or adverse effect with good patient compliance because inhibitors need to be administered for a long time.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a use of an effective amount of a composition comprising O. stamineus leaf extract to a subject suffering from angiogenesis associated disease, wherein the O. stamineus leaf extract is administered to the subject in an amount of from about 50 mg to about 1000 mg per kg body weight per day.

The present invention consists of several novel features and a combination of parts hereinafter fully described and illustrated in the accompanying description and drawings, it being understood that various changes in the details may be made without departing.from the scope of the invention or sacrificing any of the advantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The current invention will become more fully understood from the. detailed depiction provided herewith , below, and the supplementary pictures which are given by way of illustration only, and thus are not, limitative of the present invention, and wherein; Fig. 1 A is a HPLC chromatogram developed with the five markers; they are as follows, (3'-hydroxy-5, 6 _; 7, 4'- tetramethoxyflavone (T F), Sinensetin (SNS), Rqsmarinic acid (RA), Orthosiphol A (OrA) and ^' ethylripariochromene ( RC).

Fig. 1 B is a HPLC chromatogram of methanolic extract of O. stamineus leaf illustrating the peaks ^■ . corresponds to five markers with the respective retention time. . Figs. 2A-B are pictures depicting the anti-angiogenic effect of O. stamineus leaf extract in chorioallantoic ¹ membrane assays (A: control, B: treated).

Figs. 3A-D show the images demonstrating anti-angiogenic effect of O. stamineus leaf extract in e vivo rat aortic ring assay (A&B;: control, C: treated with O. stamineus leaf extract, D: treated with suramin). 5 Fig. 4 is a bar graph illustrating the dose dependent inhibitory efficacy of leaf extract of O. stamineus.on sprouting of microvessels from rat aorta ( ^* p<0.05 and ^** p<0.01 ).

Fig. 5 is a line graph depicting the inhibitory activity of O. stamineus leaf extract on VEGF-induced human umbilical vein endothelial cells (HUVECs) proliferation.

Fig. 6 is the picture of the wound created in a monolayer of endothelial cells. Standardized (50%) ethanolic extract of O. stamineus strongly inhibits the growth factor induced-HUVECs migration in a dose dependent manner. It can be seen clearly that the extract at 25 μg/ml concentration completely inhibited the migration of HUVECs, whereas at 50 μg/ml concentration including inhibition of cell, migration, the5 ; extract also caused dislodgement of monolayer of endothelial cells (indicated by the arrows). Figs. 7A-H illustrate the inhibitory effect of O. stamineus.\eai extract on the VEGF-induced tube formation by HUVECs grown on Matrigel. HUVECs (2 x 104 cells/well) were plated on atrigel precoated 96-well plates and treated with different concentrations of for 24 h. (A & E) Control; (B) _: Methanolic extract (25 g/ml) (C) Methanolic fraction of methanolic extract (50 g/ml); (D) Suramin (10 g/ml); (F) 50 % ethanolic extract (25 pg/ml); (G) 50 % ethanolic extract (50 μg/ml) and (H) Betulinic acid (10 yg/ml):

Fig. 8 is a bar graph depicting the inhibitory efficacy of O. stamineus \eai extract on VEGF 165 production by endothelial cells. Fig. 9 is a western blot image showing the dose dependent suppression of VEGF receptor-2 tyrosine kinase expression in HUVECs by O. stamineus leaf extract at various concentrations.

Fig. 10 is a bar graph showing the synergistic cytotoxic effect of O. stamineus leaf extract wit tamoxifen against estrogen dependent human breast cancer cell lines. Fig. 1 1 is the picture of tested nude mice showing the effect of O. stamineus leaf extract on human colorectal (HCT 1 16) xenografts. This figure Illustrates, the comparative effect of. two different doses of 50% ethanolic extract with control group on HCT116 tumorigenesis in nude mice (n=6, values are in Mean ± SD).

Fig. 12 is a picture of excised tumors showing the differential effects of O. stamineus leaf extract on tumor morphology. Effect of 50% ethanolic extract of O. stamineus leaves on morphology of tumors excised from mice bearing human colorectal cancer cells (HCT.116). A: Tumor harvested from control animal; B: Tumor harvested from animal treated with 100 mg/kg extract: and C: Tumor harvested from animal treated with 200 mg/kg. Arrow indicates prompt and well developed blood vessels in control whereas, the arrowhead points towards the disruption in tumor vasculature by the extract. In tumor C, density of vascularization is nil or negligible. Fig. 13 is a line graph, Illustrates the comparative effect of extract with control group on HCT116 tumorigenesis in nude mice.

Figs. 14 is line graph, showing the average tumor volumes of control group measured at the week intervals of time. Average tumor volumes of control group measured at the week intervals of time (n=6, values are in Mean ± SD).

Figs. 15 is line graph, showing the average tumor volumes of extract treated group at 100 mg/kg body weight dose, measured at the week intervals of time (n=6, values are in Mean ± SD).

Figs. 16 is line graph, showing the average tumor volumes of extract treated group at 200 mg/kg bod weight dose, measured at the week intervals of time (n=6, values are in Mean ± SD). Fig. 17 is a bar graph depicting the levels of VEGF 165 in colon tissues harvested from different treatment groups. Levels of vascular endothelial growth factor (VEGF) peptide determined quantitatively by ELISA in colon tumor tissue lysates. Tumor tissues harvested, from placebo control group showed 135.5±4 pg/100 g of tissue lysates, while the tumor tissues harvested from the animals treated with 100 and 200 mg/kg body weight showed 1 12.9±4 and 90.9±2 pg/100 g of tissue lysates, respectively, (n=6, values are in Mean ± SD).

Fig. 18 is a picture of. nude mice showing the prophylactic efficacy of O. stamineus leaf extract against breast cancer. Ethanolic : water (1 :1) extract inhibits the early-onset of breast tumor (MCF 7) in estradiol implanted nude mice.

Fig. 19 is a bar graph shows the tumor uptake ratio by respective groups. This figure depicts the inhibitory. effect of ethanolic : water (1 :1 ) extract of O. stamineus leaves on human breast tumor xenografts in nude mice (n=6, values are in. Mean ± SD). The extract at 200 mg/kg completely inhibited tumorigenesis in treated animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to use of an effective amount of a composition comprising O. stamineus leaf extract. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is. envisioned that those skilled in the art may devise various modifications and equivalents without departing from the scope of the appended claims.

It was found in the present invention that O. stamineus leaf extract exhibits the following action: inhibition of angiogenesis, down regulation of VEGF signaling pathway by inhibiting the VEGF induced HUVECs proliferation and tube formation, inhibition of VEGF protein synthesis, suppression of VEGF receptor-2 tyrosine kinase and synergistic enhancement of tamoxifen's cytotoxic potency.

Accordingly, the present invention provides the preparation and HPLC-based standardization method., comprising O. stamineus leaf extract (Fig.1). Thus, the composition of the present invention can be used for the preparation of antioxidant rich O. stamineus leaf extract. Further, the present invention provides an anti-angiogenic composition comprising O. stamineus leaf extract as active ingredient without other active ingredients, but the invention also reports an anti- tumprigenic composition comprising O. stamineus leaf extract as active ingredient with other active ingredients having the antagonistic property against estrogen receptor such as tamoxifen.

Exclusively, the current invention provides an anti-angiogenic and anti-tumorigenic composition for ; pharmaceutical, dietetic, health supplement and or nutraceutical use.

Thus, the composition of the present invention can be used for the treatment or prevention of diseases derived from angiogenesis.

Further, the present invention provides VEGF down regulation composition comprising O. stamineus leaf . ^" extract as active ingredient without other active ingredients. Furthermore, the present invention provides VEGF receptor-2 tyrosine kinase inhibitory composition comprising O. stamineus leaf extract as active ingredient without other active ingredients.

More specifically, the present invention provides .the angiogenesis-inhibitory and synergistic cytotoxic composition for pharmaceutical, dietetic, health supplement, nutraceutical, or preventive therapeutic use. Thus, the composition of the present invention can be used for the treatment or prevention of diseases . derived from over expression of VEGF protein VEGF receptor-2 tyrosin kinase activity. -

These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.

The present invention is directed to a composition comprising O. stamineus leaf extract for inhibiting angiogenesis and enhancing the cytotoxic efficacy of tamoxifen. The composition additionally comprises at least one ingredient selected from the other organic solvents extracts of O. stamineus leaf such as 00

% methanolic extract, (9:1 ) methanolic and aqueous extract, (8:2) methanolic and aqueous extract, (7:3) methanolic and aqueous extract, (6:4) methanolic and aqueous extract, (5:5 or 1 :1 ) methanolic and aqueous extract, (4:6) methanolic and aqueous extract, (3:7) methanolic and aqueous extract, (2:8) methanolic and aqueous extract, (1 :9) methanolic and aqueous extract, methanolic extract, 100 % ^' ethanolic extract, (9:1 ) ethanolic and aqueous extract, (8:2) ethanolic and aqueous extract, (7:3) ethanolic and aqueous extract, (6:4) ethanolic and aqueous extract, (5:5 or 1 :1 ) ethanolic and aqueous extract,

(4:6) ethanolic and aqueous extract, (3:7) ethanolic and aqueous extract, (2:8) ethanolic and aqueous extract, (1 :9) ethanolic and aqueous extract, 100 % aqueous extract, petroleum ether extract, diethyl ether extract, 1 ,2-dichloroethane. extract, N-hexane extract, acetone extract, ethyl acetate extract, and chloroform extract. The composition may be a pharmaceutical composition for angiogenesis inhibition.

The composition also may be a food composition for angiogenesis inhibition. The food composition may ' be a nutraceutical or health supplement. The composition is also used for prevention and/or treatment of at least one disease selected from the group consisting of cancer metastasis, obesity, arthritis, angioma, ■ angiofibroma, diabetic retinopathy, premature infant's retinopathy, nedvascular glaucoma, corneal disease induced by angiogenesis, involutional macula, macular degeneration, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis , and pyogenic granuloma.

The present invention is also .directed to a composition comprising O. stamineus leaf extract for down regulating the human VEGF signal pathway by inhibiting the VEGF protein synthesis and suppressing the expression of human VEGF receptor ¹ ? tyrosine kinase on HUVECs. The composition additionally comprises at least one ingredient selected from the other organic solvents extracts of O. stamineus leaf such as 100 % methanolic extract, (9:1) methanolic and aqueous extract, (8:2) methanolic and aqueous extract, (7:3) methanolic and aqueous extract, (6:4) methanolic and aqueous extract, (5:5 or 1 :1 ) methanolic and aqueous extract, (4:6) methanolic and aqueous extract, (3:7) methanolic and aqueous extract, (2:8) methanolic and aqueous extract, (1 :9) methanolic and aqueous extract, methanolic extract, 100 % ethanolic extract, (9:1 ) ethanolic and aqueous extract, (8:2) ethanolic and aqueous extract, (7:3) ethanolic and aqueous extract, (6:4) ethanolic and aqueous extract, (5:5 or 1 :1 ) ethanolic and aqueous extract, (4:6) ethanolic and aqueous extract, (3:7) ethanolic and aqueous extract, (2:8) ethanolic and aqueous extract, (1 :9) ethanolic and aqueous extract, 100 % aqueous extract, petroleum ether- extract, diethyl ether extract, 1 ,2-dichloroethane extract, N-hexane extract, acetone extract, ethyl acetate extract, and chloroform extract. The composition may be a pharmaceutical composition for blocking VEGF signal pathway. The composition may also be a food composition for blocking VEGF signal pathway. The composition may further be a nutraceutical or health supplement. The composition may be used for treatment of at least one hyper vascularization related disease selected from the group, consisting of . metabolic disorder manifestations including rheumatoid arthritis, diabetic blindness, obesity, macular degeneration, Alzheimer syndrome and cancer metastasis, angioma, angiofibroma, premature infant's retinopathy, neovascular glaucoma, corneal disease induced by angiogenesis, involutional macula, pterygium, retinal degeneration, retrolental fibroplasias, granular conjunctivitis, psoriasis, telangiectasis and pyogenic granuloma. +-

The present invention is also directed to a composition comprising O. stamineus leaf extract for synergistically enhancing the cytotoxic activity of tamoxifen. The composition additionally comprises at least one ingredient selected from the other organic solvents extracts of O. stamineus leaf such as 100 % . methanolic extract, (9:1 ) methanolic and aqueous extract, (8:2) methanolic and aqueous extract, (7:3) ; methanolic and aqueous extract, (6:4) methanolic and aqueous extract, (5:5 or 1 :1 ) methanolic and aqueous extract, (4:6) methanolic and aqueous extract, (3:7) methanolic and aqueous extract, (2:8) methanolic and aqueous extract, (1 :9) methanolic and aqueous extract, methanolic extract, 100 % ethanolic extract, (9:1 ) ethanolic and aqueous extract; (8:2) ethanolic and aqueous extract, (7:3) ethanolic and aqueous extract, (6:4) ethanolic and aqueous extract, (5:5 or 1 :1 ) ethanolic and aqueous extract, (4:6) ethanolic and aqueous extract, (3:7) ethanolic and aqueous extract, (2:8) ethanolic and aqueous extract, (1 :9) ethanolic and aqueous extract, 100 % aqueous extract, petroleum ether extract, diethyl ether extract, 1 ,2-dichloroethane extract, N-hexane extract, acetone extract, ethyl acetate extract, arid chloroform extract. The composition additionally comprises other tumorigenic human cell lines such as, MCF-7, MDA-MB231 , HCT-116, CQL205, CAC02, HT-29, SW480 and SW620. More specifically the composition may be a pharmaceutical composition for antitumor activity along with tamoxifen against breast cancer. Further; the composition may also be a pharmaceutical composition for antitumor activity, along with any antagonist of the estrogen receptor against breast cancer. Generally the.composition may also be a pharmaceutical composition for antitumor activity along with any antagonist of the estrogen receptor against all estrogen dependent cancer. The composition may also be a food composition for

aqueous extract, (2:8) methanolic and aqueous extract, _. (1 :9) methanolic and aqueous extract, methanolic extract, 100 % ethanolic extract, (9:1 ) ethanolic and aqueous extract, (8:2) ethanolic and aqueous extract, (7:3) ethanolic and aqueous extract, (6:4) ethanolic and aqueous extract, (5:5 or 1 :1 ) ethanojic and aqueous extract, (4:6) ethanolic and aqueous extract, (3:7) ethanolic and aqueous extract; (2:8) ethanolic and aqueous extract, (1 :9) ethanolic. and aqueous extract, 100 % aqueous extract, petroleum ether: extract, diethyl ether extract, 1 ,2-dichloroethane extract, N-hexane extract, acetone extract, ethyl acetate extract, and chloroform extract.

In the present application, "a" and "an" are used to refer to both single and a. plurality of objects.

As used herein, "O. stamineus / leaf extract" refers to methanolic extract but without limitation to other organic solvent or gas extracts of O. stamineus \e&\ which contain at least one constituent that is identified and standardized in methanolic extract of O. sfam/neivs leaf. As used herein, "extract" refers to the concentrated preparation of the essential constituents of the O. stamineus leaf. Typically, an extract is prepared by drying and powderizing the plant material. Optionally, the plant, the dried plant material or the powderized plant material may be soaked in solvent(s) at room temperature and allow for the extraction of essential constituents by intermittently stirring the solution. Alternatively the plant, the dried plant material or the powderized plant material may be boiled in solvent(s). The extract may be used in liquid form, or it may be mixed with other liquid or solid medicinal herbal extracts. Alternatively, the medicinal herbal extract may be obtained by further precipitating solid; extracts from the liquid form. In further detail and/or alternatively, "extract" refers to a concentrated preparation of the essential constituents of O. stamineus leaf. Typically, an extract is prepared by drying and subsequently cutting or grinding the dried materiai. The extraction process may then be performed with the help of an appropriate choice of solvent, typically water, ethanol, ethanol/water mixture, methanol, methanol/water mixture, chloroform, dichlor ethane, butanol, iso-butanol; acetone, hexane, petroleum ether or other organic solvents by means of maceration; percolation, repercolation, counter-current extraction, turbo-extraction, or by carbon-dioxide hypercritical (temperature/pressure) extraction. The extract may then be further ^* evaporated and thus concentrated to yield a soft extract (extractum spissum) and /or eventually a dried extract, extracum siccum, by means of spray drying, vacuum oven drying, fluid-bed drying or freeze- drying.

As used herein, "rapid and simultaneous analysis of bioactive constituents in O. stamineus leaf extract" refers to those chromatographic analysis methods which are based on advanced and sophisticated technologies including HPLC- S, HPTLC, HPTLC- S, GLC and GLC- S.

As used herein, "angiogenesis" is meant the growth of a new bipod vessel in which, the proliferatio and/or migration of ari endothelial cell is a key step. By "inhibiting angiogenesis" or "anti-angiogenesis" is meant the inhibition of any of the steps of the process of angiogenesis that includes, without limitation, proliferation, multiplication and/or migration of endothelial cells, and it includes all aspects which cause down regulation or obstruction of the entire /EGF signal pathway.

As used herein, "angiogenesis related disease" refers to those diseases that are caused by the hypervasculogenesis i:e., excessive blood vessels formation.

As used herein, "cytotoxicity" refers to the cytotoxic effect against all estrogen dependent cell lines. As used herein, "antagonist of estrogen receptor" refers to any chemical substance which can exhibit the antagonistic effect against estrogen.

As used herein, administration "in combination with" one or more further antagonists of estrogen receptors or therapeutic agents includes simultaneous (concurrent) and consecutive administration; in. an .. order. For the synergistic cytotoxic activity, the composition of preparation of most potent optimum proportion of tamoxifen and O. stamineus leaf extract mixture is 6.25 g/ml and 25 pg/ml respectively. As such, other different concentrations of the tamoxifen and extract can be used together in various ratios. But it is understood that not all of these proportions may be necessary for the desired effect of cytotoxicity. It will be readily apparent that all of the above compositions in their alternate forms may be used alone or in combination to provide an anti-angiogenic or anti-tumorigenic herbal medicine, which when given to a ^' patient, results in the preventive or therapeutic effect against anti-tumor or angiogenesis-reiated aliments.

Depending on the specific clinical status of the disease, administration can be made via any accepted systemic delivery system, for example, via oral route or parenteral route such as intravenous, intramuscular, subcutaneous or percutaneous route, or vaginal, ocular or nasal route, in solid, semisolid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, , ■- suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium, emulsions or the like, preferably in unit dosage forms suitable for easy administration of fixed dosages. The pharmaceutical. compositions will include a conventional carrier or vehicle and, in addition, may include other medicinal agents, ^' . .. pharmaceutical agents, carriers,, adjuvants, and so on. In the invention, the carrier for the herbal composition may preferably include, a base which is nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.

If a vegetable soup or bouillon base is desired to be used as a base for the herbal composition, it. can be. readily seen that any vegetable soup or bouillon base can be used, so long as the anti-angidgenic and anticancer effects of the O. sfam/neus. leaf extract composition is maintained.

If it is desired that the base be made from extracts of berries or fruits, then it is understood that any berry or fruit base may be used so long as its use does not interfere with the anti-angiogenic and anticancer effectiveness of the O. stamineus leaf extract composition. If the inventive composition is desired to be placed into soya milk, it is understood that such a drink will need to be refrigerated to prevent toxic effects. It is further understood that the inventive composition may be placed, mixed, added to or combined with any other nutritional supplement so long as the anti- angiogenic and anticancer effects of the O. stamineus leaf extract composition is maintained. If desired, the pharmaceutical composition to be administered may also contain minor amounts of heavy metals and nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and so on.

The amount of the O. stamineus leaf extract in a formulation can vary within the full range employed by those skilled in the art, e.g., from about 0.01 weight percent (wt %) to about 99.99 wt % of the medicine based on the total formulation and about 0.01 wt % to 99.99 wt % excipient.

The preferred mode of administration, for the conditions mentioned above, is oral administration. using a convenient daily dosage regimen; which can be adjusted according to the degree of the complaint. For said oral administration, a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of the herbal composition in any of the currently used excipients, such as, for example, pharmaceutical grades of dextrin, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions take the form of powder, solutions, suspensions, tablets, effervescence tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain between 0,01 wt % and 99.99 wt %· of the active compound according to this invention.

In one embodiment, the compositions will have the form of a . sugar coated pi|l or tablet and thus they will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, and the' like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, polyvinylpyrrolidone, acacia gum, gelatin, cellulose and derivatives thereof , and the like.

The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as - corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may; contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present , as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active, composition may be incorporated into sustained-release preparations and formulations.

It is understood that by "pharmaceutical composition" or "herbal composition", it is meant that the herbal composition is formulated into a substance that is to be administered purposefully for treating or preventing anti-tumor and angiogenesis related diseases in an individual. However, it is understood that the O. stamineus leaf extract composition per se will not have a toxic effect.

As used herein, "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, experimental laboratory animals, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, and so on. Preferably, the mammal is human;

As used herein, "nutraceutical" is a combination of "nutritional" and "pharmaceutical" and refers to health supplement or food component that acts as medicines. Nutraceuticals or "functional foods" are a crude or refined specific food source that allows concentrated food therapy in a specific area of nutrition. These foods assist in the- prevention or treatment of disease. Nutraceuticals may further refer to natural products that are used to supplement the diet by increasing the total dietary intake of important nutrients. Typically, nutraceuticals derived from botanical preparations such as O. stamineus leaf extracts are as crude forms, powders or extracts, and may used in the form of for example beverages made with herbal products and other ingredients.

As used herein, "subject" is a vertebrate, preferably a mammal, more preferably a human.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment. . Γ ^' V; ^" ;

Orthosiphon stamineus Leai Extract

The present invention relates to treatment for various diseases that are related to angiogenesis and breast cancer. In this way, the inventive therapeutic composition may be administered to human patients who are either suffering from, or prone to suffer from the disease by providing compositions that inhibit angiogenesis and breast cancer. In particular, the disease is associated with a wide variety of metabolic disorder manifestations including rheumatoid arthritis, diabetic retinopathy, obesity, Alzheimer syndrome and cancer, cardiovascular diseases such as angioma, angiofibroma, vascular deformity, synechia and^ edemic sclerosis; and opthalmological diseases such as neovascularization after cornea implantation, neovascular glaucoma, angiogenic corneal disease, macular degeneration, pterygium, retinal, degeneration, retiOlental fibroplasias, and granular conjunctivitis and other chronic inflamrhatory diseases. In another embodiment, the present invention relates to treating various diseases that are characterized by excessive angiogenesis, which include but are not limited to, cancer, atherosclerosis, rheumatoid ^" arthritis, endometriosis, ocular disease or obesity.

The formulation of therapeutic compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 5 mg to about 1000 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose ma be proportionally reduced as indicated by the exigencies of the therapeutic situation.

It was found in the present invention that O. stamineus leaf extract of this invention inhibits angiogenesis not only in VEGF-induced HUVECs proliferation and tube formation assay, but also in GAM assay and rat aortic ring assay. It was also found in the present invention that O. stamineus leaf extract of this invention inhibits angiogenesis by inhibiting the VEGF signal protein synthesis and by suppressing the expression of VEGF receptor-2 tyrosine kinase. ,

The HUVECs proliferation and tube formation of assays are the in vitro experimental methods that are closely related to in vivo efficacy, and these assays investigate the effect on the migration and differentiation of human endothelial cell forming microvascular network. While the CAM assay is an in vivo; assay using fertilized eggs, angiogenesis can be quantitatively measured in mouse Matrigel model. Furthermore, an ex vivo rat aortic ring assay, is the excellent 3 dimensional angiogenesis model, it can be used to assessed the angiogenesis quantitatively by measuring the microvessel out growths from the rat aorta.

Further, it was found in the present invention that O. stamineus leaf extract inhibited VEGF signal, an . important protein synthesized and secreted by the neoplasm in order to attract new blood vessel supply towards its own direction. It was found in the present invention that O. stamineus significantly inhibits the production of the VEGF signal protein in cells.

Further, it was found that O. stamineus leaf extract suppressed the expression of VEGF receptor-2 tyrosine kinase in HUVECs. When the effect of O. stamineus leaf extract on VEGF receptor-2 tyrosine kinase in HUVECs is investigated, it drastically inhibits the expression of the receptor. The inhibitory effect of O. stamineus leaf extract on VEGF receptor-2 tyrosine kinase is not, however, limited to this signal receptor. . ^'

It is therefore clear that the composition comprising O. stamineus leaf extract of the current invention is used as an anti-angiogenic and anti-tumorigenic agent for the treatment or prevention of angiogenesis- dependent diseases and. breast cancer respectively, for pharmaceutical, preventive or chemotherapeutical, nutraceutical, or health supplement, dietetic use.

It is therefore also clear that the composition comprising O. stamineus leaf extract of the present invention _, is used as a human VEGF signal pathway-inhibitory agent, for the treatment or prevention of VEGF signal or VEGF receptor-related diseases, for pharmaceutical, dietetic or cosmetic use.

O. stamineus leaf used in the present invention can be purchased or cultivated in the farm. Commercially available O. stamineus leaf or leaf powder may also be used.

O. stamineus leaf extract ^•■ used in the present invention can be prepared with conventional methods. An example of a conventional extraction method is as follows. In brief, 10 to 20 L of aqueous water or alcohol (for example, methanol, ethanol, butanol, etc.). or the. ^" various proportional ratios of these solvents, or acetone, or any other organic solvent is added to 1 kg of dried O. stamineus leaves. The mixture is allowed to extract at a temperature ranging from 60 to 80° C, for a period ranging from 30 min to 24 hours. Alternatively, cold extraction procedure can also be followed at room temperature. The extraction process may _: be repeated 2 to 3 times with other solvents (chloroform, ethyl acetate, dichloro ethane, ketone, etc.). The resulting extract is concentrated to obtain O. stamineus leaf extract.

In the present invention, an isocratic method was used for the simultaneous analysis, of the authentic markers, which were separated within a total time of 30 min (Fig. 1). All the standards were determined in a single run of HPLC. The standards were resolved and eluted at 5.54, 6.48, 9.98, 12.36 and 16.13 min (Fig. 1A), with respect to rosmarinic acid (RA), orthosiphol A (OrA), 3'-hydroxy-5,6,7,4'- tetramethoxyflavone (TMF),. sinensetin (SEN), and methylripariochromene (MRC) (Fig. 1 B). The markers , demonstrated _, a good linearity in the range from 2.0 to 1000 ng in the calibration curves that were obtained by HPLC analysis. All the reference markers were present in the chromatographic rofiies .of the: samples from various locations when the sample solution was analyzed by HPLC. The peaks of RA, OrA, TMF, SEN and MRC were confirmed by comparison of the retention times with the reference standards: To assess the precision of these methods, standard solutions of RA, OrA, TMF, SEN and MRC were determined six times on the same day and over a six-day period. The results showed very good precision, ranging from 5 to 100 ^g/ml. The accuracy of the method was evaluated through recovery studies. The recovery experiment was performed at three concentrations of the standard added to sample solutions, in which the marker content had been determined, using, sample from Bohpr Ternak. The results for the recoveries of RA, OrA, TMF, SEN and MRC were in the range 96-103%.

As mentioned above, O. stamineus leaf extract of the present invention has in ibitory effects on angiogenesis and VEGF signal pathway. While VEGF signal pathway is responsible for angiogenesis, anti-angiogenic activity of O. stamineus leaf extract is not limited to VEGF signal pathway blocking activity. That is, though VEGF signal pathway is one of the principle factors for inducing angiogenesis, O. stamineus leaf extract can inhibit other factors of angiogenesis. Furthermore, the inhibitory activity of O. , stamineus leaf extract on VEGF signal pathway is not limited to inhibition of angiogenesis.

Furthermore, O. stamineus \eai extract of the present invention has potent synergistic effect on the cytotoxicity of tamoxifen against human breast cancer cell line MCF-7. While MCF-7 -cell. -line- is human: breast cancer ceil, anti-tumorigenic activity of O. stamineus leaf extract is not limited to . MCF-7 cell line only. That is, though MCF-7 is one of the typical estrogen dependent cell lines from human breast cancer, O. stamineus leaf extract can be a cytotoxic to other estrogen dependent tumor cell lines as well.. Furthermore, the inhibitory activity of O. stamineus leaf extract on proliferation of estrogen dependent cell lines is not limited to inhibition of tumor. As explained above, O. stamineus leaf extract of the present invention has potent synergistic effect on the cytotoxicity of tamoxifen against human breast cancer cell line MCF-7. The synergistic activity of O. stamineus leaf extract in enhancing the cytotoxicity of tamoxifen is not limited to tamoxifen only. That is, though tamoxifen is one of the antagonists of estrogen receptors and used in the treatment of human breast cancer, O. stamineus leaf extract can also be used synergistically with other antagonists of estrogen receptors. Furthermore, the synergistic activity of O. stamineus leaf' extract with antagonist of estrogen receptors is not limited to. inhibition of breast tumor. . In this present invention, O. stamineus leaf extract in combination with tamoxifen showed significant cytotoxic activity against human breast tumor cell line MCF-7. While MCF-7 cell line is human breast cancer cell, anti-tumorigenic activity of O. stamineus leaf extract is not limited to MCF-7 cell line only. That is, though MCF-7 is one of the typical estrogen dependent cell lines from human breast cancer, O. stamineus leaf extract can be a cytotoxic to other estrogen dependent tumor cell lines as well. Furthermore, the inhibitory activity of O. stamineus leaf extract on proliferation of estrogen dependent cell lines is not limited to inhibition of tumor.

There are many antagonists of estrogen receptors available commercially, and these commercially available drugs can be co-treated with O. stamineus leaf extract of present invention to synergistic effect of the composition. Furthermore, the co-treatment of O. stamineus leaf extract is not limited to only with antagonists of estrogen receptors, but as such the extract can also be treated with any cytotoxic or chemotherapeutic agents.

Specifically, combined treatment of O. stamineus leaf extract with cytotoxic or chemotherapeutic agents can be used as tumor inhibitors. Further, these compositions can be added to drugs, quasi-drugs, .foods or beverages used for anti-angiogenic and anti-tumorigenic purpose.

The anti-angiogenic activity of O. stamineus leaf extract is observed in vivo chicken egg chorillantoic , membrane (CAM) assay. Angiogenesis was significantly inhibited by the O. stamineus leaf extract. ^' Images of two chorioallantoic membranes are shown in Fig. 2. The vasculature pattern formed by the blood vessels in CAM of control group treated with vehicle was normal. The primary, secondary and tertiary vessels with the dendritic branching pattern, which is characteristic of CAMs was well established in control CAMs and can be seen clearly (Fig. 2A). Whereas, the extract (100 g/disc) treated CAM showed the distorted architecture in vasculature and significant angiogenesis inhibition in treated CAMs (Fig. 2B).

The anti-angiogenic activity of O. stamineus leaf extract is observed ex vivo rat aortic ring assay, which has been shown to correlate well with in vivo events _. of- neovascularization. In this assay, the rat aortic endothelium exposed to a three-dimensional matrix containing angiogenic factors switches to a microvascular phenotype generating branching networks of microvessels (Nicosia ef a/, -1992). As shown in Fig. 3, microvessels grew out from the rat aorta in the control (Fig. 3A and B) when cultured in the medium. The anti-angiogenic activity was quantified by measuring the cell-sprouting area around the aortas exposed to the various concentrations of O. stamineus leaf extract and the control: The anti- angiogenesis effect of extract on the sprouting of microvessels from rat aorta was significantly dose dependent (P<0.05), and microvessel growth was almost completely inhibited in the presence of 100 μg/ml extract (Fig- 3C). Quantitative image analysis confirmed the dose-dependent activity of extract against rat microvessel outgrowth. Fig. 4 depicts the dose dependent response of microvessel -inhibition towards the respective concentrations of extract.

To characterize the antNangiogenesis activity of O. stamineus leaf extract, we first determined whether the extract inhibited VEGF induced endothelial cell proliferation. Fig. 5 depicts a. dose-dependant- inhibition of endothelial cell proliferation after 48 hr. The extract showed significant inhibition ^' with IC _b0 (concentration of test substance to achieve 50% inhibition) 21.1:6 g/ml. The standard reference yincristin, exhibited very potent cytotoxicity with IC ₅₀ 0.015 g/ml. While HUVEC cell proliferation was significantly increased in response to VEGF treatment in the absence of extract, it was markedly suppressed in the presence of extract.

To characterize the inhibitory effect of the extract on endothelial cell migration process, in vitro wound healing assay was conducted. This assay represents an important step in the formation of new blood vessels, and is a straightforward and economical method to study the cell migration. A scratch wound" was created on the monolayer of cells. In order to establish new cell-cell contacts again, cells from the edge of the newly created gap tend to move toward the opening of the scratch. The extract (50 pg/ml) inhibited HUVECs migration drastically (P< 0.001 ) by 97.33 % after 12 hr. Even at lower concentration of the extract (25 μg/m ) a significant (P < 0.05) inhibitory effect was observed with 76.8 % inhibition after 12. Fig. 6 depicts the migratory inhibition properties of the extract on HUVE cell line.

To further characterize its anti-angiogenesis activity exhibited by O. stamineus leaf extract in this invention, we examined the effects of extract on VEGF induced tube formation by HUVEG on Matrigel. HUVEC cells formed tube like networks within 8 h, which might, in part, reflect the process of angiogenesis. At a concentration of 25 pg/ml, the extract absolutely abrogated endothelial tube formation, reducing the tube-like structure both in width and in length. Endothelial cells rounded up and rendered network structures incomplete and broken in the presence of. extract (Fig. 7). The IC ₅₀ value of the, extract . (13.24 μς/ml) was found to be lesser than the standard, suramin (IC ₅₀ 19.33 g/ml).

Given the observed inhibition of VEGF-induced endothelial cell proliferation and tube formation by the O. stamineus leaf extract, we confirmed inhibitory effect of extracts on VEGF by quantifying VEGF 165 levels in endothelial cell lysates. 50 % ethanolic extract caused profound inhibition of VEGF production from endothelial cells. ELISA measurements indicated that control cells showed higher levels of VEGF 165 (378±5 pg/ml per 100 mg tissue) in cell lysates than cells treated with extract. The highest extract concentration nearly halved VEGF production (21 +0:26 pg/ml per 100 mg tissue), whereas the lowest one reduced VEGF levels to 310.82 pg/ml per 100 mg tissue, as shown in Fig. 8.

In order to identify the downstream signaling pathway targeted by O. stamineus leaf treatment, we examined the expression, of VEGF-R2 kinase, one of the key signaling pathway components supporting _^ endothelial cell proliferation (Takahashi and Shibuya, 1999). Detection by western blotting revealed that the extract completely suppressed the expression of. VEGF receptor on HUVC cell lines at concentration 100 g/ml. Fig. 9 illustrates the western blot image of dose dependent suppression of VEGF receptor-2 tyrosine kinase expression on HUVECs by O. stamineus leaf extract.

In addition to angiogenesis study of O. stamineus leaf extract, in the present invention, the chemotherapeutic efficacy of O. stamineus leaf extract on human breast and colon cancer has. been assessed. O. stamineus leaf exerts, an enhanced anti-cancer effect on breast cancer cells in vitro when. it was co-administered with tamoxifen. As Fig. 10 depicts, tamoxifen at concentration 6.25 , pg/ml and O, stamineus leaf extract at concentration 25 pg/ml, when used alone, induced only 9.34 and 36.86 % of cell. growth inhibitory effect, respectively. While combined use of same dose of tamoxifen with O. stamineus^ leaf extract resulted in an inhibitory effect of 97.55 %, much significant (P<0.05) when compared to t e calculated additive inhibitory effect of 42.4 %. Thus O. stamineus leaf extract demonstrated excellent;. synergistic activity in enhancing the cytotoxic effect of tamoxifen against human breast cancer cell line ' MCF-7.

With this in vitro study, the anti-tumor activity of O. stamineus leaf extract in vivo using athymic immunocompromised nude mice of BALB/c strain has been assessed. To evaluate the in vivo anti-tumor effect of standardized extract of O. stamineus, xenografts of HCT1 16 cells were implanted into nude mice. Two doses, 100 mg extract/kg and 200 mg extract/kg of body weight, were tested for two separate groups. Control animals bearing HCTT16 tumors received placebo (distilled water). The extract was administered 2 days once for four weeks via oral gavage. Figure 11 shows dose related suppression of human colorectal tumor xenograft in mice. Both doses of the extract exhibited a marked suppression in HCT116 tumor growth relative to vehicle-treated control. The morphology of excised tumors (Fig. 1 ) revealed that the density of blood vessels was very less , in tumors from treated animals than compare to tumors from control group. Figures 13 - 16 show the tumor growth profile in the respective test groups. On the other hand, the extract at a dose of 200 mg/kg on 28 ^th post cell inoculation day, demonstrated extensively drastic tumor growth suppression (-0.45%, p<0.0.1 ). The quantitative analysis of human VEGF 165 in colon tumor tissue hqmogenates revealed that the extract significantly inhibited the production of endothelial mitogen signal by the tumor tissues. At a dose of 100 mg/kg body weight, the extract showed 112.9±4 pg of VEGF per 100 mg of tumor tissue while the higher dose (200 mg/kg) showed 90.9±2 pg/100 g. In control group, the level of VEGF signal was i35.5±4 pg/100 g of tissue lysates. Fig. 17 demonstrates the dose dependent inhibition of VEGF protein production in tumor homogenates by the extract.

Further the extract also demonstrated remarkable prophylactic efficacies against hormone (estrogen) dependent human breast cancer xenografts in nude mice. Figs. 18 and 19 shows the inhibitory activity of O. stamineus leaf extract towards the onset of hormone dependent breast tumor cell lines (MCF 7) implanted subcutaneously in nude mice. A composition comprising O. stamineus leaf extract can also comprise more than one kind of diluent including dextrose, maltodextrin, saline, buffered sajine, water, glycerol, and ethanol, but the diluent is. not limited.

Formulations containing O. stamineus leaf extract may be prepared in any form. The formulation can be prepared as injectable preparation (true solution, suspension, or emulsion) and preferably in oral dosage form (tablet, effervescence tablet, capsule, soft capsule, aqueous medicine, pill, granule) and topical preparation (gel, ointment, patch, spray, solution, and the like).

The composition comprising O. stamineus leaf extract of the present invention can be administered, by. various routes. The route of administration includes oral, intravenous, intraperitoneal, .subcutaneous;. intramuscular, intra-arterial, transdermal, rectal, nasal, ocular, and topical application.

The composition comprising O. stamineus leaf extract of the present invention may be applied differently according to the diseases and route of administration. It should be understood that the amount of active ingredient has to be determined with various factors. These factors include the severity of the patient's symptoms, other co-admiriistered drugs (e.g., chemotherapeutic agents), age, sex, body weight of the _: individual patient, food, dosing time, the chosen route of administration, and the ratio of the composition.

A daily dose of O. stamineus leaf extract is preferable from about 5 mg to 3 g, most preferably 10 to 1000 , mg. In general, 0.1 to 200 mg/kg of. O. stamineus leaf extract can be administrated in a single dose at a time for 2-3 times per day. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, , ^" various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are . intended to fall within the scope of the appended claims.; The.following examples are offered by way of illustration of the present invention, and not by way of limitation. EXAMPLES Example 1

O. stamineus leaf extract was purchased commercially and used in the following examples.

TEST 1— Anti-angiogenic effect of O. stamineus using Chorioallantoic Membrane Assays (CAM Assay)

Fertilized chicken eggs were kept in a humidified incubator at 37° C. After incubation for three days, 2-3 , ml of albumin was aspirated from the eggs with a syringe of 26-gauge needle and the egg was sealed with transparent tape. A window of a small hole was drilled at the end of the eggs. Two days later, an aliquot of 50 pg of O. stamineus lea extract dissolved in 15 μΙ of 0.1 % tween 80 was applied, to sterile Whatman filter paper No.1 discs and allowed to air dry. The discs were applied to the., chorioallantoic membrane surface through the window and covered with transparent adhesive tape. The embryos were incubated for further three days at 37° C. in a humidified incubator. An appropriate volume of lipid emulsion was injected into, the embryo chorioallantois using a 26-gauge needle so that the vascular network of the chorioallantoic membrane stood out against the white lipid background. As a control, 15 μΙ of 0.1 % tween 80 was loaded to a disc instead of O. stamineus leaf extract following the same procedure as mentioned above. The resulting blood vessels were observed and compared with treated-eggs.

In the control group (n=10), capillary vessel formation was not affected in 90% of the embryo (Fig. 2A), while the inhibition of vessel formation in the disc (brighter part of the picture) treated with O. stamineus leaf extract was significant and the inhibition of the blood vessel formation of the chorioallantois was observed in all the treated eggs (n=10, 100%, Fig. 2B).

TEST 2— Anti-angiogenesis Effect of O. stamineus- Leaf Extract using rat aortic ring assay.

The effect of O. stamineus leaf extract on angiogenesis was investigated ex vivo with rat aorta. Rat aortic ring explant cultures were prepared by modification of protocols previously described. Aortic rings were prepared from male Sprague Dawley rats. Aortas were sectioned into 1 mm-long cross sections, rinsed several times with Hanks Balanced Salt Solution containing 2^g/ml amphotericin B (Sigma-Aldrich, USA). The assay was performed in a 48-well tissue culture plate (Coster Corning, USA). 500μΙ of 3mg/ml fibrinogen (Calbiochem, USA) in serum free M199 growth medium (Gibco, UK) was added to each well. with 5mg/ml of aprotinin (Sigma-Aldrich, USA) to prevent fibrinolysis of the vessel fragments. Each tissue section was placed in the center of the well and 15μΙ of thrombin (50NIH U/ml) (Sigma-Aldrich, USA) in 0.15M NaCI. Immediately after embedding the vessel fragment in the fibrin gels, 0.5ml of medium M 199 supplemented with 20% HIFCS (Gibco, UK), 0.1 % έ^ιτιίηορΒρΓθίο acid (Sigma-Aldrich, USA), 1 % L- Glutamine (Sigma-Aldrich, USA), 1 % amphotericin (Sigma-Aldrich, USA), 0.6% gentamicin (Sigma- Aldrich, USA) were added to each well. O. stamineus leaf extract at varying concentrations ranged from 6.25 g/ml to 100 pg/ml was added to the complete growth medium. Control cultures received medium without the test substances. Suramin (Sigma-Aldrich, USA), a well recognized anti-angiogehic agent was used as a positive control. Cultures were incubated at 37 ^B C for 5 days, in humidified C0 ₂ and the medium was replaced daily. The magnitude of blood vessel outgrowth on day five of the' procedure was quantified using inverted microscope (Olympus, Japan) supplied with a digital, camera (Lieca CCD, Japan) and Leica QWin computerized imaging software.

Fig. 3 shows the inhibitory effect of O. stamineus leaf extract on the vascularization of rat aortic rings. Representative photographs of the inhibitory effect of methanolic extract (100 g/ml) on ex vivo angiogenesis using a rat aortic ring. Images of rat aortic rings (A and B) Control; (C) Methanolic extract and (D) Suramin.

Fig.4 shows the dose dependent.inhibition of rat aortic microvessels by the extract of O. stamineus leaf. Table 1. Effect of O. stamineus leaf extract on rat aortic microvessel out growth.

Out growth of rat aortic microvessel is shown in mean length (μητι) of 8 replicates ± standard deviation.

TEST 3— Effect of O. stamineus Leal Extract on VEGF-induced HUVECs Proliferation. HUVECs maintained in endothelial cell basal medium containing 10% HIFCS and 1 % penicillin/streptomycin, 20% fetal bovine serum supplemented with 40 ng/ml VFGF. The cells were seeded in 96-well plates at the density of 2x10 ⁴ cells/well in 100 μΙ growth media and allowed to attach for overnight. Cells were exposed to O. stamineus leaf extract for 48 h. After the period of incubation, the viability of HUVECs was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolim bromide) assay. 20μΙ of MTT solution (5 mg/ml in PBS) was added to each well. After incubation for 4 h, the mixed media and MTT solution were carefully discarded, and then the crystallized dye was solubilized with DMSO. Vincristin was used as reference standard. The amount of blue dye formed was determined by. measuring the absorbance at 570 nm.

Fig. 5 shows the graph of anti-proliferative effect of O. stamineus leaf extract on the VEGF-induced HUVECs. The extract inhibited ^' HUVECs proliferation in dose dependent manner with IC50 21.16 μg/mΊ.

Table 2. Effect of O. stamineus leaf extract on VEGF-induced HUVECs Proliferation.

Inhibition of HVECs proliferation by test samples is shown in mean percentage of 8 replicates ± standard deviation.

TEST 4— Effect of O. stamineus Leaf Extract on VEGF-induced endothelial cell Migration..

The assay was carried out according to Chun-Ghi et al. (2007) with minor modifications. Briefly, HUVECs were plated in 6 well plates, after formation of a confluent monolayer a wound was created using 200 μΙ micropipette tip. The free cells were removed by washing twice with PBS. Two different concentrations of 50% ethanolic extract were added to the separate wells containing cells within a fresh media containing 10 % calf serum. After 12 to 18 hr, the wounds were photographed and distances between one side of scratch and the other were measured using inverted light microscope supplied with Leica Quin computerized imaging system. 10 fields for each concentration were captured and minimum 0 readings : of distance for each field were measured.

This assay represents an important step in the formation of new blood vessels, and is a straightforward and economical method to study the cell migration. A scratch wound was created on the monolayer of cells. In order to establish new celh-cell contacts again, cells from the edge of the newly created gap tend to move toward the opening of the scratch. The extract (50 g ml) inhibited HUVECs migration drastically (P< 0.001 ) by 97.33 % after 12 hr. Even at lower concentration of the extract (25 Mg/mlj a significant (P < . 0.05) inhibitory effect was observed with 76.8 % inhibition after 12. Fig. 6 depicts the migratory inhibition properties of the extract on HUVE cell line. TEST 5— Effect of O. stamineus Leaf Extract on VEGF-induced HU VECs Tube Formation.

HUVECs (ATCC, Rockville, MD, USA) were harvested and seeded in EGM-2 medium (Science cell. USA) (2%FBS) containing VEGF (100ng/ml) onto 4-well culture plates coated with .150 μΙ Matrigel (10 mg/ml was diluted at 1 :2 v/v with ice cold serum free ECM) (BD Bioscience). The cells were treated with various concentrations of O. stamineus leaf extract and incubated at 37 ^S C for 24 h: Suramin (Sigma, Aldrich, USA) was used as a positive control (1 mg/ml) which was dissolved in DMSO and diluted to 10 pg/ml in the growth medium. After treatment, the media were discarded, and the cells were washed twice with Hank's Balanced Salt Solution and stained with Galcein (8 μg/mL) for 45 minutes at 37°C, under 5% C0 ₂ . The dye was discarded, and cells were washed twice to remove excess dye. The cells were imaged under an inverted florescence microscope at low magnification. The web junctions, defined as intersections of three or more tubes, were counted in each microscopic field. The percentage of inhibition was represented as the mean ±SD.

Fig. 7 shows the images depicting the inhibitory effect of O. stamineus leaf extract on the VEGF induced HUVECs tube formation. Fig. 7 shows that a tubular network is formed as a process of neovascularization, when they are grown on Matrigel. However, the microvascular network of HUVEC on Matrigel was disconnected by O. stamineus leaf extract: HUVECs (2 x 10 ⁴ cells/well) were plated on Matrigel precoated 96-well plates and treated with different-concentrations of for 24 h. (A & E) Control; (B) Methanolic extract (25 Mg/ml) (C) Methanolic fraction of methanolic extract (50 Mg/ml); (D) Suramin (10 g/ml); (F) 50 % ethanolic extract (25 Mg/ml); (G) 50 % ethanolic extract (50 μg/<m ) and (H) Betulinic acid (10 Mg/ml).

The area of the tube was determined by the image analysis program Image-Pro Plus® (Media Cybernetics, USA), and is summarized in Table 3. Tube formation after treatment of O. stamineus leaf extract at concentration 25 M ml was inhibited by about 98.6 % as compared with the untreated control. Table 3. Effect of O. stamineus leaf extract on VEGHnduced HUVECs Tube Formation.

; Inhibition of tube formation by test samples is shown in mean percentage of , 8 replicates ± standard . ^■ . ^' : deviation. ^'■ _. ■ i- ^' -

5 TEST 6— Quantification of VEGF protein signal by ELISA

The action of mechanism of anti-angiogenic activity of O. stamineus leaf extract was quantitatively investigated by measuring the production of VEGF protein in treated and untreated HUVECs.

For the quantitative analysis of VEGF protein, a major angiogenic signal, VEGF-165 protein from HUVE cell lysate were measured using commercial human VEGF ELISA kit (IBL, Japan) following ' the Ϊ0 instructions of the manufacturer.. HUVECs were seeded in 6-well plates at Ί ίΟ ⁶ in 3 ml ECM and : incubated for cell attachment for 24 h. Cells were treated with indicated concentrations for 6 hrs, and concentration of VEGF-165 was determined of each cell lysate. Calibration curve of VEGF standard was, used to calculate concentration of VEGF of the samples. The results are resented as average ±SD (n =3). . Y As shown in Table 4 below, control cells showed higher levels of VEGF 165 (34±3 8 pg/ml) in cell lysates, 15 on the other hand, O. stamineus leaf extract at concentration 25 μg/m I showed 17±1.2 pg/ml VEGF . protein level in HUVECs lysates.

Fig. 8 shows the inhibitory effect of O. stamineus■•leaf extract on the VEGF _^ 165 protein. production n HUVECs. Table 4. Effect of O. stamineus leaf extract on VEGF-induced HUVECs Tube Formation.

Concentration of VEGF protein is shown in mean value of 8 replicates ± standard deviation.

TEST 7 Immunoblot analysis of VEGF receptor-2 tyrosine kinase in HUVECs

In order to investigate the effect of O. stamineus leaf extract on the expression of VEGF receptor, HUVECs were harvested and seeded in ECM-2 medium. Cells were grown in cell culture flasks, and incubated at 37 ^e C, 5% C0 ₂ . After the 48 hrs incubation of cells with various concentration of extract,. cells were harvested and lysed using ice-cold cell extraction buffer (Biousource);. (10 mM Tris, pH 7.4, 100 mM NaCI, 1 mM EDTA, 1 mM NaF, 20 mM Na ₃ V0 ₄ , 1 %Trito X-100, 10% glycerol, 0.1 %SDS, 0.5% deoxycholate, 1 % protease inhibitor cocktail set 1 (Upstate), 0.1% FMSF). Protein concentrations were calculated based on Bio-Rad DC assay kit (Bio-Rad Laboratories). Sodium, dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE, 8%) was used to separate protein samples. Resolved proteins were transferred into Hybond™-ECL™ nitrocellulose membrane (Amersham Biosciences, UK). Membrane was blocked in freshly prepared 5% BSA for 1 h at room temperature. Following blocking, membrane was probed overnight with anti-VEGFR2 (diluted as 1 :1000 in freshly prepared 5% BSA). with agitation at 4 °C. Followibg the first probe, blotted membrane was washed twice and .probed ..with goat anti-rabbit peroxidase conjugated IgG secondary antibody (diluted as 1 :2000 in 5% TBS-Milk) for 1 and. half an hour at room temperature. Immunobloted membrane was subjected to _: ECL detection reagent, and bands were detected with LAS-3000 luminescent image analyzer (Fujifilm). Fig. 9 shows the Immunoblot depicting the complete suppression of VEGF receptor-2 tyrosine kinase on HUVECs by O. stamineus leaf extract at concentration 100 μg/ml. . . \

Example 2 Test 8 Synergistic activity of O. stamineus leaf extract with tamoxifen on MCF-7 breast cancer cell

In order to assess the cytotoxicity of O. stamineus leaf extract, the effect of combined treatment of tamoxifen and of O. stamineus leaf extract has been studied, this composition exerts an enhanced anticancer effect on breast cancer cells. Cell viability was examined by MTT assay on human positive estrogenic receptors MCF 7 breast cancer cell line. Cells were treated with O. stamineus leaf extract and tamoxifen. Fig. 10. shows the synergistic cytotoxicity of O. stamineus leaf extract with tamoxifen. The IG ₅₀ value for O. stamineus leaf extract and tamoxifen is 63.14pg/ml and 13.46pg/ml, respectively. As 6.25 g/ml concentration of tamoxifen,. 25 g/ml concentration of O. stamineus leaf extract, when, used : alone, induced only 9.34 % and 22.2 %, of cytotoxic effect, respectively. While combined use of same dose of tamoxifen with O. stamineus leaf extract resulted in an inhibitory effect of 97.55 % as compared to the calculated additive inhibitory effect of 42.4%.

Fig. 10 shows the synergistic toxicity of O. stamineus leaf extract with tamoxifen on MCF-7 human breast tumor cell line.

Example 3 Test 9 Anti-tumor activity of O. stamineus leaf extract in nude mice

In order to test the anti-tumor activity of O. stamineus leaf extract against breast and colon cancer in nude mice, the following experiments were undertaken. I. In vivo Human Xenograft Models in Athymic Mice

Anti-tumor activity of the extract against colorectal cancer HCT 1 16 cejls. cultured in RPMI media, harvested and were injected subcutaneoUsly into the right flank of BALB/c-nu mice (4-6 weeks bid, 20-22 g). 18 mice received inoculums (200 μΙ) of 106 HCT1 16 cells per inoculum according to the British system, and then randomly divided into three groups of six mice each. hen the average tumor size reached approximately 100 mm3, in about six days post cejl inoculation (pi), a group of mice received 0.1 - ml distilled water (control) another group received treatment with 100 mg/kg body weight extract, and _; ^, a., third group received treatment with 200 mg/kg body weight extract. All administrations were done by oral gavage. Tumor volume (mm3) was determined with the aid of a calliper applying the equation- tumor: volume (mm3) =1/2 χ L χ W2 (where L is the length and W is the width of the tumor) (Hatziantonipu et al., 2006; Dimas et al., 2007). The size of tumors was recorded every 7 days. Apart from tumor volume, the . following parameters were also calculated: %ΔΤ/ΔΟ, where, ΔΤ=Τ-Δ0 and ΔΟ=Ο-Δ0 (Δ0 is the average tumor volume at the beginning of the treatment, T and C are the tumor volumes at a specified day for treated and control groups, respectively. The optimal % ΔΤ/ΔΟ value was used as a measure of drug activity. In general, the ΔΤ/ΔΟ value in percent is used as an indication of antitumor effectiveness, and a value of ΔΤ/ΔΟ≤42% is considered as showing significant antitumor activity by the Division of Cancer; Treatment, NCI, NIH (Corbett et al., 1997). Losses of weight, neurological disorders, behavioural and dietary changes, were also recorded as markers of side-effects. After 28 days, finally, the _. mice were, sacrificed, and the tumors were removed and weighted.

II. Analysis of VEGF quantity in colon tumor tissue homogenates

After euthanizing the animals, tumor tissues were dissected out from the animals of all groups. For tissue samples, 100 mg of tumor _. tissues were homogenized in 1 ml of lysis buffer. Nude mice (n=6 mice) were killed on 28th day of post inoculation/ Tumor tissues were dissected, weighed. 100 mg of tumor tissues were homogenized in 1 ml lysis buffer (Ray Bio®, USA). Homogenates were centrifuged for TO minutes at 4°C (1 0,000g), and supernatants were assayed using a VEGF-165 enzyme-linked immunosorbent assay; (ELISA). - .

III. Prophylactic activity of the extract against hormone dependent breast cancer

^'■ ■ ^' .5 18 female BALB/c athymic mice 4 to 6 weeks of age were selected for the study. All the animals were anesthetized. using sodium pentobarbitone (30 mg/kg body weight), and a pellet of 17 -estradiol (17β-Ε2) was implanted subcutaneously at the dorsal surface of the scapular region. These pellets are designed . to. produce 80-100 pg/ml of serum estradiol (as indicated by the supplier). After one week of the pellet implantation, treatment was started with two doses of extract (100 and 200 mg/kg body weight '

10 intraperitonealy, i.p.). After one week of pretreatment, each mouse received inoculums (200 μΙ) of 1 x107 MCF7 cells grown in DMEM media; and then randomly divided into three groups of six mice each: Control group of mice received 0.1 ml distilled water intraperitonealy. Tumor volume (mm3) was determined with the aid of a calliper applying the equation- tumor volume (mm3) F1/2 ^χ L ^χ W2 (where L is the length and W is the width of the tumor) (Hatziantoniou et al., 2006; Dimas et al., 2007). The size of tumors was

15 recorded every 7 days. The tumor volume and the -percentage of inhibition of tumor uptake were calculated as described above.

Handling and experimentation of animals were consistent with guidelines of the USM. Committee for Animal Care and ..received approval from the USM Animal Ethical Committee for the present work. (Reference Number USM/PPSF/50 (084) Jld.2).

20 The O. stamineus leaf extract at 200 mg/kg body weight, exhibited significant anti-tumor activity by reducing drastically the size of human colorectal tumor. Fig. 11 illustrates the antitumor activity of 0.. stamineus leaf extract against human colon cancer implanted in nude mouse. Fig. 12 shows the changes in morphological characteristics of colorectal tumor tissues harvested from treated and untreated animals. Figs 13 - 16 are the line graphs shows the growth pattern of human colorectal tumor cells in nude mice. Inhibition of VEGF production in colon tumor tissue lysates

. Having examined the effect of 50% ethanolic extract on VEGF-165 production by the endothelial cells in vitro, we , set out to determine this growth factor qualitatively in tumor tissues harvested from the treated and control · animals. For this, . we performed EUSA based, VEGF-165 enzyme-linked immunosorbent assay. With respect to the ^' in vitro result, effect of the extract was consistent even in the tumor tissues.

Result showed a significant VEGF production inhibition in tumor tissues by the extract when compared to the control (135.5±4 pg/100 g of tissue lysate). The extract showed dose dependent reduction of VEGF levels in treated tumor tissues (Fig. 17). At 100 mg/kg dose VEGF level was 112.9+4 pg/100 g of tissue lysate, where as at 200 mg/kg VEGF level was 90.9±2 pg/100 g of tissue lysate.

Standardized extract of O. stamineus inhibits the onset of estrogen dependent breast cancer in mice.

In order to assess tumor uptake ratio, xenografts of CF 7 cells were inoculated into 17 -estradiol implanted nude mice. The animals were pretreated with two doses, 100 mg extract/kg and 200 mg extract/kg body weight. Control animals received 0.1 ml distilled water. Treatment started 7 days before tumor cell inoculation. After cell . . inoculation, the treatment was continued for another 2 weeks. Figure 18 A shows manifestation of breast tumor in control animal while, Figure.18 B depicts the suppression of tumor uptake in treated animal. Figure.19 shows the. pattern of tumor uptake in the respective tested groups. Pretreatment with extract (100 mg/kg) showed 78÷99 % inhibition in tumor uptake, whereas, there was complete inhibition of tumor uptake in animals pretreated with 200 mg/kg body weight of extract. Figs. 18 and 19 show the anti-tumorigenic effect of O. stamineus leaf extract in nude mice against human colon tumor. Table 7. Anti-tumorigenic effect of O. stamineus leaf extract in nude mice against human breast cancer.

Tumor volume is shown in mean (n=6) ± standard deviation.