WO2020250189A1 - Extracts of rhazya stricta in the treatment of cancer and metastasis - Google Patents

Abstract

A method of inhibiting metastasis of metastatic cancer cells. The method comprises administering to a subject suffering from cancer a therapeutically effective amount of a Rhazya stricta extract wherein, the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, a metastatic liver cancer, and combinations thereof

Description

Extracts of Rhazya stricta in the Treatment of Cancer and Metastasis

Technical Field

[0001] The present invention relates to therapeutic formulations and methods for inhibiting metastasis of metastatic cancer cells, the formulations including Rhazya stricta extract and optionally a chemotherapeutic agent.

Background

[0002] Free radicals are mainly produced by oxidation processes and they have an important role in the processes of food spoilage and chemical materials degradation. They also contribute to human disorders such as aging-associated diseases, cardiovascular diseases, cancer and inflammatory disease. Free radicals may also cause a depletion of the immune system antioxidants, a change in the gene expression and may induce the synthesis of abnormal proteins. About 5% or more of the inhaled oxygen (O₂) is converted to reactive oxygen species (ROS) such as O₂ , H₂O₂, and OH radicals. ROS represents the major type of free radicals in any biological system. They are produced through the mitochondrial electrons transport chain.

[0003] Antioxidants are used to neutralize the effects of free radicals. Thus, they protect humans against infection and degenerative diseases. Antioxidants can be classified into two major categories, natural and synthetic. Synthetic antioxidants include butylated hydroxy anisole (BHA), butylated hydroxyl toluenes (BHT), tertiary butylated hydroquinone and gallic acid esters. These antioxidants effectively inhibit oxidation, may serve as chelating agents such as ethylene diamine tetra acetic acid (EDTA), and can bind metals reducing their contribution to the process. However, antioxidants are thought to cause or promote negative health effects such as mutagenesis and carcinogenesis in humans. Therefore, there is a strong trend to replace the synthetic with naturally occurring antioxidants that can prevent free radical -related diseases. [0004] Natural antioxidants help in controlling the formation of free radicals and activated oxygen species or they can inhibit their reaction with biological structures. These antioxidants include antioxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, and small nonenzymatic antioxidant molecules, such as glutathione and vitamins C and E. Many herbs and spices (rosemary, thyme, oregano, sage, basil, pepper, clove, cinnamon, nutmeg, and saffron), and plant extracts (tea, grapeseed, and lemon balm) contain antioxidant components.

Summary of the Embodiments

[0005] In a first aspect, provided herein is a method of inhibiting metastasis of metastatic cancer cells. The method comprises administering to a subject suffering from cancer a therapeutically effective amount of a Rhazya stricta extract. In an embodiment, the extract is of Rhazya stricta leaves. In a further embodiment, the extract is made by extracting Rhazya stricta with a solution comprising water and ethanol. In a non-limiting embodiment, the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, a metastatic liver cancer, and combinations thereof. The therapeutically effective amount of Rhazya stricta extract may be administered orally.

[0006] In a representative embodiment, the amount of the extract administered to the subject is from about 10 mg/day to about 1000 mg/day per kg body weight of the subject. In an example embodiment, the amount of the extract administered to the subject is from about 200 mg/day to about 750 mg/day per kg body weight of the subject. In a further example embodiment, the amount of the extract is from about 250 mg/day to about 500 mg/day per kg body weight of the subject.

[0007] In another embodiment, the method further comprises administering to the subject a second therapeutic agent selected from the group consisting of carboplatin; cisplatin; methotrexate; fluorouracil; gemcitabine; goserelin; leuprolide; tamoxifen; taxanes; aldesleukin; interleukin-2; etoposide; interferon alfa; tretinoin; bleomycin; dactinomycin; daunorubicin; doxorubicin; mitomycin; vinblastine; vincristine, and combinations thereof.

[0008] In a second aspect, provided herein is an improved method of treating a liver cancer in a subject. The method comprises administering a chemotherapeutic agent to the subject, the improvement comprises administering to the subject an amount of a Rhazya stricta extract sufficient to inhibit metastasis of the cancer cells. Brief Description of the Figures

[0009] The invention can be better understood with reference to the following figures and description. The components in the figures are not necessarily to scale and are not intended to accurately represent molecules, cells, cell organelles, tissues, or their interactions, emphasis instead being placed upon illustrating the principles of the invention.

[0010] FIG. 1. Total phenolic content of T. foenum-graecum (Tf), C. acutifolia (Ca), R. stricta (Rs) and extracts determined by the Folin-Ciocalteu assay and calculated as mg GAE/g extract based on dry weight. Results are the average of triplicates + SD.

[0011] FIG. 2. DPPH radical scavenging activities of the tested extracts.

[0012] FIG. 3. Linear correlations between the amount of total phenols and DPPH- radical scavenging activity (a) and between the flavonoid content and DPPH- radical scavenging activity (b).

[0013] FIG. 4. Flavonoids content of the tested extracts.

[0014] FIG. 5. Assessment of the cytotoxic effects of Trigonella foenum-graecum (Helba), Cassia acutifolia (Holoul), and Rhazya stricta (Harmal) extracts on HepG2 in vitro (a) MTT assay results of HepG2 cells viability after treatment with increasing concentrations of Helba and Holoul for 24 hours. *P<0.05, **P<0.001, ***P<0.0001 (b) MTT assay results of HepG2 cells viability after treatment with increasing concentrations of R. stricta extract for 24 hours. *P<0.05, **P<0.001, ***P<0.0001 (c) Assessment of morphological changes of HepG2 cells after treatment with increasing concentrations of R. stricta extract for 24 hours. Cells were fixed and stained with crystal violet (scale bar= 200 pm) (d) Cell cycle progression of HepG2 cells after treatment with R. stricta extract at a dose of 30 pg over a period of 48 hours (e) Quantitative distribution of HepG2 cells in different phases of the cell cycle at different time intervals (*P< 0.05) (f) Immunoblot analysis of cell cycle regulatory proteins in HepG2 cells after treatment with R. stricta extract at a dose of 30 pg over a period of 48 hours.

[0015] FIG. 6. Rhazya stricta extract inhibits colony forming ability and wound healing of HepG2 cells in a dose-dependent manner (a) Representative images of HepG2 colonies after treatment with increasing concentrations of R. stricta extract (b) Percent of area occupied by colonies in treated and non-treated wells (representative of triplicate samples; *P<0.05, **P<0.005, ***P<0.0001) and absorbance of each treated and non-treated wells (representative of 3 biological triplicates, each in triplicate; *P<0.05, **P<0.001, ***P<0.0001). (c) Representative images and quantification (three regions of three biological triplicates; *P<0.05, **P<0.001, ***P<0.0001) of wound-healing assay results of HepG2 cells treated without or with 20 and 30 pg of R. stricta extract.

[0016] FIG. 7. Rhazya stricta extract inhibits tube formation in HUVECs on Matrigel in a dose -dependent manner. Photographs of tube formation in HUVECs on Matrigel after incubation with or without R. stricta extract at 18 h. Cells were treated with R. stricta extract at a series of concentrations (10 pg; B, 20 pg; C, 30 pg; D) or DMSO vehicle (control; A) for 18 h.

[0017] FIG. 8. Assessment of the cytotoxic effects of Trigonella foenum-graecum (Helba), Cassia acutifolia (Holoul), and Rhazya stricta (Harmal) extracts on HCT116 in vitro. MTT assay results of 9cHCTl 16 cells viability after treatment with increasing concentrations of Helba (a) Holoul (b) and Harmal (c) for 24 hours. *P<0.05, **P<0.005, ***P<0.0001.

[0018] FIG. 9 is a table reporting human equivalent dose (HED) dosage factors based on body surface area of other species according to data obtained from Food and Drug Administration draft guidelines.

Detailed Description of Specific Embodiments

[0019] A study was conducted where three hydroalcoholic extracts of traditionally medicinal plants used in the United Arab Emirates (UAE) were evaluated for their antioxidant activities, phenol and flavonoid contents. These plants are: Trigonella foenum-graecum, locally known as“Helba”, Cassia acutifolia, commonly known as“Holoul” and Rhazya stricta which is locally called“Harmal”

[0020] T. foenum-graecum seeds are traditionally used as herbal medicine for their carminative, tonic, aphrodisiac and anticancer effects. The leaves of C. acutifolia are frequently used in folk medicine as a purgative for a long time. The extracts of R. stricta leaves are traditionally used for the treatment of various disorders such as diabetes, sore throat, helminthiasis, inflammatory conditions and rheumatism.

[0021] Available treatment for hepatocellular carcinoma are mainly limited to invasive hepatectomy or chemotherapy. However, the attention has shifted in recent years to natural -based products for candidate anticancer therapeutics. In the present study, the antiproliferative effects of Trigonella foenum-graecum, Cassia acutifolia, and Rhazya stricta on hepatoma cell line HepG2 were investigated. The use of HepG2 cells to test the cytotoxic effects of a wide range of drugs has been well documented, due to their wide availability, well-differentiation, and drug metabolizing activity.

[0022] Despite playing a key role in cellular processes, free radicals pose a threat to cells by damaging DNA, proteins, and cellular membranes, leading to onset of many diseases including cancer. Thus, by decreasing free radicals and oxidative stress, antioxidants play a role in ameliorating DNA damage, reducing the rate of abnormal cell division, and decreasing mutagenesis. Therefore, many antioxidant -rich plants possess anticancer activity.

[0023] Vascular endothelial growth factor (VEGF) has been recognized to be involved in several stages of angiogenesis in malignant diseases by its multi-functional effects in activating and integrating signalling pathway networks. VEGF signalling blockade reduces new vessel growth and leads to endothelial cell apoptosis. Therefore, using tyrosine kinase inhibitors or VEGF/VEGF receptor (VEGFR) antibodies to inhibit crucial angiogenic steps is a practical therapeutic strategy when treating neovascularisation diseases. A potent angiogenesis inhibitor known as E7820 has been shown to reduce integrin a 2 mRNA expression and inhibit basic fibroblast growth factor/VEGF-induced HUVEC proliferation and tube formation. Integrin a 2b 1/a 1b 1 expression is reportedly regulated by VEGF and an inhibitory antibody against a 2b 1/a 1b 1 has been shown to inhibit angiogenesis and tumour growth in VEGF -overexpressing tumour cells. Reported herein is also a study on the effect of R. stricta leaves extract on angiogenesis utilizing HUVEC tube formation assay. In addition, the experimental work was set to determine the in vitro antioxidant activity, total phenols and flavonoids, anticancer activities of tested plants with special interest in R. stricta.

[0024] Of the extracts evaluated, that of R. stricta (Harmal) showed the greatest antioxidant, antiangiogenic and antiproliferative activities, a discovery that makes this species a promising source of anticancer agent development especially for colon and liver cancers.

[0025] RHAZYA STRICTA EXTRACT COMPOSITIONS

[0026] In a first aspect, the present application provides therapeutic methods to treat, suppress, inhibit, or reduce the severity of cancer cell metastasis in a subject by administering a therapeutically effective amount of a Rhazya stricta extract, either alone or formulated together with one or more pharmaceutically acceptable carrier(s), diluent(s), or excipient(s). The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof.

[0027] Unless otherwise specified, the terms “composition including Rhazya stricta extract” and “formulation of Rhazya stricta extract” as used herein are intended to cover compositions and formulations including extracts of one or more parts of the Rhazya stricta plant, for example, but not limited to, roots, stem, leaves, buds, flowers, fruits, and seeds. Typically, a plant -derived material is first dried then brought into contact with a solvent that extracts one or more components from the material, to form a precursor solution. The precursor solution is separated from the extracted material, for example by filtration, then subjected to solvent evaporation, to yield the extract. In an exemplary embodiment, Rhazya leaves are macerated in water. In another embodiment, the leaves are extracted in a mixture of water with a water - miscible solvent, such as ethanol. Organic solvents and their mixtures are also contemplated, as is supercritical fluid extraction (SFE).

[0028] Compositions featuring Rhazya stricta extracts may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

[0029] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0030] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0031] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of Rhazya stricta extract which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, the particular mode of administration. The amount of an active ingredient which can be combined with a carrier material to produce a single dosage form will usually be that amount of the compound which produces a therapeutic effect. Usually, out of one hundred per cent, this amount will range from about 1 wt% to about 99 wt% of active ingredient, preferably from about 5 wt% to about 70 wt%, most preferably from about 10 wt% to about 30 wt%.

[0032] In certain embodiments, a formulation of Rhazya stricta extract includes an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and poly anhydrides; and an active ingredient that may be Rhazya stricta extract and/or one of its pharmaceutically acceptable derivatives. In certain embodiments, an aforementioned formulation renders orally bioavailable a Rhazya stricta extract or its derivative.

[0033] Methods of preparing these formulations or compositions include the step of bringing into association Rhazya stricta extract with the carrier and, optionally, one or more accessory ingredients. Usually, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0034] Liquid dosage forms for oral administration of Rhazya stricta extract include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0035] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [0036] Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A formulation of Rhazya stricta extract may also be administered as a bolus, electuary or paste.

[0037] In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically- acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0038] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0039] The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro -encapsulated form, if appropriate, with one or more of the above- described excipients.

[0040] The tablets, and other solid dosage forms of the formulation of Rhazya stricta extract, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical -formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g. , freeze -dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above -described excipients.

[0041] Formulations of the pharmaceutical compositions of Rhazya stricta extract for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing Rhazya stricta extract with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

[0042] Dosage forms for the topical or transdermal administration of Rhazya stricta extract include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The extract may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an extract, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0043] Powders and sprays can contain, in addition to an extract, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0044] Transdermal patches have the added advantage of providing controlled delivery of Rhazya stricta extract to the body. Such dosage forms can be made by dissolving or dispersing an extract in the proper medium. Absorption enhancers can also be used to increase the flux of the extract or dispersing the extract in a polymer matrix or gel. [0045] Pharmaceutical compositions suitable for parenteral administration include one or more components of a Rhazya stricta extract in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0046] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0047] Regardless of the route of administration selected, Rhazya stricta extract may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Rhazya stricta extract may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

[0048] In certain embodiments, the above -described pharmaceutical compositions include Rhazya stricta extract, a chemotherapeutic agent, and optionally a pharmaceutically acceptable carrier. Alternatively, the terms“chemotherapeutic agent” or“therapeutic agent” include, without limitation, platinum-based agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU) and other alkylating agents; antimetabolites, such as methotrexate; purine analog antimetabolites; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP-16), interferon alfa, and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca alkaloid natural antineoplastics, such as vinblastine and vincristine.

[0049] METHODS OF METASTASIS INHIBITION

[0050] The above Rhazya stricta extract compositions may be used in novel therapeutic methods of inhibiting metastasis in cancer patients. The methods include administering to a subject an effective amount of a pharmaceutical Rhazya stricta extract composition. In representative embodiments, the subject suffers from a liver cancer. In specific embodiments, the type of liver cancer is a hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or a metastatic liver cancer.

[0051] As anticipated above, Rhazya stricta extract may be administered by any appropriate route, for example orally, parenterally, topically, or rectally. It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the Rhazya stricta extract and the cancer to be treated. In certain embodiments, the extract may be especially suitable for the preparation of pharmaceuticals for intravenous administration, such as intravenous injection or infusion, provided that it does not contain components with serum-precipitating and/or haemagglutinating properties which disturb such an application. The extract may therefore be provided in the form of ampoule preparations which are directed to intravenous administration. In still other embodiments, the method comprises systemic administration of a subject composition to a subject.

[0052] Also provided are methods of treating cancer, for example liver cancer, which include administering Rhazya stricta extract in conjunction with a chemotherapeutic agent to a subject. Conjunctive therapy includes sequential, simultaneous and separate, or co-administration of the Rhazya stricta extract and the chemotherapeutic agent in a way that the therapeutic effect of the chemotherapeutic agent is not entirely disappeared when the Rhazya stricta extract is administered. In certain embodiments, Rhazya stricta extract and the chemotherapeutic agent may be compounded together in the same unitary pharmaceutical composition including both entities. Alternatively, the combination of Rhazya stricta extract and chemotherapeutic agent may be administered separately in separate pharmaceutical compositions, each including one of the Rhazya stricta extract and chemotherapeutic agent in a sequential manner wherein, for example, Rhazya stricta extract or the chemotherapeutic agent is administered first and the other second.

[0053] Exemplary doses of Rhazya stricta extract fall in the range from about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, or 750 to about 1000 mg/day per kg body weight of the subject. In certain embodiments, the dose of Rhazya stricta extract will typically be in the range of about 100 mg/day to about 1000 mg/day per kg body weight of the subject, specifically in the range of about 200 mg/day to about 750 mg/day per kg, and more specifically in the range of about 250 mg/day to about 500 mg/day per kg. In an embodiment, the dose is in the range of about 50 mg/day to about 250 mg/day per kg. In a further embodiment, the dose in the range of about 100 mg/day to about 200 mg/day per kg. In an embodiment, the dose is in the range of about 15 mg/day to 60 mg/day per kg. In a further embodiment, the dose is in the range of about 20 mg/day to 50 mg/day per kg. In an additional embodiment, the dose is in the range of about 25 mg/day to 45mg/day per kg.

[0054] The combined use of Rhazya stricta extract and other chemotherapeutic agents may reduce the required dosage for any individual component because the onset and duration of effect of the different components may be complementary. In such combination therapies, the different active agents may be delivered together or separately, and simultaneously or at different times within the day.

[0055] The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. For example, effective dosages achieved in one animal species may be extrapolated for use in another animal, including humans, as illustrated in the conversion table of FIG. 9 where human equivalent dose (HED) dosage factors based on body surface area of other species are reported. The dosage of any supplement, or alternatively of any components therein, lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For Rhazya stricta extract or combinations of Rhazya stricta extract and other chemotherapeutic agents, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0056] KITS

[0057] The present invention provides kits for inhibiting metastasis of cancer cells. For example, a kit may include one or more pharmaceutical compositions of Rhazya stricta extract as described above. The compositions may be pharmaceutical compositions comprising a pharmaceutically acceptable excipient. In other embodiments involving kits, this invention provides a kit including Rhazya stricta extract, optionally a chemotherapeutic agent, and optionally instructions for their use in the treatment of cancer. In still other embodiments, the invention provides a kit comprising one more pharmaceutical compositions and one or more devices for accomplishing administration of such compositions. For example, a subject kit may comprise a pharmaceutical composition and catheter for accomplishing direct intraarterial injection of the composition into a cancer. In an embodiment, the device is an intraarterial catheter. Such kits may have a variety of uses, including, for example, therapy, diagnosis, and other applications.

[0058] EXPERIMENTAL

[0059] Chemicals

[0060] All solvents were analytical grade. Agilent Cary 60 UV-Vis Spectrophotometer was used in all spectrophotometric measurements. Ascorbic acid, ferric chloride, aluminum chloride, potassium acetate, quercetin, DPPH reagent, Folin-Ciocalteu reagent, gallic acid, sodium carbonate, methanol and ethanol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Millipore deionized water was used throughout. Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich, USA), Dimethyl Sulfoxide (Sigma Aldrich, USA).

[0061] Plant samples [0062] Dried leaves of C. acutifolia, R. stricta and T. foenum-graecum were purchased from the local market. The taxonomic authentication of all the plants was carried out by Dr. Fatima Al-Ansari at the Biology Department, College of Science, United Arab Emirates University. Voucher specimens were deposited at the herbarium of the Biology Department (voucher reference numbers: BA2018-1, BA2018- 2, BA2018-3).

[0063] Preparation of plant extracts

[0064] The leaves of the medicinal plants were crushed separately in a grinder. A sample of 10 g of each plant was extracted with 150 mL of 70% ethanol and 30% water as it showed the best extraction yield [30]. The crushed plants were macerated for 48 h at 4 °C. The resulting mixture was then filtered under vacuum and concentrated under reduced pressure in a rotary evaporator at 40 °C. The extracts were further dried using a Telstar Cryodos freeze dryer machine then kept at -20 °C for further analysis. A solution of 30 mg/mL of each plant was prepared in 50% ethanol for the following tests.

[0065] Determination of total polyphenol content

[0066] The total phenolic content (TPC) was determined by using the Folin-Ciocalteu reagent. A 10% solution was prepared from the stock solution (30 mg/mL) using 50% ethanol. 100 pi of this solution was mixed with 200 mΐ of the Folin-Ciocalteu reagent and 2 mL of de -ionized water then incubated at room temperature for 3 min. A sample of 20% aqueous sodium carbonate (w/w, 1 mL) was then added to the mixture. The total polyphenols were determined after 1 h of incubation at room temperature. A negative control sample was also prepared using the same procedure. The absorbance of the resulting blue color was measured at 765 nm. Results were expressed in mg gallic acid equivalents (GAE) per g dry weight of plant material using an equation obtained from gallic acid calibration curve (5x 1

M - 5xl0⁴ M). The samples were analyzed in triplicate.

[0067] Free-radical scavenging activity

[0068] The antioxidant activity of the extracts was assessed based on their ability to scavenge the stable 1,1 -diphenyl-2 -picrylhydrazyl (DPPH) radical as described previously. Various concentrations of the three extracts in methanol were prepared (0.15 to 1.5 mg/mL). A methanolic solution of DPPH (3.8 mL, 60 pg/mL) was rapidly mixed with the plant extract (200 pi, 30 mg/mL) in a test tube, with methanol serving as the blank sample and a control was also assayed simultaneously. The contents of the tubes were swirled then allowed to stand for 30 min at room temperature in the dark. The absorbance was measured at 517 nm in a spectrophotometer. The scavenging ability of the plant extract was calculated using this equation: DPPH Scavenging activity (%) = [(Abs control-Abs sample)]/ (Abs control)] xlOO, where Abs control is the absorbance of DPPH + methanol; Abs sample is the absorbance of DPPH radical + sample (sample or standard). The ECso value (pg/mL). the effective concentration at which DPPH- radicals are scavenged by 50%, was determined graphically. The total antioxidant activity was expressed as ascorbic acid equivalent/g dry extract. The assay was done in triplicates.

[0069] Determination of total flavonoids

[0070] The total flavonoids content in the extracts was determined using the aluminum chloride colorimetric method. A known concentration (600 pg/mL) of each extract in methanol was prepared. A 500 pi of the extracts were mixed separately with 0.1 mL of 10% (w/v) aluminum chloride solution, 0.1 mL of 1 M potassium acetate solution, 1.5 mL of methanol and 2.8 mL of distilled water. The solutions were thoroughly mixed and incubated at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm using a spectrophotometer. The total flavonoids content was determined using a standard curve with quercetin (1 to 25 pg/mL) as the standard. The mean of three readings was used and expressed as mg of quercetin equivalents (QE)/ g of the dry extract.

[0071] Cell culture

[0072] A human hepatocellular carcinoma (HCC)-derived cell line (HepG2) was cultured in RPMI 1640 medium containing 1% antibiotic cocktail and supplemented with 10% fetal bovine serum. Cells were incubated at 37°C in 5% CO2 humidified incubator. Cells were passaged every 2-3 days using 0.25% trypsin-EDTA.

[0073] Cytotoxicity assay [0074] HepG2 were seeded at a density of 5000 cells/well in a 96-well plate, and were allowed to attach overnight. Thereafter, cells were treated with various concentrations of the plant extracts for 24 hours. To assess the cytotoxic effect of the three plants extracts, MTT (3 -[4,5 -dimethyl thiazol-2-yl] -2,5- diphenyltratrazolium bromide) assay was carried out. Briefly, cells treated with the plant extracts were exposed to tetrazolium MTT at a concentration of 5 mg/mL. Viable active cells reduced yellow MTT salt to insoluble purple formazan, which was dissolved using DMSO. The absorbance of the colored solution was measured at a wavelength of 570 nm using Epoch microplate spectrophotometer (BioTek). The obtained absorbance at 570 nm of both control and treated cells was used to calculate percentage of cell viability. Assuming 100% viability in control cells, percentages of treated cells viability were calculated accordingly:

[0075] Percent of viable cells = (Abs. of treated cells/ Abs. of control cells) X 100

[0076] Assessment of morphological changes

[0077] HepG2 cells were seeded at a density of 0.25 x 10⁶ cells/ well in a 6-well plate, and were allowed to attach overnight. After which, cells were treated without (0 pg. control) or with increasing concentrations of R. stricta extract (10, 20, 30, 50, 70 pg) for 24 hours. The morphology of the cells was assessed after being fixed and stained with 0.5% crystal violet using bright -field microscopy (200 x magnification, scale= 200 pm).

[0078] Colony formation assay

[0079] To assess the effects of R. stricta on cell survival, the colony formation assay was carried out in vitro. Briefly, HepG2 cells were seeded at a density of 1000 cells/ well in a 6-well plate, and were incubated for 24 hours to allow attachment. The second day, the cells were treated without (0 pg, control) or with increasing concentrations (10, 20, 30 pg) of the extract for 24 hours. Thereafter, the medium was replaced with fresh complete growth medium without the extract, and cells were left to incubate until visible colonies were formed; while changing the medium every 3-4 days. The experiment was carried out in triplicates. Colonies were fixed with absolute methanol, then stained with 0.5% crystal violet. Results are represented as the percentage of the well area that is covered by colonies (colony area percentage). Analysis has been carried out using ImageJ plugin Colony Area . In addition, an absorption-based method was carried out to validate the earlier results, by which the absorption of the crystal violet dye in each well is measured after being dissolved. Briefly, the samples that had been analyzed using ImageJ were subjected to 10% acetic acid solution, then were placed on an orbital shaker for 15 minutes. After which, 100 pL of each triplicate sample was transferred to a 96-well plate (in triplicates), and absorbance was measured using Epoch microplate spectrophotometer (BioTek).

[0080] Wound-healing assay

[0081] To assess the ability of HepG2 to migrate after the treatment with R. stricta, wound-healing assay was carried out in vitro. Cells were seeded at a density of 0.5 x 10⁶/ well in 6-well plate, and were allowed to attach overnight. A scratch in the cell monolayer was made using a sterile plastic pipetting tip, and then the monolayer was washed with PBS. The cells were treated without (0 pg) or with 20, 30 pg of the extract. Images were taken at 0, 24, 48, 72 hours using bright-field microscopy (40 x magnification). Analysis was carried out using ImageJ, percent of open area was calculated according to the following formula [T_x = T24, T48, or T72 (at time 24, 48, or 72 hours, respectively]: Percent of open area = (open area at T_x/ open area at T) X 100

[0082] Experiment was carried out in triplicates, data is representative of 3 random regions in each triplicate of each sample.

[0083] Cell cycle analysis

[0084] Effect of R. stricta extract on cell cycle progression of HepG2 cells was analyzed as previously described. Cells were treated without or with 30 pg of R. stricta extract at different time intervals (6 - 48 h), collected by trypsinization, washed twice with PBS, fixed in 70% ethanol, treated with RNase, stained with propidium iodide and then cell cycle distribution was analyzed in BD Accuri C6 cytometer and software (BD Biosciences, USA).

[0085] Western blotting [0086] HepG2 were seeded at a density of lxlO⁶ in 60 mm dish and allowed to attach overnight. Cells were treated without or with 30 pg of extract for 6, 12, 24, 48 hours. Cells were lysed and total protein was quantified using BCA. 20 pg of total protein was separated on SDS-PAGE, transferred onto nitrocellulose membranes that were blocked using 5% BSA TBST. Primary antibodies against Cdc2 (1:000; cell signaling), p-Cdc2 (1 :1000; cell signaling), Cyclin B1 (1 :1000; cell signaling), Cyclin A1 (1:000; Abeam) were used. GAPDH (1: 15000; Abeam) was used as loading control. Proteins were detected using LI COR C DiGit Chemiluminescence Western Blot Scanner.

[0087] Matrigel capillary tube formation

[0088] 96-well plate was coated with Matrigel matrix (Coming, NY, USA) at 50 pl/well and allowed to polymerize for 60 min at 37 °C. HUVEC cells were then seeded on the Matrigel at a concentration of 2 x 10⁴ cells/well without (0 pg) or with (10, 20, 30 pg) R. stricta extract. After incubation for 18 h, tubules were imaged using an inverted microscope and analyzed with ImageJ software.

[0089] Statistical analysis

[0090] All data were expressed as mean + standard deviation (SD) of three independent experiments. Correlation analysis of antioxidants versus the total phenolic and flavonoid contents were carried out using the regression analysis, with GraphPad Prism 6.0 and Microsoft Excel 2016. P< 0.05 was considered to indicate a significant difference.

[0091] Results and Discussion

[0092] Medicinal plants have been of great interest as a source of natural antioxidants used for health promotion. The therapeutic activity of plants is mostly due to their biologically active polyphenolic substances, mostly flavonoids and phenolic acids. These substances exhibit antioxidant, anti -lipoxygenase and anticancer activities. The present study elaborates on the antioxidant activity, polyphenolic and flavonoid contents of three folk plants from the UAE; T. foenum-graecum, C. acutifolia and R. stricta. The antiproliferative effect of such plants was studied against human cancer cells HepG2 in an attempt to find a correlation with the antioxidant activity of those extracts that are based on their phenolic and flavonoid contents.

[0093] Plant extraction

[0094] Different solvents have been used in the literature for the preparation of plant extracts [36]. In this study, we used 70% ethanol in water as the extraction solvent. The amorphous solid of the leave extracts under investigation was obtained by complete evaporation of the ethanol/water solvent. The yield of each extract was calculated as w/w percent yield. The yields of T. foenum-graecum, C. acutifolia and R. stricta extracts were 25%, 23% and 30% respectively.

[0095] Total polyphenol content of the extracts

[0096] Polyphenols are aromatic secondary plant metabolites and are widely spread throughout plants. They have been associated with color, sensory qualities, and nutritional and antioxidant properties of food. It is reported that there is a strong relationship between total polyphenol contents and antioxidant activity. Without being bound to any particular theory, it appears that the hydroxyl groups in phenols have a strong scavenging ability for free radicals. Therefore, the total polyphenol contents of plants may directly contribute to their antioxidant activity. The Folin-Ciocalteu reagent is commonly used in the literature to quantify phenolic compounds. This reagent reacts with phenolic compounds and produces a blue color complex that absorbs radiation and allows quantification. The total phenolic content for the ethanolic extracts of C. acutifolia, R. stricta and T. foenum-graecum was determined by the Folin-Ciocalteu method using gallic acid as a standard. The calibration curve showed linearity for gallic acid in the range of 0.5 - 26 pg/mL, with a correlation coefficient (R²) of 0.984. R. stricta contained the highest total polyphenols (11.5 + 0.013 mg GAE/g extract), followed by C. acutifolia (10.8 + 0.025 mg GAE/g extract) and T. foenum-graecum (9.7 + 0.008 mg GAE/g extract) (FIG. 1). Belguith-Hadriche et al (2013) reported the total phenolic content of various extracts of T. foenum-graecum ranged between 9.42 + 0.50 in hexane and 78.1 + 0.90 mg GAE/g dry weight extracts in methanol. A study conducted in Saudi Arabia showed the total phenolic content of R. stricta extracts ranged between 62.5 + 0.2 and 66.63 + 0.03 mg GAE/g extract. [0097] The DPPH radical scavenging activity

[0098] The antioxidant activity of each plant was also assessed based on its ability to reduce the stable DPPH radical according to the method reported by Lim. The DPPH radical (DPPH·) is a stable radical and has the ability to accept an electron or hydrogen radical and form a stable diamagnetic molecule producing a color change from blue to yellow. The color change of DPPH has been widely used to measure the radical scavenging activity because of its stability, simplicity, and reproducibility. The free radical scavenging capacity of the ethanolic extracts of the three plants were assayed based on the remaining amount (%) of DPPH· as a function of time (30 min). The total antioxidant activity was expressed as ascorbic acid equivalent/g dry extract.

[0099] The calibration curve of ascorbic acid showed linearity in the range of 5 - 20 pg/mL, with a correlation coefficient (R²) of 0.994. The percentage inhibitions of DPPH scavenging activity in all the extracts were dose-dependent (FIG. 2). The DPPH scavenging activity by the T. foenum-graecum extract was (89.7%+ 1.54) at 1.5 mg/mL and (28.6%+ 2.07) at 0.15 mg/mL. DPPH of C. acutifolia was (86.3%+ 0.64) at 1.5 mg/mL and (30.0%+ 1.37) at 0.15 mg/mL while that of R. stricta was (89.9%+ 0.51) at 1.5 mg/mL and (28.7%+ 1.27) at 0.15 mg/mL. The results showed that there is no clear difference between the obtained DPPH scavenging activities of the three extracts. These results are almost in agreement with their total polyphenol contents. Consequently, the antioxidant activity of these plants might be related to their contents of phenolic compounds.

[00100] In addition, the DPPH radical scavenging ability of the extracts was evaluated as EC50

(pg/mL) value. The smallest EC50 values indicates the best free radical scavenging activity. The highest scavenging activity was exerted by R. stricta (EC50 = 241.8 pg/mL) which contained the highest amount of total polyphenol content (11.5 pg/mL), followed by C. acutifolia (EC50 = 244.8, TPC = 10.8pg/mL). The lowest radical scavenging activity was exhibited by the T. foenum-graecum extract (EC50 = 245.9, TPC = 9.7 pg/mL). The relationship between total phenol content and free radical scavenging activity (using EC50) was also studied using linear regression analysis (FIG. 3a). The results showed a significant negative correlation (R² = -0.856, P-value <0.05) between EC50 (DPPH- scavenging) and total phenolic content suggesting that the presence of the phenolic compounds contributed significantly to the antioxidant activity of the tested plants. These results are consistent with previous works that showed a liner correlation between the total phenolic content and the reducing antioxidant capacity of some plant extracts.

[00101] Total flavonoid contents

[00102] Flavonoids are a class of secondary plant phenolics. Flavonoids and their derivatives have a wide range of biological actions including anticancer activity. The anticancer activity of flavonoids is attributed to their potent antioxidant effects which include metal chelation and free -radical scavenging activities. Flavonoids present in herbs were found to significantly contribute to their antioxidant properties. The flavonoid content was obtained using aluminum chloride assay which based on the formation of a complex between the aluminum ion, A1 (III), and the carbonyl and hydroxyl groups of flavones and flavonols that produce a yellow color. Flavonoid content was calculated from the regression equation of quercetin calibration curve and was expressed as quercetin equivalents. The calibration curve showed linearity in the range of 1 - 25 pg/mF, with a correlation coefficient (R²) of 0.999.

[00103] FIG. 4 shows the flavonoids contents in all the extracts. Using the standard curve generated by quercetin, the total flavonoids content of the T. foenum-graecum extract was (14.6 + 0.21 mg QE/g), whereas, the C. acutifolia extract was (20.8 + 0.40 mg QE/g) and finally that of R. stricta was (9.2 + 0.22 mg QE/g).

[00104] The relationship between the total flavonoids content and the free radical scavenging activity (using EC50) was studied using linear regression analysis (FIG. 3b). The results showed a positive correlation (R² = 0.460, P-value <0.05) between EC50 (DPPH scavenging) and total flavonoids content. The obtained correlation was moderate suggesting that other compounds may be participating in the radical scavenging activity of these plant extracts.

[00105] Effects of extracts on cell viability in HepG2 cells [00106] HCC remains among the leading cause of cancer -related death worldwide [47, 48].

Although therapeutic approaches for advanced HCC are limited to the use of multikinase inhibitors, such as sorafenib, only modest survival benefits have clinically been reported. Thus, identifying new compounds with promise antitumor activity against HCC is exceedingly needed. Natural products, especially from plants, are often better tolerated than their synthetic analogs used in cancer treatments. They contain a wide spectrum of bioactive secondary metabolites that are the foundation of the recently introduced notion of broad-spectrum integrative approach for cancer prevention and treatment.

[00107] Effects of tested extracts were investigated against human hepatoma (HepG2) cancer cell line. A dose-dependent reduction in cell viability was reported in cells treated with all tested extracts (FIG. 5). The IC50 values of extracts ranged from 30 pg/mL to 200 pg/mL. Treatment with R. stricta significantly enhanced the mortality of cancer cells at the lowest concentration (30 pg/mL). T. foenum-graecum was however less potent with IC50 of 200 pg/mL where 50% of HepG2 cancer cells were eradicated at 200 pg/mL. A fenugreek-enriched diet decreased colon tumor incidence and hepatic lipid peroxidation in liver cancer-induced rats in addition to increasing the endogenous antioxidant activities in liver. Li et al (2010) showed that diosgenin, fenugreek’s main active ingredient, down regulated the expression of various S TAT 3 -regulated genes, inhibited proliferation and potentiated the apoptotic effects of paclitaxel and doxorubicin, suggesting that diosgenin could be a novel and potential treatment option for HCC and other cancers. Therefore, the role of fenugreek extract and its active principles as supplements in diet-based preventive/therapeutic strategies to improve health care continues to be a fast -growing field of research. The correlation between the antioxidant activities (free radical scavenging activity) and the anticancer activities (cell viability) of C. acutifolia, R. stricta and T. foenum-graecum extracts was studied using linear regression analysis. The results showed a significant positive correlation (R² = -0.933, 0.997 and 0.797, P- value <0.05) for C. acutifolia, R. stricta and T. foenum-graecum extracts respectively.

[00108] At a concentration range of (100-200 pg/mL) Cassia acutifolia % extract was similarly cytotoxic to HepG2 cells. Alkaloids extracted from Senna species reduced cell viability in a concentration- dependent manner of different tumor cell lines including HepG2. Senna alkaloids showed important antiproliferative activity on HepG2 cells that was mediated by ERK inactivation and down-regulation of cyclin D1 expression. Similarly, extracts of different Cassia species were able to inhibit growth of colorectal (DLD1), among other, human cancer cell lines. Thus, Senna’s extract may represent a potential new antitumor and/or adjuvant treatment against liver and colorectal cancer and further investigations should be conducted to unravel its molecular mechanism. R. stricta' s extract was by far the most effective cytotoxic agent tested at the present study at as little as 30 pg/mL. The d-tocopherol and alkaloid fraction of R. stricta' s leaves extract have been shown to delay many angiogenic and inflammatory activities and to inhibit cell viability of HepG2, hence potentially useful against cancer. As R. stricta extract showed the most promising antiproliferative activity, its antiproliferative effects were tested against other GI cell line. The present study also showed that R. stricta extract inhibited cell proliferation in colorectal cancer cells (HCT116), in a dose -dependent manner (FIG. 8). To get insight into the molecular effects of R. stricta on HepG2 cells, its effect on cell cycle progression was analyzed. R. stricta treatment induced arrest of cells in the G2/M phase (12 and 48 h time points) and in Gl/S phase (24 h time point) (FIG. 5 d and e). This cell cycle arrest reflects the cytotoxic effects of R. stricta on HepG2 cells where the cells are arrested at these phases to repair the toxic lesions induced by R. stricta or to be removed by death pathways such as apoptosis. The significant increase of the fraction of cells in G2/M after long term treatment (48 h) may indicate permanent arrest of cells in this phase (quiescence). These findings are consistent with western blot data, where cell cycle promoting proteins cdc2 and its cyclin partners (cyclin A1 and cyclin B l) are down regulated post R. stricta treatment (FIG. 5f). Cdc2 activity is regulated through two mechanisms, first by binding to its cyclin partners, and second by being phosphorylated at Tyrl5, and subsequently inhibited. Cdc2/cyclin complexes contribute to cell cycle progression through phosphorylation of target proteins. As shown, Cdc2, and its partners cyclin A and cyclin B were downregulated in a time-dependent manner (FIG. 5f). In addition, treatment with R. stricta extract resulted in a slight increase in Tyrl5 phosphorylated Cdc2

(FIG. 5f).

[00109] R. stricta extract inhibited the colony formation of HepG2 cells [00110] This assay measures the ability of tumor cells to survive and grow to form colonies after treatment with cytotoxic agents. To assess clonogenicity, cells were plated onto 6-well plates and incubated with 0.0, 10, 20, and 30 pg of R. stricta extract and treated cultures were maintained in culture for an additional 10 days to allow formation of colonies. The results from that assay showed that R. stricta extract inhibited the colony formation of HepG2 cells in a dose -dependent manner (0, 10, 20, 30 pg). ImageJ analysis results were consistent with results obtained from the absorption-based method where R. stricta extract also inhibited the survival of HepG2 cells in a dose-dependent manner (FIG. 6a and 6b).

[00111] R. stricta extract inhibits the migration ability of HepG2 cells

[00112] Many cancer patients die of tumor mobilization which often lead to metastatic foci in distant part/s of the body. Therefore, attempts to prevent or slowdown progression and metastasis of cancer cells are crucial. Earlier study showed that R. stricta extract exerts potent anti-cancer ability against human breast cancer. To date, however, limited information is available regarding R. stricta extract effect on cell migration in any cancer type. The potential role of R. stricta extract on HepG2 cells’ migration was assessed using wound healing assay. Cell migration was inhibited in a dose -dependent manner (0, 20 and 30 pg) starting from 24-72 hrs post R. stricta extract treatments compared to the control (FIG. 6c).

[00113] R. stricta extract inhibits endothelial cell tube formation

[00114] Cell migration and invasion are crucial steps in various physiological processes such as morphogenesis, angiogenesis, wound healing and inflammation. Invasion and migration are critical events for tumor progression and tumor recurrence. HUVEC cells had greater numbers of branching tube networks after 18 hours of incubation with no R. stricta extract (FIG. 7A). This tube branching was attenuated by R. stricta extract treatments in a dose-dependent manner (FIG. 7B and 7C). Colony formation results demonstrate that colony numbers in all dose groups were decreased. These results suggest that R. stricta extract reduced cell migration and colony formation capacity in HepG2 cancer cells. R. stricta extract may, therefore, be introduced as a novel agent to treat/prevent HCC and, more generally, cancer metastasis, either as a single agent or in combination with other drugs. It would also be of interest to identify R. stricta extract bioactive molecules and assess their effects against cancer, particularly HCC. Further studies are currently underway to identify and characterize R. stricta extract bioactive ingredients and to unravel their molecular mechanism against cancer.

[00115] The obtained antiproliferative results of the three extracts against HepG2 cancer cell line were found to be consistent with their antioxidant activity, free radical scavenging ability and phenolic content. Out of the three extracts, R. stricta showed considerable antiproliferative activity at low concentration and was the most potent among the tested plants with IC50 = 30 pg/rnL. In addition, R. stricta showed the highest total polyphenols (11.5 + 0.013 mg GAE/g extract). It has also the most percentage inhibitions of DPPH scavenging activity (89.9%+ 0.51) at 1.5 mg/ml and (28.7%+ 1.27) at 0.15 mg/ml. This indicates that the antiproliferative effect of the three extracts, in particular R. stricta, may be attributed to their antioxidant polyphenolic and flavonoid contents. Phenolic compounds have been found to counteract cancer either by means of antioxidant effect or by inhibiting the formation of carcinogenic metabolites that damage the vital biomolecules.

[00116] Finally, the investigated UAE plants have shown a strong reducing antioxidant capacity and free radical scavenging ability. These findings validate the use of these plants in folk medicine for the treatment of certain diseases. Oxidative cell damage events are frequently correlated with the oxidative stress. We show here that both of those properties are present in the ethanolic extract of R. stricta, which has significant antiproliferative activity and a great antioxidant activity. The results reported here are very promising indicators for the potential application of this plant for preventive and therapeutic purposes.

[00117] DEFINITIONS

[00118] As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:

[00119] As used herein,“treatment” is understood to refer to the administration of a drug or drugs to a patient suffering from cancer. [00120] As used herein, the term“therapeutically effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

Claims

CLAIMS What is claimed is:

1. A method of inhibiting metastasis of metastatic cancer cells, which comprises administering to a subject suffering from cancer a therapeutically effective amount of a Rhazya stricta extract.

2. The method of claim 1, where the extract is of Rhazya stricta leaves.

3. The method of claim 1, where the Rhazya stricta is extracted with a solution comprising water and ethanol.

4. The method of claim 1, where the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, a metastatic liver cancer, and combinations thereof.

5. The method of claim 1, where the therapeutically effective amount is administered orally.

6. The method of claim 1, where the amount of the extract is from about 10 mg/day to about 1000 mg/day per kg body weight of the subject.

7. The method of claim 1, where the amount of the extract is from about 200 mg/day to about 750 mg/day per kg body weight of the subject.

8. The method of claim 1, where the amount of the extract is from about 250 mg/day to about 500 mg/day per kg body weight of the subject.

9. The method of claim 1, further comprising administering a second therapeutic agent selected from the group consisting of carboplatin; cisplatin; methotrexate; fluorouracil; gemcitabine; goserelin; leuprolide; tamoxifen; taxanes; aldesleukin; interleukin-2; etoposide; interferon alfa; tretinoin; bleomycin; dactinomycin; daunorubicin; doxorubicin; mitomycin; vinblastine; vincristine, and combinations thereof.

10. In a method of treating a liver cancer in a subject, the method comprising administering a chemotherapeutic agent to the subject, the improvement comprising administering to the subject an amount of a Rhazya stricta extract sufficient to inhibit metastasis of the cancer cells.