Description:PREAMBLE TO THE DESCRIPTION:
[0001] The following specification particularly describes the invention, and the manner in which it has to be performed:
DESCRIPTION OF THE INVENTION

Technical field of the invention

[0002] The present invention relates generally to a liquid formulation of natural ingredients in combination to inhibit glycolytic pathway for the treatment of cancer. The formulation is a combination of natural ingredients, which exhibits synergistic effect in mitigating the cell cycle and inducing apoptosis of cancer cells. The present invention also discloses a process for preparation of the formulation.
Background of the invention
[0003] Cancer refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs and is referred to as metastasis. The widespread metastases are the primary cause of death from cancer.
[0004] Cancer arises from the transformation of normal cells into tumour cells in a multi-stage process that generally progresses from a pre-cancerous lesion to a malignant tumour. These changes are the result of the interaction between a person's genetic factors and other categories of external agents including physical carcinogens, such as ultraviolet and ionizing radiation, chemical carcinogens, such as asbestos, components of tobacco smoke, alcohol, aflatoxin, and arsenic and biological carcinogens, such as infections from certain viruses, bacteria, or parasites.
[0005] Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. The most common types of cancer leading to death are breast cancer, lung cancer, colon and rectal cancer, prostate cancer, skin cancer and stomach cancer.
[0006] Glycolysis is a linear metabolic pathway of enzyme-catalyzed reactions that converts glucose into two molecules of pyruvate in the presence of oxygen or two molecules of lactate in the absence of oxygen. Glycolysis is achieved in two basic ways either in the presence or in the absence of oxygen. When oxygen is present, NADH can pass its electrons into the electron transport chain, regenerating NAD + for use in glycolysis.
[0007] In the absence of oxygen, cells may use other, simpler pathways to regenerate NAD+. In these pathways, NADH donates its electrons to an acceptor molecule in a reaction that doesn’t generate ATP but does regenerate NAD+ to continue the glycolysis and the process is termed as fermentation.
[0008] Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions.
[0009] Cancer therapies have been developed that target transporters and enzymes in the glycolytic pathway, such as glucose transporter 1 (GLUT1), hexokinase etc. As the tumor glycolysis also plays a significant role in chemoresistance of cancer cells, glycolytic inhibitors therefore have the potential to sensitize tumor cells and to improve the outcome of conventional chemotherapy.
[0010] Cancer cells have increased need for both energy and biosynthetic intermediates to support their growth and invasiveness. However, their high proliferation rate often generates regions that are insufficiently oxygenated. Therefore, their carbohydrate metabolism must rely mostly on a glycolytic process that is uncoupled from oxidative phosphorylation. This metabolic switch, also known as the Warburg effect, constitutes a fundamental adaptation of tumor cells to a relatively hostile environment, and supports the evolution of aggressive and metastatic phenotypes.
[0011] As a result, tumor glycolysis may constitute an attractive target for cancer therapy. This approach has often raised concerns that antiglycolytic agents may cause serious side effects toward normal cells. The key to selective action against cancer cells can be found in their hyperbolic addiction to glycolysis, which may be exploited to generate new anticancer drugs with minimal toxicity.
[0012] Cancer cells have different metabolic properties from normal cells. Mostly, the normal cells rely upon the process of mitochondrial oxidative phosphorylation to produce energy whereas cancer cells have developed an altered metabolism that allows them to sustain higher proliferation rates. Cancer cells could predominantly produce energy by glycolysis even in the presence of oxygen.
[0013] Recent progress indicates that glycolytic pathway of cancer cells could be a critical target for drug discovery. With a long history in cancer treatment, herbal ingredients are recognized as a valuable source for seeking bioactive anticancer compounds. Great progress has been made to identify active compounds from herbal ingredients targeting on glycolysis for cancer treatment.
[0014] The Patent Application No. CN101099756B entitled “Anti-tumor traditional Chinese medicinal composition and preparation method and medicinal preparation thereof” discloses a picrasma quassioides extract obtained by using Chinese medicinal material picrasma quassioides as raw material and adopting solvent extraction and resin separation and purification processes. The total alkaloid content in said extract is above 50%. The invention also provides a pure Chinese medicine preparation containing said picrasma quassioides extract for resisting tumor and its concrete preparation method.
[0015] The Patent Application No. CN104189557A entitled “Traditional Chinese medicine composition and tablet for treating lung cancer and preparation method thereof” discloses a traditional Chinese medicine composition and tablet for treating lung cancer and a preparation method thereof. The preparation method of the tablet comprises the following steps: preparing the traditional Chinese medicine composition from the following components in parts by weight: 5-10 parts of medicated leaven, 10-25 parts of tortoise-plastron glue, 10-25 parts of dracontomelon duperreanum, 20-50 parts of pleione bulbocodioides, 5-10 parts of brucea javanica, 5-10 parts of safflower carthamus, 5-15 parts of glomeris nipponica kishida, 5-15 parts of trogopterus dung, 20-50 parts of rhizoma phragmitis, 5-15 parts of asparagus cochinchinensis, 3-10 parts of hydrargyri oxydum rubrum, 20-50 parts of common bombax flower, 5-15 parts of humifuse euphorbia herb, 3-10 parts of picrasma quassioides and 5-15 parts of antifebrile dichroa; and adding 20-40 parts of sorbitol, 10-20 parts of guaiac gum, 5-15 parts of polysorbate-80 and 3-8 parts of polyglycerol esters of fatty acids and finally preparing into tables. The traditional Chinese medicine composition is capable of effectively treating lung cancer, has the advantages of good effect, quick response, no toxic or side effect and high bioavailability, can cure lung cancer without replase and can be absorbed favorably by a human body.
[0016] The Patent Application No. CN102526041A entitled “Application of picrasma quassioides alkaloid serving as STAT3 (Signal Transducer and Activator of Transcription 3) signal specificity inhibitor” discloses a novel medical application of picrasma quassioides alkaloid, i.e., an application of picrasma quassioides alkaloid serving as a STAT3 (Signal Transducer and Activator of Transcription 3) signal specificity inhibitor. As proved by a cell model screening experiment and Western-Blot detection, the picrasma quassioides alkaloid has high STAT3 inhibiting activity and can be used for effectively inhibiting the expression of relevant genes regulated and controlled by STAT3, so that the growth of tumor cells dependent on STAT3 signals can be inhibited effectively. The picrasma quassioides alkaloid is taken as an active ingredient for preparing an antitumor medicament based on the target spot of STAT3; and antitumor medicaments such as various oral preparations, injections and the like of various dosage forms are prepared from pharmaceutically acceptable processes and auxiliary materials by taking picrasma quassioides alkaloid as an active ingredient.
[0017] The Patent Application No. CN110025643A entitled “Anti-cancer active compound and preparation method and applications thereof” discloses anti-cancer active compound and a preparation method and applications thereof. The anti-cancer active compound is prepared from the following raw materials: a meliosma cuneifolia extract and a picrasma quassioides root extract. The preparation method comprises the following steps: S1, preparation of extracts: respectively preparing the meliosma cuneifolia extract and thepicrasma quassioides root extract by using ethanol; and S2, mixing of the extracts: uniformly mixing the meliosma cuneifolia extract and the picrasma quassioides root extract to obtain the anti-cancer active compound. In the S1, the meliosma cuneifolia extract comprises meliosma cuneifolia leaf extract and meliosma cuneifolia wood extract, meliosma cuneifolia leaf powder, meliosma cuneifolia woodpowder and picrasma quassioides root powder are respectively extracted by ethanol according to a solid-liquid volume ratio of 1:(1-20), and the obtained extractive solution is dried to obtain the meliosma cuneifolia leaf extract, the meliosma cuneifolia wood extract and the picrasma quassioides root extract respectively. The anti-cancer active compound of the solution has good anti-cancer effect.
[0018] In order to inhibit the growth of cancer cells, the agents targeting the specific steps in the glycolytic pathway are required. Even though state of the art discloses the use of agents targeting the glycolytic pathway, but the inhibition of specific step of glycolysis, which acts as a check point, is not observed. Hence, there is a need for inhibition of specific steps of glycolysis for effective inhibition of cancer cell growth.
Summary of the Invention
[0019] The present invention overcomes the drawback of the state of art by providing the formulation for the treatment of cancer. The invention discloses a liquid formulation of natural ingredients in combination to inhibit glycolytic pathway for the treatment of cancer. The ingredients exhibit synergistic effect in combination in mitigating the cell cycle and inducing apoptosis of cancer cells. The present invention also discloses a process for preparation of the formulation.
[0020] The formulation comprises the fruit of Carum carvi at a concentration in a range between 6g to 8g, fruit of Piper nigrum at a concentration in a range between 3g to 5g, fruits of Morus indica Griff at a concentration in a range between 19g to 21g, root of Withania somnifera at a concentration in a range between 3g to 5g, bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g, seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g, sorbitol at a concentration in a range between 10g to 21g, glycerin at concentration in a range between 14g to 16g, sodium benzoate at a concentration of 0.2g, sodium methyl paraben at a concentration of 0.2 g, sodium propyl paraben at a concentration of 0.1 g, bronopol at a concentration of 0.1 g, mint or orange flavour at a concentration of 0.1 g, and distilled water.
[0021] The present invention also discloses a process of preparation of the formulation. The process of preparation starts with mixing all the ingredients in the specified concentration. The ingredients include fruit of Carum carvi at a concentration in a range between 6g to 8g, fruit of Piper nigrum at a concentration in a range between 3g to 5g, fruits of Morus indica Griff at a concentration in a range between 19g to 21g, root of Withania somnifera at a concentration in a range between 3g to 5g, bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g, seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g. After mixing, the ingredients are dissolved in suitable amount of water and heated to 75°C for 30 minutes to form a uniform solution. This is followed by the dissolving the preservatives namely sodium benzoate at a concentration of 0.2g, sodium methyl paraben at a concentration of 0.2 g, sodium propyl paraben at a concentration of 0.1 g, bronopol at a concentration of 0.1 g separately. Sorbitol, glycerin and the extracts are mixed by stirring for 5 to 10 minutes to form a syrup and the previously dissolved preservatives are added, and volume is made up to the quantity of 100 ml. Finally, the flavor is added, and the mixture is stirred for 5 minutes, and the pH of the formulation is adjusted between 5.5 to 6.5. The flavors used include mint or orange at a concentration of 0.1 g.
[0022] The formulation is safe without inducing any toxicity in rats and also exhibited in vitro cytotoxicity in various cancer cells including breast carcinoma, colorectal carcinoma, colorectal adenocarcinoma and chronic myelogenous leukemia.
[0023] The inhibition of specific steps of glycolysis by the use of the formulation due to the presence of ingredients are useful for targeting inhibition of glycolysis. Thus, targeting specific steps, which switches the growth of normal cells to cancer cells is effective in halting the proliferation of cells and thus reducing growth of cancer cells.
[0024] The formulation is prepared in the form of liquid and is safe, effective as an add on therapy to cancer. The presence of natural ingredients does not indue any side effects in long term usage.
Brief description of the drawings
[0025] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0026] FIG 1 tabulates the ingredients of the liquid formulation according to an embodiment of the invention.
[0027] FIG 2 illustrates a process for preparation of the liquid formulation according to an embodiment of the invention.
[0028] FIG 3 tabulates the percentage of growth inhibition of breast carcinoma.
[0029] FIG 4 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation.
[0030] FIG 5 tabulates the percentage of growth inhibition of colorectal carcinoma.
[0031] FIG 6 indicates the percentage of inhibition of growth of colorectal carcinoma cells by the formulation.
[0032] FIG 7 tabulates the percentage of growth inhibition of colorectal adenocarcinoma.
[0033] FIG 8 indicates the percentage of inhibition of growth of colorectal adenocarcinoma cells by the formulation.
[0034] FIG 9 tabulates the percentage of growth inhibition of chronic myelogenous leukemia.
[0035] FIG 10 indicates the percentage of inhibition of growth of chronic myelogenous leukemia cells by the formulation.
[0036] FIG 11 tabulates the results of the effect of the formulation on tumor volume.
[0037] FIG 12 indicates the results of the effect of the formulation on tumor volume.
[0038] FIG 13 indicates the results of the effect of the formulation on tumor volume.
[0039] FIG 14 indicates the epithelial neoplasia driven by loss of Scrib tumors in Drosophila.
[0040] FIG 15 indicates the dose dependent anticancerous effect of the liquid formulation of the present invention in Drosophila.
Detailed description of the invention
[0041] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0042] The term “Glycolysis” refers to cytoplasmic pathway, which breaks down glucose into two three-carbon compounds and generates energy.
[0043] The term “Cytotoxicity” refers to the toxicity caused due to the action of chemotherapeutic agents on living cells.
[0044] The present invention discloses a formulation with a combination of the natural ingredients to inhibit glycolytic pathway for the treatment of cancer. The formulation is targeted to mitigate the glycolytic pathway of the cancer cells by inducing apoptosis, cell cycle arrest and inhibiting the metastasis thus exhibiting anticancer activity.
[0045] According to an embodiment of the invention, the formulation comprises combination of different ingredients at a specified concentration.
[0046] FIG 1 tabulates the ingredients of the liquid formulation according to an embodiment of the invention. The formulation comprises the fruit of Carum carvi at a concentration in a range between 6g to 8g, fruit of Piper nigrum at a concentration in a range between 3g to 5g, fruits of Morus indica Griff at a concentration in a range between 19g to 21g, root of Withania somnifera at a concentration in a range between 3g to 5g, bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g, seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g, sorbitol at a concentration in a range between 10g to 21g, glycerin at concentration in a range between 14g to 16g, sodium benzoate at a concentration of 0.2 g, sodium methyl paraben at a concentration of 0.2 g, sodium propyl paraben at a concentration of 0.1 g, bronopol at a concentration of 0.1 g, mint or orange flavour at a concentration of 0.1 g, and distilled water.
[0047] The ingredient of the formulation exhibits synergistic effect in mitigating the glycolysis and the cell cycle. The presence of the add on ingredients are focused to combat the nausea and vomiting associated with chemo cytotoxic effects. The addition of other ingredients with Picrasma Quassioides as adjuvant ingredients optimize digestive juice and positive effect of gut biome thus enhancing the immune system further.
[0048] Carum carvi is habitat from Kashmir, Godhwal and border states. The shrubs grow 0.75 to 1 meter in height and cultivated as a cold season crop in plains of India and as summer crop in hilly areas of Kashmir, Kumaon, Garhwal and Chamba. It contains volatile oil, which gives a specific scent to black cumin. Orally, it improves taste, appetite and digestion, constipation and anti-flatulent, therefore useful in bad breath, anorexia, vomiting, loss of appetite, diarrhea, indigestion, flatulence and dysentery. The fruits are used in the formulation and are greenish brown, slightly curved, elongated, mericarps, usually separate, free from the pedicel, carpophores, up to 7 mm long, 2 mm broad almost equally five sided, narrow, tapering to each end, arcuate, glabrous, brown with five very narrow, yellowish primary ridges' endosperm, orthospermous, odour and taste, aromatic and characteristic.
[0049] Piper nigrum is a fully mature dried fruit, which is a climber, cultivated from Konkan Southwards, especially in North Konkan Kerala, and also in Assam, fruits ripen from December to March, depending upon climatic conditions, fruits harvested from December to April. The fruits are used in the formulation of the present invention that appear green in colour when tender, turns red on ripening and black on drying. Fruits are hard, wrinkled, 0.4-0.5 cm in diameter, odour, aromatic; taste, pungent. The skin of the fruit contains piperine a volatile compound, piperidine aromatic oil and fatty acids. The fruit pulp has bitter resin chavicin, starch, oil gums, fats, protein and alkaloids. It clears the deposit of kapha on the tongue and chest, it is livers stimulant, vermifuge.
[0050] Morus indica Griff is an evergreen tree or shrub of 3 to 6 m high, with bright green ovate and deeply veined leaves which are 10 to 30 cm long. The fruit grows in size up to 12 cm or more and has a lumpy surface covered by polygonal-shaped sections. It is a native range extends across Southeast Asia and Australasia. The fruit is rich in vitamin C, biotin, folate, vitamin E and plant-based flavonoids, organic acids with antioxidant properties. These beneficial components aid in building a powerful defense mechanism to keep disease at bay, revitalizing skin, resolving dandruff in hair, scalp, aside from ensuring healthy red blood cell synthesis and circulation. The fruits comprise a number of phytochemicals, including lignans, oligo- and polysaccharides, flavonoids, iridoids, fatty acids, scopoletin, catechin, beta-sitosterol, and alkaloids.
[0051] Withania somnifera is a perennial shrub, found in waste land, cultivated field and open grounds throughout India, widely cultivated in certain areas of Madhya Pradesh and Rajasthan , roots are collected in the winterseason, washed and cut into short pieces. The roots are straight, unbranched and the thickness varies with age. The chemical constituents mainly include alkaloids, steroidal lactones and saponins. It exhibits anxiolytic effect and improves energy levels and mitochondrial health. In addition, the roots also exhibit anti-inflammatory and anti-arthritic activities and is effective against tuberculosis, asthma, and may increase thyroid hormone levels, which could cause fatigue, anxiety, short ness of breath.
[0052] Picrasma Quassioides is a species of Picrasma native to temperate regions of southern Asia from the northeast of Pakistan along the Himalaya and through southern, central and eastern China to Taiwan and Japan. It is a deciduous shrub or small tree growing to 10–15 m (rarely 20 m) tall with a trunk up to 50 cm diameter. The bark is smooth and dark grey and brown. The leaves are 15–40 cm long, pinnate, with 7–15 leaflets 2.5–10 cm long and 1.5–4.5 cm broad, with a coarsely and irregularly toothed margin. The flowers are green to yellow green with four or five sepals and petals, produced in cymes 8–15 cm long in mid to late spring. The fruit is an ovoid to globose red to black drupe 6–7 mm diameter. The bark is used in herbal medicine as a bitter flavoring and antibacterial agent.
[0053] Caesalpinia bonduc is an extensive, shrubby, wild perennial climber distributed through tropical parts of India. The seeds are globose or rounded, smooth, shiny, 1.2 to 2.5 cm in diameter slightly flattened on one side due to close pressing of adjacent seeds. It contains bitter substances namely phytosterenin, bonducin, saponin, phytosterol, fixed oil, starch and sucrose. In addition, the seeds also contain α, β, γ, δ and ζ caesalpins, which possess antibacterial, antidiarrheal, and cytotoxic activities.
[0054] The formulation of the present invention is prepared in the form of liquid. The present invention also discloses the specific process for preparation of the formulation.
[0055] FIG 2 illustrates a process for preparation of the formulation. The process (200) of the present invention starts with a step of (201) mixing all the ingredients in the specified concentration. The ingredients include fruit of Carum carvi at a concentration in a range between 6g to 8g, fruit of Piper nigrum at a concentration in a range between 3g to 5g, fruits of Morus indica Griff at a concentration in a range between 19g to 21g, root of Withania somnifera at a concentration in a range between 3g to 5g, bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g, seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g. At step (202), the ingredients are dissolved in suitable amount of water and heated to 75°C for 30 minutes to form a uniform solution. At step (203), the preservatives namely sodium benzoate at a concentration of 0.2g, sodium methyl paraben at a concentration of 0.2 g, sodium propyl paraben at a concentration of 0.1 g, bronopol at a concentration of 0.1 g are dissolved in separate containers in small amount of water. At step (204), sorbitol, glycerin and the extracts are mixed by stirring for 5 to 10 minutes to form a syrup. At step (205), the previously dissolved preservatives are added, and volume is made up to the quantity of 100 ml. At step (206), the flavor is added, and the mixture is stirred for 5 minutes, and the pH of the formulation is adjusted between 5.5 to 6.5. The flavors used in the formulation include mint or orange at a concentration of 0.1 g.
[0056] The present invention discloses the formulation that targets the glycolytic pathway. The blocking of conversion of glyceraldehyde-3-phosphate to 1, 3-bisphosphoglycerate during the payoff phase of glycolysis in the presence of glyceraldehyde phosphate dehydrogenase is achieved by glyceraldehyde phosphate dehydrogenase inhibitor or glyceraldehyde-3-phosphate inhibitor. The blockade of conversion of glyceraldehyde-3-phosphate to 1, 3-bisphosphoglycerate inhibits the glycolysis pathway thus preventing the accumulation of NAD+. The present invention discloses an agent targeting oxidation of NADH. The inhibition of oxidation of NADH reduces the accumulation of NAD thus halting the glycolysis and preventing the growth of cancer cells. Finally, at the end of glycolysis, the pathway shifts to an adaptive pathway converting pyruvate to lactate in the presence of pyruvate dehydrogenase providing a constant pool of NAD+ to continue glycolytic pathway aiding the growth of cancer cells. The blockade of conversion of pyruvate to lactate by pyruvate inhibitors inhibits glycolysis thus preventing the accumulation of NAD+. The absence of NAD+ reduces the proliferation of cancer cells thus limiting the growth of the cancer cells and prevents carcinogenesis.
[0057] The inhibition of specific steps of glycolysis by glyceraldehyde phosphate dehydrogenase inhibitor or glyceraldehyde-3-phosphate inhibitor, pyruvate inhibitor and oxidation inhibitors are useful for targeting inhibition of glycolysis. Thus, targeting specific steps, which switches the growth of normal cells to cancer cells is effective in halting the proliferation of cells and thus reducing growth of cancer cells.
[0058] The formulation is effective in inhibiting cancer growth by inducing the apoptosis of cancer cells of both solid and liquid tumors, inducing cell cycle arrest. The formulation is effective in halting metastasis by inhibiting the passing through the extracellular matrix, interaction with host lymphoid cells, and adhesion to basement membranes to form metastases.
[0059] The formulation is prepared in the form of a liquid for oral administration and is consumed twice a day as an add-on therapy for cancer.
[0060] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner:
Example 1: Evaluation of the toxicity of the formulation in rats

[0061] The formulation of the present invention prepared by said process is analyzed for its toxicity in Sprague Dawley. The animal studies are conducted in compliance with the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animals facility published in the gazette of India, 1998 and Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
[0062] The formulation is prepared at different dose levels of 500, 1000 and 2000 mg/kg b.w./day at a concentration of 50, 100 and 200 mg/mL. The rats are divided into four groups and are administered with 500, 1000 and 2000 mg/kg b.w./day at a same time for 14 consecutive days. The formulation of the present invention is administered orally. The parameters such as body weight, hematology, clinical chemistry, coagulation is analyzed.
[0063] The results indicated that the formulation is safe and does not induce any toxicity in rats.
[0064] The cytotoxicity of the formulation of the present invention is analyzed in various cell lines.
Example 2: Evaluation of the cytotoxicity of the formulation in breast adenocarcinoma

[0065] The formulation of the present invention is analyzed for the cytotoxicity in MDA-MB-231 cell line. The cytotoxicity is analyzed using MTT assay. The formulation is analyzed with various concentration using doxorubicin as reference drug. The formulation is used at the concentrations of 7.8 µg/mL, 15.6 µg/mL, 31.3 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL. The percentage of inhibition of growth of the adenocarcinoma cells are analyzed.
[0066] FIG 3 tabulates the percentage of growth inhibition of breast adenocarcinoma. The results indicated that the formulation in the form of liquid is effective in inhibition of the growth of the breast cancer cells with 64% of growth inhibition by the formulation at 1000 µg/mL, 56% of growth inhibition by the formulation at 500 µg/mL, 43% of growth inhibition by the formulation at 250 µg/mL, 44% of growth inhibition by the formulation at 125 µg/mL, 42% of growth inhibition by the formulation at 62.5 µg/mL, 32% of growth inhibition by the formulation at 31.3 µg/mL, 24% of growth inhibition by the formulation at 15.6 µg/mL and 0% of growth inhibition by the formulation at 7.8 µg/mL.
[0067] FIG 4 indicates the percentage of inhibition of growth of breast adenocarcinoma cells by the formulation. The formulation exhibited cytotoxicity IC50 of 501.1 µg/mL in contrast to IC50 of 4.9 µM for doxorubicin indicating an effective rate of inhibition.
Example 3: Evaluation of the cytotoxicity of the formulation in colorectal carcinoma

[0068] The formulation of the present invention is analyzed for the cytotoxicity in HCT-116 cell line. The cytotoxicity is analyzed using MTT assay. The formulation is analyzed with various concentrations using 5-fluorouracil as reference drug. The formulation is used at the concentrations of 7.8 µg/mL, 15.6 µg/mL, 31.3 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL. The percentage of inhibition of growth the colorectal carcinoma cells are analyzed.
[0069] FIG 5 tabulates the percentage of growth inhibition of colorectal carcinoma. The results indicated that the formulation in the form of liquid is effective in inhibition of the growth the colorectal cancer cells with 53% of growth inhibition by the formulation at 1000 µg/mL, 41% of growth inhibition by the formulation at 500 µg/mL, 28% of growth inhibition by the formulation at 250 µg/mL, 30% of growth inhibition by the formulation at 125 µg/mL, 25% of growth inhibition by the formulation at 62.5 µg/mL, 24% of growth inhibition by the formulation at 31.3 µg/mL, 18% of growth inhibition by the formulation at 15.6 µg/mL and 11% of growth inhibition by the formulation at 7.8 µg/mL.
[0070] FIG 6 indicates the percentage of inhibition of growth of colorectal carcinoma cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 727.6 µg/mL in contrast to IC50 of 22.4 µM for 5-fluorouracil indicating an effective rate of inhibition.
Example 4: Evaluation of the cytotoxicity of the formulation in colorectal adenocarcinoma

[0071] The formulation of the present invention is analyzed for the cytotoxicity in HT-29 cells line. The cytotoxicity is analyzed using MTT assay. The formulation is analyzed with various concentrations using 5-fluorouracil as reference drug. The formulation is used at the concentrations of 7.8 µg/mL, 15.6 µg/mL, 31.3 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL. The percentage of inhibition of growth the colorectal adenocarcinoma cells are analyzed.
[0072] FIG 7 tabulates the percentage of growth inhibition of colorectal adenocarcinoma. The results indicated that the formulation in the form of liquid is effective in inhibition of the growth the colorectal cancer cells with 71% of growth inhibition by the formulation at 1000 µg/mL, 54% of growth inhibition by the formulation at 500 µg/mL, 38% of growth inhibition by the formulation at 250 µg/mL, 31% of growth inhibition by the formulation at 125 µg/mL, 24% of growth inhibition by the formulation at 62.5 µg/mL, 19% of growth inhibition by the formulation at 31.3 µg/mL, 15% of growth inhibition by the formulation at 15.6 µg/mL and 9% of growth inhibition by the formulation at 7.8 µg/mL.
[0073] FIG 8 indicates the percentage of inhibition of growth of colorectal adenocarcinoma cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 711.0 µg/mL in contrast to IC50 of 22.4 µM for 5-fluorouracil indicating an effective rate of inhibition.
Example 5: Evaluation of the cytotoxicity of the formulation in chronic myelogenous leukemia

[0074] The formulation of the present invention is analyzed for the cytotoxicity in K562 cells line. The cytotoxicity is analyzed using MTT assay. The formulation is analyzed with various concentrations using doxorubicin as reference drug. The formulation is used at the concentrations of 7.8 µg/mL, 15.6 µg/mL, 31.3 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL. The percentage of inhibition of growth of the chronic myelogenous leukemia cells are analyzed.
[0075] FIG 9 tabulates the percentage of growth inhibition of chronic myelogenous leukemia. The results indicated that the formulation in the form of liquid is effective in inhibition of the growth the colorectal cancer cells with 68% of growth inhibition by the formulation at 1000 µg/mL, 59% of growth inhibition by the formulation at 500 µg/mL, 40% of growth inhibition by the formulation at 250 µg/mL, 33% of growth inhibition by the formulation at 125 µg/mL, 21% of growth inhibition by the formulation at 62.5 µg/mL, 16% of growth inhibition by the formulation at 31.3 µg/mL, 17% of growth inhibition by the formulation at 15.6 µg/mL and 11% of growth inhibition by the formulation at 7.8 µg/mL.
[0076] FIG 10 indicates the percentage of inhibition of growth of chronic myelogenous leukemia cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 418.3 µg/mL in contrast to IC50 of 3.4 µM for doxorubicin indicating an effective rate of inhibition.

[0077] The dosing derived from comparative data with allopathic cytotoxic drugs including doxorubicin interpreted a specific balance between the cell kill and as a result of cell kill, the antigen released lead to immune activate of innate cells i.e. the Toll pathway leading to insulin pathway blockage.
Example 5: Evaluation of the anti-tumor efficacy of the formulation in FaDu xenograft model of head and neck cancer in mice

[0078] The formulation of the present invention is analyzed for the anti-tumor activity in FaDu xenograft model of head and neck cancer in athymic nude mice. The study is approved by the Institutional Animal Ethics committee of Anthem Biosciences (CPCSEA Registration No. 1192/PO/RcBt/S/08/CPCSEA). The experimental procedures would be performed in accordance with the protocol approved by the IAEC (Protocol No. ABD/IAEC/PR/189-20-23) and the CPCSEA guidelines for animal experimentation. The animals are divided into five groups with six animals in each. Cisplatin in the form of Active Pharmaceutical Ingredient is used as reference drugs in the study. Group 1 is administered with vehicle alone, Group 2 animals are administered with Cisplatin (API) at a concentration of 5 mg/kg, Group 3 animals are administered with the formulation of present invention at a concentration of 500 mg/kg, Group 4 animals are administered with the formulation of present invention at a concentration of 1000 mg/kg, Group 5 animals are administered with the formulation of present invention at a concentration of 3000 mg/kg.

[0079] The formulation is administered orally once daily for 14 days. The reference compounds are administered twice weekly for 14 days.

[0080] The animals of all the groups are tested for different parameters such as body weight, tumor growth and the signs of morbidity and mortality. The tumor growth is measured twice weekly by using a digital Vernier caliper. Tumor volume will be calculated as follows:
Tumor Volume = [Length (L) × Width (W) 2] /2
Where length (L) is the largest diameter of the tumor, Width (W) is the smallest diameter of the tumor.
[0081] Tumor growth inhibition (TGI) is calculated based on the following formula:
% TGI = {(TVvehicle Final - TVvehicle Initial) - (TVtreatment Final - TVtreatment Initial)} x 100 (TVvehicle Final - TVvehicle Initial)

[0082] FIG 11 tabulates the results of the effect of the formulation on tumor volume. The efficacy of the formulation is analyzed on tumor volume and percentage of tumor growth index from day 0 to day 14. The results indicated that the formulation in the form of liquid is effective in inhibiting tumor volume and the percentage of tumor growth index.

[0083] FIG 12 indicates the results of the effect of the formulation on tumor volume. The results indicated that the formulation in the form of liquid is effective in inhibiting tumor volume and the percentage of tumor growth index.

[0084] FIG 13 indicates the results of the effect of the formulation on tumor volume. The results indicated that the formulation in the form of liquid at various concentrations is effective in inhibiting tumor volume and the percentage of tumor growth index for 14 days.

Example 6: In vivo validation of the formulation using Drosophila tumor model

[0085] The formulation of the present invention is analyzed for anticancer activity in Drosophila tumor model. Tumor is induced in the developing wing epithelium of the larvae of Drosophila . Scribble (scrib) is a tumor suppressor gene (TSG) which is responsible for the maintenance of apical-basal polarity of the cells, upon loss of which disrupts cell polarity eventually leading to tumor induction in Drosophila.

[0086] FIG 14 indicates the epithelial neoplasia driven by loss of Scrib tumors in Drosophila. FIG 14 (A-C) displays the apico-basal polarity of wild type Drosophila wing epithelium and localization of a scribble tumor suppressor protein to maintain healthy polarized epithelium.

[0087] FIG 14 (D-F) indicates that the cells lose polarity and becomes tumorous upon loss of scrib tumor suppressor.

[0088] FIG 14 (G) illustrates the induction of scrib tumor in Drosophila by using the UAS-Gal4 system in Drosophila wing epithelium in a tissue specific manner.

[0089] FIG 14 (H) indicates the induction of lethality by vg>scrib-i tumors and drug optimization to restore the survival in Drosophila.
Example 6: The formulation of the present invention eliminates the tumor cells through induction of apoptotic cell death pathway.
[0090] The epithelial neoplasia driven by loss of Scrib tumor suppressor i.e. actin in vestigial domain of larval wing disc using vg>scrib-RNAi (GFP) lead to pseudo-peupal lethality at room temperature. Drosophila are fed with different concentrations of the formulation of the present invention i.e. 0.4 µl/ml, 0.01 µl/ml and 0.04 µl/ml.

[0091] FIG 15 indicates the dose dependent anticancerous effect of the liquid formulation of the present invention in Drosophila. The results indicated that .4 µl/ml (vg>scribi) and the control vg>scrib-I tumor bearing Drosophila exhibited pseudo-peupal lethality. In contrast, Drosophila fed with 0.01 µl/ml and 0.04 µl/ml of the formulation exhibited tumor suppression marked by cell death (caspase) restoration of the wing epithelium (actin) and resulted in the restoration of Drosophila 62.05% and 62.07% pharate and eclosion respectively.

[0092] The anticancer mechanism may be due to cancer resistance upon oncogenic hit i.e. loss of Scrib by vg>scrib>RNAi and the mutant cells are eliminated through the tissue homeostasis by immune healthy cells by inducing apoptotic cell death pathway.

[0093] The liquid formulation of the present invention is used as metronomic dosing to achieve effective cell kill, minimize toxicity and activate the immune system especially innate macrophages.

[0094] The formulation of the present invention does not affect the body weight, histological parameters and hence is safe and effective as an add on therapy for cancer.

[0095] The formulation of the present invention with a combination of specific herbal ingredients is safe, effective in exhibiting the synergistic effect in inducing cytotoxicity in different carcinoma cell lines and tumor growth thus promising wide range of application.

, Claims:We Claim:
1. A formulation with a combination of herbal ingredients for the treatment of cancer, the formulation comprises:
a. fruit of Carum carvi at a concentration in a range between 6g to 8g;
b. fruit of Piper nigrum at a concentration in a range between 3g to 5g;
c. fruits of Morus indica Griff at a concentration in a range between 19g to 21g;
d. root of Withania somnifera at a concentration in a range between 3g to 5g;
e. bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g;
f. seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g;
g. sorbitol at a concentration in a range between 10g to 21g;
h. glycerin at concentration in a range between 14g to 16g;
i. sodium benzoate at a concentration of 0.2g;
j. sodium methyl paraben at a concentration of 0.2 g;
k. sodium propyl paraben at a concentration of 0.1 g;
l. bronopol at a concentration of 0.1 g;
m. a flavour at a concentration of 0.1 g; and
n. distilled water
wherein said ingredients are blended in a sequential manner to obtain uniform solution.

2. The formulation as claimed in claim 1, wherein the flavour used in the formulation is selected from mint or orange.

3. The formulation as claimed in claim 1, wherein the formulation is in the form of liquid for oral administration.

4. The formulation as claimed in claim 1, wherein the volume of the formulation is made up to 100 ml with said ingredients.

5. The formulation as claimed in claim 1, wherein the formulation exhibits cytotoxic activity in a carcinoma cell.

6. The formulation as claimed in claim 1, wherein the carcinoma cells are selected from a group of breast adenocarcinoma, colorectal carcinoma, colorectal adenocarcinoma and chronic myelogenous leukemia.

7. The formulation as claimed in claim 1, wherein the formulation inhibits cancer growth by inducing apoptosis of cancer cells of both solid and liquid tumors, inducing cell cycle arrest.

8. A process for preparation of the formulation of herbal ingredients, the process (200) comprising the steps of:
a. mixing the plurality of the ingredients in a specified concentration (201);
b. dissolving the mixed in a suitable amount of water and subjecting to heating to 75°C for 30 minutes to form a uniform solution (202);
c. dissolving one or more preservatives in separate containers using distilled water (203);
d. mixing sorbitol, glycerin and the ingredients by stirring for 5 to 10 minutes to form a syrup (204);
e. adding the dissolved preservatives of step c and making up the volume to the quantity of 100 ml (205); and
f. adding the flavor and stirring the mixture for 5 minutes and adjusting the pH of the formulation between 5.5 to 6.5 (206).

9. The process as claimed in claim 8, wherein the herbal ingredients include fruit of Carum carvi at a concentration in a range between 6g to 8g, fruit of Piper nigrum at a concentration in a range between 3g to 5g, fruits of Morus indica Griff at a concentration in a range between 19g to 21g, root of Withania somnifera at a concentration in a range between 3g to 5g, bitter wood of Picrasma Quassioides at a concentration in a range between 3g to 5 g, seed of Caesalpinia bonduc at a concentration in a range between 0.5g to 1.5g.

10. The process as claimed in claim 8, wherein the preservatives are selected from a group comprising sodium benzoate at a concentration of 0.2g ,sodium methyl paraben at a concentration of 0.2 g, sodium propyl paraben at a concentration of 0.1 g, bronopol at a concentration of 0.1 g.