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 topical 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 topical 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 is a genetic disease and is caused by changes to genes that control the way the cells function, especially the manner in which the cells grow and divide. Cancer cells differ from normal cells in many ways including grow in the absence of signals telling them to grow, the normal cells only grow when they receive such signals, ignore signals that normally tell cells to stop dividing or to die i.e., programmed cell death, or apoptosis, invade into nearby areas and spread to other areas of the body, normal cells stop growing when they encounter other cells, and most normal cells do not move around the body, tell blood vessels to grow toward tumors, these blood vessels supply tumors with oxygen and nutrients and remove waste products from tumors, hide from the immune system, the immune system normally eliminates damaged or abnormal cells, trick the immune system into helping cancer cells stay alive and grow, for instance, some cancer cells convince immune cells to protect the tumor instead of attacking it, accumulate multiple changes in their chromosomes, such as duplications and deletions of chromosome parts. Some cancer cells have double the normal number of chromosomes. rely on different kinds of nutrients than normal cells. In addition, some cancer cells make energy from nutrients in a different way than most normal cells. This lets cancer cells grow more quickly.
[0005] A cancer that has spread from the place where it first formed to another place in the body is called metastatic cancer. The process by which cancer cells spread to other parts of the body is called metastasis. Metastatic cancer has the same name and the same type of cancer cells as the original, or primary, cancer. For example, breast cancer that forms a metastatic tumor in the lung is metastatic breast cancer, not lung cancer.
[0006] There is a constant demand for new therapies to treat and prevent cancer. Scientific and research interest is drawing its attention towards naturally derived compounds as they are considered to have less toxic side effects compared to current treatments such as chemotherapy. The Plant Kingdom produces naturally occurring secondary metabolites that are being investigated for their anticancer activities leading to the development of new clinical drugs. With the success of these compounds that have been developed into staple drugs for cancer treatment new technologies are emerging to develop the area further.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] Though it is know the role of herbs to possess effective anti-cancer activities, however till date they have not been combined together to prepare an effective herbal anti-cancer formulation or a composition for use in the treatment of cancer patients. There is a necessity to harness the anti-cancer activity of all these important herbs to prepare an effective herbal anti-cancer formulationIn 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
[0012] The present invention overcomes the drawback of the state of art by providing the topical formulation for the treatment of cancer. The invention discloses a topical formulation as a cream consisting of combination of natural ingredients that targets 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.
[0013] The formulation comprises the leaf of Picrasma quassioides at a concentration in a range between 15g to 17g, root of Withania somnifera at a concentration in a range between 1g to 2g, seeds of Myristica fragrans Houtt at a concentration in a range between 0.5 to 1.5g, leaves of Aloe vera at a concentration in a range between 1g to 3g, rhizome of Curcuma longa at a concentration in a range between 1g to 3g, nuts of Vitellaria paradoxa at a concentration in a range between 1g to 3g, fruits of Psoralea corylifolia at a concentration in a range between 1g to 3g, hyaluronic acid at a concentration of 0.2g, glycerin at a concentration in a range between 2g to 4g, GMS at a concentration in a range between 5g to 6g, imidurea at a concentration in a range between 0.2g to 0.4g, bronopol at a concentration of 0.1g, BHT at a concentration of 0.1g and distilled water.
[0014] The formulation is prepared as a cream for topical administration. The formulation of Picrasma quassioides with the combination of other ingredients aids in combating the nausea and vomiting associated with chemo cytotoxic effects.
[0015] The present invention also discloses a process for preparation of the topical formulation, The process of the preparation of the formulation starts with the step of mixing all the ingredients in dissolved water and heated to 75°C to form a uniform solution. The ingredients oils and wax namely GMS at a concentration of 5.5g, imidurea at a concentration of 0.3g, bronopol at a concentration of 0.1g, BHT at a concentration of 0.1g are dissolved in separate containers and heated up to 75 °C. Then, the wax phase is mixed to aqueous phase after attaining the equilibrium temperature with constant stirring. The preservatives namely imidurea and bronopol are added separately with minimal amount of distilled water, and the volume is made up to the quantity of 100 ml.
[0016] The formulation of the present invention is effective in inducing in vitro cytotoxicity in various cancer cells including breast carcinoma, colorectal carcinoma, colorectal adenocarcinoma and chronic myelogenous leukemia.
[0017] The formulation is prepared in the form of cream and is safe, effective as 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
[0018] 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.
[0019] FIG 1 tabulates the ingredients of the topical formulation according to an embodiment of the invention.
[0020] FIG 2 illustrates a process for preparation of the topical formulation according to an embodiment of the invention.
[0021] FIG 3 tabulates the percentage of growth inhibition of breast carcinoma.
[0022] FIG 4 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation.
[0023] FIG 5 tabulates the percentage of growth inhibition of colorectal carcinoma.
[0024] FIG 6 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation.
[0025] FIG 7 tabulates the percentage of growth inhibition of colorectal adenocarcinoma.
[0026] FIG 8 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation.
[0027] FIG 9 tabulates the percentage of growth inhibition of chronic myelogenous leukemia.
[0028] FIG 10 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation.
[0029] FIG 11 tabulates the results of the effect of the formulation on tumor volume.
[0030] FIG 12 indicates the results of the effect of the formulation on tumor volume.
[0031] FIG 13 indicates the results of the effect of the formulation on tumor volume.
Detailed description of the invention
[0032] 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.
[0033] The term “Glycolysis” refers to cytoplasmic pathway, which breaks down glucose into two three-carbon compounds and generates energy.
[0034] The term “Cytotoxicity” refers to the toxicity caused due to the action of chemotherapeutic agents on living cells.
[0035] The present invention discloses a formulation with a combination of the natural ingredients to inhibit glycolytic pathway for the treatment of cancer. The topical 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.
[0036] According to an embodiment of the invention, the formulation comprises combination of different ingredients at a specified concentration.
[0037] The concentration of the natural ingredients is standardized and comprises Picrasma Quassioides, Withania somnifera, Myristica fragrans, Aloe vera, Curcuma longa, Vitellaria paradoxa, Psoralea corylifolia, hyaluronic acid, glycerin, GMS, imidurea, bronopol and BHT.
[0038] FIG 1 tabulates the ingredients of the formulation according to an embodiment of the invention. The formulation comprises the leaf of Picrasma Quassioides at a concentration in a range between 15g to 17g, root of Withania somnifera at a concentration in a range between 1g to 2g, seeds of Myristica fragrans Houtt at a concentration in a range between 0.5 to 1.5g, leaves of Aloe vera at a concentration in a range between 1g to 3g, rhizome of Curcuma longa at a concentration in a range between 1g to 3g, nuts of Vitellaria paradoxa at a concentration in a range between 1g to 3g, fruits of Psoralea corylifolia at a concentration in a range between 1g to 3g, hyaluronic acid at a concentration of 0.2g, glycerin at a concentration in a range between 2g to 4g, GMS at a concentration in a range between 5g to 6g, imidurea at a concentration in a range between 0.2g to 0.4g, bronopol at a concentration of 0.1g, BHT at a concentration of 0.1g and distilled water.
[0039] The ingredient of the formulation exhibits synergistic effect in mitigating the glycolysis and the cell cycle. The leaves of Picrasma Quassioides commonly known as Nigaki 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. 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 leaves are used as anti-inflammatory, antioxidant, anti-malarial, anti feedant, anti-cancer, anti-ulcer agents.
[0040] 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. The roots are collected in winter, washed and cut into short pieces. Withania somnifera mainly consists of alkaloids, steroidal lactones and saponins Withania somnifera exhibits anxiolytic effect and improves energy levels and mitochondrial health. It is an anti-inflammatory and anti-arthritic, tumors, antistress, adaptogenic properties tuberculosis, asthma, and increase thyroid hormone levels, which could cause fatigue, anxiety, and shortness of breath.
[0041] Myristica fragrans consists of the endosperm of dried seeds (of Myristica fragrans Houtt. (Fam. Myristicaceae), dioecious or occasionally monoecious aromatic tree, about 10-20 m high, found mostly in Tamil Nadu and to some extent in Kerala, Andhra Pradesh and Assam. The seed are ellipsoid, 20-30 mm long and about 20 mm broad, externally greenish brown sometimes marked with small irregular dark brown patches or minute dark points and lines slightly furrowed reticulate, a small light-colored area at one end indicating the position of the radical a groove running along the line of raphe to the darker chalaza at the opposite end, small with two widely spreading crumpled cotyledons and a small radicle odour, strong and aromatic, taste, pungent and aromatic. It contains essential oil and fixed oil. Myristica fragrans contains 5% to 15% volatile oil, lignin, stearin, starch, gum, colouring matter, and 0.08% of an acid substance. The volatile oil contains clemicine, myristicin, geraniol, borneol, pinene, camphene, and dipentene. It also contains eugenol, safrol, p-cymene and isoeugenol in small quantity, which is rich source of antioxidants, which help protect against the signs of aging and serious conditions such as cancer, heart disease, and liver disease, the aromatic properties are used in carminative, flavoring agent, flatulence, in allay nausea and vomiting.
[0042] Aloe vera is a perennial under shrub about 2-4 feet in height with fleshy succulent leaves having horny prickles on their margins. It consists of leaves of Aloe vera Tourn. ex Linn, (Fam. Liliaceae), shrub planted in many Indian gardens and found growing throughout India. Aloe vera consists of amino acids, anthraquinones, enzymes, minerals, vitamins, lignins, monosaccharide, polysaccharides, salicylic acid, saponins, and sterols. The antibacterial and antifungal properties of Aloe Vera help to increase blood flow to the affected area, thus it helps in the healing process.
[0043] Curcuma longa is a perennial herb extensively cultivated in all parts of the country. The crop is harvested after 9-10 months when lower leaves turn yellow rhizomes carefully dug up with hand-picks between October-April and cured by boiling and dried. Annual shrub, rhizome grows underground. The flowering of the plant occurs in the beginning of the rainy season. Curcuma comprises 1% volatile oil, resin and the pigment curcumin are responsible for its color. Curcumin exhibits anti-cancer and anti-proliferative property by inducing cancer cell death and prevents the spread of cancer cells respectively. Curcumin also has anti-inflammatory property and prevents tumor development in colorectal cancer.
[0044] Vitellaria paradoxa is a key non-timber forest product occurring largely in off-reserve forests in many parts of Africa. Shea butter tree is a mesopharenophyte and is abundantly found in the wide belt of savannah including African countries. It belongs to the Sapotaceae family and two species of the genus Vitellaria exist: V. paradoxa in West Africa and V. nilotica in East Africa. The mean height of the species is 10 m but can reach 15 m with a mean stem diameter of 50 cm. The species is productive from 15 to 20 years, with an annual kernel production of 2.2 kg per tree. Shea fruit has kernel which contain a great percentage of butter, which had been used over the years for different purposes such as cosmetic, medicinal, biodiesel production and soap processing industries. Shea Butter is known to treat Vata and Pitta Dosha related skin concerns. Shea Butter protects the skin from environmental damage; it has Ultra Violet-B protection which makes it a wonderful ingredient for sun protection. Enriched with Vitamin F and fatty acids, it soothes rough, dry and chapped skin and is also known as a great anti-aging butter. Shea Butter also has anti-inflammatory and healing properties that can calm redness and sensitive skin. The composition of shea butter includes oleic acid, stearic acid and linoleic acid. It is rich in vitamins A, E and C. Shea Butter helps in the management of arthritis by reducing inflammation due to its anti-inflammatory properties. It also has an analgesic property that reduces the pain associated with muscle soreness,
[0045] Psoralea corylifolia is an erect, 0.3-1.8 m high annual herb, distributed throughout India, found commonly in Uttar Pradesh, Bengal and Maharashtra. It contains essential oil, fixed oil, Psoralen, psoralidin, isopsoralen and bakuch oil that are used in the treatment of chemo protective, antioxidant, antimicrobial, and anti-inflammatory properties, thus Bakuchi extract may be found to be effective in destroying the cancer cells of osteosarcoma and lung cancer.
[0046] The formulation of the present invention is prepared in the form of cream for topical administration. The present invention also discloses the specific process for preparation of the formulation for topical administration.
[0047] 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 leaf of Picrasma Quassioides at a concentration in a range between 15g to 17g, root of Withania somnifera at a concentration in a range between 1g to 2g, seeds of Myristica fragrans Houtt at a concentration in a range between 0.5 to 1.5g, leaves of Aloe vera at a concentration in a range between 1g to 3g, rhizome of Curcuma longa at a concentration in a range between 1g to 3g, nuts of Vitellaria paradoxa at a concentration in a range between 1g to 3g, fruits of Psoralea corylifolia at a concentration in a range between 1g to 3g. At step (202), the ingredients are mixed in dissolved water and heated to 75°C to form a uniform solution. At step (203), the ingredients oils and wax namely GMS at a concentration of 5.5g, imidurea at a concentration of 0.3g, bronopol at a concentration of 0.1g, BHT at a concentration of 0.1g are dissolved in separate containers and heated up to 75 °C. At step (204), the wax phase is mixed to aqueous phase after attaining the equilibrium temperature with constant stirring. At step (205), the preservatives namely imidurea and bronopol are added separately with minimal amount of distilled water, and the volume is made up to the quantity of 100 ml.
[0048] 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.
[0049] The ingredient of the formulation exhibits synergistic effect in mitigating the glycolysis and the cell cycle.
[0050] 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.
[0051] 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.
[0052] The cytotoxicity of the formulation of the present invention is analyzed in various cell lines.
Example 1: Evaluation of the cytotoxicity of the formulation in breast carcinoma

[0053] 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 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 the breast carcinoma cells are analyzed.
[0054] FIG 3 tabulates the percentage of growth inhibition of breast carcinoma. The results indicated that the formulation in the form of cream is effective in inhibition of the growth the breast cancer cells with 85% of growth inhibition by the formulation at 1000 µg/mL, 73% of growth inhibition by the formulation at 500 µg/mL, 62% of growth inhibition by the formulation at 250 µg/mL, 57% of growth inhibition by the formulation at 125 µg/mL, 49% of growth inhibition by the formulation at 62.5 µg/mL, 47% of growth inhibition by the formulation at 31.3 µg/mL, 42% of growth inhibition by the formulation at 15.6 µg/mL and 32% of growth inhibition by the formulation at 7.8 µg/mL.
[0055] FIG 4 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation. The formulation exhibited cytotoxicity IC50 of 264.8 µg/mL in contrast to IC50 of 4.9 µM for doxorubicin indicating an effective rate of inhibition.
Example 2: Evaluation of the cytotoxicity of the formulation in colorectal carcinoma

[0056] 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.
[0057] FIG 5 tabulates the percentage of growth inhibition of colorectal carcinoma. The results indicated that the formulation in the form of cream is effective in inhibition of the growth the colorectal cancer cells with 67% of growth inhibition by the formulation at 1000 µg/mL, 58% of growth inhibition by the formulation at 500 µg/mL, 52% of growth inhibition by the formulation at 250 µg/mL, 40% of growth inhibition by the formulation at 125 µg/mL, 36% of growth inhibition by the formulation at 62.5 µg/mL, 36% of growth inhibition by the formulation at 31.3 µg/mL, 29% of growth inhibition by the formulation at 15.6 µg/mL and 29% of growth inhibition by the formulation at 7.8 µg/mL.
[0058] FIG 6 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 308.6 µg/mL in contrast to IC50 of 22.4 µM for 5-fluorouracil indicating an effective rate of inhibition.
Example 3: Evaluation of the cytotoxicity of the formulation in colorectal adenocarcinoma

[0059] 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.
[0060] FIG 7 tabulates the percentage of growth inhibition of colorectal adenocarcinoma. The results indicated that the formulation in the form of cream is effective in inhibition of the growth the colorectal cancer cells with 87% of growth inhibition by the formulation at 1000 µg/mL, 81% of growth inhibition by the formulation at 500 µg/mL, 54% of growth inhibition by the formulation at 250 µg/mL, 37% of growth inhibition by the formulation at 125 µg/mL, 33% of growth inhibition by the formulation at 62.5 µg/mL, 30% of growth inhibition by the formulation at 31.3 µg/mL, 23% of growth inhibition by the formulation at 15.6 µg/mL and 17% of growth inhibition by the formulation at 7.8 µg/mL.
[0061] FIG 8 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 405.5 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 chronic myelogenous leukemia

[0062] 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.
[0063] FIG 9 tabulates the percentage of growth inhibition of chronic myelogenous leukemia. The results indicated that the formulation in the form of cream is effective in inhibition of the growth the colorectal cancer cells with 94% of growth inhibition by the formulation at 1000 µg/mL, 93% of growth inhibition by the formulation at 500 µg/mL, 73% of growth inhibition by the formulation at 250 µg/mL, 63% of growth inhibition by the formulation at 125 µg/mL, 54% of growth inhibition by the formulation at 62.5 µg/mL, 51% of growth inhibition by the formulation at 31.3 µg/mL, 41% of growth inhibition by the formulation at 15.6 µg/mL and 22% of growth inhibition by the formulation at 7.8 µg/mL.
[0064] FIG 10 indicates the percentage of inhibition of growth of breast carcinoma cells by the formulation. The formulation exhibited cytotoxicity of IC50 of 104.3 µg/mL in contrast to IC50 of 3.4 µM for doxorubicin indicating an effective rate of inhibition.
Example 5: Evaluation of the anti-tumor efficacy of the formulation in FaDu xenograft model of head and neck cancer in mice

[0065] 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 drug 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.

[0066] The formulation is administered orally once daily for 14 days. The reference compounds are administered twice weekly for 14 days.

[0067] The animals of all the groups are tested for different parameters such as tumor volume, body weight, 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.
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)

[0068] FIG 11 tabulates the results of the effect of the formulation on tumor volume. The efficacy of the formulation is analyzed on tumour volume and percentage of tumour growth index from day 0 to day 14. The results indicated that the formulation in the form of cream is effective in inhibiting tumour volume and the percentage of tumour growth index.

[0069] FIG 12 indicates the results of the effect of the formulation on tumor volume. The results indicated that the formulation in the form of cream is effective in inhibiting tumour volume and the percentage of tumour growth index.

[0070] FIG 13 indicates the results of the effect of the formulation on tumor volume. The results indicated that the formulation in the form of cream at various concentrations is effective in inhibiting tumour volume and the percentage of tumour growth index for 14 days.

[0071] 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.

[0072] 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 thus promising a wide range of application.
[0073] 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.
[0074] 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. The presence of other ingredients with Picrasma quassioides optimize digestive juice and induces the positive effect of gut microbiome thus enhancing the immune system especially the innate immune system.
[0075] The formulation is prepared in the form of a cream for topical application on abdomen as an add on therapy for cancer.
, Claims:We Claim:
1. A formulation with a combination of herbal ingredients for the treatment of cancer, the formulation comprises:
a. leaf of Picrasma Quassioides at a concentration in a range between 15g to 17g;
b. root of Withania somnifera at a concentration in a range between 1g to 2g;
c. seeds of Myristica fragrans Houtt at a concentration in a range between 0.5 to 1.5g;
d. leaves of Aloe vera at a concentration in a range between 1g to 3g;
e. rhizome of Curcuma longa at a concentration in a range between 1g to 3g;
f. nuts of Vitellaria paradoxa at a concentration in a range between 1g to 3g;
g. fruits of Psoralea corylifolia at a concentration in a range between 1g to 3g;
h. hyaluronic acid at a concentration of 0.2g;
i. glycerin at a concentration in a range between 2g to 4g;
j. GMS at a concentration in a range between 5g to 6g;
k. imidurea at a concentration in a range between 0.2g to 0.4g;
l. bronopol at a concentration of 0.1g;
m. BHT at a concentration of 0.1g; and
n. distilled water
wherein said ingredients are blended in a sequential manner to obtain uniform formulation.

2. The formulation as claimed in claim 1, wherein the formulation is in the form of a cream for topical administration.

3. The formulation as claimed in claim 1, wherein the quantity of the formulation is made up to 100 g with said ingredients.

4. The formulation as claimed in claim 1, wherein the formulation exhibits cytotoxic activity in one or more carcinoma cells selected from a group of breast adenocarcinoma, colorectal carcinoma, colorectal adenocarcinoma and chronic myelogenous leukemia.

5. 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.

6. 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 the ingredients oils and wax namely GMS at a concentration of 5.5g, imidurea at a concentration of 0.3g, bronopol at a concentration of 0.1g, BHT at a concentration of 0.1g in separate containers and subjecting to heating up to 75 °C (203);
d. mixing the wax phase into an aqueous phase after attaining the equilibrium temperature with constant stirring (204); and
e. adding the preservatives separately with minimal amount of distilled water, and the volume is made up to the quantity of 100 ml (205).

7. The process as claimed in claim 8, wherein the herbal ingredients include leaf of Picrasma Quassioides at a concentration in a range between 15g to 17g, root of Withania somnifera at a concentration in a range between 1g to 2g, seeds of Myristica fragrans Houtt at a concentration in a range between 0.5 to 1.5g, leaves of Aloe vera at a concentration in a range between 1g to 3g, rhizome of Curcuma longa at a concentration in a range between 1g to 3g, nuts of Vitellaria paradoxa at a concentration in a range between 1g to 3g, fruits of Psoralea corylifolia at a concentration in a range between 1g to 3g.

8. The process as claimed in claim 8, wherein the preservatives are selected from a group comprising namely imidurea at a concentration of 0.3g, bronopol at a concentration of 0.1g and BHT at a concentration of 0.1g.