TITLE OF THE INVENTION: SYNERGISTIC POLY-HERBAL COMPOSITION FOR INHIBITING PROLIFERATION OF CANCER CELLS BY INDUCING PROGRAMMED CELL DEATH
FIELD OF THE INVENTION
The present invention relates to, a synergistic poly-herbal composition comprising of extracts) of medicinal plant(s) used either in whole, or by employing portion(s) inclusive of, stem and leave(s). Further, the present invention relates to a composition, comprising of medicinal plant extract(s), said composition employed in inhibiting proliferation of cancer cells. Still further, the said composition induces programmed cell death.
BACKGROUND OF THE INVENTION:
In man and animals, in the normal scheme, cell growth and death occur on a periodic basis. Such periodic cell growth and cell death are however, within the control parameters) and are part of normal and healthy growth. The parameters of control for cell growth and cell death alike, are either intrinsic, inclusive of factors, such as, genetic, or occur under triggers from external entities. External signals direct cell division and as a result, cell-to-cell signaling occurs. Programmed cell death is called "apoptosis". Under certain circumstances, however, cell growth and cell death may occur beyond the existing parameters of control.
When cell growth occurs beyond the existing parameters, the condition is medically termed as "Cancer". Unlike regular cells, "Cancer" cells do not experience programmed death and instead continue to grow and divide. The manifestation of "Cancer" is generally observed as, extra growth of cells. Such extra growth is medically termed as, "tumor*. Cancer harms the body when damaged cells divide uncontrollably to form the "tumor(s)" (except in the case of leukemia where cancer prohibits normal blood function by abnormal cell division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and circulatory systems, and they can release hormones that alter body function. Based on the tendency to expand, tumor(s) are classified as either malignant or benign. Tumors that stay in one spot and demonstrate limited growth are generally considered to be benign. Benign tumors are harmless, after being surgically operated and generally do not leave residues of cancerous tissue.
However, malignant tumors spread Cancer to other part(s) of the body and are fatal. The cells within malignant tumor(s) have the ability to invade neighboring tissues and organs, thus spreading the disease. It is also possible for Cancerous cells to break free from the tumor and enter the

bloodstream, in turn spreading the disease to other organs. Many things are known to increase the risk of cancer, including tobacco use, certain infections, radiation, lack of physical activity, obesity, and environmental pollutants. These can directly damage genes or combine with existing genetic faults within cells, to cause the disease. Approximately, five to ten percent of cancers are entirely hereditary.
While Cancer can affect people of all ages, the risk of developing Cancer generally increases with age. Incidence(s) are rising as more people live to old age and as mass lifestyle changes occur in the developing world. Typical symptoms of cancer include, the presence of an unusual lump in the body, changes in a mole on the skin, a persistent cough or hoarseness, a change in bowel habits, such as unusual diarrhea or constipation, difficulty in swallowing or continuing indigestion, any abnormal bleeding.
Cancer can affect any part of the body, however, certain parts of the body are more prone to being affected by Cancer than the other parts. For instance, Skin is the most prone to being affected by Cancer. Amongst the parts of the body affected by Cancer, Colorectal cancer is the third most common type of cancer.
A person skilled in the art, for example, a medical practitioner is very well aware that, Colon is the last part of the digestive system in most vertebrates; it extracts water and salt from solid wastes before they are eliminated from the body, and is the site in which flora-aided (largely bacterial) fermentation of unabsorbed material occurs.
Unlike the small intestine, the Colon does not play a major role in absorption of foods and nutrients. However, the Colon does absorb Water, Sodium and some Fat soluble Vitamins. Colorectal cancer, commonly known as Colon Cancer or Bowel Cancer, is a Cancer from uncontrolled cell growth in the Colon or Rectum (parts of the large intestine), or in the appendix. Symptoms typically include rectal bleeding and anemia which are sometimes associated with weight loss and changes in bowel habits. Most Colorectal cancer occurs due to lifestyle and increasing age with only a minority of cases associated with underlying genetic disorders. It typically starts in the lining of the bowel and if left untreated, can grow into the muscle layers underneath, and then through the bowel wall.
Process of spreading Cancer is called Metastasis. The symptoms and signs of Colorectal Cancer depend on the location of tumor in the bowel, and whether it has spread elsewhere in the body (Metastasis). The classic warning signs include: worsening constipation, blood in the stool, weight loss, fever, loss of appetite, and nausea or vomiting in patients over 50 years old. While rectal bleeding or anemia are high-risk features in those over the age of 50, other commonly described

symptoms including weight loss and change in bowel habit are typically only concerning if associated with bleeding. When Cancer has metastasized and has affected other areas of the body, the disease is still referred to the organ of origination.
Therefore, there exists a need in the art to prevent proliferation of cells in the tumour by killing the cancerous cells causing the tumor.
Conventionally, treatment(s) based on Chemotherapy are available and are practiced in the state of the art. The most common Chemotherapy agent(s) act by killing cells that divide rapidly, one of the main properties of most cancer cells. However, this also means that chemotherapy also harms cells that divide rapidly under normal circumstances: cells in the Bone marrow, Digestive tract, and Hair follicles. This results in the most common side-effects of Chemotherapy, like myelo-suppression (decreased production of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss). As these drugs cause damage to cells, they are termed cytotoxic.
Chemo drugs are very strong medicines that can also affect healthy cells in the body. Doctors give the drugs in cycles, with each period of treatment followed by a rest period to allow the body time to recover. Chemotherapy cycles generally last about 2 to 4 weeks, and people usually get at least several cycles of treatment. The drugs most often used for colorectal cancer include: 5-Fluorouracil (5-FU), which is often given with the vitamin-like drug leucovorin (also called folic acid). Once in the body, it is changed to 5-FU when it gets to the tumor site. However, it has been found that, when chemotherapy is employed, cells which are non-cancerous and are thereby healthy, are also destroyed. This causes devastating side effects to the patient.
Since the Chemotherapy mode of treatment In the case of Cancer in the Colon and Rectum causes dysfunctioning of the intestine and thereby the bowel movements and the functioning of the organ itself, there exists a need in the art, arrest the growth of the tumors by a chemical composition which does not cause harm to the beneficial bacteria, such as, lactobacilli and bifidus bacteria in the digestive tract and the side effects normally caused by Chemotherapy.
In addition to chemotherapy, other alternative medicines are also available in the state of the art. The available alternative medicines have certain herbal components. For example, Indian Patent application no. IN 193609 reveals a process for preparation of a novel herbal medicinal composition for cancer treatment. It contains Tricliopus zeylanicus leaf and root of Janakla arayalpalia.

It is observed that, the herbal alternatives available in the state of the art are effective to a certain degree.
However, in developing countries, for a disease like cancer, which is painful in certain cases, require treatment for longer duration, sometimes with expensive medicines and surgical procedures involving high risk, there is a requirement for an affordable and effective treatment procedure with no or minimum side effects.
OBJECTIVES:
The present invention as embodied by, synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing Apoptosis (programmed cell death) in colon-rectal cancer. The objectives of the present invention are, as broadly enumerated in the present disclosure. However, it would be perfectly clear to a person skilled in the art that, in as much as the objectives have been enumerated, such objectives are only indicative of the scope and general coverage of the present invention.
This statement of enumerated objectives should be considered as merely indicative, and is no way restrictive of the scope and ambit of the present invention in its entirety. Not only the enumerated objectives should be considered as indicative of the scope and ambit of the present invention, in addition, any combination(s), variation(s), functional equivalent^) and/or any structural altemative(s) should also be construed to be within the broad scope of the present invention, even though, such a scope of the present invention has not been explicitly stated herein and elsewhere in the present disclosure.
Cancer as is known to people skilled in the medical art is of benign and malignant types. Malignant tumors have a tendency of spreading. Process of spreading of Cancer is called Metastasis. Therefore, an objective of the present invention is, to inhibit proliferation of cancer cells of the tumor, before it spreads to the rest of the body and is detrimental to the survival of the patient. Another objective of the project is to induce programmed cell death.
Yet another objective of the present invention is, to inhibit proliferation of cancer cells and induce programmed cell death by a herbal composition which does not cause harm to the beneficial bacteria in the digestive tract such as, lactobacilli and bifidus bacteria, and side effects normally caused by chemotherapy.

Still another objective of the present invention is, to provide herbal alternative which is effective in treating cancer.
A further objective of the present invention is an easily accessible and affordable treatment for treating cancer.
A further objective of the present invention is a herbal composition, which eases the pain of the patient in an expedited manner.
A further objective of the present invention is a herbal composition which acts on cancer cells by inhibiting proliferation of cancer cells and by inducing programmed cell death, but not restricted to, the colon-rectal cancer.
SUMMARY:
The present invention is embodied by, a synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, in parts of the body inclusive but not restricted to, the Colon and Rectum so as to fulfill the need(s) in the art, giving rise to the above-mentioned objectives sought to be fulfilled by the present invention. A parson skilled in the medical art, is very well aware that, the objective(s) as enumerated, described and embodied in the present invention, are merely illustrative of the scope and purview of the present invention, as described. However, such an illustration may not be construed to restrict the present invention in any manner. It will be very clear to a person skilled in the art that, any additional variation(s), functional equivalent(s) and/or any structural alternative(s) to the present invention, may also be understood to be within the scope and purview of the present invention, as described in the present disclosure. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof.
In this context, the present invention relates to a synergistic poly-herbal composition employed for inhibiting proliferation of cancer cells by inducing programmed cell death, but not restricted to, the Colon and Rectum. The synergistic poly-herbal composition as embodied in the present invention comprises three herbal extract(s) which, in combination acts to induce programmed cell death (Apoptosis) in parts of the body inclusive but not restricted to, the Colon and Rectum.
The plants forming part of the synergistic poly-herbal composition as embodied by the present invention, are "Trichopus zeylanicus" commonly known as, "Arokiyapachchai", "Andragraphis paniculata" commonly known as "Nilavembu" and "Cissus quadrangularis" commonly known as

"Pirandai". To achieve the synergistic poly-herbal composition of the present invention, as a matter of example, 50g of Andragraphis paniculata, 100g of Trichopus zeylanicus and 100g of Cissus quadrangularis are shade dried and their extract is subjected to fermentation after soaking in quality control (QC) approved food grade solution. Persons skilled in the field of preparing and administering herbal medicine will be aware that, the values stated above are merely illustratory of the quantity of herbal extracts to be employed for achieving the herbal composition as embodied in the present invention and generally usage of the plant Andragraphis paniculata in a ratio by weight half that of the other plants used will suffice. The medicinal effect of the synergistic poly-herbal composition of present invention is achieved by the leaves of Andragraphis paniculata, the leaves of Trichopus zeylanicus and the stem of Cissus quadrangularis. The herbal composition is not only quick but also extremely effective. The herbal composition of the present invention eliminates 50% of cancerous cells in cellular material in a time duration of 24hours.
BRIEF DESCRIPTION OF DRAWINGS:
FIGURE 1 illustrates the large intestine, with the magnified view of the Colon and Rectum in normal condition, when the organ is in healthy state.
FIGURE 2 illustrates the large intestine, with the magnified view of the Colon and Rectum in diseased condition, when the organ is affected by Polyp and Cancer.
FIGURE 3 illustrates the process of Cell apoptosis
FIGURE 4 illustrates the toxic side effects on the various organs, when the conventional treatment based on Chemotherapy, is employed to remove Cancerous tissue.
FIGURE 5 illustrates the diagram of the plant Trichopus zeylanicus, the leaves of which, are used in dry/powdered form to achieve the herbal composition of the present invention.
FIGURE 6 illustrates the diagram of the plant Andrographis paniculata, the leaves of which, are used in dry/powdered form to achieve the herbal composition of the present invention.
FIGURE 7 illustrates the diagram of the plant Cissus quadrangularis, the stem of which, is used in the preparation of herbal composition, in an embodiment of the present invention.

FIGURE 8 illustrates the chemical structure of (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) which is employed in assay to determine number of cells in a tissue inclusive of but not restricted to cancerous cellular material.
FIGURE 9 (a to h) describes the procedure involved in MTT assay to determine number of cells in a tissue inclusive of but not restricted to cancerous cellular material, as employed in the present invention which uses the synergistic poly-herbal composition.
DETAILED DESCRIPTION OF DRAWINGS:
The animal body especially mammals for the purpose of example, absorbs nutrients and generates energy on a layman view of the operations in the intricate anatomy. However the generation of energy from the absorption of nutrients involves the assimilation system inclusive of the mouth, gullet and ancillary organs in the oral cavity, processing of the absorbed nutrients in the digestive system in the organs inclusive of stomach, and finally last part of digestion namely excretion via the excretory system inclusive of kidneys, large and small intestine. Though Cancer affects any portion of the body, its effect on the large intestine affects the excretion system and may also cause mixing of toxic fluids/solids in the blood. In this context it is pertinent to describe the anatomy of the large intestine inclusive of colon, rectum and other ancillary organs when the organ is not affected by Cancer and illustrate via FIGURE 1 illustrates the large intestine, with magnified view of the Colon and Rectum in normal condition(s). Although obvious to a person skilled in the medical art inclusive of medical practitioners, the colon is the last part of the digestive system in most vertebrates; it extracts water and salt from solid wastes before they are eliminated from the body, and is the site in which flora-aided (largely bacterial) fermentation of unabsorbed material occurs. Unlike the small intestine, the colon does not play a major role in absorption of foods and nutrients. The colon (C)consists of four sections: the ascending colon (AC), the transverse colon (TC), the descending colon (DC), and the sigmoid colon (SC) (the proximal colon usually refers to the ascending colon and transverse colon). The colon (C), rectum (R) and anus (A) make up the large intestine.
The colon works as the organ for storing waste products, reabsorbing water from wastes and maintaining water balance in the body. Apart from these, it serves at the site for the growth of beneficial bacteria and other microorganisms, that help to ferment undigested and unabsorbed food material, and synthesize some vital nutrients like, folic acid. These friendly bacteria also help to check the growth of harmful bacteria, and maintain the pH balance in the body. The colon contains large numbers of bacteria. The most important of these are the Lactobacilli and Bifidus bacteria, which live in a symbiotic (mutually beneficial) relationship with the colon. They ferment the soluble fiber in food,

forming valuable short-chain fatty acids that nourish intestinal cells, help regulate production of
cholesterol. Beneficial bacteria e.g., Lactobacilli and Bifidus bacteria also help to keep harmful
bacteria in check. In addition, intestinal bacteria synthesize vitamin K, important in blood clot
formation.
However, the most crucial function of the intestine inclusive of colon is the elimination of toxic waste material in the form of stool or feces through the rectum. Its muscles contract in a wave-like manner, to pass the stool down the rectum. Generally, the waste material or stool is liquid, when it enters the first part of the colon. As the liquid waste material move through this organ, all excess water is absorbed from it and by the time the stool reaches the descending colon, it becomes semi-solid. In its descending part, the waste material is stored, until it is emptied into the rectum. Defecation takes place, when the muscles of the sigmoid colon contracts to increase the pressure inside the colon, so that the stool can move and enter the rectum.
When the normal functions of this organ are disrupted, one can experience several health problems, mainly due to accumulation of the toxic waste material within the colon, and impairment of the absorption of some vital nutrients. Cancer can occur in any portion of the body. When an uncontrolled cell growth is observed in the Colon, it is manifested as Colon Cancer (CC) and illustrated in FIGURE 2 illustrates the large intestine, with magnified view of the Colon (C) and Rectum (R) in diseased condition(s), when the otyan is affected by Polyp and Cancer. Tumors of the colon (C) and rectum (R) are growths arising from the inner wall of the large intestine. Benign tumors of the large intestine are called polyps. Malignant tumors of the large intestine are called cancers. Benign polyps do not invade nearby tissue or spread to other parts of the body. Benign polyps can be easily removed during colonoscopy and are not life-threatening. If benign polyps are not removed from the large intestine, they can become malignant (cancerous) over time. Most of the cancers of the large intestine are developed from polyps (CP). Cancer of the colon (CC) and rectum (also referred to as colorectal cancer) can invade and damage adjacent tissues and organs. Cancer cells can also break away and spread to other parts of the body (such as liver and lung) where new tumors form. The spread of cancer e.g. colon to distant organs is called metastasis of the cancer. Once metastasis has occurred in colorectal cancer (CC), a complete cure of the cancer is difficult. When cell communication breaks down, uncontrolled cell growth can occur and often lead to cancer. Cancer often begins when a cell gains the ability to grow and divide even in the absence of a signal from the environment. Ordinarily, the unregulated growth triggers a signal for self destruction, otherwise known as Apoptosis. However, when a cell also loses the ability to respond to self-destruct signals, the cell divides uncontrollably and, consequently, forms a tumor. So, one method of curing Cancer is by promoting (inducing) Cell Apoptosis explained in further detail with FIGURE 3. Cell death is also possible by Necrosis.

Therefore one method of curing cancer could also be achieved by promoting Necrosis. In the present disclosure the term Apoptosis is predominantly used. However, the said invention may also achieve the purpose of cancer cure by inducing Necrosis. Mere mention of Apoptosis would not restrict the scope and purview of the present invention to exclude Necrosis.
FIGURE 3 illustrates the process of Cell Apoptosis, which is programmed cell death, and is needed to maintain the shape of the organism. When a cell is not repairable, it signals its own death. Apoptosis occurs when a cell (Ce) is damaged beyond repair, infected with a virus, or undergoing stressful conditions such as starvation. Damage to DNA from ionizing radiation or toxic chemicals can also induce apoptosis via the actions of the tumor-suppressing gene p53. The "decision" for apoptosis can come from the cell itself, from the surrounding tissue, or from a cell that is part of the immune system. In these cases Apoptosis functions to remove the damaged cell, preventing it from sapping further nutrients from the organism, or halting further spread of viral infection. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Apoptosis is nevertheless necessary for proper healthy development and needed to destroy cells that threaten the organism.
In Apoptosis the cell shrinks and pulls away from its neighbor. Apoptosis causes the cells develop bubble like beads (MB) on their surface. The DNA in the nucleus condenses (CCn) and breaks into regular sized fragments (NC), and soon the nucleus itselt, followed by the entire cell, disintegrates. The chromatin in the cell begins to degrade and mitochondria break down with the release of cytochrome "c". Several known methods are available for treatment of Cancer, based on cell apoptosis. One such conventional treatment is based on Chemotherapy and described in FIGURE 4.
FIGURE 4 illustrates the side effects of conventional treatment that is employed to remove Cancerous tissue by means of Chemotherapy.
Chemotherapy is the treatment of cancer with an antineoplastic drug or with a combination of such drugs into a standardized treatment regimen. There are a number of strategies in the administration of chemotherapeutic drugs used today. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms.
Combined modality chemotherapy is the most commonly used method of treatment using drugs with other cancer treatments, such as radiation therapy or surgery. Combination chemotherapy is a similar therapy that involves treating a patient with a number of different drugs simultaneously. Several drugs

can be used to treat colorectal cancer. Often, two or more of these drugs are combined to try to make them more effective.
Chemo drugs are very strong medicines that can also affect some healthy cells in the body. Doctors give the drugs in cycles, with each period of treatment followed by a rest period to allow the body time to recover. Chemotherapy cycles generally last about 2 to 4 weeks, and people usually get at least several cycles of treatment. Chemo drugs work by attacking cells that are dividing quickly, that is why they work against cancer cells. But other cells in the body, such as those in the bone marrow, the lining of the mouth and intestines, and the hair follicles, also divide quickly. These cells are also likely to be affected by chemo, which can lead to certain side effects.
The side effects of chemo depend on the type and dose of drugs given and the length of time they are taken. Common side effects due to usage of chemo-drugs can include, Hair loss (HL), Mouth Ulcer (MU), and Diarrhea occurs due to side effect in colon (C), Nausea and vomiting occurs due to side effect in stomach (S), and Low white blood counts (LWBC). Chemotherapy can affect the blood forming cells of the bone marrow, leading to low blood cell counts. This can lead to increased chance of infections (from low white blood cell counts), easy bruising or bleeding (from low blood platelet counts) and fatigue (from low red blood cell counts). Due to the above mentioned side effects, certain herbal alternatives are being tried. The present invention is a synergistic poly-herbal composition consisting of Trichopus zeylanicus, Andrographis paniculata and Cissus quadrangularis, each of which is described in conjunction with the subsequent Figures (Figure 5, Figure 6 and Figure 7).
FIGURE 5 illustrates the diagram of the plant Trichopus zeylanicus, the leaves of which are used in dry/powdered form to achieve the herbal composition of the present invention.
The vernacular name of the plant is "Arogya pacchai", literally meaning "the green that gives strength". Detailed chemical and pharmacological investigations showed that the leaf of the plant contained flavonoid glycosides, glycolipids and some other non-steroidal compounds. In the present invention, the leaves are used in dried/powdered form.
FIGURE 6 illustrates the diagram of the plant Andrographis paniculata, the leaves of which, are used in dry/powdered form to achieve the herbal composition of the present invention.
Andrographis paniculata commonly known as Kalmegh and Nilavembu (in Tamil) is found in the plains of India, Pakistan, Sri Lanka and West Indies. It belongs to the family Acanthaceae. It has been used

for centuries as a medicinal plant for the treatment of upper gastrointestinal tract and upper chronic diseases.
FIGURE 7 illustrates the diagram of the plant Cissus quadrangularis, the stem of which, is used in the preparation of herbal composition, in an embodiment of the present invention.
Cissus quadrangularis (Linn) also known as Vitis quadrangularis Wall, belongs to family Vitaceae. It is a common perennial climber, which is distributed throughout India particularly in tropical regions. The plant is commonly known as Vajravalli in Sanskrit, Hadjod in Hindi, Perandi in Tamil. The stem being used as the portion for achieving synergistic poly-herbal composition of the present invention. It is a climbing plant. The Cissus quadrangularis contains high amount of Carotene A, anabolic steroidal substances and Calcium. It contains carotenoids, triterpenoids and ascorbic acid.
FIGURE 8 illustrates the chemical structure of MTT which is employed in assay to determine number of cells in a tissue inclusive of but not restricted to cancerous cellular material.
The Figure shows the chemical structure of MTT whose IUPAC chemical name is 3 -(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide). The MTT assay is conventionally used to determine the number of cells. The reduction of tetrazolium salts is a reliable way to examine cell proliferation in Cancerous tissue or Cell apoptosis in cancer cure. In the present invention, the MTT assay is employed to determine the amount of cell death i.e. cell apoptosis to gauge cure of cancer. The procedure adopted is described in detail with the following Figures, namely FIGURE 9(a-h). The yellow tetrazolium MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means.
The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields tow background absorbance values in the absence of cells. For each cell type the linear relationship between cell number and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell proliferation.
Evaluation of Cytotoxicity using MTT assay in the present invention involves using Caco2 Colon carcinoma cell lines for examining the cytotoxicity of synergistic poly-herbal composition, Eagle's

minimum essential medium (MEM), trypsin/EDT A, L-glutamine, non-essential amino acids, penicillin-streptomycin-amphotericin B solution and Hank's balanced salt solution (HBSS). MTT [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyl tetrazoiium bromide], sulfuric acid, potassium iodide, ammonium molybdate, sodium thiosulfate, and Multiwell tissue culture plates area also used.
Passaging the cells includes culturing in monolayer in 100 and/or 20 mm, two plastic dishes at 37°C and under 5% C02 and 95% air. Medium is changed every 3 days, and the cells were passed once they reached approximately 80% confluence. Trypsin (2.5%)/EDTA (0.38 g/l) is used to dislodge the cells. The medium from the culture flask is aspirated. The flask is rinsed with 2 ml of PBS and aspirated quickly. 1.5 - 2 ml of trypsin-EDTA solution is added and incubated at 37°C for about 3-5 minute until cells started lifting. As soon as the cells were detached, using a transfer pipette the trypsinized cells were transferred to a tube containing equal volume of 10% FBSDMEM. Then, the medium containing cells is centrifuged at 1000 rpm for 5 minute and carefully the medium is aspirated off. Care is taken not to put the pipette tip in the bottom of the tube, where the cells were pelleted. The cells were gently resuspended in fresh 10% FBS-DMEM by pipetting up and down 5-8 times. From the cell suspension, a drop is placed to the edge of the cover slip of Neubauer haemocytometer. The drop is let to run under the cover slip by capillary action. Care is taken not to "force" the liquid and the entry of air bubble is avoided. Then the cells from the E1, E2, E3, E4 and E5 squares were counted under microscope. The number of cells is calculated using the formula:
No. of cells = No. of cells counted x 50,000 = X cells/ml
For cell cycle analysis, 5 * 105 cells seeded in 3 mL total volume in 6-well multidishes were incubated as described above for 24 h. Flow cytometric analyses were conducted using a FACScan. At the end of incubation, the cells were rinsed twice with PBS and trypsinized in trypsine- 0.02% EDTA mixture. After centrifugation for 10 minute at 600*g at 4°C, the supernatant is removed, the pellet resuspended in 300 uL of PBS, then 700 ul_ of cold methanol is added and the mixture kept at -20°C for 30 minute. After centrifugation for 5 minute at 600*g and at 4°C, the pellet is treated with 2mg/mL RNase A at 37°C during 30 minute and stained with 50 ug/mL propidium iodide containing 0.1% Triton X-100 and EDTA 0.02mg/mL The percentage of cells in each stage of the cell cycle is determined by counting 104 cells, using a cellquest software. Cell cultures were washed twice with sample buffer (phosphate-buffered saline plus 1 g glucose/L filtered through a 0.22-um Watman filter), dislodged by trypsin/EDTA incubation, centrifuged at 400g * 10 minute at 22°C, and resuspended in sample buffer. Cell concentrations were adjusted to 1 to 3 * 106 cells/ml with sample buffer, and 1 ml of the cell suspension is centrifuged at 400g x 10 minute at 22°C. All of the supernatant except 0.1 ml/106 cells were removed, and the remaining cells were mixed on a vortex mixer In the remaining fluid for 10 s.

Next, 1 ml of ice-cold ethanol [70% (v/v)] is added to the sample in a dropwise manner, with samples being mixed for 10 s between drops. Tubes were capped and fixed in ethanol at 4°C. For flow cytometric measurements, 106 Caco2 were pelleted, resuspended in 200 ml of PBS, and stained with 2 ml of 1003 stock of AO-EB in PBS. The final concentrations of AO (Aldrich, Milwaukee, Wis.) and EB (Sigma) were 0.1 and 0.25 mM, respectively. All samples were stained and analyzed immediately at room temperature. Flow cytometric analysis is performed on Coulter Profile II and FACScan benchtop cytometers with standard argon ion laser settings (488 nm) and filter sets Analyses were performed with 20,000 events per sample using the ModFit LT version 2 for Macintosh data acquisition software package Cells were sorted using a FACS 440 cell sorter equipped with a single argon ion laser and modified with CICERO (high-speed acquisition and sort electronics. AO and EB were excited at 488 nm and emission detected with a 525/20-nm filter (FL1) and a 635/20-nm filter (FL3), respectively. Four-parameter list mode data were acquired for analysis and sorting. Light scatter parameters were used to gate on single cells, with an acquisition threshold set on the forward scatter (FS) signal. Irradiated cells tended to have smaller cell debris that is included above the FSC threshold so as not to include them in the sort window and thus contaminate the sort population. Fluorescence signals were logarithmically amplified, and fluorescence compensation is determined from unlabeled and single-labeled cell controls. Samples were run for several seconds before datum acquisition to allow dye equilibration in the FACS 440 sample tubing. The initial few seconds of sample flow occasionally showed artifacts in staining intensity because of higher background fluorescence from increased sample volume at the interrogation point sorting is done with two drop packets and under conditions to maximize purity over recovery.
Cyclin D protein expression in Caco2 cells were assessed by Western blot analysis as described by Sudhakar et al. (2001). Immediately after the treatment period, 1ml of ice-cold RIPA buffer with protease inhibitor is added to 100 mm petri dish of all the groups. Cells were scrapped using rubber policeman. Then supernatant is aspirated into fresh tubes and protein concentration is determined.
The amount of protein in the tissue homogenate is estimated by Lowry's method using bovine serum albumin as standard. To 0.1 ml of suitably diluted Caco2 lysate, 0.9 ml of water and 4.5 ml of alkaline copper reagent were added and kept at room temperature for 10 minutes. Then 0.5 ml of Folin's reagent is added and the color developed is read after 20 minutes at 640 nm. The levels of protein are expressed as mg/ml. Reagents for Western blot analysis include:
1. Radioimmuno precipitation buffer (RIPA): (150 mM NaCI, 50 Tris, 1 mM EDTA, 1 % NP-40,0.5% sodium deoxycholate, 0.1% DS, pH 7.4).

For 500 ml
Tris base - 3.029 g
NaCI-4.383 g
EDTA-0.186 g
Na-deoxycholate - 2.500 mg
SDS- 500 mg
NP-40-5.00g All these chemicals were dissolved in 400 ml double distilled water, pH is adjusted to 7.4 with HCI and then the volume is made up to 500 ml. The buffer is stored in a refrigerator.
2. Protease inhibitor cocktail: One tablet of protease inhibitor (from Roche Pharmaceuticals, USA) is dissolved in 2 ml double distilled water and stored in -20°C.
3. Running gel buffer: (1.5M Tris, pH 8.8): For 250 ml, 45.375 g Tris is dissolved in 200 ml double distilled water, pH is adjusted with HCI and the volume is made up to 250 ml and stored at room temperature.
4. Staking gel buffer (0.5 M Tris, pH 6.8): For 250 nil solution, 15 g Tris is dissolved in double distilled water and pH is adjusted with HCI before adjusting the volume up to 250 ml.
5. SDS 10%: For 100 ml solution, 10 g SDS is dissolved in double distilled water and stored at room temperature.
6. Acrylamide 30%: 29 g acrylamide (29%) and 1 g bis-acrylamide (1%) were dissolved in 100 ml double distilled water.
7. Ammonium per sulfate 10%: 100 mg ammonium per sulfate is dissolved in 1 ml double distilled water.
8. 10x SDS Electrophoresis buffer (2.5 M Tris, 1.92 M Glycine and 1 % SDS): For one liter solution, 30 g Tris, 144 g glycine and 10 g SDS were dissolved in 800 ml double distilled water and volume is made up to one liter and stored at room temperature.
9. 1x SDS Electrophoresis Buffer For 500 ml, 50 ml 10X SDS electrophoresis buffer is mixed with 450 ml of double distilled water.

10. 10x Transfer buffer (250 mM Tris and 1.92 M Glycine): For one liter, dissolved 30.3 g Tris and 144 g glycine in 800 ml double distilled water and adjusted the volume up to one liter and stored at room temperature.
11. 1x Transfer Buffer with 20% methanol: For 500 ml, 50 ml 10x transfer buffer, 350 ml double distilled water and 100 ml methanol were mixed and kept cold until use.
12. 2x Sample Buffer with reducing agent: (125 mM Tris-HCI (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.004% bromophenol blue). For 10 ml, 2.5 ml 0.5 M Tris-HCI (pH 6.8),4.0 ml of 10% SDS, 2.0 ml glycerol, 1.0 ml 2-mercaptoethanol and 0.4 mg bromophenol blue were added and made up the volume to 10 ml with double distilled water. 1 ml aliquots were made and stored at -20°C.
13. 10x Phosphate buffered saline (0.1 M Sodium phosphate): For one liter solution; 13g NaH2P04.4H20 dissolved in double distilled water, pH adjusted to 7.2 with NaOH, the volume made up to one liter and stored at room temperature.
14. Blocking buffer: For 500 ml: To 500 ml of 1x PBS add 900 mg l-Block tm (Tropix, Bedford, MA, USA), dissolved by heating/stirring (care is taken not to exceed temperature 100°C, while heating). Then added 0.5mL Tween-20 and stored in refrigerator.
15. Tris buffered saline (TBS): (20 mM, Tris, 500 mM NaCI, pH 7.5): For 500 ml: 1.21g Tris and 14.62 NaCI were dissolved in 400 ml double distilled water and adjusted pH to 7.5 with HCI and made up to 500 ml.
16. T-TBS (0.2% Tween-20 in TBS): For 100 ml: Added 200 pi Tween-20 to 100 ml of TBS. The solution is kept cold.
17. Prestained SDS-PAGE standard: Commercially available prestained protein standards were obtained and the standard is stored at -20°C. Low range molecular weight standards:

1) D-galactosidase118,000
2) BSA 85,000
3) Ovalbuminute 47,000
4) Carbonic anhydrase 36,000
5) tt-lactoglobin 26,900
6) Lysozyme 20,400

Running gel (12%) includes:
Acrylamide (30%) 3.96 ml Running gel buffer 3.0 ml DDH204.8ml SDS(10%)0.12ml APS (10%) 0.12 ml TEMED0.012ml
Stacking gel (5%) includes:
Acrylamide (30%) 0.5 ml Stacking gel buffer 0.38 ml DDH202.1ml SDS(10%)0.03ml APS (10%) 0.03 ml TEMED 0.003 ml
Fifty micrograms of total protein is mixed with 2x sample buffer and boiled for 5 minute.
The sample mixture is run on 12% SDS-PAGE gels in 1x running gel buffer at 100 V for 2.5 hour and
electrotransferred to a polyvinylidene difluoride (PVDF) membrane at 30 V for 1 hour.
The membrane is blocked in blocking buffer (l-Blocktm) for overnight.
After overnight blocking, membrane is incubated with a rabbit polyclonal primary antibody for cyclin D
(1:2000) dilution for 5 hour or rabbit polyclonal primary antibody for Cyclin D for 5 hour or primary
antibody for lactin (1:1000) for 3 hour.
After incubation with the primary antibody, the membrane is washed three times with blocking buffer
for 10 minute each.
Membrane is incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000
dilution) for 45 minute.
1. The membrane is then washed in TBS and T-TBS thrice.
2. Then, the signals were detected and visualized using the enhanced chemiluminescence system.
3. The band is visualized and subjected to densitometric scanning using a densitometer (Biorad Gel Documentation).
4. The band intensity for Cyclin D is normalized with that of the internal control lactin.

Total RNA is isolated from Caco2 cells by using total RNA isolation reagent (TRIR), following the method of Chomczynski and Sacchi (1987). The total RNA isolated is non-degraded and free from protein and DNA contamination reagents. TRIR kit has the following components: Guanidium, isothiocyanate, phenol, urea, detergents, buffering agents and stabilizers.
1. Chloroform (Molecular Biology Grade)
2. Isopropanol (Molecular Biology Grade)
3. 75% Ethanol (Molecular Biology Grade)
4. 0.2% Diethylpyrocarbonate (DEPC)
Total RNA from the Caco2 cells were isolated according to the manufacturer's instruction (Trizol, One step RNA isolation kit, Medox Biotech Pvt Ltd.). After the treatment period, the 100 mm petri dishes of all the groups were swirled off with 1 ml of TRIzol reagent. The resulting lysate is transferred into a 1.5 ml micro centrifuge tube followed by the addition of 200 ml of ice cold chloroform (to dissipate different cellular components - lipids, proteins and NA) to the lysate and shaken well (vigorously) for 15-25 sec and incubated at 15-30°C for 3-5 minutes and then centrifuged for 15 minutes at 12,000 x g. (After this 3 layers are obtained -the aqueous - upper layer with NA, followed by lipids and protein). Then, the aqueous layer is transferred into 1.5 ml micro centrifuge tube and 500 ml of ice-cold isopropanol (RNA precipitating agent) is then added. The tube is kept for precipitation of RNA at -20°C for 1-2 hrs and centrifuged for 10-15 minutes at 12,000 x g. Then, the RNA pellet is washed twice by adding 1 ml of 75% ethanol and centrifuged at 7,500 x g for 5-10 minutes. The pellet is allowed to air dry, and then the RNA pellet is dissolved in 50-100 ml RNase free water (DEPC- Diethyl pyrocarbonate). The RNA is stored at-80°C. All the reactions were carried out at 4"C unless otherwise stated.
Subsequently, the purity and yield of RNA is done by measuring the absorbance of RNA solution at 260 nm and 280 nm (Absorbance ratio of 260/280 ranges from 1.6-1.8 is taken for further reaction) by using the 'Beer-Lambert' law: A230 =e260 c I and A x 40 = ug/ml (where l=1cm) where, A is the absorbance at 260nm, is the molar absorption coefficient, c is the molar concentration and I is the optical path length (usually 1 cm). For a protein free and DNA free solution of RNA the ratio of A/A should be 1.82. Any proteinacios contamination present in the preparation would decrease the ratio < 1.8. After checking the purity and concentration 5ul of the stock solution is mixed with 45ul of milli Q water (DEPC water) [the concentration must be approximated to 6 uM]. 5ul of this solution is taken and is added to the RNA. Then it is kept in the per chamber for about 3.5 h.
cDNAs were synthesized using cDNA synthesis kit (Qiagen) from RNA isolated from different groups. Real-time PCR is performed using an ABI 7000 (PE Applied-Biosystems) in the presence of SYBR-green for FasL and FADD. The optimization of the real time PCR reaction is performed according to

the manufacturers instructions but scaled down to 20 pi per reaction. The PCR conditions were standard (SYBR-Green from Invitrogen). After optimisation, nucleotide primers were used at various concentrations for the detection and quantification of 18s rRNA signal.
Efficiency of amplifications is determined by running a standard curve with serial dilutions of cDNA. For each measurement, a threshold cycle value (CT) is determined. This is defined as the number of cycles necessary to reach a point in which the fluorescent signal is first recorded as statistically significant above background. In this study, the threshold value is determined with a baseline set manually at 100 relative fluorescence units (RFU). Results were analyzed using the comparative critical threshold (AACT) method in which the amount of target RNA is adjusted to a reference (internal target RNA). The fold changes were calculated using the conventional method. The data were subjected to Students'? test to assess the significant difference.
Based on the Cell viability study, it is found that treatment of 10ul/ml of given synergistic poly-herbal composition for 24 h result in 50% cell death of Colon carcinoma cell line. The result is presented in Figure 10(b). The cell viability on Caco2 cells were analyzed for various doses ranging from 0.1 ul, 0.5 ul/ml, 1 ul/ml, 5 ul/ml and 10 ul/ml. The results showed a dose-dependent increase in the cell death of Caco2 cells. Based on the MTT analysis we found that the ID50 is 10 ul/mL at 24 h.
Caco2 cell strain is an established cell line, which has been extensively used for cytotoxicity assays. Studies conducted in this laboratory showed that the cytotoxicity of synergistic poly-herbal composition on caco2 cell line is found to be 10 ul/ml for 24 hours. The results obtained from the MTT assay are presented in Figure 9(a). Significant inhibitory effects on cell proliferation were detected at and above the dose of 10 ul/mL, and the longer exposure period generally increased the cytotoxicity of synergistic poly-herbal composition. The 50% inhibitory doses of synergistic poly-herbal composition at 24 and 48 hours were estimated at 10 ul/ml. It should be recognized that the cytotoxicity detected in in vitro cell culture systems would only be applicable when the agent reaches the cytotoxic concentration in target tissues. Therefore, the results obtained from the present study are inadequate to determine the clinical significance of the cytotoxicity of synergistic poly-herbal composition. Hence, the pro-apoptotic effect of synergistic poly-herbal composition is evaluated using cell count and cell cycle analysis by FACS.
Cell cycle analysis is performed by flow cytometry using propidium Iodide in caco2 cells treated with different concentrations of synergistic poly-herbal composition extract for 24 h. As shown in Figure 9(c) and 9(d). Gradual dose-dependent decreases in Go/G1 phase population (46% - 23%) were observed as compared with negative control (69%). Though, an increasing trend in S phase (18 -

27%) is observed with increasing dosage, significant dose-dependent decrease is observed in G2-M ranging from 4% to less than 1% when compared to 6% in Control. In addition to that, a significant dose-dependent increase is observed in percentage of dead cells when compared to controls which shows an increasing degree of apoptosis in a dose-dependent manner.
Acridine orange and ethidium bromide staining of cells is then measured by flow cytometry to more clearly differentiate live, apoptotic and necrotic cells (Liegler et al., 1995). An example of the flow cytometry output is shown in Figure 9(e) and 9(f). Live cells stained brightly for the cell permeable acridine orange (green), with low cell impermeant ethidium bromide influx (orangered). Apoptotic cells stained with a lower concentration of acridine orange and low ethidium bromide. Apoptotic cells are distinguished from live cells mainly due to apoptosis-induced DNA fragmentation, but may also reflect cell shrinkage or formation of apoptotic bodies, all of which will reduce cellular fluorescence (orange). Necrotic cells stained both low and high with acridine orange and high for ethidium bromide (red), indicative of deteriorating cells with permeable plasma membranes. As shown in Figure 9(e) and 9(f), the viability of control cells is very high, with apoptosis and necrosis evident in a near equal higher concentration after treatment with synergistic poly-herbal composition in Caco2 cells. Cellular debris were excluded from analysis gates when possible. Populations were routinely plotted on logarithmic scales as dual-parameter dot plots with AO on the x axis (FL1) and EB on the y axis (FL2). Cell populations differentially stained with AO and EB were routinely gated as follows. Live cells (Figure 9(f), Lower Left Quadrant) stain with acridine orange and fluoresce green with low orange-red signal from ethidium bromide. Necrotic cells (Figure 10(f), Upper Left Quadrant) lose the ability to exclude ethidium bromide and, as a result, fluoresce as bright orange-red. Apoptotic cells (process explained in Figure 3) (Figure 9(f), Upper Right Quadrant) exhibit a reduced green fluorescence but maintain a low ethidium bromide signal. The reduction in acridine orange signal is likely due to nuclear fragmentation and cell shrinkage. Treatment of Caco2 cells with synergistic poly-herbal composition resulted in a increase in the both apoptotic populations and necrotic populations in higher dose when compared to high necrotic population in low dose than apoptosis. On the basis of reports using these DNA-staining dyes in studies of cell death, it is plausible that apoptosis predominate in 10 ul of synergistic poly-herbal composition treatment. These studies confirm the apoptotic and necrotic potential of synergistic poly-herbal composition comprising Cissus quadrangularis, Andragraphis paniculara, Trichopus zeilanicus.
Because of the effect of synergistic poly-herbal composition on cell cycle progression, we examined the expression of Cycline D whose synthesis is initiated during 61 and drives the G1/S phase transition. Cyclin D is one of the major cyclins produced in terms of its functional importance, it interacts with four Cdks: Cdk2, 4, 5, and 6. In proliferating cells, cyclin D-Cdk4/6 complex

accumulation is of great importance for cell cycle progression. In present study, as shown in Figure 9(g), the expression of cyclin D is found to be decreased significantly in dose-dependent manner which prevents the cell cycle progression thereby proliferation.
Fas ligand or FasL is a homotrimeric type II transmembrane protein. It signals through trimerization of FasR, which spans the membrane of the "target" cell. This trimerization usually leads to apoptosis, or cell death. Soluble Fas ligand is generated by cleaving membrane-bound FasL at a conserved cleavage site by the external matrix metalloproteinase MMP-7. The Figure 9(h)shows the effect of synergistic poly-herbal composition on mRNA expression of FASL and FADD. The treatment of 5 uL and 10 uL shows the dose-dependent increase in the upregulation of mRNA. The result is consistent with the expression of its downstream signaling molecule FADD. Fas- Associated protein with Death Domain (FADD) is an adaptor molecule that bridges the Fas-receptor, and other death receptors, to caspase-8 through its death domain to form the death inducing signaling complex (DISC) during apoptosis. Thus the synergistic poly-herbal composition in a dose dependent manner induces apoptosis in colon carcinoma cell line (caco2) using extrinsic pathway. Thus the synergistic poly-herbal composition at the concentration of 10 pi induced apoptosis and necrosis in colon carcinoma cell line which is evident from our study and has the potential to be developed as a therapeutic agent for treating colon cancer.
ADVANTAGES:
The below-mentioned advantage(s) arising out of the application in conjunction with the enclosed drawing(s) are merely indicative and are not to be considered as exhaustive of the entire advantage(s) arising by the deployment of the present invention. It will be amply clear to persons skilled in the art that, any primary advantage(s) arising out of the present invention as against the problem(s) in the conventional methods are to be construed to be well within the scope and purview of the present invention. Further, any secondary advantage® arising as a result of the primary advantage(s) are also to be construed to be well within the scope and purview of the present invention. Even though it has been mentioned in the context of colon carcinoma predominately, the present invention can be employed for other types of cancer as well.
In this context, conventional chemotherapy based treatments for Cancer cause a great deal of discomfort to the patient. Although the conventional treatments based on chemotherapy are meant to cure the patient, in addition to the targeting of cancerous cells, the conventional chemotherapy based treatment methods also destroy healthy non-cancerous tissue and beneficial bacteria symbiotically coexisting in the intestine. Since the tissue to be destroyed is in the colon and rectum area which falls

in the purview of the food digestion and excretion system the destruction of the cells in the area will cause dysfunctioning of the digestion and excretion system thereby adding to the discomfort of the patient who is already facing pain due to effect of the cancer. In contrast the synergistic herbal composition of the present invention causes no side effect and does not target the non-cancerous and healthy cells thereby enabling the digestion and excretion system of the patient to be intact.
The present invention is a herbal composition and is devoid of side effects and toxicity and is also focused on targeting cells with cancerous tendency only as against conventional chemo based treatments.
The present invention is capable of destructing only cancerous cells. The present invention is economically viable and does not financially burden the patient already facing the agony of the disease. Since the herbal composition is made of only 3 plants and for the preparation of the composition no organic solvent is involved, the cost of production of the herbal composition is less, thus making it affordable to common man.
The primary desire of the patient is to be free of the disease quickly and the present invention fulfills the desire by destroying 50% of the cellular material with cancerous tendency in a matter of 24 hours. The synergistic ploy-herbal composition has excellent anti cancer activity, at the concentration of 10ul induced apoptosis and necrosis in colon carcinoma cell line and has the potential to be developed as a therapeutic agent for treating cancer.
We claim:
1. A synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing
programmed cell death, the said composition comprising of:
(a) extract of a portion of "Trichopus zeylanicus";
(b) extract of a portion of" Cissus quadrangularis";
(c) extract of a portion of" Andragraphis paniculata", said portion of" Andragraphis paniculata" being in the range of half the weight of "Trichopus zeylanicus" and" Cissus quadrangularis", individually;
(d) quality controlled food grade liquid solution.

2. The synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 1, wherein, portion of "Trichopus zeylanicus" is inclusive of leaves.
3. The synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 1, wherein, portion of "Cissus quadrangularis" is inclusive of stem.
4. The synergistic poly-herbal composition inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 1, wherein, portion of "Andragraphis paniculata", said portion of "Andragraphis paniculata" being inclusive of leaves.
5. The synergistic poly - herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 1, wherein, quality controlled food grade liquid solution is a liquified form of "Trichopus zeylanicus" "Cissus quadrangularis" and "Andragraphis paniculata".
6. The synergistic poly - herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 1, wherein said composition is used in a specific concentration to achieve Apoptosis and Necrosis of cellular material with cancerous tendency is 50% in a time duration.
7. The synergistic poly - herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 6, wherein Apoptosis and Necrosis of cellular material with cancerous tendency is 50% in a time duration, said time duration being 24 hours.

8. The synergistic poly-herbal composition for inhibiting proliferation of cancer cells by inducing programmed cell death, as claimed in Claim 6, wherein said composition is used in 10 ul concentration to induce apoptosis and necrosis in cellular material with cancerous tendency