IntroductionCancer is the second leading cause of death worldwide. In the United States in 1999, it is estimated that over 1.2 million persons will be diagnosed with invasive forms of cancer, and over 1,500 people will die as a result of cancer each day. Cancer is an ailment that affects more or less 200 types of cells. The major characteristic is the lack of control of the cell proliferation, differentiation and health, invading organs and tissues. There are many difficulties in the treatment but the more frequently are the drug resistance,less toxicity and low specificity. Medicinal plants play a key role in human healthcare. About 80% of the world population relies on the use of traditional medicine, which is predominantly based on plant materials.Scientific studies indicate that the promising phytochemicals can be developed from the medicinal plants for many health problems. Natural products from plants have been valuable sources for anticancer drug discovery. Often the different components in a herb have synergistic activities or buffer toxic effects.

Mixtures of herbs are even more complex and so might have more therapeutic or preventive activity than single products alone. In fact, several studies have demonstrated that extracts from several herbal medicines or mixtures had an anticancer potential in vitro or in vivo.There is thus increased interest in alternative treatment modalities that include chemotherapy, hormonal supplements, surgery, radiation therapy, complementary or alterative medicine, used alone or in combination. The administration of herbal drugs by patients without a physician's prior counseling is increasing globally and there is a possibility of herbdrug interactions too. Besides this Herbal drugs or extracts themselves contain a combination of active constituents, which interact within themselves and also between other prescribed pharmaceutical drugs to either enhance (synergize) or decrease (antagonize) the therapeutic effect. All phytochemicals tend to increase the therapeutic effect by blocking one or more targets of the signal transduction pathway, by increasing the bioavailability of the other drug or, by stabilizing the other drug in the system. It has been a common observation that herbal formulations are better than the synthetic drugs

as they do not possess serious side effects and chronic toxicity. Plant secondary metabolites also show promise for the cancer chemoprevention, which has been defined as the use of non-cytotoxic nutrients or pharmacological agents to enhance physiological mechanisms that protect the organism against mutant clones of malignant cells. Phenolic and flavonoids contents provide antioxidant activities that may underlie the anticancer potential. Several plants have reputed applications and are deliberately used in treatment of cancer and inflammatory diseases. Plant derived natural products such as flavonoids, terpenes, alkaloids and so on have received considerable attention in recent years due to their diverse pharmacological properties including cytotoxic and cancer chemopreventive effects. The rich and diverse plant sources of India are likely to provide effective anticancer agents. One of the best approaches in search for anticancer agents from plant resources is the selection of plant based on ethno medical leads and testing the selected plants efficacy and safety in light of modern science[1] .

AssayCytotoxicity assays are widely used in in vitro toxicology studies. The LDH leakage assay, a protein assay,the neutral red,trypan blue assay and the MTT assay are the most common employed for the detection of cytotoxicity or cell viability following exposure to toxic substances[2]. The predictive value of in vitro cytotoxicity tests is based on the idea of basal cytotoxicity that toxic chemicals affect basic functions of cells which are common to all cells, and that the toxicity can be measured by assessing cellular damage. The development of in vitro cytotoxicity assays has been driven by the need to rapidly evaluate the potential toxicity of large numbers of compounds,to limit animal experimentation whenever possible,and to carry out tests with small quantities of compound. There are 3 basic parameters upon which these measurements are based:-

1.The LDH leakage assay is based on the measurement of lactate dehydrogenase activity in the extra cellular medium. Reliability, speed and simple evaluation are some of the characteristics of this assay .It has been employed as an indicator of cytotoxicity in Jurkat cells following exposure to Herbal extracts in different concentration.The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage[2] .

LDH cytotoxicity assay measures cell death in response to chemical compounds using a coupled two step reaction.In the first step, LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of

lactate to pyruvate.In the second step of the reaction,diaphorase uses the newly formed NADH and H to catalyze the reduction of a tetrazolium salt(INT) to highly- coloured formazan which absorbes Strongly at 490520 nm.The amount of formazan produced is proportional to the amount of LDH released into the culture medium as a result of cytotoxicity.[3]

2.The MTT assay is another cell viability assay often used to determine cytotoxicity following exposure to toxic substances. It has been used in jurkat cells after exposure to herbal extracts. MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water soluble tetrazolium salt,which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes and therefore it accumulates in healthy cells. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. When the amount

of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of cells can be deduced,through the production of a dose-response curve. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple MTT formazan crystals which are insoluble in aqueous solutions. The crystals can be dissolved in acidified isopropanol. The resulting purple solution is spectrophotometrically measured. An increase in cell number resultsin an increase in the amount of MTT formazan formed and an increase in absorbance[4] .

This is a colorimetric assay that measures the reduction of yellow 3-(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (eg. isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells[5].

3.The Trypan Blue assay is used to measure cell viability. It has been used as an indicator of cytotoxicity in Jurkat cell lines and other cell lines. Trypan Blue is a vital stain used to selectively color dead tissue or cells.It is diazo dye. Living cells or tissues with intact cell membrane are not coloured. Since cells are very selective in the compounds that pass through the membrane,in a viable cells trypan blue is not absorbed;however it traverses the membrane in the dead cells.Hence dead cells are shown as a distictiveblue colour under the

microscope.Since living cells are excluded from staining,this staining method is also described as a Dye Exclusion Method[6].

Dye exclusion is a simple and rapid technique measuring cell viability but it is subject to the problem that viability is being determined indirectly from cell membrane integrity.Thus, it is possible that a cells viability may have been compromised (as measured by capacity to grow or function) even though its membrane integrity is (at least transiently) maintained. Conversely, cell membrane integrity may be abnormal yet the cell may be able to repair itself and become fully viable[7] .

Toxicological Parameters 1. IC50 The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. IC50 represents the concentration of a drug that is required for 50% inhibition in vitro[8] The IC50 is a measure of how effective a drug is. It indicates how much of a particular drug or other substance is needed to inhibit a given biological process,in this case growth of Jurkat Cells[9].The IC50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist on reversing agonist activity. IC50 values can be calculated for a given antagonist by determining the concentration

needed to inhibit half of the maximum biological response of the agonist[8].

2. LD50 LD stands for "Lethal Dose". LD50 is the amount of a material, given all at once, which causes the death of 50% (one half) of a group of test animals or cells. The LD50 is one way to measure the short-term poisoning potential (acute toxicity) of a material. In toxicology, the median lethal dose, LD50 of a toxin, radiation, or pathogen is the dose required to kill half the members of a tested population after a specified test duration. LD50 figures are frequently used as a general indicator of a substance's acute toxicity. This type of test is also referred to as a "quantal" test because it is measures an effect that "occurs" or "does not occur"[10].

3. EC50 The term half maximal effective concentration (EC50) refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after some specified exposure time. It is commonly used as a measure of drug's potency. The EC50 of a graded dose response curve therefore represents the concentration of a compound where 50% of its maximal effect is observed. The EC50 of a quantal dose response curve represents the concentration of a compound where 50% of the population exhibit a response, after a specified exposure duration. Concentration measures typically follow a Sigmoidal curve, increasing rapidly over a relatively small change in concentration. The point at which the effectiveness slows with increasing concentration is the IC50[11].

4. Percentage cytotoxicity It is another measure of drug potency. It measures the extent to which cell died after exposure to drug for specific duration of time

in a given conditions.It is the ratio of absorbance of extract treated cell divided to the absorbance of untreated or control cells substracted from 100.

% cytotoxicity = 100-[(A540 in extract treated cells/A540 in untreated cell)100]

5. Lymphocyte proliferation Index It is relative measure of lymphocyte proliferation in untreated cell compared to extract treated cell.It is simply the ratio of absorbance of extract treated cell to the absorbance of untreated cells at 540nm.

LPI = A540 in extract treated cell/A540 in untreated cells 100

This parameter is calculated using MTT assay.

Plant Material

1. Piper longum

Piper longum is of South Asian origin and is found almost all over India. Its a slender aromatic climber With perennial woody roots.Piper longum has diverse pharmacological uses including nerve depressant & antagonistic effect on electro-shock and chemo-shock seizures as well as muscular in-coordination and anticarcinogenic effects.Pepper long is the dried fruit of Piper longum which is a slender, aromatic plant with creeping jointed stems and perennial woody .Pepper contains volatile oil, the crystalline alkaloids: piperine, piperidine and piperettine, and a resin[12]. Uses of Piper longum : y Piper longum is widely used in Ayurvedic and Unani systems of medicine

particularly for diseases of respiratory tract most of them s cough, bronchitis, in cough,bronchitis, asthma etc and has also anticarcinogenic effects

y Long pepper is locally applied to counter-irritant and act as analgesic for muscular pains and inflammation. y Long pepper acts as a general tonic and hematinic and widely used in Ayurveda as good rejuvenator (Rasayana). y Piper longum is known to enhance the bio-availability of food and drugs. In fact Piper Longum is taken along with Quinine. y Piper longum is widely used as a carminative. The Piper longum fruit contain 1% volatile oil,resin, a waxy alkaloid. It is used for several medicinal properties. It has much pharmacological action such as antifungal, anti-inflammatory, antioxidant and anti cancer effect and it is known to have insecticidal activity against mosquitoes and flies[13].The plant grows all over India, in evergreen forests and is cultivated in Assam, Tamil Nadu and Andhra Pradesh. Piper longum contains a component called Piperine. Piperine is an alkaloid found naturally in plants belonging to the pyridine group of Piperaceae family, such as Piper nigrum and Piper longum. It is widely used in various herbal

cough syrups and it is also used in anti inflammatory, anti malarial, anti leukemia treatment.Piperine is widely used in various herbal cough syrups for its potent anti-tussive and bronchodilator properties.Recent medical studies have shown that it is helpful in increasing the absorption of certain vitamins, selenium, beta-cartene, also increase the bodys natural thermogenic activity[14]. Piperine is known to exhibit a variety of biological activities which include anti-pyretic anti-metastatic antithyroid and antidepressant. Piperine exhibits a toxic effect against hepatocytes and cultured hippocampal neurons.Simultaneous supplementation with black pepper or piperine in rats fed high fat diet lowered thiobarbituric acid reactive substances (TBARS) and conjugated dienes levels and maintained superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX),glutathione-S- transferase (GST) and glutathione (GSH) levels close to control rats. It has also been observed that supplementation of piperine caused inhibition of Phase I and II enzymes, elevation of glutathione metabolizing enzymes, reduction in DNA damage and DNA protein cross-links in benzo (a)

Figure (1): Piperine pyrene induced lung carcinogenesis in mice[15]. Piperine prevent the formation of oral carcinoma, probably due to its antilipidperoxidative and antioxidant potential as well as its modulating effect on the carcinogen detoxification process.Many scientist observed that piperine displayed an anti-proliferation effect at 24 hours and statistically significant inhibition at 48 and 72 hours at 100 - 200 M concentration against cultured human colon cancer cells (DLD-1). Also has been investigated the influence of piperine on chromosomes in rat bone marrow. piperine administered to Wister male rats at dose of 100, 400 and 800 mg/kg, b wt, (p.o.) for 24 hrs then challenged with cyclophosphamide at a dose of 50 mg/kg, b wt, (i.p.). They demonstrated that piperine at a dose of 100 mg/kg, gave a statistically significant reduction in chromosomal aberration[16].

Piperaldehyde is one of the important constituent of piper longum Linn. It was isolated from the fruits of the piper longum by extracting with methanol as solvent. Studies shows that the pet alcoholic extract and piperaldehyde shows significant DPPH scavenging activity. The extract and piperaldehyde were also found to exert protective effective in the myocardial narcotic rats.They have protected myocardium from the harmful effects of lipid per oxidation and even maintained the gluthione levels to normal. Hence it can be concluded that the alcoholic extract as well as piperaldehyde are useful in exerting protective activity in case of myocardial ischemia in treated animals. Piperaldehyde showed dose dependent inhibitory activities on platelet aggregation induced by collagen, AA, and PAF, except for that induced by thrombin. Piperaldehyde had the most potent antiplatelet effect. Piperaldehyde inhibited platelet aggregation induced by collagen[17].

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Figure (2): Piperaldehyde

Another compound isolated from metahanolic extract of Piper longum , piperettine, has properties similar to that of piperine.

Figure : Piperettine

2. Piper Nigrum Black pepper (Piper nigrum) is a flowering vine in the family Piperaceae, cultivated for its fruit, which is usually dried and used as a spice and seasoning. The fruit, known as a peppercorn when dried, is approximately 5 millimetres (0.20 in) in diameter, dark red when fully mature, and, like all drupes, contains a single seed. Peppercorns, and the powdered pepper derived from grinding them, may be described simply as pepper, or more precisely as black pepper, white pepper, or green pepper. Green peppercorns are simply the immature black peppercorns[18]. Black peppers are native to India and are extensively cultivated there and elsewhere in tropical regions. Currently Vietnam is by far the world's largest producer and exporter of pepper, producing 34% of the world's Piper nigrum crop as of 2008[19]. Dried ground pepper has been used since antiquity for both its flavor and as a medicine. Black pepper is the world's most traded spice. It is one of

the most common spices added to European cuisine and its descendants. The spiciness of black pepper is due to the chemical piperine. It may be found on nearly every dinner table in the industrialized world, often alongside table salt.Piper Nigrum or Black pepper oil can be used to help in the treatment of pain relief, rheumatism, chills, flu, colds, increase circulation, exhaustion, muscular aches, physical and emotional coldness, nerve tonic and fevers. It furthermore increases the flow of saliva, stimulates appetite, encourages peristalsis, tones the colon muscles and is a general digestive tonic. Sometimes it is used in place of cubebs for gonorrhoea. As a gargle it is valued for relaxed uvula, paralysis of the tongue. On account of its stimulant action it aids digestion and is especially useful in atonic dyspepsia and turbid condition of the stomach. It will correct flatulence and nausea. It has also been used in vertigo, paralytic and arthritic disorders. It has also been advised in diarrhoea, cholera, scarlatina and in solution for a wash for tinea capititis. Externally it is used for its rubefacient properties and as a local application for relaxed sore throat and some skin diseases. Its oleoresin has bacteriostatic and fungistatic properties. In Ayurvedic

medicine, this herb is used for treatment of various disorders such as asthma, colon toxins, obesity, chronic indigestion, sinus congestion, fever, cold extremities, intermittent fever, cholera, colic pain, diarrhea, gastric ailments, worms and piles. Various health benefits of black pepper are described here in this article[19]. Health Benefits of Piper nigrum Some of the medicinal properties of Piper nigrum are as follows:

y

Appetizer Piper nigrum is an effective home remedy for various digestive disorders. The herb increases production of gastric juices and saliva. To increase appetite, mix powdered black pepper with malted jaggery. To get relief from indigestion and stomach heaviness, you can take piper powder mixed in the buttermilk.

y

Dentrifice Mix piper powder and salt to prevent foul breath, dental caries, toothache, bleeding and painful gums. Mix pepper powder with clove oil and apply it on the caries to prevent toothache.

y

Fever Piper is effective for cure of fever and severe cold. To get relief from cold in the head, 20 grams of powdered pepper boiled in milk and added a pinch of turmeric can be taken once a day for three days.

y

Rheumatism Small amount of pepper powder fried in a little amount of sesame oil. This mixture can be applied as an analgesic for treatment of rheumatic and myalgia pains.

y

Potency 6 peppers are eaten with 4 almonds with milk to treat impotency and it is also a never tonic[20]. Many of the secondary metabolites isolated from the piper nigrum has been found to show anticancer properties. Pellitorine , an compound isolated from the roots of Piper nigrum, showed strong cytotoxic activities against many cancer cell line. Two other alkaloids were also found from Piper nigrum. They are (E)-1-[3,4 (methylenedioxy) cinnamoyl]piperidine and 2,4-tetradecadienoic acid isobutyl amide. These compounds,having anticarcinogenic properties, were isolated

Figure (2): Pellitorine using chromatographic methods and their structures were elucidated using MS, IR and NMR techniques[21].These compounds are found to have anti proliferative activity and showed cytotoxicity activity against cancer cell line.Piper nigrum contains small quantities of chemopreventive and the antimutagenic activity of black pepper could be related to the large number of these potent chemopreventive compounds such as -carotene,piperine, tannic acid and capsaicin[22]. Black pepper has been reported to be rich in glutathione peroxidase, glucose-6-phosphate dehydrogenase, and vitamin E. Both water extract and ethanol extract of black pepper exhibited a strong antioxidant activity. Piperine, an active alkaloid, is known to possess several pharmacological actions such as antimetastatic , antimutagenic and antioxidant. Besides this Piperine modulates benzo[a]pyrene metabolism

by direct interaction with the cytochrome P4501A1 enzyme and it counteracts aflatoxin B1 toxicity by suppressing cytochrome P-450mediated bioactivation of the mycotoxin. The recent studies suggested that piperine might protect human from various types of cancer . It reduces experimentally induced colon, liver, and pulmonary cancers . some studies has reported reported the protective effect of piperine against benzo[a]pyrene -induced experimental lung carcinogenesis in Swiss albino mice by altering the tricarboxylic acid (TCA) cycle enzymes, reducing phase I enzymes and by enhancing the activity of glutathione-metabolizing enzymes. In addition, piperine plays an important role against benzo[a]pyrene induced lung carcinogenesis in Swiss albino mice by protecting the glycoprotein levels in serum and tissue, as it is one of the indicators of tumorigenesis.Piperine demonstrates not only chemopreventive effect , but also genoprotective effects against benzo[a]pyrene induced mutagenesis in Swiss albino mice. It can induce apoptosis in benzo[a]pyrene induced lung carcinogenesis in Swiss albino mice. Furthermore, black pepper supplementation can protect the colon by decreasing the activity of

bacterial enzymes -glucuronidase and mucinase, in the presence of the procarcinogen 1,2-dimethyl hydrazine (DMH) in rats . Piperine,one of the constituent of piper nigrum, has been demonstrated in in vitro studies to protect against oxidative damage by inhibiting or quenching free radicals and reactive oxygen species. Black pepper or piperine treatment has also been evidenced to lower lipid peroxidation in vivo and beneficially influence cellular thiol status, antioxidant molecules and antioxidant enzymes in a number of experimental situations of oxidative stress.However, Piperine is known to induce antiproliferative proapoptotic effects in a diverse range of tumor cells by arresting G2/M, down-regulating NF-kappa B, Akt, cyclin D, c-myc and initiating PARP cleavage/DNA fragmentation.This herb root is also known to contain sapogenins, phytosterols, beta-sitosterol and campesterol, which may contribute in part to the tumoricidal properties[23].

Figure (3): Phytosterols

3. Zingiber officinale Ginger is the rhizome of the plant Zingiber officinale, consumed whole as a delicacy, medicine, or spice. It lends its name to its genus and family (Zingiberaceae). Ginger cultivation began in South Asia and has since spread to East Africa and the Caribbean. It is sometimes called root ginger to distinguish it from other things that share the name ginger. The characteristic odor and flavor of ginger is caused by a mixture of zingerone, shogaols and gingerols, volatile oils that compose one to three percent of the weight of fresh ginger. In laboratory animals, the gingerrols increase the motility of the gastrointestinal tract and have analgesic, sedative, antipyretic and antibacterial properties. Ginger oil has been shown to prevent skin cancer in mice and a study at the University of Michigan demonstrated that gingerols can kill ovarian cancer cells[24]. [6]-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone) is the major pungent principle of ginger. The chemopreventive potentials

of [6]-gingerol present a promising future alternative to expensive and toxic therapeutic agents.Ginger contains up to three percent of a fragrant essential oil whose main constituents are sesquiterpenoids, with (-)zingiberene as the main component. Smaller amounts of other sesquiterpenoids ( -sesquiphellandrene, bisabolene and farnesene) and a small monoterpenoid fraction ( -phelladrene, cineol, and citral) have also been identified but its principal constituent are zingiberene, and zingiberol[25].Ginger is valued in medicine as a carminative and stimulant to the gastro-intestinal tract.It is much in vogue as a household remedy for indigestion dyspepsia,flatulence and colic. Ginger is reported to contain an antihistaminic factor.It is included among antidepressants and it forms an ingredient of some anti-narcotic preparations. Ginger is primarily used to treat nausea, but it is also used as an antiinflammatory, a pain remedy, a warming remedy and a cholesterollowering herb. Randomized controlled trials support its use in preventing nausea[26].

Figure (4): Gingerols

Gingerols isolated from Zingiber officinale inhibit growth & spread of various cancers including that of the ovary, cervix, colon, rectum, liver, urinary bladder, oral cavity, neuroblastoma and leukaemia by inducing apoptosis. The most active individual component, 6-shogaol, isolated from Zingiber officinale, inhibit growth & spread of many cancers particularly the ovarian cancer by blocking formation of new blood vessels and by inducing apoptosis & autophagy. It is effective even in chemotherapy resistant ovarian cancer. Zingiber officinale also possesses antioxidant, antimutagenic and anti-inflammatory properties and reduces side effects of chemotherapy & radiotherapy[27]. The pungent vanilloids, gingerol and paradol found in ginger, are very highly

effective in killing cancer cells. They achieve this both by direct cytotoxic activity against the tumour and indirectly by inducing apoptosis in the cancer cells[28]. Ginger was also shown to have anticancer effect by inducing apoptosis in rat liver cancer cells via upregulation of the expression of pro-apoptotic protein, caspase-8 and downregulation of the expression of antiapoptotic protein Bcl-2. bElemene, an anticancer drug extracted from the ginger plant, triggered apoptosis in non-small-cell lung cancer cells through a mitochondrial release of the cytochrome c-mediated apoptotic pathway.[6]-gingerol a major phenolic compound derived from ginger, inhibited TRAIL induced NF-kB activation by impairing the nuclear translocation of NFkB, suppresses cIAP expression and increased TRAIL-induced caspase3/7 activation in gastric cancer cells. On the other hand, [6]-shogaol alone reduced viability by damaging microtubules, arrested cell cycle in G2/M phase and reduced viability in a caspase-3/7-independent manner in gastric cancer cells.The other compound, zerumbone antagonises the processes of both tumour initiation and promotion. It does this by inducing antioxidant enzymes and by weakening the pro-inflammatory

signalling pathways associated with communication between cancer cells. Zerumbone found in subtropical ginger Zingiber zerumbet exhibits antiproliferative and antiinflammatory activities. Zerumbone mediates its activity through the modulation of NF- B activation, zerumbone suppressed NF- B activation induced by tumor necrosis factor (TNF), okadaic acid, cigarette smoke condensate, phorbol myristate acetate, and H2O2 and that the suppression was not cell type specific[29].

Figure (5): zerumbone

Cancer is often associated with inflammatory processes and gingers potent anti-inflammatory activity reduces the risk of inflammationinduced malignancy. Ginger is an effective COX-2 inhibitor, curtailing the activity of potentially damaging COX-2 enzymes, the overproduction of which may cause harm to several tissue

types[30].Ginger is listed among the herbs possessing the highest antitumor activities. The observed evidence from in vitro experiments warrant further investigations into the potential anti-tumor activity of ginger. This antitumor activity was shown to be related to its vanilloids, [6]-gingerol and [6]-paradol. Both compounds were shown to block the epidermal growth factor responsible for cell transformation, thus inhibiting cellular proliferation.Ginger may also produce its antitumor effect by inducing programmed cell death, also known as apoptosis, in cancer cells[31]. In one study, the cytotoxic effect of [6]-gingerol and

Figure (6): Paradols

[6]-paradol was associated with the induction of apoptosis in human promyelocytic leukemia cells (HL-60). A third mechanism of gingers antitumor protection is its modification to the carcinogen-metabolizing enzymes in the liver. Both glutathione S-transferase and aryl hydrocarbon hydroxylase activity in the liver were elevated following administration of ginger oil to mice for 14 consecutive days. [6]-paradol induced apoptosis in cancer cells , but concentrations of 50 M or greater resulted in apparent necrotic cell death . Therefore, [6]-paradol appears to exert its primary inhibitory effect on cell transformation through the induction of apoptosis. The application of [6]-gingerol to the shaven backs of mice prior to applying cancer-promoting agents significantly inhibited skin cancer formation. gingerol has been shown to inhibit cell adhesion, invasion, and motility in ER-negative (estrogen independent) human breast cancer cells in the laboratory.[6]-gingerol and[ 6]-paradol block EGF-induced cell transformation and although closely related structurally, act through different mechanisms.[ 6]Gingerol inhibit EGF-induced AP-1 transactivation by blocking EGFinduced AP-1 DNA binding activity in a concentration-dependent

manner, and in contrast, [6]-paradol had no effect on AP-1 activation. Therefore, in exerting their antitumorigenic effects,[ 6]-gingerol appears to act through inhibition of AP-1 activation, whereas [6]-paradol appears to act through induction of apoptosis[32].

4.Glycyrrhiza glabra

Glycyrrhiza glabra, also known as licorice and sweetwood, is native to the Mediterranean and certain areas of Asia. Historically, the dried rhizome and root of this plant were employed medicinally by the Egyptian, Chinese,Greek, Indian, and Roman civilizations as an expectorant and carminative. In modern medicine, licorice extracts are often used as a flavoring agent to mask bitter taste in preparations, and as an expectorant in cough and cold preparations. Licorice extracts have been used for more than 60 years in Japan to treat chronic hepatitis, and also have therapeutic benefit against other viruses, including human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Herpes simplex. Deglycyrrhizinated licorice (DGL) preparations are useful in treating various types of ulcers, while topical licorice preparations have been used to sooth and heal skin eruptions, such as psoriasis and herpetic lesions[33]. A number of components have been isolated from licorice, including a water-soluble, biologically active complex that accounts for

40-50 percent of total dry material weight. This complex is composed of triterpene saponins, flavonoids, polysaccharides, pectins, simple sugars, amino acids, mineral salts, and various other substances[34].Glycyrrhizin, a triterpenoid compound, accounts for the sweet taste of licorice root. This compound represents a mixture of potassium-calcium-magnesium salts of glycyrrhizic acid that varies within a 2-25 percent range. Among the natural saponins, glycyrrhizic acid is a molecule composed of a hydrophilic part, two molecules of glucuronic acid, and a hydrophobic fragment, glycyrrhetic acid. The yellow color of licorice is due to the flavonoid content of the plant, which includes liquiritin, isoliquiritin (a chalcone), and other compounds[35]. The isoflavones glabridin and hispaglabridins A and B have significant antioxidant activity,and both glabridin and glabrene possess estrogen-like activity.The beneficial effects of licorice can be attributed to a number of mechanisms.Glycyrrhizin inhibit hepatic metabolism of aldosterone and suppress 5-reductase, properties responsible for the well-documented pseudoaldosterone syndrome. The similarity in structure of glycyrrhetic acid to the structure of hormones secreted by the adrenal cortex accounts

for the mineralocorticoid and glucocorticoid activity of glycyrrhizic acid[36].

Figure :(7) glycyrrhizic acid

Figure :(8) Glycyrrhetinic acid

Both compounds possesses a great variety of pharmacological and biological properties such as anti-inflammatory, anti-ulcerogenic, antiallergic, anti-oxidant, anti-hepatotoxic, anti-tumor, and antiviral activities[37].

Certain licorice constituents possess significant antioxidant and hepatoprotective properties. Glycyrrhizin and glabridin inhibit the generation of reactive oxygen species (ROS) by neutrophils at the site of inflammation. In vitro studies have demonstrated licorice isoflavones, hispaglabridin A and B,inhibit Fe3+-induced mitochondrial lipid peroxidation in rat liver cells. Other research indicates glycyrrhizin lowers lipid peroxide values in animal models of liver injury caused by ischemia reperfusion. Licorice constituents also exhibit hepatoprotective activity by lowering serum liver enzyme levels and improving tissue pathology in hepatitis patients[38].

Figure (9): Glycyrrhizin Glycyrrhizin and other licorice components appear to possess anticarcinogenic properties as well. Although the exact mechanisms are still under investigation,research has demonstrated they inhibit abnormal cell proliferation, as well as tumor formation and growth in breast, liver, and skin, cancer. Glycyrrhizin is a triterpenoid saponin found in Glychyrrhiza glabra (licorice). Chemically, glycyrrhizin is a sulphated polysaccharide named (3-beta,20-beta)-20-carboxy-11-oxo-30-norolean12-en-3-yl 2-O-beta-D-glucopyranuronosyl-alpha-D glucopyranosiduronic acid[39]. It is considered to the active constituent of

the drug and the standardization of licorice is based on glycyrrhizin content. The standardized extracts of licorice sold in the market contain 20% of glychrrhizin. Glycyrrhizin is converted into glychyrrhetic acid by an enzyme, glycaronidase.Constituents of licorice includes triterpenoids, such as glycyrrhizin and its aglycone glycyrrhizic acid, various polyphenols, and polysaccharides. A number of pharmaceutical effects of licorice are known or suspected (anti-inflammatory, antivirus, antiulcer, anticarcinogenesis, and others). Licorice and its derivatives protect against carcinogen-induced DNA damage and may be suppressive agents as well. Glycyrrhizic acid is an inhibitor of lipoxygenase and cyclooxygenase, inhibits protein kinase C, and downregulates the epidermal growth factor receptor. Licorice polyphenols induce apoptosis in cancer cells. These and other activities of licorice are reviewed, and a rationale is suggested for combinations of agents in preventive clinical trials. G.glabra extract has been used in herbal formulations for treatment of cancers.G.glabra appear to induce the bcl-2 phosphorylation and G2/M cycle arrest in tumor cell lines.70% methanol soluble fraction of G.glabra extract was found to induce

apoptosis in human monoblastic leukaemic U937 cells.The compound was identified to be licocoumarone also responsible for antioxidant and antimicrobial activity.Activator protein-1 (AP-1) is a nuclear transcription factor.Blocking of tumour promoter induced AP-1 activity could be used to arrest the induced cellular transformation.It was found that Glycyrrhizin induced AP-1 activity in untreated cells whereas inhibited TPA induced AP-1 activity in TPA treated cells[40]. A variety of phytochemicals present in root extract of glycyrrhiza exhibit a potential antioxidant activity.Licochalcones B and D exhibit a potential activity by inhibiting the microsomal lipid peroxidation.Retrochalcones exhibit mitochondria lipid peroxidation and prevent red blood corpuscles from oxidative hemolysis.Isoflavones like glabridin & hispaglabridin A present in glycyrrhiza were found to have a very potential antioxidant activity against NADH dependent peroxidation injury[41].

Figure: Licochalcones

4. Phyllanthus emblica Amla (phyllanthus emblica) is a medium-sized deciduous tree. It is also named Emblica officinalis. It belongs to the plant family Euphorbiaceae.The fruits of P.emblica L. known as amla, are consumed as fruit, or in the form of food products. The species is native to India and also grows in tropical and subtropical regions. It primarily contains tannins, alkaloids, and phenolic compounds, but flavonoids derived from amla shows maximum beneficial in medicinal aspect. Ayurveda and Siddha systems of medicine have recognized the importance of this plant. It is one of the strongest rejuvenatives among Indian medicinal plants due to its antimicrobial , antifungal , radioprotective , chondroprotective , antimutagenic, and anticancer properties , but its most extraordinary features are its anti-inflammatory and antioxidative properties . A clinical study has also found that amla showed significant antioxidant properties . The fruits of Emblica officinalis are rich in tannins. The fruits have 28% of the total tannins distributed in the whole plant. The fruit contains two hydrolysable tannins Emblicanin A and B,

which have antioxidant properties, one on hydrolysis gives gallic acid, ellagic acid and glucose wherein the other gives ellagic acid and glucose[42]. Phyllanthus emblica is a medicinal fruit used in many Asian traditional medicine systems for the treatment of various diseases including cancer. Eighteen main compounds, including four norsesquiterpenoids and 14 phenolic compounds isolated from Phyllanthus emblica, together with a main constituent, proanthocyanidin polymers identified from the roots, were found to be highly effective for their antiproliferative activities against many cancer cell line.Norsesquiterpenoid glycosides, phyllaemblicins B and C , exhibited significant inhibitory effects on many cancer cell proliferation[43].

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Figure (10): Norsesquiterpenoid glycosides All phenolic compounds (518 )showed inhibitory activity against the growth of the many tumor cell lines besides their antioxidant properties.The image part with relationship ID rId88 was not found in the file.

Research showed that the antitumor activity of polyphenols might be linked to their anti-inflammatory properties.Since P. emblica has been used widely for its anti-inflammatory and antipyretic effects by local people in its growing areas. Phylanthus emblica significantly inhibited hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in

experimental animals.20 In addition to its hepatoprotective activities, Phylanthus emblica appears to be functional in acute necrotizing pancreatitis, reducing inflammation and the damage to acinar cells[44]. The studies have showed that Emblica officinalis has significant ulcer protective and healing effects and this might be due to effects both on offensive and defensive mucosal factors.27 The breast cancer is one of the most common cancers in women.Lipid-metabolizing enzymes, lipids and lipoproteins have been associated with the risk of breast cancer.The elevated levels of free cholesterol, total cholesterol,triglycerides, phospholipids and free fatty acids and decreased levels of ester cholesterol in plasma, kidney and liver found in cancer suffering animals were reverted back to near normal levels on treatment with methanolic extracts of Emblica officinalis.Besides it antiproliferative properties Phylanthus emblica has been found to enhance natural killer cell activity and antibody dependent cytotoxicity in tumor bearing mice, enhancing lifespan to 35% beyond the control animals. Phylanthus emblica has been shown to significantly reduce the cytotoxic effects of sodium arsenite when administered orally in experimental animals. Amla

resulted in an enhanced cell survival, decreased free radical production and higher antioxidant levels similar to that of control cells. Phylanthus Emblica enhance cell survival, increases phagocytosis and gammainterferon ( -IFN) production. Phylanthus Emblica has beneficial effects such as memory improving property, cholesterol lowering property and anticholinesterase activity.Phyllembin, isolated from the ethanolic extract of the fruit pulp has been found to potentiate the action of adrenaline in vitro and in vivo. It showed a mild depressant action on Central Nervous System and also had a spasmolytic activity. Phyllembin antagonize the spasmogenic effect of acetylcholine, bradykinin and serotonin on the ileum. It also antagonize serotonin and acetylcholineinduced contractions of oestrogenised uterus[45]. It increased the amplitude of cardiac contraction and heart rate transiently.Alcoholic extract of a plant (1g/kg) has shown to increase the cardiac glycogen and a decrease in serum GOT, GPT and LDH rats, suggesting a cardioprotective action[46].

5. Trikatu Trikatu is a traditional Ayurvedic herbal formulation consisting of three herbs, Black pepper(Piper nigrum), Long pepper (P. longum) and Ginger (Zingiber officinale). Trikatu means the three pungents .It consists three crude drug black pepper (Piper nigrum Linn.), dried fruits of long pepper (Piper longum Linn.) and dried rhizomes of ginger (Zingiber Officinalis Rosc) in the ratio of (1:1:1; w:w). It is an essential ingredient of numerous formulations and prescriptions of Ayurvedic medicine, used for a wide range of diseases.It is traditionally used for lung and nasal problems with the hot spices drying excessive mucus production. Trikatu is also used as a food supplement to help maintain healthy respiratory, digestive and circulatory systems. It is used in Ayurveda as part of a weight loss regime to increase metabolism, digest fats and balance appetite.Trikatu works n helpfully t prevent n allergies. It h ur skin nd t very

anti-inflammatory nd anti-

helminthes properties. It helps n removing th worms out f th body nd thus improving th digestive functions. Trikatu helps t maintain th

healthy digestive system nd removes th toxins fr m th body. It helps n rejuvenating ur body cells nd l helps n preventing n natural

infection f inflammation f th digestive organs. Trikatu h anti-histamine nd thus t helps t prevent n infection nd inflammation f th gastric mucosa. It prevents

ur body fr m n r

allergic reaction. Regular intake f th product helps n increasing appetite. Th digested food used b r body cells t give

energy t perform various activities. Trikatu enhances th metabolic activity nd rapid absorption f th nutrients. Trikatu product t treat th b d odor fr m mouth. Trikatu l th normal flow f blood n l a very g d

helps t maintain

ur body cells.Trikatu mainly has

antioxidant properties. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules.Trikatu contains combined properties of ginger,piper longum and piper nigrum as has been discussed.

Composition and constituents

As mentioned above, Trikatu consists of equal parts Black pepper (Piper nigrum dried fruit), Long pepper (P.longum dried fruit) and Ginger (Zingiber officinale dried rhizome). Both Black and Long pepper contain as their major active constituent the alkaloid piperine, which is chiefly responsible for the pungency of these peppers.

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Figure : Piperine

Black pepper contains 5-9% of the alkaloids piperine and piperettine and 1-2.5% of volatile oil, the major constituents of which are alpha- and beta-pinene, limonene and phellandrene. In one study, the essential oil of Black pepper was found to comprise 33.7% beta-caryophyllene. Long pepper was found to contain about 1.25% piperine as well as about 1% volatile oil, the major constituents of which were beta-caryophyllene

(17%), pentadecane (17.8%) and beta-bisabolene (11.16%). Long pepper also contains an amide, which has demonstrated coronary vasorelaxant activity.The major pungent compound in dried Ginger rhizome is [6]shogaol, the dehydration product of [6]-gingerol, which is the primary pungent compound in fresh Ginger. [6]-Shogaol is more pungent than [6]-gingerol. Ginger also contains a volatile oil, which shows considerable variation depending on geographical origin. Ginger from India typically yields a volatile oil containing high levels of zingiberene and ar-curcumene[46].

REFERENCES:[1]ijprd.com/IN%20VITRO%20CYTOTOXICITY%20OF%20CASSIA%20ITALICA%20MIL LER.pdf [2] linkinghub.elsevier.com/retrieve/pii/S037842740900085X [3] http://www.nshtvn.org/ebook/molbio/Current%20Protocols/CPI/ima03b.pdf