Medicinal plants

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Medicinal Plants have played a significant role in maintaining human health and improving the quality of human life for thousands of years and have served human well as valuable components of seasoning, beverages, cosmetics, dyes and medicines. Several herbs that may enhance immunity system as well, due to presence of flavonoids, vitamin C or carotenoids which enhance immune function. The flavonoids rich herbs may also possess mild anti-inflammatory action (Bruneton 1995; Tyler, 1994).

Recently, great attention has been focused on the role of the antioxidative defense system in oxidative stress. Endogenous antioxidants in medicinal herbs may play an important role in antioxidative defense against oxidative damage, possibly protecting the biological functions of cells. There is an increasing interest in the protective biological function of natural antioxidants contained in Chinese medicinal herbs, which are candidates for the prevention of oxidative damage (Shi et al., 2006).

2.1 Phytochemicals and Oxidative stress

Medicinal plants play important role in individual health. These plants composed of some chemical substances that produce a definite physiological action on the human body. Most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds (Hill, 1952). Many of these indigenous medicinal plants are used as spices and food plants. They are also sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes (Okwu, 1999, 2001). Today many natural products extracted from medicinal plants are being tested for presence of new drugs with new modes of pharmacological action. Special features of higher plants are their capacity to produce a large number of secondary metabolites (Castello et al., 2002). Recent studies are involved in the identification and isolation of new therapeutic compounds of medicinal importance from higher plants for specific diseases (Erturk et al., 2006; Mohantaet al., 2007). Some bioactive plants derived from plants are include tannins, alkaloids, cardiac glycosides , flavinoids, sterol, triterpenes  and anthraquinones which play main role in nutrition, physiology and control of various diseases (Sofowara, 1993). Tannins play important role in many biological activities including spasmolytic activity of smooth muscles (Tona et al., 1999), free radical scavengers (Bharani et al., 1995), protect the body against oxidative stress (Yoshizawa et al., 1987). Saponins control Na+ efflux in body and Na+−Ca+ balance thus minimizes oxidative stress, and cardiac failure (Schneider and wolfling, 2004) while Glycosides take part in cardiac degerative diseases, and hypoglycemic dysfunction (Akhter et al. , 1981).

Some fat soluble natural product obtained from medicinal plants are best antioxidants, such as fat soluble vitamin E and more hydrophilic flavonoids, possess free radical scavenging properties. The interaction of these natural antioxidants with reactive oxygen species implicated in inflammation. Recent evidence suggests that vitamin E and its analogies may not only protect cells from free radical damage but also induce apoptotic cell death in malignant cell lines and also act as antitumour in vivo. Flavonoids are a type of polyphenols that commonly occur in plants, and more than 4000 flavonoids and polyphenolic have been found and are frequent in components of human diet (Henry et al., 2006). Currently, due to antioxidant potential of phenolic compounds, its study become of great interest. Dietary plant phenolic compounds have been described to exert a variety of biological actions such as free-radical scavenging, metal chelating, modulation of enzymatic activity and more recently to affect signal transduction, activation of transcription factors and gene expression. They received particular attention in the past 10 years because of their putative role in the prevention of several human diseases (Srinivasan et al., 2005).

Therefore, it has become a task to prevent oxidative stress and cancer by eliminating free radicals and lipid peroxidation through the use of potent phytochemicals and natural antioxidant (Adewole et al., 2007).

2.2 Control of microbes and medicinal plants

In ancient times people used spices and herbs in their food not only as flavoring agents, but also as folk medicine and food preservatives (Beuchat, 1994; Cutler, 1995). In addition to flavoring agents, many herbs, Spices also possessed free radical scavenging antioxidant activities and antimicrobial activities like bactericidal and bacteriostatic (Beuchat and Golden, 1989). Today world wide a large number of medicinal plants, spices and herbs are in use for their antimicrobial activities in addition to salad, food flavoring, and fragrance. In addition to fresh use of medicinal plant, spices and herbs, their extracts are being used as alternative medicines for microbial control, food preservation, pharmaceuticals, and natural therapies (Lis-Balchin and Deans, 1997).

Medicinal plant possessed many bioactive compounds including phenolic compounds which play important role in detoxification of oxidative stress and antimicrobial activities (Hara-Kudo et al., 2004). Liu et al., (2009) studied the chemical composition and antimicrobial activity of essential oil of Ganoderma japonicum against eighteen microbes and found that this essential oil possessed significant antimicrobial activity and concluded as natural antibiotic. Duraipandiyan and Ignacimuthu, (2009) characterized the antibacterial and antifungal activity of bioactive compound flindersine isolated from the traditional medicinal plant, Toddalia asiatica (L.) Lam. using disc-diffusion method and minimum inhibitory concentrations (MICs). Runyoro et al., (2009) four Ocimum species (Ocimum basilicum, Ocimum kilimandscharicum, Ocimum lamiifolium, Ocimum suave) of Tanzania for their photochemical and antimicrobial properties, found eighty one compounds that were possessed best antibacterial properties. Antimicrobial activities of 46 extracts from dietary spices and medicinal herbs were evaluated through agar-well diffusion method against five food borne bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella anatum). Their Phytochemicals were also studied and found that many herb and spice extracts contained high levels of phenolics and exhibited antibacterial activity (Shan et al., 2007). More et al., (2008) studied the antimicrobial activities of ethanolic extracts of eight plant species used traditionally in South Africa were investigated for in vitro antimicrobial activity against oral pathogens namely Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Actinomyces israelii, Candida albicans, Porphyromonus gingivalis, Privotella intermedia and Streptococcus mutans using the disk diffusion through Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using micro dilution. For the first time, both antioxidant and antimicrobial activities were studied in polar fractions of halophytic plants. Chloroformic and methanolic extracts of the halophytes Eryngium maritimum L., Crithmum maritimum L. and Cakile maritima Scop were characterized to investigate antimicrobial activities, in addition, to radical scavenging antioxidant activities and phytochemistry and concluded that chloroformic fraction was more active than methanolic fraction (Meot-Duros et al., 2008).

Webster et al., (2009) assesed antifungal activity of aqueous extract of 14 plants against five species of human pathogenic fungus and concluded these plant extract significantly controlled the fungal strains. 70% ethanolic classical and ultrasound solvent extract of Echinacea purpurea L. (Asteraceae) was compared for phytochemical, radical scavenging antioxidant and antimicrobial activity and concluded that  extracts obtained by classical extraction was more active than those by ultrasound extraction (Stanisavljevi et al.,2009).The essential oil and methanolic extract of Ziziphora clinopodioides Lam. harvested in the Eastern part of Turkey were evaluated for phytochemical composition and antibacterial activity against 52 Gram-positive and Gram-negative bacteria. 18 compounds were determined through GC-MS analyses; the main constituents of the essential oils were (+)-pulegone, 1, 8-cineole, limonene, menthol, menthone, piperitenone and piperitone, concluded that the essential oil and methanolic extract possessed significant antimicrobial activity against pathogens in broth microdilution bioassays (Ozturk and Ercisli., 2007).

2.3 Cytotoxic characterization of medicinal plants

Plants are used worldwide for the treatment of many diseases, and novel drugs continuously developed from plants. There are more than 20,000 species of plants used in traditional medicines, and all of these bioactive fractions for development of new drugs (Hamamouchi, 2002). In industerlized countries advance medicine research replaced the medicinal plant in developing countries; the majority of the world's population cannot afford pharmaceutical drugs and use their own plant-based indigenous medicines. Therefore traditional medicinal plants have received comparatively more attention because of the presence of their bioactive components leads to new drugs (Kim, 2006).    During the past decade, many cancer therapeutic agents induce a cell death process known as programmed cell death, or apoptosis. Although the mechanisms by which chemotherapeutic agents can kill tumors via apoptotic pathways have been controversial, the killing of tumors through the induction of apoptosis has been now recognized as a novel strategy for the identification of anticancer drugs (Panchal, 1998; McConkey et al., 1996).  Rahman et al., (2008) characterized the medicinal importance of crude extract and

2α,3β,21β,24β,28-pentahydroxy-olean-12-ene isolated from the root of Laportea crenulata exhibited remarkable cytotoxic activities through brine shrimps in addition to antimicrobial activities,  LD50 value of the compound was found to be 27.54 μg/ml. Cytotoxic activities of 80% methanolic extract of 45 medicinal plants collected in Sankuru (Democratic Republic of Congo) through MRC-5 cell lines respectively. Cell-lines MRC-5 were cultured in MEM medium supplemented with 20mM l-glutamine, 16.5mM NaHCO3, 5% foetal calf serum and 2% P/S solution. After 4 h incubation, cell proliferation/viability was spectrophotomecally assessed at 540 nm after addition of MTT. In each assay, the IC50 value for each sample was derived by the drug concentration-response curves (Mesia et al., 2009).

Kilani et al., (2009) studied the cytotoxic and apoptotic activities of tubers infusion and extracts of Cyperus rotundus. It was observed that due to presence of cytotoxic compounds in ethyl acetate extracts suppressed growth and proliferation of L1210 cells derived from murine lymphoblastic leukemia. Mothana et al., (2008) characterized the methanolic extract of Yemeni Medicinal Plants against FL-cells lines cytotoxicity and found that activities of plant extracts are due to presence of some bioactive compounds present in the extract. Twenty medicinal plants crude methanolic extracts possesses the antimicrobial activities was characterized in vitro for cytotoxic activity on MCF7 (human breast epithelium) cell line though MTT assay, found after 72 hrs incubation with various concentration of the extracts on MCF7 cells were positive (Shamim et al., 2009).

Abu-Dahab and Afifi et al., (2007) characterized 76 bioactive active ethanolic extract from Jordanian flora for their antiproliferative activity on a breast cancer cell line (MCF7). The cells were cultured in RPMI 1640 medium and incubated with the extracts for 72 hours using SRB cytotoxicity assay.

2.4 Chromatographic characterization of Flavonoids

Flavonoids are important and largest group of a class of natural compounds with wide distribution in plant kingdom. Approximately more than 4000 flavonoids have been identified to date, in the leaves, seeds, bark and flowers of plants. These compounds provide protection against ultraviolet radiation, pathogens, and attract pollinating insects due to presence of anthocyanin are pigments in flowers and also responsible for fruits colures (Harborne and Williams, 2000).

Flavonoids are benzo-γ-pyrone derivatives consisting of phenolic and pyrane rings (Figure 1) and are classified according to substitutions. Flavonoids are classified into six classes, which differ in their chemical structure, flavanols, flavones, flavonols, flavanons, isoflavons and anthocyanidins. While the most dietary common flavonoids occur in food as 3-O-glycosides and polymers, but they can also exist in aglycon forms. Many beneficial health effects are attributed to flavonoids, mostly due to their antioxidant and chelating Abilities, antimycotic, antiviral, antiinflamatory, and ezyme inhibitor (Heim et al., 2002).

Saputera et al., (2006) characterized hexane soluble fraction of Indonesian croton tiglium seeds through GC/MS and found that it possessed 17 peaks. Flavonoids content of seed and root exudates of Lotus pedunculatus was analyzed  using  capillary zone electrophoresis coupled to a UV spectral array detector (CZE-UV), high performance thin-layer chromatography with densitometry (HPTLC-UV) and gas chromatography with mass spectrometry (GC-MS), found that seed extract composed of  catechin, naringenin, kaempferol, quercetin aglycone and 3 different glycosides of quercetin were detected while  sterile root exudates contained all of these in addition to apigenin, kaempferol (Steelea et al., 1999). Flavonoids of Ginko biloba was characterized through HPLC including quercetin, kaempferol, isorhamnetin using Hypersil BDS C18 5μm 4 250 mm; column temperature: 35°C; mobile phase: 46% methanol-54% water; flow rate: 0.7 ml/min; detection wavelength: 360nm (Zhang and Xiang , 2002). Four different flavonoids were quantified using standard by Zu et al., (2006) by RP-HPLC method using photodiode array detection (PAD) in Pigeonpea Cajanus cajan (L.) Millsp. leaves with standard protocol while Cristea et al., (2003) Identified and quantitatified three main flavonoids of weld (luteolin, luteolin-7-glucoside and luteolin-30,7-diglucoside) through HPLC analysis in weld (Reseda luteola L.). Ye et al., (2002) Assessed flavonoids content in 40 samples of Semen Cuscutae collected from areas all around China were investigated and five flavonoids including quercetin 3-O-β-D-galactoside-7-O-β-D-glucoside, quercetin3-O-β-D-apiofuranosyl-(1→2)-β-Dgalactoside, hyperoside, quercetin and kaempferol were analyzed simultaneously by using a reversed phase liquid chromatograph system with 0.025 M phosphoric acid-methanol as mobile phase, found Cuscuta australis contained a much higher content of kaempferol than C.chinensis, and few flavonoids were detected in C. japonica.

2.5 Allelopathy and crop production

In recent years, scientists have focused to increase the production of food needed for the fast expansion of world population, but unfortunately, crop yield losses occur due to insects and plant diseases caused by fungi, bacteria and viruses (Fletcher et al., 2006). Fungi and bacteria have also unfavorable effects on quality, safety and preservation of food. Therefore various synthetic chemicals are widely used in the control of plant diseases. However, these chemicals may cause toxic residues in treated products (Barnard et al., 1997; Isman, 2000) and environmental imbalance and slow biodegradation (Barnard et al., 1997; Misra and Pavlostathis, 1997. Weeds are another major problem in crop production because they losses in crop yield every year. Therefore, farmers have increased chemical spray called herbicide. While, intensive use of synthetic herbicides can result in soil and groundwater contamination, and development of weed resistance (Duke et al., 2000). Therefore herbicides at high concentrations can also increase the risk of toxic residues in agricultural products. Therefore, researchers have focused on new potential of allelochemicals and bio-herbicides, having different and selective herbicidal mechanisms in comparison to their synthetic herbicides (Dudai et al., 1999; Duke et al., 2000). Allelopathy is the science that studies any process involving secondary metabolites produced by plants, algae, bacteria, coral and fungi that influence the growth and development of agricultural and biological systems. The biomolecules are called allelochemicals and are produced by some plants as secondary metabolites. When the allelochemicals are released into the environment, they inhibit the development of neighboring weed plants which reduces the production of crop yields (IAS, 1996).

Kordali et al., (2008) studied the chemical composition of essential oil isolated by hydro distillation from the aerial parts of Origanum acutidens by GC-MS and their impact on production of crop as allelopathic chemical. The oil, carvacrol and thymol completely inhibited the germination of seed and seedling growth of Amaranths retroflexus,Chenopodium album and Rumex crispus while p-cymene did not show any phytotoxic effect. These findings of the present study suggest that herbicidal properties of the oil can be due carvacrol, and these agents have a potential to be used herbicide for crop and food to meet need of day. The allelopathic potential of Tetrapleura tetraptera (Schum and Thonn.)Taub, was studied by using aqueous leaf extracts at different concentrations. The extracts stimulated production of lateral roots in Lycopersicon esculentum Mill. But inhibited root growth in Abelmoschus esculentum L. and similarly reduced shoot length at 25% concentration and above in Amaranthus spinosus L. while the extract significantly stimulated it at the same concentrations in L. esculentum and A. esculentum. However, both shoot and root lengths of Capsicum annum L. were significantly inhibited at all extract concentrations, showed that root length is a more sensitive indicator of phytotoxic activity (Amoo et al., 2008).

2.6 Effect of Carbon tetrachloride on Histology

Carbon tetra chloride is used as a hapatotoxic chemical in experimental animals. Tissue damage by carbon tetrachloride depends upon the amount of dosage and duration of exposure of the experimental animals to this toxicin, causes many dysfunctions of kidneys, lungs, testis and brain as well as in blood by generating free radicals.

According to Shenoy et al. (2001) the administration of CCl4 to rat causes hydrophilic changes in centrilobular hapatocyts with single cell necrosis surrounded by neutrophils. Congestion of central vein and sinusoids were seen with acute and chronic inflammatory cells infiltrating sinusoids mainly in central zone. The midzonal and periportal hapatocyts showed mild to moderate degree of fatty changes. Okada et al. (2003) reported the effect CCl4 on liver of rats and observed that CCl4 causes severe parenchymal damages, delay in regeneration of hepatocytes and prominent proliferation in mesenchymal cells and fibrosis in growth hormone deficient rats as compared to normal rats. Vascular degeneration in cetrilobular to midzonal hapatocytes and necroses of hapatocytes was also observed. In addition, an accumulation of agrophilic reticular fibers was observed along with infiltration of small epithelial like cells. In other reported studies it was revealed that CCl4 imposed focal necrosis, fatty changes, ballooning degeneration and lymphocytes around the central vein. Long chronic exposure to CCl4 also causes hepatocellular carcinoma in rats, but in most cases the benign tumour and hepatic nodules are reported in rats. The development of hepatocellular carcinoma, benign tumour, hepatic nodules and hepatic injuries depends the type of strain used during the experiment. Several studies reported the production of malignant tumors in rats following subcutaneous injections and oral administrative of CCl4. Tumour production in rats has been demonstrated in number of strains in both sexes (California Public Health Goal (PHG), 2000).Administration of CCl4 at a dose of 1.2 g/kg body weight 3 times per week for 3 weeks led to the development of hepatic injury in rats. The histopathalogical changes induced by the treatment of CCl4 alone caused multi dimensional changes includes diffuse fatty degeneration and multiple foci of hepatocellular necrosis, sinusoids dilations by erythrocytes was seen. Central vein was also found congested (Omolola et al., 2009).

According to Jayakumar et al., (2008) treatment of CCl4 significantly effects the histology of kidney, heart and brain. These changes includes distortion of the renal corpuscles and distention of the proximal and distal convoluted tubules in the kidneys, disruption of blood capillaries in the heart and vacuolization of the cerebral cortical cells in the brain tissue were observed. Administration of 2 ml/kg body weight CCl4 for 16 weeks caused marked changes in histopathology of testicular tissue. These changes includes complete atrophy of seminiferous tubules while in other areas of the section the tubular basement membranes of seminiferous tubules were identified, but most of the germ cells were degenerated, especially the ones involving highly differentiated germ cells along with deformed sperms. Partially the ground substance within the interstitium also disappeared and replaced by fibroblast and inflammatory cells (Khan and Ahmed, 2009).

Ogeturk et al., (2005) studied the effects of CCl4 toxicity in rat oxidative stress and nephrotoxicity.  Administration of CCl4 affected glomeruli with mild dilatation of Bowman's capsule, glomerular atrophy, congestion in the capillary loops with an adhesion between visceral and parietal layers of Bowman's capsule, interstitial inflammatory cell infiltrations, fibrosis and congestion in the peritubular vessels. Treatment of CCl4 caused marked marked histological changes in both cortex and medulla. Cortex was more severely affected due to the CCl4 toxicity as compared to medulla. The renal sections showed marked tubular degeneration, tubular dilatation, interstitial fibrosis, glomerular atrophy, glomerular hypertrophy, glomerular degeneration and congestion in capillaries (Khan et al., 2009).

2.7 Effect of Carbon tetra chloride at Molecular level

CCl4 also affects the macromolecules like DNA, RNA, and Protein. It causes the DNA damages leading to the micro or macro mutation in the cell. DNA damages are due to the free oxy radical's formation of CCl4 with oxygen molecules. Carbon tetrachloride induces reactive oxygen species (ROS) and oxidative DNA damages, with the formation of DNA adducts, genetic mutation, strand breakage and chromosomal alterations. DNA strand breaks are especially important in inducing mutations, such as deletions and translocations, in affected cells undergoing replication with error-prone repair or without proper repair. Moreover, extensive DNA strand breaks without prompt repair may cause cell death and compensatory cell regeneration (Jia et al 2002).

According to Kamalakkannan et al., (2005) carbon tetrachloride metabolized intotrichloromethyl, (CCl3*) free radical in the presence cytochrome P450 oxygenase system of the endoplasmic reticulum. These free radical reacts with amino acids, nucleotides and fatty acids, as well as proteins, nucleic acids and lipids. In the presence of oxygen, the CCl3* radical is converted to the trichloromethyl peroxy radical (CCl3OO*). This radical is more reactive and is capable of abstracting hydrogen from polyunsaturated fatty acids (PUFA) to initiate the process of lipid per oxidation (Weber et al., 2003).

Telomeres are repetitive (TTAGGG) non-coding DNA regions at the ends of chromosomes, which are progressively shortened because of incomplete replication of the 3-ÙŽends. In vertebrates, telomerase is the main mechanism presently known that can stabilize the loss of telomeres. High telomerase activity is observed in malignant tumors, neoplasias and hyperplasias, whereas low or no telomerase activity is observed in normal somatic cells. These findings suggest that telomerase activation is a crucial step in cell immortalization and oncogenesis (Segawa et al., 2003). Administration of 3ml/kg b.w. CCl4 for 16 weeks activates telomerase enzyme, where as it was found absent in control non treated group, justify telomerase enzyme activity (Khan et al., 2009)

2.8 Effect of Carbon tetra chloride at Biochemical level

CCl4 causes variation in biochemical level. It also effect the level of marker enzymes in serum as well as the level of antioxidant enzymes and antioxidant compounds like Vitamin C, E and other compounds which were recently investigated. The membrane marker enzymes are amino transaminase (AST), Alanine transaminase (ALT), alkaline phasphatase (ALP), gamma glutamyl transaminase (γ-GT), increases bilirubin, total serum protein, globulin and creatnine while decreases albumin and creatnine clearance showing abnormality of liver and kidney. When liver cell plasma is damaged a variety of enzymes located normally in cytosol is released into the blood, thereby causing increased enzyme level in the serum. The estimation of enzymes in the serum is a useful quantitative marker of the extent and types of hepatocellular damage (Jadon et al., 2007). (Sreelatha et al., 2009) reported that administration of 1 ml/kg b.w. CCl4 in rats significantly elevated the serum marker enzyme level including alkaline phasphatase (ALP), alkaline transaminase (ALT), amino transaminase (AST), acid phasphatase (ACP), serum total protein and hyperbilirubinaemia indicating sever necrosis of liver. Secretion of antioxidant enzymes like superoxide dismutase, catalase, Glutathione peroxidase, and TBARs indicating increase in lipid peroxidation. According to Kamalakkannan et al. (2005) the excessive liver damage and oxidative stress caused by CCl4 depleted the levels of GSH, vitamin C and vitamin E. Oxidative stress induced by CCl4 results in the increased utilization of GSH and subsequently the levels of GSH is decreased in plasma and tissues. Utilization of vitamin E is increased when oxidative stress is induced by CCl4 and this shows the protective role of vitamin E in mitigating the elevated oxidative stress. Vitamin C scavenges and destroys free radicals in combination with vitamin E and glutathione. It also functions cooperatively with vitamin E by regenerating tocopherol from the tocopheroxyl radical. A decrease in the levels of vitamin C may indicate increased oxidative stress and free radical formation in CCl4-induced liver injury. A major defense mechanism involves the antioxidant enzymes including super oxide dismutase (SOD), Catalase (CAT) and GPx which convert active oxygen molecules into non-toxic compounds. CCl4-administration decreased the activities of these antioxidant enzymes and GSH concentration in the tissues.

Singh et al., (2008) studied the protective effects of potato peel extract against CCl4 induced toxicity in rats. Level of serum marker enzymes like ALT, ALP, AST, and LDH was significantly increased in CCl4 treated rat which was recovered by various doses of extract. Similarly secretion of antioxidant enzymes and TBARS was reversed by to control level, proving the protective effects of potato peel extract against hepatotoxicity in rats. Bhattacharya et al., (2005) evaluated toxicity of CCl4 in liver and kidney and found that significant increase in Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phasphatase (ALP), lactate dehydrogenase (LDH), and blood urea nitrogen (BUN). This study indicates that the enzymatic activity was comparatively higher in the liver than kidneys, suggesting that the liver is the target organ of CCl4 toxicity. Carbon tetrachloride reduce the total protein amount in blood as well as induce the depletion of hepatic and renal antioxidant enzymes such as catalase (CAT), peroxidase (POD) , super oxide dismutase (SOD), Glutathione peroxidase, and TBARs indicating increase in lipid peroxidation, creatnine and blood urea nitrogen (BUN). The depletion of these antioxidant enzymes are due to the controlling action against oxy radicals produce by the CCl4 (Adewole et al., 2007).  Jayakumar et al., (2008) reported the protective effect of the oyster mushroom, Pleurotus ostreatus on carbon tetrachloride (CCl4)-induced toxicity in male Wistar rats. CCl4 significantly reduced the level of of reduced glutathione (GSH), vitamins C and E and elevated the level of malondialdehyde (MDA) in kidneys, heart and brain of rats exposed to CCl4, when compared to values in control rats. Quantitative and qualitative analysis of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (Gpx) and glutathione-S-transferase (GST) revealed lower activities of these antioxidant enzymes in the kidneys, heart and brain of rats exposed to CCl4. While extract of P. ostreatus was used to treat rats with CCl4-induced toxicity, it lowered the mean level of MDA, elevated the mean levels of GSH and of vitamins C and E and enhanced the mean activities of CAT, SOD, Gpx and GST near to control rats suggests that an extract of P. ostreatus is able to alleviate the oxidative damage caused by CCl4 in the kidneys, heart and brain of Wistar rats. Khan and Ahmed et al., (2009) reported the protective effects of Digera muricata against CCl4 induced toxicity in rats. Treatment of CCl4 (2 ml/kg body weight i.p.) in rats increased the body weight and relative testis weight. It also markedly increases the level of TBARS and nitrites along with corresponding decrease in reduced glutathione and various antioxidant enzymes in testis, i.e., catalase, peroxidase, superoxide dismutase and glutathione peroxidase. Serum level of testosterone, luteinizing hormone and follicle stimulating hormone was significantly decreased while estradiol and prolactin were increased. Orally supplementation of Digera muricata in treated rats reversed the CCl4 intoxication in rats near to control group, provides evidence that Digera muricata possesses some bioactive compounds which showed theraphitic role in detoxification.

2.9 Potassium bromate (KBrO3) and cancer

An imbalance in the pro-oxidant-anti-oxidant level leading to oxidative stress. The oxidative stress of KBrO3 caused damage in a cell, tissue or organ caused by the reactive oxygen species (ROS). These ROS react with cellular membrane, lipids, nucleic acids, proteins and enzymes resulting in cellular damage and degeneration. They are involved in many diseases including diabetes mellitus, carcinogenesis, mutagenesis and ageing (Sun,

1990). Potassium bromate (KBrO3) is a potent nephrotoxic and hepatotoxic widely used as a food additive in the bread-making process for the maturation of flour and cold-wave hair lotions. It is also used in food products, beverages and found in drinking-water samples as a by-product of ozone disinfection (Kurokawa et al., 1982). KBrO3 induces renal cell tumors and carcinogenesis, mesotheliomas of the peritoneum, and follicular cell tumors of the thyroid and cause chromosomal aberrations and micronucleus formation both in vivo and in vitro (Kurokawa et al., 1990). KBrO3 administration along with N-ethyl-N-hydroxyethyl-nitrosamine initiated renal tumors in rats (Umemura et al., 1995).

Khan and Sultana, (2004) studied the modulatory effects of soy isoflavones on KBrO3 induced renal oxidative stress and cell proliferation in rats. Administration of  KBrO3 (125 mg/kg body weight, intraperitoneally) caused reduction in renal glutathione content, activities of renal anti-oxidant enzymes, and phase-II metabolizing enzymes with enhancement in xanthine oxidase, lipid peroxidation, γ-glutamyl transpeptidase and hydrogen peroxide (H2O2, blood urea nitrogen, serum creatnine and tumor promotion markers, viz., ornithine decarboxylase (ODC) activity and thymidine [3H] incorporation into renal DNA which was reversed by  treatment of soy isoflavones, shows that soy isoflavones acts as potent chemopreventive agent against KBrO3 induced renal oxidative stress, and cell proliferation in rats.

Delker et al., (2006) investigated the contribution of oxidative stress in KBrO3-induced cancer; KBrO3 was administered in rat for 2-100 weeks. mRNA transcripts was expressed in bromate treatment included multiple cancer, cell death, ion transport and oxidative stress genes. Multiple glutathione metabolism genes, 8-Oxodeoxyguanosine glycosylase (Ogg1) mRNA were up-regulated in kidney but not thyroid. Similarly KBrO183 orally gavage proved the enrichment in liver followed by kidney, thyroid and testis. Cadenas and Baria, (1999) studied the protective effects of melatonin, resveratrol, vitamin E, butylated hydroxytoluene , 2-mercaptoethylamine, and the a-phenyl-N-tert-butyl nitrone (PBN) against oxidative DNA damage of carcinogen potassium bromate (KBrO3) in kidney. Oxidative damage of kidney DNA was estimated 6 hours afterwards by measuring 8-oxo-7,8-dihydro-29-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high performance liquid chromatography with electrochemical-coulometric and ultraviolet detection, was significantly increased in KBrO3, prevented by melatonin, resveratrol, vitamin E, butylated hydroxytoluene , 2-mercaptoethylamine, and the a-phenyl-N-tert-butyl nitrone (PBN). Matsuoka et al., (2007) evaluate protective effects of resveratrol and its analogue in a 6-month feeding test in young adult mice, found that KBrO3 doesn't change any parameter of serum biochemistry and MDA level.

2.10 Oxidative stress and DNA damages

More than 200 years after the first reports of chemically induced cancer in humans, many carcinogenic chemicals have been identified in the environment. Chemical carcinogenesis typically requires chronic exposure, followed by a period of years during which a complex series of events, involving DNA damage and alterations in gene expression, take place. The earliest carcinogen-induced events typically include DNA structural damage, which often occurs as the result of covalent binding of carcinogens to DNA (DNA-adduct formation). This results in DNA mutations, leading to alterations in protein structure and function that can result in tumorigenesis. Many human cancers are associated with exposure to genotoxic chemicals. There is typically a long period (years) between early events that include initial carcinogen exposure, the onset of DNA damage and the fixation of mutations, and the subsequent appearance of a tumor (Miller, 1970). Normal cells DNA damage is an important first step in this carcinogenic process. Chemical carcinogens can cause the formation of carcinogen-DNA adducts or induce other modifications to DNA, such as oxidative damage and alterations to DNA ultra structure (DNA-strand cross linking, DNA-strand breakage, chromosomal rearrangements and deletions). Although cells posses mechanisms to repair many types of DNA damage, these are not always completely effective, and residual DNA damage can lead to the insertion of an incorrect base during DNA replication, followed by transcription and translation of the mutated templates, ultimately leading to the synthesis of altered protein. Mutations in an oncogene, tumor-suppressor gene or gene that controls the cell cycle can result in a clonal cell population with a proliferative or survival advantage. The development of a tumor requires many such events, occurring over a long period of time, and for this reason human cancer induction often takes place within the context of chronic exposure to chemical carcinogens. In experimental models of chemical carcinogenesis, DNA adducts have been shown to be necessary, but not sufficient, for tumorigenesis (Poirier, 2004).

Exogenous chemicals such as heavy metals, KBrO3, CCl4 and alcohol or endogenous factors such as estrogen and infections can induce reactive oxygen species (ROS) and oxidative DNA damage, with formation of DNA adducts genetic mutations, strand breakage and chromosomal alterations. DNA strand breaks are especially important in inducing mutations, such as deletions and translocations, in affected cells undergoing replication with error-prone repair or without proper repair. Moreover, extensive DNA strand breaks without prompt repair may cause cell death and compensatory cell regeneration. In addition, diverse types of DNA damage like DNA strand breaks can trigger the induction of p53 signals. Many investigators consider that p53 accumulation in the early stage of hepatic-carcinogenesis may be used as an indicator of DNA damage (Jia et al., 2002).

Jia et al. (2002) clarify the significance of oxidative DNA damage and interaction between oxidative DNA damage, apoptosis and neoplastic cell growth, used the mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver due to mutations of the ATP7b gene encoding copper ATPase. In LEC rats, hepatitis spontaneously develops at around 20 weeks of age and then HCC at more than 50 weeks of age. Oxidative stress, including lipid per oxidation, and increased formation of oxidative DNA, p53, proliferation adducts in the liver of LEC rats are known to be linked with various types of mutations such as base substitutions, deletions and insertions. Therefore, the induction of DNA strand breaks, the expression of p53 and the development of preneoplastic foci while monitoring the kinetic changes of cellular proliferation and apoptosis during overall hepatocarcinogenesis was examined in the experimental models (Sone  et al., 2000).

Damage to DNA from reactive oxygen species (ROS) is a consequence of oxidative stress, and several oxidative DNA adducts, including 8-oxodG, have been implicated in the tumorigenic process (Beckman and Ames, 1997). Oxidative stress exists when pro-oxidants such as ROS exceed antioxidant capabilities. This environment can result from increased generation of ROS as well as impaired removal of ROS by antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme systems (Fig. 1). Differences in ROS generation or antioxidant enzyme activities between the small intestine and colon may alter the levels of oxidative DNA damage, thus contributing to the variation in cancer susceptibility at these two intestinal sites (Sanders et al., 2004).

Cellular antioxidant enzyme defenses against reactive oxygen species. Superoxide radicals (O2 .- ) can be generated from O2 via cellular oxidase or mitochondrial electron transport. Superoxide dismutase (SOD) converts superoxide radicals to the non-radical reactive species, H2O2. H2O2 can be removed enzymatically by glutathione peroxidase (GPx) or CAT to form water and/or O2. Alternatively, H2O2 can react with iron (Fe) or copper (Cu) via a Fenton reaction to form hydroxyl radicals (OH- that can directly damage DNA. The balance of the activity of these enzymes in the generation and removal of H2O2 is crucial in maintaining the oxidative status of the cell (Sanders et al., 2004).

Continuous cell death and regeneration in the course of chronic hepatitis results in accumulation of DNA damage and eventually leads to development of hepatocellular carcinomas (HCCs).The causes of hepatitis in humans are mainly hepatitis C or B viruses or environmental chemicals.  Recent reports have shown that oxidative stress induced by endogenous or exogenous oxidants is closely associated with tumor generation, by contributing to the chronic hepatitis phase (Yamamoto et al., 1998).

Chipman et al., (1998) studied that either potassium bromate directly oxidizes DNA or it is linked to lipid peroxidation, treated the calf thymus DNA with potassium bromate (KBrO3) and glutathione (GSH), caused an increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured.DNA oxidation is GSH-dependent and was associated with loss of GSH during incubation. 8-oxodG was not found to be elevated significantly in either total tissue DNA or mitochondrial DNA isolated from rat kidney perfused in situ with KBrO3, with no increase in the level of renal lipid peroxidation or reduced or oxidised GSH. 100 mg/kg KBrO3 in rats gave evidence for oxidative stress in the kidney, increased lipid peroxidation and oxidised GSH. Pretreatment of rats with diethylmaleate (DEM) to deplete GSH, elevated the toxicity of 100 mg/kg KBrO3. However, 20 mg/kg, showed no change of kidney oxidative stress either with or without DEM pretreatment with the exception of a small but increase in mitochondrial 8-oxodG when KBrO3 was given following DEM pretreatment. DNA oxidation in the kidney is therefore not inhibited by GSH depletion and requires exposure of toxic concentration of KBrO3 which is associated with lipid peroxidation and GSH oxidation. The results do not support a role, in rat kidney, of a direct, GSH-mediated mechanism for KBrO3-induced DNA oxidation as seen in vitro.

Khan et al., (2009) studied protective effects of Digera muricata against CCl4 oxidative DNA damages in rats. 3 ml/kg CCl4 significantly oxidizes the renal DNA of rat, studied by DNA fragmentation and verify by DNA ladder assay but this oxidative damages was significantly reversed by methanolic fraction of Digera muricata, due to may be posses some bioactive compounds.

2.11 Telomerase activity and cancer

Telomerase is an important ribonucleoprotein that acts as an enzyme for the maintenance of telomeres during cell division. It is an RNA dependent DNA polymerase that synthesises the telomeric DNA repeats by using an RNA template (termed "hTR") subunit of the telomerase holoenzyme. The enzyme is inactive in adult somatic cells, except for germ cells, activated lymphocytes, and stem cells of regenerative tissues. Elevation of telomerase activity has been selectively demonstrated in a large number of human tumours (Soria et al., 1998). Telomerase enzyme was first identified in the ciliated protozoan Tetrahymena. This organism has a large number of telomeres and is a relatively rich source of telomerase. Telomerase contains an RNA component. The RNA has a short template sequence that is copied into DNA, which extends, and thus lengthens, the chromosomal DNA. It is this addition of telomeric DNA in increments to the ends of chromosomes that offsets and counter balances the shortening of chromosome ends (Kirk et al., 2005).

Telomeric Repeats Amplification Protocol (TRAP assay) has been first time used by Kim et al. (1994) in vitro by using radio-labeled phosphorus to measure the telomerase activity from the intensity of radioactivity. In case of cancerous cells telomerase activity is much greater as compared to normal cells, due to which telomerase activity is used as a prognostic marker for malignancy. A number of methods for determining telomerase activity have been developed. The detection of telomerase activity using the conventional assay, which requires a large amount of cell or tissues, has largely been replaced by the telomeric repeat amplification protocol (TRAP) described by Kim et al. (1994). Because of several disadvantages, numerous improvements have been made to the original TRAP protocol, making the assay more sensitive and reliable for potential clinical application (Durusoy and Ozturk, 2001).

The TRAP assay includes preparation of a protein exctract by cell lysis and adding a primer and dNTPs. If telomerase is active in the extract, it elongates the added primer, and the reaction product (templates) is amplified by PCR. This technique is highly sensitive but can provide only qualitative (presence/absence) evaluation. For quantitative analysis, the area or intensity of 6bp ladders appearing in an X-ray film must be measured by densitometry with a computer program, but the result still depends on conditions of quantitative autoradiography that do not easily produce precise linearity and reproducibility (Heros et al., 1997).

Number of scientists have been tried to improve the results has been modified the TRAP assay. Aldous and Grabill, (1997) have introduced a fluorescent method of detecting telomerase to alleviate the problems including incorporation of radio nucleotides detected by autoradiography and the  time required to complete the assay.

Tatematsu et al. (1996) has beeen developed Stretch PCR, a modification of TRAP, for quantitation of telomerase activity. The introduction of steps for purification of telomerase products before PCR reaction and the use of specially designed primers that contain unrelated internal sequences of the templates contributes significantly to the quantitative accuracy of the assay.

Gelmini et al., (1998) presented a modification of the TRAP method that can provide quantitative information without requiring time-consuming post-PCR procedures such as gel electrophoresis with radioactive materials and autoradiography. The detection and measurement of telomerase activity is performed by evaluating the amount of double-strand DNA generated in the telomerase reaction and PCR amplification; with the use of the fluorescent dye Pico Green, which selectively binds double strand DNA.

Torre et al. (2002) introduce a new method of TRAP-silver staining; a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines is a best and less hazardous method to measure telomerase activity.

Gao and Chen, (2006) investigated the effect of telomerase inhibition with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-A (TNF-ά)-induced apoptosis in prostate cancer cells (PC3), found in vitro  experiment on cell line thathTERT Antisense phosphorothioate oligodeoxynucleotide can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNFά-induced apoptosis of PC3 cells.

2.12 Association of nucleolar organizer region and cancer (AgNORs assay)

Nucleolar organizer regions (NORs) are composed of chromosomal sites endowed with ribosomal DNA (rDNA), transiently clustered in order to synthesize ribosomal RNAs (rRNA). rRNAs are the main constitutive elements of ribosomes, responsible for all protein synthesis in the cell. It is also defined as nucleolar components where ribosomal genes are complexed with a set of non-histone protein characterized by a high affinity for silver (AgNORs proteins) (Trere, 1996). Increased number of nucleoli (nucleolar organizer regions, NORs) with abnormal shapes and sizes, including small dots, has been used as prognostic tools to evaluate tumor proliferation levels and troublesome borderline lesions. NOR patterns of cancers were performed in the search of a valuable prognostic method to complement other histological procedures. Silver-staining techniques have been developed to reveal NOR did sites in metaphase chromosomes and interphase nuclei base on the detection of ribosomal proteins, nucleolin, and RNA polymerase I associated to rDNA transcription. The staining intensity, shape, and number of NORs are indicative of cell activity and proliferation levels (Hofstadter et al., 1995). Human ribosomal cistrons are located on secondary constrictions of chromosome pairs 13, 14, 15, 21, 22, which frequently associate to give rise to a single NOR cluster (nucleolus), round in shape. In cancer cells, this arrangement is often disrupted, with increased number of NORs of abnormal shapes and different sizes, including small dots. Some studies have used NOR patterns as prognostic tools to evaluate proliferation levels (Evans et al., 1991) and tumor progression. Increased staining and alterations in shape, size, and number of NORs account for extra amounts of ribosome's needed by highly proliferating cancer cells.

Three types of solid human skin cancers are currently described: basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and malignant melanoma (MM). These tumors arise from epidermal basal cells, squamous cells, and melanocytes, respectively, showing different biological behavior concerning metastasis potential. BCC tumors may be grouped according to the degree of histopathological aggressivity as superficial multicentric, solid, adenoid or cystic, and sclerosing. SCC and MM tumors develop in situ and may present several invasive grades (Rosana et al., 2005). Changes in copy number and transcription of ribosomal cistrons have been reported, showing altered NOR patterns on skin cancers (Hofstadter et al., 1995) However, superficial, compared to solid BCC, tumors showed no differences in NOR/nucleus-related areas. MM cells presented scattered NORs (dots) and increased number of clusters. Statistical differences were found concerning the number of NORs between premalignant lesions and MM, but not between benign and dysplastic nevus (Li et al., 2003). Furthermore, increased number of NOR clusters and dots were observed in MMs more than 1.5-mm width, correlated to a worse prognosis. Patients bearing MM tumor stage I, with a high number of NORs, developed metastasis some months earlier than those with fewer NORs. Nevertheless, there are discrepancies among different studies regarding the prognostic value of AgNOR counts in MM (malignant melanoma) (Ronan et al., 1994).

Papadhimitriou et al., (2007) investigate the prognostic value of (AgNORs) in multiple myeloma 55 newly diagnosed patients, no clear prognostic value for the AgNOR count was found in multiple myeloma. Instead, the results indicate that the AgNOR count might be an index for M protein synthesis rate. This is consistent with other findings in tissues with low proliferative potential and high protein synthetic activity, and calls for a cautious interpretation of AgNORs in malignancies with similar features.

Phytochemical and Pharmacological Evaluation of S. asper and L. procumbens