Phytochemical screening


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5.1. Phytochemical screening

Phytochemical screening provides basic information about the medicinal importance of the plant extract. In this study phytochemical screening and quantitative estimation of the chemical constituents of S. asper and L. procumbens shows the presence of various metabolites including reducing compounds and free radicals scavenging compounds such as flavonoids, alkaloids, terpenoids, saponins, coumarins, tannins, cardiac glycosides anthraquinones, and phlobatinins. Tannins possessed spasmolytic activity in smooth muscles cells, free radical scavenger and antioxidant (Tona et al., 1999). Flavonoids are polyphenolic compounds like quercetin 3'-O-glucoside, rutin have antioxidant and antimicrobial properties while saponins are glycosides possessed antimicrobial and inhibit Na+ efflux, by blockage of the entrance of the Na+ out of the cell, reducing congestive heart failure (Abou-Donia et al., 2008). Various studies revealed that natural and synthetic derivates of alkaloids and have possessed medicinal importance includes analgesic, antisplasmodic and bactericidal activities, antioxidant and are useful in renal disorder (Okwu and Okwu, 2004). Our results agree with investigation of Sofowara, (1993) during characterization of medicinal plants.

The total phenolic contents of Sonchus asper and Launaea procumbens was measured using Folin-Ciocalteu method. Phenolics compounds present in fruits and vegetables have received considerable attention because of their potential antioxidant activities (Pan et al., 2008). Phenolic compounds react with phosphotungstic and phosphomolybdic acids present in the folin-ciocalteu reagent (Amin & Yazdanparast, 2007), amino acids, proteins, organic acids, sugars and aromatic amines causing interference in determination (Meda et al., 2005; Roura, Andres-Lacueva, Estruch, & Lamuela-Ravents, 2006) but in this investigation, S. asper and L. procumbens were dried before extraction while ascorbic acid was lost during drying process and amino acids, proteins and sugars can be removed from the extraction solvents. Thus, interference from ascorbic acid or other compounds like amino acids, proteins and sugars should be very little. In the present study these plants possessed contents of phenolic compounds in ranges from (88.3±2.1) to (432.8±2.93) mg gallic acid equivalent (GAE mg/g extract) in LPHE and LPME followed by (95.6±1.7) to (332±1.53) in SAHE and SAME respectively. Similarly high flavonoids content was found in methanolic extract of Sonchus asper (15.2±0.76) followed by LPME (13.98±0.87) mg rutin equivalent/g extract. The presence of phenolic and flavonoids compounds, possess diverse biological activities such as anti-inflammatory, anti-carcinogenic and anti-atherosclerotic activities. These activities might be related to their antioxidant activity (Chung, Wong, Huang, & Lin, 1998). Phenolic compounds may contribute to antioxidative action (Duh et al., 1999), inhibitory effects on mutagenesis and carcinogenesis in humans (Tanaka et al., 1998). Several phenolic compounds like tannins present in the cells of plants are inhibitors of many hydrolytic enzymes such as proteolytic macerating enzymes used by plant pathogens. Other compounds like saponins also have antifungal properties (Aboaba and Efuwape, 2001; Mohanta et al., 2007). This study indicates that the presence of these bioactive compounds in the various fractions of plant might be responsible for their antioxidant, anticancer, antimicrobial, allelopathic and cytotoxic activities.

5.2. Elemental and nutritional analysis of medicinal plants

Wild growing leafy vegetables play an important role in the diet of inhabitants of different parts of the world. The proximate analysis of Sonchus asper and launaea procumbens shows that their leaves are a good source of metallic elements, protein and sugar. Due to the possession of these constituents, consumers are more benefited when they use these as a substitute of sugar in various food preparations. These values were found to be comparable with or higher than those of commonly used vegetables such as spinach, lettuce and cabbage. Potassium, calcium, magnesium and sodium are nutritionally important, were found in reasonable amount in the leaf. These inorganic compounds play an important role in the maintenance of normal glucose-tolerance and   in the release of insulin from beta cells of islets of langerhans (choudary and Bandyopadhyay, 1999). Some other metallic elements such as selenium, zinc and manganese are considered as potent antioxidant micronutrients increases immunity and prevent many diseases mediated with oxidative stress and free radicals. Iron is essential element in the synthesis of hemoglobin, presence of appropriate concentration in these plants play important role in nutritional disorder such as iron deficiency anemia (Talwar et al., 1989). Similar investigation was reported by Tadhani, M and Subhash, (2006) during the preliminary studies on stevia rebaudiana leaves for phytochemical and mineral screening. Undoubtedly, there is growing interest in natural sources of nutrients and health-promoting compounds. Within these compounds, polyphenols and antioxidants have special attention, which is understandable because of their role as potential protective and preventive molecules against chronic ailments, such as atherosclerosis and cardiovascular diseases, ischemic heart disease, Alzheimer's disease, Parkinson's disease, cancer, osteoporosis and in the entire aging process (Aruoma, 2003; Coruh, Celep, & Ozgokce, 2007; Dasgupta & De, 2004).

5.3. Chromatographic evaluation of flavonoids

Flavonoids are rarely present in free in plant extract. These are found in combination as esters, glycosides or are bound to the cell wall. For this reason, before HPLC analysis, hydrolysis of glycosides or esters was necessary, so that phenolic compounds can be identified, since a considerable fraction is in bounded form. Moreover, BHT, a powerful antioxidant, was added to prevent degradation of phenolic during hydrolysis (Nardini and Ghiselli, 2004). In the present study the thin layer chromatography investigation was justified by high performance liquid chromatogram which reveal that SAME composed of three compounds orientin, rutin, hypersoid, SAEE showed vitexin, orientin, rutin while SABE hypersoid, vitexin. SACE possesses vitexin and SAWE possessed hypersoid however SAHE showed one for hypersoid. Chromatogram of LPME composed of five compounds catechin, orientin, rutin, hypersoid, myercitin. LPCE showed five known compounds catechin, rutin, vitexin, hypersoid, and myercitin while LPEE showed two compounds orientin, hypersoid and LPWE showed 2 compounds catechin, vitexin. LPBE showed 2 compounds catechin and vitexin, while LPHE shows unknown compounds. Results of Gudej and Tomczyk, (2004); Male et al., (2006) are in accordance to our study. The TLC results of Poukens-Renwart et al., (1992) showed the presence of vitexin, orientin and aglycone flavonoids in the bagasse and leaf extracts, and in the juice samples, which supports our results.

Previous studies indicated that flavonoids were a potent antioxidant agent, quenching radicals, singlet oxygen and hydrogen peroxide (Bourgou et al., 2008). For that, natural antioxidants such as polyphenols are often added to foods to stabilizethem and prevent off-flavor development and have considerable interest for their potential role as functional foods or nutraceuticals (Espin et al., 2007). The mechanism by which antioxidants protect food from oxidation is by scavenging of free radicals via donation of an electron or a hydrogen atom, or by deactivation of metal ions and singlet oxygen. Gallic acid, for example, has been widely used as additives to avoid the degradation of foods and is known to have anti-inflammatory, antimutagenic, and anticancer activity (Soong and Barlow, 2006). In addition, vanillic acid is a phenolic derivative known to possess antimicrobial, anti-filarial and hepatoprotective activities (Singh et al., 2006).  The results reported by Ksouri et al., (2009) show similarty with our results during polyphenolic characterization of Tamarix gallica L. using same mobile phase.

5.4. Antimicrobial and antitumor potency of extracts

The results of our screening assays justify the use of the investigated plants in the Pakistani ethnomedicine. The findings shows that Gram negative bacteria such as Staphylococcus areus was inhibited by MIC of LPCE (2.5ug/ml), LPME (1ug/ml), LPEE (2.5 ug/ml) and SABE (5ug/ml), SAME (2.5 ug/ml), SAWE (5 ug/ml) respectively while MIC of E.coli includes LPME (2.5 ug/ml), LPEE (2.5 ug/ml) and SAME (5 ug/ml) however Klebsiella pneumoniae growth was by MIC range of LPEE , LPBE , SAHE , SAME were (1ug/ml) and SAEE was (5ug/ml) respectively. In case of gram positive bacteria LPME, LPBE (5ug/ml), SAME (2.5ug/ml) showed MIC against Micrococcus lutes, while Bacillus subtillus was inhibited by LPBE, LPME, LPEE, SAME and SAEE with MIC (1ug/ml), however LPBE, LPME, SAME and SAEE inhibited the growth of Enterobacter aerogenes with MIC (5ug/ml) respectivelythat the plant possesses MIC of antibacterial, antifungal and antitumor potential. LPME and LPWE of Launaea procumbens and SAME of Sonchus asper showed activity against E.coli. Staphylococcus areus was inhibited by LPCE, LPME, LPWE and SABE, SAME, SAWE respectively. Fractions of Launaea procumbens including LPHE, LPEE, LPME, and LPWE markedly inhibited the effects of Klebsiella pneumoniae while SAHE, SAME, SAWE showed inhibition against them. Bacillus subtillus was inhibited by LPBE, LPME, LPWE and SAME, SAWE while Micrococcus lutes and Enterobacter aerogenase was controlled to by LPME, LPWE, SAME and LPWE, SABE respectively. Our MIC results agree with results reported by Ndhlala et al., (2009) during characterization of Antimicrobial, anti-inflammatory and mutagenic investigation of the South African tree aloe (Aloe barberae). Narod et al. (2004) reported that antibacterial activity of hexane, methanol and water extract of leaf and stem of Toddalia asiatica were active against Gram-negative and Gram positive bacteria. They found that the MIC of methanol extract against Staphylococcus aureus was 2 mg/ml.

Duraipandiyan and Ignacimuthu, (2008) reported the same results during screening of antibacterial activities of various fractions of traditional medicinal plant, Toddalia asiatica (L.) Lam. The MIC values of the Flindersine compound isolated from same plant against bacteria Bacillus subtilis (31.25 ug/ml), Staphylococcus aureus (62.5 ug/ml), Staphylococcus epidermidis (62.5 ug/ml), Enterococcus faecalis (31.25 ug/ml), Pseudomonas aeruginosa (250 ug/ml) respectively. Algiannis et al. (2001) proposed a classification based on MIC values of antifungal activities of extracts, where extracts with MIC up to 500 ug/ml are considered as strong inhibitors, 600-1500 ug/ml as moderate inhibitors and those with MIC values above 1600 as weak inhibitors. In this study replicate results of antifungal show that all fractions showed some extent of antifungal activity at 200 ug/ml concentration of various fractions, however growth of Aspergillus niger was markedly inhibited by methanolic and ethyl acetate fraction of both plants, while F.solani was inhibited by butanolic, methanolic and water fraction of Launaea procumbens and Sonchus asper. Similarly Aspergillus flavus was inhibited by butanolic, methanolic, water fraction of Launaea procumbens and while water fraction of Launaea procumbens, ethyl acetate and methanolic fractions of Sonchus asper showed siignificant growth inhibition respectively. These results suggest that methanolic extracts of Sonchus asper and Launaea procumbens were more efficient to inhibit bacterial growth than fungal one, probably in relation to their active molecules. Several studies attributed the inhibitory effect of plant extracts against bacterial pathogens to their phenolic composition (Baydar et al., 2004; Rodriguez Vaquero et al., 2007) and might be presence of saponins which have antifungal properties (Aboaba and Efuwape, 2001; Mohanta et al., 2007). Kabuki et al. (2000) reported that the antimicrobial spectrums of the crude catechins were more effective against gram-positive bacteria than gram-negative bacteria. This tendency of tannin could be explained by that the structures of cell envelope, including cytoplasmic membrane and cell wall component, are different between gram-positive and gram-negative bacteria. Gram-negative bacteria possess an outer membrane surrounding the cell wall, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. Without outer membrane, the cell wall of gram-positive bacteria can be permeated more easily and tannins can disturb the cytoplasmic membrane, disrupt the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Burt, 2004). Therefore, the structural difference of bacteria plays an important role in their susceptibility.

Crown-gall is a neoplastic disease of plants, in which autonomous plant tumor cells are produced from normal, wounded plant cells by the action of bacteria-borne tumor inducing plasmids. The method is independent of antibiosis (Fadli et al., 1991). It is caused by a specific strain of Gram-negative bacterium Agrobacterium tumefaciens (Pelczar and Reid, 1965). As certain mechanisms of tumorigenesis, such as the intracellular incorporation of extraneous nucleic acids, are common in both plants and animals (McLaughlin, 1991), the fundamental concept of developing this method was that the antitumor drugs might inhibit the initiation and growth of tumors in both animal and plant systems. Data of the present study reveal that all fractions of L.procumbens and S.asper show antitumor activity with exception of water and chloroform fractions of S.asper however methanolic fraction of L.procumbens with IC50 (13±0.2 µg/ml) and S.asper (45±1.7 µg/ml) were markedly control crown-gall tumor formation and their IC50 value is near to control indicated the use this plant species for the prevention and/or treatment of cancer. Hussain, Zia and Mirza, (2007) reported that methanolic extract of Fagonia cretica L. shows potent antitumor activities as compare to control, which supports our results. The results documented by Turker and Camper, (2002); DAS et al., (2007) are in accordance to our results. The results of present study supports the traditional usage of the studied plants and suggests that some of the plant extracts possess compounds with antimicrobial and anticancer properties that may be used as antimicrobial and anticancer agents in new future drugs for the therapy of cancer and infectious diseases caused by pathogens.

5.5. Phototoxic and cytotoxicity effects of plans

Cytotoxicity screening models provide important preliminary data to select plant extracts with potential antineoplastic properties for future work (Cardellina et al., 1999). In the present study the order of LD50 of brine shrimps LPBE > LPME > LPEE > LPHE > LPWE > LPCE and SAME >SABE > SAEE > SAWE > SAHE > SACE was reported. Our results showed that the brine shrimp survival is inversely proportional to the concentration of the extract used. The extracts with LD50 values higher than 200 mg/l in the brine shrimp test can be considered inactive (Anderson et al., 1991), but in the present study almost all of our extracts possesses LD50 less than 200 mg/l and are markedly effective in removing foreign cell and could be used as chemopreventive anticancer, however chloroform fraction of Launaea procumbens, n-hexane and water fraction of Sonchus asper was inactive, showed no significance cytotoxicity activity and LD50 value are greater than 200 ug/ml (200 mg/l).

Our results agree with the reports inferred by Hussain et al., (2008) during screening various fraction of Nepeta juncea through brine shrimps cytotoxicity that only water fraction showed activity. Zaidi et al., (2006) studied that methanolic fraction of Arceuthobium oxycedri exhibited 100% cytotoxicity for brine shrimps at high dose level, with LC50 of 8.3μg/ ml. its cytotoxicity might be responsible for the damage it causes to this fossil species. Cyrus et al., (2008) reported no correlation between antibacterial and cytotoxic activity during screening of Kenyan medicinal plants. The plant fraction that was lethal to brine shrimp nauplii showed low inhibitory activity against bacteria. From the plant extracts, only Ajuga remota Benth (LC50 < 100 μg/ml) was considered toxic, while other species had (LC50 < 1000 μg/ml) non-toxic (Santos et al. 2003). These reports support our results as there was successive correlation between antibacterial and cytotoxic activity of various plant fractions.

Recently scientist have focused to increase the crop production to meet the needed of world population, but unfortunately, crop yield losses every year due to insects and plant diseases caused by various pathogens (Fletcher et al., 2006), slow biodegration of herbicides (Barnard et al., 1997). To control these shortcomings researchers have focused on allelochemicals and bio-herbicides, produced by plants themselves. The phyototoxic results of all fractions of both plants shows that they inhibited the growth of reddish root as well as shoot as compare to non treated control plant. Water, methanolic and butanolic fractions showed marked growth inhibition of root and shoot while n-hexane and ethyl acetate fraction of both plants showed moderate effects. Our findings showed similarity with results reported by Javaid et al., (2009) that water extract of Withala somnifera and Datura alba possessed some bioactive compounds which significantly inhibited the growth of root and shoot of Rumex dentatus L. highly competitive weed in wheat during allelopathic screening. Similar investigations was found by Kordali et al., (2008) that essential oil isolated from Turkish Origanum acutidens and their phenolic compounds completely inhibited the growth of seedling and roots and possessed antifungal activity when compare to standards compounds.

5.6. Scavenging of free radicals and medicinal plants

Oxidation is a necessary process for human beings for energy production; however during normal metabolism oxygen consumption, through many enzymatic systems produces reactive oxygen species (ROS) such as superoxide anion radical, hydroxyl radical and hydrogen peroxide. In small amounts, these ROS are beneficial in signal transduction and growth regulation. However large amount of ROS produced oxidative stress, attack many molecules such as protein, DNA and lipids (Halliwell & Gutteridge, 1999).

Human beings possessed a system of antioxidant enzymes which scavenge and terminate directly ROS radical-mediated oxidative reactions they may be used a method of prevention of aging-associated diseases and health problems. In addition to antioxidant enzymes, non enzymatic compounds also take part in removing of ROS such as vitamin c, vitamin E and α-topherol in addition to exogenous antioxidant compounds such as polyphenols. This has led to an accelerated search for antioxidant compounds, the identification of natural resources, and the isolation of active antioxidant molecules. Antioxidants have been detected in a number of agricultural and food products including cereals, fruits, vegetables and oil seeds (Adom, Sorrells, & Liu, 2003; Netzel, Netzel, Tian, Schwartz, & Konczak, 2007; Yu, Perret, David, Wilson, & Melby, 2002). Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PG) have been widely used as antioxidants in the food industry (Nawar, 1996). However, the safety of these synthetic antioxidants has been questioned. BHA has shown to be carcinogenic and BHT has been related to internal and external hemorrhaging at high doses in rats and guinea pigs (Ito et al., 1986). These findings increased the interest of consumer in natural food additives for effective uptake of antioxidant compounds as alternative source for scavenging of antioxidant compounds. Data of the present study revealed that various fractions of both plant show marked scavenging potential. Among them methanolic fraction of both plants indicated that this is more active in scavenging of 1, 1-diphenyl 1-2-picryl-hydrazyl, 2,2 azobis-(3-ethylbenzothiozoline-6-sulphonic acid), reduction of reduction of Mo (VI) to Mo (V) and inhibiting the oxidation of linoleic acid and subsequent bleaching of b-carotene. Similarly superoxide radical, hydroxyl radical and hydrogen peroxide most reactive and induces severe damage to adjacent biomolecules are scavenges effectively, as well as possesses a good metallic reducing and iron chelating properties. Our result shows similarity with the investigation of Hagerman et al. (1998) and Falleh et al., (2008). The results obtained by Duenas et al., (2006) and Kilani et al., (2008) also support our investigations.

5.7. Free radicals and changes of body and tissue weight

The present study revealed that highly significant variation was observed in body weight and % increase in body weight. CCl4 and KBrO3 treatment of rats significantly decreased the body weight as compare to the non treated control group. Okada et al., (2003) reported the significant decrease in the body weight of rats by oral administration of CCl4. These results are in accordance with Lin and Lin, (2006) that CCl4 treatment causes a significant decrease in the body weight of the rats as compared with control rats. The results of Noyan et al. (2006) were also similar to our findings, in which they find the significant decrease in the body weight of mice groups treated with CCl4 intraperatonialy.

Khan and Ahmad, (2009) reported that the treatment of male rats with CCl4 for 16 weeks caused a significant loss in the body weight, which was restored by methanolic extract of Digera muricata. These studies justify the decrease in the body weight of rats due to CCl4 and KBrO3 which was significantly recovered in the present study with the treatment of various fractions of Sonchus asper and Launea procumbens. Our result opposes to the findings reported by Farombi et al., (2002) that KBrO3 treatment to rat decreases the body weight non significantly as compare to control group.  Cadenas and baria, (1999) reported that 80 mg/kg b.w. KBrO3 administration for 8 hours in rats non significantly decreased the body weight as compare to control rat, but our results controverses than these findings due chronic administration of KBrO3 as well as kidney weight of the rats was significantly increased when compared to control. This increase of tissue weight was balanced by administration of Resveratrol, Meltonin, vitamin E and PBN .

Data of the present study revealed that tissue weight of various organs was increased in the CCl4 and KBrO3 treated rats as compared to control groups and DMSO treated rats. These increases were significantly ameliorated by post-treatment of various fractions of Sonchus asper and Launaea procumbens. Lin et al., (2008) injected 20% CCl4 for six weeks twicely, reported that liver and spleen weight was significantly increased as compared to control. They studied that these elevation was significantly restored by feeding rats with 200-1000mg/kg b.w. Solanum nigrum plant extract. Jodynis-Liebert et al., (2008) reported that Aquilegia vulgaris extract attenuates carbon tetrachloride-induced liver fibrosis and increase of liver weight in rats. Farombi et al., (2001) reported that a single dose of KBrO3 (300 mg/kg b.w.) insignificant changes the liver weight, as was compared to control group rats.

Results of Adewole et al. (2007) are also in accordance to our study, in which CCl4 treatment significantly increased the weight of the kidney as compared to control. Our result was supports by treatment of rats with KBrO3 three times a week for 4 weeks resulted significant increase in kidney weight as compared to control rat which was modulated by biflavonoid (kolaviron) isolated from Garcinia kola (Farombi et al., (2001). Administration of various concentration of in the Big Blue rats showed that high doses of the chemical caused significant decreased in body weight while kidney of the rats was significantly increased when compared to control, however less amount of dose showed insignificant variations prove that low of amount of KBrO3 is not toxic as well as mutagenic (Yamaguchi et al., (2008). Decrease in body weight with CCl4 and KBrO3 treatment in this experiment might occur due the degeneration/necrosis of body tissues while increase in the weight of various tissues accounts due to the fatty accumulation, lipidperoxidation as well as fibrosis and inflammatory response of the organs. Response of the cells might change and adaptation increase by the protective effects of the rutin and various fractions, results in the recovery of the bodily and organs weight.

5.8. Serum marker enzymes and oxidative stress

The areas of dietary modification and chemoprevention show considerable effective approaches for oxidative stress and are a focus on the research of these days. It has been found that some edible plants as a whole or their some portion have protective effects against various disorders including oxidative stress and cancer (Aruoma, 2003). Various studies have shown that several mutagens and carcinogens cause generation of oxygen free radicals, which play a major role in emergence of cancer especially promotion stages of carcinogenesis (Sun, 1990).

We are of the opinion that for the assessment of protective effects of pure chemical or extracts, it is more important to change the response of the cells rather than priming the cells against the insult. On this base rats were treated with CCl4 (3 ml/kg b.w.) and KBrO3 1 ml/kg b.w. (20 mg/ml) and after 48 h with various fractions of S. asper, L.procumbens and rutin.

In the present study, biweekly treatment of CCl4 and KBrO3 for 4 weeks resulted in acute pathological levels of ALT, AST, ALP, LDH and lipids profile in serum. It was suggested that CCl4 and KBrO3 metabolites through lipid peroxidation of hepatocytes cause acute injuries, cell membrane integrity was altered and the enzymes in hepatocytes leaked out. Similar elevated enzymes activities were and changes in lipids profile observed by (Sreelatha et al., 2008) in serum of rats treated with chemicals and these levels relapsed towards the normal level in rats treated with CCl4 plus rutin and various fractions of S. asper and L.procumbens. Similar investigations were reported by Singh et al. (2008) while working on hepatoprotective effects of potato peel against CCl4 induced hepatic injury in rats. Farombi et al., (2002) studied the protective effects of kolaviron against KBrO3 induced hepatotoxity and nephrotoxicity in rats, repoted that kolaviron significantly reversed the enzymatic changes of serum level.

Serum albumin is the predominant serum protein, which reflects the synthetic function of the liver. Data of the present study reflected that CCl4 and KBrO3 intoxication produced a significant reduction in albumin serum levels compared to control group similar to other findings (Venukumar and Latha, 2002). In the present study administration of rutin and various fractions of plants produced a significant increase in protein and albumin level. This coincides with the other studies (Dang et al. 2007; Lin et al. 2008) where plant extracts had reversed the protein and albumin towards the normal level.

Bilirubin is a brownish yellow substance found in bile produced during old RBCs breakage. Total and direct bilirubin tests are used for identification of liver damages. Increased levels of total or direct bilirubin may be due to jaundice, liver tubes blockage or bile ducts, cirrhosis. Our results showed that CCl4 and KBrO3 significantly (P<0.01) elevated total and direct bilirubin of serum which was significantly recovered by post-treatment of various fractions of S. asper and L. procumbens as well as rutin. Same result was reported in other studies (Rafatullah et al., 2006). Similar investigations were reported by Singh et al. (2008) and Sreelatha et al., (2008) while working on hepatoprotective effects of extracts against CCl4 induced hepatic injury in rats. Results of different studies have shown that various enzymatic and non enzymatic systems have been developed by eukaryotic cells to cope with oxygen free radicals (Recknagel et al., 1989; Halliwell and Gutteridge, 2000). However when oxidative stress is developed then the defense system become insufficient (Halliwell and Gutteridge, 2000). Medicinal plant materials also composed of different amounts of antioxidants which play main role in controlling various pathological condition including cancer, cardiovascular diseases, liver diseases and lipid peroxidation (Martin et al., 1993, Agbor et al., 2001). These results indicated that various fractions and rutin are helping in response and adaptation of the hepatocytes possibly via the presence of flavonoids, saponins, phenols and tannins in various fractions.

Urine analysis may provide information regarding the status of kidney function and acid base balance (Free and Free, 1972). During normal condition urobilinogen are not excreted into the urine unless any pathogenesis. Urobilinogen; is the end product of conjugated bilirubin after it has passed through the bile ducts and been metabolized in the intestine. The presence of high levels of urobilinogen, urea, creatinine and albumin in urine are the main indication of kidney injuries induced through CCl4 and KBrO3 treatment (Pels et al., 1989; Farombi et al., 2002; Ozturk et al., 2003; Ogeturk et al., 2005; Simerville et al., 2005). Specific gravity and pH of urine correlates with urine osmolality and was affected with chemical administration (Khan et al., 2009).

Our results showed that CCl4 and KBrO3 significantly increased serum urea, serum globulin, RBC and WBC showing renal injuries. Glomerular haematuria is typically associated with erythrocyte cases, dysmorphic red blood cells and significant proteinuria. The serum creatinine level does not rise until at least half of the kidney nephrons are damaged or destroyed (Bhattacharya et al., 2005). The glomerular capillary wall is permeable only to substances with a low molecular weight. Once filtered, low-molecular-weight proteins are reabsorbed and metabolized by the proximal tubule cells. High level of proteinurea and haematuria in urine of this study showed the nephrotoxicity induced with CCl4 (Ogawa et al. 1992). The present study revealed that oral administration of different fractions significantly improved creatinine and urobilinogen, and decreased the elevated levels of proteinuria and haematuria.

Present study revealed that administration of KBrO3 and CCl4 caused marked impairment in renal function along with significant oxidative stress in the kidneys. Serum creatinine, urobilinogen, BUN, total bilirubin, direct bilirubin concentrations were significantly higher in the CCl4 treated rats which are consistent with lower creatinine clearance (Adewole et al., 2007; Bhadauria et al., 2008). Rutin and various fractions of L. procumbens and S.asper significantly improved creatinine clearance and decreased the elevated levels of creatinine, BUN, total bilirubin and direct bilirubin. In addition, elevated level of urinary albumin and reduced level of serum albumin in KBrO3 and CCl4-treated rats might have resulted from remarkable leakage due to injuries in glomeruli and tubules. On the other hand, rutin and various fractions protect kidney tissues against oxidative damages induced by CCl4. Earlier studies have also shown that different plant extracts comprehensively ameliorated the renal injuries induced through CCl4 intoxication (Ogeturk et al., 2005; Adewole et al., 2007; Khan et al., 2009).

Our study revealed that KBrO3 and CCl4 treatment caused a significant increase in serum protein content of rat. CCl4 administration resulted in oxidative damage of proteins (Abraham et al., 1999) and their accumulation (Srinivansan et al., 2005) due to poor degradation by proteasomal and lysosomal pathways, causing metabolic dysfunction of kidneys (Dalle-Donne et al., 2006). L. procumbens and S.asper erased the CCl4 and KBrO3 intoxication and protein contents reverted towards normal.

5.9. Antioxidant enzymes and oxidative stress

Oxidative stress was characterized by increased lipid peroxidation and/or altered non-enzymatic and enzymatic antioxidant systems. Cumulative evidence suggested that various enzymatic and non-enzymatic systems have been developed by mammalian cells to cope with ROS and other free radicals. Toxicity induced with CCl4 and KBrO3 results from its metabolism by the cytochrome P450 enzyme system in to the reactive intermediates which leads to oxidative stress and tissue injuries. In this study KBrO3 and CCl4-treatment caused marked deterioration of endogeneous antioxidant profile as evidenced by decrease in CAT, POD and SOD activities in the different tissues. These results were consistent with the literature (Khan et al., 2009). CAT, POD and SOD are the supportive team against the ROS defense (Sreelatha et al., 2009). Superoxide dismutases (SODs) are a class of closely related enzymes that catalyze the breakdown of the superoxide anion into oxygen and hydrogen peroxide. Catalases are enzymes that catalyze the conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor. Peroxidases catalyze the reduction of hydrogen peroxide, organic hydroperoxides, as well as peroxynitrite. During renal injury ROS generate at the site of damage, and SOD is exhausted as a result of oxidative stress caused by CCl4 and KBrO3 that further leads to accumulation of free radicals. Szymonik-Lesiuk et al. (2003) have shown that CCl4 intoxication can lead to alteration in gene expression and depletion of CAT and SOD levels in kidneys. Farombi et al., (2001), Khan and Sultana., (2005) reported the induction of KBrO3 in rats significantly reduces the activities of antioxidant enzymes like SOD and catalase, which has been restored by supplementation of Ficus racemosa and a natural antioxidant kolaviron. Post-administration of various fractions protected the antioxidant machineries of the tissues as revealed by the enhanced level of CAT, POD and SOD in this experiment. The ameliorating effects of different fractions might be due to the presence of flavonoids and saponins against the toxicity of KBrO3 and CCl4. This has also been documented in various studies (Manna et al., 2006; Hassan et al., 2007). 

The glutathione system includes reduced glutathione, glutathione reductase, glutathione peroxidase and glutathione-S-transferase. Glutathione peroxidase is an enzyme containing four selenium-cofactors that catalyzes the GSH-dependant reduction of H2O2 and other peroxides and protects the organism from oxidative damage. There are at least four different glutathione peroxidase isozymes in animals. In addition, the glutathione-S-transferases show high activity with lipid peroxides. Glutathione detoxifies toxic metabolites of drugs, regulates gene expression, apoptosis, and transmembrane transport of organic solutes. We obtained from this study significant depletion of GST, GSR, GSH-px and quinone reductase, while increase in the activity of γ-GT in various tissues with KBrO3 as well as CCl4 treatment of rats. These results support the view that extensive oxidative stress takes place in tissues. Decrease in the activity of GST might be due to the decreased availability of GSH contents as a result of lipid peroxidation. GST binds to lipophilic compounds and act as an enzyme for GSH conjugation reactions. Administration of different fractions of S.asper and L. procumbens ameliorated the chemicals induced toxicity, thereby increases the activity of GST, GSR, GSH-px and quinone reductase, whereas the activity of γ-GT was decreased as compared to the CCl4 and KBrO3 treated groups. Similar observations were also reported during administration of melatonin against chemically induced oxidative stress in rats (Adewole et al., 2007) and accordance with result obtained by Farombi et al., (2001), Khan and Sultana., (2005); Bhadauria et al., (2008).

This study also reported significant increase in the nitrite contents in rats treated with CCl4 and KBrO3 as compared to control group. This increase in nitrite contents might be ascribed due to two reasons. Firstly, CCl4 and KBrO3 intoxication directly damages the tissue and nitrite level is elevated. Secondly, constriction of blood capillaries further causes injuries in tissues thereby increasing the nitrite contents. Similarly oxidative stress can promote the formation of a variety of vasoactive mediators that can affect renal function directly by causing renal vasoconstriction. The increase in nitrite contents with CCl4 and KBrO3 treatment in tissues might be due to direct damages which cause constriction of blood capillaries. Ameliorated effects with various fractions of S. asper and rutin have been established in this study similar to other findings (Khan and Ahmed, 2009; Khan et al., 2009).

The involvement of xanthine oxidase in ROS-mediated diseases has been proposed as a result of the generation of O2 and H2O2 during hypoxanthine and xanthine oxidation. Xanthine dehydrogenase prefers NAD+ but will use molecular oxygen in the absence of NAD+. Xanthine oxidase utilizes oxygen as an electron acceptor and has very little activity towards NAD+ which promotes generation of ROS. In this study xanthine oxidase activity was elevated with CCl4 treatment while it reversed towards the normal level with the co-treatment of rutin, S.asper and L. procumbens fractions.

CCl4 significantly decreased the GSH contents while increased the TBARS and H2O2 contents as compared to the control group in this experiment. Similar observations were found in rats treated with KBrO3. TBARS is a major reactive aldehyde resulting during the peroxidation of polyunsaturated fatty acids (PUFA). It is a useful indicator showing tissue damages including a series of chain reactions (Ohkawa et al., 1979). GSH is an important protein thiol which coordinates body defense system against oxidative stress. GSH effectively scavenge free radicals and other reactive oxygen species (e.g., hydroxyl radical, lipid peroxy radical, proxy nitrite and H2O2) directly or indirectly through enzymatic reactions (Fang et al., 2002). Reactive oxygen species of CCl4 decreases the activity of phase II metabolizing enzymes by depletion of GSH contents (Singh et al., 2008). In such reactions, GSH oxidized to form GSSG, which is then reduced to GSH by the NADPH-dependent glutathione reductase. In addition, glutathione peroxidase catalyzes the GSH-dependent reduction of H2O2 and other peroxides and protects the organism from oxidative damage. Administration of different fractions significantly elevated the GSH and decreased the TBARS and H2O2 contents. Similar results were also reported by co-administration of Digera muricata and Ficus racemosa extract against oxidative stress of CCl4 and KBrO3 in kidneys (Khan et al., 2009; Khan and Sultana., (2005).

5.10. Reactive oxygen species and Hormones

Liver cirrhosis, a highly prevalent chronic disease, is frequently associated with endocrine dysfunctions, notably in the thyroid, adrenal and gonadal axis. During the present study, low dose of CCl4 and KBrO3 was administered for a longer period of time to establish the liver cirrhosis in rat. Intraperatonialy administration of CCl4 and KBrO3 to rats at a lower dose and for longer period of time allowed the development of liver cirrhosis in all the treated animals (Gonzales et al., 2007).

Reproductive hormones are secreted under the control of hypothalamus-pituitary-gonadal axis. Hypothalamus secreting gonadotropin releasing hormone (GnRH), which in turn elicits the release of FSH and LH from the pituitary gland (Conn, 1986). FSH binds with receptors in the sertoli cells and stimulates spermatogenesis; however LH stimulates spermatogenesis in Leydig cells (Conn, 1986). In the present study, potassium bromate and carbon tetrachloride treatment decreased the serum level of testosterone, FSH and LH. Secretion of testosterone is probably impaired due to excessive oxidative stress and the degeneration of Sertoli cells and Leydig cells (Santos et al., 2004). The decrease in plasma testosterone may have resulted either from the direct effect of chemical on Leydig cells or indirectly as a consequence of decreased LH levels thereby affecting steroidogenesis. It may also have been attributed due to increased plasma cortisol. The latter possibility is consistent with previous reports suggesting that a high cortisol level causes a reduction in testosterone levels (Hall, 1994). Toxic effects caused by potassium bromate and carbon tetrachloride result in the imbalance of hypothalamus-pituitary axis, which caused testicular dysfunction leading to infertility.

Data of the present study shows that administration of potassium bromate and carbon tetrachloride increases the estradiol and prolactin in all treated groups, either through direct mechanism of toxicity, imbalance, prolactinemia, hypothalamic dysfunction being associated, as observed in hypogonadism. This later mechanism may be partly responsible for the central hypogonadism in our study. Treatment of rats with various fractions of both plant as well as rutin ameliorated the toxic effects of chemical increased the level of testosterone, FSH and LH while by contrast the serum level of estradiol and prolactin was decreased significantly in a dose dependent manner.

Our results show that CCl4 and KBrO3 treatment causes significant increase in the secretion of epinephrine, nor epinephrine and cortisol. These changes are markedly restored by orally treatment with various fractions of plant extracts and rutin. Stern and Brody, (1963) reported that the oral administration of 2.5 ml/kg CCl4 in peanut oil to rats elevated free epinephrine and nor epinephrine levels in plasma and urine. The elevated urinary free catecholamine levels were probably the result of increased secretion, rather than decreased metabolism or tissue depletion, which supports our results. Similarly results of Rubinstein (1962) are accordance to our investigations, reported that intraduodenal administration of carbon tetrachloride to rats for two hours caused increase in serum epinephrine level.

Thyroid hormones are essential for normal growth of organs, development, function and also regulate hepatocytes metabolism while liver in turn metabolizes thyroid hormones. It means that liver and thyroid hormones are connected with one another, dysfunction of one causes disturbance of other, (regulated by negative feedback mechanism of hypothalamus, pituitary and thyroid gland Malik and Hodgson, 2002).

The hypothalamus releases thyrotrophic-releasing hormone (TRH), which stimulates pituitary gland to releases thyroid stimulating hormone (TSH), which in turn promotes thyroid cells to produce thyroid hormones. When level of thyroid hormone is low than TSH and TRH are high, try to increase thyroid hormone and causes risk of thyroid tumor in rats   (Hill et al., 1989). Many toxic chemicals PTU (propylthiouracil) and PB (Phenobarbital) produce antityhroid effect that reduces thyroid hormones (T4, T3) and increases Thyroid stimulating hormones (Hard, 1998; Glatt et al., 2005).

T3, the active form of THs, appears to be produced largely by peripheral enzymatic monodeiodination of T4 mainly in the liver and other tissues (Van der Geyten et al., 1998). In general, a decline in plasma T3 levels is mostly due to a drop in thyroidal T4 production and secretion and/or changes in peripheral TH metabolism. Previous investigation has shown that hepatic damage reduces hepatic T4 deiodination in animals (Liu and Wang, 1991), which in turn causes a decline in peripheral T3 production. Potassium boromate and carbon tetrachloride are potent hepatotoxin causes severe hepatic lesions in rats (Abraham et al., 1999; sleathra et al., 2008). This Stress could provoke an acute drop in T3 levels by decreasing hepatic outer ring deiodination activity in addition to physiological stress (Walpita et al., 2007).

Our results showed that administration of KBrO3 and CCl4 depleted the secretion of thyroid hormones ie T3 and T4 and elevated TSH level in serum of rats which was recovered by various fractions of L.procumbens and S.asper. Similar results were reported by administration of 10mg/kg PTU (propylthiouracil) and 100 mg/kg PB (Phenobarbital) in rats, which caused depletion of thyroid hormones and elevated thyroid stimulating hormone (Glatt et al., 2005). Jatwa and Kar (2008); Hamdy et al., (2009) documented results in accordance to our study.

Data of the present study reveal that administration of CCl4 and KBrO3 significantly (P<0.01) decreased the secretion of insulin, and enzymatic activity of amylase while increased the serum level of glucose and activities of lipase, which was reversed by the treatment of rutin and with various fraction of S.asper and L.procumbens. Coskun et al., (2005) showed similar result during study of chemical induced diabetes, found that chemical causes elevation of serum glucose while decreased insulin secretion which was restored by co-treatment with flavonoids (quercitin). Jatwa and Kar (2008) show controversies in their investigation that administration of carbon tetrachloride induced hyperglycemia with increased blood glucose level as well as the insulin resistance. Observations on fasting serum glucose following CCl4 administration indicated a hyperglycemic condition as reported by earlier workers (Meyer-Alber et al., 1992; Castilla- Cortazar et al., 1997).

The results obtained from various hormonal study revealed that various fractions of S.asper and L.procumbens markedly modulates the hormonal detoxification, in addition to enzymatic changes, which might be the presence of bioactive constituent in plant extract including flavonoids, saponins and phenolic compounds. It might be considered that aside from its scavenging activity it might directly vitalize the central nervous system.

5.11. Oxidative stress and DNA damages

Carbon tetrachloride metabolism begins with the formation of the tri chloro methyl free radical, CCl3* through the action of the mixed function cytochrome P450 oxygenase system of the endoplasmic reticulum. The CCl3* radical reacts with various biologically important substances such as amino acids, fatty acids, 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 peroxidation. Lipid hydroperoids are the initial product of unsaturated fatty acid oxidation. These products are mostly short lived and are either reduce by glutathione peroxidases to unreactive fatty acids alcohols or they react with metals to produce a veriety of reactive products such as epoxides and aldehydes etc. The major aldehyde products of lipid peroxidation are malondialdehyde (MDA) and 4-hydroxynonenal (HNE) (Schauenstein and Esterbauer, 1978; Marnett, 2000). MDA is mutagenic in bacterial and mammalian cells and carcinogenic in rats (Yau, 1979; Basu and Marnett, 1983; Spalding, 1988 and Marnett, 2000) while HNE is weakly mutagenic but major toxic product of lipid peroxidation and has powerful effect on signal transduction pathway which has a major effect on the phenotypic characteristic of cells (Esterbauer et al., 1991).

According to the Marnett (2000) and Dedon et al. (1998) the product of lipid peroxidation, MDA react with DNA to form the adducts M1G, the mutagenic pirimedopurinone adduct of deoxyguanosine. According to Shimoda et al. (1994) it is very important to identify risk factors for genomic instability which is responsible for the occurrence of genetic alterations for carcinogenesis. 8-Hydroxydeoxyguanosine (oh8dG) is a promutagenic DNA lesion produced from deoxyguanosine by oxygen radicals (Kasai et al., 1984). Formation of oh8dG in DNA induces targeted G:C-to-T:A transversions unless repaired prior to DNA replication not only in vitro (Shibutani et al., 1991) but also in vivo (Wood et al., 1990; Moriya et al., 1991; Cheng et al., 1992). Transversions of G: C-to-T: A frequently occurs in the p53 gene with the development of hepatocellular carcinoma (Hollstein et al., 1991; Oda et al., 1992).

The data of the present study revealed that the treatment of CCl4 and KBrO3 causes significant oxidative DNA damage in various tissue of rats which are visualize on agarose gel by staining with ethidium bromide. Treatment with various fractions of L.procumbens, S.asper plants extracts as well as rutin an antioxidant reference chemical significantly reduce these damages. Similar investigation was reported by Khan et al., (2009) during study of protective effects against carbon tetrachloride induced toxicity in rats. Farombi et al., (2001), Khan and Sultana., (2005) reported the induction of KBrO3 cause oxidative DNA damages, bases transversion as well as genetic alteration of 8-Hydroxydeoxyguanosine (oh8dG) in rats which supports to our investigation. These results show that the extract of both plants contain bioactive compounds which play important role in DNA repair.

5.12. Histopathological changes and chemical toxicity

Histopathological variations play key role in justification of biochemical investigation of present study. Microscopic study of the lungs shows marked histopathological variations among the treatments. In carbon tetrachloride and KBrO3 treated group, the lung tissues are extensively damaged having destructed alveolar septa and congested blood capillaries with several inclusions in their lumen. Due to the constriction of blood capillaries, blood cells were also gathered at various places in the lung tissues producing the endemic conditions. Most of the alveolar septa become hard with increased number of type II alveolar cells. The fibroblasts were also increased in number which is responsible for the accumulation of collagen fibers at the junction of various alveolar walls which become hard and are ruptured. Alveolar macrophages were present at various places in alveolar septa, but they were also severely or moderately damaged. Similar results were reported in lungs tissues by administration of CCl4 in rats (Boyd et al., 1980; Farombi et al., (2001); Zakaria et al., 2004; Yao et al., 2005; Zhang et al., 2007; Toklu et al., 2008). Post-administration of different fractions of S. asper and L.procumbens markedly recovered the histopathology near to control group.

Histopathological studies of the liver reveals that administration of CCl4 as well as KBrO3 caused marked increased in fatty changes, cellular hypertrophy, and necrotic foci, inflammatory cells infiltrations, degeneration of the lobular shape and the formation of septa, dilation of central vein and congestion of blood vessels were observed. Post-administration of various fraction of Sonchus asper attenuated the hepatic injuries with very less or no fatty changes, dilation of blood vessel and uniform morphology of hepatocytes near to control group was found. Similar observations were recorded by post-treatment of rutin while no abnormal changes were found in the morphology of control group. This result suggested a good correlation with the results of the serum aminotransferase activities, hepatic antioxidant enzyme activities and hepatic lipid peroxidation. Similar observations were documented by the investigation of Farombi et al., (2001); Sreelatha et al., (2009).

CCl4 and KBrO3 mediated lipid peroxidation causes damages in histopathological levels of kidney. Our results suggested that CCl4 treatment caused marked characteristic morphological changes such as interstitial fibrosis, glomerular and tubular degeneration, and interstitial mononuclear cell infiltration. The vasoconstriction induced by CCl4 and KBrO3 produces an ischemic local environment, which leads to a number of cellular damages such as deterioration in membrane integrity the marked histological changes are prominent in the outer cortex and inner medullary region of the kidney. Tubular changes including vacuolization, atrophy and finally detachment of epithelial cells, indicated tubular necrosis in kidneys of rats treated with CCl4 and KBrO3. Similar histopathological changes were recorded in other studies of CCl4 treatment (Doi et al., 1991; Ozturk et al., 2003). It is believed that with these histopathological changes the capacity of tubular absorption may have been altered, thus bringing about functional overload of nephrons with subsequent renal dysfunction (Adewole et al., 2007). Treatment with various fractions of L. procumbens and rutin recovered these changes up to normal level. Similar observations were also observed by co-treatment of kolaviron a natural antioxidant (Farombi et al., 2001), caffeic acid phenyl ester (Ogeturk et al., 2005), Digera muricata (Khan et al., 2009) and Ficus racemosa (Khan and Sultana., 2005) against CCl4 and KBrO3 treatment.

5.13. Telomerase activity

Telomeres are specialized protein-DNA structures at chromosomal ends of highly conserved G rich sequence TTAGGG all eukaryotic cells that are lost gradually during each cell division. Telomerase is a key enzyme for synthesis of telomeric repeats. In immortal cancer cell, telomerase was found to be reactivated in response to the onset of tumorigenesis (Wen et al.,1998). To date, measurement of telomerase activity has been considered as a diagnostic maker for malignant tumors (Lin et al., 1997; Yoshida et al., 1997) and in cell lines (Chakraborty et al., 2006). Most of the reports indicate that the telomerase activity was found to be specific to tumors with malignant potential, except germ cells and hematopoietic stem ce1ls (Counter et al., 1995; Nouso et al., 1996).

The results inferred from the present study shows that the low chronic dose of CCl4 and KBrO3 induces telomerase activity in rats. The highly sensitive assay TRAP assay was used to detect the telomerase activity. The rat treated with CCl4 and KBrO3 showed amplification of telomeres which was significantly controlled by administration with various fractions of L.procumbens and S.asper. Similar results were obtained by our lab group of researcher that CCl4 induced telomeric activity was reversed by post-treatment with various fractions (Khan et al., 2009). Similar results have been documented in various studies (Ramachandran et al., 2002; Eitsukaa et al., 2004). This antitelomeric and anticancer effect of various fractions showed that this might be possible due the presence of bioactive natural telomerase inhibitors compounds, which needs further isolation and purification.

5.14. AgNORs count and chemical toxicity

Silver stained nucleolar organelles (NORs) per cell and chemical toxicity are directly correlated each other. Various studies indicated that the quantity of protein AgNORs is related to the rapidity of cell proliferation and there is evidence of a relationship between AgNORs counts and the prognosis of malignant tumors. Thus, the quantification of these proteins has been a useful method in diagnostic pathology especially in the differential diagnosis between benign and malignant tumors and helpful in recognizing limitrophic lesions (Ghazizadeh et al. 1997); Irazusta et al.1998). Oz (1992) reported the possible contribution of the silver colloid technique for staining nucleolar organizer regions in the distinction between benign and malignant adrenocortical neoplasms was investigated. It was found that the AgNORs technique can help in differential diagnosis between benign and malignant adrenocortical lesions and thus may have a prognostic value. In oncology, Ploton et al. (1986) demonstrated that human prostate cancer cells are characterized by a higher number of interphase AgNORs compared to corresponding benign or hyperplastic cells. Trere (1989) reported that the mean number of AgNORs per nucleus accurately correlates with mitotic rate in tumor cell lines. Charpin (1992) observed that the stained complex corresponds ultrastructurally to the binding sites of RNA polymerase 1 and associated proteins regulating transcription and posttranslational modification of ribosomal RNA. In a series of experiments carried out in human cultured cancer cell lines, a highly significant correlation between AgNORs numbers and the speed of cell replication has been found (Trere, 1993).

In present study statistically significant difference was observed between the numbers of AgNORs in nuclei of cells in the rats treated with CCl4 and KBrO3. Higher number of AgNORs with CCl4 and KBrO3 administration indicated the presence of invasive neoplasia (Wilkinson et al., 1990).  In the control group number of nucleoli was ranging from 2-3 per cell. Nuclei were uniform in size and shape. In chemical treated group the numbers of nucleoli per cell were ranged from 5-10 along with abnormal increase in nuclear size and shape. This was reduced to 2-3 by administration with various fractions of rutin, L.procumbens and S.asper in rats.

Khan et al., (2009) recently reported that CCl4 toxication in rats significantly increases the numbers of AgNORs in the nuclei of glomerular and tubular cells in the renal tissues and were scattered through out the nucleus. Higher number of AgNORs with CCl4 administration indicated the presence of invasive neoplasia which was there by decreased orally administration of methanolic and n-hexane fraction of Digera muricata. This study was strongly in support of our investigation that medicinal plant take part in reduction of AgNORs.

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