Here the study has been carried out for exploring the pharmacological activities and phytochemical screening of the indian plants like T.arjuna, C.sinensis, C.sativum, P.longum, F.glomerata, T.foenum which belongs to our study. These plants are useful in reducing the cholesterol level in blood. So, the parts of the plants which are reported for their hypocholesterolemic activity were chosen and extracts of that parts were done with methanol and ethanol and are selected to screen for inhibition of cholesterol esterase enzyme.
These plants has been used traditionally by the local people and tribals in india to treat several diseases which are related to lungs, wound healing, in kapha, pitta,as diuretic, skin diseases and heart diseases.
Phytochemical screening of crude extracts of the plants revealed the presence of flavonoids, phenolics, tannins and glycosides. Flavonoids are 3-ring phenolic compounds consisting of double ring attached by a single bond to a third ring. They are a group of poly phenolic compounds responsible for the various biological effects such as hypocholestrolemia, antihepatotoxic, antiulcer, antidiabetic, antithrombotic and anti-cancer activities. They also inhibit enzymes like xanthine oxidase and aldose reductase. Flavonoids, tannins and phenolics found to possess anti oxidant activities. Phenols are important constituents because of their scavenging ability due to their hydroxyl group.
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Cholesterol plays a vital role in causing cardiovascular diseases like atherosclerosis, hypertension, ischemic heart diseases etc. and was supplied to the body by two sources. In this, diet is the major source for cholesterol intake into the body. After a meal containing high amounts of fat, the serum lipids begin to rise within two hours reaching a maximum concentration in approximately four to five hours, then again returning to basal level. The serum cholesterol concentration is influenced by the type as well as by the amount of dietary fat. (Katrina et al., 1997)
Cholesterol esterase is an enzyme which raises the cholesterol level by converting the esters of cholesterol to cholesterol and increases the cholesterol absorption in the intestine. Mechanism-based inhibitors of CEase map prevent absorption of dietary cholesterol into the blood stream and thereby be of therapeutic value in the treatment of atherosclerosis. Inhibition of cholesterol esterase will decrease the cholesterol levels in the body and useful to reduce the occurrence of cardiovascular diseases. Bovine pancreatic cholesterol esterase obtained from sigma Aldrich was used in our study (Sohl et al.,1988).
CEase hydrolyzes both water soluble and hydrophobic esters and its structure places it evolutionarily between the triglyceride lipases and the esterases. The triglyceride lipases preferentially hydrolyze hydrophobic esters and lipids, while esterases such as acetylcholinesterase function mainly on water soluble substrates. For CEase to accommodate larger, more hydrophobic esters, bile salt is required, in a dual role as a molecule binding specifically to CEase to open the active site to these bulkier molecules and as a surfactant to solubilize the hydrophobic esters, phospholipids, and triglycerides. Therefore, the bile salt dependence of CEase allows the enzyme to accommodate both hydrophilic and hydrophobic substrates (Chen et al.,1998).
The presence of bile salts effectively prevents trypsin and chymotrypsin inactivation of cholesterol esterase. Protective effect of taurocholate against proteolytic inactivation of this enzyme was due to the formation of a specific bile acid-enzyme complex, no enzymatic hydrolysis of the substrate occurred until taurocholate was added. In our study this complex is useful to the enzyme to undergo hydrolysis with the substrate pNPB. This protection was due to prevention of an irreversible denaturation of the enzyme. It was also reported that the loss in enzyme activity in the absence of bile salt could not be restored by subsequent addition of taurocholate. Thus, cholic acid and its conjugates appear to be cofactors for pancreatic juice cholesterol esterase, even though general proteolysis of pancreatic proteins was not effected. (George et al., 2002).
Orlistat drug is the reference standard used in the current study. This is an anti obesity drug which is a potent and specific inhibitor of the pancreatic lipases, which is responsible, in conjunction with a pancreatic co-lipase for the breakdown of dietary triglcerides into the absorbable fatty acids and monoglycerides. This inhibition decrease the absorption of fats by >30%. In healthy human it leads to >90% enzyme inhibition with no activity against trypsin. Orlistat binds covalently to the active site and forms a stable complex. The complex induces a conformational change in the enzyme that leads to a lid-like structure on the lipase, hence exposing the catalytic active site. This operation leads to acylation of a hydroxyl group on serine residue burden on the active site of the enzyme making it inactive as lipase (Suwailem et al., 2006)
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Some plant phytoconstituents have desired hypocholesterolemic potentials which include saponins, flavonoids, tea polyphenols , plant sterols, β-carotenes, fibers, plant indoles etc. these phytoconstituents have the cholesterol reducing capacity ( Pulok M, 2003)
It is evident from the current study that the plants investigated are rich sources of flavonoids, saponins, plant sterols and poly phenols. Recent epidemiological studies suggest that flavonoids have a good influence on different cardiovascular diseases, which might play an important role in inhibiting the cholesterol esterase enzyme. The inhibition caused by flavonoids can be attributed to their ability to form hydrophobic bonds with the active site present in the enzyme.
All the plant extracts of our study were screened for cholesterol esterase inhibition and their IC50 values were calculated. Except FGMFE all other extracts of our study are having slightly higher IC50 values than that of standard and are good inhibitors of CEase but the one mentioned is having more IC50 value than that of standard, which shows that extract is not a potential CEase inhibitor.
Free radicals are known to play a vital role in an extensive pathological manifestation. Anti-oxidants fight against the free radicals by protecting us from various diseases and scavenges of reactive oxygen radicals or protects the anti-oxidant defense mechanism. Reactive oxygen species are capable of damaging biological macromolecules such as DNA, carbohydrates and proteins. Human disease is believed to be due to imbalance between oxidative stress and anti- oxidant defense, it is possible to limit oxidative tissue damage and hence prevent disease progression by anti-oxidant defense supplements. In addition anti-oxidant activity may be regarded as a fundamental property important for life. Convincing evidence point out that increased intake of antioxidants relating to diet or fruits and vegetables with these properties may give enhancement in quality of life by stopping onset and decreasing the risk of degenerative diseases related to aging (Nagulendran et al., 2007).
Major active oxygen species causing lipid oxidation and huge biological damage are hydroxyl radicals. Incubation of Ferric-EDTA with H2O2 and ascorbic acid was done at pH 7.4. They were formed in free solution and were noticed by their capacity to degrade 2-deoxy-2-ribose into fragments and formation of pink chromogen occurs on heating with TBA at low pH (Kumar et al., 2006). Hydroxyl radical is highly reactive oxygen centered radical produced from the reaction of various hydro peroxides with transition metal ions. It attacks proteins, DNA, polyunsaturated fatty acid in membranes and most biological molecule it contacts and is known to be capable of abstracting hydrogen atoms from membrane lipids and brings about peroxidic reaction of lipids. The crude extracts of the plants of this study exhibit the concentration dependent scavenging activity against the hydroxyl radical generated in fenton's reaction. All the six extracts when added to the reaction mixture, scavenge the hydroxyl radicals by preventing the decomposition of deoxyribose.
Nitric oxide is a vital bio-regulatory molecule necessary for several physiological processes like neural signal transmission, immune response, control vasodialation and control of blood pressure etc. NO is generated from the terminal guanido nitrogen atom of L-arginine by various NADPH-dependent enzymes called NO synthases(NOS). NO has an unpaired electron, hence is a free radical (NO). NO becomes nitrosonium cation (NO+) or nitroxyl anion (NO−) by donating or accepting an electron, respectively. NOS is synthesized in a variety of cell types from multiple mammalian species and can produce consistent, high concentrations of NO upon induction with cytokines and or bacterial lipopolysaccharide (LPS) The plant or plant products may have the property to neutralize the effect of NO formation and in turn may be of significant interest in avoiding the ill effects of extreme NO generation in vivo. In our study, extracts of the plants was investigated for its inhibitory effect on nitric oxide generation. Curcumin was used as a reference standard. Concentration needed for 50% inhibition was calculated. The results were found statistically significant, (Jagetia et al., 2004). In the present study all the six extracts reduced the quantity of nitrite formed from the breakdown of sodium nitroprusside. This might be because of the antioxidant constituents in the extract which participate with oxygen to react with NO· thus inhibiting the production of nitrite.
Superoxide anions damage biomolecules directly or indirectly by forming H2O2, .OH, peroxy nitrite or singlet oxygen during aging and pathological events such as ischemic reperfusion injury. Superoxide has also been observed to directly initiate lipid peroxidation. Superoxide anions can be generated by both enzymatic and non-enzymatic system . In our study we adopted the non-enzymatic method to generate superoxide anion radicals. In this assay PMS /NADH -NBT system was used to generate and estimate super oxide anions. Super oxides obtained from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The reduced absorbance with antioxidants indicates consumption of super oxide anion in the reaction mixture. The superoxide scavenging activity of extracts was increased markedly with the increase in concentrations. (Nagulendran et al., 2007) The decline in absorbance at 560 nm and antioxidants shows the utilization of superoxide anion in the reaction. The degree of discoloration indicates the scavenging potential of the plant extracts. As expected, the lower the IC50 values the higher the percentage of NBT radical inhibition of the samples (Salar and Dhall, 2010).
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Ferrozine can quantitatively form complexes with Fe2+ but in the presence of ion chelating agents, complex formation is disrupted resulting in a reduction in the red colour of the complex. Measurement of the rate reduction of the colour allows estimation of the chelating activity of the co-existing chelator. The absorbance of Fe2 - Ferrozine complex was linearly decreased dose dependently (from 10 to 160 ïg/ml). Extracts captured ferrous ion earlier than ferrozine and thus, have ferrous chelating ability. In this assay extracts and standard compound interfered with the formation of ferrous complex with the reagent ferrozine, suggesting that it has chelating activity (Jayaprakasha et al., 2002). The findings of our study expressed that the extracts have valuable ability for iron binding, suggesting its antioxidant potential. Moreover, the metal chelating capacity of the fractions established that they decrease the concentration of the catalysing transition metal concerned in the peroxidation of lipids.
The important effect of flavonoids is the scavenging of oxygen-derived free radicals (Umamaheswari et al., 2009). The total flavonoid content in the extract was expressed as µg quercetin equivalent per mg. All the extracts showed high flavonoid content which has added directly to the antioxidant activity by neutralising the free radicals. In the present study CSMLE, TFMSE and TAEBE was found to possess significant amount, when compared to other extracts.
Thus, from the present investigation it can be said that all the plant extracts exhibited remarkable anti oxidant property in various in vitro assay systems.
From the results of the current study, it is evident that all the plant extracts were capable of inhibiting the enzyme cholesterol esterase and they are good sources of antioxidants which have beneficial effects in preventing oxidative damage to membranes. They showed direct relationship between the concentration of the tested plant extract and its activity which was done. They might be useful in the clinical field with low side effects (in vivo and toxicology tests have been performed). Further studies are required to assess their effects and mechanisms of actions.