CHOLESTEROL ESTERASE ENZYME INHIBITORY AND ANTIOXIDANT ACTIVITIES OF LEAVES OF CAMELLIA SINENSIS (L.) KUNTZE. USING IN VITRO MODELS

CHOLESTEROL ESTERASE ENZYME INHIBITORY AND ANTIOXIDANT ACTIVITIES OF LEAVES OF CAMELLIA SINENSIS (L.) KUNTZE. USING IN VITRO MODELS

A. Pradeep Kumar*, A. T. Sivashanmugam M. Umamaheswari, V. Subhadradevi and P. Jagannath

Department of Pharmacology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, 395, Sarojini Naidu Road, Siddhapudur, Coimbatore-641044, Tamil Nadu, India

ABSTRACT

The present study was to evaluate the in vitro cholesterol esterase enzyme inhibitory and in vitro antioxidant activity of the methanol extract of the leaves of Camellia sinensis (L.).  Phytochemical screening of the extract shows the presence of flavonoids, phenolics and terpenoids. The extract shows ability to inhibit the enzyme with IC₅₀ (82.46±0.74µg/ml) whereas that of standard, Orlistat (24.15±0.59µg/ml). Antioxidant and free radical scavenging activity were also assessed by using the methods, IC₅₀ values for Nitric oxide radical scavenging activity (396.83±0.83µg/ml), whereas for standard curcumin (260.38±0.66µg/ml), hydroxyl radical scavenging activity (47.04±2.26µg/ml) and for quercetin (70.99±1.31µg/ml).  Moreover, the extract was found to scavenge the superoxide with 50% inhibition at 308.17±23.25µg/ml and standard ascorbic acid at 225.08±2.44µg/ml, IC₅₀ for ferrous chelating ability assay (44.12±4.63µg/ml) and of ascorbic acid (47.25±.89µg/ml). Total content of flavonoids present in 1mg of extract was 19.8±0.11 µg quercetin equivalents/mg. Results indicated that the extract shows potential bioactive compounds which might have a beneficial impact on diseases related to cholesterol synthesis and showed potential antioxidant and free radical scavenging activities.

Free radical scavenging activity, Flavonoids

INTRODUCTION: Dietary cholesterol comprised of free and esterified cholesterol. In diets rich in meats, significant percent of cholesterol is esterified. Hydrolysis of cholesteryl ester in the lumen is catalyzed by cholesterol esterase (CEase).

Cholesterol esterase (CEase) also is a lipolytic enzyme with wide substrate specificity capable of hydrolysing cholesteryl esters, triacylglycerol, phospholipids and lysophospholipids. It is synthesized primarily by the pancreas and is found as a component of pancreatic juice. This enzyme is also found in the milk of numerous species. Milk CEase is postulated to substitute for the pancreatic enzyme in the neonatal gastrointestinal tract before maturation of the pancreas ¹. Pancreatic cholesterol esterase is also known as bile salt-activated lipase, nonspecific lipase, phospholipase A₁ lysophospholipase, bile salt-dependent lipase, bile salt stimulated lipase, carboxyl ester lipase, and carboxyl ester hydrolase ².

A number of studies have suggested a possible role for CEase in the absorption of free cholesterol at the brush border membrane of the small intestine ³. Cholesterol and its esters are the major risk factors for the development of various cardiovascular diseases and are known to be one of the leading causes of death in developing and developed countries. Free radical is an atom with at least one unpaired electron in the outer most shell; this has the capability of independent existence. It is easily formed when, breakage of covalent bond occurs between entities and remaining of one unpaired electron in each new atom. They are extremely reactive due to the presence of unpaired electrons. Free radicals are implicated in various diseases. Free radicals act directly on the cells and effect cholesterol levels by an increase in cholesterol biosynthesis and esterification, decrease in cell cholesterol ester hydrolysis, reduced cholesterol efflux from the cells ⁴.

Antioxidants counteract free radicals by donating one of their own electrons, ending the carbon-stealing reaction. They act as scavengers, helping to prevent cell and tissue damage that directs to cellular damage and disease.

Camellia sinensis belongs to the family Theaceae, and also known as tea. Tea is native to mainland China, Southern Asia, but it is today cultivated across the world in tropical and sub tropical regions. Leaves are 4-15 cm long and 2-5 cm broad ⁵. Fresh leaves have 4% caffeine. Light green and young leaves are preferably produced for tea production. Leaves of Camellia sinensis reported antimicrobial properties, they contain flavonoids, their analysis and their functions ⁶, they inhibits the enzyme ribonuclease A ⁷, antioxidant activity ⁸, antibacterial activity ⁹, tea polyphenols used in the herbal infusions ¹⁰, protection against reactive oxygen species induced degradation of lipids, proteins and 2-deoxyribose by tea catechins ¹¹, green tea polyphenols having antiatherogenic properties ¹².

There is need to develop plant derived CEase inhibitory and antioxidant compounds as the synthetic drugs available are all found to have adverse effects.  Various studies have been conducted to explore the potential CEase inhibitory and antioxidant compounds from plant extracts.

MATERIALS AND METHODS:

Materials: Leaves of Camellia sinensis used in the present study were procured from Tamil Nadu, India, during the month of July 2010. Plant was recognized and authenticated by Dr. G.V.S. Murthy, Joint Director, C-I/C, Botanical Survey of India and bearing no.- BSI/SRC/5/23/10-11/Tech.- 828. Bovine pancreatic cholesterol esterase (C3766) and pNPB (p-nitro phenyl butyrate) were purchased from the Sigma Aldrich, USA. Acetonitrile and taurocholate were purchased from Loba Chemicals, Mumbai. Orlistat drug was purchased from Annanya chemicals, Hyderabad. Quercetin and 2-deoxy-2-ribose, sodium nitroprusside and curcumin were purchased from SRL, Mumbai.  Ascorbic acid, nitro blue tetrazolium (NBT) and EDTA were purchased from Hi Media labs. Pvt. Ltd., Mumbai. All the other drugs and chemicals used in this study were commercially obtained and were of analytical grade.

Plant collection and extraction procedure: Fresh green leaves were collected as shoot on fields in Ooty. 100g of fresh leaves were cut to 1-1.5mm size then immerse in solvent methanol (1:5 to 1:15 g/ml) for a certain time (0-90 minutes). Then it was transferred to flask and brewed, in to keep temperature not rise above 70^oC. After that, the infusion was let cool down to room temperature, filtered to separate solid. Final infusion was stored in refrigerator at 4°C ¹³.

Methods: Phytochemical screening of the methanolic leaves extract of Camelia sinensis was done for tannins and phenolics, flavonoids, terpenoids & steroids, glycosides and alkaloids ¹⁴.

Cholesterol esterase Enzyme Inhibitory Activity: Cholesterol esterase enzyme inhibitory activity was evaluated and performed in the presence of sodium taurocholate with p-nitrophenyl butyrate as chromogenic substrate ¹⁵. Stock solution of CEase (19.5ng/ml) and taurocholate (12mM) were prepared by using 100mM sodium phosphate buffer of PH (7.O). Stock solution of pNPB (200μM) & inhibitors were prepared by using Acetonitrile (6%). A final volume of 1ml is taken into a cuvette containing 430µl of assay buffer, 500µl of TC solution, 40µl of acetonitrile, 10µl of pNPB solution and 10 µl of an inhibitor solution were added and thoroughly mixed. Incubation for 2 min at 25°C, the reaction was initiated by adding 10µl of the enzyme solution. Uninhibited enzyme activity was determined by adding acetonitrile instead of the inhibitor solution.   Absorbance was measured at 405 nm. The percentage inhibition was calculated by using, ¹⁶.

CEase inhibition % I = [1- a] x 100

a= enzyme activity with inhibitor /enzyme activity without inhibitor

Anti-oxidant Models:

Hydroxyl Radical Scavenging Assay: Hydroxyl radical scavenging activity of the extract was assessed by using 2-Deoxy 2-Ribose method ¹⁷. This assay was quantified by studying the competition between deoxyribose and extract for hydroxyl radical generated by Fe3⁺-EDTA– Ascorbate-H₂O₂ system (Fenton reaction).The reaction mixture in a final volume of 1.0 ml contained 100μl of 2-deoxy2-ribose (28 mM in 20 mM KH₂PO₄ buffer, pH 7.4), 500μl of the extract at various concentrations (10-160μg/ml) in buffer, 200μl of 1.04 mM EDTA and 200 μM FeCl₃ (1:1v/v), 100μl of 1.0 mM hydrogen peroxide (H₂O₂) and 100μl of 1.0 mM ascorbic acid.

Test samples were kept at 37^oC for 1 h. The free radical damage imposed on the substrate, deoxyribose was measured using the thiobarbituric acid test. One ml of 1% thiobarbituric acid (TBA) and 1.0 ml 2.8% trichloroacetic acid (TCA) were added to the test tubes and were incubated at 100⁰C for 20 min. After cooling, the absorbance was measured at 532 nm against a blank containing deoxyribose and buffer. Quercetin (10-160μg/ml) was used as the positive control. Percentage inhibition (%I) was calculated by comparing the results of the test with that of control.

Total Flavonoid Content: Samples were analyzed for total flavonoid content according to the aluminium nitrate method using quercetin as a standard ¹⁸. Different concentrations of quercetin in the range of (10-320µg/ml) were made with 80% ethanol to construct a standard graph. Known concentration of extract (1000 μg) was added to 1ml of 80% ethanol. An aliquot of 0.5 ml is added to test tubes containing 0.1 ml of 10% aluminium nitrate, 0.1 ml of 1 M potassium acetate and 4.3 ml of 80% ethanol. The absorbance of the supernatant is measured at 415 nm after 40 min of incubation at room temperature.

Ferrous Chelating Ability: Fe²⁺ forms complexes with ferrozine quantitatively. But in the presence of ion chelating agents, the complex development is disturbed, following a reduction in the red color of the complex ¹⁹. The reaction mixture containing different concentrations of the extract were added to 2mM ferrous chloride (0.1 ml) and 5mM ferrozine (0.2 ml) to initiate the reaction and the mixture is shaken vigorously and left to stand at room temperature for 10 min. The absorbance of the solution is measured at 562 nm. The percentage chelating effect of ferrozine-Fe²⁺ complex formation is calculated. The positive control used was ascorbic acid

Superoxide Anion Radical Scavenging Assay: Superoxide anions were generated in a non-enzymatic phenazine methosulfate- nicotinamide adenine dinucleotide (PMS- NADH) system through the reaction of PMS, NADH, and oxygen ¹⁹. The scavenging activity towards superoxide anion radicals of the extract was measured. In this assay  superoxide anions were generated in a final volume of 3 ml of 100 mM Tris-Hydrochloric acid buffer (pH 7.4) containing 0.75 ml of 300µM NBT solution, 0.75 ml of 936 µM NADH solution and 0.3 ml of various concentrations of the extract. The reaction was started by adding 0.75 ml of PMS (120 μM) to the solution. The mixture after incubation for 5 min at 25^oC, absorbance was measured at 560 nm in UV spectrophotometer. The positive control used was ascorbic acid ²⁰ in different concentrations (50-800µg/ml). The super oxide anion scavenging activity was calculated according to the following equation:

% Inhibition = [(A_o-A₁) / A_o × 100]

Where A₀ was the absorbance of the control (blank, without extract) and A₁ was the absorbance in the presence of the extract.

Nitric Oxide Scavenging Assay: Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction. Sodium nitroprusside in water at physiological pH on impulse instinctively generates nitric oxide, which interacts with oxygen to generate nitrite ions that can be estimated by Greiss reagent ²¹. Various concentrations of the extract and sodium nitroprusside (5mM) in phosphate buffer saline (0.025 M, pH 7.4) in a final volume of 3 ml are incubated at 25^o C for 150 min.

Control experiments without the test compounds but with equivalent amount of buffer are prepared in the same manner as done for the test. There after, 0.5 ml of incubation solution is removed and diluted with 0.5 ml Griess’ reagent (1% sulphanilamide, 2% O-Phosphoric acid and 0.1% naphthyethylene diamine dihydrochloride) and allowed to react for 30 min. The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and subsequent coupling with naphthyethylene diamine dihydrochloride is read at 546 nm. The positive control used was curcumin and the percentage inhibition was calculated.

Statistical analysis: The data were analyzed by one way analysis of variance (ANOVA) to study the CEase inhibitory and antioxidant activity of the extract and between the concentrations. Probability level was fixed to ^*P < 0.001.

RESULTS AND DISCUSSION: In present study, plant known for their therapeutic properties of the traditional medicine was selected for evaluating their potential as an anti-hyperlipidemic agent by testing the inhibitory effect against cholesterol esterase. Methanol extract of leaves of Camellia sinensis was prepared. Phytochemical screening of crude extract of the plant revealed the presence of flavonoids, phenolics, tannins and alkaloids. In addition steroids and terpenoids were found to be present in the leaves extract of Camellia sinensis. Flavonoids, tannins and phenolics were reported to possess antioxidant and other important pharmacological activities including anti-hyperlipidemic activity ²².

Cholesterol esterase Inhibitory Activity: CEase inhibitory activity can be attributed to the presence of flavonoids in the extract. Flavonoids have been reported to irreversibly bind with the cholesterol esterase enzyme in its active fatty acid pocket at Serine 194. It was also suggested that the flavonoids act as suicide substrates ahead of cholesterol esters ²³. The inhibitory effect of the extract against bovine pancreatic cholesterol esterase was performed, which showed good activity and the same was comparable with that of the standard used, Orlistat.

Extract exhibited good Cholesterol esterase activity ranging from 17.8 to 81.2% inhibition in comparison to corresponding values of standard Orlistat ranges from 15.89 to 93.9% (Table 1 & 2), activity increasing with higher concentrations. IC₅₀ value of the extract was found to be 82.46µg/ml. These results indicate that the plant extract had a noticeable effect on the enzyme cholesterol esterase. The differences among the samples were found to be extremely significant as per analysis of variance ^*** (P < 0.001).

TABLE 1: CHOLESTEROL ESTERASE INHIBITION BY THE STANDARD, ORLISTAT

Values are expressed as mean ± S.E.M. Mean values of six readings with three replications

TABLE 2: CHOLESTEROL ESTERASE INHIBITION BY METHANOL EXTRACT OF CAMELLIA SINENSIS LEAVES

Values are expressed as mean ± S.E.M. Mean values of six readings with three replications

Anti-oxidant activity:

Superoxide Radical Scavenging Activity: Superoxide anions indirectly initiated lipid oxidation as a result of superoxide and hydrogen peroxide serving as precursors of singlet oxygen and hydroxyl radicals ¹⁹. Various studies reported that the anti-oxidant properties of flavonoids are effective mainly via the scavenging of superoxide anion. In this assay, decline in absorbance at 560 nm indicates that anti oxidants shows the utilization of superoxide anion in the reaction ²⁴. Superoxide scavenging activity of extracts was increased markedly with an increase in concentrations having the percentage inhibition in the range from 14.2 to 74.8% and that of standard ascorbic acid starts from 13.2 to 85.2%. IC₅₀ values of the extract and the standard was expressed in Table 3.

Hydroxyl Radical Scavenging Activity: Hydroxyl radicals are the major active oxygen species causing lipid oxidation and enormous biological damage. Ferric-EDTA was incubated with H₂O₂ and ascorbic acid at pH 7.4. Hydroxyl radicals were formed in free solution and were detected by their ability to degrade 2-deoxy-2-ribose into fragments that on heating with TBA at low pH form a pink chromogen ^{17, 18, 20}.  In this assay, the extract, prevented the decomposition of deoxyribose in a dose dependent manner. They exhibited concentration dependent scavenging activity against the hydroxyl radical generated in Fenton’s reaction. Percentage inhibition of the extract increased distinctly with that of concentrations used in the range from 21.9 to 74.3% in comparison with that of standard quercetin ranges from 11.9 to 81.5% and the IC₅₀ values were tabulated in the Table 3.

Ferrous Chelating Ability: In this assay, extract and standard compound interferes with the formation of ferrous complex with the reagent ferrozine, suggesting that it has chelating activity. The results of our study expressed that the extracts have ability to bind iron indicates its antioxidant potential ^{19, 20}. Moreover, the metal chelating capacity of the extracts established that they decrease the concentration of the catalyzing transition metal concerned in the peroxidation of lipids with the increase in the concentration of the extract in the solution and the percentage inhibition of the extracts ranges from 19.5 to 71.2%  in  similarity to that of the standard ascorbic acid having the inhibition values 13.7 to 79.1% and the IC₅₀ values of both are mentioned in the Table 3.

Nitric Oxide Radical Scavenging Activity: It is well known that nitric oxide has an important role in various types of inflammatory processes in the animal body ^{17, 18}. In the present study, crude extract of the leaves of the plant reduced the quantity of nitrite formed from the breakdown of sodium nitroprusside. This might be due to the presence of antioxidant constituents in the extract which participate with oxygen to react with NO· thus inhibiting the production of nitrite. Different concentrations of the extract was checked for its activity and compared with that of the standard and percentage inhibition of the extract starts from 18.4% at 50 µg/ml concentration and ends up at 68.9% at 800 µg/ml concentration.IC₅₀ values for both were expressed in Table 3.

TABLE 3: IN VITRO ANTIOXIDANT ACTIVITY OF THE METHANOL EXTRACT OF THE LEAVES OF CAMELLIA SINENSIS

Mean values of five readings with three replications. Values are expressed as mean± S.E.M

Total Flavonoid Content: In aluminum nitrate colorimetric method, aluminum nitrate forms acid stable complex with the keto group and either the hydroxyl group in A or C ring of flavonoids ²⁵ in addition it forms acid labile complexes with orthodihydroxyl groups in the A or B ring of flavonoids ¹⁸. The aluminum nitrate complexes of flavonoid compounds show strong absorbance at 415 nm and flavonoids with more functional groups absorb stronger at 415 nm. We used quercetin as a standard compound because it is one of the widely spread flavonoid in most of the plant extracts. Total flavonoid content of the methanolic extract of the leaves of the Camellia sinensis was 19.8±1.26µg quercetin equivalents/mg.

CONCLUSION: From the results of our study, we can conclude that the plant extract was capable of inhibiting the enzyme cholesterol esterase, but at varied potencies and were found to be good sources of antioxidants which have beneficial effects in preventing oxidative damage to membranes. It showed direct relationship between the concentration of the tested plant extract and its activity which was evaluated. It might be useful in the clinical field and since being plant extracts can be expected to exhibit lower incidence of side effects. Isolation of phyto- constituents from the extract would help in determining the active principles responsible for the activities, even though it could be suggested that flavonoids might be the potential agent. The existence of poly phenols and flavonoids can be a reason for the prevalence of these activities. Further, in vivo studies are required to assess possible clinical potential and confirm its mechanisms of action.

ACKNOWLEDGEMENTS: The author gratefully acknowledges Annanya chemicals, Hyderabad, for providing standard (orlistat) for the project work.

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