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Bovine pancreatic cholesterol esterase 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. Trichloroacetic acid (TCA), thiobarbituric acid (TBA), ferric chloride, sulphanilamide, phosphoric acid were purchased from SD Fine Chemicals Ltd., Mumbai. Ascorbic acid, nitro blue tetrazolium (NBT) and EDTA were purchased from Hi Media labs. Pvt. Ltd., Mumbai. All other drugs and chemicals used in the study were obtained commercially and were of analytical grade.
All the parts of the above plants were collected in and around Coimbatore & Nilgiri districts, Tamil Nadu, 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, Tamil Nadu Agricultural University Campus, Coimbatore, Tamilnadu, India.
4.5 EXTRACTRACTION PROCEDURES:
4.5.1 T.arjuna bark extract:
The plant material was collected and dried under the shade for seven days. Dried bark of T.arjuna was cut into small pieces and then they were ground into powder in a powder mill. The powder was refluxed with 70% (v/v) ethanol in water for about four hours using Soxhlet apparatus. The extract was filtered and the filtrate was evaporated to dryness. Brownish red crystals were obtained with a yield of 14.7%. ( Pasenjit et al., 2007)
4.5.2 P.longum fruit extract:
The fruits were washed thoroughly by using tap water and was cut into small pieces and dessicated under sunlight. Plant material was subsequently pulverized and macerated in absolute methanol, (at the ratio of 1 kg of plants per 3L of methanol). The supernatants were collected after 7 days and filtered and the solid was macerated again. This procedure was repeated two times. Whole methanol extract from plant material was filtered and evaporated to dryness, at 55Â°C. The extracts were then stored at 4Â°C until use. Methanol extraction of PLF gave 12.4% yield.( Nongyao et al., 2004)
4.5.3 F.glomerata fruit extract:
Fruits were collected and cut into small pieces and dried in sunlight. Dried fruits were taken and made into a coarse powder and sieved through. 20, which was repeatedly extracted in a 1000 ml round bottomed flask with solvent, methanol. The reflux time for solvent was 4 h. The extract was cooled to room temperature, filtered and evaporate to dryness. The residues yielded were stored at 4- 8Â°C.
( Naghma et al., 2004)
4.5.4 C.sinensis leaves extract:
Fresh green tea was collected as shoot on fields. 100g of fresh green tea 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 70oC. After that, the infusion was let cool down to room temperature, filtered to separate solid. Final infusion was stored in refrigerator at 4Â°C. ( Quan et al., 2009)
4.5.5 T.foenum seed extract:
Seeds of this plant are collected and washed thoroughly with water to remove dirt. They are dried in light till the moisture content in the seeds is minimum. 250gm of powdered dried seeds were macerated with 500ml of methanol for 6days. After that the decoction of the seeds was collected and filtered twice. The filtrate was concentrated by dessicating the the solvent at temperature not more than 50Â°C.The dried residue was stored in refrigerator at 4Â°C. ( Tayyaba et al., 2001)
4.5.6 C.sativum seed extract:
Seeds of the plant are collected and dried under shade and extracts were obtained by stirring 100g of dry powder with 500ml of pure methanol for 30 min. Extraction was carried out using maceration at room temperature for 24 h followed by filtration through Whatman No.1 filter paper and evaporation to dryness. Percentage yield of evaporated dried extract was 5%. Sample was stored at 4°C.( Manel et al.,2010)
The above stored extracts were used for in vitro cholesterol esterase inhibitory and anti-oxidant studies.
4.6 PHYTOCHEMICAL SCREENING
Chemical tests were carried out for all extracts of plants for the presence of phytochemical constituents (Trease and Evans, 2007; Khandelwal, 2004)).
4.6.1 Test for tannins and phenolics
Ferric chloride test
To the solution of the extract, 3 to 4 drops of 0.1% ferric chloride was added and observed for bluish-black colouration or brownish green.
4.6.2 Test for saponins
About 10 ml of the extract was mixed with 5 ml of distilled water and vigorously shaken for a steady persistent-froth. The frothing was mixed with 3-4 drops of olive oil and shaken vigorously and observed for the development of an emulsion.
4.6.3 Test for flavonoids
a) To a portion of the extract, concentrated H2SO4 was added. A yellow colouration observed indicates the presence of flavonoids. The yellow coloration disappeared on standing.
b) Few drops of 1% AlCl3 solution was added to a portion of extract. A yellow coloration indicates the presence of flavonoids.
c) A portion of the dried extract was heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken normally with 1 ml of dilute ammonia solution.Yellow coloration indicate a positive test for flavonoids.
4.6.4 Test for tri terpenoids and steroids
Small amount of extract was dissolved in 5 ml of chloroform separately. Different tests are done to detect the phytosterol presence with this solution.
Above prepared solution was treated with few drops of concentrated
sulphuric acid followed by few drops of diluted acetic acid, 3ml of acetic anhydride. Phytosterols presence can be indicated by the appearance of bluish green colour.
To 1ml of above prepared chloroform solution, few drops of concentrated sulphuric acid was added to the solution. Brown colour produced shows the presence of phytosterols
4.6.5 Test for alkaloids
A small portion of the extract was stirred with few drops of
dil HCl and filtered.
Dragendroff's reagent: To the filtrate, potassium bismuth iodide
solution was added and an orange brown precipitate indicates the presence of alkaloids.
Mayer's reagent: To the filtrate, potassium mercuric iodide solution was added and a cream precipitate indicates the presence of alkaloids.
Hager's test : To the filtrate, saturated solution of picric acid was added, yellow precipitate formed indicates the presence of alkaloids.
Wagner's test : To the filtrate , potassium iodide solution was added and formation of reddish brown precipitates indicates the presence of alkaloids.
4.7 In vitro CHOLESTEROL ESTERASE ENZYME INHIBITORY ACTIVITY
Cholesterol esterase inhibition activity was assayed by using p-nitro phenyl butyrate (pNPB) as substrate.
Hydrolyses were performed in the presence of a high enzyme concentration, and products were identified spectrophotometrically.
Assay buffer used was 100mM sodium phosphate buffer pH (7.0) which was made by adding 100mM sodium phosphate with 100mM Nacl in deionized water.
Inhibition assay was performed in the presence of sodium Taurocholate with p-nitrophenyl butyrate as chromogenic substrate. (Markus et al., 2004)
Preparation of stock solutions
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 (200Î¼M), extracts and standard of different concentrations (10-320Âµg/ml) were prepared by using Acetonitrile(6%).
A final volume of 1ml is taken into a cuvette containing 430 Âµl of assay buffer, 500 Âµl of TC solution, 40 Âµl of acetonitrile, 10 Âµl of pNPB solution and 10 Âµl of an inhibitor solution were added and thoroughly mixed. Incubation for 2 min at 25Â°C, the reaction was initiated by adding 10 Âµl of the enzyme solution. The absorbance was measured at 405nm against blank. Percentage inhibition was calculated by using the formula,
%I CEase = I x 100
where ; I= 1-a
a = enzyme activity with inhibitor/ enzyme activity without inhibitor
Uninhibited enzyme activity was determined by adding acetonitrile instead of the inhibitor solution. Control absorbance was measured by adding 100mM sodium phosphate pH 7.0, instead of enzyme. Orlistat is used as positive control.
4.8 In vitro ANTI OXIDANT METHODS
4.8.1 Superoxide anion scavenging activity assay
The scavenging activity towards superoxide anion radicals was measured.
Superoxide anions were generated in a non-enzymatidc phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS, NADH, and oxygen.
It was assayed by the reduction of nitroblue tetrazolium (NBT). In these experiments the superoxide anion was generated in 3 mL of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 mL of NBT (300 ÂµM) solution, 0.75 mL of NADH (936 ÂµM) solution and 0.3 mL of different concentrations of the extract. The reaction was initiated by adding 0.75 mL of PMS (120 ÂµM) to the mixture. After that 5 min of incubation is done at room temperature, the absorbance was measured at 560 nm by using spectrophotometer. The super oxide anion scavenging activity was calculated according to the following equation:
% Inhibition = [(Ao-A1) / Ao Ã- 100],
where Ao was the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract.( Nagulendran et al., 2007)
4.8.2 Metal chelating activity assay
The chelating activity of the extracts for ferrous ions Fe2+ was measured according to the method . To 0.5 mL of extract, 1.6 mL of deionized water and 0.05 mL of FeCl2 (2 mM) was added. After 30 s, 0.1 mL ferrozine (5 mM) was added. Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. This was kept for 10 min at room temperature, the absorbance of Fe2+ Ferrozine complex was measured at 562 nm. The chelating activity of the extract for Fe2+ was calculated as
Chelating rate (%) = (A0 - A1) / A1 Ã- 100
where A0 implies the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract.( Nagulendran et al.,2007)
4.8.3 Total ï¬‚avonoid content by aluminum nitrate method
Total soluble flavonoid content of the extracts was determined by aluminium nitrate method using quercetin as standard. One mg of the fraction is added to 1ml of 80% aqueous ethanol. Aliquots of diluted extracts (0.5ml) were added to test tubes and mixed with 0.1 ml of 10% aluminum nitrate, 0.1ml of 1M aqueous potassium acetate and 4.3ml of 80% ethanol. After incubation for 40 min at room temperature, absorbance of the reaction mixtures was measured at 415nm. Different concentrations of Quercetin in the range of( 10 -320Âµg/ml )was made with 80% ethanol to construct a standard graph. By using the graph total flavonoid content as Âµg of quercetin was determined. ( shiva et al., 2006)
4.8.4 Hydroxyl Radical Scavenging Activity
This activity was quantified by studying the competition between deoxyribose and extracts for hydroxyl radical generated by Fe3+-EDTA- Ascorbate-H2O2 system (Fenton reaction).
This reaction mixture include a final volume of 1.0 mL, in which 500 ÂµL of the various concentrations of extracts,100Âµl of 2-deoxy-2-ribose (28 mM) & different concentrations of standard were diluted in KH2PO4-KOH buffer,( 20mM), pH 7.4, 200 Âµl of (1.04 mM) EDTA & (200 ÂµM) FeCl3 (1:1 v/v), 100 Âµl of 1.0 mM H2O2 and 100 Âµl of 1.0 mM ascorbic acid was incubated at 37°C for 1 h. 1.0 mL of 1% thiobarbituric acid(TBA) and 1.0 mL of (2.8%)trichloroacetic acid(TCA) were added to the test tubes and were incubated at 100°C for 20 min. After cooling, absorbance was calculated at 532 nm against control containing deoxyribose and buffer. Quercetin(10-160Âµg/ml) was used as a standard. Reactions were done in triplicate. The percentage inhibition (%I) was calculated by comparing the results of the test with control compounds. (Ramanathan et al., 2006)
4.8.5 Nitric oxide generation and assay of nitric oxide scavenging.
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 use of Greiss reagent.
Scavengers of nitric oxide compete with oxygen leading to lesser generation of nitric oxide.
Sodium nitroprusside (5mM) in phosphate-buffered saline was mixed with different concentrations of the extracts dissolved in the appropriate solvent systems and incubated at 25Â°C for 150min. Control experiments were done without the test compound but with equal amount of buffer. The samples from above was reacted with Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthyl ethylene diamine dihydrochloride). The absorbance of the chromophore which is formed during the diazotization of nitrite with sulphanilamide and later coupling with napthyl ethylene diamine was measured at 546nm and compared with the absorbance of standard solutions of potassium nitrite treated in the same way with Griess reagent.The experiment is done in triplicate using Curcumin as standard. (Ganesh et al., 2004)
4.9 Statastical analysis:
All the experiments were done in triplicate and the values were expressed as mean Â± standard error of mean (S.E.M).