Bovine pancreatic cholesterin 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, Na nitroprusside and curcumin were purchased from SRL, Mumbai. Trichloroacetic acid ( TCA ) , thiobarbituric acid ( TBA ) , ferrous chloride, sulphanilamide, phosphorous acid were purchased from SD Fine Chemicals Ltd. , Mumbai. Ascorbic acid, nitro bluish tetrazolium ( NBT ) and EDTA were purchased from Hi Media labs. Pvt. Ltd. , Mumbai. All other drugs and chemicals used in the survey were obtained commercially and were of analytical class.
All the parts of the above workss were collected in and around Coimbatore & A ; Nilgiri territories, 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 study of India, Tamil Nadu Agricultural University Campus, Coimbatore, Tamilnadu, India.
4.5 EXTRACTRACTION Procedures:
4.5.1 T.arjuna bark infusion:
The works stuff was collected and dried under the shadiness for seven yearss. Dried bark of T.arjuna was cut into little pieces and so they were land into pulverization in a pulverization factory. The pulverization was refluxed with 70 % ( v/v ) ethyl alcohol in H2O for about four hours utilizing Soxhlet setup. The infusion was filtered and the filtrate was evaporated to dryness. Brownish ruddy crystals were obtained with a output of 14.7 % . ( Pasenjit et al. , 2007 )
4.5.2 P.longum fruit infusion:
The fruits were washed exhaustively by utilizing tap H2O and was cut into little pieces and dessicated under sunshine. Plant stuff was later pulverized and macerated in absolute methyl alcohol, ( at the ratio of 1 kilograms of workss per 3L of methyl alcohol ) . The supernatants were collected after 7 yearss and filtered and the solid was macerated once more. This process was repeated two times. Whole methyl alcohol infusion from works stuff was filtered and evaporated to dryness, at 55A°C. The infusions were so stored at 4A°C until usage. Methanol extraction of PLF gave 12.4 % output. ( Nongyao et al. , 2004 )
4.5.3 F.glomerata fruit infusion:
Fruits were collected and cut into little pieces and dried in sunshine. Dried fruits were taken and made into a harsh pulverization and sieved through. 20, which was repeatedly extracted in a 1000 milliliter unit of ammunition bottomed flask with dissolver, methyl alcohol. The reflux clip for dissolver was 4 h. The infusion was cooled to room temperature, filtered and vaporize to dryness. The residues yielded were stored at 4- 8A°C.
( Naghma et al. , 2004 )
4.5.4 C.sinensis foliages infusion:
Fresh green tea was collected as shoot on Fieldss. 100g of fresh green tea were cut to 1 – 1.5mm size so immerse in solvent methyl alcohol ( 1:5 to 1:15 g/ml ) for a certain clip ( 0 – 90 proceedingss ) . Then it was transferred to flask and brewed in to maintain temperature non lift above 70oC. After that, the extract was let cool down to room temperature, filtered to divide solid. Concluding extract was stored in icebox at 4A°C. ( Quan et al. , 2009 )
4.5.5 T.foenum seed infusion:
Seeds of this works are collected and washed exhaustively with H2O to take soil. They are dried in visible radiation till the wet content in the seeds is minimal. 250gm of powdered dried seeds were macerated with 500ml of methyl alcohol for 6days. After that the decoction of the seeds was collected and filtered twice. The filtrate was concentrated by dessicating the the dissolver at temperature non more than 50A°C.The dried residue was stored in icebox at 4A°C. ( Tayyaba et al. , 2001 )
4.5.6 C.sativum seed infusion:
Seeds of the works are collected and dried under shadiness and infusions were obtained by stirring 100g of dry pulverization with 500ml of pure methyl alcohol for 30 min. Extraction was carried out utilizing maceration at room temperature for 24 H followed by filtration through Whatman No.1 filter paper and vaporization to dryness. Percentage output of evaporated dried infusion was 5 % . Sample was stored at 4°C. ( Manel et al.,2010 )
The above stored infusions were used for in vitro cholesterin esterase inhibitory and anti-oxidant surveies.
4.6 PHYTOCHEMICAL Screening
Chemical trials were carried out for all infusions of workss for the presence of phytochemical components ( Trease and Evans, 2007 ; Khandelwal, 2004 ) ) .
4.6.1 Test for tannic acids and phenoplasts
Ferric chloride trial
To the solution of the infusion, 3 to 4 beads of 0.1 % ferrous chloride was added and observed for bluish-black color or chocolate-brown viridity.
4.6.2 Test for saponins
About 10 milliliter of the infusion was assorted with 5 milliliter of distilled H2O and smartly shaken for a steady persistent-froth. The frothing was assorted with 3-4 beads of olive oil and agitate smartly and observed for the development of an emulsion.
4.6.3 Test for flavonoids
a ) To a part of the infusion, concentrated H2SO4 was added. A xanthous color observed indicates the presence of flavonoids. The xanthous colour disappeared on standing.
B ) Few beads of 1 % AlCl3 solution was added to a part of infusion. A xanthous colour indicates the presence of flavonoids.
degree Celsius ) A part of the dried infusion was heated with 10 milliliters of ethyl ethanoate over a steam bath for 3 min. The mixture was filtered and 4 milliliter of the filtrate was shaken usually with 1 milliliters of dilute ammonium hydroxide solution.Yellow colour indicate a positive trial for flavonoids.
4.6.4 Test for tri terpenoids and steroids
Small sum of infusion was dissolved in 5 milliliter of trichloromethane individually. Different trials are done to observe the phytosterol presence with this solution.
Liebermann-Burchard ‘s trial
Above prepared solution was treated with few beads of concentrated
sulfuric acid followed by few beads of diluted acetic acid, 3ml of acetic anhydride. Phytosterols presence can be indicated by the visual aspect of blue green coloring material.
To 1ml of above prepared chloroform solution, few beads of concentrated sulfuric acid was added to the solution. Brown coloring material produced shows the presence of phytosterols
4.6.5 Test for alkaloids
A little part of the infusion was stirred with few beads of
dil HCl and filtered.
Dragendroff ‘s reagent: To the filtrate, K Bi iodide
solution was added and an orange brown precipitate indicates the presence of alkaloids.
Mayer ‘s reagent: To the filtrate, K mercurous iodide solution was added and a pick precipitate indicates the presence of alkaloids.
Hager ‘s trial: To the filtrate, concentrated solution of picric acid was added, xanthous precipitate formed indicates the presence of alkaloids.
Wagner ‘s trial: To the filtrate, K iodide solution was added and formation of ruddy brown precipitates indicates the presence of alkaloids.
4.7 In vitro CHOLESTEROL ESTERASE ENZYME INHIBITORY ACTIVITY
Cholesterol esterase suppression activity was assayed by utilizing p-nitro phenyl butyrate ( pNPB ) as substrate.
Hydrolyses were performed in the presence of a high enzyme concentration, and merchandises were identified spectrophotometrically.
Assay buffer used was 100mM Na phosphate buffer pH ( 7.0 ) which was made by adding 100mM sodium phosphate with 100mM Nacl in deionized H2O.
Inhibition check was performed in the presence of Na 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 utilizing ( 100mM ) Na phosphate buffer of pH ( 7.O ) .
Stock solution of pNPB ( 200I?M ) , infusions and criterion of different concentrations ( 10-320Aµg/ml ) were prepared by utilizing Acetonitrile ( 6 % ) .
A concluding volume of 1ml is taken into a cuvette incorporating 430 Aµl of check buffer, 500 Aµl of TC solution, 40 Aµl of acetonitrile, 10 Aµl of pNPB solution and 10 Aµl of an inhibitor solution were added and exhaustively assorted. Incubation for 2 min at 25A°C, the reaction was initiated by adding 10 Aµl of the enzyme solution. The optical density was measured at 405nm against space. Percentage suppression was calculated by utilizing the expression,
% 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 alternatively of the inhibitor solution. Control optical density was measured by adding 100mM Na phosphate pH 7.0, alternatively 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 groups was measured.
Superoxide anions were generated in a non-enzymatidc phenazine methosulfate-nicotinamide A dinucleotide ( PMS-NADH ) system through the reaction of PMS, NADH, and O.
It was assayed by the decrease of nitroblue tetrazolium ( NBT ) . In these experiments the superoxide anion was generated in 3 milliliter of Tris-HCl buffer ( 100 millimeter, pH 7.4 ) incorporating 0.75 milliliter of NBT ( 300 AµM ) solution, 0.75 milliliter of NADH ( 936 AµM ) solution and 0.3 milliliter of different concentrations of the infusion. The reaction was initiated by adding 0.75 milliliter of PMS ( 120 AµM ) to the mixture. After that 5 min of incubation is done at room temperature, the optical density was measured at 560 nanometers by utilizing spectrophotometer. The ace oxide anion scavenging activity was calculated harmonizing to the undermentioned equation:
% Inhibition = [ ( Ao-A1 ) / Ao A- 100 ] ,
where Ao was the optical density of the control ( clean, without infusion ) and A1 was the optical density in the presence of the infusion. ( Nagulendran et al. , 2007 )
4.8.2 Metal chelating activity assay
The chelating activity of the infusions for ferric ions Fe2+ was measured harmonizing to the method. To 0.5 milliliter of infusion, 1.6 milliliter of deionized H2O and 0.05 milliliter of FeCl2 ( 2 millimeter ) was added. After 30 s, 0.1 milliliter ferrozine ( 5 millimeter ) was added. Ferrozine reacted with the divalent Fe to organize stable magenta complex species that were really soluble in H2O. This was kept for 10 min at room temperature, the optical density of Fe2+ Ferrozine composite was measured at 562 nanometer. The chelating activity of the infusion for Fe2+ was calculated as
Chelating rate ( % ) = ( A0 – A1 ) / A1 A- 100
where A0 implies the optical density of the control ( clean, without infusion ) and A1 was the optical density in the presence of the infusion. ( Nagulendran et al.,2007 )
4.8.3 Entire i¬‚avonoid content by aluminium nitrate method
Entire soluble flavonoid content of the infusions was determined by aluminum nitrate method utilizing quercetin as standard. One milligram of the fraction is added to 1ml of 80 % aqueous ethyl alcohol. Aliquots of diluted infusions ( 0.5ml ) were added to prove tubings and assorted with 0.1 milliliters of 10 % aluminium nitrate, 0.1ml of 1M aqueous K ethanoate and 4.3ml of 80 % ethyl alcohol. After incubation for 40 min at room temperature, optical density of the reaction mixtures was measured at 415nm. Different concentrations of Quercetin in the scope of ( 10 -320Aµg/ml ) was made with 80 % ethyl alcohol to build a standard graph. By utilizing the graph entire flavonoid content as Aµg of quercetin was determined. ( shiva et al. , 2006 )
4.8.4 Hydroxyl Radical Scavenging Activity
This activity was quantified by analyzing the competition between deoxyribose and infusions for hydroxyl extremist generated by Fe3+-EDTA- Ascorbate-H2O2 system ( Fenton reaction ) .
This reaction mixture include a concluding volume of 1.0 milliliter, in which 500 AµL of the assorted concentrations of extracts,100Aµl of 2-deoxy-2-ribose ( 28 millimeter ) & amp ; different concentrations of criterion were diluted in KH2PO4-KOH buffer, ( 20mM ) , pH 7.4, 200 Aµl of ( 1.04 millimeter ) EDTA & A ; ( 200 AµM ) FeCl3 ( 1:1 v/v ) , 100 Aµl of 1.0 millimeters H2O2 and 100 Aµl of 1.0 millimeters ascorbic acid was incubated at 37°C for 1 h. 1.0 milliliter of 1 % thiobarbituric acid ( TBA ) and 1.0 milliliter of ( 2.8 % ) trichloroacetic acid ( TCA ) were added to the trial tubing and were incubated at 100°C for 20 min. After chilling, optical density was calculated at 532 nanometers against control incorporating deoxyribose and buffer. Quercetin ( 10-160Aµg/ml ) was used as a criterion. Chemical reactions were done in triplicate. The per centum suppression ( % I ) was calculated by comparing the consequences of the trial with control compounds. ( Ramanathan et al. , 2006 )
4.8.5 Nitric oxide coevals and check of azotic oxide scavenging.
Nitric oxide was generated from Na nitroprusside and measured by the Greiss reaction.
Sodium nitroprusside in H2O at physiological pH on impulse instinctively generates azotic oxide, which interacts with O to bring forth nitrite ions that can be estimated by usage of Greiss reagent.
Scavengers of azotic oxide compete with O taking to lesser coevals of azotic oxide.
Sodium nitroprusside ( 5mM ) in phosphate-buffered saline was assorted with different concentrations of the infusions dissolved in the appropriate solvent systems and incubated at 25A°C for 150min. Control experiments were done without the trial compound but with equal sum of buffer. The samples from above was reacted with Greiss reagent ( 1 % sulphanilamide, 2 % H3PO4 and 0.1 % napthyl ethylene diamine dihydrochloride ) . The optical density of the chromophore which is formed during the diazotization of nitrite with sulphanilamide and subsequently matching with napthyl ethylene diamine was measured at 546nm and compared with the optical density of standard solutions of K nitrite treated in the same manner with Griess reagent.The experiment is done in triplicate utilizing Curcumin as criterion. ( Ganesh et al. , 2004 )
4.9 Statastical analysis:
All the experiments were done in triplicate and the values were expressed as mean A± criterion mistake of mean ( S.E.M ) .