Introduction. On the other hand, human pancreatic

Introduction. Obesity is a severepublic health problem in all industrialized countries.

According to WorldHealth Organization worldwide obesity has nearlytripled since 1975.  Statistical analysesof 2016 showed that   more than 1.9 billion adults were overweight.

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Of these over 650 million were obese. (Obesity and overweight Fact sheet Updated October 2017 ). Obesity often is a direct resultof fat -rich diet.

On the other hand, human pancreatic lipase (triacylglycerolacyl hydrolase EC is an enzyme responsible for fat digestion and subsequentabsorption by human organism. Therefore dietary fat absorption can be reduced by partialinhibition of lipase activity and this is one of the main strategies infighting obesity(??????????).Thousands of plants have been screened for the purpose of finding out naturallipase inhibitors(??????????).  Various foodstuffs such as cereals, soybean, medicinalplants, grapevine fruits, green tea or a leguminous plant have been shownto consist of phytochemicals with anti-lipase activity (??????????).  Here are some classesof phytochemicals that reveal anti-lipase activity:  alkaloids, carotenoids, glycosides,   polysaccharides, saponins, terpenes,polyphenols (??????????).

Nevertheless, nowadaysOrlistat (Xenical) is the only pancreatic lipase inhibitor that is in clinicaluse.  Orlistat  is a hydrogenated derivative of lipstatinisolated from Gram-positive bacterium  Streptomyces toxytricini . But some adverse effects of orlistat such as   liquid stools, diarrhea, steatorrhea fecalurgency etc.

significantly limit use of this medication (??????????).  Thus, there is a huge call for newer naturalsources of phytochemicals with high anti-lipase activity. Withreference to this, it is worth to mention that Georgian wines are known toconsist of huge amount of polyphenols and therefore can be considered aspotential natural inhibitors of pancreatic lipase. For instance, totalpolyphenol content in white Georgian wines produced according to the special”Kakhetian” technology was found to be between 2000 and 2290 mg per liter. Inred wines total polyphenols content ranged between 2848-4416 mg per liter(Shalashvili et al.

Bulletin of the Georgian academy of sciences, 2007). Winery and winemakingin Georgia starts from early sixthmillennium BC and Georgia is believed to be a cradle of wine.  (Patrick McGoverna et al. EarlyNeolithic wine of Georgia in the South Caucasus. Proceedings of the nationalacademy of sciences of the united states of america. November 28, 2017 vol.

114 no.48 Wine consumption playeda significant role in formation of dietary culture of Georgians.

 Consumption of wine in every day diet ofGeorgians is obviously related with many beneficial and well-known healthpromoting properties of wines and their chemical constituents particularlyanti-lipase activity amongst them (?? ?????????? ????),. Numerous and variouskind of researches of Georgian wines have been carried out so far (?? ??????????????), but despite of this, thereare no data available in scientific literature about anti-lipase activity ofGeorgian wines. Hence, such data together with information about Georgia’straditional cousin may be useful from the point of view of revealing role ofwine consumption in case of high fat diet of humans in general. Basedon the above-mentioned the objective of this work was to study in vitro anti-lipase activityof several sorts of Georgian wines and to identify relationship betweenanti-lipase activity, polyphenol content and antioxidant activity of theGeorgian wines. Materials and methods ChemicalsAscorbicacid, Olive Oil, Sodium Hydroxide and Potassium dihydrogen phosphate werepurchased from Sigma – Aldrich (Steinheim, Germany), TPTZ-2,4,6-Tris(2-pyridyl)-s-triazine (Sigma – Aldrich, Switzerland), Folin – Ciocalteureagent (Appli Chem, Germany), hydrochloric acid, formic acid and phosphoricacid were provided by Merck (Darmstadt, Germany), Sodium carbonate waspurchased from Chem Cruz (Chem Cruz Biochemicals, USA), Ethyl acetate andmethanol (Sigma – Aldrich Steinheim, Germany) were HPLC grade.

Detergent Tween80 was obtained from Ferak Berlin GmbH (Germany).  Lipase concentrate -HP was purchased forIntegrative Therapeutics, LLC (USA). All other reagents were commerciallyavailable at the local market and were of analytical grades.Sample preparation for chemical analyses Preparation of sample for individualpolyphenols determination by HPLC (Varian e Prostar – 500, USA, detector – UVVarian Prostar, Australia, column – 250 mm 4.6 mm, dp ¼ 5 mm, Symmetry, Waters,Ireland) method was done according to Koyuncu et al. (2010) 27. Briefly, 10mL samples were adjusted to pH 1.

5 with 10 mL phosphoric acid-water (2%, v/v).The samples were filtered through filter paper 45 mm (Whatman, UK). Then, 1.5mL buffer (0.01 M KH2PO4, pH 8.

0) was added to 1.5 mL sample, 1 mL (vitamin C)of preferred mixtures were loaded on to C 18 cartridges (Agilent, Bond Elut,USA). After loading, 3.0 mL water adjusted to pH 1.5 with 2.0 mL phosphoricacid-water (2%, v/v) were passed through the cartridges??? MeasuresDeterminationof pH pHvalue of the berry fruits was measured using a pH-meter (PHS-3C, ShanghaiRotech Pharmaceutical Engineering Co.

, Ltd, China) 30.Total Dry Matter.For measurement of nonvolatile dry matter in wines, 50mL of wine sample was poured out on porcelain basin; the basin was placed on waterbath until evaporation of moisture, alcohol and other volatile compounds.Finally moisture wasevaporated from the samples by oven drying at 105 0C for 16 hours.

Total dry matter was determined gravimetrically as residue remaining afterdrying. Alcohol contentAlcohol content in wines wasdetermined by simple distillation method (?? ????????? ????). Ferric reducing ability of plasma (FRAP)assay Theantioxidant capacity was determined following the procedure described by Benzieet al. (1996) 32 with modifications. The FRAP reagent was freshly prepared byadding 10 mM 2,4,6- tripyridyl-s-triazine (TPTZ) (dissolved in 40 mM of HCl),20 mM of FeCl3 in water and 300 mM of acetate buffer (pH 3.

6) in theratio of 1:1:10. The FRAP reagent was warmed to 37 C for 15 min. Then, 100 mLof sample was added to 3.0 mL reagent blank. The absorbance was recorded at 593nm. The reaction was monitored for 4 min. FRAP values of samples were comparedto that of ascorbic acid and expressed as vitamin C equivalents per 1 L of wines.Determinationof total phenolic compounds (TPC) Determination of TPC was performed by Bond etal.

(2003) 31. An aliquot of 1.0 mL of diluted sample extract was vortexedwith 10 mL DI water and 1.0 mL Folin-Ciocalteau reagent, and 1.0 mL deionizedwater was used as control. After equilibration at room temperature for 8 min,the solutions were mixed with 4 mL of 7.

5% (w/v) Na2CO3. The samples andstandards (Gallic acid dilute working standard solutions: 10e50 mg mL1 ) wereequilibrated at room temperature for 60 min. The absorbance of the samples andstandards were measured spectrophotometrically (UV/Vis spectrophotometer,A Lab Co LTD, UK) at 765 nm, with a 10 mm path length cell. TPC wascalculated as mg of gallic acid equivalents per 100 g fresh weight of sample.Lipase activity Lipaseactivity unit definition:   One unit willhydrolyze 1.0 micro equivalent of fatty acid from a triglyceride in one hour atpH 7.

2 at 37 ° C.  Procedure fordetermination of lipase activity was as follows as reported by M. Stoicheva et al Current AnalyticalChemistry, 2012, 8, 400-407. Analytical Methods for Lipases  Activity Determination.  Margarita Stoytcheva 1,* , Gisela Montero 1 ,Roumen Zlatev 1 , José Ángel León 1  and VelizarGochev 2  and as described in (1993) Reagent Chemicals ACSSpecification, 8th ed., 95 with little modification.

  Briefly,to prepare initial reaction mixture 3 ml of olive oil was added by 1 ml 200 mMTris HCl buffer, pH 7.2 and 0.5 ml detergent (Tween 80) and mixed rigorously bymagnetic mixer for 15 min. Then 150 mg lipase preparation was added to theemulsified mixture and incubated at 37 0C for 30 min.

At the end ofincubation 3 ml 95% alcohol was added to the incubated mixture and the final reactionmixture was titrated with 50mM sodium hydroxide solution using automaticpotentiometric titrator ZDJ-4A ( Hanheng Instrument Co..China). End point of titration was set at pH 9.0. Blank titrationwas carried out the same way but without lipase . Lipase activity wascalculated according to the following formula:Units =(NaOH)  Volume (in milliliters)  used for test titration minus (NaOH)  volume (in milliliters) used for blanktitration X1000X2X(df) Here: 1000 = Conversion factor from milliequivalent to microequivalent preparation2 = Time conversion factor from 30 minutes to 1hour,   df = Dilution factor Measuring percent of Inhibition of lipaseactivity.1 ml of wine was added to the initial reactionmixture.

The following procedures were identical to those described above. Atthe end of the process enzyme activity was calculated in the presence of wineas inhibitor and compared to that of without inhibitor. Accordingly, percent ofinhibition was calculated.     Statistical analysis Thedata represents the mean of three replicates ± standard deviation (SD). Datawere subjected to the one-way ANOVA and Tukey’s HSD tests. All calculationswere performed with Microsoft Excel 2007 (Microsoft Corp., Redmond, WA, USA)with PHstat 2 version 3.11 add-in assistance


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