Introduction. plant have been shown to consist of phytochemicals

Introduction. Obesity is a severe
public health problem in all industrialized countries. According to World
Health Organization worldwide obesity has nearly
tripled since 1975.  Statistical analyses
of 2016 showed that   more than 1.9 billion adults were overweight.
Of these over 650 million were obese. (Obesity and overweight Fact sheet Updated October 2017 
http://www.who.int/mediacentre/factsheets/fs311/en/ ). Obesity often is a direct result
of fat -rich diet. On the other hand, human pancreatic lipase (triacylglycerol
acyl hydrolase EC 3.1.1.3) is an enzyme responsible for fat digestion and subsequent
absorption by human organism. Therefore dietary fat absorption can be reduced by partial
inhibition of lipase activity and this is one of the main strategies in
fighting obesity
(??????????).
Thousands of plants have been screened for the purpose of finding out natural
lipase inhibitors
(??????????).  Various foodstuffs such as cereals, soybean, medicinal
plants, grapevine fruits, green tea or a leguminous plant have been shown
to consist of phytochemicals with anti-lipase activity (??????????).  Here are some classes
of phytochemicals that reveal anti-lipase activity:  alkaloids, carotenoids, glycosides,   polysaccharides, saponins, terpenes,
polyphenols (??????????). Nevertheless, nowadays
Orlistat (Xenical) is the only pancreatic lipase inhibitor that is in clinical
use.  Orlistat  is a hydrogenated derivative of lipstatin
isolated from Gram-positive bacterium  Streptomyces 
toxytricini . But some adverse effects of orlistat such as   liquid stools, diarrhea, steatorrhea fecal
urgency etc. significantly limit use of this medication (??????????).  Thus, there is a huge call for newer natural
sources of phytochemicals with high anti-lipase activity.

With
reference to this, it is worth to mention that Georgian wines are known to
consist of huge amount of polyphenols and therefore can be considered as
potential natural inhibitors of pancreatic lipase. For instance, total
polyphenol content in white Georgian wines produced according to the special
“Kakhetian” technology was found to be between 2000 and 2290 mg per liter. In
red wines total polyphenols content ranged between 2848-4416 mg per liter
(Shalashvili et al. Bulletin of the Georgian academy of sciences, 2007). Winery and winemaking
in Georgia starts from early sixth
millennium BC and Georgia is believed to be a cradle of wine.  (Patrick McGoverna et al. Early
Neolithic wine of Georgia in the South Caucasus. Proceedings of the national
academy of sciences of the united states of america. November 28, 2017 
vol. 114 no.
48      http://www.pnas.org/cgi/doi/10.1073/pnas.1714728114 Wine consumption played
a significant role in formation of dietary culture of Georgians.  Consumption of wine in every day diet of
Georgians is obviously related with many beneficial and well-known health
promoting properties of wines and their chemical constituents particularly
anti-lipase activity amongst them (?? ?????????? ????),. Numerous and various
kind of researches of Georgian wines have been carried out so far (?? ??????????
????), but despite of this, there
are no data available in scientific literature about anti-lipase activity of
Georgian wines. Hence, such data together with information about Georgia’s
traditional cousin may be useful from the point of view of revealing role of
wine consumption in case of high fat diet of humans in general.

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Based
on the above-mentioned the objective of this work was to study in vitro anti-lipase activity
of several sorts of Georgian wines and to identify relationship between
anti-lipase activity, polyphenol content and antioxidant activity of the
Georgian wines.

Materials and methods

Chemicals

Ascorbic
acid, Olive Oil, Sodium Hydroxide and Potassium dihydrogen phosphate were
purchased from Sigma – Aldrich (Steinheim, Germany), TPTZ-2,4,6-Tris
(2-pyridyl)-s-triazine (Sigma – Aldrich, Switzerland), Folin – Ciocalteu
reagent (Appli Chem, Germany), hydrochloric acid, formic acid and phosphoric
acid were provided by Merck (Darmstadt, Germany), Sodium carbonate was
purchased from Chem Cruz (Chem Cruz Biochemicals, USA), Ethyl acetate and
methanol (Sigma – Aldrich Steinheim, Germany) were HPLC grade. Detergent Tween
80 was obtained from Ferak Berlin GmbH (Germany).  Lipase concentrate -HP was purchased for
Integrative Therapeutics, LLC (USA). All other reagents were commercially
available at the local market and were of analytical grades.

Sample preparation for chemical analyses

 Preparation of sample for individual
polyphenols determination by HPLC (Varian e Prostar – 500, USA, detector – UV
Varian 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, 10
mL 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, 1.5
mL 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 phosphoric
acid-water (2%, v/v) were passed through the cartridges???

Measures

Determination
of pH

pH
value of the berry fruits was measured using a pH-meter (PHS-3C, Shanghai
Rotech Pharmaceutical Engineering Co., Ltd, China) 30.

Total Dry Matter.

For measurement of nonvolatile dry matter in wines, 50
mL of wine sample was poured out on porcelain basin; the basin was placed on water
bath until evaporation of moisture, alcohol and other volatile compounds.
Finally moisture was
evaporated from the samples by oven drying at 105 0C for 16 hours.
Total dry matter was determined gravimetrically as residue remaining after
drying. 

Alcohol content

Alcohol content in wines was
determined by simple distillation method (?? ????????? ????).

Ferric reducing ability of plasma (FRAP)
assay

The
antioxidant capacity was determined following the procedure described by Benzie
et al. (1996) 32 with modifications. The FRAP reagent was freshly prepared by
adding 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 the
ratio of 1:1:10. The FRAP reagent was warmed to 37 C for 15 min. Then, 100 mL
of sample was added to 3.0 mL reagent blank. The absorbance was recorded at 593
nm. The reaction was monitored for 4 min. FRAP values of samples were compared
to that of ascorbic acid and expressed as vitamin C equivalents per 1 L of wines.

Determination
of total phenolic compounds (TPC)

 Determination of TPC was performed by Bond et
al. (2003) 31. An aliquot of 1.0 mL of diluted sample extract was vortexed
with 10 mL DI water and 1.0 mL Folin-Ciocalteau reagent, and 1.0 mL deionized
water 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 and
standards (Gallic acid dilute working standard solutions: 10e50 mg mL1 ) were
equilibrated at room temperature for 60 min. The absorbance of the samples and
standards were measured spectrophotometrically (UV/Vis spectrophotometer,
A Lab Co LTD, UK) at 765 nm, with a 10 mm path length cell. TPC was
calculated as mg of gallic acid equivalents per 100 g fresh weight of sample.

Lipase activity

Lipase
activity unit definition:   One unit will
hydrolyze 1.0 micro equivalent of fatty acid from a triglyceride in one hour at
pH 7.2 at 37 ° C.  Procedure for
determination of lipase activity was as follows as reported by M. Stoicheva et al Current Analytical
Chemistry, 2012, 8, 400-407. Analytical Methods for Lipases  Activity Determination.  Margarita Stoytcheva 1,* , Gisela Montero 1 ,
Roumen Zlatev 1 , José Ángel León 1  and Velizar
Gochev 2  and as described in (1993) Reagent Chemicals ACS
Specification, 8th ed., 95 with little modification.  Briefly,
to prepare initial reaction mixture 3 ml of olive oil was added by 1 ml 200 mM
Tris HCl buffer, pH 7.2 and 0.5 ml detergent (Tween 80) and mixed rigorously by
magnetic mixer for 15 min. Then 150 mg lipase preparation was added to the
emulsified mixture and incubated at 37 0C for 30 min. At the end of
incubation 3 ml 95% alcohol was added to the incubated mixture and the final reaction
mixture was titrated with 50mM sodium hydroxide solution using automatic
potentiometric titrator ZDJ-4A ( Hanheng Instrument Co..China). End point of titration was set at pH 9.0. Blank titration
was carried out the same way but without lipase . Lipase activity was
calculated according to the following formula:

Units =(NaOH)  Volume (in milliliters)  used for test titration minus (NaOH)  volume (in milliliters) used for blank
titration X1000X2X(df)

Here: 
1000 = Conversion factor from milliequivalent to microequivalent preparation

2 = Time conversion factor from 30 minutes to 1
hour,   df = Dilution factor

 Measuring percent of Inhibition of lipase
activity.

1 ml of wine was added to the initial reaction
mixture. The following procedures were identical to those described above. At
the end of the process enzyme activity was calculated in the presence of wine
as inhibitor and compared to that of without inhibitor. Accordingly, percent of
inhibition was calculated.   

  Statistical analysis

The
data represents the mean of three replicates ± standard deviation (SD). Data
were subjected to the one-way ANOVA and Tukey’s HSD tests. All calculations
were performed with Microsoft Excel 2007 (Microsoft Corp., Redmond, WA, USA)
with PHstat 2 version 3.11 add-in assistance