Anthocyanin and Carbohydrate Content in Selective Extracts btained from Black Grape Varieties SILVIA ISUB 1, CRISTINA SARE 2, ILEANA RAU 1, AURELIA MEGHEA 1 * 1 University Politehnica of Bucharest, Department of Inorganic Chemistry, Physical Chemistry and Electrochemistry, 1-7 Polizu Str., 011061, Bucharest Romania 2 University of Pitesti, 1 Targul din Vale, Pitesti, Romania A series of comparative results are presented in relation to the main bio-active components of some selective natural extracts obtained from grape peel. Extracts of three black grape varieties, Cabernet Sovignon, Merlot, and Pinot Noir have been obtained and characterized for their anthocyanin and carbohydrate contents. Separation and purification of ethanolic extracts have been performed by reverse-phase C18 HPLC chromatography. The composition of the carbohydrates contained in the grapes, with glucose and fructose as major constituents have been identified by thin layer chromatography, and spectrophotometric Dubois method. The main monoglucoside derivatives of anthocyanins with high antioxidant activity: malvidin, peonidin, delphinidin, petunidin, cyanidin have been also identified. Keywords: anthocyanins, carbohydrates, separation, selective extracts, grape peels Pharmaceutical research increasingly focuses its objectives towards obtaining various active compounds from selective plant extracts, including grapes. Among their benefic actions the antioxidant activity of flavanols (catechins, epicatechins, anthocyans) can be mentioned. Fig 2. Structure of Resveratrol Flavan-3-ols, Polyphenols colorless Flavanone (Flavonols), Naringenin, Yelow color Another important derivative of anthocyanin malvidin with a more complex structure is 8.8-methylmethine-(epi) cathechin-malvidin-3-glycoside (fig.3), which was highlighted by reverse-phase HPLC chromatography C 18, [3-6]. Leucocyanidin, colorless + Anthocyans, Cyanidin Red color, UV Protector Fig.1. The bio synthese of anthocyanins from flavanols The compounds from the flavanols group are formed in grapes through biosynthesis of the base compound derivatives, such as flavan-3-ol. These biochemical processes take place under the action of various enzymes like: phenylalanine ammonia lyase, tyrosine ammonia lyase, cinnamate 4-hydroxylase, 4-coumaroyl CoA-ligase, chalcone synthase, chalcone isomerase, flavonoid 3 hydroxylase, flavonone 3-hydroxylase, dihydroflavanol 4- reductase, anthocyanidin synthase [1,2]. In this way, different compounds with complex structures and special functions on the plant protection are synthesized, such as anthocianins and their derivates. In figure 1 is presented the process of biosynthesis of chromophore with extended conjugated systems able to protect the plant against the UV radiation, which absorb at wavelengths around λ = 530 nm. Resveratrol a polyphenol from stilbene class - is also bio synthesised in grapes and has special pharmacological properties as cardiac protector, (fig.2). Fig.3. Structure of 8,8-methylmethine-(epi)-cathechine-malvidin-3- glicoside The morphological structure of the grapes is represented by three main parts: pulp, peel and seeds. In these structures the main compounds are specifically bioaccumulated. Indeed, in the grape pulp carbohydrates are found in the following average concentrations: cca 56% glucose, 40% fructose, 0.05% sucrose. In the seeds the maximum concentrations of fatty acids found are as follows: linoleic acid 70%, oleic acid 16%, palmitic acid, 7%, and 4% stearic acid. In our study only grape peels have been used to obtain selective extracts. Indeed, the maximum concentrations of anthocyanins and other polyphenols of physiological importance found in the grape peels are: total anthocians 0.085%, carbohydrates 0.5%, resveratrol 7 ppm. In this paper both similarities and differentiations have been studied during separation, purification and analysis * email: a.meghea@gmail.com; Tel. 0213154193 1078
of the main bioactive components identified in the selective ethanolic extracts obtained from the grape peels of three varieties: Cabernet Sovignon (CS), Merlot (M) and Pinot Noir (PN). Experimental part Materials and methods In order to obtain extracts, black grapes have been utilized, matured in ctober 2011, from the Cabernet Sovignon, Merlot, and Pinot Noir varieties provided by Murfatlar Romania. Solvents and reagents of high purity (Merck) were used. The thin layer chromatography (TLC silica gel), and reverse-phase chromatography HPLC (column Hypersep C18 Thermo Scientific) has been used for separation and purification of components. The absorption spectra of anthocyanins and glucides compounds (Dubois method) have been registered in the 350-800 nm spectral domain, using the UV-VIS Spectrophotometer SPECRD S 600. For the qualitative and quantitative determination of carbohydrates contained in selective extracts obtained from the grape peels, the dialysis separation of low molecular weight carbohydrates has been used by the regenerated celulose membrane MWC 3500 Daltons (Molecular Weight Cut ff) - Spectra Por 3, Standard RC membranes - Spectrum Laboratories, Inc. Procedure for obtaining the ethanolic extracts Due to the significant content of anthocyanins existent in black grape peels we opted for mechanical isolation of these parts of grapes from the other parts (pulp and seeds). Rapid washing with distilled water at a temperature of 4 C was performed to remove impurities adhering to the peel, followed by drying of grape peels in warm air flow (max. 60 o C). In every process of obtaining ethanolic extract an amount of 100 g of dry peels from each grape variety: CS, M and PN has been used as raw material. For anthocyanin extraction from grapes the solid-liquid extraction has been applied with a volume of 100 ml of ethanol / hydrochloric acid 2M (99/1), at a maximum temperature of 20 o C. Separation by dialysis of low molecular weight carbohydrates Filtered extracts were placed in Spectra Por 3 dialysis membrane MWC 3.5 kda (regenerated Cellulose Dialysis Membrane Spectra / Por 3 Spectrum Laboratories Inc.), 34 mm diameter tubulars and volumetric capacity of 93 ml/ 10 cm. We proceeded to the dialysis of extracts in 2 L of distilled water for 2 h and then distilled water was replaced. Carbohydrate content of the permeate was refractometrically tested and remained constant after 3 cycles of dialysis. The remnant solutions have been gathered in a single solution from which samples were taken for chromatographic analysis of carbohydrates, in thin layer of silicagel TLC and total carbohydrate analysis was carried out through Dubois method. Separation and identification of sugars contained in the permeate have been performed by thin layer chromatography using standards of monoglucide and saccharose, stationary phase silica gel plates 60. The eluent used was a mixture of 1-propanol / ethyl acetate / water in a volume ratio: 7/2/1. The final image obtained on chromatographic plate is shown in figure 4, and separation parameters are presented in table 1. The carbohydrates were thermally dehydrated in the presence of sulfuric acid and derivatives of furfural were obtained. These derivatives form with phenol red compounds that can be quantitatively determined by Fig. 4. TLC chromatographic plate image. Standard sugars from dialysis samples of ethanol extracts obtained from grape peels from CS, M and PN Fig. 5. Reaction scheme for total sugar determination spectral Dubois method at a wavelength of 490 nm, reported as total sugar content [7]. Results and discussions From the analysis of retention parameters for ethanolic fractions separated and purified as compared to the standards of some mono- and di-glucides, the values specified in the table 1 have been obtained. ne can observe that the total sugar content as determined by Dubois method, is quite similar in the three grape varieties, around 4 mg/g, with a decreasing tendency in the series: CS > M > PN. A detail analysis on components carried out by TLC chromatography revealed as major components glucose and fructose as expected, accompanied by a minor contribution of arabinose, mannose, and galactose. Chemical composition of major components and anthocianins separation The ethanolic extracts obtained from the three varieties of grapes have been obtained and processed in order to separate, purify and characterize the components according to the methods described in the literature [8, 9]. The purification of the selective extracts was performed in two main phases according to the scheme presented in the figure 6. The liquid-liquid extraction (mixture hexane/ water) was utilized in order to remove non-polar components. The next Solid Phase Extraction (SPE) is performed by reverse-phase chromatography HPLC C18, using as mobile phase: ethanol / hydrochloric acid 1M: (90/10). REV. CHIM. (Bucharest) 64 No. 10 2013 1079
Table 1 CARBYDRATE CMPSITIN F THE THREE GRAPE VARIETIES Fig. 6. Stages for obtaining selective grape peel extracts CS, M and PN Fig.7. Monoglucoside derivatives of the main anthocyanins identified The main anthocianins identified in the obtained extracts are: petunidin, delfinidin, malvidin, peonidin, cyanidin, and their monoglycoside derivatives are presented in figure 7. Characterization through UV-Vis spectrophotometry of the fractions Separated fractions are concentrated in a vacuum environment for characterization by HPLC chromatography and spectral methods. Due to the fact that the spectral properties are correlated with the conjugated chromophore structure of the system represented by anthocyanins, their molecular absorption 1080 bands observed in the UV-Vis spectra are very similar (table 2). However, some batochromic spectral shifts are observed with increasing molecular weight. Quantitative determination of five representative anthocyanin monoglucoside extracts is presented in table 2, while their distribution in investigated extracts of the three grape varieties is illustrated in figure 8. ne can observe that in all the three grape varieties the dominant monoglucoside anthocyanin belongs to malvidin, followed by petunidin and delphinidin for CS and M, and peonidin for M and PN. Besides these compounds identified in HPLC separated fractions, other fractions have been also detecteded, with
Table 2 SPECTRAL CHARACTERISTICS AND CNCENTRATINS F ANTCYANIN MNGLUCSIDE DERIVATIVES Fig. 8. Distribution of the main anthocyanins contents in extracts CS, M, and PN (mg/100ml extract) Fig. 9. Esters of malvidin-3-glucosides with acetic, coumaric, and caffeic acids Fig.10. Content of the anthocianin glucoside esters (mg/100g grape peels) absorption bands located at higher wavelengths. The compounds from these fractions have been associated with anthocianin glucoside esters with different acids: acetic, caffeic, and coumaric, [10]. Some representatives of these pigments derived from malvidine chromophore are presented in the figure 9. By correlating the spectral data obtained referring to the type of the esters derivatives of anthocianins glucosides with the above mentioned acids, a distribution can be obtained on two esters, acethyl and cumaryl as compared with nonacylate derivative (fig. 10). It is interesting to notice that despite the dominant content of nonacylated malvidin present in Pinot Noir, its esthers are almost absent, which corroborated with the lower total sugar content, probably makes the difference in organoleptic properties of these high quality wines. Conclusions The objectives of this work have allowed a comparative assessment of the contents of anthocyanins and sugars as bioactive constituents existent in grape peels extracts obtained from three Romanian varieties: Cabernet Sovignon, Merlot and Pinot Noir. As referring to the total sugar content as determined by Dubois method, this is quite similar in the three grape varieties, around 4 mg/g, with a decreasing tendency in the series: CS > M > PN. It was established that in all the three grape varieties the dominant monoglucoside anthocyanin belongs to malvidin, followed by petunidin and delphinidin for CS and M, and peonidin for M and PN. Some esters with different acids: acetic, caffeic, and coumaric of anthocyanin derivatives have been also identified, the relative contribution of all these bioactive components being REV. CHIM. (Bucharest) 64 No. 10 2013 1081
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