Extraction, Separation And In Vitro Antioxidative Effects Of Proanthocyanidins In Radix Sanguisorbae

As a kind of polyphenol mixtures,proanthocyanidins exist in a wide range of plant tissues. Proanthocyanidins have great capacity of trapping and scavenging oxygen free radicals and reduce risk of some diseases(stroke,tumor and cancer,etc.).Through comparing the content of proanthocyanidins,Radix Sanguisorbae ranks the highest among 52 different plant materials.There are abundant plant resources of Radix Sanguisorbae which are widely distributed in many provinces of China.To make full use of Radix Sanguisorbae and its value-added processing,it is of great importance to extract and analyze the proanthocyanidins from Radix Sanguisorbae.In this paper,the following contents of proanthocyanidins from Radix Sanguisorbae were studied:Firstly,the quantitative method for proanthocyanidins was researched.Then the suitable extracting solvent was chosen and the optimum extraction technique of proanthocyanidins from Radix Sanguisorbae was established.Secondly,the suitable macroporous absorbent resin was chosen to separate and purify proanthocyanidins from Radix Sanguisorbae and the adsorption and desorption characteristics were studied.Then the purified compound was analyzed by infrared spectroscopy.Thirdly,Sephadex LH-20 was used to further separate of proanthocyanidins from Radix Sanguisorbae,and the separated components were detected by high performance liquid chromatography(HPLC).At last,mainly researched on the in vitro antioxidative activities of proanthocyanidins from Radix Sanguisorbae,and the inhibition of bacteria was referred.The main results are as follows:1.The content of proanthocyanidins is quantitatively determined by Vanillin-H₂SO₄ assay.In this paper,(+)-catechin was used as the standard substance,and there is a very good linearity when the concentration of(+)-catechin changing from zero to 0.35mg·mL^-1.The standard curve equation is Y_A500=2.0458X+0.0093,and the R² is 0.9953.The Vanillin-H₂SO₄ assay is suitable especially for the new plant resources which do not have the mature product of their standard substance for its sufficient precision and ruggedness.And this assay is very sensitive for the water content of reacting system.In the present work,the reaction medium was strictly free of water for the sharp decline of A₅₀₀ along with the increase of water content. 2.The extraction capacity of acetone,ethanol and methanol are:acetone＞ethanol＞methanol. Acetone was selected as the best one regarding its highly selective and strong extraction capacity of proanthocyanidins.3.Quadratic regression orthogonal rotary experiments for acetone extraction of proanthocyanidins were used in this paper.And the results were analyzed by DPS.The results indicated that the 3 factors in the experiments-extraction time(X₁),acetone concentration(X₂),ratio between solvent and material(X₃)-had strong influences to the extraction effects.And quadratic term of extraction time(X₁²),quadratic term of acetone concentration(X₂²),quadratic term of ratio between solvent and material(X₃²),the interaction between acetone concentration and ratio between solvent and material(X₂X₃) were also very strong.The influencing extents of the 3 factors were:acetone concentration(X₂)＞extraction time(X₁)＞ratio between solvent and material(X₃).The best extraction conditions were:extraction time(X₁) was 20min,acetone concentration(X₂) was 70%,ratio between solvent and material(X₃) was 25:1.Under these conditions,the average extraction amount was 78.11 mg·g^-1,and it's about 4.34%higher than the theoretical calculation value.The equation which showed the relationship between the extraction effect and the 3 factors is:Y=75.31591+2.20690X₁+1.54104X₂+3.25183X₃-3.13318X₁²-2.10788X₂²-1.98944X₃² +1.97750X₂X₃4.The results of static adsorption,desorption and the kinetics of static adsorption of AB-8, LSA-800B macroporous absorbent resin showed that LSA-800B resin was better than AB-8 resin with higher absorbing and eluting capacities.The absorbing and eluting rate of LSA-800B resin was 83.4%and 88.0%respectively,which was better than those of AB-8 resin.The two resins were able to greatly adsorb proanthocyanidins within 6 hours and reach equilibrium.In the following experiments,LSA-800B resin was chosen to purify proanthocyanidins from Radix Sanguisorbae.5.Through dynamic desorption of proanthocyanidins absorbed on the LSA-800B resin,the results showed that absolute ethanol had a strong eluting effect and the proanthocyanidins can be fully eluted about 4BV of absolute ethanol.The symmetry of elution peak was quite well and the recovery rate was 93.2%.After vacuum freeze-drying,the purity of proanthocyanidins can reach 95.6%.6.By analysis of the Infrared(IR) Spectra of purified proanthocyanidins from Radix Sanguisorbae,the skeleton vibration mainly occurs at 3600-3000cm^-1,1700-1000cm^-1 and 850-650cm^-1.Among them,the IR vibration peaks include the stretching vibration of hydroxy group, the skeleton vibration of benzene ring,the stretching vibration of C-O-C and the stretching vibration of C-H. 7.Through separated by Sephadex LH-20 and analyzed by HPLC,the results showed that Sephadex LH-20 had a good separation effect of proanthocyanidins from Radix Sanguisorbae and four separated components(F-1,F-2,F-3,F-4) were obtained.The retention times of chromatographic peaks were almost within 17-45min.Compared with standards,(+)-catechin, (-)-epicatechin could be identified.8.In vitro antioxidative experiments indicated that proanthocyanidins from Radix Sanguisorbae had strong antioxidative activity.The reducing power increased with the growing concentration of pranthocyanidins,and the Ferric Reducing/Antioxidant Power(FRAP) value was 8.629g·mmol^-1 FeSO₄.Proanthocyanidins from Radix Sanguisorbae showed a high scavenging activity of 1, 1-diphenyl-2-picryl-hydrazyl([DPPH·]),and the 50%inhibitory concentration(IC₅₀) was 0.245mg·mL^-1.The scavenging activity for hydroxyl radical([·OH]) increased with the growing concentration of pranthocyanidins,and the IC₅₀ was 1.117mg·mL^-1.Furthermore,Proanthocyanidins from Radix Sanguisorbae revealed powerful inhibitory effects on superoxide anion radical([O₂^-·]). At a low concentration level,it gave a high scavenging activity for[O₂^-·],and the IC₅₀ was 0.015mg·mL^-1.9.The results of minimum inhibitory concentration(MIC) of proanthocyanidins from Radix Sanguisorbae for Staphylococcus aureus,Bacillus subtilis,Escherichia coli,Bacillus cereus showed that proanthocyanidins from Radix Sanguisorbae had strong inhibition for these four bacteria.