China. Keywords: Synergistic Effects, Antioxidant, Ginkgo Leaves Flavonoid, Loquat Leaves Flavonoid, Response Surface Method(RSM).

Transcription

1 2016 International Conference on Energy Development and Environmental Protection (EDEP 2016) ISBN: Antioxidant Synergistic Effect and Formulation Optimization of Ginkgo Leaves Flavonoid and Loquat Leaves Flavonoid Xiao-Lei CHEN 1,a, Yi-Hua JIANG 1,b,*, Xin-Long JIANG 1,2,c, Zhe-Zi JIN 1,d, Ning-Hong MAO 1,e, Zhi-You ZHI 1,f 1 Zhejiang Shuren University, Hangzhou, China 2 Institute of Food and Fermentation Engineering, Zhejiang Shuren University, Hangzhou, China a @qq.com, b hzjyih828@163.com, c jxinl2003@163.com, d @qq.com, e @qq.com, f @qq.com * Corresponding author Keywords: Synergistic Effects, Antioxidant, Ginkgo Leaves Flavonoid, Loquat Leaves Flavonoid, Response Surface Method(RSM). Abstract. In order to understand the synergetic effect between ginkgo leaves flavonoid and loquat leaves flavonoid, synergetic effect and optimal formulation of compound antioxidant were determined with the response surface method under DPPH system. The results showed the component ratio of optimal compound antioxidant was ginkgo leaves flavonoid 8.67µg ml -1 and loquat leaves flavonoid µg ml -1,the DPPH scavenging rate of the system was 44.82%. Compound antioxidant of ginkgo leaves flavonoid and loquat leaves flavonoid had antagonistic action. Introduction Ginkgo leaves and loquat leaves are garden wastes.loquat leaves is a well known traditional chinese medicine used as antitussive and anti-inflammatory agent for acute and chronic bronchitis[1]. Phytochemical studies with this species have led to the isolation of many natural products including flavonoids, sesquiterpene and triterpenes glycoside[2-4].chemical components of ginkgo leaf mainly is flavonoids, alkaloids, post, etc, which has the function of the medicinal composition is mainly flavonoids[5].the garden wastes of loquat leaves and ginkgo leaves are rich in flavone and showed a higher scavenging activity [6-7]. DPPH method has been widely used in phytomedicine for the assessment of scavenging activities of bioactive fractions[8]. In this paper,the loquat leaves flavonoid and ginkgo leaves flavonoid as typical representative of natural garden wastes flavonoids,the DPPH scavenging rate as evaluation index of antioxidant activity, the synergetic effect and optimal formulation were determined using the response surface optimization method under DPPH scavenging system. Material and Methods Materials, Reagents Treatment process for test materials: fresh ginkgo leaves and loquat leaves(collected from our campus in March, 2015) cleaning drying(40 C) powdering eliminating impurities

2 with petroleum ether drying again grinding and sieving from mesh screen for use. Rutin standard sample was purchased from chinese medicine group shanghai chemical reagent company,dpph was purchased from Sigma company,other reagents were of analytic grade. Distilled water was used throughout the experiment. Methods The Preparation of Flavonoid Accurate weighing dry garden wastes powder adding a certain amount of ethanol solution extracting agent extraction filtering macroporous absorbent resins(d-101) purification collected eluent constant volume measuring. Product Analysis According to the reported method in the article[9],content of flavonoids in the extract was determined using rutin as standard sample, the regression equation C (mg)=0.6503a between concentration of flavonoids and absorbance was established, the linear range was 0 ~ 1.0mg,the correlation coefficient r was Antioxidant Test DPPH method is used to evaluate a natural antioxidant activity with sensitive,simple, rapid and feasible.dpph radical-scavenging activity was performed by the method described of Ying QL [8]. The stock solution ( mol/l) was diluted to a dilution to mol/l with 60% (v/v) ethanol. An aliquot of each dilution (1.0 ml) was mixed with solution of DPPH (5 ml, mol/l). The mixtures were shaken vigorously and incubated in the dark at 37 C for 30 min. A control containing 60% (v/v) ethanol (1.0 ml) and solution of DPPH (5 ml, mol/l) was run at the same time. The absorbance was measured at 517 nm against 60% (v/v) ethanol (5.0 ml) mixed tested extract solution(1.0 ml)as a blank. The percentage of DPPH scavenging was calculated as follows: P% =[1-(Ai-Aj)/A 0 ] 100, where A 0 is absorption of control, Aj is absorption of blank, Ai is absorption of tested extract solution. Synergy Analysis Method The effect produced exceeds the sum of their respective alone when used in combination of several substances, the synergistic effect is produced. The synergy evaluation method of antioxidant in vitro have direct comparison method, the addition method and the response surface method commonly. Response Surface Methodology (RSM) is a effective for responses that can reflect the experimental method of synergistic effect between each factor[10], it can optimize process parameters by combination of mathematics and statistics method to fit the factors and response value.comparison of the response coefficients in the quadratic regression equations, can be quantified analysis factors influence on the composite antioxidant activity from the view of mathematics [11]. The subject chosen response surface methodology (RSM). Response Surface Test Method The Single Factor Test The flavonoid sample solution (1 mg/ml) was preparated with 60% (v/v) ethanol. The DPPH solution (5 ml, mol/l) was mixed with sample solution. To add a small amount the sample solution at first, increases gradually, observe the fade of solution while still

3 mixing. When the solution basic color faded, remembered the concentration of sample.the maximum concentration was this concentration. Based on the maximum concentration, the single factor test was tested according to the antioxidant test method. Based on the coordination of the two factors, so choose to DPPH clearance is less than 50% of the three levels. The Response Surface Experimental Design According to the single factor experiment results, the concentration of ginkgo leaves flavonoid and loquat leaves flavonoid as independent variables,response surface methodology experiments of Box-Behnken design with 2 factors and 3 levels were chosen for formulation optimization. The factors and levels were listed in Table 1. Table 1. Levels of factors in response surface methodology. Factor Level A ginkgo leaves flavonoid [µg ml -1 ] B loquat leaves flavonoid [µg ml -1 ] Result and Analysis The Single Factor test The DPPH Scavenging Rate of the Ginkgo Leaves Flavonoid. Fig. 1 showed, it had a high dose effect relationship between ginkgo leaves flavonoid and scavenging DPPH free radical. The ability of scavenging DPPH increased with increasing concentration in the range of 100 µg/ml. The change of the scavenging DPPH ability tended to be stable when the concentration was more than 50 µg/ml. Three levels were of 6, 8, 10 µg/ml were determined at ginkgo leaves flavonoid concentration. The DPPH Scavenging Rate of the Loquat Leaves Flavonoid. Fig. 1 showed, the ability of scavenging DPPH increased with increasing concentration in the 100 µg/ml range of sample concentration. Three levels were of 20, 60, 100 µg/ml were determined at loquat leaves flavonoid concentration. P/% Ginkgo leaves total flavonoid concentration/µg ml -1 P/% Loquat leaves flavonoid concentration/µg ml -1 Figure 1. Effects of different factors on the DPPH scavenging rate.

4 The Test Results of RSM Based on the results of single factors experiments, the formulation optimization of ginkgo leaves flavonoid and loquat leaves flavonoid experiments were carried out by RSM design according to Table 2. Analysis of Variance Table 2. Data of RSM. No. A B P(%) Analysis of variance was carried out by design expert. The results showed that the p values of loquat leaves flavonoid (B) was less than 0.05 and more than 0.01, which showed the factor had important influence on the scavenging DPPH. The results showed that the factors and responses were not linear relationship. Sourc of Squares Sum of Square Table 3. Analyze of mean square. Degree of freedom Mean Square F Value Prob > F Model A B AB A B Residual Lack of Fit Pure Error Cor Total Fitting the Model to Establish and Antioxidant Synergy Analysis To fit the response surface test data, to obtain the coding variable regression equation of two order polynomia. P = A-0.14B-0.70AB-1.19 A B 2

5 The p vale of the lack of fit value was of (not significant), the model was of (significant), which showed that the model fit the RSM data significantly, the equation was of the good mathematic model to fit the synergetic antioxidant effect between the ginkgo leaves flavonoid and loquat leaves flavonoid. According to the regression equation, the interaction coefficient of ginkgo leaves flavonoid and loquat leaves flavonoid was negative value, reflecting the synergistic effect between two factors was negative cooperativity. Using the antioxidants concentration of ginkgo leaves flavonoid(a) and loquat leaves flavonoid(b) as X and Y and the cavenging rate of DPPH as the Z, make the corresponding 3D figure. The interaction between ginkgo leaves flavonoid and loquat leaves flavonoid had great influence on the response, which showed relatively steep curve in the figures and it was consistent with the regression analysis results. The Optimal Process Validation The equivalent linear regression model was shown in Fig. 2. The maximum value of model was calculated by the software. The component ratio of optimal compound antioxidant was loquat leaves flavonoid µg ml -1, ginkgo leaves flavonoid 8.67 µg ml -1, the DPPH scavenging rate was 44.82%, the verified value could reach above 44.8%, it was consistent with the predicted value and verified the validity of the model. The single component test with combination formulations of ginkgo leaves flavonoid 8.67µg ml -1, loquat leaves flavonoid µg ml -1, the actual production of DPPH scavenging rate were: 42.00%%, 38.63%, the total scavenging rate of %, respectively. It was significant more than the combined formula. It showed that combined formula antioxidant activity was poorer than single antioxidant and consistent with the regression model analysis. Figure 2. The equivalent linear regression model and P=f (A, B) response surface. Conclusion In this study, synergetic effect and optimal formulation of compound antioxidant between the ginkgo leaves flavonoid and loquat leaves flavonoid were determined with the response surface method. The optimum formula of compound antioxidants were: ginkgo leaves flavonoid 8.67 µg ml -1 and loquat leaves flavonoid µg ml -1, the DPPH scavenging rate was 44.82%. Comparison the antioxidant activity between compound antioxidants and single antioxidant, concluded that mixed antioxidants significant poorer than single antioxidant. There were antagonistic action of antioxidant.

6 Acknowledgments This work was supported by National innovation and entrepreneurship training program (No ). References [1] Qin Ren-hui, Zhang meng-jun, Wu shi-rong, et al. Chemical Extraction and Medical Application of Ginkgo flavonoids: Medical Application[J]. Journal of changsha university, 2004, 60 (6): [2] He Y Z Research advances in the extractive composition, pharmacologic effect of Eriobotrya japonica (Thunb.) Lindl.[J].Journal of Guangxi University of Technology, 18 (2): [3] Guo Y, Wu S J, Piao H S Advance on the chemical composition and pharmacological activity of loquat [J].Lishizhen Medicine and Materia Medica Research, 17(6): [4] Chuan Y X F Study on the ingredients of loquat[j].foreign Medical Sciences:Chinese Medicine, 25(5):316. [5] Chen Xi-juan, Wang cheng-zhang,ye jian-zhong. Research Progress of Chemical Constituents of Ginkg obiloba Linn.Leaves and Their Application[J]. Biomass Chemical Engineering, 2008, 42 (4): [6] LI Hong-jun. Study on Extraction of Flavonoids from Ginkgo biloba Leaves and Antioxidant Action in vitro[j].northern Horticulture, 2013, (05): [7] FU Xiao-dan, TANG Chun-feng, LIU Rang-lian,et al. Antioxidant effect of flavonoids in extracts from the Eriobotrya Japonica( Thunb.) Lindl. leaves[j].science and Technology of Food Industry, 2014, 36(01): [8] Ying QL, Study on debinding and antioxidant technology eel[d].hangzhou:zhejiang Gongshang University [9] L.J. Zhang, W.H. Zhang, H.J. Zhang, Determination of the total content of flavones in Qianshan Semen Cassiacs. Food Sci, 2003, 24(12): [10] S.J. Kalil, F. Maugeri, M.I. Rodrigues. Response surface analysis and simulation as a tool for bioprocess design and optimization[j].process biochemistry, 2000,35: [11] Tan Z Q, Wu W L, Pan L Y, Research on Compound Antioxidants on low energy of cocoa butter equivalent antioxidant[j].science and Technology of Food Industry, 2012, 33(19): , 291.