球面设计优化金线莲黄酮类物质提取工艺及糖耐受性的研究

余 杰,崔仕超

汕头大学生物学系,汕头 515063

球面设计优化金线莲黄酮类物质提取工艺及糖耐受性的研究

余 杰＊,崔仕超

汕头大学生物学系,汕头 515063

以金线莲为材料,利用统计学实验设计方法对黄酮类物质的提取工艺进行了优化。选定液料比、乙醇浓度和时间三个因素的五个水平进行球面设计实验,建立黄酮提取率的二次回归方程,通过回归分析及岭脊分析得到优化提取组合条件。研究结果表明,当提取工艺条件为:液料比 24,乙醇浓度 63%,提取时间 48 h时,黄酮提取率的最大预测值为 1.107%,验证值为 1.05%。葡萄糖耐受性试验结果表明:金线莲黄酮类萃取物能够提高正常小鼠对糖的耐受性。

金线莲;黄酮类;球面设计;糖耐受

Introduction

The genus Anoectochilus(Orchidaceae),comprising more than 35 species in the word,is a perennial herb[1].It is distributed from India,the Himalayas, Southeast Asia,and Indonesia to New Caledonia and Hawaii[1].Of the species,twenty are found in china,includingAnoectochilus for m osanus,Anoectochilus inabai, Anoectochilus koshunensis,Anoectochilus roxburghiiand so on.Anoectochilus spp.are now expensive medicinal plants used to treat tumor,hyperliposis,hepcititis,nephitis and so on[2].Flavonoids are a group ofmore than 4000 polyphenolic compounds that occur naturally in foods of plant origin.These compounds possess a common phenylbenzopyrone structure(C6-C3-C6),and they are categorized according to the saturation level and opening of the central pyran ring,mainly into flavones,flavanols,isoflavones,flavonols,flavanones and flavanonols[3].The flavonoids have long been recognized to possess anti-inflammatory,antioxidant[4,5],antihemslytic[6],hepatoprotective[7],antiviral[7]and anticarcinogenic activities[8].Recently,some reports show flavonoids can regulate glycemic control in the body.In the present work,we attempted to investigate the extraction of flavonoids fromAnoectochilus roxburghii.In parallel,the glucose tolerance was tested.

Materials andM ethods

Materials

Anoectochilus roxburghiiwhich was producted from Yunnan Province,was obtained from Chinese medicinal herbs shop of Xiamen.Female Balb/c mice were obtained from theMedical College of Shantou University. The mice were maintained at room temperature under alternating natural light/dark photoperiod,and had access to standard laboratory food and fresh water.All other chemicals and solvents used were of analytical grade.

Total flavonoid content

The total flavonoid content was determined using a modified version of the method described by Hyung-Kyoon Choi(2008)[9].Each test sample(100μL) was added to a volumetric flask(15 mL volume).Six minutes after adding 0.4 mL of 5%NaNO2,0.4 mL of 10%AlCl3was added.After 6 min,4 mL of 1 mol/L NaOH was added and the volume was made up to 10 mL with distilledwater.After 10 min,the absorbance of the solution wasmeasured against a blank at 510 nm using a spectrophotometer.

Exper imental design

In order to obtain the optimum condition for the production of flavonoids,three variables,such as ethanol concentration,ratio of ethanol to raw material,and extraction time,were chosen.Thus,a spherical symmetric design-response surface methodology was used to systemically investigate the effects of these three critical variables on the production of flavonoid.The details of the design are outlined in Table 1.For each factor,the experimental range was selected,based on the resultsof preliminary exper iments and taking into consideration of the feasibility of yield of flavonoid at the extreme values.All of the formulations in these experiments were prepared in three duplicates.

Glucose tolerance test(GTT)[10]

Ten healthy mice were divided equally into 2 groups. The first group was intraperitoneally injected with 200 mg/kg body weight of the flavonoid-riched extract.The second group was intraperitoneally injected with saline. On the 10th day,after the last administration,the first and second groups were intraperitoneally injected with 2 g/kg body weight of the glucose,namely,flavonoidriched extract group,saline– glucose group.Blood sampleswere obtained from the tail veinsof the mice at 0,30,60,and 90 min,respectively.Blood glucose valueswere deter mined with Glucometer.

Results and D iscussion

Opt im ization of formulation

The spherical symmetric design-response surface methodology constitutes an alternative approach because it offers the possibility of investigating a high number of variables at different levels,perfor ming only a l imited number of experiments[11].The variables in Table 1 were chosen taking into account those in our preliminary exper iments.Table 2 shows the experimental results concerning the tested variables on the yield of flavonoid.A mathematical relationship between factors and parameterswere generated by response surface regression analysis in statistical software SAS.The following second order polynomial equation is found to explain the yield of flavonoids:

The three-d imensional response surface plots for the most statistical significant variableson the evaluated regressers are shown in Fig.1.The response surface diagrams show that the highest Y1didn’t exist,so canonical analysis was perfor med.Table 3 shows minus and positive representative values of the spherical symmetric exper iment existed.For getting the highest values, the ridge analysis was needed to perform.Table 4 was the result of the ridge analysis.In table 4,when the coded radius was 1,the highest response values was obtained at the levels of ethanol concentration of 63. 02%,ratio of ethanol to raw material of 24.25:1 and extraction time of 47.9 h.To test validity of the spherical symmetric design-response surface methodology,the flavonoids was extracted under the optimal condition and the extraction yield was 1.05%.The value was close to the theoretical predicted values of 1.11%,indicating that the experimental design matrix may better reflect the extraction parameters of the flavonoids.

Glucose tolerance test

The ascension of blood sugar induced by glucose was inhibited 30 min later in flavonoid-riched extract–glucose group and saline– glucose group(Fig.2). Fig.2 showed the blood sugar of flavonoid-riched extract– glucose group were lower than saline– glucose group after 30 min,and after 90 min,the level of blood suger of flavonoid-riched extract– glucose group was very close to the normal mice.However,the level of blood sugar of the mice in the saline– glucose group did not decrease to nor mal mice 90 min later.From Fig.2,we can see the sugar tolerance of normalmice was improved after intraperitoneal injection of flavonoid-riched extract.

Table 2 Response values of different variables for the optim ization of production of flavonoid using the spherical symmetric design-response surface methodology

Table 3 Representative values of the spherical symmetric exper iment

Fig.1 Three-d imensional resonse surface plots showing the variation in the production of flavonoids

Table 4 Est imated ridge of max imum response

Fig.2 Effects of flavonoid-riched extract on sugar tolerance of normolm ice■saline– glucose group;◆flavonoid-riched extract– glucose group

Conclusion

This present study indicates that the flavonoids fromAnoectochilus roxburghiiare able to be produced by employing boiling water extraction and improved the sugar tolerance of normal mice was improved after intraperitoneal injection of flavonoid-riched extract.The optimal conditions obtained by the spherical symmetric design-response surface methodology for production of the flavonoids fromAnoectochilus roxburghiiinclude the following parameters: ethanol concentration of 63.02%,ratio of ethanol to raw material of 24.25:1 and extraction time of 47.9 h.Under these conditions, the experimental yield was 1.05%,which is well matched with the predictive yield.

1 Hayata CC,Tsenga HF,Shanga,et al.Antitumor and immunostimulating effects ofAnoectochilus for mosanus.Phytom edicine,2006,13:366-370.

2 LiangWL,Chen RC,et al.Study on the physiological activities of Jin-Sian-Lian.For mosan Sci,1990,43:47-58.

3 Ren WY,et al.Flavonoids:Promising anticancer agents medicinal research reviews.M ed Res Rev,2003,23:519-534.

4 Choi HK,Lima YS,Kim YS,et al.Free-radical-scavenging and tyrosinase-inhibition activities of Cheonggukjang samples fermented for various times.Food Chem,2008,106:564-568.

5 Abrahama LCN,Masakuni T,Isao H,et al.Antioxidant flavonoid glycosides from the leaves ofFicus pum ilaL..Food Chem,2008,109:415-420.

6 Naim M,et al.Antioxidative and antihemslytic activities of soybean isoflavones.Food Chem,1976,24:1174-1176.

7 Sharma B,Vis wanath G,Salunke R,et al.Effects of flavonoid-rich extract from seeds ofEugenia jam bolana(L.)on carbohydrate and lipid metabolism in diabetic mice.Food Chem,2008,110:697-705.

8 Chang HL,Su JH,Yeh YT,et al.Protoapigenone,a novel flavonoid,inhibits ovarian cancer cell growthin vitroandin vivo.CancerLett,2008,267:85-95.

9 Choi HK,Lima YS,Kim YS,et al.Free-radical-scavenging and tyrosinase-inhibition activities of Cheonggukjang samples fermented for various times.Food Chem,2008,106:564-568.

10 Hana CC,Yuanb JH,Wangc YZ,et al.Hypoglycemic activity of fermented mushroom ofCoprinus com atusrich in vanadium.J Trace Elements in M ed B iology,2006,20:191-196.

11 Ge YB,et al.Opt imized preparation of daidzein-loaded chitosan microspheres andin vivoevaluation after intramuscular injection in rats.International J Phar m,2007,338:142-151.

Extraction and Glucose Tolerance of the Flavonoids-riched Extract fromAnoectochilus roxburghii

YU Jie＊,CU I Shi-chao
Depar tm ent of B iology of Shantou University,Shantou 515063,China

The extraction of flavonoids fromAnoectochilus roxburghiiwas studied in thispaper.The effectof the extraction parameters of extraction time,concentration and ratio of ethanol to raw materialwere opt imized by using statistical techniques such as a three factor,five level spherical symmetric design-response surface methodology,with a view to maximize the extraction yield underopt imal extraction parameters combination.From this study,it could be concluded that the max imum yield of the flavonoids(1.05%)can be obtained ethanol concentration of 63.02%,extraction time of 47.9 h,ratio ofwater to material of 24.25:1.Glucose tolerance test show that flavonoid-riched fromAnoectochilus roxburghilextract can improve the sugar tolerance of nor malmice.

Anoectochilus roxburghii;flavonoids;spherical symmetric design;glucose tolerance

January 4,2009;Accepted June 25,2009

R151.3;Q946.91

A

1001-6880(2010)03-0471-04

＊Corresponding author Tel:86-532-88963253;E-mail:jyu@stu.edu.cn