Optimization of Microwave Extraction of Flavonoids from Water Chestnut Skin Dregs with Response Surface Method

Lihua HUANG

Department of Food Science,Guangzhou City Polytechnic,Guangzhou 510405,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

Water chestnut is a kind of perennial sedge in shallow water.The underground bulbs of water chestnut are not only juicy,sweet and nutritious but also have certain medical value.They are excellent edible and medical fruit and vegetable[1-4].Water chestnut has been widely planted in Guangdong Province,and it is one of special industries with Guangdong’s characteristics.Water chestnut not only can be eaten directly but also can be processed into starch,dried fruit,beverage and other products.Among the products,water chestnut starch is the representative.However,the water chestnut skin dregs,a kind of by-product of water chestnut starch,are usually discarded,and they are underutilized.Although in recent years,the researches on the utilization of water chestnut skin dregs have attracted more and more attention,most of the researches focus on the component analysis of water chestnut skin dregs.There have been rare researches on the industrial utilization of water chestnut skin dregs[5-6].

Water chestnut skin dreg is rich in flavonoids.Flavonoids have a lot of physiological functions,such as biological antioxidant,anti-aging,treating cardiovascular and cerebrovascular diseases,antimicrobial,scavenging free radicals,reducing blood fat and lowering blood pressure[7-9].The extraction of flavonoids can be an important utilization way of water chestnut skin dregs[10-11].In this study,the water chestnut skin dregs were used as the material.The microwave extraction of flavonoids from water chestnut skin dregs was optimized using response surface methodology.Each technical parameter was optimized,aiming at improving the flavonoids yield and providing reference and guidance for the highly efficient development and utilization of water chestnut skin dregs resources.

Material and Methods

Material

The tested water chestnut skin dregs,by-product of water chestnut starch,were provided by the Guangzhou Pan Tang Food Co.The skin dregs were dried at 50 ℃for 24 h.Then they were crushed and passed through an 80 mesh sieve.The main reagents included rutin,sodium nitrite,ethanol,sodium hydroxide and aluminum nitrate.The rutin was biochemical reagent,and the other reagents were all of analytical grade.The main instruments included centrifuge (LG 10-2.4A),microwave oven (WD 800),rotary evaporator(RE-52A),UV-visible spectrophotometer (U-3010 Spectrophotometer) and electric grinder(DWF-100).

Methods

Determination of flavonoids content in water chestnut skin dregsThe rutin was treated as the control.The flavonoids yield in water chestnut skin dregs was determined with the complex-spectrophotometry[12].

Preparation of rutin standard solutionA certain amount (10.0 mg) of rutin standard that had been dried at 120 ℃ to constant weight was weighed accurately.The weighed rutin standard was diluted in 30% (v/v)ethanol to 100 ml.Thus the rutin standard solution was prepared.

Determination of detection wavelengthEach of 5 ml of rutin standard solution and water chestnut skin dregs extract were transferred to volumetric flasks (10 ml).Subsequently,the NaNO2-Al(NO3)3-NaOH complexing agent was added to the volumetric flasks.The volumetric flasks were then stood for 15 min.The solutions were scanned with UV-visible spectrophotometer with wavelength ranging from 400 to 700 nm,respectively.Finally,the wavelength,under which the rutin standard solution and the water chestnut skin dregs extract all had strong absorption,was selected for detection[12].

Drawing of standard curveCertain amounts (0,1.0,2.0,3.0,4.0 and 5.0 ml) of rutin standard solution were diluted with 30% ethanol to 5 ml in volumetric flasks (10 ml).Then,0.3 ml of 5% sodium nitrite was added to each of the volumetric flasks.After mixed,the solutions were stood for 6 min.And then,0.3 ml of 10% of aluminum nitrate was added to each of the volumetric flasks.After mixed,the solutions were stood for 6 min.And then,4.0 ml of 4% sodium hydroxide was added to each of the volumetric flasks.Finally,30% ethanol was added to each of the volumetric flasks to 10 ml.The absorbances of the gradient standard solutions were determined at 510 nm.The standard curve was constructed with absorbance (A) as the horizontal axis and rutin concentration(C)as the vertical axis.The regression equation was as follows:

C=0.0831A-0.00007,r=0.9981.

When the rutin concentration ranged from 0 to 0.05 mg/ml,the absorbance and rutin concentration showed a good linear relationship[12].

Determination of flavonoids content in water chestnut skin dregsThere were 2 volumetric flasks(10 ml).One contained 1 ml of water chestnut skin dregs extract and NaNO2-Al(NO3)3-NaOH complexing agent,and the other only contained 1 ml of water chestnut skin dregs extract.Then,30%ethanol was added to each of the 2 volumetric flasks to 10 ml.The 2 volumetric flasks were stood for 15 min.The solution in the non complexing agent-added flask was treated as the reference solution,and then the absorbance of the solution in the other flask was determined at 510 nm.Thus the flavonoids yield was calculated in according to the standard curve equation[9,12].

Extraction of flavonoids from water chestnut skin dregsA certain amount (2.0 g)of water chestnut skin dregs was weighed accurately and then placed in a conical flask(150 ml).The flavonoids were extracted[9].The single-factor tests on the effects of ethanol concentration,microwave time,solid/liquid ratio and microwave power on the flavonoids yield were carried out respectively.Based on the results of the single-factor tests,the microwave extraction of flavonoids from water chestnut skin dregs was optimized with response surface method.The flavonoids yield from water chestnut skin dregs was determined referring to the method above.

Results and Analysis

Effects of ethanol concentration on flavonoids yield

Certain amounts of water chestnut skin dregs were placed in 150 ml conical flasks (2.0 g/flask).The solid/liquid ratio,microwave time and microwave power were fixed at 1:20 g/ml,2.5 min and 320 W,respectively.Then different concentrations (10%,30%,45%,60%,75% and 90%) of ethanol were added to the flasks.The absorbance of each extract was determined.Then the effects of differentconcentration ethanol on flavonoids yield were compared.

As shown in Fig.1,the flavonoids yield was increased with the increase of ethanol concentration in the early period (P ＜0.05).In the microwave field,the damaging effects of ethanol on the basal cell membrane were stronger than those of water,resulting in more flavonoids released.However,when the ethanol concentration was higher than 60%,the flavonoids yield was decreased with the increase of ethanol concentration.Under conditions of high-concentration ethanol,the dissolved amounts of alcohol-soluble impurities and pigments,which competed with the flavonoids for alcohol and water molecules,were increased,so the flavonoids yield was decreased.In addition,the microwave oven is a closed system,but the boiling point of high-purity ethanol is lower,leading to a large amount of ethanol gas in the microwave oven.This is undoubtedly risky.Therefore,the ethanol concentration should be better not higher than 60%.

Effects of solid/liquid ratio on flavonoids yield

Certain amounts of water chestnut skin dregs were placed in 150 ml conical flasks (2.0 g/flask).The microwave time and microwave power were fixed at 2.5 min and 320 W,respectively.Then 60% ethanol was added to the flasks in according to solid/liquid ratios of 1:10,1:15,1:20,1:25,1:30,1:35 and 1:40 g/ml,respectively.The absorbance of each extract was determined.Then the effects of different solid/liquid ratio on flavonoids yield were compared.

Fig.2 showed the flavonoids yield was increased with the increase of solid/liquid ratio.When the solid/liquid ratio was higher than 1:25 g/ml,the flavonoids yield was stable.The further increased solid/liquid ratio almost had no effects on the flavonoids yield.The increased proportion of solvent increased the concentration difference of flavonoids between solvent and material,promoting the dissolution of flavonoids from material and reducing the residual amount of flavonoids in material,thereby increasing the flavonoids yield.With the increased proportion of solvent,the flavonoids yield was increased.However,when the solid/liquid ratio was increased to 1:25 g/ml,the flavonoids in water chestnut skin dregs was almost completely extracted,so the flavonoids yield started to be maintained at a stable level.Then the effects of solid/liquid ratio on flavonoids yield were slight.In the premise of a higher flavonoids yield,in order to reduce costs,the solid/liquid ratios of 1:20,1:25 and 1:30 g/ml were selected as the coding levels of solid/liquid ratio for the response surface optimization.

Effects of microwave time on flavonoids yield

Certain amounts of water chestnut skin dregs were placed in 150 ml conical flasks(2.0 g/flask).The ethanol concentration,solid/liquid ratio and microwave power were fixed at 60%,1:25 g/ml and 320 W,respectively.Then the microwave extraction was conducted for 0.5,1.0,1.5,2.0,2.5,3.0,3.5 and 4.0 min,respectively.The absorbance of each extract was determined.Then the effects of different microwave time on flavonoids yield were compared.

As shown in Fig.3,the flavonoids yield was increased first and then decreased with the increase of microwave time,and it reached the peak at microwave time of 3 min.When the microwave time was increased,the thermal motion of flavonoids in water chestnut skin dregs and solvent was enhanced,producing enormous heat in local place.The enormous heat would increase sharply but non-uniformly the temperature,which was even higher than the boiling point in a short time.Thus the internal volume would be expanded rapidly,damaging the basal cell membranes,and leading to the release of flavonoids.However,if the microwave time was extremely long,the temperature inside the extraction environment would be too high,leading to the decomposition and damaging of heat-intolerable components in flavonoids and the evaporation loss of solvent,thereby reducing the flavonoids yield.

Effects of microwave power on flavonoids yield

Certain amounts of water chestnut skin dregs were placed in 150 ml conical flasks(2.0 g/flask).The ethanol concentration,solid/liquid ratio and microwave time were fixed at 60%,1:25 g/ml and 3 min,respectively.Then the microwave extraction was conducted at microwave power of 160,320,480,640 and 800 W,respectively.The absorbance of each extract was determined.Then the effects of different microwave power on flavonoids yield were compared.

Fig.4 showed the flavonoids yield was increased and then decreased with the increase of microwave power,and it reached the peak at microwave power of 320 W.When the microwave power was increased,the diffusion speeds of material and solvent were increased.Moreover,the destroying effect of increased microwave power was enhanced,and this would be more conducive to the extraction of flavonoids.However,if the microwave power was too high,the extraction temperature would fluctuate greatly.The instant high temperature would lead to the complete or partial decomposition of heat-intolerable components in flavonoids,thereby lowering flavonoids yield.Therefore,the microwave powers of 160,320 and 480 W were selected as the coding levels of microwave power for the response surface optimization.

Optimization of microwave extraction using response surface methodology

The test was designed based on the response surface methodology[14-15].The ethanol concentration,solid/liquid ratio and microwave power were treated as the variables,and the flavonoids yield was treated as the response.The 3 factors × 3 levels response surface analysis method was adopted (Table1).The analysis results were shown in Table2.There were a total of 15 tests,including 3 central tests and 12 factorial tests.The results in Table2 were calculated with Design Expert 7.1.3 and analyzed with RSREG.After fitting with quadratic regression,the response function was obtained.The regression equation was as follow:

The results of variance analysis of response surface test were as follows:Fmodel= 60.87,Pmodel= 0.000 1; FA=88.81,PA= 0.000 2; FB= 355.25,PB＜0.000 1;FC=15.92,PC=0.010 4;FAB=7.78,PAB= 0.038 5; FAC= 1.21,PAC=0.322 3; FBC= 4.821E -004,PBC=0.983 3;FA2=4.48,PA2=0.087 9;FB2=26.35,PB2=0.003 7;FC2=56.50,PC2=0.000 7;Flackoffit=14.15,Plackoffit=0.066 7.Based on the analysis results above,it could be concluded the simulation regression of flavonoids yield was significant (P = 0.000 1); the effects of A,B,C,AB,B2and C2on flavonoids yield were significant,but the effects of A2,AC and BC on flavonoids yield were not significant; the lack of fir item was not significant; the Adj R2(0.974 7)was close to Pred R2(0.860 9).The variance analysis results showed the fitting effect between actual test and regression equation was relatively good; there were good linear relationships between flavonoids yield and ethanol concentration,solid/liquid ratio and microwave power; among the ethanol concentration,solid/liquid ratio and microwave power,the effects ranked as solid/liquid ratio ＞ethanol concentration ＞microwave power.

Fig.5 - Fig.7 were drawn based on the regression analysis results.The comparison of the 3 maps showed the effects of solid/liquid ratio on flavonoids yield were greatest and the graphic surface was steepest; the effects of ethanol concentration on flavonoids yield were relatively small;the effects of microwave power on flavonoids yield were not significant.The variance analysis results were consistent with the analysis results of the response surface maps.The theoretically optimum extraction conditions were obtained using Design Expert 7.1.3(Table3).

Under conditions of the optimum extraction conditions,the flavonoids yield was predicted to be 2.380%.However,due to the restriction of test conditions,the flavonoids yield was predicted to be 2.379% at ethanol concentration of 58.8%,solid/liquid ratio of 1:30 g/ml and microwave power of 320 W.In order to save cost,the ethanol concentration,solid/liquid ratio and microwave power were selected at 55.4%,1:30 g/ml and 320 W,and thus the flavonoid yield was 2.376%.The ethanol concentration of 55.4%,the solid/liquid ratio of 1:30 g/ml and the microwave power of 320 W were also the optimum combination under test conditions.Under the optimum test conditions,the flavonoids yield was determined 3 times repeatedly with flavonoids yield of 2.365%,and the predicted flavonoids yield with response surface methodology was 2.376%.The relative error between the actual values and the theoreticallypredicted values was 0.461%.It is indicated the detection results of flavonoids yield with response surface methodology-optimized method are reliable.The response surface methodology-optimized microwave extraction method has certain practical value.

Table1 Experimental factors and coding levels for the optimization of microwave extraction of flavonoids from water chestnut skin dregs with response surface method

Table2 Box-benhnken test results

Table3 Optimum conditions predicted by response surface method

Conclusion

The flavonoids yield was treated as the evaluating index.Based on the test results of single-factor tests,the microwave extraction of flavonoids from water chestnut skin dregs was optimized with response surface methodology.A 3 factors × 3 levels Box-behnken-based response surface test was conducted.The Design Expert 7.1.3 was used for calculation and programming.The obtained optimum extraction conditions were as follows:ethanol concentration of 55.4%,solid/liquid ratio of 1:30 g/ml,microwave power of 320 W,microwave time of 3 min and 2 duplications.Under the optimum extraction conditions,the flavonoids yield could reach 2.365%.Different from the traditional extraction method,the microwave extraction method utilizes the microwave-absorption difference of different structural substances in the microwave field and heats selectively certain areas in substances or certain components in extraction system,separating the extracted substances from the substrate or system.Microwave extraction is a new modern extraction technology.It is characterized by protecting active ingredients,saving time,high extraction efficiency,safety and saving energy.Microwave extraction method has good application prospect[9].

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