Study on Response Surface Methodology Optimization for Extraction Process of Cyclocarya paliurus Extract and Its Blood Sugar-lowering Effect

Yufeng LI Xiayun LIAO Gang WANG Lichun ZHAO

Abstract [Objectives] This study was conducted to discuss the mechanism of the blood sugar-lowering effect of Cyclocarya paliurus extract. [Methods] With C. paliurus leaves as the raw material， the ethyl acetate-butyl acetate microwave extraction process of C. paliurus was studied. With the yield of C. paliurus extract as an evaluation index， single-factor experiments were carried out on such 6 factors as the dry C. paliurus leaf powder size， solid-to-liquid ratio， extraction time， microwave frequency， microwave power， and microwave extraction time， and the extraction and purification process was further optimized by response surface analysis. The normal mice and the hyperglycemic mice modeled by alloxan were injected with C. paliurus extract to study the blood sugar-lowering effects of different groups of C. paliurus extract. [Results] The optimal process combination for the extraction of C. paliurus leaves was obtained： dry C. paliurus powder size 90-100 mesh， solid-to-liquid ratio 1∶20 （g/ml）， extraction time 192 min， microwave frequency 2 500 MHz， microwave power 490 W， and extraction time 248 s. Under these conditions， the yield of C. paliurus extract reached the maximum， 95.10%， reaching 99.83% of the predicted value. The blood sugar-lowering test on mice showed that the C. paliurus extract had a good effect on lowering blood sugar， indicating that C. paliurus extract can improve the free radical scavenging capacity to a certain extent. [Conclusions] This study provides certain reference for the in-depth study of the physiological effects of C. paliurus extract and the comprehensive development and utilization of C. paliurus extract.

Key words Cyclocarya paliurus; C. paliurus extract; Yield; Blood sugar lowering; Radical scavenging

Cyclocarya paliurus， also known as Qingqianli and Yaoqianshu， is a plant of Cyclocarya in Juglandaceae of Dicotyledoneae. C. paliurus is a tall tree， widely distributed in Jiangxi， Zhejiang， Jiangsu， Fujian， Taiwan， Guangdong， Guangxi and other areas south of the Yangtze River[1]. It is a single species of plant unique to China and one of the endangered plants under national key protection. It belongs to the three types of protected plants. For a long time， the folks use its tender leaves to make tea for consumption. The tea has the effects of promoting the secretion of saliva or body fluid and slaking thirst， eliminating summer-heat by cooling， reducing blood pressure and strengthening the heart， and prolonging life. It can be effectively used to prevent and treat diabetes， hypertension， coronary heart disease， neurasthenia and other diseases[2-3]. Since the 1980s， a large number of health care products and functional foods with green C. paliurus leaves as the main raw material have been on the market. Preliminary animal and clinical experiments conducted by scientific research institutions and hospitals in some domestic colleges and universities have shown that these products do have a variety of physiological and pharmacological functions beneficial to the human body[4]. Studies have suggested that the steroids， terpenoids and lactones contained in C. paliurus may have the effect of inhibiting the increase in blood sugar[5]. Meanwhile， the trace element zinc plays an important role in lowering blood sugar. There are also studies that believe that selenium has an insulin-like effect[6]. Other elements such as chromium， nickel， vanadium， magnesium and some rare earth elements are also significantly related to lowering blood sugar. Yi et al.[7] investigated the blood sugar-lowering effect of Jiangxi Meishan Hypoglycemic Tea on rats with alloxan diabetes， and the results showed that after giving the Meishan Hypoglycemic Tea （3.6 g） to rats with alloxan diabetes for 4 consecutive weeks， the fasting blood glucose of rats was significantly reduced， and glucose tolerance was increased， but the Hypoglycemic Tea had no significant effect on the blood sugar of normal rats and mice.

In this study， a new extraction and purification process of C. paliurus leaves was established and optimized by single factor experiments and the response surface method， and the blood sugar-lowering effect of the C. paliurus extract was tested on mice. In the test， mice in different groups were injected with the C. paliurus extract to study the blood sugar-lowering effect of the C. paliurus extract， and the blood sugar-lowering mechanism of the C. paliurus extract was preliminarily studied by measuring the capacity of mouse liver to scavenge free radicals before and after the injection of C. paliurus extract. This study provides certain reference for the in-depth study on the physiological effects of C. paliurus extract and the comprehensive development and utilization of C. paliurus extract.

Materials and Methods

Materials and Reagents

C. paliurus leaves （Guangxi Alili Health Science and Technology Co.， Ltd.）; mice （Animal Experiment Base of Guangxi Medical University）; alloxan （Sigma A7413）; glucose kit （Changchun Huili Biotech Co.， Ltd.）; 1，1-dipheny-2-picryhydrazyl free radical （DPPH） （Sigma）; Meibida Glipizide Tablets （Hainan Zambon Pharmaceutical Co.， Ltd）; other reagents， all domestically produced， analytically pure.

Instruments and equipment

W-501S rotary evaporator （Guangzhou Xingshuo Instrument Co.， Ltd.）; electronic balance （Mettler-Toledo Instrument （Shanghai） Co.， Ltd.）; pulverizer （Shanghai Hannuo Instruments Co.， Ltd.）; electric heating jacket （Tianjin Taisite Instrument Co.， Ltd.）; NN-K587WS type microwave oven （Panasonic Co.， Ltd.）.

Experimental methods

Extraction process of C. paliurus leaves

The extraction process of C. paliurus extract included the steps of ① sun-drying， crushing， sieving， ② reflux extraction， ③ filtration， concentration， ④ microwave extraction of ethyl acetate-butyl acetate mixture， ⑤ mixing of extracts， and ⑥ vacuum rotary steaming.

Optimization of extraction conditions

The dry C. paliurus powder size， solid-to-liquid ratio， extraction time， microwave frequency， microwave power， and microwave extraction time were selected as the six factors investigated. The conditions of each factor were as follows： dry C. paliurus powder size： below 60， 60-80， 80-100， 100-120， and above 120 mesh; solid-to-liquid ratio： 1∶10， 1∶15， 1∶ 20， 1∶25， 1∶30; extraction time： 60， 120， 180， 240， 300 min; microwave frequency： 2 000， 2 250， 2 500， 2 750， 3 000 MHz; microwave power： 400， 450， 500， 550， 600 W; microwave extraction time： 180， 210， 240， 270， 300 s. The experiment was repeated 3 times for each factor， and the results were averaged.

Single factor experiments were carried out using a small amount of dried C. paliurus leaves as raw material with the extraction yield of C. paliurus as target while fixing other factors （dry C. paliurus powder size， solid-to-liquid ratio， extraction time， microwave frequency， microwave power， microwave extraction time）. Then， according to the results of single factor extraction， the dry C. paliurus powder size， extraction time， microwave power， and microwave extraction time were selected for response surface test design. With the yield of C. paliurus extract as the response value， the optimal conditions of C. paliurus extraction process were determined. The experiments were carried out according to Table 1.

Yield of C. paliurus extract （%）=C. paliurus extract/C. paliurus leaves×100

Test on hypoglycemic activity of C. paliurus extract in mice[8]

Treatments of normal mice

The mice were divided into 4 groups： control group 1， control group 2， group A1 and group B1， each including 10 mice. A1 was given an intraperitoneal injection of C. paliurus leaf extract solution at a dose of 100 mg/kg. After 6 and 24 h， blood was collected from the tail vein of A1 mice to measure blood glucose. Group B1 was given an intraperitoneal injection of the same amount of distilled water. After 6 and 24 h， blood was collected from the tail vein of group B1 to measure blood glucose.

Establishment and treatment of hyperglycemic mouse model

All 30 mice were given a tail vein injection of 10 mg/ml alloxan at a dose of 50 mg/kg. After 7 d， blood was collected from the tail vein to measure blood sugar. Mice with blood sugar levels above 10 mmol/L were successfully modeled hyperglycemic mice. The hyperglycemic mice were divided into 6 groups， including the hyperglycemia groups A2 and B2， 10 mice in each group. The hyperglycemic mice in group A2 were given an intraperitoneal injection of C. paliurus leaf extract solution at a dose of 100 mg/kg. Hyperglycemic mice in group B2 were given an intraperitoneal injection of Meibida Glipizide Tablets solution at a dose of 100 mg/kg. Blood sugar was measured 6 h later.

Blood sugar determination by the GOD-POD method

Pipette tips and 0.5 ml Effendorf tubes were rinsed with saturated potassium oxalate solution （anticoagulant）， and used to collect blood. The blood was centrifuged at 5 000 r/min for 20 min， obtaining the supernatant which was determined for blood sugar with a glucose kit.

Determination of the capacity of mouse liver to scavenge free radicals by the DPPH method

Preparation of liver tissue homogenate

Five mice were randomly selected from the normal group and the hyperglycemia group before and after the injection of C. paliurus leaf extract， respectively. They were sacrificed by cervical dislocation， and dissected. The liver was placed in cold normal saline， and after removing the surface water， it was weighed and added with phosphate buffer （pH 7.2） at a mass-to-volume ratio of 1∶10， and ground to a slurry in an ice bath. The mixture was then centrifuged at 5 000 r/min for 10 min， obtaining the supernatant which was determined for the free radical scavenging rate.

Determination method of free radical scavenging rate

Two test tubes were taken， and added with 2 ml of 0.2 mmol/L DPPH solution， respectively. The standard tube was added with 1 ml of phosphate buffer （pH 7.2）， and 1 ml of tissue fluid was added into the sample tube. After standing for 30 min at room temperature， centrifugation was performed at 3 000 r/min for 5 min. After zero setting， the OD values were determined at 520 nm， and recorded as Astandard and Asample， and the free radical scavenging rate was calculated according to following calculation formula： Free radical scavenging rate （%） = （1-Asample/Astandard）×100.

Data analysis

OrignPro 8.0 and Design Expert 8.06 software were used for data analysis and processing. There parallel experiments were carried out each time， and the significance test was performed by one-way analysis of variance. The measurement data was expressed as x±s， and multiple comparisons was performed by the LSD method in the one-way analysis of variance for the significance of differences between groups， while P≤0.05 meant that the difference was significant.

Results and Analysis

Single-factor experiments

Effect of dry C. paliurus powder size on extraction yield

The results are shown in Fig. 1. As the dry C. paliurus powder size decreased， the yield of C. paliurus extract first increased and then decreased， and the maximum appeared in the range of 100-120 mesh. Therefore， the dry C. paliurus powder size was selected to be 100-120 mesh.

Effect of solid-to-liquid ratio on extract yield

The results in Fig. 2 showed that the larger the solid-to-liquid ratio， the higher the yield， but when the solid-to-liquid ratio reached 1∶20， the yield increase would decrease. Therefore， the solid-to-liquid ratio 1∶30 was appropriate.

Effect of extraction time on extract yield

The results in Fig. 3 showed that the longer the extraction time， the higher the yield， but after the extraction time reached 180 min， the yield increase would decrease. Therefore， the extraction time should be selected as 180 min.

Effect of microwave frequency on extract yield

The results are shown in Fig. 4. As the microwave frequency increased， the yield of C. paliurus extract showed a trend of first increasing and then decreasing， reaching a maximum at around 2 500 MHz. Therefore， the microwave frequency should be selected at 2 500 MHz.

Yufeng LI et al. Study on Response Surface Methodology Optimization for Extraction Process of Cyclocarya paliurus Extract and Its Blood Sugar-lowering Effect

Effect of microwave power on extract yield

The results in Fig. 5 showed that with the increase of microwave power， the yield of C. paliurus extract showed a trend of first increasing and then decreasing， reaching a maximum at 500 W. Therefore， the microwave power was preferably selected as 500 W.

Effect of microwave extraction time on extract yield

The results are shown in Fig. 6. The longer the microwave extraction time， the higher the yield， but after the extraction time reached 240 s， the yield increase would decrease. Therefore， the extraction time was chosen as 240 s.

Response surface optimization for the optimal extraction and purification conditions of C. paliurus leaves

Box-Behnken design and experimental results

According to the single factor experiment results， the dry C. paliurus powder size， extraction time， microwave power， and microwave extraction time were selected as the four factors of the Box-Behnken design. The Box-Behnken design and the yield results of C. paliurus extract are shown in Table 2.

The resulting response value regression model function expression is as follows：

Y=94.4-2.20X1+1.71X2-1.38X3+1.92X4+0.33X1X2+0.77X1X3+0.15X1X4+0.65X2X3-0.35X2X4+0.28X3X4-3.80X21-3.47X22-4.13X23-3.49X24

According to the analysis of variance in Table 3， the model was extremely significant; the lack of fit of the model was not significant; the coefficient of determination was R2=0.982 5; and the adjusted coefficient of determination was R2Adj=0.965 1. It indicated that the equation had good fit and high reliability. According to the analysis of the results， the primary terms X1， X2， X3， X4 were extremely significant， and the secondary terms X21， X22， X23， and X24 were also extremely significant. It showed that the model fitted well， and it was feasible to use this model to optimize the extraction process of C. paliurus leaves. The response surfaces and contour lines of the interaction between various factors of the extraction conditions are shown in Fig. 7.

It can be seen from Fig. 7 that the interaction diagrams of dry C. paliurus powder size with extraction time， dry C. paliurus powder size with microwave power， dry C. paliurus powder size with microwave extraction time， extraction time with microwave power， microwave extraction time with extraction time， and microwave power with microwave extraction time are close to a circle， indicating that the interaction of the six was not significant.

Determination and verification of the extraction conditions of C. paliurus

The variance analysis of the model showed that the regression model had a maximum value. The optimal conditions for the extraction process were as follows： dry C. paliurus powder size 94-106 mesh， extraction time 192 min， microwave power 491.5 W， and extraction time 247.5 s. Under these conditions， the yield of C. paliurus leaf extract was 95.26%. In order to facilitate the operation， the verification test was carried out with the dry C. paliurus powder size of 90-100 mesh， extraction time of 192 min， microwave power at 490 W and extraction time of 248 s， and the extraction yield was 95.10%， reaching 99.83% of the predicted value.

Study on hypoglycemic activity of C. paliurus leaf extract

Blood sugar-lowering effect of C. paliurus extract in normal mice at 6 and 24 h

Table 1 shows the blood sugar-lowering effect of C. paliurus leaf extract in normal mice at 6 and 24 h. It can be seen from Table 1 that in group A1， the extract of C. paliurus leaves had a significant effect of lowering blood sugar. The blood sugar-lowering effect of C. paliurus leaf extract at 6 h after treatment was extremely significant compared with that of the control group， and the average blood sugar reduction rate reached 7%. The average blood sugar reduction rate of C. paliurus leaf extract was 2% at 24 h after treatment. Therefore， in the subsequent experiments， the 6 h treatment with C. paliurus leaf extract was selected as the detection time standard.

Blood sugar-lowering effect of C. paliurus leaf extract on hyperglycemic mice

The experimental results are shown in Table 5. The blood sugar level of the hyperglycemic mice after modeling was significantly higher than that of the normal mice. After the hyperglycemic mice were injected with C. paliurus leaf extract， their blood sugar levels fell within the range of normal blood sugar levels， and the blood sugar-lowering effect was better than that of western medicine Meibida Glipizide Tablets. Compared with the mice in the hyperglycemia group and the control group， the blood sugar-lowering effect of C. paliurus leaf extract reached a very significant level of 1%， suggesting that C. paliurus leaf extract has a significant blood sugar-lowering effect on mice with hyperglycemia.

Effect of C. paliurus leaf extract on the capacity of liver to scavenging free radical

Experiment on the capacity of liver to scavenging free radical in mice given C. paliurus leaf extract

As shown in Table 6， the liver's free radical scavenging capacity of hyperglycemic mice was significantly lower than that of normal mice. After the normal mice were injected with the C. paliurus leaf extract， the capacity of liver to scavenge free radicals increased slightly， and the scav enging rate increased by approximately 5.41%. After the hyperglycemic mice were injected with the C. paliurus leaf extract， the liver's capacity to scavenge free radicals was greatly improved， which was close to the scavenging rate of free radicals in the liver of normal mice. Compared with the hyperglycemia group， the scavenging rate increased by 19.30%.

Conclusions and Discussion

Through single factor experiments and response surface analysis， the optimal process combination for the extraction of C. paliurus leaves was obtained： dry C. paliurus powder size 90-100 mesh， solid-to-liquid ratio 1∶20 （g/ml）， extraction time 192 min， microwave frequency 2 500 MHz， microwave power 490 W， and extraction time 248 s. Under these conditions， the yield of C. paliurus extract reached a maximum， 95.10%， reaching 99.83% of the predicted value.

When fasting （no sugar and any sugary food intake within 8 h） blood sugar is higher than the normal range， it is called hyperglycemia. Under normal circumstances， the human body can ensure the balance between the source and outlet of blood sugar through the two major regulatory systems of hormone regulation and neuromodulation， so that blood sugar is maintained at a certain level. However， under the combined action of genetic factors （such as family history of diabetes） and environmental factors （such as unreasonable diet， obesity， etc.）， the two major regulatory functions are disturbed， and blood sugar levels will rise. Long-term hyperglycemia can cause pathological changes in various tissues and organs of the body， leading to acute and chronic complications， such as pancreatic failure， dehydration， electrolyte imbalance， nutritional deficiencies， decreased resistance， impaired renal function， neuropathy， and fundus diseases.

Alloxan is a pancreatic β-cytotoxic agent that can produce free radicals and selectively attack pancreatic β-cells， resulting in decreased insulin secretion， leading to increased blood sugar. It can also affect other organs， such as the liver. It inhibits the glycogen synthesis and glucose utilization functions of liver， which leads to experimental diabetes. Therefore， in this study， we used alloxan to create a hyperglycemic mouse model[9-10].

The results showed that the extract of C. paliurus leaves had a certain effect on lowering blood sugar in normal mice and mice with hyperglycemia caused by alloxan. The study on the liver's capacity to scavenge free radicals showed that the liver's free radical scavenging capacity of mice in the normal group and the hyperglycemia group was improved to varying degrees after the injection of the C. paliurus leaf extract. It can be seen that the blood sugar-lowering activity of C. paliurus leaf extract is to protect the liver by improving the free radical scavenging capacity of the liver of hyperglycemic mice. It accelerates the synthesis of liver glycogen， and simultaneously reduces the damage of free radicals to islet cells and increases the secretion of insulin， thereby regulating blood sugar. Therefore， the extract of C. paliurus leaves can regulate blood sugar from the perspective of organ protection， and achieve the effect of lowering blood sugar. C. paliurus is expected to be developed into a hypoglycemic product， and its exact mechanism of action and functional components need to be further studied.

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