The Optimization of Ultrasonicassisted Extraction of Polysaccharide from Sipunculus nudus by Response Surface Methodology and Study on Antioxidant Activity

Wen CHEN Xiangjun WANG Yujie WANG Sihui LIU Jiahui WEN Ruzheng CAI Weiqiang LIU Liuhuan LI

Abstract［Objectives］ The contents of seven components (gallinic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, rutin, and caffeic acid) in Vidal blanc grape were determined.

［Methods］ The L9(34) orthogonal design method was used to optimize the extraction process. High performance liquid chromatography (HPLC) was used to determine gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, rutin and caffeic acid in Vidal blanc grape.

［Results］ The contents of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, rutin and caffeic acid in Vidal blanc grape were 155 1 μg/g, 20.55 mg/g, 77.11 mg/g, 0.738 8, 0.910 0, 164.5 and 175.8 μg/g, respectively.

［Conclusions］ HPLC method for the determination of the contents of seven components in the Vidal blanc grape can better control the quality of the variety.

Key wordsVidal blanc; Phenolic substances; Content determination; Orthogonal test



Received: June 8, 2018Accepted: October 19, 2018

Supported by College Students Practice Innovation Training Program of Liaoning Province (201811430070); Scientific Research Project of Liaoning Education Department (L2017lkyfwdf05); Project of Liaoning Science and Technology Department (2016003003, 20180551223).

Yetong LIAO (1995- ), female, P. R. China, devoted to research about pharmaceutical engineering.

*Corresponding author. Email: chubotany@sina.cn.

Grape, also known as Caolongzhu, smells sweet, and is neutral in nature, nontoxic. Compendium of Materia Medica records that grapes mainly treat arthritis with fixed pain casued by dampness, and can enhance Qi and delay aging after longterm eating［1］. Vidal blanc is a French hybrid, a kind of wine grape bred by crossing Μgni blanc and Seyval blanc, which is widely planted in Canada and the eastern United States. Huanren Manchu Autonomous County is located in the eastern mountainous area of Liaoning Province, with geographical coordinates of 125°21′-125°39′E, 41°12′-41°22′N. It has a midtemperate continental humid climate and is suitable for the growth of Vidal blanc［2］. From 2000, Vidal blanc has been introduced into the Huanren Manchu Autonomous County of Liaoning Province from Canada, and has been widely planted in large areas, and ice grape planting industry has been formed and is on the right track. The main components of Vidal blanc grape are phenolic acids, which are present in the flesh, skin and seeds, and not only affect the sensory quality of the wine, but also determine the physiological activity of the wine［3］. There have been many studies on phenolic substances in wine at home and abroad, while few studies have been conducted on phenolic substances in the raw grape fruit, though the influence of above substances on the quality of wine is more important. The study of gallic acid, chlorogenic acid, ferulic acid and caffeic acid and flavonoids (epicatechin, catechin and rutin) in Vidal blanc grape will provide a theoretical basis for breeding and quality improvement for wine grapes.

Instruments and Reagents

Instruments

Agilent 1100 high performance liquid chromatography (including quaternary gradient pump, online degasser, VWD detector, Agilent HP1100 workstation); AR2140 electronic analytical balance (Shanghai ohaus); LERAB135S ultra micro balance (Switzerland); METTQE200 highspeed Chinese medicine pulverizer (Wuyi Yili Tools Co., Ltd.); KQ5200DB type digital ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); TGL16C desk centrifuge (Shanghai Anting Scientific Instrument Factory); MV1500 ultravioletvisible spectrophotometer (AOE Instruments (Shanghai) Co., Ltd.); RE52C rotary evaporator (Shanghai Yarong Biochemical Instrument Factory).

Reagents

Gallic acid reference substance (11083200302), epicatechin reference substance (878200102), catechin reference substance (877200001), ferulic acid reference substance (07739910), chlorogenic acid reference substance (110753200413) and rutin reference substance (0809303) were all purchased from National Institute for the Control of Pharmaceutical and Biological Products. Caffeic acid reference substance (Y17D6C7672) was purchased from Shanghai Yuanye Biotechnology Co, Ltd. The used water was Wahaha pure water. Acetonitrile, phosphoric acid and methanol were all chromatographically pure.

The ice grapes were collected from Huanren County, Benxi City, Liaoning Province, and the fresh grapes were dried at 50 ℃ for 72 h and smashed.

Methods and Results

Chromatographic conditions

Diamosil C18 column (4.6 mm × 150 mm, 5 μm) was used and operated at room temperature. The mobile phase for epicatechin, catechin, ferulic acid, chlorogenic acid and caffeic acid was acetonitrile0.05% phosphoric acid (20∶80, V/V)［4-8］, and the mobile phase for gallic acid and rutin was methanol0.5% phosphoric acid (15∶85, V/V)［9-10］. The HPLC was performed at a flow rate of 1.0 ml/min under an injection volume of 10 μl. The detection wavelength of catechin and catechin was 280 nm［11-12］; that of ferulic acid, chlorogenic acid and caffeic acid was 325 nm［13-15］; and those of gallic acid and rutin were 270 and 356 nm, respectively［16-17］.

Preparation of reference solutions

Proper amounts of the catechin reference substance, catechin reference substance, ferulic acid reference substance, chlorogenic acid reference substance, caffeic acid reference substance, gallic acid reference substance and rutin reference substance were accurately weighed and added with methanol solution, obtaining solutions containing 1.101, 4.102 g, 60.01 μg, 0.200 5 mg, 9.11 mg, 215.1 μg, and 9.003 mg per 1 ml of corresponding substances, respectively［18-20］.

Preparation of test solutions

The sample powder was weighed, and prepared according to the experimental conditions to a sample, which was filtered with a 0.45 μm micropore membrane, obtaining the filtrate as the test solution.

Three factors including materialliquid ratio (A), ethanol concentration (B) and ultrasonic extraction time (C) were selected. Each factor was set at 3 levels. The levels of test factors are shown in Table 1. The experimental schedule is shown in Table 2. The variance analysis is shown in Table 3. The analysis of the orthogonal test is shown in Table 4.

During the preparation of the test solutions of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, caffeic acid and rutin in the Vidal blanc grape sample, best processes corresponding to each component were adopted.

Table 1Factor levels

Level A Solidliquid ratioB Ethanol concentration∥%C Extraction time∥minD Blank

11∶168060

21∶136530

31∶105015 

Table 2Experimental design and results

ColumnFactor1Solidliquidratio2Ethanolconcentration3Extractiontime4Blank

Results

Gallic acidcontent Epicatechincontent Catechincontent Chlorogenicacid content Caffeic acidcontent Ferulic acidcontent Rutincontent

11111851.810.7866.710.840 1155.30.730 1101.80

21222923.212.7266.360.650 2154.90.560 484.33

313331 209.020.5577.110.740 1123.90.660 3139.50

421231 051.013.0147.650.670 2143.60.230 2113.20

52231803.311.1444.570.870 2153.00.430 1164.50

623121 000.018.5348.840.800 4118.60.330 4 96.72

731321 109.014.0351.460.820 1150.20.270 2139.60

83213998.312.6852.830.910 3175.80.310 1101.10

933211 551.019.9551.840.640 4128.40.210 181.11

Table 3Variance analysis

Factor Square of devianceDegree of freedom F ratio F critical value Significance

Gallic acidSolidliquid ratio 1.241×105213.3019

Ethanol concentration 1.907×105220.4219*

Extraction time 7.696×10428.24319

Error 9 3372

EpicatechinSolidliquid ratio 2.72520.775 219

Ethanol concentration 106.4230.2519*

Extraction time 3.05920.870 119

Error 3.5202

Catechin Solidliquid ratio 880.7223.2219*

Ethanol concentration 38.2621.00919

Extraction time 9.13420.241 119

Error 37.932

Ferulic acidSolidliquid ratio 0.256 1232.0219*

Ethanol concentration 0.002 00320.251 119

Extraction time 0.030 0123.75119

Error 0.010 022

Chlorogenic acid Solidliquid ratio 0.004 00124.00119

Ethanol concentration 0.011 01211.0219

Extraction time 0.065 02265.0119*

(Continued)

(Table 3)

Factor Square of devianceDegree of freedom F ratio F critical value Significance

Error 02

Caffeic acid Solidliquid ratio 257.123.90419

Ethanol concentration 2 231233.8919*

Extraction time 113.921.73119

Error 65.822

Rutin Solidliquid ratio 575.422.78419

Ethanol concentration 274.121.32619

Extraction time 5 377226.0219*

Error 206.72

Table 4Analysis on results of orthogonal test

Phenolic substanceSignificanceOrder Optimal process

Gallic acidEthanol concentrationEthanol concentration>solidliquid ratio>extraction time Solidliquid ratio at 1∶12, 50% ethanol, 30 min of ultrasonic treatment

EpicatechinEthanol concentrationEthanol concentration>extraction time> solidliquid ratioSolidliquid ratio at 1∶16.5, 50% ethanol, 15 min of ultrasonic treatment

CatechinSolidliquid ratioSolidliquid ratio>ethanol concentration>extraction timeSolidliquid ratio at 1∶16.5, 60% ethanol, 60 min of ultrasonic treatment

Ferulic acidSolidliquid ratioSolidliquid ratio>extraction time>ethanol concentrationSolidliquid ratio at 1∶16.5, 60% ethanol, 60 min of ultrasonic treatment

Chlorogenic acidExtraction methodExtraction time> ethanol concentration>solidliquid ratioSolidliquid ratio at 1∶12, 80% ethanol, 60 min of ultrasonic treatment

Caffeic acidEthanol concentrationEthanol concentration>solidliquid ratio>extraction timeSolidliquid ratio at 1∶12, 80% ethanol, 60 min of ultrasonic treatment

RutinExtraction methodExtraction time>solidliquid ratio>ethanol concentration Solidliquid ratio at 1∶10, 80% ethanol, 15 min of ultrasonic treatment

Agricultural Biotechnology2019

Investigation of linear relation

2, 4, 6, 8, 10 and 20 μl of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, caffeic acid and rutin reference solutions were taken and determined according to the "Chromatographic conditions", respectively, obtaining chromatograms, where were recorded. The peak area Y was linearly regressed to the concentration X, and the results are shown in Table 5.

Table 5Investigation of linear relation

Component Linear equation RLinear range

Gallic acid Y=6 937 504.368X-16 583.5420.999 70.430 1-4.301 mg

Epicatechin Y=701 026.123X-1 507.4120.999 82.202 0-22.02 mg

Catechin Y=2 150 813.806X-19 973.8260.999 68.201 0-82.01 mg

Ferulic acid Y=4 032.131X+32.3750.999 50.120 3-12.03 μg

Chlorogenic acid Y=11 279.098X-11.1250.999 30.400 4-4.004 μg

Caffeic acid Y=220 803.898X-3 347.0830.999 718.21-182.1 μg

Rutin Y=2 394 280.108X-409.0830.999 418.03-180.3 μg

It could be seen from investigation of linear relation that the peak area and concentration of each reference substance showed a good linear relationship.

Precision test

10 μl of each reference solution under "Preparation of reference solutions" was accurately measured and injected for 6 times continuously, and determined under the above chromatographic conditions, obtaining peak areas. The RSD values of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, caffeic acid and rutin were 0.7%, 0.5%, 0.6%, 0.7%, 0.4%, 0.8% and 0.7%, respectively, indicating that the precision of each instrument was good.

Stability test

Each test solution was taken at room temperature and measured at 0, 2, 4, 8, and 24 h, respectively. The peak areas of each sample were recorded, and the RSD was calculated. Specifically, the RSD values of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, caffeic acid and rutin were 0.5%, 0.5%, 0.6%, 0.8%, 0.7%, 0.6% and 0.7%, respectively, indicating that each sample was stable within 24 h.

Reproducibility test

An appropriate amount of ice grape sample powder was measured and prepared according to the method under "Preparation of test solutions" into 6 parts of test solutions, which were determined in parallel according to the content determination method. The result showed that the RSD values of gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, caffeic acid and rutin contents in the sample were 0.7%, 0.6%, 0.6%, 0.7%, 0.5%, 0.6% and 0.5%, respectively, indicating that the method has good reproducibility test.

Recovery test

1.0 g of ice grape sample powder (containing gallic aid 1 550 μg, epicatechin 20.55 mg, catechin 77.11 mg, ferulic acid 0.738 8 μg, chlorogenic acid 0.910 0 μg, rutin 164.5 μg and caffeic acid 175.8 μg) was accurately measured, and added with gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, rutin and caffeic acid reference solutions, respectively. The obtained mixtures were prepared according to"Preparation of test solutions" into solutions which were then determined, giving chromatograms. Recoveries were calculated, as shown in Table 6.

Table 6Results of recovery test (n=5)

Phenolic substance Sample amount∥μg Added amount∥μgMeasured value∥μgRecovery∥%Mean∥%RSD∥%

Gallic acid 1 4831 5042 988100.10

1 4901 5042 92595.36

1 5071 5042 97897.7897.921.9

1 6091 5043 11099.81

1 6121 50433 06096.53

Epicatechin1.953×1041.993×1043.950×104100.20

2.015×1041.993×1044.014×104100.30

2.056×1041.993×1043.044×10498.7598.591.6

2.105×1041.993×1044.052×10497.69

2.157×1041.993×1044.071×10496.01

Catechin7.323×1047.701×10415.05×104100.30

7.498×1047.701×10414.98×10497.14

7.689×1047.701×10415.38×10499.8799.131.1

7.887×1047.701×10415.48×10498.65

8.006×1047.701×10415.68×10499.69

Ferulic acid 0.680 90.716 61.38698.27

0.694 30.716 61.412100.10

0.722 10.716 61.43299.0598.411.1

0.746 50.716 61.44597.69

0.752 40.716 61.44696.91

Chlorogenic acid 0.848 50.882 71.70596.89

0.869 50.882 71.754100.20

0.887 60.882 71.76799.6598.811.2

0.907 70.882 71.77297.93

0.926 80.882 71.80499.37

Rutin 151.7159.6311.5100.10

154.8159.6310.897.65

158.9159.6318.399.8898.831.0

163.2159.6320.698.63

166.5159.6322.697.91

Caffeic acid163.2170.8331.198.23

165.8170.8337.3100.40

167.9170.8338.599.8798.911.1

169.7170.8336.597.67

179.4170.8338.498.35

Table 7Determination results of contents of 7 components in Vidal grape

Batch Component123Mean

Gallic acid∥μg/g1 548.001 610.001 492.001 550.00

Epicatechin∥mg/g20.3920.9220.3420.55

Catechinic acid∥mg/g77.0170.1970.1377.11

Ferulic acid∥μg/g0.748 80.720 10.747 50.738 8

Chlorogenic acid∥μg/g0.890 70.900 80.938 50.910 0

Rutin∥μg/g162.4164.8166.3164.5

Caffeic acid∥μg/g176.4174.9176.1175.8

Determination of contents

10 μl of each of the reference solutions and the test solutions was accurately taken and injected into the liquid chromatography, to determine the peak area. The determination results of the seven components in ice grapes (gallic acid, epicatechin, catechin, ferulic acid, chlorogenic acid, rutin and caffeic acid) are shown in Table 7.

Conclusions and Discussion

In this study, orthogonal analysis method was applied to select the optimal processes for extracting different components in ice grapes (the specific process description as shown in Table 4). It was demonstrated that extraction time, ethanol concentration and solidliquid ratio all affected the determination of different components in ice grape to varying degrees. Among them, solidliquid ratio had a greater influence on the extraction of ice grape components, and extraction time less influenced the extraction of ice grape components. The orthogonal analysis method is suitable for this study, and lays a foundation for the followup work of this study.

Ice grapes as raw materials for traditional Chinese medicines are rich in flavonoids and phenolic acids. Three kinds of flavonoids including epicatechin, catechin and rutin and four kinds of phenolic acids such as gallic acid, chlorogenic acid, ferulic acid and caffeic acid have been detected in the ice grapes selected in this study. There are significant differences in the contents of different components in ice grapes. This study selected the ice grapes from Huanren County, Dandong City, Liaoning Province, which contain catechin the most, and less chlorogenic acid and ferulic acid.

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