Optimization of Extraction Process for Longan (Dimocarpus longan Lour.) Leaves Using Multi-indicator Comprehensive Evaluation Method

Jie LIANG, Jinyu WEI, Dongfang HUANG, Xiaosi CHEN, Jing LIN, Guangqiang HUANG, Hua ZHU,2*

1. Guangxi University of Chinese Medicine, Nanning 530200, China; 2. Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine, Nanning 530200, China; 3. Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Nanning 530200, China

Abstract [Objectives] This study aimed to optimize the extraction process of longan (Dimocarpus longan Lour.) leaves by multi-indicator comprehensive evaluation method. [Methods] The contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol in longan leaves were determined simultaneously by HPLC. Using the multi-indicator comprehensive evaluation method, the methanol concentration, solid to liquid ratio and extraction time for extraction of longan leaves were optimized by orthogonal test. [Results] The optimal extraction process of longan leaves are as follows: methanol concentration of 100%, solid to liquid ratio of 1∶5, and extraction time of 50 min. [Conclusions] The optimized process is simple and feasible, and it can be used to determine the contents of different ingredients in longan leaves.

Key words Longan leaf, Dimocarpus longan Lour., Orthogonal test, Multi-indicator weighed comprehensive evaluation method, Extraction process

1 Introduction

Longan (DimocarpuslonganLour.) leaves or shoots are mainly produced in Guangdong, Guangxi, Fujian and Taiwan, as well as in Sichuan, Hainan, Yunnan and Guizhou. They have the functions of clearing heat, detoxifying and drying dampness, and are mainly used for the treatment of colds, fever, malaria, acne and eczema[1]. Based on a previous study[2], the contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol in longan leaves were determined simultaneously, the extraction process for longan leaves was optimized using multi-indicator comprehensive evaluation method combined with orthogonal test, and the differences in the contents of the five ingredients in longan leaves from 10 different producing areas in Guangxi were compared in this paper, in order to evaluate longan leaves more comprehensively and provide reference for the intrinsic quality control of longan leaves.

2 Instruments and materials

2.1 InstrumentsThe used apparatus and instruments mainly included high-performance liquid chromatograph (1260, Agilent, America), ultrasonic apparatus (KQ-500DA, Kunshan Ultrasonic Instrument Co., Ltd., China) and electronic balance (SQP, Sartorius, China).

2.2 MaterialsThe used materials mainly included gallic acid ethyl ester reference substance (purity > 99%, self-made)[3], quercetin standard (batch No.100081-201610, China Food and Drug Administration Research Institute, China), astragalin standard (batch No.170326, Ronghe, China), luteolin standard (batch No.170626, Ronghe, China), kaempferol standard (batch No.170517, Ronghe, China), methanol (chromatographically pure, Fisher, America), acetonitrile (chromatographically pure, Fisher, America) and ultrapure water. The rest reagents used were analytically pure. The sources of the longan leaves used in this study are shown in Table 1. They were identified by the associate professor Teng Jianbei of the School of Pharmacy, Guangxi University of Chinese Medicine.

Table 1 Sources of the 10 batches of longan leaves

Sample No.Collection siteCollection timeS1WuzhouAugust, 2017S2HezhouAugust, 2017S3YulinAugust, 2017S4QinzhouAugust, 2017S5NanningAugust, 2017S6LiuzhouAugust, 2017S7BeihaiAugust, 2017S8PingnanAugust, 2017S9ChongzuoAugust, 2017S10FangchenggangAugust, 2017

3 Methods and results

3.1 Preparation of reference solutionAppropriate amounts of the standards that have been dried to constant weight at 105 ℃ were mixed in a volumetric flask and dissolved to 10 mL with methanol to prepare the mixed reference solution in which the concentrations of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were 4.06, 0.12, 4.06, 0.12 and 0.841 mg/mL, respectively.

3.2 Preparation of test solutionAn accurate amount (around 2.0 g) of the coarse powder of longan leaves was added with 10 mL of methanol-hydrochloric acid (8∶1) solution and weighed. The mixed solution was then subjected to ultrasonic extraction (500 W, 40 kHz) for 50 min and cooled. The lost weighed was supplemented with methanol-hydrochloric acid (8∶1) solution. Then, the solution was filtered, and the filtrate was centrifuged at 13 000 r/min for 10 min to collect the supernatant, which was the prepared test solution. A certain volume (10 μL) of the test solution was injected into the high-performance liquid chromatograph to determine the contents of related ingredients.

3.3 Chromatographic conditionsThe chromatographic conditions were as follows: column, Kromasil C18column (250 mm×4.6 mm, 5 μm); detection wavelength, 280 and 360 nm; mobile phase, methanol (A) -0.2% phosphoric acid solution (B); gradient elution (0-20 min, 15%-40% A; 20-50 min, 40%-70% A); flow rate, 1.0 mL/min; column temperature, 30 ℃; injection volume, 10 μL. Under these chromatographic conditions, gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were baseline separated from other components. The resolution was greater than 1.5, and the number of theoretical plates was not less than 5 000. The chromatogram is shown in Fig.1.

Note: 1. gallic acid ethyl ester; 2. astragalin; 3. quercetin; 4. luteolin; 5. kaempferol.

Fig.1 High-performance liquid chromatograms of mixed reference solution (A) and longan leaf sample (B)

3.4 Investigation on linear relationshipAccurate volumes (0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL) of the mixed reference solution were diluted to 10 mL, respectively. Under the chromatographic conditions described in Section3.3, the contents of the related ingredients were determined. Taking the concentration of the reference solution (ng/μL) as abscissa and the peak area as ordinate, the regression equation of each standard was obtained (Table 2). It is indicated that the five standards have a good linear relationship within their concentration ranges.

Table 2 Results of investigation on linear relationship

StandardRegression equationrLinear range∥ng/μLGallic acid ethyl esterY1=28.4X1+640.999 9162.4-568.4AstragalinY2=23.5X2+0.20.999 74.8-16.8QuercetinY3=34.9X3+82.80.999 9162.4-568.4LuteolinY4=434.6X4+1.80.999 94.8-16.8KaempferolY5=33.9X5+1.40.999 732.4-113.4

3.5 Stability testAn accurate amount (around 2.0 g) of the coarse powder (S1) of longan leaves from the same producing area was prepared into test solution according to the description in Section3.2. After standing at room temperature for 0, 4, 8, 12, 16 and 24 h, 10 μL of the test solution was submitted to chromatographic analysis under the chromatographic conditions described in Section3.3, respectively. TheRSDvalue of the peak areas of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol was 0.1%, 1.1%, 0.4%, 1.3% and 0.1%, respectively, indicating that the test solution prepared according to the method described above has good stability within 24 h.

3.6 Precision testThe mixed reference solution was subjected to chromatographic analysis 6 times repeatedly (10 μL for each) under the conditions described in Section3.3. TheRSDvalue of the peak areas of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol was 0.9%, 1.4%, 0.8%, 0.8% and 0.8%, respectively, indicating that the precision of the instrument is good.

3.7 Reproducibility testSix portions (about 2.0 g for each) of the coarse powder (S1) of longan leaves from the same producing area were accurately weighed and prepared into test solutions, respectively according to the method described in Section3.2. The prepared test solutions were detected chromatographically under the conditions described in Section3.1(10 μL for each). TheRSDvalue of the peak areas of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol was 0.1%, 1.0%, 0.2%, 0.9% and 0.6%, respectively, indicating that the method is reproducible.

3.8 Recovery testA total of nine portions (around 1.0 g for each) of coarse powder (S1) of longan leaves with known contents of the ingredients were weighed accurately. They were added with high, middle and low-dose mixed reference solution, respectively (i.e. 150%, 100% and 50% of the sample content), three for each dose. According to the description in Section3.2, test solutions were prepared. Under the chromatographic conditions described in Section3.3, the contents of the ingredients were determined. The average recovery rate of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol was 100.52%, 100.68%, 100.00%, 101.48% and 100.40%, respectively, and theirRSDvalue was 0.3%, 1.2%, 0.6%, 1.3% and 0.8%, respectively.

3.9 Content determination of components in longan leaves from different habitatsAn accurate amount (around 2.0 g) of coarse powder of longan leaves from each producing area was prepared into test solution according to the description in Section3.2and detected under the chromatographic conditions described in Section3.3, respectively. The contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were calculated, and the results are shown in Table 3. The ingredients mentioned above had the highest contents in longan leaves from Wuzhou, followed by those from Pingnan, and had the lowest contents in longan leaves from Qinzhou.

Table 3 Contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol in longan leaves from different producing areas in Guangxi (mg/g,n=3)

SourceGallic acidethyl esterAstragalinQuercetinLuteolinKaempferolS12.004 10.054 32.127 50.040 40.486 9S22.001 50.049 5 2.104 10.031 70.322 0S31.986 80.031 8 1.994 30.030 10.297 4S41.956 80.030 51.980 90.023 50.288 2S52.001 93.049 72.004 10.032 70.314 1S61.971 70.031 71.982 60.029 20.294 1S72.001 50.048 92.093 00.031 90.318 3S82.002 70.049 92.109 40.037 10.324 1S92.000 80.047 92.001 40.031 30.314 1S102.001 10.046 02.102 30.034 40.314 8

3.10 Design of orthogonal testTaking the contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol as evaluation indicators, a L9(34) orthogonal test was conducted to optimize the methanol concentration, solid to liquid ratio and extraction time for the extraction of longan leaves. The test factors and levels are shown in Table 4.

Table 4 Factors and Levels of the orthogonal test

LevelMethanolconcentration (A)∥%Solid to liquidratio (B)∥g∶mLExtractiontime (C)∥min1801∶05302901∶084031001∶1050

3.11 Comprehensive evaluation method and results analysis

The extraction rates of the five ingredients in the longan leaves were comprehensively evaluated.

Extraction rate=Actual amount of certain ingredient extracted/Content of certain ingredient in the sample×100%. According to Table 3, the total content of the five ingredients in longan leaves (S1) was 4.713 2 mg/g, in which the percentages of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were 42.52% (2.004 1/4.713 2), 1.15% ( 0.054 3/4.713 2), 45.14% ( 2.127 5/4.713 2), 0.86% (0.040 4/4.713 2) and 10.33% ( 0.486 9/4.713 2). Therefore, the weights of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were set as 42%, 2%, 45%, 1% and 10%. The comprehensive score of each test is the sum of extraction rate and weight product of each ingredient. The comprehensive score of No.1 test=0.490 7/2.004 1×100×42%+0.016 9/0.054 3×100×2%+0.452 9/2.127 5×100×45%+0.010 1 /0.040 4×100×1%+0.140 4/0.486 9×100×10%=23.619 9. The scoring results were analyzed statistically by ANOVA. The test arrangement and results are shown in Table 5 and Table 6.

As shown in Table 5, the influencing intensity of the three factors on the extraction of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol from longan leaves ranked as methanol concentration > solid to liquid ratio > extraction time. As shown in Table 6, methanol concentration had a significant effect (P<0.05), while solid to liquid ratio and extraction time had no significant effects (P>0.05) on the extraction of the five ingredients. The optimal extraction process was A3B1C3, that is, methanol concentration of 100%, solid to liquid ratio of 1∶5 and extraction time of 50 min.

Table 5 Design and results of L9(34) orthogonal test

No.Methanolconcentration(A)Solid toliquid ratio(B)Extractiontime(C)Error(D)Content∥mg/gGallic acidethyl esterAstragalinQuercetinLuteolinKaempferolComprehensivescore111110.490 70.016 90.452 90.010 10.140 423.619 9212220.482 00.014 40.440 90.009 70.139 723.067 4313330.456 60.012 80.427 60.008 20.128 521.927 6421230.952 20.030 80.805 80.015 10.278 444.226 8522310.851 70.028 40.795 60.013 30.263 441.463 7623120.778 10.026 60.774 40.012 00.257 239.247 0731321.999 30.049 82.127 20.039 80.465 299.268 6832131.815 80.040 42.115 30.038 20.465 294.518 4933211.798 80.039 91.932 30.031 60.441 089.879 6K122.87255.70552.46251.654K241.64653.01752.39153.861K394.55650.35154.22053.558K71.6845.3541.8292.207

Table 6 Results of variance analysis

Source of varianceSum of squaresfVariancePMethanol concentration (A)8 290.4882965.920<0.05Solid to liquid ratio (B)42.99325.009>0.05Extraction time (C)6.4420.750>0.05Error8.582

Note:F0.05(2, 2)=19.00.

3.12 Verification testTo further examine the reliability and stability of the optimized process, three portions (around 2.0 g for each) of the coarse powder of longan leaves (S1) were weighed accurately and extracted under the optimal conditions determined in Section3.11, respectively. The contents of gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were determined, and the results are shown in Table 7. It is indicated that the process is stable and reliable.

Table 7 Results of verification test (mg/g,n=3)

Ingredient123MeanRSD∥%Gallic acid ethyl ester2.002 42.002 02.006 92.003 80.14Astragalin0.054 70.054 40.055 00.054 70.48Quercetin2.131 12.134 12.136 22.133 80.12Luteolin0.040 80.040 20.041 00.040 71.01Kaempferol0.486 30.486 90.487 60.486 90.13

4 Discussion

This study compared Agilent, Hypersil, and Kromasil C18columns. The peaks of the Kromasil C18(250 mm×4.6 mm, 5 μm) column were sharp and the resolution of each ingredient reached 1.5. The theoretical plate number was greater than 5 000. The mobile phases of acetonitrile-water, methanol-water, methanol-0.2% phosphoric acid solution and acetonitrile-0.2% phosphoric acid solution, the flow rates of 0.8, 1.0 and 1.2 mL/min, and the column temperatures of 25, 30 and 35 ℃ were compared. The results show that under the conditions of mobile phase of methanol-0.2% phosphoric acid solution, flow rate of 1.0 mL/min and column temperature of 30 ℃, the peak shape of each chromatogram is good, the baseline is stable, and the separation effect is better.

This study compared the effects of impregnation method, ultrasonic method, reflux extraction method and Soxhlet extraction method on the extraction of the ingredients from longan leaves. The results show that the differences between reflux extraction, Soxhlet extraction and ultrasonic extraction were not obvious. Considering the cumbersomeness of experimental apparatus and instruments, ultrasonic extraction method was finally selected. Through full-wavelength scanning and literature review[4-5], it is found that the detection wavelength of gallic acid ethyl ester is different from those of astragalin, quercetin, luteolin and kaempferol, so in this study, the test was carried out by changing the wavelength.

In this study, gallic acid ethyl ester, astragalin, quercetin, luteolin and kaempferol were used as evaluation indexes, the orthogonal design is reasonable, convenient and simple, the sample extraction process is reliable, the results of the methodological investigation are accurate, and the method is reproducible. The multi-indicator comprehensive scoring method directly reflects the scientificity and rationality. The process optimized by the multi-indicator scoring method avoids the difference in the processes optimized from single indicators. When using the orthogonal test for optimizing the process, the choice of survey indicators and their weights should be analyzed on a case-by-case basis. The contents of astragalin and luteolin in longan leaves are low, so their weights in this paper were assigned as 2% and 1%, respectively. This method is reasonable. It can provide a more comprehensive theoretical basis for comprehensive evaluation of longan leaf quality.