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Abstract［Objectives］This study was conducted to study the extraction process and the content determination of flavonoids in ginkgo (Ginkgo biloba L.) leaves.
［Methods］ Ethanol extraction and methanol extraction of total flavonoids in ginkgo leaves were studied, and the optimal extraction conditions for flavonoids were determined by orthogonal test; and with quercetin as reference substance, total flavonoid content in ginkgo leaves was determined by UV spectrophotometry.
［Results］ The optimal extraction process was 4 h of Soxhlet extraction with methanol; and the total flavonoid contents had a good linear relation in the range of 0.006 5-0.039 mg/ml (R2=0.999 9), the average content was stabilized at 1.135%, and the average recovery of the method was 102.0%.
［Conclusions］ This study selected the optimal extraction process for total flavonoids in ginkgo leaves. The test method is simple with high accuracy and precision, and is suitable for the extraction and determination of total flavonoids in ginkgo leaves.
Key wordsGinkgo biloba L.; Total flavonoids; Extraction process; Content determination
Received: August 29, 2018Accepted: November 21, 2018
Supported by Guilin Science and Technology Bureau Project (20100305); Guangxi "2011 Collaborative Innovation Center"Zhuang Yao Medicine Collaborative Innovation Center Project (G2013［20］); Special Fund for Traditional Medical Science and Technology of Department of Public Health of Guangxi Zhuang Autonomous Region (GZMZ1212).
Dengfeng ZOU (1975-), male, P. R. China, professor, devoted to research about natural medicine.
*Corresponding author. Email: 437593157@qq.com.
Ginkgo (Ginkgo biloba L.) is also known as Gongsunshu, "living fossil" and "panda in the plant world"［1］. Flavonoids in ginkgo leaves are composed of flavones and their glycosides, biflavones and catechins［2］. The aglycones of ginkgo flavonoids have quercetin and kaempferol as their main components［3］. Modern pharmacological research has shown that flavonoids have a variety of biological activity, and are the most important active components of ginkgo leaves［4］. Flavonoids have the effects of improving cardiovascular and cerebrovascular circulation, resisting platelet activating factor and lowering cholesterol［5-6］, and are mainly used for the treatment of coronary heart disease, angina pectoris and hyperlipidemia［7］. Therefore, the content of flavonoids in ginkgo is an important indicator for the evaluation of ginkgo pharmacognosticals and its related products［8］. The extraction rate of flavonoids in leaves can be improved by studying the optimal extraction conditions of flavonoids in ginkgo leaves, so as to maximize the medicinal and economic value of ginkgo leaves and more effectively utilize abundant ginkgo tree resources in China. In this study, the absorbance of the extract was determined with quercetin reference substance as a standard, and the total flavonoid content in ginkgo leaves was calculated. Experimental Materials
Materials
Ginkgo leaves were collected from the Botanical Garden of Dongcheng Campus of Guilin Medical University in September, 2017, and identified as G. biloba leaves by associate professor Huang from the Teaching and Research Section of Pharmacognosy, Guilin Medical University. After sampling, the samples were taken back to the laboratory within 30 min, then cleaned, and placed in an oven at 105 ℃ for 15 min. The leaves were then dried at 80 ℃ to constant weight. All samples were pulverized and sieved with a 50-60 mesh sieve for later use.
Reagents
Methanol, chloroform, hydrochloric acid, and anhydrous ethanol, all of which were analytical pure and purchased from Tianjin Baishi Chemical Industry Co., Ltd.; quercetin standard, National Institute for the Control of Pharmaceutical and Biological Products, purity≥98%.
Instruments
Electronic balance (Beijing Sartorius Instrument System Co., Ltd.); UV spectrophotometer (Shimadzu, Japan); SHBIII circulating water multipurpose vacuum pump (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.); thermostat water bath (Jintan Hengfeng Instrument Factory).
Experimental Methods
Extraction process of total flavonoids in ginkgo leaves
Preparation of quercetin standard solution
A certain amount of quercetin standard (13.35 mg, with a quercetin content of 97.4%, equivalent to quercetin 13 mg) was accurately weighed, and added with an appropriate amount of methanol to dissolve it. The solution was transferred to a 100 ml volumetric flask, diluted to constant volume with methanol, and shaken to obtain 0.13 mg/ml quercetin standard solution.
Determination of maximum absorption wavelength of quercetin standard solution
According to the structural characteristics and spectral distribution of flavonoids, spectral scanning was performed in the range of 200-700 nm under a UV spectrophotometer. It could be seen from the above test that the quercetin standard solution had the highest absorption peak at 259.5 nm, so 259.5 nm was chosen as the measurement wavelength.
Drawing of quercetin standard curve
(1) Drawing of quercetin standard curve using methanol as solvent［9］
At first, 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 ml of quercetin standard solution were accurately pipetted into 10 ml volumetric flasks, respectively, followed by diluting to constant volume with methanol and shaking uniformly. The absorbance values of the obtained solutions were determined at 259.5 nm with methanol as a blank reference solution. A standard curve was drawn with the absorbance as the ordinate and the concentration as the abscissa. The concentration and absorbance values are shown in Table 1. The mass concentration of quercetin X (μg/ml) was in good linear relation with the absorbance Y in the range of 6.50-39.0 μg/ml. The linear regression equation of quercetin concentration X (μg/ml) and absorbance Y was Y=0.034 4X-0.020 1, R2=0.999 9. The linear relation was good. Table 1Quercetin standard solution concentration and absorbance (with methanol as solvent)
Standard solvent∥mlConcentration∥μg/mlAbsorbance
0.506.500.202 5
1.0013.00.426 1
1.5019.50.645 4
2.0026.00.882 2
2.5032.51.098 9
3.0039.01.315 0
Fig. 1Quercetin standard curve (with methanol as solvent)
(2) Drawing of quercetin standard curve using ethanol as solvent
The quercetin standard solution was prepared with ethanol according to the method in "Preparation of quercetin standard solution", and the quercetin standard curve was plotted as "Drawing of quercetin standard curve using methanol as solvent" with ethanol as the solvent. The concentration and absorbance values are shown in Table 2. The mass concentration of quercetin X (μg/ml) was in good linear relation with the absorbance Y in the range of 6.50-39.0 μg/ml. The linear regression equation of quercetin concentration X (μg/ml) and absorbance Y was Y=0.072 1X+0.001, R2 = 0.999 9. The linear relation was good.
Orthogonal test on extraction process of total flavonoids from ginkgo leaves［10］
Three factors including extraction temperature, ethanol volume fraction and extraction time were selected to investigate their effects on absorbance, so as to determine the correlative factors and their ranges for later orthogonal test. The factors and levels of the orthogonal test are shown in Table 3. The orthogonal test design is shown in Table 4, and the results and analysis of the orthogonal test are shown in Table 5. In the orthogonal test, the effects of the various factor on absorbance ranked as A>C>B, i.e., extraction time>ethanol concentration>extraction temperature. In the ethanol extraction method, the optimal extraction process was A2B3C2: the extraction time of 90 min, the extraction temperature at 80 ℃, and the ethanol concentration of 70%.
Table 2Quercetin standard solution concentration and absorbance (with ethanol as solvent)
Standard solvent∥mlConcentration∥μg/mlAbsorbance
0.506.500.459 8
1.0013.00.947 2
1.5019.51.407 1
2.0026.01.873 8
2.5032.52.354 6
3.0039.02.801 1
Fig. 2Quercetin standard curve (with ethanol as solvent)
Table 3Factors and levels of orthogonal test
Ethanol concentration A∥%Temperature B∥℃Time C∥min
60(1)60(1)60(1)
70(2)70(2)90(2) 80(3)80(3)120(3)
Table 4Orthogonal test design
Factor level TimeA∥minTemperatureB∥℃Ethanol concentrationC∥%
1606060
2607070
3608080
4906070
5907080
6908060
71206080
81207060
91208070
Table 5Results and analysis of orthogonal test
Factor level Time A∥minTemperature B∥℃Ethanol concentration C∥%Weight∥gAbsorbance Content∥%
16060601.022 30.681 50.461 6
26070701.017 10.785 90.535 1
36080801.016 80.940 10.640 4
49060701.002 81.131 10.781 4
59070801.001 80.950 50.657 2
69080601.004 30.884 80.610 2
712060800.998 60.556 70.385 8
812070601.002 70.848 80.586 3
912080701.006 11.037 60.714 4
K10.545 70.542 90.552 7
K20.682 90.592 90.677
K30.562 10.655 00.561 2
R0.137 20.112 10.124 3
Order A＞C＞B
Optimal level 908070
Standard curve y=0.0721x+0.001 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves.
Dengfeng ZOU et al. Extraction Process and Content Determination of Total Flavonoids in Ginkgo (Ginkgo biloba L.) Leaves
Comparison of ethanol extraction and methanol extraction
Since the orthogonal test used ethanol as the solvent to study the optimal extraction conditions in the ethanol extraction method, the ethanol extraction rate and methanol extraction rate were compared to determine the optimal extraction scheme. The ethanol extraction results are shown in Table 6, and the methanol extraction results are shown in Table 7. To compare which of the ethanol extraction method and the methanol extraction method was better, the results were statistically tested. When comparing these two methods, whether the two population variances were the same should be determined at first, that is, the homogeneity of the variance, and F test was required. If the two population variances are equal, t test is used directly. If not, t test or variable transformation or rank sum test might be used. The results of the F test are shown in Table 8.
Table 6Results of ethanol extraction
Weight of ginkgo leaves∥gAbsorbanceContent∥%
0.999 00.976 90.677 4
0.997 81.095 70.760 8
1.000 31.198 40.830 1
1.003 30.939 8 0.648 8
1.003 60.900 60.621 5
1.005 40.987 50.680 4
Standard curve y=0.034 4x+0.020 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves. wherein C is the concentration, m is the weight of ginkgo leaves.
F<F0.05/2, P>0.05, it indicated that there was no significant difference in total flavonoid content between ethanol extraction and methanol extraction. Then, t test was directly used, and the results of t test are shown in Table 9. t>t0.05/2(10), P<0.05, it indicated that there was a significant difference in total flavonoid content between ethanol extraction and methanol extraction; and t>t0.05(10), P<0.05, then, the content of total flavonoids obtained by methanol extraction was better than that by ethanol extraction, so methanol extraction was selected.
Table 7Results of methanol extraction
Weight of ginkgo leaves∥gAbsorbance Content∥%
1.022 20.786 31.147
1.037 80.809 21.161
1.006 40.781 81.158
0.996 40.752 71.127
1.011 40.725 61.072
1.004 00.766 31.138
Standard curve y=0.034 4x+0.020 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves.
Preparation of test solution
About 1 g of leaf powder was accurately weighed, placed in a Soxhlet extractor and added with chloroform, followed by 2 h of reflux extraction. The chloroform was discarded, and the residue was evaporated and added with methanol, followed by 4 h of reflux extraction. The extract was evaporated to dryness, and the residue was added with 25 ml of methanol25% hydrochloric acid solution (4∶1) mixture, and refluxextracted for 30 min. After cooling, the extract was transferred to a 50 ml volumetric flask, added with methanol to constant weight, and shaken uniformly.
Reproducibility test［11］
According to the preparation method of the test solution, 6 parts of ginkgo leaves were treated under the same conditions, and six test solutions were obtained, respectively. Then, 1.00 ml of the test solutions were accurately pipetted and placed in 10 ml volumetric flasks, respectively. Methanol was then added into the volumetric flasks, followed by shaking uniformly. The absorbance was measured at the maximum absorption wavelength of 259.5 nm. Total flavonoid contents were calculated, RSD=2.6%, indicating good reproducibility. The results are shown in Table 10.
Table 8Results of F test
Source of variationSample meanSample varianceDegree of freedomFF0.05/2
Ethanol extraction method 0.703 20.0065 5.459.60 Methanol extraction method 1.1350.0015
Table 9Results of t test
Source of variation Sample mean Sample variance Degree of freedomStt0.05/2(10)t0.05(10)
Ethanol extraction 0.703 20.00650.084 3 8.880 22.206 01.859 5
Methanol extraction 1.1350.0015
Table 10Reproducibility test of total flavonoid determination
No. Weight of Ginkgo biloba∥gAbsorbance Total flavonoid content∥%Mean of total flavonoid content∥%RSD∥%
11.022 20.786 31.1471.1352.6
21.037 80.809 21.161
31.006 40.781 81.158
40.996 40.752 71.127
51.011 40.725 61.072
61.004 00.766 31.138
Precision test［12］
Six parts of the same quercetin reference solution (1.00 ml) were accurately weighed, filled in 10 ml volumetric flasks, and added with methanol to constant weight, followed by shaking uniformly. The absorbance values were measured at the maximum absorption wavelength of 259.5 nm, RSD=0.12%, indicating that the precision was good. The results are shown in Table 11.
Table 11Precision test
No. 123456
Absorbance 0.437 00.438 10.437 20.438 10.438 30.437 3
Mean0.437 7
RSD∥%0.120 0
Stability test［13］
At first, 1.00 ml of the test solution was accurately pipetted into a 10 ml volumetric flask, and added with methanol to constant volume, followed by shaking uniformly. The absorbance at the maximum absorption wavelength of 259.5 nm was measured at 0, 1, 2, 4, 8, 24 and 48 h, respectively. The total flavonoid contents were calculated, and the RSD was 1.0% within 8 h and 3.4% within 48 h. It indicated that the test solution was stable within 8 h, but unstable within 48 h. Therefore, the test solution could not be stood for a long time, and it is best to measure within 8 h. The results are shown in Table 12.
Table 12Stability test of the test solution
No1234567
Time∥h 012482448
Absorbance 0.766 30.765 50.757 00.767 80.746 40.736 70.686 7
Total flavonoid content∥%1.1401.1391.1261.1421.1111.0971.025
Table 13Results of recovery test
Sample No.Weight of ginkgoleaves∥gTotal flavonoidcontent∥mgAddition amountof quercetin∥mgAbsorbanceMeasuredvalue∥mgRecovery∥%Averagerecovery∥%RSD∥%
10.502 35.7033.590.610 19.1698.8102.02.4
20.501 25.6913.490.615 09.23104.1
30.500 65.6843.490.608 99.14101.7
40.501 25.6915.650.759 611.33102.6 50.499 85.6755.540.765 911.42106.5
60.498 65.6615.750.765 911.42102.9
70.499 75.6747.290.848 512.6297.9
80.499 45.6707.190.866 112.88103.0
90.500 85.6867.190.855 212.72100.5
Recovery (%)=(Measured valueTotal flavonoid content)/(Addition amount of quercetin×97.4%)×100%
Recovery test［14］
Nine parts of the sample with known content (11.35 mg/g), each 0.5 g, were accurately weighed, accurately added with appropriate amounts of quercetin reference substance, respectively, and prepared according to the preparation method of test solution, obtaining nine test solutions. Then, 1.00 ml of each solution was accurately pipetted into a 10 ml volumetric flask, and added with methanol to constant weight, followed by shaking uniformly. The absorbance values were measured at the maximum absorption wavelength of 259.5 nm, with an average recovery rate of 102.0%, and the RSD was 2.4%. The results are shown in Table 13.
Results
A certain amount of ginkgo leaf sample (1.004 0 g) was accurately weighed, and prepared according to the preparation method of test solution, obtaining an extract. The absorbance of the ginkgo leaf extract was measured at 259.5 nm to be 0.766 3, which was substituted to the linear equation, obtaining the quercetin concentration of 22.86 μg/ml, and the total flavonoid content in ginkgo leaves was calculated to be 1.138%.
Discussion and Conclusions
At present, the methods for extracting flavonoids mainly include organic solvent extraction method and enzyme extraction method. In this study, the organic solvent extraction method was studied. The best extraction process of total flavonoids in ginkgo leaves was extracting with methanol after chloroform degreasing for 4 h. In this study, quercetin was selected as the reference substance, and the content of total flavonoids was determined after hydrolysis of the extract. It could be seen from the experimental results that the UV spectrophotometry is simple with good reproducibility and high accuracy, and is suitable for determination of total flavonoids in ginkgo leaves.
References
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［2］ WANG CZ, CHEN X, TAN WH, et al. Chemical constituents and analytical methods of flavonoids in the leaves of Ginkgo biloba L.［J］. Chemistry and Industry of Forest Products, 1998, 18(1): 83-88. (in Chinese) ［3］ WANG FJ. Review of the investigation on flavonoids of ginkgo leaves［J］. Northwest Pharmaceutical Journal, 2010, 25(2): 155-156. (in Chinese)
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［5］ LIU SH, LIANG JY, CHEN JA, et al. Effects of gender, tree age and crown area on flavoniods content in Ginkgo biloba leaves［J］. Hubei Agricultural Sciences, 2011, 50(6): 1169-1171. (in Chinese)
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［14］ XIE FF, YAN PH, LI L, et al. Determination of chlorogenic acid in Zhuang medicine Artemisia scoparia［J］. Lishizhen Medicine and Materia Medica Research, 2017, 28(12): 2898-2900. (in Chinese)
［Methods］ Ethanol extraction and methanol extraction of total flavonoids in ginkgo leaves were studied, and the optimal extraction conditions for flavonoids were determined by orthogonal test; and with quercetin as reference substance, total flavonoid content in ginkgo leaves was determined by UV spectrophotometry.
［Results］ The optimal extraction process was 4 h of Soxhlet extraction with methanol; and the total flavonoid contents had a good linear relation in the range of 0.006 5-0.039 mg/ml (R2=0.999 9), the average content was stabilized at 1.135%, and the average recovery of the method was 102.0%.
［Conclusions］ This study selected the optimal extraction process for total flavonoids in ginkgo leaves. The test method is simple with high accuracy and precision, and is suitable for the extraction and determination of total flavonoids in ginkgo leaves.
Key wordsGinkgo biloba L.; Total flavonoids; Extraction process; Content determination
Received: August 29, 2018Accepted: November 21, 2018
Supported by Guilin Science and Technology Bureau Project (20100305); Guangxi "2011 Collaborative Innovation Center"Zhuang Yao Medicine Collaborative Innovation Center Project (G2013［20］); Special Fund for Traditional Medical Science and Technology of Department of Public Health of Guangxi Zhuang Autonomous Region (GZMZ1212).
Dengfeng ZOU (1975-), male, P. R. China, professor, devoted to research about natural medicine.
*Corresponding author. Email: 437593157@qq.com.
Ginkgo (Ginkgo biloba L.) is also known as Gongsunshu, "living fossil" and "panda in the plant world"［1］. Flavonoids in ginkgo leaves are composed of flavones and their glycosides, biflavones and catechins［2］. The aglycones of ginkgo flavonoids have quercetin and kaempferol as their main components［3］. Modern pharmacological research has shown that flavonoids have a variety of biological activity, and are the most important active components of ginkgo leaves［4］. Flavonoids have the effects of improving cardiovascular and cerebrovascular circulation, resisting platelet activating factor and lowering cholesterol［5-6］, and are mainly used for the treatment of coronary heart disease, angina pectoris and hyperlipidemia［7］. Therefore, the content of flavonoids in ginkgo is an important indicator for the evaluation of ginkgo pharmacognosticals and its related products［8］. The extraction rate of flavonoids in leaves can be improved by studying the optimal extraction conditions of flavonoids in ginkgo leaves, so as to maximize the medicinal and economic value of ginkgo leaves and more effectively utilize abundant ginkgo tree resources in China. In this study, the absorbance of the extract was determined with quercetin reference substance as a standard, and the total flavonoid content in ginkgo leaves was calculated. Experimental Materials
Materials
Ginkgo leaves were collected from the Botanical Garden of Dongcheng Campus of Guilin Medical University in September, 2017, and identified as G. biloba leaves by associate professor Huang from the Teaching and Research Section of Pharmacognosy, Guilin Medical University. After sampling, the samples were taken back to the laboratory within 30 min, then cleaned, and placed in an oven at 105 ℃ for 15 min. The leaves were then dried at 80 ℃ to constant weight. All samples were pulverized and sieved with a 50-60 mesh sieve for later use.
Reagents
Methanol, chloroform, hydrochloric acid, and anhydrous ethanol, all of which were analytical pure and purchased from Tianjin Baishi Chemical Industry Co., Ltd.; quercetin standard, National Institute for the Control of Pharmaceutical and Biological Products, purity≥98%.
Instruments
Electronic balance (Beijing Sartorius Instrument System Co., Ltd.); UV spectrophotometer (Shimadzu, Japan); SHBIII circulating water multipurpose vacuum pump (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.); thermostat water bath (Jintan Hengfeng Instrument Factory).
Experimental Methods
Extraction process of total flavonoids in ginkgo leaves
Preparation of quercetin standard solution
A certain amount of quercetin standard (13.35 mg, with a quercetin content of 97.4%, equivalent to quercetin 13 mg) was accurately weighed, and added with an appropriate amount of methanol to dissolve it. The solution was transferred to a 100 ml volumetric flask, diluted to constant volume with methanol, and shaken to obtain 0.13 mg/ml quercetin standard solution.
Determination of maximum absorption wavelength of quercetin standard solution
According to the structural characteristics and spectral distribution of flavonoids, spectral scanning was performed in the range of 200-700 nm under a UV spectrophotometer. It could be seen from the above test that the quercetin standard solution had the highest absorption peak at 259.5 nm, so 259.5 nm was chosen as the measurement wavelength.
Drawing of quercetin standard curve
(1) Drawing of quercetin standard curve using methanol as solvent［9］
At first, 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 ml of quercetin standard solution were accurately pipetted into 10 ml volumetric flasks, respectively, followed by diluting to constant volume with methanol and shaking uniformly. The absorbance values of the obtained solutions were determined at 259.5 nm with methanol as a blank reference solution. A standard curve was drawn with the absorbance as the ordinate and the concentration as the abscissa. The concentration and absorbance values are shown in Table 1. The mass concentration of quercetin X (μg/ml) was in good linear relation with the absorbance Y in the range of 6.50-39.0 μg/ml. The linear regression equation of quercetin concentration X (μg/ml) and absorbance Y was Y=0.034 4X-0.020 1, R2=0.999 9. The linear relation was good. Table 1Quercetin standard solution concentration and absorbance (with methanol as solvent)
Standard solvent∥mlConcentration∥μg/mlAbsorbance
0.506.500.202 5
1.0013.00.426 1
1.5019.50.645 4
2.0026.00.882 2
2.5032.51.098 9
3.0039.01.315 0
Fig. 1Quercetin standard curve (with methanol as solvent)
(2) Drawing of quercetin standard curve using ethanol as solvent
The quercetin standard solution was prepared with ethanol according to the method in "Preparation of quercetin standard solution", and the quercetin standard curve was plotted as "Drawing of quercetin standard curve using methanol as solvent" with ethanol as the solvent. The concentration and absorbance values are shown in Table 2. The mass concentration of quercetin X (μg/ml) was in good linear relation with the absorbance Y in the range of 6.50-39.0 μg/ml. The linear regression equation of quercetin concentration X (μg/ml) and absorbance Y was Y=0.072 1X+0.001, R2 = 0.999 9. The linear relation was good.
Orthogonal test on extraction process of total flavonoids from ginkgo leaves［10］
Three factors including extraction temperature, ethanol volume fraction and extraction time were selected to investigate their effects on absorbance, so as to determine the correlative factors and their ranges for later orthogonal test. The factors and levels of the orthogonal test are shown in Table 3. The orthogonal test design is shown in Table 4, and the results and analysis of the orthogonal test are shown in Table 5. In the orthogonal test, the effects of the various factor on absorbance ranked as A>C>B, i.e., extraction time>ethanol concentration>extraction temperature. In the ethanol extraction method, the optimal extraction process was A2B3C2: the extraction time of 90 min, the extraction temperature at 80 ℃, and the ethanol concentration of 70%.
Table 2Quercetin standard solution concentration and absorbance (with ethanol as solvent)
Standard solvent∥mlConcentration∥μg/mlAbsorbance
0.506.500.459 8
1.0013.00.947 2
1.5019.51.407 1
2.0026.01.873 8
2.5032.52.354 6
3.0039.02.801 1
Fig. 2Quercetin standard curve (with ethanol as solvent)
Table 3Factors and levels of orthogonal test
Ethanol concentration A∥%Temperature B∥℃Time C∥min
60(1)60(1)60(1)
70(2)70(2)90(2) 80(3)80(3)120(3)
Table 4Orthogonal test design
Factor level TimeA∥minTemperatureB∥℃Ethanol concentrationC∥%
1606060
2607070
3608080
4906070
5907080
6908060
71206080
81207060
91208070
Table 5Results and analysis of orthogonal test
Factor level Time A∥minTemperature B∥℃Ethanol concentration C∥%Weight∥gAbsorbance Content∥%
16060601.022 30.681 50.461 6
26070701.017 10.785 90.535 1
36080801.016 80.940 10.640 4
49060701.002 81.131 10.781 4
59070801.001 80.950 50.657 2
69080601.004 30.884 80.610 2
712060800.998 60.556 70.385 8
812070601.002 70.848 80.586 3
912080701.006 11.037 60.714 4
K10.545 70.542 90.552 7
K20.682 90.592 90.677
K30.562 10.655 00.561 2
R0.137 20.112 10.124 3
Order A＞C＞B
Optimal level 908070
Standard curve y=0.0721x+0.001 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves.
Dengfeng ZOU et al. Extraction Process and Content Determination of Total Flavonoids in Ginkgo (Ginkgo biloba L.) Leaves
Comparison of ethanol extraction and methanol extraction
Since the orthogonal test used ethanol as the solvent to study the optimal extraction conditions in the ethanol extraction method, the ethanol extraction rate and methanol extraction rate were compared to determine the optimal extraction scheme. The ethanol extraction results are shown in Table 6, and the methanol extraction results are shown in Table 7. To compare which of the ethanol extraction method and the methanol extraction method was better, the results were statistically tested. When comparing these two methods, whether the two population variances were the same should be determined at first, that is, the homogeneity of the variance, and F test was required. If the two population variances are equal, t test is used directly. If not, t test or variable transformation or rank sum test might be used. The results of the F test are shown in Table 8.
Table 6Results of ethanol extraction
Weight of ginkgo leaves∥gAbsorbanceContent∥%
0.999 00.976 90.677 4
0.997 81.095 70.760 8
1.000 31.198 40.830 1
1.003 30.939 8 0.648 8
1.003 60.900 60.621 5
1.005 40.987 50.680 4
Standard curve y=0.034 4x+0.020 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves. wherein C is the concentration, m is the weight of ginkgo leaves.
F<F0.05/2, P>0.05, it indicated that there was no significant difference in total flavonoid content between ethanol extraction and methanol extraction. Then, t test was directly used, and the results of t test are shown in Table 9. t>t0.05/2(10), P<0.05, it indicated that there was a significant difference in total flavonoid content between ethanol extraction and methanol extraction; and t>t0.05(10), P<0.05, then, the content of total flavonoids obtained by methanol extraction was better than that by ethanol extraction, so methanol extraction was selected.
Table 7Results of methanol extraction
Weight of ginkgo leaves∥gAbsorbance Content∥%
1.022 20.786 31.147
1.037 80.809 21.161
1.006 40.781 81.158
0.996 40.752 71.127
1.011 40.725 61.072
1.004 00.766 31.138
Standard curve y=0.034 4x+0.020 1, R2=0.999 9; percentage content (%)=C×10×50/m×100%, wherein C is the concentration, m is the weight of ginkgo leaves.
Preparation of test solution
About 1 g of leaf powder was accurately weighed, placed in a Soxhlet extractor and added with chloroform, followed by 2 h of reflux extraction. The chloroform was discarded, and the residue was evaporated and added with methanol, followed by 4 h of reflux extraction. The extract was evaporated to dryness, and the residue was added with 25 ml of methanol25% hydrochloric acid solution (4∶1) mixture, and refluxextracted for 30 min. After cooling, the extract was transferred to a 50 ml volumetric flask, added with methanol to constant weight, and shaken uniformly.
Reproducibility test［11］
According to the preparation method of the test solution, 6 parts of ginkgo leaves were treated under the same conditions, and six test solutions were obtained, respectively. Then, 1.00 ml of the test solutions were accurately pipetted and placed in 10 ml volumetric flasks, respectively. Methanol was then added into the volumetric flasks, followed by shaking uniformly. The absorbance was measured at the maximum absorption wavelength of 259.5 nm. Total flavonoid contents were calculated, RSD=2.6%, indicating good reproducibility. The results are shown in Table 10.
Table 8Results of F test
Source of variationSample meanSample varianceDegree of freedomFF0.05/2
Ethanol extraction method 0.703 20.0065 5.459.60 Methanol extraction method 1.1350.0015
Table 9Results of t test
Source of variation Sample mean Sample variance Degree of freedomStt0.05/2(10)t0.05(10)
Ethanol extraction 0.703 20.00650.084 3 8.880 22.206 01.859 5
Methanol extraction 1.1350.0015
Table 10Reproducibility test of total flavonoid determination
No. Weight of Ginkgo biloba∥gAbsorbance Total flavonoid content∥%Mean of total flavonoid content∥%RSD∥%
11.022 20.786 31.1471.1352.6
21.037 80.809 21.161
31.006 40.781 81.158
40.996 40.752 71.127
51.011 40.725 61.072
61.004 00.766 31.138
Precision test［12］
Six parts of the same quercetin reference solution (1.00 ml) were accurately weighed, filled in 10 ml volumetric flasks, and added with methanol to constant weight, followed by shaking uniformly. The absorbance values were measured at the maximum absorption wavelength of 259.5 nm, RSD=0.12%, indicating that the precision was good. The results are shown in Table 11.
Table 11Precision test
No. 123456
Absorbance 0.437 00.438 10.437 20.438 10.438 30.437 3
Mean0.437 7
RSD∥%0.120 0
Stability test［13］
At first, 1.00 ml of the test solution was accurately pipetted into a 10 ml volumetric flask, and added with methanol to constant volume, followed by shaking uniformly. The absorbance at the maximum absorption wavelength of 259.5 nm was measured at 0, 1, 2, 4, 8, 24 and 48 h, respectively. The total flavonoid contents were calculated, and the RSD was 1.0% within 8 h and 3.4% within 48 h. It indicated that the test solution was stable within 8 h, but unstable within 48 h. Therefore, the test solution could not be stood for a long time, and it is best to measure within 8 h. The results are shown in Table 12.
Table 12Stability test of the test solution
No1234567
Time∥h 012482448
Absorbance 0.766 30.765 50.757 00.767 80.746 40.736 70.686 7
Total flavonoid content∥%1.1401.1391.1261.1421.1111.0971.025
Table 13Results of recovery test
Sample No.Weight of ginkgoleaves∥gTotal flavonoidcontent∥mgAddition amountof quercetin∥mgAbsorbanceMeasuredvalue∥mgRecovery∥%Averagerecovery∥%RSD∥%
10.502 35.7033.590.610 19.1698.8102.02.4
20.501 25.6913.490.615 09.23104.1
30.500 65.6843.490.608 99.14101.7
40.501 25.6915.650.759 611.33102.6 50.499 85.6755.540.765 911.42106.5
60.498 65.6615.750.765 911.42102.9
70.499 75.6747.290.848 512.6297.9
80.499 45.6707.190.866 112.88103.0
90.500 85.6867.190.855 212.72100.5
Recovery (%)=(Measured valueTotal flavonoid content)/(Addition amount of quercetin×97.4%)×100%
Recovery test［14］
Nine parts of the sample with known content (11.35 mg/g), each 0.5 g, were accurately weighed, accurately added with appropriate amounts of quercetin reference substance, respectively, and prepared according to the preparation method of test solution, obtaining nine test solutions. Then, 1.00 ml of each solution was accurately pipetted into a 10 ml volumetric flask, and added with methanol to constant weight, followed by shaking uniformly. The absorbance values were measured at the maximum absorption wavelength of 259.5 nm, with an average recovery rate of 102.0%, and the RSD was 2.4%. The results are shown in Table 13.
Results
A certain amount of ginkgo leaf sample (1.004 0 g) was accurately weighed, and prepared according to the preparation method of test solution, obtaining an extract. The absorbance of the ginkgo leaf extract was measured at 259.5 nm to be 0.766 3, which was substituted to the linear equation, obtaining the quercetin concentration of 22.86 μg/ml, and the total flavonoid content in ginkgo leaves was calculated to be 1.138%.
Discussion and Conclusions
At present, the methods for extracting flavonoids mainly include organic solvent extraction method and enzyme extraction method. In this study, the organic solvent extraction method was studied. The best extraction process of total flavonoids in ginkgo leaves was extracting with methanol after chloroform degreasing for 4 h. In this study, quercetin was selected as the reference substance, and the content of total flavonoids was determined after hydrolysis of the extract. It could be seen from the experimental results that the UV spectrophotometry is simple with good reproducibility and high accuracy, and is suitable for determination of total flavonoids in ginkgo leaves.
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