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Abstract [Objectives] This study was conducted to determine the content of total flavonoids in ginkgo （Ginkgo biloba L.） leaves.
[Methods] The content of total flavonoids from ginkgo leaves was determined by reversed phase high performance liquid chromatography （RP HPLC）. The flavonoid glycosides were first extracted with methanol， and hydrolyzed with hydrochloric acid solution to prepare a test solution. Platisil ODS C 18 column （150 mm×4.6 mm， 5 μm） and the mobile phase V methanol ∶V water （0.4% phosphoric acid solution）=85∶15 were selected for HPLC separation. The HPLC separation was performed with the column at a column temperature of 25℃ using the mobile phase at a flow rate of 1 ml/min. The sample size was 10 μl， and detection was performed with an Agilent HPLC ultraviolet detector at 360 nm.
[Results] The reference substance， quercetin， had good linearity in the range of 0.002 6-0.052 0 g/L， with a correlation coefficient of 0.999 7 ; and the RSD was 1.26%.
[Conclusions] The determination method has rapid and simple operation with accurate results and is good in repeatability. This method is suitable for the determination of content of total flavonoids in ginkgo leaves.
Key words Ginkgo leaves; Flavonoids; High performance liquid chromatography; Content determination
Ginkgo leaves refer to dry leaves of Ginkgo biloba L. in Ginkgoaceae. Ginkgo is also known as "living fossil"[1] and "panda in the plant world"， and is mainly distributed in Shandong， Guangxi and Yunnan[2]. Ginkgo leaves are sweet， bitter， astringent and neutral in nature and taste. They have the effects of promoting blood circulation， relieving pain and lowering blood fat， and are used for blood stasis， stroke and hemiplegia[3]. Modern chemistry research shows that[4-6]， ginkgo leaves mainly contain flavonoids， anthraquinones （ginkgo terpene lactones）， alkaloids， polysaccharides， phenols， etc.[7]， and especially， ginkgo flavonoids and ginkgo terpene lactones have received extensive attention due to their unique pharmacological activity. Japanese scholar Furukawa Ka Youbi first extracted biflavonoids from ginkgo leaves in 1932. In 1941， Nakazawa Koichi separated ginkgetin. Subsequently， Borker et al. isolated two flavonoids （iso glucagonin and bilobetin） from a mixture of ginkgetin. Internationally， the standard of ginkgo leaves is based on EGB761 produced by the patent process of German Schwbe pharmaceutical factory under the quality control index that ginkgo flavonoids are higher than 24%. The Chinese Pharmacopoeia （2015 edition） also adopts this standard[8]. According to various structural characteristics of ginkgolides， ultraviolet visible spectrophotometry， dual wavelength spectrophotometry and the like could be selected for determination. However， due to the limitations of the measurement characteristics of the two methods， it is often difficult to guarantee the accuracy. The molecular structure of flavonoids from ginkgo has strong absorption at 360 nm， so it can be combined with HPLC to determine flavonoid contents accurately.
Experimental Materials
Ginkgo leaves were collected from the Medicinal Plant Garden of Dongcheng Campus of Guilin Medical University， and identified as G. biloba leaves by associate professor Huang from Guilin Medical University.
Instruments： Soxhlet extractor; Agilent 1100 chromatograph; Agilent chemical station; ODS column （150 mm×4.6 mm， 5 μm）.
Reagents： 99.9% methanol （analytical pure）; ultrapure water; chromatographically pure methanol and phosphoric acid.
Experimental Methods
Chromatographic conditions
Platisil ODS C 18 column （150 mm×4.6 mm， 5 μm） and the mobile phase V methanol ∶V water （0.4% phosphoric acid solution）= 85∶15 were selected. The HPLC separation was performed with the column at a column temperature of 25℃ using the mobile phase at a flow rate of 1ml/min. The sample size was 10 μl， and detection was performed with an Agilent HPLC ultraviolet detector at 360 nm.
Preparation of quercetin standard solution
A certain amount of quercetin standard （0.005 2 g） was accurately weighed to a 100 ml volumetric flask. The quercetin standard was dissolved and diluted to constant volume with methanol， and shaken to obtain 0.052 0 g/L quercetin stock solution.
Drawing of standard curve[9]
Certain amounts of the quercetin standard stock solution （0.5， 1.0， 2.0， 4.0 and 8.0 ml） were pipetted into 50 ml volumetric flasks， respectively， and diluted with anhydrous methanol to constant volume. Then， 10 μl of each solution was injected and determined for peak area. The test results showed that the peak area of quercetin A was in good linear relation with concentration C in the range of 0.002 6-0.052 0 g/L. The regression equation was A=5.069 8×104c-41.704， R2=0.999 7.
Preparation of test solution
About 1 g of leaf powder was accurately weighed， placed in a Soxhlet extractor and added with petroleum ether， followed by 1 h of reflux extraction. The petroleum ether was discarded， and the residue was evaporated and added with a proper amount of trichloromethane， followed by 2 h of reflux extraction. The solvent was discarded， and the residue was evaporated to dryness 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 methanol 25% hydrochloric acid solution （4∶1） mixture， and reflux extracted for 40 min at 85 ℃. After cooling， the extract was transferred to a 50 ml volumetric flask， added with methanol to constant weight， and shaken uniformly. The solution was filtered with 0.45 μm filter membrane before injection.
Calculation of total flavonoids in ginkgo leaves
After the sample was analyzed by HPLC， the main components in the sample were quercetin， isorhamnetin and kaempferol， which had the peak areas A， B， and C， respectively. According to the quercetin standard curve and average conversion factor （2.51[10]） and the regression equation， the content of flavonoid glycosides can be obtained， and the calculation formula is given as below：
C T%=[（A+B+C）×（1.972×10 -5 ）+0.000 822]× 2.51 ×50/m s×100%
Where in 1.972×10 -5 is the slope of the quercetin standard curve; 0.000 822 is the intercept of the quercetin standard curve; 50 is the volume of the sample solution （ml）; m s is the mass of the sample （mg）; and 2.51 is the average conversion factor.
Methodological investigation
Repetitive experiment
The dried and pulverized ginkgo leaves were extracted in parallel for five times according to the experimental methods， and five parts of the test solutions were prepared. The total flavonoid contents were 0.56%， 0.56%， 0.57%， 0.56% and 0.55%， respectively， with an average value of 0.56% ， RSD=1.26%. It could be seen that the reproducibility of the analytical method is better. Reproducibility is mainly controlled by the reproducibility of the extraction operation， and the operation should be quantitative and proficient especially.
Precision test
The reference solution was prepared according to the method under "Preparation of quercetin standard solution"， and injected for 4 times according to the chromatographic conditions under "Chromatographic conditions"， and the chromatogram was recorded. According to the relative peak area of quercetin and the peak width， peak height and symmetrical factor， the RSD of the peak area was 0.215%， and the results are shown in Table 2. The test results showed that the precision of this method is good.
Stability test
The prepared sample solution was determined at 0， 4， 8， 16 and 24 h， respectively， and the relative peak area （RSD<4%） was investigated. It was further known that the differences in content were almost inconspicuous. Therefore， the stability of the sample solution was good within 24 h.
Results and Analysis
Determination of total flavonoid content in ginkgo leaves
According to the test method， the same test solution was injected for five times with a sample size of 10 μl， and the total flavonoid contents were 0.56%， 0.56%， 0.57%， 0.56% and 0.55% ， respectively， with an average value 0.56% >0.40%， RSD=1.26%. The content of total flavonoids in ginkgo leaves was in accordance with the standards in Chinese Pharmacopoeia. Discussion
System suitability
The reference solution， the test solution and the blank solution were determined according to the chromatographic conditions under "Chromatographic conditions". The chromatograms are shown in the figures as below：
In the determination of the reference solution， the peak shape was complete， and there was no interference peak. For the test solution， the peaks of the main components were completely separated， with a resolution of 2.24， and the theoretical plate number was 2 689. The blank solution had no interference on the determination of the content in the test solution.
Selection of detection wavelength
The two wavelengths， 360 nm and 370 nm， were selected for pre determination， and it is found that the results measured at 360 nm were better than those at 370 nm under the existing measurement conditions and environment. Therefore， 360 nm was selected as the detection wavelength.
Exploration of mobile phase
Under the existing experimental conditions， according to the polarity characteristics of the effective components， the acetonitrile water system was selected and tested. The obtained results showed that the separation effect was better under the V methanol ∶V water （0.4% phosphoric acid aqueous solution）=85∶15 system. Therefore， this solution ratio was selected as the experimental mobile phase.
Determination of conversion factor in calculation method
Three kinds of aglycones， quercetin， isorhamnetin and kaempferol， were obtained through hydrolysis of ginkgo leaves and purification. It could be seen from the pharmacopoeia and a large number of literatures and the statistical analysis of the results in many previous studies， the conversion factor was selected to be 2.51. With quercetin （2.51）， kaempferol （2.61） and isorhamnetin （2.39） as the control， the determined results were relatively lower， so the average coefficient was selected for calculation.
Determination of hydrolysis solution concentration[11]
In view of the stability of the sample， it is necessary to pay attention to the selection of the concentration of the hydrolysis solution， so as to avoid large deviation of the results caused by excessive loss. According to the chemical nature of the parent nucleus of total flavonoid glycosides， too high or too low acidity of hydrochloric acid would lead to a lower content. Chinese Pharmacopoeia selects methanol 25% hydrochloric acid solution （4∶1）， and the mixed solution achieves a good hydrolysis effect. It is also possible to add 5 ml of 5 mol/L HCl to 30 ml of methanol as a hydrolysis condition. Hydrolysis time
According to literature reports and pharmacopoeia standards， flavonoid glycosides can be completely hydrolyzed into aglycones under the conditions of this study， and there were no significant differences in the content of main aglycones obtained after 60 to 120 min of hydrolysis. However， it is common to adopt a hydrolysis time of 30 min. After several times of extraction for improving the conditions， taking accidental error into account， it was finally decided to hydrolyze the sample for 40 min.
Hydrolysis temperature[12]
It has been reported in literatures that the best suitable hydrolysis temperature is 85 ℃， as shown in Table 4.
Conclusions
HPLC combines the advantages of many determination methods and is a very good choice for the determination of compounds with strong UV absorption such as total flavonoids in ginkgo leaves. And it has strong practicability， and is generally applicable and capable of satisfying the measurement needs at most occasions. The results showed that in the determination of total flavonoids in ginkgo leaves， HPLC satisfied the requirements of methodological investigation. The determination method has rapid and easy operation with accurate results， and provides a reliable experimental basis for the development of ginkgo.
References
[1] LIANG LX. Chinese Ginkgo biloba L.[M]. Jinan： Shandong Science and Technology Publishing House， 1988： 2.
[2] HAO LQ， HAO W. Prospects for the development of Yunnan ginkgo industry[J]. Resource Development & Market， 1999， 15 （4）： 210-212.
[3] Chinese Pharmacopoeia Commission. Chinese pharmacopoeia[S]. Beijing： Chinese Medical Science and Technology Press， 2010： 296-297.
[4] XU YF， ZHANG LJ， SONG XB. Research progress of Ginkgo biloba extract[J].Drug Evaluation Research， 2010， 33（6）： 352-356.
[5] WANG JT. Medicinal and edible value of Ginkgo biloba extract[J]. Food and Drug， 2006， 5（8）： 73-74.
[6] KANG W. Study on chemical constituents of ginkgo leaves and their medicinal application[J].China Health Industry， 2014， 11（2）： 194-195.
[7] ZHANG LY. Review on the progress of pharmacology research and clinical application of ginkgo leafage[J]. Chinese Pharmaceutical Affairs， 2006， 20（2）： 114-117.
[8] Chinese Pharmacopoeia Commission. Ginkgo biloba L.[M]. 2015.
[9] XIA Q， LIU Y. Study on HPLC fingerprint of Herba Bidentis[J]. Chinese Journal of Experimental Traditional Medical Formulae， 2013， 19（7）： 73-77.
[10] LIU SH， YANG YC， ZHAO H， et al. Determination of total flavonoids in Ginkgo biloba by HPLC[J]. Chinese Journal of Pharmaceuticals， 1999， 25（1）：25.
[11] QIAN ZY， FU J， HU M. HPLC detection of ginkgo flavonoids and its Discussion[J]. Journal of China Pharmaceutical University， 1996， 27（4）： 253
[12] FENG P， SUN M， ZHANG LP. Study on extraction technics of five flavonoids in ginkgo leaves[J]. Applied Chemical Industry， 2013， 42（6）： 1090.
[Methods] The content of total flavonoids from ginkgo leaves was determined by reversed phase high performance liquid chromatography （RP HPLC）. The flavonoid glycosides were first extracted with methanol， and hydrolyzed with hydrochloric acid solution to prepare a test solution. Platisil ODS C 18 column （150 mm×4.6 mm， 5 μm） and the mobile phase V methanol ∶V water （0.4% phosphoric acid solution）=85∶15 were selected for HPLC separation. The HPLC separation was performed with the column at a column temperature of 25℃ using the mobile phase at a flow rate of 1 ml/min. The sample size was 10 μl， and detection was performed with an Agilent HPLC ultraviolet detector at 360 nm.
[Results] The reference substance， quercetin， had good linearity in the range of 0.002 6-0.052 0 g/L， with a correlation coefficient of 0.999 7 ; and the RSD was 1.26%.
[Conclusions] The determination method has rapid and simple operation with accurate results and is good in repeatability. This method is suitable for the determination of content of total flavonoids in ginkgo leaves.
Key words Ginkgo leaves; Flavonoids; High performance liquid chromatography; Content determination
Ginkgo leaves refer to dry leaves of Ginkgo biloba L. in Ginkgoaceae. Ginkgo is also known as "living fossil"[1] and "panda in the plant world"， and is mainly distributed in Shandong， Guangxi and Yunnan[2]. Ginkgo leaves are sweet， bitter， astringent and neutral in nature and taste. They have the effects of promoting blood circulation， relieving pain and lowering blood fat， and are used for blood stasis， stroke and hemiplegia[3]. Modern chemistry research shows that[4-6]， ginkgo leaves mainly contain flavonoids， anthraquinones （ginkgo terpene lactones）， alkaloids， polysaccharides， phenols， etc.[7]， and especially， ginkgo flavonoids and ginkgo terpene lactones have received extensive attention due to their unique pharmacological activity. Japanese scholar Furukawa Ka Youbi first extracted biflavonoids from ginkgo leaves in 1932. In 1941， Nakazawa Koichi separated ginkgetin. Subsequently， Borker et al. isolated two flavonoids （iso glucagonin and bilobetin） from a mixture of ginkgetin. Internationally， the standard of ginkgo leaves is based on EGB761 produced by the patent process of German Schwbe pharmaceutical factory under the quality control index that ginkgo flavonoids are higher than 24%. The Chinese Pharmacopoeia （2015 edition） also adopts this standard[8]. According to various structural characteristics of ginkgolides， ultraviolet visible spectrophotometry， dual wavelength spectrophotometry and the like could be selected for determination. However， due to the limitations of the measurement characteristics of the two methods， it is often difficult to guarantee the accuracy. The molecular structure of flavonoids from ginkgo has strong absorption at 360 nm， so it can be combined with HPLC to determine flavonoid contents accurately.
Experimental Materials
Ginkgo leaves were collected from the Medicinal Plant Garden of Dongcheng Campus of Guilin Medical University， and identified as G. biloba leaves by associate professor Huang from Guilin Medical University.
Instruments： Soxhlet extractor; Agilent 1100 chromatograph; Agilent chemical station; ODS column （150 mm×4.6 mm， 5 μm）.
Reagents： 99.9% methanol （analytical pure）; ultrapure water; chromatographically pure methanol and phosphoric acid.
Experimental Methods
Chromatographic conditions
Platisil ODS C 18 column （150 mm×4.6 mm， 5 μm） and the mobile phase V methanol ∶V water （0.4% phosphoric acid solution）= 85∶15 were selected. The HPLC separation was performed with the column at a column temperature of 25℃ using the mobile phase at a flow rate of 1ml/min. The sample size was 10 μl， and detection was performed with an Agilent HPLC ultraviolet detector at 360 nm.
Preparation of quercetin standard solution
A certain amount of quercetin standard （0.005 2 g） was accurately weighed to a 100 ml volumetric flask. The quercetin standard was dissolved and diluted to constant volume with methanol， and shaken to obtain 0.052 0 g/L quercetin stock solution.
Drawing of standard curve[9]
Certain amounts of the quercetin standard stock solution （0.5， 1.0， 2.0， 4.0 and 8.0 ml） were pipetted into 50 ml volumetric flasks， respectively， and diluted with anhydrous methanol to constant volume. Then， 10 μl of each solution was injected and determined for peak area. The test results showed that the peak area of quercetin A was in good linear relation with concentration C in the range of 0.002 6-0.052 0 g/L. The regression equation was A=5.069 8×104c-41.704， R2=0.999 7.
Preparation of test solution
About 1 g of leaf powder was accurately weighed， placed in a Soxhlet extractor and added with petroleum ether， followed by 1 h of reflux extraction. The petroleum ether was discarded， and the residue was evaporated and added with a proper amount of trichloromethane， followed by 2 h of reflux extraction. The solvent was discarded， and the residue was evaporated to dryness 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 methanol 25% hydrochloric acid solution （4∶1） mixture， and reflux extracted for 40 min at 85 ℃. After cooling， the extract was transferred to a 50 ml volumetric flask， added with methanol to constant weight， and shaken uniformly. The solution was filtered with 0.45 μm filter membrane before injection.
Calculation of total flavonoids in ginkgo leaves
After the sample was analyzed by HPLC， the main components in the sample were quercetin， isorhamnetin and kaempferol， which had the peak areas A， B， and C， respectively. According to the quercetin standard curve and average conversion factor （2.51[10]） and the regression equation， the content of flavonoid glycosides can be obtained， and the calculation formula is given as below：
C T%=[（A+B+C）×（1.972×10 -5 ）+0.000 822]× 2.51 ×50/m s×100%
Where in 1.972×10 -5 is the slope of the quercetin standard curve; 0.000 822 is the intercept of the quercetin standard curve; 50 is the volume of the sample solution （ml）; m s is the mass of the sample （mg）; and 2.51 is the average conversion factor.
Methodological investigation
Repetitive experiment
The dried and pulverized ginkgo leaves were extracted in parallel for five times according to the experimental methods， and five parts of the test solutions were prepared. The total flavonoid contents were 0.56%， 0.56%， 0.57%， 0.56% and 0.55%， respectively， with an average value of 0.56% ， RSD=1.26%. It could be seen that the reproducibility of the analytical method is better. Reproducibility is mainly controlled by the reproducibility of the extraction operation， and the operation should be quantitative and proficient especially.
Precision test
The reference solution was prepared according to the method under "Preparation of quercetin standard solution"， and injected for 4 times according to the chromatographic conditions under "Chromatographic conditions"， and the chromatogram was recorded. According to the relative peak area of quercetin and the peak width， peak height and symmetrical factor， the RSD of the peak area was 0.215%， and the results are shown in Table 2. The test results showed that the precision of this method is good.
Stability test
The prepared sample solution was determined at 0， 4， 8， 16 and 24 h， respectively， and the relative peak area （RSD<4%） was investigated. It was further known that the differences in content were almost inconspicuous. Therefore， the stability of the sample solution was good within 24 h.
Results and Analysis
Determination of total flavonoid content in ginkgo leaves
According to the test method， the same test solution was injected for five times with a sample size of 10 μl， and the total flavonoid contents were 0.56%， 0.56%， 0.57%， 0.56% and 0.55% ， respectively， with an average value 0.56% >0.40%， RSD=1.26%. The content of total flavonoids in ginkgo leaves was in accordance with the standards in Chinese Pharmacopoeia. Discussion
System suitability
The reference solution， the test solution and the blank solution were determined according to the chromatographic conditions under "Chromatographic conditions". The chromatograms are shown in the figures as below：
In the determination of the reference solution， the peak shape was complete， and there was no interference peak. For the test solution， the peaks of the main components were completely separated， with a resolution of 2.24， and the theoretical plate number was 2 689. The blank solution had no interference on the determination of the content in the test solution.
Selection of detection wavelength
The two wavelengths， 360 nm and 370 nm， were selected for pre determination， and it is found that the results measured at 360 nm were better than those at 370 nm under the existing measurement conditions and environment. Therefore， 360 nm was selected as the detection wavelength.
Exploration of mobile phase
Under the existing experimental conditions， according to the polarity characteristics of the effective components， the acetonitrile water system was selected and tested. The obtained results showed that the separation effect was better under the V methanol ∶V water （0.4% phosphoric acid aqueous solution）=85∶15 system. Therefore， this solution ratio was selected as the experimental mobile phase.
Determination of conversion factor in calculation method
Three kinds of aglycones， quercetin， isorhamnetin and kaempferol， were obtained through hydrolysis of ginkgo leaves and purification. It could be seen from the pharmacopoeia and a large number of literatures and the statistical analysis of the results in many previous studies， the conversion factor was selected to be 2.51. With quercetin （2.51）， kaempferol （2.61） and isorhamnetin （2.39） as the control， the determined results were relatively lower， so the average coefficient was selected for calculation.
Determination of hydrolysis solution concentration[11]
In view of the stability of the sample， it is necessary to pay attention to the selection of the concentration of the hydrolysis solution， so as to avoid large deviation of the results caused by excessive loss. According to the chemical nature of the parent nucleus of total flavonoid glycosides， too high or too low acidity of hydrochloric acid would lead to a lower content. Chinese Pharmacopoeia selects methanol 25% hydrochloric acid solution （4∶1）， and the mixed solution achieves a good hydrolysis effect. It is also possible to add 5 ml of 5 mol/L HCl to 30 ml of methanol as a hydrolysis condition. Hydrolysis time
According to literature reports and pharmacopoeia standards， flavonoid glycosides can be completely hydrolyzed into aglycones under the conditions of this study， and there were no significant differences in the content of main aglycones obtained after 60 to 120 min of hydrolysis. However， it is common to adopt a hydrolysis time of 30 min. After several times of extraction for improving the conditions， taking accidental error into account， it was finally decided to hydrolyze the sample for 40 min.
Hydrolysis temperature[12]
It has been reported in literatures that the best suitable hydrolysis temperature is 85 ℃， as shown in Table 4.
Conclusions
HPLC combines the advantages of many determination methods and is a very good choice for the determination of compounds with strong UV absorption such as total flavonoids in ginkgo leaves. And it has strong practicability， and is generally applicable and capable of satisfying the measurement needs at most occasions. The results showed that in the determination of total flavonoids in ginkgo leaves， HPLC satisfied the requirements of methodological investigation. The determination method has rapid and easy operation with accurate results， and provides a reliable experimental basis for the development of ginkgo.
References
[1] LIANG LX. Chinese Ginkgo biloba L.[M]. Jinan： Shandong Science and Technology Publishing House， 1988： 2.
[2] HAO LQ， HAO W. Prospects for the development of Yunnan ginkgo industry[J]. Resource Development & Market， 1999， 15 （4）： 210-212.
[3] Chinese Pharmacopoeia Commission. Chinese pharmacopoeia[S]. Beijing： Chinese Medical Science and Technology Press， 2010： 296-297.
[4] XU YF， ZHANG LJ， SONG XB. Research progress of Ginkgo biloba extract[J].Drug Evaluation Research， 2010， 33（6）： 352-356.
[5] WANG JT. Medicinal and edible value of Ginkgo biloba extract[J]. Food and Drug， 2006， 5（8）： 73-74.
[6] KANG W. Study on chemical constituents of ginkgo leaves and their medicinal application[J].China Health Industry， 2014， 11（2）： 194-195.
[7] ZHANG LY. Review on the progress of pharmacology research and clinical application of ginkgo leafage[J]. Chinese Pharmaceutical Affairs， 2006， 20（2）： 114-117.
[8] Chinese Pharmacopoeia Commission. Ginkgo biloba L.[M]. 2015.
[9] XIA Q， LIU Y. Study on HPLC fingerprint of Herba Bidentis[J]. Chinese Journal of Experimental Traditional Medical Formulae， 2013， 19（7）： 73-77.
[10] LIU SH， YANG YC， ZHAO H， et al. Determination of total flavonoids in Ginkgo biloba by HPLC[J]. Chinese Journal of Pharmaceuticals， 1999， 25（1）：25.
[11] QIAN ZY， FU J， HU M. HPLC detection of ginkgo flavonoids and its Discussion[J]. Journal of China Pharmaceutical University， 1996， 27（4）： 253
[12] FENG P， SUN M， ZHANG LP. Study on extraction technics of five flavonoids in ginkgo leaves[J]. Applied Chemical Industry， 2013， 42（6）： 1090.