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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 1  |  Page : 47-53

A simple high-performance liquid chromatography method for the assay of flavonoids in Ginkgo biloba Leaves


State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China

Date of Submission05-Oct-2020
Date of Acceptance03-Dec-2020
Date of Web Publication8-Mar-2021

Correspondence Address:
Prof. Hua Yang
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210 009
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_9_21

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  Abstract 


Objective: Ginkgo biloba leaves, as an herbal medicine or dietary supplement, have been widely used worldwide. In this study, an integrated analytical method was established for the comprehensive analysis of flavonoids in G. biloba leaves. Materials and Methods: A practical chromatographic method combining high-performance liquid chromatography fingerprint analysis and quantitation was used to simultaneously determine 11 flavonoids (6 flavonol glycosides and 5 biflavones) in G. biloba leaves from different regions. Results: A total of 11 characteristic peaks were identified accurately, and the similarity of fingerprints ranged from 0.944 to 0.996. Methodology validation revealed appropriate linearity ( R2 ≥ 0.9997), precision, repeatability, stability, and recovery. The total contents of the six flavonol glycosides and five biflavones were within the range of 2.142-8.378 mg/g and 3.759-5.675 mg/g in 19 batches of samples, respectively. Among them, two coumaroyl flavonol glycosides were the predominant components. Conclusions: In this study, a convenient and reliable approach was successfully employed for the comprehensive evaluation of flavonoids in G. biloba leaves, which also provided a reference for its quality standard.

Keywords: Ginkgo biloba leaves, Flavonoids, Flavonol glycosides, HPLC, Fingerprint


How to cite this article:
Wu DD, Qu C, Liu XG, Li P, Gao W, Yang H. A simple high-performance liquid chromatography method for the assay of flavonoids in Ginkgo biloba Leaves. World J Tradit Chin Med 2021;7:47-53

How to cite this URL:
Wu DD, Qu C, Liu XG, Li P, Gao W, Yang H. A simple high-performance liquid chromatography method for the assay of flavonoids in Ginkgo biloba Leaves. World J Tradit Chin Med [serial online] 2021 [cited 2021 Apr 21];7:47-53. Available from: https://www.wjtcm.net/text.asp?2021/7/1/47/310933




  Introduction Top


Ginkgo biloba L. a relic plant of Ginkgophyta, existing on the Earth for 180 million years, has been known as a living fossil.[1] Currently, the extract of G. biloba leaves (GBL) has been widely used as an herbal medicine (HM) or dietary supplement for treating cardio-cerebrovascular disease,[2],[3] tinnitus,[1] cognitive impairment,[4],[5] Alzheimer's disease,[1],[6] and other diseases in clinical practice. Flavonoids were considered as the most abundant components of GBL including flavonol glycosides, aglycones and biflavones.[7],[8]According to a most recently review, 110 flavonoids were reported with unambiguous structures from GBL, which belonged to seven classes including: 59 flavonol or their glycosides; 14 flavone glycosides; three flavanones or their glycoside; three isoflavones or their isoflavone glycoside; four flavan-3-ols; 13 biflavonoids; and 9 biginkgosides.[9]

Among these flavonoids, flavonol glycosides were considered as the important bioactive ingredients of GBL, most of which are formed by linking aglycones such as quercetin, kaempferol, and isorhamnetin with glucose or rhamnose in different amounts or positions.[7],[8],[10] The ingredients are also the control indicators of current international quality standards for GBL preparations. Due to the lack of available flavonol glycosides standards and the diversity of flavonoid glycoside glycosylation, the common method for quantitation of flavonol glycosides was by indirectly determining aglycones from acid hydrolysis (including current methods applied in United States Pharmacopeia and European Pharmacopoeia).[2],[6],[10],[11] Although the indirect quantification is simple and easy, the results can not directly reflect the content of flavonoid glycosides.[1] Moreover, biflavones mainly including bilobetin, ginkgetin and isoginkgetin, are also characteristics constituents in Gymnosperm,[1],[2] which is of great significance for the quality identification of leaves.[12],[13],[14],[15],[16] Thus, it is worthwhile to develop a method to comprehensively and directly characterize the content of both flavone glycosides and biflavones for the quality evaluation of flavonoids in GBL.

In this study, we propose a quality control strategy combining a direct quantitative assay of 11 flavonoids (6 flavonol glycosides and 5 biflavones) and high-performance liquid chromatography (HPLC)-based fingerprint analysis, which was applied successfully in 19 batches of GBL samples. Direct assay of flavonoids and fingerprint analysis could be a simple strategy for the quality control of GBL, which can provide a more direct correlation with its bioactive components.


  Materials and Methods Top


Reagents and standards

Rutin (1, purity ≥91.7%), kaempferol-3- O-rutinoside (3, purity ≥91.4%), isorhamnetin-3- O-rutinoside (4, purity ≥93.1%), and amentoflavone (7, purity ≥97.7%) were obtained from the National Institutes for Food and Drug Control (Beijing, China). Quercetin-3- O-glucopyranosyl-(1-2)-rhamnoside (2), quercetin-3- O-2”-(6”- p-coumaroyl)-glucosyl-rhamnoside (5), and kaempferol-3- O-2”-(6”- p-coumaroyl)-glucosyl-rhamnoside (6) were provided by Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Bilobetin (8), ginkgetin (9), isoginkgetin (10), and sciadopitysin (11) were obtained from Chengdu Biopurify Phytochemicals Co., Ltd. (Chengdu, China). The purities of the other seven standards were >97%. The chemical structures of the 11 compounds are shown in [Figure 1].
Figure 1: The chemical str uctures of the 11 flavonoids (1-6 flavonol glycosides and 7-11 biflavones) in Ginkgo biloba. 1 rutin, 2 quercetin-3- O-glucopyranosyl-(1-2)-rhamnoside, 3 kaempferol-3- O-rutinoside, 4 isorhamnetin-3- O-rutinoside, 5 quercetin-3- O-2”-(6”- p-coumaroyl)-glucosyl-rhamnoside, 6 kaempferol-3- O-2”-(6”- p-coumaroyl)-glucosyl-rhamnoside, 7 amentoflavone, 8 bilobetin, 9 ginkgetin, 10 isoginkgetin, 11 sciadopitysin

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HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from Merck (Darmstadt, Germany). HPLC-grade formic acid was obtained from ROE (Newark, USA). Ultrapure water (18 MΩ) was obtained from a Milli-Q system (Millipore Corporation, MA, USA) Other reagents were of analytical grade.

Preparation of samples

Nineteen batches of samples were purchased from Jiangsu, Shandong, Yunnan, Henan, Hubei, Anhui, Zhejiang, Jiangxi, Guangxi, Guizhou, Sichuan, Gansu, Liaoning province, and Shanghai city (China). All specimens were morphologically authenticated by Professor Hua Yang. The information of all the samples is listed in [Table S1]. The voucher species have been preserved in the State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China.



The prepared sample powder (0.5 g, 65-mesh) was precisely weighed and extracted ultrasonically (40 KHz, 500 W) with 10 mL of MeOH for 30 min. The residue was re-extracted with 10 mL of 75% MeOH. The merged extracts were filtered through a 0.22 μm filter and stored at 4°C before HPLC analysis.

Standard solution preparation

Flavonoids were accurately weighed and dissolved in MeOH to the corresponding standard solutions. Biflavones were first prepared with dimethyl sulfoxide. Then, solutions of appropriate concentration range were prepared by diluting the standard solutions with MeOH for calibration curves and stored at 4°C or −20°C before use.

Instrument and chromatographic conditions

The experiment was performed on an Agilent 1260 Infinity II HPLC system equipped with a quaternary pump, an autosampler, column compartment, and diode array detector (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on an Agilent Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm) at 40°C. The mobile phase comprised 0.05% formic acid aqueous solution (A) and ACN/MeOH (8:2, v/v, B) with the following gradient program: 0–18 min, 15% B; 18–19 min, 15%–16% B; 19–33 min, 16% B; 33–34 min, 16%–20% B; 34–45 min, 20% B; 45–53 min, 20%–26% B; 53–65 min, 26% B; 65–66 min, 26%–42% B; 66–80 min, 42% B; 80–81 min, 42%–50% B; 81–90 min, 50% B; 90–100 min, 50%–90% B; and 100–105 min, 90% B. The flow rate was 0.3 mL/min, and 2 μL sample solution was injected into the instrument for further analysis. The detection wavelength of flavonoids was set at 360 nm with special ultraviolet absorption.

Data analysis

The chromatograms of the 19 batches of samples were exported as American Instrumentation Association files and processed with the Similarity Evaluation System for Chromatographic Fingerprint of TCM software (version 2012, Chinese Pharmacopoeia Committee, Beijing, China).


  Results and Discussion Top


Optimization of chromatographic conditions

To achieve better separation of all tested analytes, several different columns, including Agilent ZORBAX Plus C18 (2.1 × 100 mm, 1.8 μm), Agilent InfinityLab Poroshell 120 EC-C18 (2.1 × 100 mm, 1.9 μm), and Waters ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm), were examined [Figure S1]. The Agilent ZORBAX Plus C18 was selected owing to the high resolution of quantitative components, especially for quercetin-3- O-glucopyranosyl-(1-2)-rhamnoside and kaempferol-3- O-rutinoside. Moreover, the organic phase (70% ACN/30% MeOH, 80% ACN/20% MeOH, 90% ACN/10% MeOH, and 100% ACN, v/v), concentration of formic acid in aqueous phase (0.05% and 0.1%), flow rate (0.28, 0.30, and 0.32 mL/min), and temperature (35°C, 40°C, and 45°C) were optimized to improve the peak shape and resolution [Figure S2],[Figure S3],[Figure S4],[Figure S5]. Finally, the HPLC conditions were set as follows: the mobile phase comprised 0.05% formic acid (A) and 80% ACN/20% MeOH (B), a flow rate at 0.3 mL/min, temperature of 40°C, and detection wavelength of 360 nm were selected. Typical LC chromatograms are shown in [Figure 2].

Figure 2: Fingerprints of the 19 batches of Ginkgo biloba samples and the reference fingerprint (R) obtained by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software (The peaks marked with 1–11 represent the 11 characteristic common peaks).

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Optimization of extraction conditions

The extraction solvents (EtOH, MeOH, 25% MeOH, 75% MeOH), extraction times (once and twice), extraction method (ultrasonic and reflux), extraction time (15, 30, and 45 min), and material–liquid ratio (1:20, 1:40, and 1:60) based on the method of single factor investigation were compared to maximize the extraction efficiency, respectively [Figure S6]. The results indicated that two-step extraction of 0.5 g powder with 10 mL of MeOH and 75% MeOH ultrasonically for 30 min was the most efficient extraction method.



Method validation

Fingerprint analysis

The established method was verified by determining the precision, repeatability, and stability. Peak 1 (S1) and peak 8 (S2) were chosen as the reference peaks for flavonoid glycosides and biflavones, respectively. Precision was analyzed with six replicates at each level continuously, and six independent sample solutions were prepared for repeatability. The same sample (YX-YN-1) was injected at 0, 2, 4, 6, 8, 10, 12, and 24 h to evaluate stability, respectively. The relative standard deviation (RSD) values of the relative retention time (RRT) and relative peak areas (RPA) did not exceed 3%, demonstrating that the chromatographic method was reliable [Table S2] and [Table S3].



Quantitative analysis

The results of linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability, precision, stability, and recovery are shown in [Table 1]. All compounds showed an appropriate linearity ( R2 ≥ 0.9997) within the test ranges, and the LOD and LOQ values were 0.016–0.259 and 0.048–0.345 μg/mL, respectively. The RSD values of repeatability and stability analysis were not above 2.40%. The intra- and inter-day precision was assessed within the same day and on 3 consecutive days, and the RSD value did not exceed 2.62%. Recovery analysis was performed by adding the same level of reference solution as the sample during the extraction process to evaluate the accuracy. The recoveries of the targeted compounds varied from 92.89% to 106.64% with RSD <2.00%.

Establishment of fingerprints and similarity analysis

The chemical fingerprints of 19 batches of GBL samples are shown in [Figure 3]. The reference fingerprint (R) was generated using the median method, and the 11 characteristic common peaks were marked as 1–11. The RRT of the characteristic peaks was calculated to determine the location of each peak in different batches [Table S4]. In addition, the similarity values of 19 batches of GBL samples were in the range of 0.944–0.996, indicating that various samples possessed similar chromatographic patterns [Table 2].
Figure 3: Fingerprints of the 19 batches of Ginkgo biloba leaves and the reference fingerprint (r) obtained by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software (11 characteristic common peaks marked with 1-11)

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Table 2: The similarity of 19 batches of Ginkgo biloba leaf samples

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Quantitative analysis of flavonoids

The proposed HPLC analytical method was applied to simultaneously analyze 6 flavonol glycosides and 5 biflavones from 19 batches of samples [Figure 4] and [Table 3]. The content of 11 compounds varied significantly among the 19 batches of samples. The highest content of 11 flavonoids was 14.053 mg/g (YX-YN-3), while the lowest was 6.529 mg/g (YX-AH-1). Two coumaroyl flavonol glycosides (5 and 6) were the most abundant flavonol glycosides with mean values of 1.061 and 0.981 mg/g, respectively.
Figure 4: The contents of flavonoids in 19 batches of Ginkgo biloba leaf samples

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Table 3: The content of flavonoids in 19 batches of Ginkgo biloba leaf samples

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Among the five biflavones, sciadopitysin (11) exhibited the highest content in GBL, with an average content of 1.745 mg/g. The content of other four biflavones were low, with mean values of 1.418 mg/g for isoginkgetin (10), followed by ginkgetin (9, 0.870 mg/g), bilobetin (8, 0.429 mg/g), and amentoflavone (7, 0.043 mg/g).

In addition, the total content of 11 flavonoids in samples from Pizhou city and standardized planting base of Yunnan province ranged from 9.039 to 14.053 mg/g and were slightly higher than other production areas (6.529–10.036 mg/g), indicating that natural geographical advantages and standardized planting were beneficial to the accumulation of chemical components.





In our study, a convenient analytical method combining chemical fingerprints with quantification of flavonoids was performed for quality analysis of GBL from different geographic locations. Eleven common compounds (six flavonol glycosides and five biflavones) were selected as characteristic peaks to establish HPLC fingerprints. The various samples had similar chemical information, while the content of 11 flavonoids varied significantly among the 19 batches of samples. Based on this study, the established method could be applied to detect adulteration and for comprehensive quality control and evaluation of GBL in the future.

Acknowledgments

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 81722048).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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