2.1. Plant Materials
G. biloba leaf samples of different ages were collected from Tancheng (Shangdong province, China), and samples of different genders from 20-year-old trees were also picked from Tancheng (Shangdong province, China); samples of different cultivation sources collected from the 20-year-old trees were collected from Lingchuan (Guangxi province), Anji (Zhejiang province), Hefei and Ningguo (Anhui province), Yangzhou, Nanjing, Suzhou, Taixing, Pizhou and Nantong (Jiangsu province), Yongzhou (Hunan province), Changting (Fujian province), Shijiazhuang (Hebei province), Zunyi and Guiyang (Guizhou province), Dandong (Liaoning province), Luoyang (Henan province), Chengdu, Tainan (Sichuan province), Tancheng and Taian (Shangdong province), China. All samples were collected in November after fruit harvest season. After collection, the leaves were dried at 50 °C for seven days. Their botanical origins were identified by the second corresponding author, and the voucher specimens were deposited at the Herbarium in Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, China.
2.2. Chemicals and Reagents
The seven chemical standards ginkgolide C (1), bilobalide (2), ginkgolide A (3), ginkgolide B (4), quercetin (5), kaempferol (6), and isorhamnetin (7) were previously isolated from G. biloba leaves in our laboratory. Their structures were determined by 1H-NMR, 13C-NMR, MS and UV spectra (purities > 98%). Each reference compound was precisely weighed and dissolved in methanol as stock solution. The chemical structures of these reference compounds are shown in . Methanol was HPLC-grade from Merck (Darmstadt, Germany) and deionized water was purified by an EPED superpurification system (Eped, Nanjing, China). Other reagent solutions were of analytical grade (Nanjing Chemical Plant, Nanjing, China).
Chemical structures of the terpene lactones and flavonol aglycones in the G. biloba leaves as chemical markers.
2.3. Preparation of Standard Solutions
2.3.1. For Terpenoids
A mixed standard stock solution containing ginkgolides A–C and bilobalide was prepared in methanol. The concentration of each compound in the stock solution was 4.800 mg/mL for ginkgolide C (1), 8.360 mg/mL for bilobalide (2), 6.640 mg/mL for ginkgolide A (3), 3.640 mg/mL for ginkgolide B (4), respectively. Working standard solutions were prepared by diluting the mixed standard stock solution with methanol to give six different concentrations within the ranges: 1, 0.1208–4.800 mg/mL; 2, 0.2095–8.360 mg/mL; 3, 0.1668–6.640 mg/mL and 4, 0.0911–3.640 mg/mL for calibration curves.
2.3.2. For Flavonol Aglycones
Standard solutions of kaempferol, isorhamnetin and quercetin were prepared in methanol. The concentration of each compound in the stock solution was 0.4805 mg/mL for quercetin (5), 0.4805 mg/mL for kaempferol (6), and 0.3844 mg/mL for isorhamnetin (7). Working standard solutions were prepared by diluting the mixed standard stock solution with methanol to give six different concentrations within the ranges: 5, 0.0301–0.4805 mg/mL; 6, 0.0301–0.4805 mg/mL and 7, 0.024–0.3842 mg/mL for calibration curves.
All of the solutions containing different concentrations of the analytes were injected in triplicate and all solutions were stored in a refrigerator at 4 °C before analysis.
2.4. Sample Preparation
2.4.1. For Terpene Lactones
After being dried at 50 °C for seven days, the leaves were pulverized to homogeneous powders (40 meshes). The dried powder (1.5 g) was weighed accurately, and then put in a soxhlet with petroleum ether (30–60 °C) for 1 h, the leaves were discarded and dried in an oven, and then refluxed in the soxhlet with MeOH for 6 h. The extract was filtered through analytical filter paper, and the filtrate was evaporated in vacuo
and dissolved in 10 mL MeOH under sonication, then 5.0 mL of the solution was transferred to a solid-phase extraction (SPE) column containing 3 g acidic Al2
The column was eluted with 25 mL MeOH, and then evaporated in vacuo
. The residue was dissolved with a 55% MeOH solution (v/v) in a 10 mL volumetric flask. The resulting solution was filtrated through a syringe filter (0.45 μm) before being injected into the HPLC system for analysis with an ELSD detector [17
2.4.2. For Flavonol Aglycones
The dried powder (1.0 g) was weighed accurately and then put into a soxhlet with CHCl3
for 2 h, the leaves were discarded and dried in an oven, and then refluxed in the soxhlet with MeOH for 4 h. The extract was evaporated to dryness by using a rotary evaporator in vacuo
. The residue was dissolved with 25 mL of MeOH-25% HCl solution (4:1, v/v) and then refluxed for 0.5 h. The acidic extract was diluted with MeOH in a 50 mL volumetric flask and then filtered through a 0.45 μm membrane filter before being injected into the HPLC system for analysis with a PDA detector [17
2.5. Apparatus and Chromatographic Conditions
Analysis was performed on a Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, and an autosampler. The raw data of terpenoids were detected by a Waters 2424 ELSD, and flavonol aglycones were detected by a Waters 2998 PDA detector. HPLC separation of terpenoids was achieved using a Dikma Platisil C18 column (150 mm × 4.6 mm, 5 μm). The mobile phase was prepared by mixing water, methanol and tetrahydrofuran in a ratio of 65:25:10 (v/v/v). The flow rate of the mobile phase was 1.0 mL·min−1, and the column temperature was maintained at 35 °C. The analytes were monitored with ELSD. The drift tube temperature of the ELSD was set at 80 °C, and the nitrogen flow rate was 2.7 L·min−1. A Hanbon Kromasil C18 column (200 mm × 4.6 mm, 5 μm) was used for the HPLC separation of flavonol aglycones. The mobile phase was prepared by mixing water, methanol and phosphoric acid at a ratio of 48:50:2 (v/v/v). The column temperature was maintained at 35 °C. PDA detection wavelength was set at 360 nm.
2.6. Validation of the Methods
The dilute solution of the reference compounds was further diluted to a series of concentrations with methanol to assess the limits of detection (LOD) and quantification (LOQ). The LOD and LOQ were determined as signal-to-noise (S/N) ratio of three and 10, respectively. The intraday and interday precision was determined by analyzing calibration samples during a single day and on three consecutive days, respectively. To confirm the repeatability, five different working solutions were analyzed. The R.S.D. was taken as a measure of precision and reproducibility. A recovery test was used to evaluate the accuracy of the method. In the test, reference compounds were added to the sample from Lingchuan, and then were analyzed as described above. The average recoveries were estimated by the formula: recovery (%) = (amount found − original amount)/amount added × 100%, and R.S.D. (%) = (S.D./mean) × 100%.