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Agricultural and Environmental Chemistry
Determination and comparison of 4#-O-methylpyridoxine analogs in Ginkgo biloba seeds in different growth stages Hao Gong, Caie Wu, Gong-jian Fan, Ting-ting Li, Jia-hong Wang, and Tao Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02522 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018
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Journal of Agricultural and Food Chemistry
Determination and comparison of 4ʹ-O-methylpyridoxine analogs in Ginkgo biloba seeds in different growth stages Hao Gong1, 2, Cai-E Wu1, 2*, Gong-Jian Fan1, 2, Ting-Ting Li1, 2, Jia-Hong Wang1, 2, Tao Wang2, 3 1. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 2. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China; 3. Department of Chemistry Engineering, Xuzhou College of Industrial Technology, Xuzhou 221140, China. Hao Gong did the experimental, analyses of experimental data, figures drawing, critical reading of the manuscript, and writing of the manuscript; Cai-E Wu was responsible for study conception and design, analysis and interpretation of data work; Gong-Jian Fan contributed to study conception, to analysis of experimental data and to the writing of the manuscript; Ting-Ting Li, Jia-Hong Wang and Tao Wang supervised the statistical analyses and contributed to the writing of the manuscript. All authors discussed and commented the results and gave their final approval for submission.
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Abstract:
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4ʹ-O-methylpyridoxine-5ʹ-glucoside (MPNG), and vitamin B6 compounds, including
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pyridoxal (PL), pyridoxamine, pyridoxine, pyridoxal-5ʹ-phosphate (PLP), and,
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pyridoxamine-5ʹ-phosphate, exist in Ginkgo biloba seeds, which are widely used as
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food and medicine. This work aimed to determine the MPN analogs in G. biloba
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seeds in different growth stages in terms of cultivars and age of trees. The highest
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total MPN contents of 249.30, 295.62, and 267.85 µg/g were obtained in the mature
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stage ripening stage of three selected G. biloba samples. The total contents of vitamin
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B6 compounds decreased significantly in the entire growth period of the three samples.
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Principal component analysis revealed that MPN and MPNG were important
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contributors in the MPN analogs metabolism of G. biloba seeds. the possible
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metabolic pathway of MPN was PLP → PL → MPN → MPNG. The influence of
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the cultivar on the content and composition of MPN analogs was greater than that of
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the age of G. biloba tree.
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Key words: Ginkgo biloba seeds; 4ʹ-O-methylpyridoxine; Vitamin B6; Principal
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component analysis.
The antivitamin B6 4ʹ-O-methylpyridoxine (MPN), its glucoside
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Journal of Agricultural and Food Chemistry
1. Introduction
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Ginkgo biloba L. is one of the oldest tree species1. G. biloba leaves extract
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(GBEs) received extensive attention due to its rich in flavonoids and triterpenoid.
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These bioactive compounds widely used as a commonly diet supplement and treated
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many disease2. In China, its seeds also have been used as traditional medicine to cure
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treat asthma, cough, and allergic inflammation3. As food, G. biloba seeds are cooked
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by boiling, microwave heating, roasting, and frying and eaten as part of daily diet. G.
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biloba seeds are also included in desserts, glazed fruits, beverages, and alcoholic
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drinks in the food processing industry4.
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Overconsumption of G. biloba seeds can lead to poisoning, which manifests as
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abdominal pain, diarrhea, clonic convulsions, and loss of consciousness5. Previous
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studies demonstrated that 4ʹ-O-methylpyridoxine (MPN; Table 1) and its glucoside
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4ʹ-O-methylpyridoxine-5ʹ-glucoside (MPNG) exist as antivitamin B6 in G. biloba
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plants, and these components are primarily responsible for poisoning due to
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overconsumption of G. biloba seeds6,7. As a derivative of vitamin B6 compounds,
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MPN is synthesized via the deoxyxylulose-independent pathway in G. biloba8. The
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administration of MPN can reduce the level of γ-aminobutyric acid (GABA;
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inhibitory neurotransmitter) by inhibiting glutamate decarboxylase (GAD), which is
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an enzyme that synthesizes GABA with pyridoxal phosphate (PLP) as a coenzyme in
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the brain9. According to a widely accepted hypothesis, a decrease in the GAD activity
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is attributed to low PLP levels because MPN has a high affinity for human pyridoxal
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kinase when MPN is administered through the intake of G. biloba seeds10. Vitamin B6 3
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compounds (VB6) are a generic term for six interconvertible pyridine compounds:
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pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their respective
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5ʹ-phosphorylated
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pyridoxamine-5ʹ-phosphate (PMP), and PLP. These compounds differ in the identity
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of the chemical group present at the 4ʹ position (Table 1). In plants, vitamin VB6
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regulates the growth and development of plants and improves their stress resistance11.
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For humans, vitamin VB6 not only plays an important role in the metabolism of amino
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acids, carbohydrates, lipids, and neurotransmitters but also modulates immune
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responses, antitumor effects, and antioxidant activities12,13. The best known form of
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vitamin B6 is PLP, which is a cofactor for 191 known enzymatic reactions, including
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proteins, lipids, DNA, and carbohydrate syntheses, in all organisms14.
forms,
namely,
pyridoxine-
5ʹ-phosphate
(PNP),
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Basic ingredients contents in G. biloba differ during plant growth and can be
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influenced by various factors, such as growing region, cultivation sources, and
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climate2. The MPN content gradually increases as seeds grown and observed that the
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highest MPN content is obtained in seed the ripening period of G. biloba seeds 15.
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However, studies have yet to examine the changes in other VB6 in G. biloba seeds in
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different growth stages. The relationship of the synthesis of MPN analogs with that of
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VB6 in different growth stages has yet to be described.
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This work aimed to compare the composition and contents of MPN analogs,
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namely, MPN, MPNG, PMP, PLP, PM, PL, and PN, in G. biloba seeds from different
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samples in terms of cultivars and age of trees in various growth stages. The
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interrelation among the seven MPN analogs and the differences in three samples in 4
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terms of the MPN analog contents were analyzed through principal component
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analysis (PCA).
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2. Materials and methods
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2.1 Chemicals and standards
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MPN and MPNG standards were synthesized by Kangbei Biochemical Co.
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(Ningbo, China) with 98% purity through high-performance liquid chromatography
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(HPLC). The standards, including PLP, PMP, pyridoxal hydrochloride, pyridoxamine
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dihydrochloride, and pyridoxine hydrochloride (≥ 98%), were purchased from Yuanye
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Biological Technology Co. (Shanghai, China). HPLC-grade acetonitrile and methanol
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were acquired from Tedia Company Inc. (Ohio, USA). HPLC-grade phosphoric acid
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(≥ 85%) was obtained from Kemiou Chemical Reagent Co. (Tianjin, China).
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HPLC-grade sodium pentanesulfonate (≥ 99.5%) was procured from Yuwang
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Industrial Co. (Shandong, China). Other reagents were of analytical grade. Distilled
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water was prepared using a Milli-Q system (Millipore A10; Billerica, MA, USA).
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2.2 Sample collection
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G. biloba trees used selected in this study were grown in Nanjing, Jiangsu
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Province, China, and classified on the basis of their cultivars and ages. Three kinds of
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trees, namely, 10- and 15-year-old ‘Maling’ cultivar and 10-year-old ‘Fozhi’ cultivar
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were sampled and marked as Maling-10, Maling-15, and Fozhi-10. The detailed
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information on the sampling trees is listed in Table 2. Ten samples of G. biloba seeds
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were harvested from June 24 to September 16 in 2017. Each samples including 100
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seeds randomly collected from three trees for three different G .biloba trees
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(Maling-10, Maling-15, and Fozhi-10). Afterward, the episperm, mesosperm
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mesosphere, and endopleura were removed, and G. biloba seeds` were obtained. The 5
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dry matter of the single seeds (Figure 1: embryoid) was determined by removing the
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water from the sample in an oven at 105 °C, and the result was expressed by the
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average of 100 seeds. The growth stages of G. biloba seeds were determined on the
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basis of the changes in the dry weight of a seed. The fresh seeds collected in different
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growth stages were freeze dried, pulverized, and stored at 0 °C for further analysis.
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2.3 Determination of the MPN analogs in G .biloba seeds
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2.3.31 Extraction of MPN analogs from G. biloba seeds
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For the analysis of the contents of the MPN analogs, 150 mg of
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powders was mixed with 1.5 mL of distilled water (adjusted to pH 2.5 by
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using phosphoric acid). The tubes were incubated in a shaking table
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(Jinghong, THZ320, Shanghai, China) at 25 °C and 220 r/min for 40 min.
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Afterward, the tubes were centrifuged at 9167 × g for 30 min at 4 °C
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(Sigma, model 2-16K, Germany). The supernatant was filtered through a
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0.45 µm syringe filter (Jinglong, Tianjing, China) and injected into the
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HPLC system. All of the samples were prepared in triplicate.
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2.3.1 2 HPLC methods
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HPLC methods were applied to determine the composition and contents of the
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MPN analogs in G. biloba seeds collected in different growth stages. The HPLC
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conditions were adopted from a previously reported method with slight
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modifications16. The MPN analogs were chromatographically analyzed with a Waters
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HPLC 2695 system (Waters, Milford, USA) equipped with Waters 2475 fluorescence
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measurement (Waters, Milford, USA) ( Emission wavelength: 395 nm; excitation
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wavelength: 295 nm), and separation was performed with a Waters XBridge RP18
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column (250 mm × 4.6 mm, 5 µm; Waters Corp., USA). Mobile phase A was 5
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mmol/L potassium phosphate containing 5 mmol/L sodium pentanesulfonate adjusted
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to pH 2.5 by using phosphoric acid, and mobile phase B was acetonitrile. The column
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temperature was maintained at 30 °C, the flow rate was 1.0 mL/min, and the injection
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volume was 10 µL. The HPLC gradient program was as follows: from 4% of mobile
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phase B to 8.5% of mobile phase B at the 15th min, to 15% at the 30th min, and to 4%
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in the final 20 min. The contents of MPN analogs in G .biloba seeds was calculated
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by the standards curve. The respective standard curves of MPN, MPNG, PMP, PLP,
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PM, PL, and PN were y = 381.39x − 175944, y = 198.13x − 128585, y = 132.21x –
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10342, y = 30.47x – 942.06, y = 448.84x – 7880.2, y = 202.65x – 3093.5, and y =
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302.59x − 60183, (x: the concentration of each standard; y: peak area), and their
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respective correlation coefficients were 0.9979, 0.9999, 0.9992, 0.9999, 0.9999,
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0.9999, and 0.9999. (Tables S1). The other information about the determination
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methods was shown in Table S1-S3.
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2.3.2 Preparation of the standard curve
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The standards of the MPN analogs were diluted with distilled water (adjusted to
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pH 2.5 by using phosphoric acid) to obtain solutions at concentrations of 0.01, 0.05,
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0.1, 0.5, 1.0, and 1.5 mg/mL. The samples were injected into the HPLC system under
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chromatographic conditions, and chromatograms were recorded at an excitation
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wavelength of 295 nm and an emission wavelength of 395 nm. The calibration curves
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were constructed by plotting the peak area versus the concentration, and the
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regression equations were calculated for each analysis. The respective standard curves
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of MPN, MPNG, PMP, PLP, PM, PL, and PN were y = 381.39x − 175944, y =
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198.13x − 128585, y = 132.21x – 10342, y = 30.47x – 942.06, y = 448.84x – 7880.2, y
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= 202.65x – 3093.5, and y = 302.59x − 60183, (x: the concentration of each standard; 7
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y: peak area), and their respective correlation coefficients were 0.9979, 0.9999, 0.9992,
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0.9999, 0.9999, 0.9999, and 0.9999.
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2.3.3 Extraction of MPN analogs from G. biloba seeds
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For the analysis of the contents of the MPN analogs, 150 mg of powders was
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mixed with 1.5 mL of distilled water (adjusted to pH 2.5 by using phosphoric acid).
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The tubes were incubated in a shaking table (Jinghong, THZ320, Shanghai, China) at
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25 °C and 220 r/min for 40 min. Afterward, the tubes were centrifuged at 9167 × g for
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30 min at 4 °C (Sigma, model 2-16K, Germany). The supernatant was filtered through
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a 0.45 µm syringe filter (Jinglong, Tianjing, China) and injected into the HPLC
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system.All of the samples were prepared in triplicate.
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2.5 Statistical analysis
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The influence of growth stages on all analyzed parameters for three kinds of
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Ginkgo G. biloba was evaluated through one-way ANOVA, and significant
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differences at 5% (p < 0.05) level between mean values were determined with Tukey’s
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test. Standardized data were input in the PCA matrix, and the number of principal
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components (PCs) was selected when the eigenvalues of the factors was more than
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1.00. Analyses were performed using SPSS version 20.0 (SPSS, Inc., Chicago, IL).
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3. Results
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3.1 Determination of the growth stages of G. biloba seeds
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The growth curves of the G. biloba seeds were determined from three kinds of
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samples collected from June 24 to September 16 by considering the changes in the dry
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weight of a seed as an index (Figure 2). The data revealed that the single weight of the
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three kinds of samples presented a similar growth rhythm in the entire growth cycle:
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the single weight of the seed increased rapidly from June 24 to July 28, whereas no 8
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significant increase was recorded from August 4 to August 25 (p