Subscriber access provided by NEW MEXICO STATE UNIV
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
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.
*
Tel/Fax ： +86-25-85427844,
E-mail:
[email protected],
0000-0002-1126-5464
1
ACS Paragon Plus Environment
ORCID:
Journal of Agricultural and Food Chemistry
1
Abstract:
2
4ʹ-O-methylpyridoxine-5ʹ-glucoside (MPNG), and vitamin B6 compounds, including
3
pyridoxal (PL), pyridoxamine, pyridoxine, pyridoxal-5ʹ-phosphate (PLP), and,
4
pyridoxamine-5ʹ-phosphate, exist in Ginkgo biloba seeds, which are widely used as
5
food and medicine. This work aimed to determine the MPN analogs in G. biloba
6
seeds in different growth stages in terms of cultivars and age of trees. The highest
7
total MPN contents of 249.30, 295.62, and 267.85 µg/g were obtained in the mature
8
stage ripening stage of three selected G. biloba samples. The total contents of vitamin
9
B6 compounds decreased significantly in the entire growth period of the three samples.
10
Principal component analysis revealed that MPN and MPNG were important
11
contributors in the MPN analogs metabolism of G. biloba seeds. the possible
12
metabolic pathway of MPN was PLP → PL → MPN → MPNG. The influence of
13
the cultivar on the content and composition of MPN analogs was greater than that of
14
the age of G. biloba tree.
15
Key words: Ginkgo biloba seeds; 4ʹ-O-methylpyridoxine; Vitamin B6; Principal
16
component analysis.
The antivitamin B6 4ʹ-O-methylpyridoxine (MPN), its glucoside
17
2
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
18
Journal of Agricultural and Food Chemistry
1. Introduction
19
Ginkgo biloba L. is one of the oldest tree species1. G. biloba leaves extract
20
(GBEs) received extensive attention due to its rich in flavonoids and triterpenoid.
21
These bioactive compounds widely used as a commonly diet supplement and treated
22
many disease2. In China, its seeds also have been used as traditional medicine to cure
23
treat asthma, cough, and allergic inflammation3. As food, G. biloba seeds are cooked
24
by boiling, microwave heating, roasting, and frying and eaten as part of daily diet. G.
25
biloba seeds are also included in desserts, glazed fruits, beverages, and alcoholic
26
drinks in the food processing industry4.
27
Overconsumption of G. biloba seeds can lead to poisoning, which manifests as
28
abdominal pain, diarrhea, clonic convulsions, and loss of consciousness5. Previous
29
studies demonstrated that 4ʹ-O-methylpyridoxine (MPN; Table 1) and its glucoside
30
4ʹ-O-methylpyridoxine-5ʹ-glucoside (MPNG) exist as antivitamin B6 in G. biloba
31
plants, and these components are primarily responsible for poisoning due to
32
overconsumption of G. biloba seeds6,7. As a derivative of vitamin B6 compounds,
33
MPN is synthesized via the deoxyxylulose-independent pathway in G. biloba8. The
34
administration of MPN can reduce the level of γ-aminobutyric acid (GABA;
35
inhibitory neurotransmitter) by inhibiting glutamate decarboxylase (GAD), which is
36
an enzyme that synthesizes GABA with pyridoxal phosphate (PLP) as a coenzyme in
37
the brain9. According to a widely accepted hypothesis, a decrease in the GAD activity
38
is attributed to low PLP levels because MPN has a high affinity for human pyridoxal
39
kinase when MPN is administered through the intake of G. biloba seeds10. Vitamin B6 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 28
40
compounds (VB6) are a generic term for six interconvertible pyridine compounds:
41
pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their respective
42
5ʹ-phosphorylated
43
pyridoxamine-5ʹ-phosphate (PMP), and PLP. These compounds differ in the identity
44
of the chemical group present at the 4ʹ position (Table 1). In plants, vitamin VB6
45
regulates the growth and development of plants and improves their stress resistance11.
46
For humans, vitamin VB6 not only plays an important role in the metabolism of amino
47
acids, carbohydrates, lipids, and neurotransmitters but also modulates immune
48
responses, antitumor effects, and antioxidant activities12,13. The best known form of
49
vitamin B6 is PLP, which is a cofactor for 191 known enzymatic reactions, including
50
proteins, lipids, DNA, and carbohydrate syntheses, in all organisms14.
forms,
namely,
pyridoxine-
5ʹ-phosphate
(PNP),
51
Basic ingredients contents in G. biloba differ during plant growth and can be
52
influenced by various factors, such as growing region, cultivation sources, and
53
climate2. The MPN content gradually increases as seeds grown and observed that the
54
highest MPN content is obtained in seed the ripening period of G. biloba seeds 15.
55
However, studies have yet to examine the changes in other VB6 in G. biloba seeds in
56
different growth stages. The relationship of the synthesis of MPN analogs with that of
57
VB6 in different growth stages has yet to be described.
58
This work aimed to compare the composition and contents of MPN analogs,
59
namely, MPN, MPNG, PMP, PLP, PM, PL, and PN, in G. biloba seeds from different
60
samples in terms of cultivars and age of trees in various growth stages. The
61
interrelation among the seven MPN analogs and the differences in three samples in 4
ACS Paragon Plus Environment
Page 5 of 28
Journal of Agricultural and Food Chemistry
62
terms of the MPN analog contents were analyzed through principal component
63
analysis (PCA).
64
2. Materials and methods
65
2.1 Chemicals and standards
66
MPN and MPNG standards were synthesized by Kangbei Biochemical Co.
67
(Ningbo, China) with 98% purity through high-performance liquid chromatography
68
(HPLC). The standards, including PLP, PMP, pyridoxal hydrochloride, pyridoxamine
69
dihydrochloride, and pyridoxine hydrochloride (≥ 98%), were purchased from Yuanye
70
Biological Technology Co. (Shanghai, China). HPLC-grade acetonitrile and methanol
71
were acquired from Tedia Company Inc. (Ohio, USA). HPLC-grade phosphoric acid
72
(≥ 85%) was obtained from Kemiou Chemical Reagent Co. (Tianjin, China).
73
HPLC-grade sodium pentanesulfonate (≥ 99.5%) was procured from Yuwang
74
Industrial Co. (Shandong, China). Other reagents were of analytical grade. Distilled
75
water was prepared using a Milli-Q system (Millipore A10; Billerica, MA, USA).
76
2.2 Sample collection
77
G. biloba trees used selected in this study were grown in Nanjing, Jiangsu
78
Province, China, and classified on the basis of their cultivars and ages. Three kinds of
79
trees, namely, 10- and 15-year-old ‘Maling’ cultivar and 10-year-old ‘Fozhi’ cultivar
80
were sampled and marked as Maling-10, Maling-15, and Fozhi-10. The detailed
81
information on the sampling trees is listed in Table 2. Ten samples of G. biloba seeds
82
were harvested from June 24 to September 16 in 2017. Each samples including 100
83
seeds randomly collected from three trees for three different G .biloba trees
84
(Maling-10, Maling-15, and Fozhi-10). Afterward, the episperm, mesosperm
85
mesosphere, and endopleura were removed, and G. biloba seeds` were obtained. The 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
86
dry matter of the single seeds (Figure 1: embryoid) was determined by removing the
87
water from the sample in an oven at 105 °C, and the result was expressed by the
88
average of 100 seeds. The growth stages of G. biloba seeds were determined on the
89
basis of the changes in the dry weight of a seed. The fresh seeds collected in different
90
growth stages were freeze dried, pulverized, and stored at 0 °C for further analysis.
91
2.3 Determination of the MPN analogs in G .biloba seeds
92
2.3.31 Extraction of MPN analogs from G. biloba seeds
93
For the analysis of the contents of the MPN analogs, 150 mg of
94
powders was mixed with 1.5 mL of distilled water (adjusted to pH 2.5 by
95
using phosphoric acid). The tubes were incubated in a shaking table
96
(Jinghong, THZ320, Shanghai, China) at 25 °C and 220 r/min for 40 min.
97
Afterward, the tubes were centrifuged at 9167 × g for 30 min at 4 °C
98
(Sigma, model 2-16K, Germany). The supernatant was filtered through a
99
0.45 µm syringe filter (Jinglong, Tianjing, China) and injected into the
100
HPLC system. All of the samples were prepared in triplicate.
101
2.3.1 2 HPLC methods
102
HPLC methods were applied to determine the composition and contents of the
103
MPN analogs in G. biloba seeds collected in different growth stages. The HPLC
104
conditions were adopted from a previously reported method with slight
105
modifications16. The MPN analogs were chromatographically analyzed with a Waters
106
HPLC 2695 system (Waters, Milford, USA) equipped with Waters 2475 fluorescence
107
measurement (Waters, Milford, USA) ( Emission wavelength: 395 nm; excitation
108
wavelength: 295 nm), and separation was performed with a Waters XBridge RP18
6
ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Journal of Agricultural and Food Chemistry
109
column (250 mm × 4.6 mm, 5 µm; Waters Corp., USA). Mobile phase A was 5
110
mmol/L potassium phosphate containing 5 mmol/L sodium pentanesulfonate adjusted
111
to pH 2.5 by using phosphoric acid, and mobile phase B was acetonitrile. The column
112
temperature was maintained at 30 °C, the flow rate was 1.0 mL/min, and the injection
113
volume was 10 µL. The HPLC gradient program was as follows: from 4% of mobile
114
phase B to 8.5% of mobile phase B at the 15th min, to 15% at the 30th min, and to 4%
115
in the final 20 min. The contents of MPN analogs in G .biloba seeds was calculated
116
by the standards curve. The respective standard curves of MPN, MPNG, PMP, PLP,
117
PM, PL, and PN were y = 381.39x − 175944, y = 198.13x − 128585, y = 132.21x –
118
10342, y = 30.47x – 942.06, y = 448.84x – 7880.2, y = 202.65x – 3093.5, and y =
119
302.59x − 60183, (x: the concentration of each standard; y: peak area), and their
120
respective correlation coefficients were 0.9979, 0.9999, 0.9992, 0.9999, 0.9999,
121
0.9999, and 0.9999. (Tables S1). The other information about the determination
122
methods was shown in Table S1-S3.
123
2.3.2 Preparation of the standard curve
124
The standards of the MPN analogs were diluted with distilled water (adjusted to
125
pH 2.5 by using phosphoric acid) to obtain solutions at concentrations of 0.01, 0.05,
126
0.1, 0.5, 1.0, and 1.5 mg/mL. The samples were injected into the HPLC system under
127
chromatographic conditions, and chromatograms were recorded at an excitation
128
wavelength of 295 nm and an emission wavelength of 395 nm. The calibration curves
129
were constructed by plotting the peak area versus the concentration, and the
130
regression equations were calculated for each analysis. The respective standard curves
131
of MPN, MPNG, PMP, PLP, PM, PL, and PN were y = 381.39x − 175944, y =
132
198.13x − 128585, y = 132.21x – 10342, y = 30.47x – 942.06, y = 448.84x – 7880.2, y
133
= 202.65x – 3093.5, and y = 302.59x − 60183, (x: the concentration of each standard; 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
134
y: peak area), and their respective correlation coefficients were 0.9979, 0.9999, 0.9992,
135
0.9999, 0.9999, 0.9999, and 0.9999.
136
2.3.3 Extraction of MPN analogs from G. biloba seeds
137
For the analysis of the contents of the MPN analogs, 150 mg of powders was
138
mixed with 1.5 mL of distilled water (adjusted to pH 2.5 by using phosphoric acid).
139
The tubes were incubated in a shaking table (Jinghong, THZ320, Shanghai, China) at
140
25 °C and 220 r/min for 40 min. Afterward, the tubes were centrifuged at 9167 × g for
141
30 min at 4 °C (Sigma, model 2-16K, Germany). The supernatant was filtered through
142
a 0.45 µm syringe filter (Jinglong, Tianjing, China) and injected into the HPLC
143
system.All of the samples were prepared in triplicate.
144
2.5 Statistical analysis
145
The influence of growth stages on all analyzed parameters for three kinds of
146
Ginkgo G. biloba was evaluated through one-way ANOVA, and significant
147
differences at 5% (p < 0.05) level between mean values were determined with Tukey’s
148
test. Standardized data were input in the PCA matrix, and the number of principal
149
components (PCs) was selected when the eigenvalues of the factors was more than
150
1.00. Analyses were performed using SPSS version 20.0 (SPSS, Inc., Chicago, IL).
151
3. Results
152
3.1 Determination of the growth stages of G. biloba seeds
153
The growth curves of the G. biloba seeds were determined from three kinds of
154
samples collected from June 24 to September 16 by considering the changes in the dry
155
weight of a seed as an index (Figure 2). The data revealed that the single weight of the
156
three kinds of samples presented a similar growth rhythm in the entire growth cycle:
157
the single weight of the seed increased rapidly from June 24 to July 28, whereas no 8
ACS Paragon Plus Environment
Page 8 of 28
Page 9 of 28
Journal of Agricultural and Food Chemistry
158
significant increase was recorded from August 4 to August 25 (p
