Discovery of Neolignan Glycosides with Acetylcolinesterase Inhibitory Activity from Huangjinya Green Tea Guided by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Data and Global Natural Product Social Molecular Networking

Article abstract of DOI:10.1021/acs.jafc.9b05605

Global Natural Product Social feature-based networking was applied to follow the phytochemicals, including nine flavonoid glycosides, six catechins, and three flavonols in Huangjinya green tea. Further, a new 8-O-4′-type neolignan glycoside, camellignanoside A (1), and 15 known compounds (2-16) were isolated through a variety of column chromatographies, and the structure was elucidated extensively by ultra performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry, ¹H and ¹³C nuclear magnetic resonance, heteronuclear single-quantum correlation, heteronuclear multiple-bond correlation, ¹H-¹H correlation spectroscopy, rotating frame nuclear Overhauser effect spectroscopy, and Nuclear Overhauser effect spectroscopy, and circular dichroism spectroscopies. Compounds 1 and 2 showed acetylcolinesterase inhibition activity, with IC₅₀ = 0.75 and 0.18 μM, respectively.

Full text of DOI:10.1021/acs.jafc.9b05605

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Bioactive Constituents, Metabolites, and Functions
Discovery of Neolignan Glycosides with Acetylcolinesterase
Inhibitory Activity from Huangjinya Green Tea Guided
by UPLC-MS2 data and GNPS Molecular Networking
Hao-Yue Wu, Jia-Ping Ke, Wei Wang, Ya-Shuai Kong, Peng Zhang, Tie-Jun Ling, and Guan-Hu Bao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b05605 • Publication Date (Web): 08 Oct 2019
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Journal of Agricultural and Food Chemistry
Discovery of Neolignan Glycosides with Acetylcolinesterase
Inhibitory Activity from Huangjinya Green Tea Guided by
UPLC-MS2 data and GNPS Molecular Networking
Hao-Yue Wu,† Jia-Ping Ke,† Wei Wang,† Ya-Shuai Kong,† Peng Zhang,† Tie-Jun
Ling,† Guan-Hu Bao†,*
Natural Products Laboratory, State Key Laboratory of Tea Plant Biology and
Utilization, Anhui Agricultural University, Hefei, Anhui Province 230036, China
*Corresponding author. Phone: +86-551-65786401. Fax: +86-551-65786765; E-mail:
baoguanhu@ahau.edu.cn (G.-H. Bao).
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Abstract: Global Natural Product Social (GNPS) feature based networking was
applied to follow the phytochemicals including 9 flavonoid glycosides, 6 catechins,
and 3 flavonols in Huangjinya green tea. Further, a new 8-O-4′-type neolignan
glycoside, camellignanoside A (1), together with 15 known compounds (2-16), were
isolated through a variety of column chromatography, and the structure was
4
5
6
1
7
elucidated extensively by UPLC-Q-TOF-MS2, H and 13C NMR, HSQC, HMBC,
8
1H-1H CCOY, ROESY, and NOESY, and circular dichroism (CD) spectroscopies.
Compounds 1 and 2 showed acetylcolinesterase (AChE) inhibition activity with IC50
= 0.75 and 0.18 μM, respectively.
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Keywords: Huangjinya, green tea, Camellia sinensis, neolignan glycosides, GNPS,
Acetylcholinesterase
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INTRODUCTION
Tea, made of leaf or bud plucked from the plant Camellia sinensis, is popularly
consumed all round the world. It is classified into six major types as green, yellow,
white, oolong, black, or dark tea.1-3 Among them, green tea has been studied the most
extensively because of its significant health benefits.
Huangjinya, a natural tea mutant, is one special light-sensitive albino cultivar
(Camellia sinensis var. sinensis cv. Huangjinya). The tender shoots are albino under
sun-light, whereas turn green with different shading conditions, which is just the
opposite to the case of Anji white tea.4,5 This albino mutant has high content of free
amino acids, improving drinking quality. Previous researches focused on the
characteristics and underlying formation mechanism of albino tea,6 emphasized on the
analysis of changes in transcriptome and proteome,7 and the expression of
photoreceptor genes.8 However, purification of phytochemicals from this albino
mutant has never been approached.
Here, we used GNPS to automatically analyze the phytochemicals from Huangjinya
green tea extract (HGTE) based on the tandem mass (MS/MS, MS2) data. Three major
clusters including catechins, flavonoid glycosides, as well as neolignan glycosides
were achieved according to the MS2 similarity, further supervising the isolation and
elucidation of phytochemicals in HGTE. One new 8-O-4′-type neolignan glycoside,
camellignanoside A, together with 15 known compounds (2-16) were isolated and
identified (Figure 1). Their structures were determined by analyses of their
UPLC-Q-TOF-MS/MS, 1D and 2D NMR, IR and circular dichroism (CD)
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spectroscopic data as well as comparison with those in literature.
Neolignans had been reported from various food-related plants including tea. They are
biosynthesized through the shikimic acid pathway and possess a variety of
bioactivities including antitumor,9 antioxidant,10 anti-inflammation,11 antipsychotic,12
neuroprotective,13 and anti-acetylcholinesterase activity.14
Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder, usually
associated with neuron loss in the central nervous system.13 Discovery of agents able
to inhibit the activity of acetylcholinesterase (AChE) has been reported as one
effective approach to treat AD.15 We found that tea polyphenols, especially catechins
esterzied with hydroxycinnamic acid, have strong AChE inhibition activity.15 As such,
we also tested the AChE inhibitory activity of 1 and 2, indicating that 2 showed
stronger while 1 showed less inhibition against AChE than the positive AChE
inhibitor huperzine A (hup A).
MATERIALS AND METHODS
Chemicals. HPLC grade of methanol (MeOH), acetonitrile (AcN), formic acid
(HCOOH) (DUKSAN, Ansansi, Korea) was used for HPLC analysis and purification.
Analytical grade of petroleum ether (PE), ethyl acetate (EtOAc), MeOH,
dichloromethane (CH2Cl2), were used for extraction and isolation (Chengdu Kelong
Chemical Reagent Co., Ltd, Chengdu, China). Chloroform-d (CDCl3) and dimethyl-d6
sulfoxide (DMSO-d6) were bought from Cambridge Isotope Laboratories, Inc.
(Andover, MA, USA). Column chromatography (CC) were performed with silica gel
(Yantai Jiangyou Silicon Development Co., Ltd., Shandong, China), MCI-Gel
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CHP20P (Mitsubishi Ltd., Japan), Toyopearl HW-40F (Tosoh Bioscience Shanghai
Co., Ltd., Shanghai, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB,
Sweden). All the flowing reagents were used in the AChE inhibitory activity assay: 5,
5'-dithiobis (2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide (ATCI) (Fluka,
Shanghai, China), AChE (500 U, Type VI-S Electric Eel) (Sigma-Aldrich, Shanghai,
China), hup A (Herbest, Shanxi, China) and phosphate buffer saline (PBS) (Splarbio,
Beijing, China).
NMR spectra including one dimensional (1H, 13C NMR, and DEPT-135) and two
dimensional (1H-1HCOSY, HSQC, HMBC, ROESY, and NOESY) were acquired in
DMSO-d6 with Agilent DD2 600 MHz. IR spectra were recorded on an FTIR-650
spectrometer purchased from GangDong Sci. & Tech. Development Co., Ltd. (Tianjin,
China). The Agilent 6210 UPLC-Q-TOF-MS/MS system has a photodiode array
detector (PAD), coupled to a HR-TOF-MS, with electrospray ionization (ESI) source.
Optical rotations were obtained with MCP 100 modular circular polarimeter (Anton
Paar GmbH, Graz, Austria). JASCO J-815 spectropolarimeter (JASCO, Tokyo, Japan)
was used to measure circular dichroism spectra.
Plant Sample. Huangjinya (Camellia sinensis var. sinensis cv. Huangjinya) green tea
was purchased from Anji, Zhejiang Province, China, collected in April, 2017. The tea
sample was deposited at the Natural Products Laboratory of Anhui Agricultural
University.
LC-MS2 Detection for Molecular Networking. Tea sample (160 mg) was extracted
with 70% MeOH (8 mL) in 10 mL glass flask volumetric by ultrasonic twice for 2 h
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each time at room temperature. The extract was centrifuged at 10,000 rpm for 10 min.
Took out the supernatant and filtered it with 0.22 μm syringe filters for LC-MS
analysis. The tea extracts were monitored using an Agilent 6210
UPLC-Q-TOF-MS/MS. The chromatographic separation was equipped with
ACQUITY UPLC BEH Shield RP18 column (2.1 × 150 mm, i.d., 1.7 μm). Column
temperature was set at 25 °C. The UPLC parameters were as follows: The injection
volume was 3 μL. The flow rate was 0.3 mL/min. The eluent was composed of A
(water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). The
gradient elution was as follows: 90% A for 0–1, 90-85% A for 1-2.5, 85-82% A for
2.5-7, 82-90% A for 7-8, 90-84% A for 8-10, 84-82% A for 10-10.5, 82-78% A for
10.5-12, 78-77% A for 12-14, 77-5% A for 14-15, and 5% A for 15-18, 5-90% A for
18-21 min. The UV was detected with a PAD at full-length scan ranging from 190 to
400 nm.
Molecular Networking. The complete molecular networking of HGTE was created
through
the
online
workflow
at
GNPS
platform
(https://gnps.ucsd.edu/ProteoSAFe/index.jsp?task=25d18427537b4d93b8df362c533f8
e2b). 0.02 Da parent mass and MS2 fragment ion tolerance was used to cluster the
data and create consensus spectra. Additionally, we discarded the consensus spectra
containing less than 4 spectra in the experiments. A network with edges filtered to
have a cosine score above 0.5 was then created.
Purification Procedure. Huangjinya green tea (8 kg) was ground and extracted with
PE (15 L), EtOAc (15 L), and MeOH (20 L) for three times at room temperature,
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successively. The tea leaves were entirely soaked by the solvent during all the
extraction process. The crude extract was concentrated in vacuo and fractions A-C
were got: fraction A (PE fraction, 36 g), fraction B (EtOAc fraction, 85 g), and
fraction C (MeOH fraction, 1123 g). Fraction C was dissolved in water and extracted
with CH2Cl2. The resulting water residue (800 g) was further divided into eight
fractions (C1 to C8) by MCI gel CC (60 cm × 7 cm, length × internal diameter,
abbreviated as 60 × 7 cm) eluted with MeOH-H2O (0:1 to 1:0, v/v). Fraction C4 (19.6
g) was further separated with a Toyopearl CC (45 × 3.5 cm) by methanol/water (0:1
to 1:0, v/v), 1200 mL per gradient, leading to further fractions D1 to D10. Fraction D1
was applied to a LH-20 CC (60 × 1.5 cm) eluted with MeOH (500 mL) to yield E1 to
E3. Compounds 1 (7 mg) and 2 (21 mg) from E1 were achieved through repeated
HPLC performed on an X-Bridge Prep C18 (10 × 250 mm i.d., 5 μm) at 30 °C. The
HPLC parameters were as follows: injection volume, 30 μL; flow rate, 2.0 mL/min.
The mobile phase was composed of water (A) and AcN (B). The timeline gradient of
A was (min, %, Table S1): 0–35, 87, 35-36, from 87-70, then kept at 70 for 2 min,
38-39, from 70-87%, then kept at 87% for 6 min. The UV was detected at wavelength
210 and 280 nm (Figure S1).
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Fraction C2 was applied on LH-20 CC (50 × 3.5 cm), Toyopearl CC (60 × 3.5 cm) to
yield nine fractions, F1-F9. F2 was 11 (16.5 mg), F4 was 4 (21.4 mg), F5 was 3 (28.5
mg), F6 was 5 (72.4 mg). F3 was applied on Toyopearl with MeOH to yield 8 (15.6
mg).
Fraction C3 was separated by Toyopearl CC (50 × 3.6 cm), LH-20 CC (60 × 2.5 cm)
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to yield fraction G1 to G7. Fraction G4 was subject to LH-20 CC to get 6 (62.5 mg), 7
(16.3 mg). Fraction G2 was separated by silica gel CC eluted with EtOAc-MeOH
(10:1 to 0:1) to yeild 10 (22.3 mg). Fraction G3 was applied on Toyopearl CC with
MeOH to yield 9 (13.2 mg).
Fraction B (50 g) was divided into seven fractions B1 − B7 by a MCI CC with
MeOH-H2O (1:0 to 0:1). Fraction B5 (10.5 g) was separated by a silica gel CC eluted
with CH2Cl2-MeOH solution with increasing polarity (50:1 to 0:1) to obtain eight
fractions, H1-H8. H5 was compound 12 (243.1 mg). Compound 14 (80.5 mg) was
purified from fraction B3 using Toyopearl CC. Compound 13 (175.3 mg) was isolated
from fraction B4 (8 g) using silica gel CC with a mixture of CH2Cl2 and MeOH in a
gradient elution (30:1 to 0:1) and LH-20 CC with MeOH.
Fraction D5 was applied on LH-20 CC (60 × 1.5 cm) with MeOH to give 15 (38.6 mg)
and 16 (15.6 mg).
Camellignanoside A (1), UV λmax (MeOH) nm 204.7, 228.2, 279.2. [α]25D = +4.5 (c
0.4, MeOH); CD (c 0.05, 70% aqueous CH3CN, nm (Δε)), 207 (- 4.84), 238 (+ 5.62).
IR (KBr): νmax 3385, 1605, 1511, and 1029 cm-1. HR-ESI-MS m/z 539.2119 [M-H]-
(C26H35O12, calcd 539.2129). 1H and 13C NMR data was shown in Table 1.
D
Compound 2, UV λmax (MeOH) nm, 205.8, 228.2, 279.2. [α]25 = +2.6 (c 0.08,
MeOH); CD (c 0.05, 70% aqueous CH3CN, nm (Δε)), 211 (- 1.15), 237 (+ 11.96). IR
(KBr): νmax 3373, 1604, 1513, and 1077 cm-1. HR-ESI-TOF m/z 539.2110 [M-H]-
(C26H35O12, calcd 539.2129). 1H and 13C NMR data was shown in Table 1.
Enzymatic Hydrolysis of 1 and 2. The enzymatic hydrolysis reaction was assessed
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by Zhao’s procedure with some modifications.16 Briefly, at 37 °C for 24 h, about 1.8
mg of 1 and 2 mg of 2 were respectively hydrolyzed by 40 mg β-glucosidase in 2 mL
H2O. Then the hydrolysate was extracted three times with EtOAc. The EtOAc phases
were isolated and purified to obtain aglycones 1a and 2a, separately. The aqueous
phases were isolated and purified to give glucose. The glucose moiety was identified
by GC-MS with the authentic β-D-glucose (Figure S40) using the method reported in
our previous paper.1
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Inhibition of AChE. Inhibition of AChE was conducted in accordance with the
method previously used with some modifications.15,17 The solution was composed of
160 μL of 0.001 M DTNB, 50 μL of 0.5 U mL-1 AChE, 20 μL of the test samples.
series concentration of 1 and 2 was 0.01, 0.1, 0.5, 1, 10, 100 μM. The reaction
solution was incubated at 37 °C for 20 min. Additional ATCI (20 μL, 0.0075 M) was
put in the solution and incubated again for 5 min. To determine the blank value,
AChE was replaced by the buffer (50 μL), the tested samples were replaced by 20 μL
buffer solution. Only the test compounds solution was replaced by 20 μL buffer as the
reference value. Each experimental concentration was determined trice by
SpectraMax 190 microplate reader (25 °C, 412 nm). The percent inhibition was
calculated by the formula:
AChE inhibitory activity (%) = (Ar - At) / (Ar - Ab) × 100 %, where Ar is
absorbance of reference, At is absorbance of test and Ab is absorbance of blank. Hup
A was the positive control.
Statistical Analysis. The assay experiments were conducted for at least three times,
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and the results were given as mean ± standard deviation (SD). GraphPad Prism
software (version 6.0) was the software for statistical analysis.
RESULTS AND DISCUSSION
Molecular Networking of Tea Polyphenols from Huangjinya Green Tea.
Molecular networking of HGTE on the basis of the similarity of the MS2 spectra was
generated by GNPS, including 43 clusters (node ≥ 2) and 728 single nodes.
Additional
details
could
be
referred
to
the
GNPS
platform
(https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=25d18427537b4d93b8df362c533f
8e2b).
The results revealed the presence of 1 neolignan and 3 distinct tea polyphenol clusters.
As shown in the black frame of Figure 2, GNPS generated four clusters efficiently
including neolignans (Cluster I), flavonoid glycosides (Cluster II), catechins (Cluster
III), and flavonol aglycones (Cluster IV), which can be clearly separated from each
other by the MS2 fragments. The three flavonol aglycones were identified to be
kaempferol, quercetin, and myricetin through the GNPS library (labeled as pink in
Figure S41), which were also confirmed through later purification.
The reference compound with m/z 561.1914 [M + Na]+, was identified to be a known
neolignan but was found from tea for the first time18 (labeled as green in Figure 2)
through the GNPS library and the MS2 data, suggesting that these compounds were
8-O-4′-type neolignans. Thus, neolignans are the major composition of Cluster I
(Figure 2). The compound with m/z 563.2072 [M + Na]+ was also clustered, which
was further purified and elucidated as a new neolignan and the details were presented
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and discussed in the next section. In addition, this phenomenon also shows that
compounds with the same skeleton type are clustered together.
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MS2 of flavonoid glycosides (Cluster II), was clustered into a distinct group of nodes
(Figure 3). With the help of the reference compounds quercetin- and kaempferol-
3-O-rutinoside, the m/z 773.209, 757.214, 449.106 were tentatively identified as
quercetin- and kaempferol- 3-O-galactosylrutinoside, kaempferol-3-O-glucoside
(Figure 3).19 Another series of ions with m/z 481.093, 465.103 were also clustered,
which were tentatively identified as quercetin 3-O-galactoside, myricetin
3-O-glucoside.
To study the structure of the phytochemicals in the cluster III, four catechins
including epicatechin (EC), epicatechin 3-O-gallate (ECG), epigallocatechin
3-O-gallate (EGCG), epiafzelechin 3-O-gallate (EAG) were used as references for
GNPS analysis (Figure 2). The ions of m/z 307.081 and 473.105 corresponded to
epigallocatechin (EGC) and epigallocatechin 3-O-gallate-4′′-methyl (EGCG4′′Me).
Separation and Purification of 1-16. Huangjinya tea sample was grounded and
extracted with PE, EtOAc and MeOH for three times, successively. The MeOH
fraction (fraction C) was separated by opened CC, then further by semipreparative
HPLC (Figure S1) to give a new neolignan glycoside (1).
Compound 1 was got as a colourless powder, with the [M-H]- signal at m/z
539.2119 (C26H35O12, calcd 539.2129) in negative mode, suggesting that it has a
1
molecular formula C26H36O12, with 9 degrees of unsaturation. Firstly, H NMR (600
1
MHz, DMSO-d6) combined with H-1H COSY and HSQC spectra (Table 1) showed
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two sets of ABX aromatic ring signals, δH 6.99 (s), 6.95 (d, J = 7.8 Hz), 6.78 (d, J =
7.8 Hz), and δH 6.73 (s), 6.66 (d, J = 7.8 Hz), 6.62 (d, J = 7.8 Hz). 1 aslo has
trimethylene protons [δH 1.67 (2H, m), 2.48 (2H, t, J = 1.8 Hz), 3.37 (2H, t, J = 5.4
Hz)], two methoxyl group protons [δH 3.68 (3H, s), 3.72 (3H, s)], two methіne protons
[δH 4.76 (1H, d, J = 4.2 Hz), 4.42 (1H, m)], a β-D-glucopyranosyl anomeric proton
[δH 4.08 (1H, d, J = 7.8 Hz)]. The presence of two C6-C3 units arising from neolignan
and a β-D-glucopyranoside moiety was suggested according to the 13C NMR
spectrum (Table 1). The HMBC correlations between glucopyranosyl H-1′′ and C-9,
C-1′′ and H-9 suggested that the β-D-glucopyranoside was attached at postion 9-OH.
More HMBC correlations (Figure 4) from H-8′ to C-1′, H-6′ to C-7′, H-8 to C-4′, H-8
to C-1, H-9 to C-7, H-7 to C-9, H-7 to C-2, further suggested that 1 is a
8-O-4′-neolignan 9-O-glucoside.
A large and small J value for H-7 and H-8 of 8-O-4′-neolignan diastereoisomers
correspond to the threo (around 7 Hz) and erythro (around 4 Hz) form, respectively.20
Hydrolysis of 1 with β-glucosidase produced 1a and D-glucose. JH-7,8 value of 1a is
3.6 Hz, implying that 1 could be determined to have relative erythro-configuration.21
The further CD and ROESY spectroscopic evidences could establish its absolute
configurations at C-7 and C-8. The positive Cotton effect (+5.62) at 238 nm indicated
that 1 has an 8S configuration (Figure S33).22 Clear ROESY correlation between H-7
and H-8, H-8 and H-5′ as shown in Figure 4, suggesting the H-7 has the same
β-orientation as that of H-8. Consequently, the structure of 1 was considered to be as
shown in Figure 4 with 7R, 8S-configuration.
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Compound 2 is an amorphous powder, with the same molecular formula, C26H36O12,
as that of 1, its [M-H]- signal at m/z 539.2110 (C26H35O12, calcd 539.2129) in negative
mode, with 9 degrees of unsaturation. The NMR data of compound 2 is very similar
to those of 1 (Table 1). The only difference is that the glucose group is connected at
position 7-OH for compound 2, which can be confirmed by the HMBC correlations
between H-1′′ and C-7, C-1′′ and H-7. Hydrolysis of 2 with β-glucosidase produced
2a and D-glucose. The large JH-7,8 value (8.4 Hz) (Table 1) of the aglycone (2a) and a
positive Cotton effect near 237 nm suggested that the absolute configurations at C-7
and C-8 of 2 could be established as 7S, 8S.21
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From above evidence, compounds 1 and 2 were identified as (+)
-(7R,8S)-erythro-4,7,9′-trihydroxy-8-O-4′-neolignan-9-O-β-D-glucopyranoside and
(7S,8S)-threo-4,9,9′-trihydroxy-8-O-4′-neolignan-7-O-β-D-glucopyranoside.
The other compounds were confirmed as EC (3), EGC (4), ECG (5), EGCG (6),
EAG
(7),
epicatechin-3-O-gallate-3′′-methyl
(8),
epigallocatechin
3-O-gallate-3′′-methyl (9), gallate (10), methyl gallate (11), kaempferol (12),
quercetin (13), myricetin (14), kaempferol 3-O-rutinoside (15), and quercetin
3-O-rutinoside (16).
AChE Inhibitory Assay. Compound 2 showed significant inhibition activity with
IC50 value at 0.18 μM, compared with the positive control hup A (IC50 = 0.29 μM). 1
also showed strong activity (IC50 = 0.75 μM).
We have studied the chemical constituents from green,2,23,24 white,25 and black tea,26
while paid little attention to albino tea mutants. Under certain environment, leaves of
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the albino tea cultivars become bleaching and their amino acid contents increase
obviously compared to common green leaves. However, studies on the chemical
constituents are seldom reported. Here, we first used GNPS to guide the isolation and
identification of compounds from HGTE. GNPS is a recent published Molecular
networking, which is developed as the world’s largest repository and data analysis
tool for MS2 data.27 Assisted by GNPS, we can systematically compared and
categorize large numbers of chemicals according to the structural similarities, high
throughput dereplicating natural products. Nowadays, GNPS is becoming an efficient
tool to trace natural products for purification or metabolomic purpose. In 2017, Ge et
al. extracted triterpenoid saponins from Eleutherococcus senticosus leaves (ESL),
using MS2 data networking for rapid tracking 106 triterpene saponins.28 In this paper,
with the help of GNPS, three major clusters including neolignan glycosides, flavonoid
glycosides and catechins were achieved according to the MS2 similarity, leading to
speedy discovery and structural elucidation of phtochemicals in HGTE for the first
time. Recently, phenylpropanoidated catechins,2 flavonol glycosides,1,23
flavoalkaloids,24,25 and dimeric alkaloids,3,26 have been reported from different kinds
of tea, however, neolignan glycosides have seldom been discovered from tea.
A growing body of evidence continues to emerge, demonstrating a variety of potential
health benefits of neolignans. In 2018, Şöhretoğlu et al. tested AChE, tyrosinase,
α-glucosidase, and butyrylcholinesterase (BuChE) inhibitory effects of
(-)-4-O-methyldehydrodiconiferyl alcohol 9'-O-β-glucopyranoside, which suggested
that it had good tyrosinase inhibition, however, very little inhibition against AChE,
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BuChE, and α-glucosidase.29 Kantham et al. reported that honokiol, a biphenyl
neolignan, showed moderate-to-weak cholinesterases inhibition. Further, honokiol
shared higher stability than EGCG.13 Zhao et al. isolated five pairs of 8-O-4′
neolignans from the fruit of Crataequs pinnatifida Bge, most of which exhitited
noticeable neuroprotective acitvity against H2O2-induced damage in SH-SY5Y cells.30
Sun et al. isolated a new neolignan glycoside from the roots of Spiraea salicifolia L.,
which showed significant anti-inflammatory effects.31 As such, the neolignans may
also contribute to the health benefits of tea.
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In summary, we reported the purification and structural elucidation of a new
8-O-4′-type neolignan glycoside, camellignanoside A, together with 15 known
compounds (2-16) from Huangjinya green tea guided by GNPS. The considerable
AChE inhibition of 1 and 2 suggested that this special tea albino mutant may be
potentially developed as potential agent for treatment of AD or related
neurodegenerative disorder.
ASSOCIATED CONTENT
Supporting Information
The Supporting data is available free of charge on the ACS Publications website at
DOI:
HPLC preparation method (Table S1), AChE inhibitory assay result (Table S2),
HPLC purity detection (Figure S1-2), UV spectra of 1 and 2 (Figure S3-4), NMR
spectra of 1 and 2 (Figure S5-16), HR-ESI-MS spectrum of 1-16 (Figure S17-32),
CD spectra of 1 and 2 (Figure S33-34), purity checks of 1 and 2 by HPLC with a
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chiral column (Figure S35), 1H NMR data of 1a and 2a (Figure S36-37), IR spectra
of 1 and 2 (Figure S38-39), GC-MS identification of the glucose (Glc) moiety of 1
(Figure S40), Molecular networking of flavonols (Figure S41).
AUTHOR INFORMATION
ORCID
Guan-Hu Bao: 0000-0003-4336-5678
Acknowledgements and Funding
This paper was supported by the grant from National Natural Science Foundation of
China 31972462, and Anhui Provincial Key Research and Development Plan
201904a06020011.
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Figure Captions
Figure 1. The structures of compounds 1-16 from Huangjinya green tea. Rha,
rhamnose unit; Glc, Glucose unit.
Figure 2. Clusters based molecular network of neolignan glycosides (I) and catechins
(III) in Huangjinya green tea extracts detected by the UPLC-Q-TOF-MS/MS (The
picture in black frame is the complete molecular networking of HGTE, which shows a
global overview of the spectral similarity of all MS2 spectra from this dataset.
Numbered amplified regions in the global network are shown. The nodes with color
represent the discriminating features of the project, which is at the upper right of the
figure. The arrows are showing the corresponding relationship between the structure
of compounds and the nodes in the network one by one.).
Figure 3. A: The molecular networking of flavonoid glycosides (II) in Huangjinya
green tea extracted from Figure 2 (Lables as green were compounds identified
through GNPS library and MS2 spectra (B) ). B: MS2 spectra of relative compounds.
Rha, rhamnose unit; Glc, Glucose unit.
Figure 4. Selected two dimentioanl nuclear magnetic resonance correlations (2D
NMR) including the key 1H 1H COSY (heavy solid line), HMBC (solid single
arrowhead line), and ROESY (compound 1) or NOESY (dashed double arrowhead
line) correlations of compound 2.
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Table 1. 1H and 13C NMR Data of Compounds 1 and 2.
1a
1ab
2a
δH (J, Hz)
2ab
No.
δC
δH (J, Hz)
δH (J, Hz)
δC
δH (J, Hz)
1
2
133.2
112.3
130.3
112.3
6.99 (s)
6.97 (d, 1.2)
6.87 (d, 7.8)
6.98 (s)
6.98 (d, 1.8)
3
147.4
147.3
4
5
6
7
8
146.0
115.1
120.1
146.1
115.0
6.66 (d, 7.8)
6.78 (d, 7.8)
6.66 (d, 7.8)
6.79 (d, 7.8)
4.93 (d, 4.2)
4.39 (m)
6.90 (d, 7.8)
6.93 (dd, 8.4, 1.8)
4.96 (d, 8.4)
3.97 (m)
6.82 (dd, 8.4, 1.8) 120.2
72.0 4.76 (dd, 4.2, 4.8) 4.97 (dd, 3.0, 4.2)
78.3
82.6
82.2
4.44 (m)
3.48 (dd, 11.4,
5.4)
4.11 (m)
9
68.3
3.45 (d, 1.2)
60.3
3.18 (dd, 10.8, 4.2)
3.48 (d)c
3.95 (m)
3.72 (s)
3.65 (m)
3.89 (s)
3.65 (m)
3.71 (s)
3.62 (m)
3.90 (s)
—OMe 55.9
55.9
1′
2′
135.7
113.4
135.8
6.73 (s)
6.77 (d, 1.2)
113.2
6.78 (s)
6.79 (d, 1.8)
3′
4′
149.9
146.2
149.9
146.2
5′
116.7
6.95 (d, 7.8)
6.89 (d, 7.8)
116.3
6.93 (d, 7.8)
7.03 (d, 7.8)
6′
7′
8′
9′
120.6
31.7
34.9
60.6
6.62 (d, 7.8)
2.48 (t, 1.8)
1.67 (m)
6.75 (dd, 7.8, 1.2) 120.5
6.62 (d, 7.8)
2.48 (t-like, 8.4)
1.67 (m)
6.76 (dd, 7.8, 1.8)
2.69 (t-like, 9.0)
1.89 (m)
2.69 (t-like, 7.8)
1.89 (m)
31.7
34.9
3.37 (t, 5.4)
3.68 (s)
3.69 (t, 6.0)
3.90 (s)
60.6
3.39 (t, 6.0)
3.74 (s)
3.69 (t, 6.0)
3.91(s)
—OMe 56.1
56.0
1′′
2′′
103.7
73.9
77.1
70.4
77.3
4.08 (d, 7.8)
2.95 (m)
102.7
74.7
4.39 (d, 7.8)
3.02 (m)
3′′
3.10 (m)
77.1
3.13 (m)
4′′
3.01 (m)
70.7
3.02 (m)
5′′
3.01 (m)
77.5
3.02 (m)
6′′
61.4 3.37 (m), 3.61 (m)
8.75s
61.4
3.39 (m), 3.57 (m)
8.78s
4-OH
7-OH
2′′-OH
3′′-OH
4′′-OH
6′′-OH
9′-OH
4-OH
9-OH
2′′-OH
3′′-OH
4′′-OH
6′′-OH
9′-OH
5.25 (d, 4.8)
4.96 (d, 4.8)
4.91 (d, 4.8)
4.86 (d, 3.6)
4.48 (t, 6.0)
4.42 (m)
4.62 (t, 5.4)
4.91 (d, 4.2)
4.90 (d, 4.2)
4.86 (d, 4.2)
4.28 (t, 5.4)
3.33 (m)
438
a1H at 600 MHz and 13C NMR at 150 MHz in DMSO-d6. b1H at 600 MHz in CDCl3.
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cOverlapped with other signals. s, single peak; d, double peaks; m, multipeaks.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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