Platycosides P and Q, two new triterpene saponins from Platycodon grandiflorum

Article abstract of DOI:10.1080/10286020.2018.1488835

The EtOH extract of the roots of Platycodon grandiflorum afforded two new triterpene saponins platycoside P (1) and platycoside Q (2). Their structures were elucidated based on spectroscopic means and hydrolysis products. These compounds were evaluated for inhibitory activity against LPS-induced TNF-α production in RAW 246.7 macrophages. Compounds 1 and 2 showed inhibitory activity with the inhibition ratios (%) of 38.6 and 44.1 at 50?μM, respectively.

Full text of DOI:10.1080/10286020.2018.1488835

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Platycosides P and Q, two new triterpene saponins
from Platycodon grandiflorum
Lu Qiu, Yu Xiao, Yao-Qi Liu, Lian-xin Peng, Wan Liao & Qiang Fu
To cite this article: Lu Qiu, Yu Xiao, Yao-Qi Liu, Lian-xin Peng, Wan Liao & Qiang Fu (2018):
Platycosides P and Q, two new triterpene saponins from Platycodonꢀgrandiflorum, Journal of Asian
Natural Products Research, DOI: 10.1080/10286020.2018.1488835
To link to this article: https://doi.org/10.1080/10286020.2018.1488835
Published online: 02 Jul 2018.
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Journal of asian natural Products research, 2018
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1488835
ꢀ
Platycosides P and Q, two new triterpene saponins from
Platycodon grandiflorum
a
a
a
a,b
c
a,b
Lu Qiu , Yu Xiao , Yao-Qi Liu , Lian-xin Peng , Wan Liao and Qiang Fu
ꢇ
b
cꢄꢈꢈꢉgꢉ ꢄꢊ Pꢀꢇꢆmꢇꢋy ꢇꢌꢃ Bꢅꢄꢉꢌgꢅꢌꢉꢉꢆꢅꢌg, cꢀꢉꢌgꢃꢍ uꢌꢅvꢉꢆꢂꢅꢁy, cꢀꢉꢌgꢃꢍ 610106, cꢀꢅꢌꢇ; Kꢉy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢊ
ꢋ
cꢄꢇꢆꢂꢉ cꢉꢆꢉꢇꢈ Pꢆꢄꢋꢉꢂꢂꢅꢌg, Mꢅꢌꢅꢂꢁꢆy ꢄꢊ agꢆꢅꢋꢍꢈꢁꢍꢆꢉ, cꢀꢉꢌgꢃꢍ 610106, cꢀꢅꢌꢇ; sꢁꢇꢁꢉ Kꢉy lꢇbꢄꢆꢇꢁꢄꢆy Bꢆꢉꢉꢃꢅꢌg Bꢇꢂꢉ
ꢄꢊ syꢂꢁꢉmꢇꢁꢅꢋ rꢉꢂꢉꢇꢆꢋꢀ, dꢉvꢉꢈꢄpmꢉꢌꢁ ꢇꢌꢃ uꢁꢅꢈꢅzꢇꢁꢅꢄꢌ ꢄꢊ cꢀꢅꢌꢉꢂꢉ Mꢉꢃꢅꢋꢅꢌꢉ rꢉꢂꢄꢍꢆꢋꢉꢂ, cꢄꢈꢈꢉgꢉ ꢄꢊ Pꢀꢇꢆmꢇꢋy,
cꢀꢉꢌgꢃꢍ uꢌꢅvꢉꢆꢂꢅꢁy ꢄꢊ tꢆꢇꢃꢅꢁꢅꢄꢌꢇꢈ cꢀꢅꢌꢉꢂꢉ Mꢉꢃꢅꢋꢅꢌꢉ, cꢀꢉꢌgꢃꢍ 611137, cꢀꢅꢌꢇ
ABSTRACT
ARTICLE HISTORY
rꢉꢋꢉꢅvꢉꢃ 5 apꢆꢅꢈ 2018
aꢋꢋꢉpꢁꢉꢃ 10 Jꢍꢌꢉ 2018
The EtOH extract of the roots of Platycodon grandiflorum afforded
two new triterpene saponins platycoside P (1) and platycoside Q
(
2). Their structures were elucidated based on spectroscopic means
KEYWORDS
and hydrolysis products. These compounds were evaluated for
inhibitory activity against LPS-induced TNF-α production in RAW
cꢇmpꢇꢌꢍꢈꢇꢋꢉꢇꢉ; Platycodon
grandiflorum; ꢁꢆꢅꢁꢉꢆpꢉꢌꢉ
ꢂꢇpꢄꢌꢅꢌꢂ; tnf-α pꢆꢄꢃꢍꢋꢁꢅꢄꢌ
ꢅꢌꢀꢅbꢅꢁꢄꢆy ꢇꢋꢁꢅvꢅꢁy
2
46.7 macrophages. Compounds 1 and 2 showed inhibitory activity
with the inhibition ratios (%) of 38.6 and 44.1 at 50 μM, respectively.
1
. Introduction
Platycodon grandiflorum A. DC. (Campanulaceae) is distributed widely in northeast Asia.
Its dried roots are a source of traditional Chinese medicine “Jiegeng ,” with claims being
made concerning its anti-tumor, hepatoprotective, immunoregulatory, and anti-oxidant
properties [1,2]. Chemical investigations on this plant afforded triterpene saponins, fla-
vonoids, phenolic acids, polyacetylene, and sterols [2–5]. Several biological effects, such
as anti-inflammation, anti-viral, and anti-tumor, may be attributed to the characteristic
markers of triterpene saponins based upon platycodigenin [5–7].
In our previous work, a systematic investigation of bioactive components for the treat-
ment of inflammation, particularly on the triterpene saponins from traditional Chinese
CONTACT Qꢅꢇꢌg fꢍ
pꢉꢁꢉꢆ028@126.ꢋꢄm
©
2018 iꢌꢊꢄꢆmꢇ uK lꢅmꢅꢁꢉꢃ, ꢁꢆꢇꢃꢅꢌg ꢇꢂ tꢇyꢈꢄꢆ & fꢆꢇꢌꢋꢅꢂ Gꢆꢄꢍp

2
L. QIU ET AL.
medicine were implemented [8–10]. As part of our systematic investigation, a research on
the roots and rhizomes of P. grandiflorum led to the isolation of two new triterpene sapo-
nins (Figure 1). Although P. g randiflor u s aqueous extracts showed a clear inhibitory effect
on LPS-induced inflammation and a decrease in the expression of the inflammatory genes
TNF-α, iNOS, and COX-2, no investigation on anti-inflammation bioactivity of triterpene
saponin from P. g randiflor u s was reported. Herein, we report the isolation, structure eluci-
dation, and anti-inflammation bioactivity of two new triterpene saponins.
2
. Results and discussion
e EtOH extract of roots of P. grandiflorum was suspended in water, and then extracted by

n-butanol. e n-butanol extract was separated by repeated column chromatography using
silica gel, Sephadex LH-20, and HPLC to give two new triterpene saponins 1 and 2 (Figure 1).
Compound 1 was obtained as white amorphous powder. e molecular formula of 1
−
was found to be C H O by the negative HR-ESIMS ion at m/z 1121.5384 [M-H] . e
5
3
86 25
Figure 1. tꢀꢉ ꢂꢁꢆꢍꢋꢁꢍꢆꢉꢂ ꢄꢊ ꢋꢄmpꢄꢍꢌꢃꢂ 1 ꢇꢌꢃ 2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
13
Table 1. h ꢇꢌꢃ c nMr ꢂpꢉꢋꢁꢆꢇꢈ ꢃꢇꢁꢇ ꢊꢄꢆ ꢇgꢈyꢋꢄꢌꢉ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢊ ꢋꢄmpꢄꢍꢌꢃꢂ 1 ꢇꢌꢃ 2 (500 Mhz, ꢅꢌ c d n).
5
5
1
2
No.
δH
δc
δH
δc
1
1.52–1.54 m
45.1
1.63–1.66 m
2.01–2.05 m
5.41 ꢃ (5.0)
4.71 ꢂ
41.3
1
.90–1.93 m
2
3
4
5
6
4.03–4.06 m
4.67 ꢂ
69.7
86.6
48.0
46.8
19.2
83.3
89.4
54.1
51.7
19.4
1.83 ꢃꢃ (6.5, 3.0)
1.52–1.55 m
2.15–2.19 m
1.27–1.30 m
2.13–2.16 m
1.41–1.45 m
1.73–1.75 m
1
.81–1.85 m
1.42–1.46 m
.62–1.64 m
7
33.6
33.6
1
8
9
40.6
47.5
37.5
23.8
40.6
48.2
37.7
24.5
1.86 ꢁ (9.0)
2.02 ꢁ (9.0)
1
0
1
1
1.94–1.97 m
1.92–1.96 m
2.04–2.07 m
5.51 ꢁ (3.0)
2
.03–2.07 m
12
13
14
15
5.62 ꢁ (3.0)
123.2
144.4
42.4
122.5
145.1
42.4
1.72–1.76 m
36.1
1.29–1.32 m
2.26–2.29 m
5.17–5.20 m
36.1
2
.30–2.34 m
16
17
18
19
5.24–5.27 m
73.9
49.7
41.5
47.4
74.0
49.6
41.5
47.2
3.54 ꢃꢃ (3.5, 13.5)
1.35–1.38 m
3.50 ꢃꢃ (3.5, 13.5)
1.33–1.36 m
2.76–2.81 m
2
.76–2.78 m
2
0
1
31.0
36.1
31.1
36.1
2
1.27–1.32 m
.35–2.39 m
2.14–2.17 m
.25–2.30 m
4.02–4.06 m
.48–4.52 m
4.11–4.15 m
.59–4.61 m
1.25–1.30 m
2.33–2.36 m
2.15–2.18 m
2.26–2.29 m
4.33–4.36 m
4.74–4.78 m
2
22
23
24
32.0
63.3
63.5
32.1
57.3
2
4
178.4
4
25
26
27
28
29
30
1.42 ꢂ
1.07 ꢂ
1.71 ꢂ
18.4
17.5
27.2
175.8
33.1
24.6
1.27 ꢂ
1.02 ꢂ
1.69 ꢂ
17.7
18.2
27.5
176.1
33.2
24.8
0.96 ꢂ
1.11 ꢂ
0.94 ꢂ
1.09 ꢂ
spectral features and physicochemical properties revealed 1 to be a triterpenoid saponin.
1

1
e H NMR spectrum displayed the signals of five tertiary methyl groups at δ 0.96, 1.07,
1
3
.11, 1.42, and 1.71 and one olefinic proton at δ 5.62 (t, J = 3.0 Hz). e C NMR spectrum
3
2
showed the presence of five sp carbons at δ 17.5, 18.4, 24.6, 27.2, and 33.1, and two sp
olefinic carbons at δ 123.2 and 144.4, and five oxygenated methylene and methine carbons at
1
13
δ 63.3, 63.5, 69.7, 73.9, and 86.6 (Table 1). Further comparison of the H and C NMR data
of 1 with reported values revealed the aglycone of 1 to be platycodigenin (2β,3β,16α,23,24-
pentahydroxyolean-12-en-28-oic acid) [5]. e downfield shif of C-3 at δ 86.6 and the
upfield shif of C-28 at δ 175.8 suggested that 1 was a bisdesmosidic saponin.
1

e H NMR spectrum of 1 exhibited four anomeric proton resonances at δ 4.94 (d,
J = 8.0 Hz), 5.17 (d, J = 8.0 Hz), 6.49 (brs), and 5.80 (brs). Acid hydrolysis of 1 yielded glu-
cose, arabinose, and rhamnose, which were detected by TLC comparison with authentic

4
L. QIU ET AL.
1
13
Table 2. h ꢇꢌꢃ c nMr ꢂpꢉꢋꢁꢆꢇꢈ ꢃꢇꢁꢇ ꢊꢄꢆ ꢂꢍgꢇꢆ mꢄꢅꢉꢁꢅꢉꢂ ꢄꢊ ꢋꢄmpꢄꢍꢌꢃꢂ 1 ꢇꢌꢃ 2 (500 Mhz, ꢅꢌ c d n).
5
5
1
2
No.
δH
δc
δH
δc
Gꢈꢋ
1
2
3
4
5
6
4.94 ꢃ (8.0)
4.04–4.09 m
4.06–4.10 m
4.01–4.03 m
3.88–3.90 m
105.5
74.1
88.2
69.8
78.1
62.3
5.21 ꢃ (8.0)
3.93–3.96 m
4.11–4.14 m
4.09–4.13 m
3.97–3.99 m
4.24 ꢃꢃ (12.0, 6.0)
4.75 ꢃꢃ (12.0, 2.0)
5.01 ꢃ (8.0)
4.01–4.03 m
4.13–4.15 m
4.14–4.17 m
3.86–3.89 m
4.27 ꢃꢃ (12.0, 6.0)
4.48 ꢃꢃ (12.0, 2.0)
6.48 bꢆꢂ
4.48–4.51 m
4.49–4.51 m
4.36–4.40 m
3.89–3.93 m
4.52–4.56 m
5.81 bꢆꢂ
105.1
74.7
78.2
71.2
77.1
70.0
4.22 ꢃꢃ (12.0, 6.0)
4
.46 ꢃꢃ (12.0, 2.0)
Gꢈꢋ′
1
2
3
4
5
6
5.17 ꢃ (8.0)
105.4
75.5
78.2
71.7
78.6
62.5
105.5
75.3
78.3
71.6
78.4
62.5
4.03–4.05 m
4.24–4.27 m
4.11–4.14 m
4.02–4.06 m
4.20 ꢃꢃ (12.0, 6.0)
4
.60 ꢃꢃ (12.0, 2.0)
aꢆꢇ
1
2
3
4
5
6.49 bꢆꢂ
93.6
75.4
70.3
66.2
63.0
93.5
75.5
70.1
66.0
63.1
4.48–4.52 m
4.49–4.51 m
4.37–4.42 m
3.91–3.95 m
4
.52–4.57 m
rꢀꢇ
1
2
3
4
5
6
5.80 bꢆꢂ
101.5
72.3
72.6
73.8
70.4
18.5
101.4
72.4
72.7
73.7
70.5
18.5
4.47–4.49 m
4.48–4.51 m
4.24–4.28 m
4.54–4.56 m
1.67 ꢃ (6.0)
4.47–4.51 m
4.48–4.52 m
4.26–4.29 m
4.54–4.57 m
1.66 ꢃ (5.5)
samples. e absolute configuration of the monosaccharides was determined to be D for
glucose, and L for arabinose and rhamnose by GC analysis of chiral derivatives of the
monosaccharides in the hydrolysate of each compound (see Section 3). e relatively large
1
coupling constants (8.0 Hz) for the anomeric protons in the H NMR spectrum of 1 sug-
gested that the glucopyranosyl moiety has a β-configuration. e α-configurations of the
rhamnopyranosyl and arabinopyranosyl moieties were determined from the broad singlets
1
observed for the anomeric protons. e large J
value of the rhamnopyranosyl moiety
H-1,C-1
(
172 Hz) confirmed that the anomeric proton was equatorial (α-pyranoid anomeric form).

e spin–spin coupling system of individual monosaccharide units was identified by
1
analysis of 1D TOCSY and 2D NMR spectra. H NMR spectral data of individual mono-
saccharide units were obtained by selective irradiation of the anomeric protons or methyl
groups of rhamnopyranosyl units in a series of 1D TOCSY experiments. Analysis of the
1
1
H- H COSY spectrum resulted in sequential assignment of all proton resonances of the
four monosaccharide units, including the identification of most of their multiple splitting
patterns and coupling constants, as shown in Table 2. In the HSQC experiment, proton
resonances were correlated with those of the corresponding carbons, and associated ano-
meric protons were correlated with their respective carbon atoms from HSQC-TOCSY data,
leading to unambiguous assignments of the carbons in each monosaccharide unit (Table
2
). Considering the known effects of O-glycosylation, 1 contains two D-glucopyranosyl
units (Glc), one L- arabinopyranosyl unit (Ara), and one L- rhamnopyranosyl unit (Rha).
In the HMBC spectrum, the anomeric proton signals at δ 4.94 (Glc-H-1), 5.17 (Glc′-H-1),
6
.49 (Ara-H-1), and 5.80 (Rha-H-1) showed cross-peaks with the carbon signals at δ 86.6

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 2. sꢉꢈꢉꢋꢁꢉꢃ hMBc ꢋꢄꢆꢆꢉꢈꢇꢁꢅꢄꢌꢂ ꢄꢊ ꢋꢄmpꢄꢍꢌꢃ 2.
(
Aglycone-C-3), 88.2 (Glc-C-3), 175.8 (Aglycone-C-28), and 75.4 (Ara-C-2), respectively.

ese signals provide evidence to determine the linkages between the sugars, and the sugar
and the aglycone. ese linkages were also confirmed by NOESY correlations. From the
above evidence, the structure of 1 was established as 3-O-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl platycodigenin 28 O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl
ester, and named platycoside P.
Compound 2 was obtained as a white amorphous powder. e molecular formula of
-
2
was found to be C H O by the negative HR-ESIMS ion at m/z 1117.5063 [M-H] .
5
3
82 25

e IR spectrum exhibited absorptions at 3433 (OH) and 1740 (γ-lactone carbonyl). e
1
13
H and C NMR spectroscopic data (Table 1) of 2 indicated that 2 has an olean-12-en
skeleton. Comparison of NMR data for 2 and 1 revealed that two compounds are similar.

e differences found were the signals of C-24 and C-2, which appeared at down-field
δ 178.4 and 83.3 in 2 instead of δ 63.5 and 69.7 in 1, respectively. e lactone carbon
of C-24 was observed to be correlated with H-2 (δ 5.41) based on the HMBC spectrum
(
Figure 2). Further comparison NMR data for 2 with reported values revealed the aglycone
of 2 to be platycogenic acid A lactone (3β,16α,23-trihydroxyolean-12-en-2,24-lacton-28-
oic acid), produced via intramolecular esterification between the 24-carboxylic acid and
the 2-hydroxy group of platycogenic acid A [5,11]. Signals due to sugar moieties linked
to C-3 and C-28 of aglycone were almost superimposable between 1 and 2 except for the
linkage site of the Glc′, which was connected to Glc-C-6 in 2 instead of to Glc-C-3 in 1
according to HMBC correlations observed between Glc′-H-1 and Glc-C-6. is was fur-
ther supported by NOE correlations observed between Glc′-H-1 and Glc-H-6. us, the
structure of 2 was characterized as 3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl
platycogenic acid A lactone 28 O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl
ester, and named platycoside Q.
Compounds 1 and 2 were evaluated for inhibitory activity against LPS-induced TNF-α
production in RAW 246.7 macrophages (See Section 3). Curcumin was used as positive
control (IC = 7.4 ± 1.4 μM). Compounds 1 and 2 showed inhibitory activity with the
5
0
inhibition ratios (%) of 38.6 and 44.1 at 50 μM, respectively.

6
L. QIU ET AL.
3
. Experimental
3
.1. General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco, Tokyo,
Japan). IR spectra were recorded on a Bruker IFS-55 plus spectrometer (Bruker, Ettlingen,
German). NMR spectra were recorded on an Inova 500 spectrometer (Bruker, Waltham,
MA, USA). HRESI/MS measurements were performed on a Bruker-Daltonics APES-III 7.0
TESLA FTMS spectrometer (Bruker, Billerica, MA, USA). High-performance liquid chroma-
tography (HPLC) separation was carried out on an octadecylsilanized column (YMC-pack
ODS-A, 250 × 10 mm, i.d. 5 μm, YMC, Kyoto, Japan) with a photo-diode array detector
(
Waters, Millford, MA, USA). GC was performed with a SHIMADZU GC-14D. Column
chromatography was performed with silica gel (Merck, Darmstadt, Germany) and C18 silica
gel (Merck).
3
.2. Plant material
e roots of Platycodon grandiflorum were collected in Aug 2016 in Taihe, Anhui Province,

China. e identification of the plant was performed by the author (Qiang Fu). A voucher
specimen (PG 201608) is maintained in the herbarium of College of Pharmacy and
Bioengineering, Chengdu University.
3
.3. Extraction and isolation

e dried roots of P. grandiflorum (5.5 kg) were extracted with 95% EtOH (40 L). Af er
removing the solvent, the residue (849.3 g) was suspended in H O and extracted with
2
n-BuOH (233.1 g). e n-BuOH extract was subjected to silica gel column chromatog-
raphy (CC) using eluent mixtures of CHCl /MeOH (10:1 → 1:2) to afford fractions 1–5.
3
Fraction 3 (23.9 g) was subjected to C silica gel chromatography and eluted in a gradient
1
8
of MeOH–H O (10:90 → 95:5) to afford sub-fractions 1–4 (3.9, 1.7, 12.7, and 1.8 g, respec-
2
tively). Sub-fraction 4 (1.8 g) was subjected to Sephadex LH-20 column chromatography
(
MeOH) and prep-HPLC (ACN-H O, 15:85 → 40:60, 2.0 ml/min, UV detection at 210 nm),
2
affording compounds 1 (33.9 mg, 24.8 min) and 2 (29.4 mg, 28.3 min).
3
.3.1. Platycoside P (1)
2
0
−1
White amorphous powder; [ꢀ] − 23.3 (c 1.0, MeOH); IR (KBr) v (cm ): 3425, 1746,
D
max
1
13
and 1072; H and C NMR spectral data, see Tables 1 and 2. HR-ESIMS: m/z 1121.5384
−
[
M-H] (calcd for C H O ,1121.5380).
5
3
85 25
3
.3.2. Platycoside Q (2)
2
0
−1
White amorphous powder; [ꢀ] − 18.4 (c 1.0, MeOH); IR (KBr) v (cm ): 3433, 1740,
D
max
1
13
and 1070; H and C NMR spectral data, see Tables 1 and 2. HR-ESIMS: m/z 1117.5063
−
[
M-H] (calcd for C H O , 1117.5067).
5
3
81 25

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3
.4. Acid hydrolysis
Compounds 1 and 2 (5.0 mg, respectively) in 2 M of trifluoroacetic acid (1.0 ml) were heated
at 110 °C for 2 h, and then dried by N gas. Af er cooling, the reaction mixture was neu-
2
tralized with Amberlite IRA-400 resin (OH–form) and the resin was filtered. Af er removal
of the solvent from the filtrate under pressure, the residue was passed through a Sep-Pak
C18 cartridge with H O and MeOH. e H O eluate was concentrated and the residue was
2
2
treated with L-cysteine methyl ester hydrochloride (2.0 mg) in pyridine (1.0 ml) at 60 °C for
h. Af er being dried under N2 gas, the residue was treated with N-(trimethylsilyl) imidazol
0.2 ml) at 60 °C for 1 h. e reaction was ended by adding water (1.0 ml), and extracted
2
(
with cyclohexane (1.0 ml, thrice). e cyclohexane layer was collected and concentrated to
1
.0 ml for GC analysis. Separations were carried out on a HP-5 column (28 m × 0.32 mm,
−1
Agilent, Santa Clara, CA, USA). Highly pure He was employed as carrier gas (1.0 ml·min
flow rate), and the FID detector was operated at 260 °C (column temp 180 °C). e retention
times of the monosaccharide derivatives were as follows: L-Rha, 7.13 min; D-Glc, 12.84 min;
and L-Ara, 6.31 min.
3
.5. Assay for inhibitory activity against LPS-induced TNF-α production
Tested compounds were separately dissolved in DMSO, and diluted with phosphate buffered
saline (PBS) to a final concentration of 10 mM. Such solution was then diluted to various
5
test concentrations. RAW 264.7 macrophages were seeded in 96 well plates (1 × 10 cells/
well). Cells were co-incubated with tested samples and lipopolysaccharide (LPS, 1.0 μg/ml)
for 24 h. TNF-α production levels were determined using a commercially available TNF-α
ELISA kit (BioLegend, Inc., CA) according to the protocol provided by the manufacturers.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was supported by the Innovation Team Foundation of Educational Commission of Sichuan
Province of China [grant number 18TD0049].
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