Water-Assisted/Water-Accelerated Photoreaction of trans-2,3,4′,5-Tetrahydroxystilbene-2- O-β- d -glucoside from the Roots of Polygonum multiflorum

Article abstract of DOI:10.1021/acs.jafc.9b04922

The photoreaction of 2,3,4′,5-tetrahydroxystilbene-2-O-β-d-glucoside (TSG) has been investigated. Water-assisted/water-accelerated photodimerization of trans-TSG favored the formation of syn-head-to-tail [2 + 2] photocyclobutane under 365 nm irradiation as a result of hydrophobic association and a fluorescent solute-solute aggregate from their excited singlet states. In contrast, irradiation with 254 nm led to [2 + 2] photocycloreversion. The two cyclobutane dimers were first obtained through straightforward photoreaction and identified as multiflorumiside A and multiflorumiside C through the detailed analysis of high-resolution electrospray ionization mass spectrometry and one- and two-dimensional nuclear magnetic resonance. Therefore, trans-TSG should be protected from light and water.

Full text of DOI:10.1021/acs.jafc.9b04922

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Bioactive Constituents, Metabolites, and Functions
Water-assisted/water-accelerated photoreaction of
trans-TSG from the roots of Polygonum multiflorum
Ni-Man Bao, Jin Dai, Ning-Lin Liao, Wen-Feng Ying, and Rong-Hua Zhao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04922 • Publication Date (Web): 14 Oct 2019
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Water-assisted/water-accelerated photoreaction of trans-TSG from the roots of
Polygonum multiflorum
Ni-Man Baoa, Jin Daia, Ning-Ling Liaoa, Wen-Feng Yinga, Rong-Hua Zhaoa,*
a Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China
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ABSTRACT
The photoreaction of 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) has
been investigated. Water-assisted/water-accelerated photodimerization of trans-TSG
favoured the formation of syn-HT (head to tail) [2+2] photocyclobutane under 365 nm
irradiation due to hydrophobic association and a fluorescent solute-solute aggregate
from their excited singlet states. In contrast, irradiation with 254 nm led to [2+2]
photocycloreversion. The two cyclobutane dimers were first obtained through
straightforward photoreaction, and identified as multiflorumiside A and
multiflorumiside C through the detailed analysis of HRESIMS, 1D/2D NMR.
Therefore, trans-TSG should be protected from light and water.
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Keywords: [2+2] photocycloreversion-photocyclobutane; trans-TSG; Polygonum
multiflorum
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1. INTRODUCTION
Trans-2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside
(trans-TSG),
a
photosensitive stilbenoid, was first isolated in 1975 by Hata et al. from the traditional
1-2
Chinese herb Polygonum multiflorum Thunb. (Polygonaceae).
It shows diverse
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bioactivities, including antitumour effects, antioxidant activity, promotion of hair
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growth, antimelanogenic activity, neuron protection, amelioration of vascular
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dysfunction, antiaggregation, antiatherosclerosis,
improvement of age-related
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cognitive impairment, and anti-inflammatory effects.
Quantification of the
main active stilbenoid trans-TSG has been documented in Chinese Pharmacopeia as a
criterion for the quality control of Polygonum multiflorum Thunb. (in Chinese
14
Heshouwu).
The photosensitivity of trans-TSG and related bioactivities have
increasingly attracted the attention of both pharmaceutical and natural product
chemists, due to the hepatotoxicity and notable biological activities reported. Previous
reports are consistent with the conclusion that trans-TSG efficiently isomerizes into
15
cis-TSG through ultraviolet light or sunlight irradiation , and cis-TSG can induce
immunological idiosyncratic hepatotoxicity in LPS-treated rats by suppressing
PPAR-γ 16.
Purification of trans-TSG from the ethanol-extraction of Polygonum multiflorum
Thunb. is performed through D-101 resin column chromatography, fllowed by
recrystallization in aqueous solution to yield trans-TSG with purity less than 95%.
Typically, either the purity or the colour of trans-TSG not only fails to make the grade
but also decrease and darkens, respectively, through repeated recrystallization.
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Additionally, it is well known that colourless trans-TSG solid dissolved into an
aqueous darkens, especially with prolonged exposure time. The phenomenon aroused
our interest in determining whether or not trans-TSG kept its initial state in water.
Based on the photochemistry of stilbene compounds, we conducted further
investigation regarding the photoreactions of trans-TSG.
2. MATERIALS AND METHODS
2.1 General experimental procedures
NMR spectra were recorded in CD3OD using a Bruker AV600 MHz
spectrometer with TMS as an internal standard. ESIMS spectra were recorded using a
Waters Xevo TQ-S ultra-high-pressure liquid chromatography triple quadrupole mass
spectrometer. HRESIMS data were obtained using an Agilent 1290 UPLC/6540
Q-TOF mass instrument. Both HPLC-UV analysis and semi-preparation were all
performed on an Agilent 1260 HPLC system (Agilent, USA) consisting of a
quaternary solvent delivery system, an online degasser, an autosampler, a column
temperature controller and an ultraviolet detector. The chromatographic separation
was performed on an Agilent Zorbax SB-C18 column (4.6×250 mm, 5 μm) at a
solvent flow rate of 1 mL/min at 30 ℃. Water and MeOH were used as mobile phases
A and B, respectively. The solvent isocratic adopted was as follows: 0-30 min, 30% B.
The detection wavelength was 220 nm, and the injection volume was 5 μL.
Semi-preparation was carried out with same conditions described above, except
chromatographic separation was performed on an Agilent Zorbax SB-C18 column
(9.4 mm×250 mm, 5 μm) at a solvent flow rate of 3 mL/min at 30 ℃, and the injection
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volume was 100 μL.
2.2 Plant Materials
The roots of P. multiflorum were collected in Simao County of Yunnan Province,
People’s Republic of China, in October of 2017. The plant material was identified by
Prof. Rong-Hua Zhao (Yunnan University of Chinese Medicine), and a voucher
specimen (No. 20171203) was deposited at the Institute.
2.3 Extraction and Isolation
The roots of P. multiflorum (25 kg) were dried, crushed, and extracted using 90%
EtOH under reflux (80 L, 2 h × 3). A residue (2.2 kg) was obtained by removing the
solvent under reduced pressure. The residue was sequentially partitioned into EtOAc
and water. The EtOAc fraction (1.0 kg) was chromatographed on a macroporous resin
(D101) column, eluting successively with H2O, 20% EtOH, 50% EtOH, 70% EtOH,
and 95% EtOH. The 20% EtOH portion (200 g) was recrystallized repeatedly to yield
trans-TSG at 95% purity, purified by semi-preparation HPLC. Trans-TSG was
dissolved into ethanol solution by stirring, followed by 365 nm irradiation for 1 h.
Then, purification was carried out through chromatographic separation with
semi-preparation (with the same conditions as in 2.1) to yield cis-TSG, which
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identified by H and C-DEPT NMR spectra, and MS data (showed in supporting
information).
2.4 Photoreactions of trans-TSG and cis-TSG
Both trans-TSG and cis-TSG were dissolved fairly effectively in water and
MeOH individually to yield a water-solution (6 mg/mL) and MeOH-solution (6
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mg/mL); both the water-solution and MeOH-solution were exposed to UV(365 nm
and 254 nm) irradiation separately, followed by HPLC-UV analysis to investigate
photoreactional adducts in varying solvent and time conditions. Photo-induced
reactional products including a photoisomer and two dimers were purified by
semi-preparative HPLC. Two photodimer aqueous solutions were irradiated
separately with UV (254 nm) light and subjected to HPLC-UV analysis.
2.5 Quantification analysis
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Table 1 Calibration Curve of trans-TSG, cis-TSG, multiflorumiside A and
multiflorumiside C.
compounds
multiflorumiside A
cis-TSG
regression equation
y = 1681.16 x + 32.77
y = 2463.58 x - 44.21
y = 1111.43 x - 8.546
y = 1354.24 x -240.73
Linearity (μg)
0.15-6.00
r2
0.9998
0.999990
0.9992
0.9997
0.22-8.80
0.10-1.20
1.40-14.00
multiflorumiside C
trans-TSG
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The calibration curve (y = a + bx) for each stilbenoid was constructed by plotting
the peak area against the concentration, and the linearity was verified by correlation
coefficients (r2). The lower limit of detection (LOD) and lower limit of quantification
(LOQ) were determined on the basis of the response at signal-to-noise ratios (S/N) of
3 and 10, respectively. All calibration curves showed good linearity (r2 > 0.999).
3. Results and Discussion
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Compound 2 was isolated as an isomerization product from 4 (trans-TSG) in the
photoreaction, and it has been reported from Polygonum multiflorum Thunb. The
structures of 2 and 4 were identified by their NMR and MS data (Table 2 and Table 3),
which are consistent with the literature 17. Compounds 1 and 3 were purified as
dimerization products due to their molecular weights, all being 835 ([M + Na]+
ESI-MS m/z); the structures were deduced by comparing spectroscopic data with
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previous literature 18. All deduced structures were confirmed by the 2D NMR spectra;
the relative configurations of compounds 1 and 3 were determined by the major
ROESY correlations (Fig. 1), which were first reported here.
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The trans-cis isomerization of TSG efficiently occurred in both aqueous and
methanol solutions under vis/uv lights (sunlight; 254 nm and 365 nm). In addition,
photodimerizations of [2+2] cyclobutane have only been observed in trans-TSG
aqueous solution within 3 h under 365 nm or sunlight irradiation, rather than in
methanol media or under 254 nm light irradiation. In contrast, 254 nm light irradiation
for more than 3 h of the trans-TSG aqueous solution resulted in lower yields of
photodimers (Fig. 3). Investigation of 254 nm and 365 nm irradiation over 3 h both in
cis-TSG aqueous and methanol solutions separately, resulted only in inefficient
isomerization rather than dimerization. In addition, investigation of
photoisomerization and photodimerization of the trans-TSG solution in varying
thermal conditions below 100 ℃ been carried out; neither dimerization nor
isomerization were observed (Fig. 2).
Since water played an important role in the TSG photoreaction, it may be
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attributed to the hydrophobic association of the stilbenoids in aqueous solution
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facilitating molecular aggregation . It is known that the degree of a hydrophobic
interaction in aqueous solution is related to the entropy of the solution and of the
solute △ S. Values of △ S (cal K-1.mol-1) when gaseous cyclohexane is dissolved in
various solvents at 298 K are as follows: -21.5 (hexane); -20.5 (benzene); -21.5
(acetonitrile); -29.3 (methanol); and -45.0 (water) 21. A large negative value of △S is
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considered to reflect a strong solute-solute interaction 22. These data indicate that
strong solute aggregation occurs in water, resulting in the enhanced photodimerization
in the aqueous solvent. Isomerization of trans-TSG efficiently occurred both in
methanol and aqueous solutions under uv/vis light irradiation, while the
photodimerization inefficiently occurred, except in the water with 365 nm or vis
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irradiation. This seems to be consistent with a previous proposition
that
photodimerization of trans-TSG only occurred from the excited singlet states and that
trans-cis isomerization is known to occur from either the excited singlet or triplet
states.
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Table 2 H (600 MHz) and C NMR (150 MHz) data of compounds 2 and 4 in
methanol-d4 (δ, ppm).
no.
4
no.
2
δC
δH
δC
δH
1
133.75, s
137.92, s
155.95, s
1
2
133.87, s
137.94, s
155.33, s
103.79, d
152.10, s
107.62, d
125,54, d
131.25, d
129.75, s
131.43, d
115.89, d
157.77, s
115.89, d
131.43, d
2
3
3
4
103.57, d 6.27, (d, 2.8)
152.03, s
4
6.20, (d, 2.9)
5
5
6
102.68, d 6.64, (d, 2.8)
121.70, d 7.72, (d, 16.5)
130.06, d 6.92, (d, 16.5)
130.84, s
6
6.10, (d, 2.9)
6.69, (d, 12.2)
6.45, (d, 12.2)
7
7
8
8
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
1′
2′
3′
4′
5′
6′
129.22, d 7.74, (d, 8.5)
116.43, d 6.78, (d, 8.5)
157.77, s
7.04, (d, 8.4)
6.59, (d, 8.5)
116.43, d 6.78, (d, 8.5)
129.22, d 7.74, (d, 8.5)
108.21, d 4.52, (d, 7.8)
6.59, (d, 8.5)
7.04, (d, 8.4)
1′′ 107.94, d
2′′ 75.35, d
3′′ 77.92, d
4′′ 70.97, d
5′′ 78.26, d
6′′ 62.21, t
4.55, (d, 7.8)
75.47, d
77.93, d
70.76, d
78.21, d
62.06, t
3.58, (overlap)
3.45, (t-like, 9.1)
3.55, (overlap)
3.29, (m)
3.45, (m)
3.38, (m)
3.44, (m)
3.17, (ddd, 9.4, 4.7, 2.5)
3.75, (dd, 11.9, 2.6)
3.68, (dd, 11.9, 4.6)
3.83, (dd, 11.8, 2.3)
3.78, (dd, 11.8, 4.0)
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Table 3 H (600 MHz) and C NMR (150 MHz) data of compounds 1 and 3 in
methanol-d4 (δ, ppm).
No.
1
No.
3
δC
δH
δC
δH
1a
137.63, s
138.74, s
151.08, s
102.37, d
155.30, s
106.99, d
42.45, d
46.08, d
133.86, s
130.33, d
115.38, d
156.08, s
115.38, d
130.33, d
137.59, s
138.96, s
151.20, s
102.37, d
155.19, s
107.34, d
42.62, d
48.11, d
133.59,s
130.76,d
115.45,d
156.01,s
115.45,d
130.76,d
108.22,d
75.55,d
1a
2a
3a
4a
5a
6a
7a
8a
9a
136.61, s
137.11, s
151.58, s
102.37, d
155.63, s
108.25, d
41.72, d
47.95, d
132.97, s
2a
3a
4a
6.08, (d, 2.8)
6.09, (d, 2.8)
6.50, (d, 2.8)
5a
6a
6.45, (d, 2.8)
4.91, (overlap)
4.41, (overlap)
7a
4.93, (overlap)
8a
4.12, (t, overlap)
9a
10a
11a
12a
13a
14a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
1′
7.04, (d, 8.2)
6.57, (d, 8.3)
10a 130.98, d
11a 115.62, d
12a 156.39, s
13a 115.62, d
14a 130.98, d
7.01, (d, 8.2)
6.56, (d, 8.3)
6.56, (d, 8.3)
7.04, (d, 8.2)
6.56, (d, 8.3)
7.01, (d, 8.2)
1b
2b
3b
4b
5b
6b
7b
8b
9b
135.90, s
138.15, s
151.50, s
102.93, d
155.34, s
107.58, d
41.34, d
48.19, d
130.98,s
6.04, (d, 2.8)
6.10, (d, 2.8)
6.32, (d, 2.8)
6.49, (d, 2.8)
5.16, (t, 9.1)
5.04, (t, 9.1)
4.12, (dd, 10.2, 7.2)
4.29, (dd, 10.4, 6.7)
7.07, (d, 8.4)
6.52, (d, 8.3)
10b 130.76,d
11b 115.56,d
12b 156.18,s
13b 115.56,d
14b 130.76,d
7.00, (overlap)
6.55, (overlap)
6.52, (d, 8.3)
6.55, (overlap)
7.00, (overlap)
4.38, (d, 7.9)
7.07, (d, 8.4)
4.34, (d, 8.0)
1′
2′
3′
4′
5′
6′
108.12,d
74.66,d
77.12,d
70.77,d
78.11,d
61.84,t
2′
3.55, (overlap)
3.42, (m)
3.56, (m)
3′
78.14,d
3.44, (t, 9.2)
4′
70.71,d
3.57, (overlap)
3.36, (m)
3.53, (overlap)
3.09, (dt, 9.7, 3.0)
3.68, (dd, 12.0, 4.1)
3.67, (dd, 12.0, 2.4)
4.38, (d, 7.9)
5′
78.24,d
6′
62.18,t
3.88, (dd, 12.0, 4.1)
3.96, (dd, 12.0, 2.4)
4.41, (overlap)
3.56, (overlap)
3.44, (m)
1′′
2′′
3′′
4′′
5′′
6′′
107.98,d
75.49,d
78.07,d
70.64,d
78.33,d
61.93,t
1′′
2′′
3′′
4′′
5′′
6′′
107.49,d
75.53,d
77.40,d
70.62,d
78.25,d
61.84,t
3.56, (m)
3.66, (m)
3.57, (overlap）
3.44, (m)
3.69, (overlap）
3.13, (ddd, 9.7, 4.3, 2.4)
3.74, (dd, 12.2, 2.4)
3.69, (overlap)
3.88, (dd, 12.0, 4.1)
3.96, (dd, 12.0, 2.4)
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Water-assisted
photodimerization
prefers
to
form
trans-syn-HT
(multiflorumiside A) products (Fig. 5) due to not only the “Breslow hydrophobic
25
effect” and the “hydrogen-bonding effect” but also the polarity effect
.
Photocycloaddition of trans-TSG is a photo-allowed and thermo-forbidden reaction
that corresponds to the conservation of orbital symmetry rules and the frontier orbitals
theory by Woodward, Hoffmann, and Fukui for a 4π electron-system 26-28. Since no
other reagent would be more sustainable than light and no other medium greener than
water, we believe that the [2+2] photocyclobutane described here has a momentous
role as a synthetic tool to construct cyclobutane in the future.
OH
OH
OH
HO
O
OH
OH
HO
O
HO
HO
OH
O
OH
OH
O
O
O
OH
HO
OH
HO
1H-1H COSY
HMBC
OH
OH
O
HO
HO
OH
HO
OH
HO
O
HO
OH
HO
1
compound
3
compound
OH
152
OH
OH
OH
HO
O
OH
OH
HO
O
HO
OH
O
OH
OH
HO
O
O
O
OH
HO
OH
HO
1H-1H ROESY
OH
OH
O
HO
HO
OH
OH
HO
O
HO
HO
OH
1
compound
3
compound
OH
HO
153
154
Fig. 1 Major HMBC, 1H-1H COSY, and ROESY correlations of compounds 1 and 3
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Interestingly, [2+2] photocycloreversion of two dimers has been switched on by
254 nm light irradiation to form trans/cis-TSG (Fig. 4). These results are consistent
with the previously proposed conclusion that trans-TSG tends to aggregate in water
21-22; 27. The [2+2] photocycloaddition-reversion between trans-TSG and the dimers
leads to a switch of the π electron channel, which is usually accompanied by a
156
157
158
159
160
161
fluorescence change from 365 nm to 254 nm (Fig. 4)
MeOH
1
(multiflorumiside A)
 100℃
trans-TSG
cis-TSG
trans-TSG
+
162
163
164
165
166
167
168
169
170
171
+
+
vis/uv
H2O
syn-HT
anti-HT
vis/uv
+
cis-TSG
3 (
multiflorumiside C)
OH
OH
OH
HO
H
OH
HO
GlcO
OH
OGlc
OGlc
OH
H
OGlc
OH
HO
H
H
OGlc
OH
HO
syn-HT
trans-TSG
4
HO
1
HO
172
173
174
175
176
177
178
179
180
181
OH
365 nm
254 nm
+
+
photo-isomerization
anti-HT
syn-HT
OH
OH
HO
cis-TSG
OH
OH
HO
OH
HO
GlcO
OH
OGlc
HO
OGlc
H
H
GlcO
2
H
OGlc
OH
H
3
HO
anti-HT
HO
OH
OH
Fig. 2 Molecular structures concerning photoreaction of the trans-TSG including
photocycloreversion-photocyclobutane and photoisomerization.
The time course for the photolysis of trans-TSG or cis-TSG in water was
monitored by High Pressure Liquid Chromatography (HPLC) (Fig. 3). The results
indicated that the dimers multiflorumiside A and multiflorumiside C were formed
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from the excitation of the trans-TSG rather than from excitation of the cis-TSG. The
time course for the photocycloreversion of dimers multiflorumiside A and
multiflorumiside C in water was monitored by HPLC (Fig. 4). This suggests that the
photocycloreversion of the two photodimers originally disassembled into trans-TSG,
followed by isomerization to form cis-TSG. Therefore, the results indicate that 254
nm irradiation switches on the photocycloreversion of the two main photodimers
within 0.5 h due to efficient trans/cis isomerization, which are related to
Heshouwu-induced liver injury. With regards to trans-TSG aggravating
cis-TSG-induced liver injury, purification of trans-TSG should be carried out
folowing the photocycloreversion of multiflorumiside A and C.
photoisomerization
photocyclodimerization
uv
cis-
trans-
ii
i
uv
193
194
195
196
197
198
199
200
201
202
203
Fig. 3 The contents of photoreaction products (compounds 1-3) of trans-TSG in
aqueous solution for varying irradiation times (3, 4, 5, 24 and 25 h). (a is compound 1;
b is compound 2; c is compound 3; d is compound 4; 245 nm and 365 nm are the
wavelengths of fluorescent light used for irradiation); (i is the phtocyclodimerization
and
ii
is
the
photoisomerization
of
trans-TSG.)
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hv
hv
254 nm
204
isomerization
iii
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
iv
Fig. 4 The contents of photocycloreversion products (2, 4) of two photodimers (1, 3)
in aqueous media for varying irradiation times (0.5, 1.5, 2.5, 4, 5, 24 and 25 h) under
254 nm light. (a is compound 1; b is compound 2; c is compound 3; d is compound 4).
(iii is the photocycloreversion of the syn-HT product multiflorumiside A and iv is the
photocycloreversion of the anti-HT product multiflorumiside C)
uv
+
+
v
2(cis-TSG)
-
trans-
3 h
1
trans
3
4(trans-TSG)
vi
254 nm
yields
yields
0%
0%
21 h
3.91% 2.59%
3.26%
365 nm
3 h
yields
yields
5.21%
1(syn-HT)
21 h
18.13% 10.87%
254 nm
+
trans-
cis-
3(anti-HT)
0.5 h
yields
yields
1
45.21% 7.80%
0.5 h
3
18.20%
7.88%
Fig. 5 v. the yields of photoreaction products of the photocycloreversion
-photocyclobutane of trans-TSG, irradiated separately for 3 and 21 h by 254 and 365
nm light; vi. HPLC-UV chromatogram of trans-TSG in aqueous media by 365 nm
light irradiation. (1 is syn-HT product multiflorumiside A; 2 is isomerization product
cis-TSG, 3 is anti-HT product multiflorumiside C; 4 is trans-TSG).
In conclusion, trans-TSG should be protected from light and aqueous media.
This suggests that the detection of photodimerization or photoisomerization should be
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233
234
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237
238
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245
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conducted by HPLC or nuclear magnetic resonance (NMR) for cyclobutane signals
[δC 42.45(d); 46.08(d); 42.62(d); 48.11(d)] in the quality control of trans-TSG. The
usage of a fluorescence photoswitch for the photocycloreversion of the two main
photodimers by 254 nm light irradiation in order to maintain high quality control of
trans-TSG should be finished within 0.5 h (Fig. 5), due to the efficient trans/cis
isomerization, which is related to Heshouwu-induced liver injury. Owing to the
efficient fluorescence photoswitching [2+2] photocycloreversion- photocyclobutane
of TSG through 254 nm and 365 nm light irradiation in aqueous media, trans-TSG
was not only used for disease therapies but also used as a photoswitchable fluorescent
materials.
The supporting information includes 1D/2D NMR, ESI-MSHRESI-MS
spectrums of compounds 1-4, and quantitative HPLC-UV analysis of 1-4 in the
photoreaction.
ASSOCIATED CONTENT
Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
* Corresponding author
Tel: +86 13888074508.
E-mail: kmzhaoronghua@hotmail.com. (Rong-Hua Zhao)
ACKNOWLEDGMENT
We gratefully acknowledge financial support from the Application and Basis
Research Project of Yunnan China (Grant no. 2017FD111), and the Southern
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Medicine Collaborative and Innovation Center (Grant no. 30270100500), the Yunnan
Key Laboratory for Dai and Yi Medicines, Yunnan University of Chinese Medicine.
NOTES
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The authors declare no competing financial interest.
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Water-assisted/water-accelerated photoreaction of trans-TSG from the roots of
Polygonum multiflorum
Ni-Man Baoa, Jin Daia, Ning-Ling Liaoa, Wen-Feng Yinga, Rong-Hua
Zhaoa,*
a Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan
Province, China
* Corresponding author: Rong-Hua Zhao; kmzhaoronghua@hotmail.com;
Tel: +86 13888074508.
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