Anti-inflammatory active components of the roots of Datura metel

Article abstract of DOI:10.1080/10286020.2020.1739660

One new phenolic glycoside, methyl 3,4-dihydroxyphenylacetate-4-O-[2-O-β-D-apisoyl-6-O-(2-hydroxybenzoyl)]-β-D-glucopyranoside (1), together with 10 known compounds (2–11), were isolated from the roots of Datura metel. The structures of these compounds we

Full text of DOI:10.1080/10286020.2020.1739660

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Anti-inflammatory active components of the roots
of Datura metel
Ze Qin, Jin Zhang, Liang Chen, Shu-Xiang Liu, Hai-Feng Zhao, Hui-Min Mao,
Hong-Yang Zhang & De-Fang Li
To cite this article: Ze Qin, Jin Zhang, Liang Chen, Shu-Xiang Liu, Hai-Feng Zhao, Hui-Min Mao,
Hong-Yang Zhang & De-Fang Li (2020): Anti-inflammatory active components of the roots of
Daturaꢀmetel, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2020.1739660
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1739660
Anti-inflammatory active components of the roots of
Datura metel
Ze Qina, Jin Zhanga, Liang Chena, Shu-Xiang Liua, Hai-Feng Zhaoa, Hui-Min
Maoa, Hong-Yang Zhanga and De-Fang Lib
b
aThe Fourth Hospital of Shijiazhuang, Shijiazhuang 050011, China; School of Integrated Traditional
Chinese and Western Medicine, Binzhou Medical University, Yantai 264003, China
ABSTRACT
ARTICLE HISTORY
Received 16 July 2019
Accepted 4 March 2020
One new phenolic glycoside, methyl 3,4-dihydroxyphenylacetate-
4-O-[2-O-b-D-apisoyl-6-O-(2-hydroxybenzoyl)]-b-D-glucopyranoside
(1), together with 10 known compounds (2–11), were isolated
from the roots of Datura metel. The structures of these com-
pounds were elucidated on the basis of their spectroscopic data.
Furthermore, the in vitro anti-inflammatory activities of com-
pounds 1–11 were evaluated.
KEYWORDS
Solanaceae; Datura metel;
phenolic glycoside; anti-
inflammatory activity
1. Introduction
The majority of anesthetic drugs employed around the world originate from
China. Thousands of years of accumulation and several effective anesthetic drug
formulas have been developed, but black henbane is one of the most extensively
used drugs [1,2]. Flos daturae, a representative of black henbane drugs, is the dried
flower of Datura metel L., from the Solanaceae family. The primary effects of
CONTACT Jin Zhang
zhangjinmorning@163.com
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
Z. QIN ET AL.
Figure 1. Structures of compounds 1–11.
F. daturae include relief of dyspnea and cough, but it also exhibits antispasmodic and
pain-relieving activities. F. daturae is mainly used to treat asthma, cough, stomach
pain and chills, wind-dampness arthralgia, and infantile convulsions, but it is also
used for surgical anesthesia [3].
Chemical composition studies have shown that the main chemical components in
F. daturae are withanolides, flavonoids, alkaloids, sesquiterpenes, lignans, and phen-
olic acids. Animal experiments and clinical observations have shown that F. daturae
has a significant effect on the central nervous system, cardiovascular system, and
respiratory system, as well as possesses anti-inflammatory, anti-itch, and cell protec-
tion activities. However, previous studies mainly studied the alkaloid component of F.
daturae, which represents only 0.43% of its total content, whereas investigations on
the anti-inflammatory components of this herbal anesthetic have been limited [4,5].
To determine the anti-inflammatory pharmacodynamics and to elucidate the active
components of the roots of D. metel, we used a bioactivity-guided separation method
to conduct a systematic chemical composition study. Eleven phenolic acids were iso-
lated and identified from the ethyl acetate fraction of a 95% ethanol solution contain-
ing the dried roots of D. metel (Figure 1), which included a new phenolic glycoside,
methyl 3,4-dihydroxyphenylacetate-4-O-[2-O-b-D-apisoyl-6-O-(2-hydroxybenzoyl)]-
b-D-glucopyranoside (1), and 10 known compounds (2–11). In this article, we report
the isolation and structural elucidation of the 11 compounds (1–11). Furthermore,
the in vitro anti-inflammatory activities of compounds 1–11 were evaluated.
2. Results and discussion
Compound 1 was obtained as a white, amorphous powder with a specific rotation of
20
½aꢀD ꢁ68.3 (c 0.1, MeOH). Its molecular formula C27H32O15 was deduced from
1
HRESIMS at m/z 619.1619 [M þ Na]þ. In H-NMR spectrum (Table 1), there was a
group of proton signals from a 1,2,4-trisubstituted benzene ring (dH 6.96 (1H, m),
6.68 (1H, d, J ¼ 1.7 Hz), and 6.40 (1H, dd, J ¼ 8.3, 1.6 Hz)), and a group of proton
signals from a 1,2-disubstituted benzene ring (dH 7.80 (1H, dd, J ¼ 8.1, 1.5 Hz), 7.53
(1H, m), and 6.96 (2H, m)). In addition, there were two anomeric carbon proton sig-
nals (dH 5.27 (1H, d, J ¼ 2.8 Hz) and 4.86 (1H, d, J ¼ 7.5 Hz)), indicating that

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) spectral data of compound 1 in CD3OD.
No.
dH
dC
No.
dH
dC
1
2
3
4
5
6
7
8
129.3
117.0
147.1
144.3
117.1
120.1
40.0
172.0
100.3
79.5
76.3
70.6
73.7
64.6
100
5.27 (d, J ¼ 2.8)
3.80 (d, J ¼ 2.6)
110.3
75.8
79.0
74.0
6.68 (d, J ¼ 1.7)
200
300
400
4.08 (d, J ¼ 9.3)
3.57 (d, J ¼ 9.0)b
3.27–3.34 (m)a
6.94–6.98 (m)a
6.40 (dd, J ¼ 8.3, 1.6)
500
63.6
113.2
160.6
117.7
136.2
119.8
130.6
168.9
52.1
3.46–3.49 (m)a
1000
2000
10
20
30
40
50
60
4.86 (d, J ¼ 7.5)
3.50 (d, J ¼ 7.7)
3.46–3.49 (m)a
3.27–3.34 (m)a
3.76–3.79 (m)
3000
6. 94–6.98 (m)a
7.52–7.55 (m)
4000
5000
6. 94–6.98 (m)a
7.80 (dd, J ¼ 8.1, 1.5)
6000
7000
4.63–4.64 (m)
8-OMe
3.57 (s)b
4.33 (dd, J ¼ 11.7, 7.4)
aOverlapped with other signals.
bOverlapped each other.
compound 1 has two glycosyl groups in its structure. Both sugars were assigned D-
configuration on the basis of GC-MS analysis of the per trimethyl silylated sugars in
the hydrolysate of 1. 13C-NMR spectrum (Table 1) showed 27 carbon atom signals,
including two anomeric carbon atoms (dC 100.3 and 110.3).
According to the NMR results and the literature [6], we inferred that compound 1
is a phenolic glycoside compound containing a b-D-glucosyl group and a b-D-apisoyl
group. A comparison with the NMR data of the known compound 4-hydroxyaceto-
phenone 4-O-(5-O-(4-hydroxybenzoyl)-b-D-apisoyl)-(1!2)-b-D-glucopyranoside [7]
revealed that compound 1 contained an o-hydroxybenzoyl group and a methyl 3,4-
1
dihydroxyphenylacetate moiety, confirmed by H-NMR resonances (Table 1) at dH
7.80 (H-6000), 7.53 (H-4000), 6.96 (H-3000, 5000,5), 6.68 (H-2), 6.40 (H-6), 3.57 (8-OMe),
3.49 (H-7), and 3.46 (H-7) as well as corresponding 13C-NMR resonances at dC 172.0
(C-8), 168.9 (C-7000), 160.6 (C-2000), 147.1 (C-3), 144.3 (C-4), 136.2 (C-4000), 130.6 (C-
6000), 129.3 (C-1), 120.1 (C-6), 119.8 (C-5000), 117.7 (C-3000), 117.1 (C-5), 117.0 (C-2),
113.2 (C-1000), 52.1 (8-OMe), and 40.0 (C-7). HSQC, COSY, and HMBC (Figure 2)
further hinted at the presence of these two moieties. The heteronuclear multiple bond
correlation (HBMC) spectrum (Figure 2) showed the correlations between H-10 (dH
4.86) and C-4 (dC 144.3) and between H-60 (dH 4.64, 4.33) and C-7000 (dC 168.9), indi-
cating that the methyl 3,4-dihydroxyphenylacetate moiety and the o-hydroxybenzoyl
group are attached at the C-10 and C-60 positions, respectively. In addition, the pres-
ence of HMBC correlation between H-20 (dH 3.50) and C-100 (dC 110.3) demonstrated
the interglycosidic linkage. Therefore, compound 1 was determined to be methyl 3,4-
dihydroxyphenylacetate-4-O-[2-O-b-D-apisoyl-6-O-(2-hydroxybenzoyl)]-b-D-gluco-
pyranoside (Figure 1).
The known compounds were identified as protocatechuic acid (2) [8], protocate-
chuic aldehyde (3) [9], caffeic acid (4) [9], caffeic acid ethyl ester (5) [10], vanillic
acid (6) [8], p-coumaric acid (7) [8], p-hydroxybenzoic acid (8) [11], p-hydroxy-ben-
zenepropanoic acid (9) [10], p-hydroxyacetophenone (10) [12], and methyl benzoate
(11) [12] by NMR analysis and a comparison with those reported, and all the com-
pounds were isolated from this plant for the first time.

4
Z. QIN ET AL.
Figure 2. Key COSY and HMBC correlations of compound 1.
Table 2. Inhibitory effect of compounds 1–11 against NO production in LPS-induced
macrophages.
a
a
Compounds
IC50 (lM)
Compounds
IC50 (lM)
1
2
3
4
5
6
21.8 1.9
32.3 1.7
12.8 1.0
15.8 1.2
21.3 2.4
15.5 2.1
7
8
9
10
11
22.0 4.6
58.4 3.3
62.7 5.6
54.6 4.3
77.9 6.4
8.8 1.4
Dexamethasoneb
aResults are the mean SD (n ¼ 3).
bPositive control.
The anti-inflammatory activity results of compounds 1–11 are shown in Table 2.
Compounds 1–11 inhibited the generation of NO and showed certain anti-inflamma-
tory effects, of which compounds 3, 4, and 6 possessed most potent anti-inflamma-
tory effects. The data suggested that the anti-inflammatory effects of D. metel are
mainly due to phenolic acids and lignans [13].
3. Experimental
3.1. General experimental procedures
The main instruments and reagents used in this study are as follows: AVANCE DRX
600 M nuclear magnetic resonance (NMR) system (Bruker Biospin Corporation,
Fallanden, Switzerland); 6210 LC/MS TOF mass spectrometer (Agilent Technologies,
Santa Clara, CA, USA); P-1020 polarimeter, FT/IR-480 Plus Fourier Transform infra-
red spectrometer (JASCO Corporation, Tokyo, Japan); 210A ultraviolet (UV) spectro-
photometer (Shimadzu, Tokyo, Japan); Prostar 210 preparative high-performance
liquid chromatography (HPLC) system (VARIAN, Palo Alto, CA, USA), Welch
Material XB-C18 semi-preparative column (10 mm ꢂ 250 mm); UltiMate 3000 analyt-
ical HPLC system (DIONEX, Sunnyvale, CA, USA); Waters XBridge C18 analytical
column (4.6 mm ꢂ 250 mm, 5 lm); ELx 800 microplate reader (BioTek, Winooski,
Vermont, USA); silica gel for column chromatography (100–200 mesh, 200–300
mesh, 300–400 mesh), silica gel GF254 for thin layer chromatography (Qingdao
Marine Chemical Factory, Qingdao, China). All other reagents were of the analytical
grade and were obtained from local vendors.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3.2. Plant material
The dried roots of D. metel were purchased from Anhui Bozhou Xianggang
Professional Chinese Medicinal Herb Planting Cooperative and were identified as
such by Professor Defang Li of the Institute of Chinese Medicine of Binzhou Medical
University. The specimens (specimen number: 2017012) were stored in the specimen
room at the Institute of Chinese Medicine, Binzhou Medical University.
3.3. Extraction and isolation
The dried and pulverized roots of D. metel (15 kg) were extracted by refluxing with
95% ethanol thrice (3 ꢂ 30 L, 2 h each time), and the solvent was recovered under
reduced pressure to obtain 1.44 kg of the extract. The extract was then resuspended
in water (2 L), and the mixture was extracted sequentially with petroleum ether
(5 ꢂ 2 L), ethyl acetate (5 ꢂ 2 L), and water-saturated n-butanol (5 ꢂ 2 L). The ethyl
acetate fraction (128 g) was subjected to normal-phase silica gel column chromatog-
raphy, and gradient elution was performed with a dichloromethane-methanol system
(50:1 to 0:1) to obtain eight fractions (Fr. 1–8). Fr. 3 (28 g) was separated using flash
octadecylsilane (ODS) with methanol–water (20:80–80:20) at 25 ml/min, and then
preparative HPLC (methanol–water, 30:70–50:50) purification was carried out at
5 ml/min and an UV detection wavelength of 254 nm to yield compounds 2 (52 mg),
3 (22 mg), 5 (67 mg), and 6 (12 mg). Fr. 4 (23 g) was separated with ODS (methanol–
water, 25:75–95:5) followed by preparative HPLC eluted with methanol–water
(30:70–75:25) at 5 ml/min and an UV detection wavelength of 254 nm to yield com-
pounds 3 (24 mg), 4 (18 mg), and 7 (11 mg). Next, Fr. 5 (16 g) was subjected to ODS
column (methanol–water) (10:90–90:10) and preparative HPLC (methanol–water)
(52:48) at 5 ml/min and an UV detection wavelength of 254 nm to yield compounds
10 (19 mg) and 11 (21 mg). In a similar manner to that described for other fractions,
Fr. 6 (18 g) yielded compounds 1 (16 mg), 8 (14 mg), and 9 (28 mg).
3.3.1. Methyl 3,4-dihydroxyphenylacetate-4-O-[2-O-b-D-apisoyl-6-O-(2-hydroxyben-
zoyl)]-b-D-glucopyranoside (1)
20
White amorphous powder; ½aꢀD ꢁ68.3 (c 0.1, MeOH); UV (MeOH): kmax (lg e): 204
(3.68), 281 (2.73), 306 (2.82) nm; IR (KBr, ꢀ): 3315 (–OH), 2922, 1717 (C ¼ O), 1674,
1
1583 cmꢁ1; H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz)
spectral data see Table 1; HR-ESI-MS: m/z 619.1619 [M þ Na]þ (calcd for
C27H32O15Na, 619.1633).
3.4. Acid hydrolysis of compound 1
According to the reported method [5], compound 1 was dissolved in 2.0 ml of
0.2 mol/l H2SO4 in a water bath at 95 ꢃC for 1 h. The reaction mixture was washed
with chloroform (2 ml ꢂ 3), and then the water phase was collected and dried
through nitrogen blow down. The residue was mixed with 0.1 ml of anhydrous pyri-
dine and 0.1 ml of 0.08 N D-cysteine methyl ester hydrochloride, and the mixture was
heated at 60 ꢃC for 1 h, and then 0.1 ml of 1-trimethylsilyl imidazole was added.

6
Z. QIN ET AL.
Subsequently, the mixture was heated at 60 ꢃC for 1 h. The mixture was heated until
dry and then extracted with n-hexane and water. The n-hexane extract was injected
into the gas chromatography/mass spectrometry (GC/MS, Shimadzu, GCMS-QP2010
Ultra) system under the following conditions. The GC conditions were as follows:
Inertcap column, 0.25 mm ꢂ 30 m; injector temperature, 260 ꢃC; initial temperature,
120 ꢃC (1 min), increased at 10 ꢃC/min to 280 ꢃC, held for 10 min; carrier gas, He,
operated in the splitting mode; and injection volume, 0.5 ll. The MS conditions were
as follows: EI voltage, 70 eV; scanned-mass range, m/z 50–1000. The spectrum of acid
hydrolyzate of 1 was then compared with those of the acid hydrolyzates of D-glucose
(17.78 min) and D-apiose (14.61 min) references to confirm the presence of D-glucose
(17.77 min) and D-apiose (14.63 min) moieties in compound 1.
3.5. Evaluation of anti-inflammatory activity
According to the method reported in the literature [5], the test compound was dis-
solved in DMSO and diluted to different concentrations of solutions with Dulbecco’s
modified Eagle medium (DMEM); the final DMSO concentration was ꢄ0.25%.
Macrophages were seeded in 96-well plates at 2 ꢂ 105/well. The above prepared test
compound solutions and LPS solution (10 ng/ml) were added to the cell culture
medium and incubated for 24 h using dexamethasone as the positive control. Then,
50 ll of cell culture supernatant were collected from each well and added to 50 ll of
Griess reagent (1% sulfanilamide, 5% phosphoric acid, and 0.1% naphthylethylenedi-
amine). The absorbance of the reaction product was measured at a wavelength of
570 nm. Logarithmic regression analysis was used to calculate the half-maximal
inhibitory concentration (IC50) value.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was financially supported by the Guidance Plan of Science and Technology
Research and Development of Shijiazhuang (No. 171461533).
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