A new indole-type alkaloid from the roots of Clematis florida var. plena

Article abstract of DOI:10.1080/14786419.2018.1510396

One new indole-type alkaloid, α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranosyl 6-methoxy-3-indolecarbonate (1), together with three known alkaloids (2–4), one aromatic acid (5) and five known saponins (6–10), was isolated from the roots of Clematis florida var. plena. Their structures were established by NMR spectroscopic analysis and acid hydrolysis. In in vivo anti-inflammatory activity, n-butanol extract was found to be potent against ear edema in mice, with inhibition rate of 48.7% at a dose of 800 mg/kg. Furthermore, compounds 8 and 9 obtained from the n-butanol extract exhibited significant anti-inflammatory activities with inhibition rates of 50.9% and 54.7% at a dose of 200 mg/kg.

Full text of DOI:10.1080/14786419.2018.1510396

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new indole-type alkaloid from the roots of
Clematis florida var. plena
Kai-Hui Sun, Xin-Hua Ma, Xian-Ming Zeng, Zhong-Yue Lin, Yu-Mei Cai, Hai-Tao
Zhang, Xiao-Yan Lin, Shi-Biao Feng, Tian-Hua Zhong & Yong-Hong Zhang
To cite this article: Kai-Hui Sun, Xin-Hua Ma, Xian-Ming Zeng, Zhong-Yue Lin, Yu-Mei Cai, Hai-
Tao Zhang, Xiao-Yan Lin, Shi-Biao Feng, Tian-Hua Zhong & Yong-Hong Zhang (2018): A new
indole-type alkaloid from the roots of Clematisꢀfloridaꢀvar.ꢀplena, Natural Product Research, DOI:
10.1080/14786419.2018.1510396
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1510396
A new indole-type alkaloid from the roots of Clematis
florida var. plena
Kai-Hui Suna, Xin-Hua Maa, Xian-Ming Zenga, Zhong-Yue Lina, Yu-Mei Caia,
Hai-Tao Zhanga, Xiao-Yan Lina, Shi-Biao Fenga, Tian-Hua Zhongb and
Yong-Hong Zhanga
aKey Laboratory of Natural Drug Pharmacology in Fujian Province, School of Pharmacy, Fujian
b
Medical University, Fuzhou, P. R. China; Key Laboratory of Marine Biogenetic Resources, Third
Institute of Oceanography, State Oceanic Administration, Xiamen, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 1 May 2018
Accepted 12 July 2018
One new indole-type alkaloid, a-L-rhamnopyranosyl-(1!6)-b-D-
glucopyranosyl 6-methoxy-3-indolecarbonate (1), together with
three known alkaloids (2–4), one aromatic acid (5) and five known
saponins (6–10), was isolated from the roots of Clematis florida
var. plena. Their structures were established by NMR spectroscopic
analysis and acid hydrolysis. In in vivo anti-inflammatory activity,
n-butanol extract was found to be potent against ear edema in
mice, with inhibition rate of 48.7% at a dose of 800mg/kg.
Furthermore, compounds 8 and 9 obtained from the n-butanol
extract exhibited significant anti-inflammatory activities with
inhibition rates of 50.9% and 54.7% at a dose of 200mg/kg.
KEYWORDS
Clematis florida var. plena;
alkaloid; saponins;
anti-inflammatory
1. Introduction
Clematis florida Thunb. var. plena D. Don is a perennial herbal plant belonging to the
family Ranunculaceae and the genus Clematis and is a variety of Clematis florida
Thunb. (Editorial Committee of Flora of China 1980). This variety has long been used
as a folk remedy named Shiershichen in She Ethnopharmacy, and is widely distributed
in southeast China. The roots and rhizomes of C. florida var. plena were popularly used
CONTACT Yong-Hong Zhang
zhangyh@mail.fjmu.edu.cn; Tian-Hua Zhong
zhongtianhua@tio.org.cn
Supplemental data for this article can be accessed here at https://doi.org/10.1080/14786419.2018.1510396.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
K.-H. SUN ET AL.
Figure 1. Chemical structures of compounds 1–10.
as an anti-inflammatory, anti-rheumatism and analgesic agent in She Ethnopharmacy
in Fujian Province, China (Huang et al. 2013). Chemical investigations of this genus
afforded triterpene saponins (Zhao et al. 2016), alkaloids (Li et al. 2013), flavonoids
(Chang et al. 2017), lignans (Xiong et al. 2014), coumarins (Xiong et al. 2014), macro-
cyclic compounds (Qiu et al. 2017), and phenolic glycosides (Zhang et al. 2015). In
order to search for anti-inflammatory active ingredients, we have performed systemat-
ically chemical and pharmacological investigations of C. florida var. plena. Here we
report the isolation and structural elucidation of one new indole alkaliod (1), together
with three known analogues (2–4), one aromatic acid (5) and five known triterpene
saponins (6–10) given in Figure 1, as well as the anti-inflammatory activities of the
extracted fraction and isolated compounds.
2. Results and discussion
Compound 1 was obtained as a light brown gummy material. The positive-ion
HRESIMS (Figure S1, supplementary material) showed the molecular ion [M þ Na]þ at
m/z 522.1576, corresponding to the formula C22H29NO12. Its IR spectrum showed the
absorption of ester carbonyl group at 1695 cmꢀ1 and aromatic ring absorptions at
1
1529 cmꢀ1. The H NMR spectrum (Figure S2, supplementary material) indicated the
presence of an ABX system of protons at dH 7.86 (1 H, d, J ¼ 8.8 Hz), 6.76 (1 H, dd,
J ¼ 2.2, 8.8 Hz), 6.87 (1 H, d, J ¼ 2.2 Hz), a singlet aromatic proton at dH 7.87 (1 H, s) and
a methoxy group at dH 3.73 (3 H, s) (Table S1, supplementary material). Its 13C NMR

NATURAL PRODUCT RESEARCH
3
spectrum exhibited eight aromatic carbons and an ester carbonyl carbon (dC 165.5)
(Figure S3 and Table S1, supplementary material). All investigations suggested that
compound 1 had a similar indole molecular skeleton to compound 4, however, an
ester methoxy group in 4 was found to be replaced by two hexoses in 1. The two
anomeric protons arising from the sugar moieties appeared at dH 5.60 (1 H, d, J ¼ 7.
8 Hz) and 4.63 (1 H, d, J ¼ 1.6 Hz), which correlated respectively with signals at dC 95.2
1
and 102.2 ppm in the H-13C HSQC spectrum (Figure S4, supplementary material). The
spin systems associated with monosaccharides were identified by a 1H-13C HSQC
1
experiment with the aid of H-1H COSY spectra (Figure S6, supplementary material). In
1
the H-13C HMBC spectrum (Figure S5, supplementary material), the anomeric proton
at dH 5.60 showed long-range correlation with the carbon at dC 165.5 (C-10), suggest-
ing the attachment of the glucosyl moiety at C-10. The anomeric proton at dH 4.63
indicated interaction with the carbon at dC 67.8 (C-6 of the glucose) confirming that
1
1
the rhamnosyl unit was linked to C-6 of the glucosyl unit. The H-13C HMBC and H-1H
COSY correlations are shown in Figure S7, supplementary material. The sugars were
confirmed by acid hydrolysis and comparison with authentic sugars on TLC.
Consequently, the structure of 1 was elucidated as a-L-rhamnopyranosyl-(1!6)-b-D-
glucopyranosyl 6-methoxy-3-indolecarbonate.
By comparing the physical and spectral data with the literature data, 9 known
compounds (2–10) were identified as: a-L-rhamnopyranosyl-(1!6)-b-D- glucopyranosyl
- 3-indolecarbonate (2) (Shi et al. 2006), methyl 6-O-a-L- rhamnopyranosyl -(1!6)-a-D-
glucopyranosyl 3-indolecarbonate (3) (Shi et al. 2006), methyl 6-methoxy-3-indolecar-
bonate (4) (Shi et al. 2006), Trans-isoferulic acid (5) (Prachayasittikul et al. 2009),
3-O-b-D-ribopyranosyl-(1!3)-a-L-rhamnopyranosyl
-
(1!2) -[b-D-glucopyranosyl-
(1!4)]-a-L-arabinopyranosyl oleanolic acid 28-O-a-L-rhamnopyranosyl-(1!4)-b-D-glu-
copyranosyl-(1!6)-b-D-glucopyranoside (6) (Zhao et al. 2014), Tomentoside A (7)
(Wang et al. 2013), Huzhangoside C (8) (Kizu et al. 1995), Clematangoticoside B (9)
(Zhao et al. 2016), Huzhangoside B (10) (Kizu et al. 1995), respectively. All compounds
were isolated from C. florida var. plena for the first time. The H and 13 C spectral data
1
of the isolated compounds (1–10) are given in Table S1 and Table S2.
In order to identify the biological activity, an in vivo anti-inflammatory model was used,
the results showed that the n-butanol extract of the plant exhibited potent anti-inflamma-
tory activity against ear edema in vivo, with inhibition rate of 48.7% at a dose of 800 mg/
kg (as shown in Table S3). The n-butanol subfraction (Fr. B4) exhibited potent anti-inflam-
matory activity with inhibition rate of 47.6% at a dose of 600 mg/kg. Furthermore, the
results showed that compounds 8–9 (triterpene saponins) obtained from the n-butanol
subfractions (Fr. B4) displayed significant anti-inflammatory activity with inhibition rate of
50.9% and 54.7% at a dose of 200 mg/kg. However, compound 1 (alkaloid) exhibited
weak anti-inflammatory activity with inhibition rate of 27.0% at a dose of 200 mg/kg.
3. Experimental
3.1. General experimental procedures
Column chromatography (CC): silica gel 60 (SiO2, 100–200 mesh and 200–300 mesh;
Qingdao Marine Chemical Factory), Sephadex LH-20 gel (40–70 lm; GE Healthcare,

4
K.-H. SUN ET AL.
Sweden). TLC: silica gel G (Qingdao Marine Chemical Factory). Prep. HPLC (with SPD-20A
detector): ODS columns (25 cm ꢁ 10 mm i.d.) at r.t. on a Pegasil ODS-II 5 lm column
(Waters Scientific Co., Ltd., USA).Optical rotation: TU-1901 spectrometer. UV Spectra: UV-
210A spectrometer; kmax in nm. IR spectra: Nicolet 170 SX FT-IR spectrometer; KBr pel-
lets; ꢀ in cmꢀ1. NMR spectra: Bruker NMR spectrometer; at 400 (1H) and 100 MHz (13C);
d in ppm, J in Hz. HR-ESI-MS: Bruker APEX II mass spectrometer, ESI-MS: LCMS-2020
mass spectrometer, in m/z.
3.2. Plant material
The roots of Clematis florida Thunb. var. plena D. Don were purchased from Ningde,
Fujian Province of China in May 2016 and identified by Prof. Yonghong Zhang, Fujian
Medical University. Clematis florida Thunb. var. plena D. Don is a variety of Clematis flor-
ida Thunb., and there are obvious difference in the shape of stamens between them.
The stamens in C. florida var. plena are petal-like, while wide linear in C. florida (Editorial
Committee of Flora of China 1980). A voucher specimen (No. zyh 20160502) has been
deposited in the Laboratory of the Natural Products, Fujian Medical University.
3.3. Preparation of the methanol extract and the fractions
Dried roots of C. florida var. plena (25 kg) were ground and extracted with methanol at
room temperature. The solvent was evaporated under vacuum to afford 1100 g crude
extract (yield, 4.4%). The methanol extract of C. florida var. plena (MCF) was then sus-
pended in water and partitioned successively with petroleum ether, ethyl acetate and
n-butanol. Each fraction was evaporated in vacuo to yield the residues of petroleum
ether 85.5 g (7.8%), ethyl acetate 93.1 (8.5%), n-butanol 391.7 g (35.6%), respectively.
3.4. Evaluation of anti-inflammatory activity
3.4.1. Animals
Male ICR mice (approximately 20 g each) were obtained from the Laboratory Animal
Center, Fujian Medical University and housed in temperature-controlled rooms
(22–23 ꢂC). The food and water were supplied ad libitum and each experimental group
contained 10 animals. Animals required adaptive feeding for a week, and fasting for
12 h before the experiment. All experimental protocols were conducted in accordance
with the National Institutes of Health Guide for Care and Use of Laboratory Animals
(Publication No. 85-23, revised 1985) and were approved by Committee of Ethics of
Fujian Medical University, China.
3.4.2. Ear edema induced by xylene
MCF and the fractions were suspended in a 0.5% (w/v) sodium carboxyl methylcellu-
lose (CMC-Na). The doses employed were expressed as mg of the dried extract per kg
body weight. All the treatments involved intragastric administration (i.g.) to the ani-
mals in weight-based dosages. Aspirin was used as the standard anti-inflammatory

NATURAL PRODUCT RESEARCH
5
drug. After the administration of extracts or standard drug for 1 h, each mouse was
externally treated with a test substance at 40 lL xylene solvent on surface of the right
ear, and the left ear served as the control. After 1 h of xylene application, the mice
were sacrificed by cervical dislocation. Circular sections (diameter: 6 mm) of both ears
of each mouse were removed and weighted using an electronic analytical balance
with 0.1 mg precision to calculate the inhibition of ear edema. The difference in
weight between the two plugs was taken as a measure of edematous response. The
percentage of protection was calculated using the following ratio:
ð
Þ
control mean ꢀ treated mean ꢁ 100%=control mean
3.4.3. Statistical analysis
The data represent the means SD (n ¼ 10). Differences between experimental groups
and control group were tested using student’s t test; P < 0.01 were considered
significant.
3.5. Purification
A portion of ethyl acetate extract (93.1 g) was isolated by silica gel (100–200 mesh)
column chromatography (8 ꢁ 50 cm, 750 g) and eluted with petroleum ether/EtOAc in
gradient mode (from 100:0 to 0:100, v/v) to afford eighteen fractions (Fr. A1-Fr. A18);
Fr. A11 (8.3 g) was subjected to C-18ODS (solvent: MeOH/H2O, 5:95-100:0, v/v) to give
six subfractions (Fr. A11-1 - Fr. A11-6); the subfraction Fr. A11-4 (60% MeOH, 1.9 g) was
seperated by Sephadex LH-20 (CH3Cl/MeOH 1:1) and purified by semi-PHPLC (CH3CN/
H2O 33:67, 2.0 mLꢃminꢀ1) to yield compound 4 (12.1 mg) and compound 5 (37.4 mg).
The n-butanol extract (391.7 g) was fractionated by MCI gel column and eluted with
H2O (Fr. B1), 20% MeOH (Fr. B2), 50% MeOH (Fr. B3), 70% MeOH (Fr. B4), 90% MeOH
(Fr. B5) and MeOH (Fr. B6). The Fr. B2 (27.3 g) was further subjected to Sephadex LH-20
eluted with CH3Cl/MeOH (1:1) and purified by semi-PHPLC (CH3CN/H2O 15:85,
3.0 mLꢃminꢀ1) to yield compounds 1 (55.2 mg), 2 (35.7 mg), 3 (33.4 mg). The Fr. B4
(60.2 g) was subjected to C-18ODS (solvent: MeOH/H2O, 5:95-100:0, v/v) to give six sub-
fractions (Fr. B4-1 - Fr. B4-6); the subfraction Fr. B4-4 (60% MeOH, 19.2 g) was purified
by semi-PHPLC (CH3CN/H2O 29:71, 3.0 mLꢃminꢀ1) to afford compounds 6 (79.5 mg), 7
(77.3 mg), 8 (1131.6 mg) and 9 (1193.2 mg). The Fr. B5 (33.0 g) was seperated using a
semi-PHPLC (MeOH/H2O 55:45-100:0, 3.0 mLꢃminꢀ1) to yield compound 10 (161.7 mg).
3.6. Acid hydrolysis of compound 1
Compound 1 and the authentic sugars were dissolved in methanol and spotted on a
silica gel plate, then the plate was fumigated by the steam of hydrochloric acid for
30 min, after this period, the plate was expanded by n-butanol/EtOAc/2-propanol/
HOAc/H2O (7:20:12:7:6). D-Glc and L-Rha were detected from compound 1 by compar-
ing with the authentic sugars. The Rf of the D-Glc and L-Rha were 0.53 and 0.70,
respectively.

6
K.-H. SUN ET AL.
a-L-rhamnopyranosyl-(1!6)-b-D-glucopyranosyl 6-methoxy-3-indolecarbonate (1):
light brown gum; [a]20D-121.5 (c 2.0, MeOH), UV (MeOH): kmax 286, 232, 211 nm; IR
(KBr): ꢀmax 3357, 2926, 1695, 1529, 1433, 1170, 1068, 980 cmꢀ1; HRESI-MS m/z:
522.1576 [M þ Na]þ (calcd for C22H29NO12Na, 522.1582); 1 H and 13 C NMR data, see
Table S1.
4. Conclusion
The result of xylene ear inflammation in mice showed that n-butanol extract and its
subfractions of powdered root of Clematis florida var. plena exerted anti-inflammatory
activity in vivo against ear edema. Bioactivity-guided isolation of the n-butanol yielded
one new alkaloid 1, together with three known analogues 2–4, one aromatic acid 5
and five known triterpene saponins 6–10. The n-butanol fraction exhibited significant
anti-inflammatory activity between 600 and 800 mg/kg, in a dose-dependent manner.
At 200 mg/kg dose, the anti-inflammatory activity of triterpene saponins (8, 9) was
superior to that of alkaloid (1) (50.9%, 54.7% vs 27.0%) and the content of triterpene
saponins was high in the plant, indicating that the triterpene saponins might be the
anti-inflammatory constituents. The triterpene saponins isolated from the genus
Clematis have been reported for their anti-inflammatory activity (Fu et al. 2010). C. flor-
ida var. plena was recognized to be a good source for anti-inflammatory activity may
be attributed to these constituents. The results demonstrate the anti-inflammatory
properties of C. florida var. plena which might justify the use of this plant for the treat-
ment of inflammatory disease in Chinese, folk and herbal medicine.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Natural Science Foundation of Fujian Province [grant
number 2016J01369], [grant number 2018J01847]; the Open Project of National Marine Bureau
Key Laboratory of Marine Biogenetic Resources [grant number HY201807], [grant number
HY201604]; the Science and technology innovation platform project of Fujian Province [grant
number 2018Y2001].
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