One new cycloartane triterpene glycoside from Beesia calthaefolia

Article abstract of DOI:10.1080/14786419.2015.1058791

One new cycloartane triterpene glycoside (1) was isolated from the whole plant of Beesia calthaefolia. Its structure was elucidated on the basis of extensive spectroscopic data analysis. Its inhibitory effect was measured by the classical pathway of the complement system, and compared with those of known related cycloartane glycosides 2 and 3, previously isolated by us from the same plant. Compounds 1 and 2 exhibited inhibitory activity of complement system with IC₅₀of 395.3 and 214?μM, respectively. The results suggested that OH at C-12, C-18 and C-15 along with the polarity could affect the inhibitory activity.

Full text of DOI:10.1080/14786419.2015.1058791

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
One new cycloartane triterpene glycoside from
Beesia calthaefolia
Jin-Yuan Zhao, Li-Hua Mu, Xian-zhe Dong, Yuan Hu & Ping Liu
To cite this article: Jin-Yuan Zhao, Li-Hua Mu, Xian-zhe Dong, Yuan Hu & Ping Liu (2015): One
new cycloartane triterpene glycoside from Beesia calthaefolia, Natural Product Research, DOI:
10.1080/14786419.2015.1058791
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Date: 06 November 2015, At: 20:34

Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2015.1058791
One new cycloartane triterpene glycoside from Beesia calthaefolia
1
Jin-Yuan Zhao, Li-Hua Mu , Xian-zhe Dong, Yuan Hu and Ping Liu*
Department of Clinical Pharmacology, General Hospital of PLA, Beijing 100853, P.R. China
(
Received 3 February 2015; final version received 30 May 2015)
One new cycloartane triterpene glycoside (1) was isolated from the whole plant of
Beesia calthaefolia. Its structure was elucidated on the basis of extensive spectroscopic
data analysis. Its inhibitory effect was measured by the classical pathway of the
complement system, and compared with those of known related cycloartane glycosides
2
and 3, previously isolated by us from the same plant. Compounds 1 and 2 exhibited
inhibitory activity of complement system with IC50 of 395.3 and 214 mM, respectively.
The results suggested that OH at C-12, C-18 and C-15 along with the polarity could
affect the inhibitory activity.
Keywords: cycloartane triterpene glycoside; Beesia calthaefolia; complement system;
classical pathway
1
. Introduction
Beesia calthaefolia (Maxim.) Ulbr. (Ranunculaceae) is an endemic plant of China and widely
grown in southwest and northwest of China. As a herbal medicine of Chinese folk, it possesses
anti-inflammatory, analgesic and detoxification functions, relieves fever and can invigorate
blood circulation. Its rhizomes or the whole plant have been used to treat colds, rheumatic
arthritis, dysentery, sore throats and headaches (Agendae Academiae Sinicae Edita 1979).
Previous chemical investigations on this plant revealed the presence of beesiosides I-IV (Sakurai
et al. 1986, 1990, 1993; Inoue et al. 1985), beesiosides A-H and beesiosides J-P (Ju, Liu, Lin, Xu,
et al. 2002) (all the compounds named as beesiosides are cycloartane triterpene glycosides).
A number of cycloartane triterpene glycosides have been reported to show immunomodulatory
effects, including anticomplement activity (Ju, Liu, Lin, Zhang, et al. 2002; Lee et al. 2012).
Five cycloartane triterpene glycosides were isolated from the whole plant, including two known
compounds (beesiosides E and F, already cited in the text), as well as three new compounds,
including compounds named herein 2 and 3, which exhibit anticomplement activity in classical
*
Corresponding author. Email: liuping301@126.com
q 2015 Taylor & Francis

2
J.-Y. Zhao et al.
complement pathway (Mu et al. 2014). Subsequent fraction of the n-BuOH fraction yielded a
new cycloartane-type saponin (compound 1). As part of our ongoing investigation on new
cycloartane triterpene glycoside from natural source, the chemical studies led to the isolation of
one new saponin (1). In our previous study, compound 2 and 3 exhibited inhibitory activities of
complement system with IC50 of 206.0 mM and over 500 mM (Mu et al. 2014). Compounds 1–3
have similar structure. In order to compare the activities of 1 with 2 and 3, ensure data accuracy
and dependability, we re-measured the anticomplement activities of compounds 2 and 3. So we
describe herein the isolation and structural elucidation of 1, as well as the anti-complement
activity in classical complement pathway of compounds 1–3 (Figure 1).
2
2
. Results and discussion
.1. Structure of compound 1
Compound 1 was obtained as white amorphous powder and the HR-ESI-MS showed an ion peak
1
þ
at m/z 805.4353 [M þ Na] , indicating a molecular formula of C H O . The H-NMR
4
1 66 14
spectrum (Table S1) showed the presence of cyclopropane methylene groups at d 0.36 (1H, d,
J ¼ 4.2 Hz) and 0.60 (1H, d, J ¼ 4.2 Hz), seven tertiary methyls (d 0.88, 1.04, 1.69, 1.70, 1.79,
1
.83 and 1.95), indicating that 1 might be a cycloartane-type triterpenoid (Mu et al. 2014). The
C NMR spectrum of 1 displayed 41 carbon signals, three signals attributable to oxygen-
1
3
bearing quaternary carbons at d 71.9, 82.8 and 110.3. The singlet at d 71.9 was assigned to C-25
by comparison with that of beesioside O (Ju, Lin, Yang and Lu, 2002). The oxygen-bearing
quaternary carbon at d 82.8 (C-20), the acetal carbon at d 110.3 (C-24) and the methine carbon at
22
21
O
R1
2
0
24
OH
R2
O
19
11
15
R3
3
30
OH
4
R O
u
Gl
O
O
Xyl
HO
OH
HO
OH
2
8
29
OH
OH
R1
R2
R3
R4
-
3
1
OH
CH3
CH3
H
H
u
Xyl Gl
-
2
3
OH
H
Xyl
-
CH OH
OH
Xyl
2
Figure 1. Structures of compounds 1–3.

Natural Product Research
3
d 73.0 (C-16) suggested the presence of a (20R, 24R or 20S, 24S)-16b, 24 and 20, 24 diepoxy
1
structure in 1, by comparison with the C NMR spectrum of beesioside IV (Sakurai et al. 1990).
3
1
13
The H and C spectra of 1 are similar to the known compound 2 (Mu et al. 2014). In the
ROESY spectrum (Figure S1), the b configuration of the C-12 hydroxyl group was confirmed by
the correlation between H-12 and H -30. The a configurations of H-16 and H-17 were confirmed
3
by the ROESY correlations between d 1.83 (H-17) and d 0.88 (H -30), d 4.60 (H-16) and d
H
H
H
3
H
1
H -30 and H-23/H -27 in the ROESY spectrum confirmed the 20S*, 24S*configuration. Further
.83 (H-17). The correlations between H -21/H-17, H -21/H-22, H-22/H-23, H-16/H-23, H-16/
3 3
3
3
1
13
comparison of the H and C NMR data assignable to the sugar chain between 1 (Table S1) and
suggested one more set of terminal D-glucopyranose moiety signals in 1. From the coupling
2
constant value of the anomeric signal d 5.33 (1H, d, J ¼ 7.8 Hz), the D-glucosyl was deduced to
H
be of b-configuration. The b-D-xylopyranose unit was shown to be attached at C-3 by the
observation of a long range cross-peak between H-1 and C-3 (d 88.4) in the HMBC spectrum.
0
The attachment point of the xylose residue was established by analysis of HMBC and ROESY
spectra. HMBC correlation was observed between d 99.4 (Glc-C-1) and dH 4.14 (Xyl-H-3).
ROESY correlation was observed between dH 5.33 (Glc-H-1) and dH 4.14 (Xyl-H-3).
Accordingly, the structure of compound 1 was established as (20S*, 24S*)-16b, 24; 20, 24-
diepoxy-9, 19-cycloanostane-3b, 12b, 25-triol-3-O-[b-D-gluc-opyranosyl-(1→3)]-b-D-xylo-
pyranoside.
2
.2. Measuring the anticomplement activity in classical complement pathway
Cycloartane glycosides 1–3 were evaluated for anticomplement activity by measuring their
inhibitory activity on the classical complement pathway (Oh et al. 2000). The results (IC50
values) are summarised in Table 1. Compounds 1 and 2 exhibited inhibitory activities of
complement system with IC50 of 395.3 and 214 mM, respectively. On the other hand, compound
3
was inactive. The structures of 1 and 2 are similar, 1 showed less anticomplement activities
than 2 suggesting that polarity affects the inhibitory activity. The structures of 2 and 3 are also
similar, 2 showed better anticomplement activities than 3 against the classical complement
pathway suggesting that OH at C-12, C-18 and C-15 affect the inhibitory activity.
3
3
. Experimental
.1. General experimental procedures
NMR spectrum was measured in pyridine d on an INOVA 600 spectrometer (Varian Associates,
5
Inc., NMR Instruments, Palo Alto, CA, USA), using TMS as internal standard. IR spectrum was
obtained on a Nicolet 6700 FT-IR (by a KBr disk method) spectrometer (Thermo-Fisher,
Wultham, MA, USA). HR-ESI-MS spectrum was recorded using Waters SYNATT and an ESI-Q-
TOF mass spectrometer. Silica gel (200-300 mesh) (from Haiyang Chemical Group Co., Qingdao,
Shandong Province, People’s Republic of China) and ODS-A-HG (YMC Co., Ltd) were used.
Table 1. Inhibitory effects of the isolated compounds 1–3 on the complement system.
Compounds
IC50 (mM)
1
2
395.3 ^ 8.5
214.0 ^ 6.9
$500.0
b
b
3
Rosmarinic acid
a
178.0 ^ 15.6
a
This compound was used as a positive control.
The reported IC50 values for 2 and 3 were 206.0 and $500 mM, respectively (Mu et al. 2014).
b

4
J.-Y. Zhao et al.
Compounds were finally isolated with the help of a medium pressure liquid chromatography
(
MPLC) (BUCHI-ODS 3.6 cm £ 45 cm, 10 mm). Thin-layer chromatography was carried out on
silica gel GF254 (0.15–0.20 mm) (from Jiangyou silica gel Group Co., Yantai, Shandong
Province, People’s Republic of China) and the spots were visualised by spraying with 10% H SO
and heating.
2
4
3
.2. Plant material
The whole plant of B. calthaefolia was collected at Wu dang, Guiyang City, Guizhou Province,
People’s Republic of China in 2009 and identified by Dr Jianxin Zhang, Institute of Medicinal
Plant Development and Chinese Science Academy. Voucher specimen (NO. 2009010) has been
deposited in the department of clinical pharmacology, General Hospital of People’s Liberation
Army, Beijing.
3
.3. Extraction and isolation
The air-dried and pulverised whole plant of B. calthaefolia (6.9 kg) was extracted two times with
5% EtOH for 2 h under reflux and then extracted two times with 50% EtOH for 2 h under reflux.
After removal of solvent, the residue (1.0 kg) obtained by 50% EtOH was suspended in water
9
(
(
1000 mL) and partitioned successively with petroleum ether (1000 mL £ 6 mL), CHCl
3
1000 mL £ 6 mL), EtOAc (1000 mL £ 6 mL) and n-BuOH (1000 mL £ 6 mL). The n-BuOH-
soluble fraction (106 g) was subjected to low-pressure column chromatography (CC) on Si gel
200–300 mesh). Gradient elution with petroleum CHCl –MeOH (15:1, 10:1) and CHCl –
MeOH–H O (8:2:0.2, 7:3:0.5) gave six fractions, A (13.0 g), B (10.0 g), C (10.4 g), D (9.9 g),
(
3
3
2
E (10.4 g) and F (9.0 g). Fraction A was isolated by LPLC over Si gel (200–300 mesh, 400 g,
cm £ 100 cm) to give seven fractions, eluting with CHCl –MeOH (12:1–4:1) and CHCl –
2 2
4
3
3
MeOH–H O (8:2:0.2). The third was separated by MPLC (MeOH: H O ¼ 10:90–70:30) at a
flow rate of 15.0 mL/min (every 14 min to collect a fraction) to afford 1 (168 mg). Compounds 2
and 3 were isolated from the CHCl -soluble fraction by silica gel and ODS CC as previously
reported (Li et al. 2013).
3
Compound 1: White amorphous powder; 27.1 (c ¼ 0.29, MeOH); IR (KBr) vmax: 3412,
2
1
þ
2
8
940, 1638, 1384, 1164 and 1052 cm ; HR-ESI-MS m/z 805.4353 ([M þ Na] , C H ; calc.
4
1 66
1
05.4350). H NMR (C D N, 600 MHz, d): 3.48 (1H, dd, J ¼ 11.4, 4.2 Hz, H-3), 3.94 (1H, m,
5 5
H-12), 4.60 (1H, m, H-16), 1.83 (1H, d, J ¼ 6.0 Hz, H-17), 1.79 (3H, s, H-18), 0.36 (1H, d,
J ¼ 4.2 Hz, Ha-19), 060 (1H, d, J ¼ 4.2 Hz, Hb-19), 1.95 (3H, s, H-21), 1.70 (3H, s, H-26), 1.69
(
3H, s, H-27), 1.33 (3H, s, H-28), 1.04 (3H, s, H-29), 0.88 (3H, s, H-30), 4.85 (1H, d, J ¼ 7.8 Hz,
0
0
0
0
H-1 ), 4.05 (1H, m, H-2 ), 4.14 (1H, t, J ¼ 9.0 Hz, H-3 ), 4.22 (1H, m, H-4 ), 3.72 (1H, t,
0
0
00
J ¼ 10.8 Hz, Ha-5 ), 4.34 (1H, dd, J ¼ 11.4, 5.4 Hz, Hb-5 ), 5.33 (1H, d, J ¼ 7.8 Hz, H-1 ), 4.07
00 00 0 0 0
(
1H, m, H-2 ), 4.21 (1H, m, H-3 ), 4.23 (1H, m, H-4 ), 3.97 (1H, m, H-5 ), 4.38 (1H, m, Ha-6 ),
0
.49 (1H, m, Hb-6 ). C NMR (C D N, 150 MHz, d): 32.2 (C-1), 30.0 (C-2), 88.4 (C-3), 41.3
5 5
13
4
(
C-4), 47.5 (C-5), 20.9 (C-6), 26.1 (C-7), 46.3 (C-8), 20.2(C-9), 26.0 (C-10), 29.4 (C-11), 72.3
C-12), 49.1 (C-13), 51.3 (C-14), 45.5 (C-15), 73.0 (C-16), 54.6 (C-17), 14.7 (C-18), 29.8
C-19), 82.8 (C-20), 40.6 (C-22), 40.9 (C-23), 110.3 (C-24), 71.9 (C-25), 26.9, 23.1, 23.4, 25.8,
(
(
0 0 0 0
1
5.4, 20.9 (C-21, 26, 27, 28, 29, 30). 107.6 (C-1 ), 75.6 (C-2 ), 79.7 (C-3 ), 71.3 (C-4 ), 67.1
0 00 00 00 00 00 00
(
C-5 ), 99.4 (C-1 ), 78.6 (C-2 ), 78.9 (C-3 ), 75.5 (C-4 ), 78.1 (C-5 ), 63.0 (C-6 ).
3.4. Acid hydrolysis of compound 1 and determination of absolute configuration of
monosaccharides
Compound 1 (5 mg) was heated in 2 M trifluoroacetic acid (5 mL) at 958C for 5 h. The reaction
mixture was extracted with CHCl3 (5 mL £ 3 mL). The remaining aqueous layer was

Natural Product Research
5
concentrated to dryness with EtOH to give a residue. After that, the residue was dissolved
in pyridine (2 mL), to which of 3 mg of L-cysteine methyl ester hydrochloride was added.
The mixture was kept at 608C for 1 h. After the reaction, the residue was added with
trimethylchlorosilane (0.5 mL) and hexamethyldisilane (1 mL) at 608C for 0.5 h. Finally, the
supernatant (0.5 mL) was analysed by GC-MS (GC-MS-QP 2012, Shimadzu Corporation,
Japan) under the following conditions: capillary column, Rtx-5MS (30 m £ 0.25 mm
£
0.25 mm), temperature gradient: 608C–3008C, rate, 208C/min; carrier, helium gas (1.0 mL/
min); and injection volume: 1.0 mL. The presence of D-xylose and D-glucose in the acid
hydrolysate of 1 was confirmed by comparison of their respective retention times with those of
standard samples. The retention times (t , min) of monosaccharide derivatives were as follows:
R
D-xylose (9.18, 9.38 min) and D-glucose (10.24, 10.64 min).
3
.5 Anti-complement bioassays
The anti-complement assay used in this study measures inhibition of sheep red blood cell
hemolysis following induction of the complement binding reaction (Kabat & Mayer 1961; Klerx
et al. 1983). This assay was carried out according to the method of Kabat and Mayer and Klerx
et al. with modifications. The rate of hemolysis was measured spectrophotometrically and
compared to the untreated control. SRBC (two percent sensitized sheep erythrocytes) were used
for the complement binding reaction of the classical pathway. A solution of complement serum
(
100 mL) came from a healthy guinea pig (female). Each sample was dissolved in DMSO, which
was also used as the negative control. The sample (25 mL) was mixed with BBS (barbitol buffer
solution, 175 mL), serum (100 mL) and SRBC (200 mL) together. The mixture was cultured at
3
78C for 30 min in a water bath. And then, the mixture was centrifuged (48C, 5000 rpm, 20 min).
Last the optical density of the supernatant (200 mL) was measured at 405 nm (Xu et al. 2007).
The anticomplement activity was determined in triplicate measurements and was expressed as
the concentration inhibiting 50% of the complement dependent hemolysis in the control (IC50
value) (Oh et al. 2000). Purity of the isolated compounds used in the assay was above 95% as
determined by analytical HPLC/ELSD.
4
. Conclusion
Compound 1 was a new cycloartane triterpene glycoside, which was isolated from the whole
plant of B. calthaefolia. Its structure was elucidated on the basis of 1D-NMR, 2D-NMR, HR-
ESI-MS, IR, GC-MS and optical rotation spectral data. Compounds 1–3 were evaluated for their
inhibitory effects on the complement system by the classical pathway.
Supplementary material
Experimental details relating to this article are available online http://dx.doi.
org/10.1080/14786419.2015.1058791, alongside Table S1 and Figures S1–S9.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by National Nature Science Foundation of China [grant number 31000152].
We are grateful to Ms. Meifeng Xu for the NMR and HR-ESI-MS measurement in the Instrumentation
Center of the Academy of Military Medical Sciences.

6
J.-Y. Zhao et al.
Note
1
. This author contributed equally to this work.
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