Cycloartane triterpenes from Beesia calthaefolia and their anticomplement structure–activity relationship study

Article abstract of DOI:10.1080/10286020.2016.1174698

Fifteen cycloartane triterpenes were isolated from Beesia calthaefolia and among them one was new cycloartane triterpenoid. The structure of new compound was determined by the application of spectroscopic analyses and chemical methods. The fifteen compoun

Full text of DOI:10.1080/10286020.2016.1174698

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Cycloartane triterpenes from Beesia calthaefolia
and their anticomplement structure–activity
relationship study
Li-Hua Mu, Jin-Yuan Zhao, Jing Zhang & Ping Liu
To cite this article: Li-Hua Mu, Jin-Yuan Zhao, Jing Zhang & Ping Liu (2016): Cycloartane
triterpenes from Beesia calthaefolia and their anticomplement structure–activity relationship
study, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2016.1174698
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1174698
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Date: 06 May 2016, At: 19:48

Journal of asian natural Products research, 2016
ꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1174698
ꢀ
Cycloartane triterpenes from Beesia calthaefolia and their
anticomplement structure–activity relationship study
a
a1
b
a
Li-Hua Mu , Jin-Yuan Zhao , Jing Zhang and Ping Liu
ꢈ
b
dꢉpꢈꢅꢁmꢉꢆꢁ ꢃꢇ cꢋꢄꢆꢄꢌꢈꢋ Pꢀꢈꢅmꢈꢌꢃꢋꢃgy, Gꢉꢆꢉꢅꢈꢋ hꢃꢊpꢄꢁꢈꢋ ꢃꢇ Pla, Bꢉꢄjꢄꢆg 100853, cꢀꢄꢆꢈ; sꢀꢈꢆxꢄ uꢆꢄvꢉꢅꢊꢄꢁy ꢃꢇ
tꢅꢈꢂꢄꢁꢄꢃꢆꢈꢋ cꢀꢄꢆꢉꢊꢉ Mꢉꢂꢄꢌꢄꢆꢉ, tꢈꢄyꢍꢈꢆ 030024, cꢀꢄꢆꢈ
ABSTRACT
ARTICLE HISTORY
Fifteen cycloartane triterpenes were isolated from Beesia calthaefolia
and among them one was new cycloartane triterpenoid. The
structure of new compound was determined by the application
of spectroscopic analyses and chemical methods. The fifteen
compounds were evaluated for their anticomplement activity by
classic pathway. The structure–activity relationship analysis indicated
that the configurations of 12-OH is preferable to be α than β, and
rꢉꢌꢉꢄvꢉꢂ 22 oꢌꢁꢃbꢉꢅ 2015
aꢌꢌꢉpꢁꢉꢂ 1 apꢅꢄꢋ 2016
KEYWORDS
Beesia calthaefolia;
ꢌyꢌꢋꢃꢈꢅꢁꢈꢆꢉ gꢋyꢌꢃꢊꢄꢂꢉ;
ꢈꢆꢁꢄꢌꢃmpꢋꢉmꢉꢆꢁ ꢈꢌꢁꢄvꢄꢁy;
ꢊꢁꢅꢍꢌꢁꢍꢅꢉ–ꢈꢌꢁꢄvꢄꢁy
ꢅꢉꢋꢈꢁꢄꢃꢆꢊꢀꢄp
18-OH can decrease while 15-OH can increase the anticomplement
activity, but saponin with both 15-OH and 18-OH lost most of its
activity. The glycosyl moiety of most isolated cycloartane triterpenes
is xylosyl. When xylosyl was substituted by glucosyl or galactosyl, their
anticomplement activities were decreased or increased, respectively.
Further structure–activity relationship (SAR) studies must be carried
out to achieve general conclusions regarding the effect of further
functionalizations on the anticomplement saponins.
1
. Introduction
Saponins are natural surfactants, found in many plants. Chemically, saponins are glyco-
sides with a distinctive foaming characteristic. ey consist of a polycyclic aglycone that
is either a steroid or triterpenoid attached at C-3 with a sugar side chain. Saponins possess
a wide range of structural diversity and versatile biological activities including anticancer,
hypocholesterolemic, immunomodulatory, antiviral, hypoglycemic, and antioxidant activity
[
1–3]. e diverse activity of saponins could be explained by its complexity of chemical
natures. Cycloartane-type triterpene glycosides are a kind of triterpenoid saponins with
,19-cycloartane. A number of cycloartane-type triterpene glycosides have been reported to
9
show immunomodulatory effects, including anticomplement activity [4, 5]. Beesia calthaefo-
lia (Maxim.) Ulbr. (Ranunculaceae) is widely distributed in the southwest and northwest of
China. Its rhizomes or the whole plant is used to treat colds, rheumatic arthritis, dysentery,
sore throats, and headaches [6]. We recently isolated 15 cycloartane triterpenes, including 14
known cycloartane triterpenes (1–14) [7–9] and 1 new cycloartane triterpenoid (15) from
CONTACT Pꢄꢆg lꢄꢍ
ꢌpꢄ301@163.ꢌꢃm
tꢀꢉ ꢊꢍppꢋꢉmꢉꢆꢁꢈꢅy ꢂꢈꢁꢈ ꢇꢃꢅ ꢁꢀꢄꢊ pꢈpꢉꢅ ꢈꢅꢉ ꢈvꢈꢄꢋꢈbꢋꢉ ꢃꢆꢋꢄꢆꢉ ꢈꢁ ꢀꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1174698.
tꢀꢄꢊ ꢈꢍꢁꢀꢃꢅ ꢌꢃꢆꢁꢅꢄbꢍꢁꢉꢂ ꢉqꢍꢈꢋꢋy ꢁꢃ ꢁꢀꢄꢊ wꢃꢅk.
1
©
2016 iꢆꢇꢃꢅmꢈ uK lꢄmꢄꢁꢉꢂ, ꢁꢅꢈꢂꢄꢆg ꢈꢊ tꢈyꢋꢃꢅ & fꢅꢈꢆꢌꢄꢊ Gꢅꢃꢍp

2
L.ꢀH. Mꢁ ꢂT AL.
Figure 1. sꢁꢅꢍꢌꢁꢍꢅꢉ ꢃꢇ ꢌꢃmpꢃꢍꢆꢂ 15.
the whole plant of Beesia calthaefolia (Figure 1). Structurally, cycloartane-type triterpene
glycosides have a general backbone structure. However, a complexity of carbohydrate and
various hydroxyls attached on the aglycone make them show different bioactivities. As part
of our ongoing study on discovery of new immunoregulative compounds from natural
source, we report here the isolation and structure identification of the new cycloartane
triterpenoid from Beesia calthaefolia. In addition, the in vitro immunosuppressive activities
of all the saponins 1–15 were assayed and the structure–activity relationship was concluded
by comparison of the structure and anticomplement activities between these compounds.
2
. Results and discussion
Compound 15 was obtained as a white powder. Its molecular formula (C H O ) was
3
6
60 11
+
deduced from the analysis of NMR and HR-ESI-MS data {m/z: 669.4222 [M+H] (calc.
1
for C H O , 669.4208)}. e H NMR spectrum of 1 (Table 1) showed the presence of
3
6
61 11
two characteristic cyclopropane protons signals at δ 0.52 and 0.21 (each d, J = 4.2 Hz),
six methyl singlets characteristic at δ 0.98, 1.21 1.28, 1.34, 1.38 and 1.51, indicating that
1
5 is a cycloartane-type triterpene. A comparison of the spectroscopic data of 15 with
those of beesioside E [10] showed that, structurally, 15 closely resembles beesioside E,
with the main differences of the sugar unit. e sugar obtained af er acid hydrolysis was
identified as ꢀ-glucose by comparing with the standard. From the coupling constants of
the anomeric signal (δ 4.95 1H, d, J = 7.8 Hz), the ꢀ-glucosyl was deduced to be β-con-
H
figuration. A correlation was observed between H-1′ (δ 4.95 1H, d, J = 7.8 Hz) and C-3
H
(
δC 88.6), suggesting that the sugar unit was attached at the C-3. In the ROESY spectrum
and ChemDraw 3D modeling using MM2 (Figure 2), H-3 showed a correlation with H-5
′
and H-1 suggesting an α-orientation of the H-3. Significant cross-peaks between H-17/H -
3
3
0, H-16/H -30, H-8β/H-15, and H-24α/Me-21/Me-27 indicated the presence of OH-15α,
3
OH-16β, and (20S*, 24R*) configurations. us, the structure of 15 was determined as (20S*,
4R*)-epoxy-9,19-cyclolanostane-3β,15α,16β,18,25-pentaol-3-O-β-ꢀ-glucopyranoside.
e isolated cycloartane glycosides 1–15 were evaluated for anticomplement activity by
measuring their inhibitory activity on the classical complement pathway [11]. e results
IC values) are summarized in Table 2, and the aglycon of compounds 1–4 is type I, while
2

(
5
0

JOꢁRꢃAL OF ASꢄAꢃ ꢃATꢁRAL PROꢅꢁꢆTS RꢂSꢂARꢆH
3
1
13
ꢈ
Table 1. h-nMr (600 Mhz) ꢈꢆꢂ c-nMr (150 Mhz) ꢊpꢉꢌꢁꢅꢈꢋ ꢂꢈꢁꢈ ꢃꢇ 15 ꢄꢆ pyꢅꢄꢂꢄꢆꢉ-d .
5
Position
δH
δꢆ
Position
δH
δꢆ
1
2
3
4
5
6
7
8
9
1.21–1.25 m,1.47–1.49 m
1.81–1.88 m,2.37–2.38 m
32.3
30.4
88.6
41.2
47.7
21.0
26.7
48.4
20.4
26.5
26.6
20
21
22-α
22-β
23-α
23-β
24
25
26
27
28
29
30
1′
–
1.38ꢊ
86.3
26.3
37.0
3.49ꢂꢂ(11.4,4.2)
1.65–1.68 m
2.48–2.51 m
1.92–1.98 m
2.37–2.38 m
3.96–3.99 m
–
–
1.31–1.34 m
0.63q(12.6)1.49–1.51 m
1.25–1.29 m,2.11–2.13 m
24.6
85.4
70.6
26.3
28.5
25.7
15.4
13.6
106.8
75.8
78.7
71.8
78.2
63.0
2.09–2.11 m
–
1.21ꢊ
1.51ꢊ
1.28ꢊ
0.98ꢊ
1
1
1
0
1-α
1-β
–
2.15–2.17 m
1.01–1.05 m
1.68–1.73 m;2.01–2.05 m
–
12
13
14
15
16
17
18
19
29.7
53.1
49.7
86.9
82.5
53.3
65.9
29.9
1.34ꢊ
4.95ꢂ(7.8)
4.02–4.03 m
4.22–4.24m
4.22–4.24m
3.95–3.97 m
–
2′
3′
4′
5′
b
4.95ꢂ(3.6)
4.72–4.75 m
2.54ꢂ(8.4)
b
4.32ꢂꢂ(4.8,12.0)4.55ꢂ(12.0)
0.21ꢂ(4.2)0.52ꢂ(4.2)
6′
4.39–4.41 m,4.53–4.57 m
ꢈ
aꢊꢊꢄgꢆmꢉꢆꢁꢊ bꢈꢊꢉꢂ ꢃꢆ tocsY, hMQc, ꢈꢆꢂ hMBc ꢉxpꢉꢅꢄmꢉꢆꢁꢊ.
ovꢉꢅꢋꢈppꢉꢂ ꢊꢄgꢆꢈꢋꢊ.
b
Figure 2. sꢉꢋꢉꢌꢁꢉꢂ roesY ꢌꢃꢅꢅꢉꢋꢈꢁꢄꢃꢆꢊ ꢇꢃꢅ ꢌꢃmpꢃꢍꢆꢂ 15.
5
–15 is type II. e structure–activity relationships were inferred by comparing of the
structure and anticomplement activities. Saponins 1 and 2 have the same sapogenin parts,
while 1 is one more β-ꢀ-glucopyranose than 2. e activities of saponin 2 > 1 suggested
that the glucosyl moiety at C-3 of xylosyl (Xyl) can decrease the anticomplement activity.
Compound 2 showed much more anticomplement activity than 3, which means both the

4
L.ꢀH. Mꢁ ꢂT AL.
Table 2. iꢆꢀꢄbꢄꢁꢃꢅy ꢉffꢉꢌꢁꢊ ꢃꢇ ꢌꢃmpꢃꢍꢆꢂꢊ 1–15 ꢃꢆ ꢁꢀꢉ ꢌꢃmpꢋꢉmꢉꢆꢁ ꢊyꢊꢁꢉm.
ꢄꢆ50 (μM)a
ꢃo.
Aglycon
R1
R2
R3
R4
R5
1
2
3
4
5
6
7
8
9
i
i
i
oh
oh
h
h
h
oh
h
h
h
h
oh
oh
oh
oh
oh
oh
oh
oh
–
h
h
oh
h
oh
oh
oh
oh
oh
oh
h
h
h
oh
oh
–
Xyꢋ-Gꢋꢍ
Xyꢋ
Xyꢋ
oaꢌ
oh
h
oh
oh
oaꢌ
oh
oh
oh
oh
oh
oh
–
–
–
–
395.3 ± 15.1
206.0 ± 21.4
naꢌ
120.2 ± 11.5
209.9 ± 18.6
naꢌ
200.1 ± 19.1
315.0 ± 25.6
407.1 ± 30.1
367.0 ± 34.2
248.0 ± 22.5
naꢌ
343.3 ± 17.8
467.4 ± 29.2
naꢌ
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
–
β-oh
β-oh
β-oh
α-oh
α-oh
β-oh
β-oh
β-oh
β-oh
α-oh
h
Xyꢋ
Xyꢋ
Xyꢋ
Xyꢋ
Xyꢋ
Xyꢋ
Xyꢋ
Gꢈꢋ
Xyꢋ
Xyꢋ
Xyꢋ
Gꢋꢍ
–
10
11
12
13
14
15
h
–
b
rꢃꢊmꢈꢅꢄꢆꢄꢌ ꢈꢌꢄꢂ
181.8 ± 13.9
ꢈ
Vꢈꢋꢍꢉꢊ ꢈꢅꢉ mꢉꢈꢆꢊ ± sd, n = 3, ic50 ꢄꢆ μM.
Pꢃꢊꢄꢁꢄvꢉ ꢌꢃꢆꢁꢅꢃꢋ.
b
ꢌ
ic50 > 500 μM.
number and location of OH at C-12, C-15, and C-18 are essential for the anticomplement
activities. To further infer the contributions of 12-OH, 15-OH, and 18-OH, more type I
cyclorartane glycosides need to be isolated. e only differences of 8 and 10, 12 and 13
are the configurations of 12-OH, 8 and 13 showed better anticomplement activity than 10
and 12, respectively, suggesting the configurations of 12-OH are preferable to be α than
β. Saponins 10 and 8 are one more 18-OH than 5 and 7, respectively, but the activities of
5
1
8
> 10 and 7 > 8 suggested that 18-OH can decrease the anticomplement activity. Saponins
0 and 8 are one more 15-OH than 12 and 13, respectively, the activities of 10 > 12 and
> 13 suggesting that 15-OH can increase the anticomplement activity. Comparing with
the activity of 9 and 10, 5 and 6, the 16-OH could increase, while 16-OAc could decrease
the anticomplement activities. Saponins (11 and 12) and saponins (14 and 15) with the
same sapogenin part showed different activities (11 > 12 and 14 > 15), indicating that
C-3 glycosyl moieties contribute differently to the anticomplement activities (Gal > Xyl,
Xyl > Glu). It is of interest to note that 4 showed stronger inhibitory activity (IC 120.2 μM)
50
than positive control (Rosmarinic acid, IC 181.8 μM). Comparing with 4, compound 9
5
0
is two more OH at C-15 and C-18, but 9 almost showed no anticomplement activity. is
may be ascribed to the steric hindrance of 15-OH and 18-OH groups. As a conclusion,
further SAR studies must be carried out to achieve general conclusions regarding the effect
of further functionalizations on the anticomplement saponins.

JOꢁRꢃAL OF ASꢄAꢃ ꢃATꢁRAL PROꢅꢁꢆTS RꢂSꢂARꢆH
5
3
. Experimental
3
.1. General experimental procedures
Optical rotations were determined on a PolAAr-3005 digital polarimeter (OA Co., Ltd,
Cambridgeshire, UK). IR spectra were obtained on a Vertex 70 FT-IR (by a KBr disk
method) spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were measured in pyri-
dine-d on a Varian INOVA 600 spectrometer (Palo Alto, CA, USA). HRESI mass spectra
5
were recorded using a Waters SYNAPT (Q-TOF) mass spectrometer. Silica gel (200–300
mesh, from Haiyang Chemical Group Co., Qingdao, China) and RI-102 ODS-A-HG (Ymc
Co., Kyoto, Japan) were used. Compounds were finally isolated with the help of a Hanbon
Sci. & Tech NP7000 preparative HPLC system equipped with a Shodex detector using an
ODS column (YMC-ODS, 20 × 250 mm, 5 μm). TLC was carried out on silica gel GF254
(
0.15–0.20 mm) (from Jiangyou silica gel Group Co., Yantai, China) and the spots were
visualized by spraying with 10% H SO and heating.
2
4
3
.2. Plant material

e whole plant of Beesia calthaefolia was collected at Wudang, Guiyang City, Guizhou
Province, China in 2009 and identified by Dr. Jianxin Zhang, key laboratory of Chemistry
for Natural Products of Guizhou Province and Chinese Academy of Sciences. e voucher
specimen (collection No 196) is deposited in the Department of Clinical Pharmacology,
PLA General Hospital.
3
.3. Extraction and isolation
e air-dried and pulverized whole plant of B. calthaefolia (6.8 kg) was extracted two times

with 95% EtOH for 2 h under reflux (80 L × 2) and then extracted with 50% EtOH for 2 h
under reflux (80 L × 2). Af er removal of solvent, the residue (1.0 kg) obtained by 50%
EtOH was suspended in water (1000 ml) and partitioned successively with petroleum ether
(
1000 ml × 6), CHCl (1000 ml × 6), EtOAc (1000 ml × 6), and n-BuOH (1000 ml × 6).
3

e CHCl -soluble fraction (170 g) was subjected to low-pressure column chromatog-
3
raphy on silica gel (200–300 mesh). Gradient elution with petroleum ether–EtOAc (1:4,
1
:6, 1:9,), EtOAc, EtOAc–MeOH (20:1, 15:1, 10:1), and MeOH gave nine fractions, A (8 g),
B (9 g), C (10 g), D (12 g), E (10 g), F (15 g), G (12 g), H (15 g), and I (9 g). e fraction D
further separated by Silica gel (200–300 mesh) eluting with CHCl –MeOH (40:1–8:1) and
3
preparative HPLC at a flow rate of 15.0 ml/min to afford 2 (300 mg, t = 19.8 min, 75%
R
MeOH). e fraction E was further separated by silica gel (200–300 mesh) eluting with
CHCl –MeOH (30:1–12:1) and preparative HPLC at a flow rate of 15.0 ml/min to afford 13
3
(
11 mg, t = 7.5 min, 75% MeOH). Fraction F was fractionated by LPLC over Silica gel H to
R
give six smaller fractions, eluting with CHCl –MeOH (40:1–8:1). e fif h was further sepa-
3
rated by ODS column chromatography [MeOH:H O(65:35→68:32→70:30→75:25→80:20)] to
2
afford 9 (9.9 mg) and by preparative HPLC at a flow rate of 15.0 ml/min to afford 4 (105 mg,
t = 21.4 min, 75% MeOH) and 3 (45 mg, t = 6.8 min, 75% MeOH). Fraction H was iso-
R
R
lated by LPLC over Silica gel H, eluting with CHCl –MeOH (40:1–9:1) to give four small
3
fractions. e fourth fraction was further separated by preparative HPLC at a flow rate of

6
L.ꢀH. Mꢁ ꢂT AL.
1
7
5.0 ml/min to afford 10 (36.7 mg, t = 4.5 min, 75% MeOH), 12 (24.7 mg, t = 12.75 min,
R
R
5% MeOH), and 14 (149 mg, t = 11.5 min, 75% MeOH), respectively.
R
e n-BuOH-soluble fraction (106 g) was subjected to low-pressure column chromatog-
raphy on Silica gel (200–300 mesh). Gradient elution with 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
3
3
2
(
9.9 g), E (10.4 g), and F (9.0 g). Fraction A was isolated by LPLC over Si gel (200–300 mesh)
to give seven fractions, eluting with CHCl –MeOH (12:1–4:1) and CHCl –MeOH–H O
3
3
2
(
8:2:0.2). e third was separated by MPLC (MeOH: H O = 10:90–70:30) at a flow rate of
5.0 ml/min to afford 1 (168 mg). Fraction B was separated by ODS column chromatog-
2
1
raphy (MeOH: H O = 10:90–70:30) at a rate of 15.0 ml/min to afford 100 fractions. e
2
7
7–78 fractions were further separated by preparative HPLC at a flow rate of 15.0 ml/min
to afford 5 (86.7 mg, t =11.0 min, 75% CH CN), 7 (52.0 mg, t =15.0 min, 75% CH CN),
R
3
R
3
and 8 (14.9 mg, t =12.9 min, 28% CH CN). Fraction C was separated by ODS column
R
3
chromatography (MeOH: H O = 30:70–85:15) at a rate of 15.0 ml/min to afford 6 (63 mg).
2
Fraction D was separated by ODS column chromatography (MeOH: H O = 15:85–70:30)
2
at a rate of 15.0 ml/min to afford 85 fractions. e 73–79 fractions were further separated
by preparative HPLC at a flow rate of 15.0 ml/min to afford 11 (66.7 mg, t =10.0 min, 75%
R
MeOH) and 15 (108.0 mg, t =13.5 min, 75% MeOH). e purity of all compounds was
R
assessed by HPLC as more than 95%.
3
.3.1. Compound 15
2
0
White amorphous powder (MeOH).[α ] + 30.6(c 0.16, MeOH); IR (KBr) vmax: 3412, 2940,
D
−
1
1
13
1
1
637, 1383, 1164 and 1051 cm ; H-NMR (C D N, 600 MHz) and C-NMR (C D N,
5 5 5 5
+
50 MHz) spectral data are given in Table 1. HR-ESI-MS: m/z 669.4222 [M + H] (calcd
for C H O , 669.4208).
3
6
61 11
3
.4. Hydrolysis of compound 15
Compound 15 (5 mg) was heated in 2 M trifluoroacetic acid (5 ml) at 95°C for 5 h. e
reaction mixture was extracted with CHCl (5 ml × 3). e remaining aqueous layer was
3
concentrated to dryness to give a residue, which was dissolved in pyridine (2 ml), to which
3
1
mg L-cysteine methyl ester hydrochloride was added. e mixture was kept at 60 °C for
h. Af er the reaction, the residue was added with trimethylchlorosilane (0.5 ml) and hex-
amethyldisilane (1 ml) at for 0.5 h. Finally, the supernatant (0.5 ml) was analyzed by GC-MS
GC-MS-QP 2012, Shimadzu Corporation, kyoto, Japan) under the following conditions:
(
capillary column, Rtx-5MS (30 m × 0.25 mm inner diameter, 0.25-mm film), detector
temperature: 60 °C-300 °C, programmed increase, 20 °C/min; carrier, helium gas (1.0 ml/
min); and injection volume: 1.0 μl. ꢀ-glucose was confirmed by comparing the retention
time with that of standard sample.
3
.5. Anticomplement bioassays
e anticomplement assay used in this study measures inhibition of sheep red blood cell

hemolysis following induction of the complement binding reaction. is assay was carried
out according to the method of Kabat and Mayer and Klerx et al. [12, 13] with modifica-
tions. e rate of hemolysis was measured spectrophotometrically and compared to the

JOꢁRꢃAL OF ASꢄAꢃ ꢃATꢁRAL PROꢅꢁꢆTS RꢂSꢂARꢆH
7
untreated control. Primary EA (sensitized erythrocytes) were used for the complement
binding reaction of the classical pathway. A diluted solution of normal guinea pig serum
(
complement serum, 100.0μl) collected from healthy guinea pigs (female) was mixed with
barbitol buffer solution (BBS, 200.0 μl) with and without sample. Each sample was dis-
solved in DMSO, which was also used as the negative control, followed by the addition of
sensitized erythrocytes (sheep red blood cells, 200.0μl). e mixture was preincubated at
3
7 °C for 30 min. Af er incubation under identical conditions, the mixture was centrifuged
4 °C, 1500 rpm), and the optical density of the supernatant (200 μl) was measured at
05 nm [14]. e anticomplement activity of each sample was determined as a mean of triple
measurements and expressed as the IC values from complement-dependent hemolysis
(
4
5
0
of the control [15]. Purity of the isolated compounds used in the assay was above 95% as
determined by analytical HPLC/ELSD.
Acknowledgments
We are grateful to Dr. Mei-Feng Xu for the NMR and HR-ESI-MS measurement in the Instrumentation
Center of the Academy of Military Medical Sciences.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was financially supported by the National Nature Science Foundation of China [grant
number 31000152].
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