A new sulfated triterpene glycoside from the sea cucumber Colochirus quadrangularis, and evaluation of its antifungal, antitumor and immunomodulatory activities

Article abstract of DOI:10.1016/j.bmc.2021.116188

Our continuing search for marine bioactive secondary metabolites led to the screening of crude extracts of sea cucumbers by the model of Pyricularia oryzae. A new sulfated triterpene glycoside, coloquadranoside A (1), together with four known triterpene glycosides, philinopside A, B, E and pentactaside B (2–5) were isolated from the sea cucumber Colochirus quadrangularis, and their structures were elucidated using extensive spectroscope analysis (ESI-MS, 1D and 2D NMR) and chemical methods. Coloquadranoside A possesses a 16-acetyloxy group in the holostane-type triterpene aglycone with a 7(8)–double bond, a double bond (25,26) at its side chain, and two β-D-xylose in the carbohydrate chain. Coloquadranoside A exhibits in vitro some antifungus, considerable cytotoxicity (IC₅₀ of 0.46–2.03 μM) against eight human tumor cell lines, in vivo antitumor, and immunomodulatory activity.

Full text of DOI:10.1016/j.bmc.2021.116188

Bioorg. Med. Chem. 41 (2021) 116188
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A new sulfated triterpene glycoside from the sea cucumber Colochirus
quadrangularis, and evaluation of its antifungal, antitumor and
immunomodulatory activities
Wen-Sheng Yanga, Xin-Rui Qia, Qiang-Zhi Xub, Chun-Hong Yuanc, Yang-Hua Yib,
,
,*
Hai-Feng Tangd, Li Shena *, Hua Hana
a School of Medicine, Tongji University, Shanghai 200092, China
b Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
c Department of Food Production and Environmental Management, Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan
d Department of Pharmacy, Xijing Hospital, Fourth Military University, Xi’an 710032, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Our continuing search for marine bioactive secondary metabolites led to the screening of crude extracts of sea
cucumbers by the model of Pyricularia oryzae. A new sulfated triterpene glycoside, coloquadranoside A (1),
together with four known triterpene glycosides, philinopside A, B, E and pentactaside B (2–5) were isolated from
the sea cucumber Colochirus quadrangularis, and their structures were elucidated using extensive spectroscope
analysis (ESI-MS, 1D and 2D NMR) and chemical methods. Coloquadranoside A possesses a 16-acetyloxy group in
the holostane-type triterpene aglycone with a 7(8)–double bond, a double bond (25,26) at its side chain, and two
β-D-xylose in the carbohydrate chain. Coloquadranoside A exhibits in vitro some antifungus, considerable cyto-
Sea cucumber
Colochirus quadrangularis
Triterpene glycoside
Coloquadranoside A
Bioactivity
toxicity (IC50 of 0.46–2.03
μ
M) against eight human tumor cell lines, in vivo antitumor, and immunomodulatory
activity.
1. Introduction
Moreover, the glycosides sulfated at C-6 hydroxy groups of glucose and
3-O-methylglucose, were proved to be a universality, although other
positions of sulfates in different sugars rarely occur.12–15
Triterpene glycosides (saponins) are the predominant secondary
metabolites of sea cucumbers. More than 700 of them are described from
various species of holothurians existing chemical structures of sulfated,
non-sulfated and acetylated glycosides, characterized by the tremen-
dous structural diversity.1–3 A broad spectrum of biological activities are
exhibited in them, such as hemolysis, cytotoxicity, antifungus, anti-
bacterium, antivirus, antiinflammation, cholesterol-lowering, immuno-
regulation, antioxidation, antitumor and antidementia.3–10 Therefore,
the search for new representatives of these triterpene glycosides and
studies of their biological activities appear to be relevant. The majority
of the glycosides are usually the lanostane with an 18(20)-lactone (i.e.
the holostane-type triterpene) and with a sugar chain of up to six
monosaccharide units [principally D-glucose (Glu), D-xylose (Xyl), D-
quinovose (Qui), and their methylated forms] linked to C-3 of the
aglycone.11 As a rule, the first sugar is identified as xylose, whereas the
methylated monosaccharides are always the terminal sugars. The car-
bohydrate chains are sulfated, at least at C-4 of the first xylose residue.
As part of our ongoing search for new bioactive compounds from
holothurians,16–24 we have already described the isolation and the
structure elucidation of a number of glycosides thoroughly from the sea
16–20
cucumber Colochirus quadrangularis
(known formerly as its syno-
nym, Pentacta quadrangularis) collected from the South China Sea, near
Guangdong Province, China.25,26 These glycosides usually form an
extremely complicated mixture in the organism producer, and the
reinvestigation aims to purify, characterize and evaluate these conge-
ners from C. quadrangularis. We report herein the isolation and struc-
tural elucidation of a new triterpene glycoside from C. quadrangularis,
coloquadranoside A (1), and its bioactivities in vitro and in vivo.
* Corresponding authors at: School of Medicine, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
E-mail addresses: shenli2007@tongji.edu.cn (L. Shen), hanhua@tongji.edu.cn (H. Han).
https://doi.org/10.1016/j.bmc.2021.116188
Received 5 February 2021; Received in revised form 15 April 2021; Accepted 26 April 2021
Available online 30 April 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
2. Results and discussion
atoms at δC 105.2, 105.4, 105.1, 105.6, and four anomeric protons at δH
4.81, 5.12, 4.96, 5.46). The large vicinal coupling constants (3JH-1/H-2
=
2.1. Chemical investigation
7.1–7.9 Hz) of each anomeric proton indicated a trans-diaxial orienta-
tion with respect of their coupling partners (β-configuration).4 Com-
pound 1 was treated with aqueous 2 mol/L trifluoroacetic acid to give D-
Xyl, D-Qui and D-3-O-methylglucose (Glu3Me) in the ratio 2: 1: 1. The
monosaccharides were identified by GC–MS in the form of correspond-
ing standard aldononitrile peracetates. Fragments corresponding to the
loss of sugars and SO3Na+ from the [M+Na]+ peak were also observed at
1121 [Mꢀ SO3Na+Na+H]+, 945 [Mꢀ SO3Na+Na+H - Glu3Me]+, 791
[Mꢀ SO3Na+2H - Glu3Me - Xyl]+, and 667 [Mꢀ SO3Na+2H - Glu3Me -
Xyl - Qui]+ showing that its Glu3Me must be terminal (Fig. 2). The
common D-configuration for the four carbohydrate units were assumed
also according to those most often encountered among triterpene gly-
cosides from sea cucumbers.4 The 1H–1H COSY experiment allowed the
sequential assignment of most of the resonances for each sugar ring,
starting from the easily distinguished signals due to anomeric protons.
Complete assignment was achieved by combination of 1H–1H COSY and
TOCSY results. The HMQC experiment correlated all proton resonances
with those of their corresponding carbons. Data from the above exper-
iments indicated (Table 2) four sugar residues existing in their pyranose
forms. The location of the interglycosidic linkages was deduced from the
chemical shifts of Xyl1 C-2 (δC 83.6), Qui2 C-4 (δC 86.2) and Xyl3 C-3 (δC
87.3), which were downfield relative to shifts expected for the corre-
sponding methyl glycopyranosides. These evidences suggested that 1
possesses the same tetrasaccharide chain as philinopside A (2) and
philinopside E (4).16,17 Finally, direct support of the sequence of the
sugars and binding sites came from the results of HMBC experiment: a
cross peak between C-3 of the aglycone and H-1 of Xyl1 indicated that
Xyl1 was connected to C-3 of the aglycone; the linkage of Qui2 at C-2 of
Xyl1 was indicated by the cross peak Qui2 H-1/Xyl1 C-2. Similarly, the
linkages of the terminal Glu3Me4 at the C-3 of Xyl3 in turn linked to C-4
of Qui2 were indicated by cross peaks Glu3Me4 H-1/Xyl3 C-3, Xyl3 H-1/
Qui2 C-4. This conclusion was confirmed by the correlations between H-
3 of aglycone and Xyl1 H-1, between Qui2 H-1 and Xyl1 H-2, between
Xyl3 H-1 and Qui2 H-4, and between Glu3Me4 H-1 and Xyl3 H-3, in the
NOESY spectrum (Fig. 3). Fragment ion peak at m/z 1121
[Mꢀ SO3Na+Na+H]+ in ESI-MS indicated the presence of a sulfate
groups in 1 which was confirmed by the IR spectrum with absorption
bands at 1233 cmꢀ 1. The site of the sulfo group linked to the sugar unit
in 1 was also demonstrated by its solvolysis with dioxane/pyridine to
desulfocoloquadranoside A (1a). The sugar moiety of 1a was identical to
that of desulfated intercedenside A from Mensamria interceden and
desulfophilinopside B from C. quadrangularis.17,28 When the 13C NMR
signals of the sugar moiety of 1 were compared with those of 1a, an
The EtOH extract of C. quadrangularis (5 kg, dry weight) was
sequentially passed through column chromatography (DA-101 resin and
silica gel), and the fraction containing coloquadranoside A (1) was ob-
tained. The new compound 1 and other known compounds 2–5 (phi-
linopside A, B, E and pentactaside B) were further isolated and purified
by reversed-phase HPLC on Zobax SB C-18 (Fig. 1).
Coloquadranoside A (1), a white amorphous powder, was positive in
the Liebermann-Burchard and Molish tests. Its molecular formula was
determined as C55H85NaO25S from pseudomolecular ion peak at m/z
1223 [M+Na]+ (positive-ion mode ESI-MS) and m/z 1177 [Mꢀ Na]ꢀ
(negative-ion mode ESI-MS). The IR spectrum showed the presence of
hydroxyl (3419 cmꢀ 1), carbonyl (1747 cmꢀ 1), and olefinic (1647 cmꢀ 1
groups.
)
The 1H- and 13C NMR spectra of compound 1 suggested the presence
of a triterpene aglycone with two olefinic bonds, one acetoxyl and one
lactone carbonyl group, one hydroxyl group bonded to an oligosaccha-
ride chain composed of four sugar units. Comparing the spectral data of
1 (Table 1) with those of the known compounds indicated that its
aglycone part was identical to that of philinopside B (3).16 Two C-atoms
of 1 at δ 145.6 (C-25) and δ 110.6 (C-26), along with two olefinic
C
signals atCδC 38.2 and 22.0 (C-24 and C-27), confirmed the presence of a
characteristic C(25) = C(26) bond. The 1H- and 13C NMR spectra of 1
showed resonances for a 7(8)-double bond (δ 145.5, C-8 and 120.1, C-7;
C
δ
H 5.74 br.s, H-7) and 16β-acetyloxy group (δC 170.2 and 21.3, CH3; δ
1.98, s) closely related to those of frondoside D isolated from CucumariHa
frondosa and philinopside A (2).16,27 Location of the AcO group at C-16
was deduced from chemical shift of the H-16 signal at δH 6.00 (ddd, J =
7.8, 8.4, 9.0), which showed coupling to signals at δH 2.69 (d, J = 9.3, H-
17), 2.66 (dd, J = 8.1, 12.0, Hα-15), and 1.84 (m, Hβ-15) in the 1H–1H
COSY plot, and correlation with the carbonyl carbon signal at δ 170.2 in
the HMBC spectrum. The 16β configuration of the AcO group was
confirmed by NOESY and the coupling constants for H-16 with Hα-17
(9.3 Hz) and Hα-15 (8.1 Hz), which was also close to the data of pen-
tactaside B (5),19 and different with those of philinopside E (4) without
the 16β-acetyloxy.17 Analysis of 1H–1H COSY, HMQC and NOESY data
allowed the assignment of all 1H- and 13C NMR resonances and estab-
lished the relative configuration of all chiral centers of the aglycone.
Thus, the aglycone of 1 was determined to be 16β-acetoxy-holosta-7,25-
diene-3β-ol.
The sugar portion of 1 displayed 1H- and 13C NMR resonances,
suggesting the presence of four monosaccharide units (four anomeric C-
Fig. 1. Structure of compounds, extracted from C. quadrangularis. (The new compound in this paper: 1, coloquadranoside A; the related structurally known com-
pounds 16,17,19: 2, philinopside A; 3, philinopside B; 4, philinopside E; 5, pentactaside B).
2

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
Table 1
1H- and 13C NMR data and select HMBC correlations for the aglycon moiety of
coloquadranoside A (1) (δ in ppm, J in Hz).a
δH (J in Hz)b
1H-1HNOESY
HMBC (H
a
Position
δC
→ C)
1
2
36.2,
1.42 (m)
CH2
27.4,
1.87–2.05 (m)
3.33 (dd, J = 4.2, 11.4)
0.99 (t, J = 7.8)
2.00 (m)
H-19
CH2
3
4
5
89.3, CH
39.5, C
48.1, CH
H-5, H-31, Hα-1
Hα-3, H-31, H-7
C-1 (Xyl1)
C-4, C-30,
C-31
6
7
23.4,
CH2
120.1,
CH
5.74 (br. s)
H-17, H-31, H-32,
H-5
Fig. 2. Sketch map of ESI-MS fragmentation of coloquadranoside A (1) with
positive-ion mode.
8
145.5, C
47.2, CH
36.0, C
22.5,
9
3.56 (bd, J = 14.2)
1.51–1.76 (m)
10
11
Table 2
1H- and 13C NMR data and key HMBC correlations for the sugar moiety of
CH2
12
31.6,
2.06–2.25 (m)
coloquadranoside A (1) (δ in ppm, J in Hz).
CH2
a
b
Position
δC
δH
HMBC (H → C)
13
14
15
59.3, C
47.5, C
43.7,
4-NaSO3-Xyl1 (1 → C-3)
1.84 (m, Hβ); 2.66 dd
(8.1, 12.0, Hα)
6.00 (ddd, J = 7.8, 8.4,
9.0)
1
2
3
4
5
105.2, CH
83.6, CH
76.4, CH
75.4, CH
64.3, CH2
4.81 (d, J = 7.2)
4.05–4.13 (m)
4.01–4.12 (m)
5.17–5.24 (m)
3.79–3.86 (m)
4.70–4.78 (m)
C-3 (Aglycone)
CH2
16
17
75.1, CH
Hα-17, H-32
C-17,
MeCO
54.7, CH
2.69 (d, J = 9.3)
Hα-16, H-7, H-32,
H-21
C-13, C-21
18
19
179.6, C
24.1,
CH3
Qui2 (1 → 2)
1.31 (s)
Hβ-2, H-30,
1
105.4, CH
75.6, CH
75.8, CH
86.2, CH
71.9, CH
18.0, CH3
5.12 (d, J = 7.6)
3.99–4.10 (m)
4.28–4.36 (m)
3.66–3.70 (m)
4.03–4.11 (m)
1.83 (d, J = 6.2)
C-2 (Xyl1)
C-4 (Qui2)
2
20
21
85.1, C
28.3,
CH3
3
1.66 (s)
H-17
4
5
22
23
24
38.4,
CH2
1.78–2.33 (m)
1.95–2.04 (m)
1.97 (t, J = 7.8)
6
Xyl3 (1 → 4)
22.8,
CH2
1
2
3
4
5
105.1, CH
74.0, CH
87.3, CH
69.1, CH
66.5, CH2
4.96 (d, J = 7.8)
3.78–3.86 (m)
4.23–4.28 (m)
4.15–4.23 (m)
4.37 (m, Hα)
38.2,
CH2
C-26, C-27
25
26
145.6, C
110.6,
CH2
4.77 (t)
1.67 (s)
1.13 (s)
1.23 (s)
1.04 (s)
3.72–3.77 (m, Hβ)
Glu3Me4 (1 → 3)
27
30
31
32
22.0,
CH3
1
105.6, CH
75.1, CH
88.2, CH
70.5, CH
78.4, CH
62.0, CH
54.7
5.46 (d, J = 7.9)
4.27–4.35 (m)
3.73–3.83 (m)
4.10–4.19 (m)
4.05–4.13 (m)
4.26–4.36 (m); 4.55 (m)
3.86 (s)
C-3 (Xyl3)
C-4 (Xyl3)
2
17.2,
CH3
H-19
C-3, C-5,
C-31
3
4
28.7,
CH3
H-3, H-5, H-7
H-16, H-17, H-7
C-3, C-5,
C-30
5
6
32.5,
CH3
C-8, C-13,
C-14
OMe
a
b
MeCO
MeCO
170.2,
21.3,
Measured at 150 MHz in C5D5N/D2O 4:1.
Measured at 600 MHz in C5D5N/D2O 4:1.
1.98 (s)
a
Measured at 150 MHz in C5D5N/D2O 4:1.
Measured at 600 MHz in C5D5N/D2O 4:1.
b
2.2. Antifungal activity and cytotoxicity
Some triterpene glycosides hitherto isolated from sea cucumbers
showed antifungal activity.29 Compounds 1–5, extracted from
C. quadrangularis, exhibited selective antifungal activities against four
strains, while 1, 3 and 4 possessing C(25) = C(26) showed more
considerable growth inhibitory activities among them, with MIC80 of
esterification shift (from 70.8 to 75.4) (Table 3) was observed at the
signals of C-4 (Xyl1). Data from NOESY and HMBC experiments were
combined to check the sequence of sugars, which was also indicated by
ESI-MS, and to determine the attachment points of interglycosidic. The
sequence of the sugar residues of 1, determined from HMBC cross-peaks
at H-1′/C-3, H-1′′/C-2′, H-1′′′/C-4′′, and H-1′′′′/C-3′′′, should be as
Glu3Me-(1 → 3)-Xyl-(1 → 4)-Qui-(1 → 2)-Xyl-(1 → 3)-aglycone.
The structure of compound 1 was finally elucidated on the basis of
extensive spectroscopic analysis and chemical evidence as 16β-acetoxy-
3-O-[3-O-methyl-β-D-glucopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 →
4–16
μ
g⋅mLꢀ 1 (Table 4). Cytotoxicity of compounds 1–5 against eight
human tumor cell lines (A-549, MKN-28, MCF-7, HCT-116, BEL-7402,
HO-8910, HL-60, SPC-A4) in vitro were examined using the sulforhod-
amine (SRB) assay with HCP as a positive control.30 It was interesting
that 1 and 3 with the both presence of C(25) = C(26) and AcO exhibited
more considerable cytotoxicity against some tumor cell lines (Table 5)
after exposures. As previous studies reported, the membranolytic ac-
tivity plays a substantive role for cytotoxicity and antifungal activity of
the lanostane glycosides with an 18(20) lactone and a sugar chain linked
4)-β-D-quinovopyranosyl-(1
→
2)-4-O-sodiumsulfate-β-D-xylopyr-
anosyl]-holosta-7,25-diene-3β-ol.
3

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
Fig. 3. Key NOESY correlations for coloquadranoside A (1), while its absolute configuration remains to be determined.
to C-3 of the aglycone,1,2 and the component of the carbohydrate chain
and the double bond position in the aglycone moiety, as well as the side
chains, are assumed to be important for cytotoxic activity among these
glycosides against human tumor cells.19–23,28 Our analysis suggested
that the Δ25 terminal double bond and 16β-acetyloxy group of colo-
quadranoside A (1) contribute to its cytotoxicity and antifungal activity.
Based on these initially promising results, coloquadranoside A deserves
further study as a potential antifungal and antitumor compound,
although more extensive studies are needed before a clear structure-
activity relationship can be reached.
Table 3
13C NMR data for the aglycon and sugar moiety of desulfocoloquadranoside A
(1a) (δ in ppm, J in Hz).
Aglycon
a
a
δC
Sugar unit
δC
1
36.2, CH2
27.3, CH2
89.2, CH
39.6, C
Xyl1 (1 → C-3)
2
1
2
3
4
5
105.1, CH
84.5, CH
78.6, CH
70.8, CH
66.8, CH2
3
4
5
48.0, CH
23.4, CH2
120.5, CH
145.6, C
47.0, CH
35.8, C
6
7
8
Qui2 (1 → 2)
2.3. Antitumor activity in vitro
9
1
105.5, CH
76.6, CH
75.6, CH
86.1, CH
71.7, CH
17.6, CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30
31
32
MeCO
MeCO
2
22.7, CH2
31.5, CH2
59.2, C
3
When tested for bioactivity by inducing morphological deformation
of mycelia (Table 6), coloquadranoside A showed deformation against
P. oryzae with MMDC ≤ 16 µg/mL.31,32 It indicated that the compound
has the potential antitumor probably, consistent with its observation in
cytotoxicity assay. Then, HCT-116 was selected to characterize its
antitumor activity in vitro. Transwell migration assay demonstrated that
the migration capacity of the 24 h treated cells was significantly
diminished after administration (Fig. 4). Hoechst results revealed that
when exposed to coloquadranoside A, the cells underwent the typical
morphological changes that are associated with apoptosis, as indicated
by dense granular fluorescence (Fig. 4). The cells in vehicle group rarely
exhibited the apoptotic nuclei, while the apoptotic nuclei cells were
easily seen in the coloquadranoside A groups (Fig. 4). Quantitatively,
the compound significantly increased the number of apoptotic nuclei in
HCT-116 cells. Besides, the apoptosis induced by coloquadranoside A
was further confirmed by Annexin V and propidium iodide double-
staining assay. It showed that the compound significantly decreased
the percentages of live cells (Q4) with increased percentages of the early
and late apoptotic cells (Q3 and Q2) and necrotic cells (Q1) in HCT-116
cells (Fig. 4). And thereby it was evident that the compound induces
apoptosis in cancer cells. The overall results demonstrated that colo-
quadranoside A has the ability to suppress the migration of cancer cells,
meanwhile accelerating their apoptosis, revealing its potential anti-
tumor activity in vitro.
4
5
47.4, C
6
43.7, CH2
74.9, CH
54.6, CH
179.5, C
24.0, CH3
85.2, C
Xyl3 (1 → 4)
1
2
3
4
5
105.5, CH
73.7, CH
87.3, CH
69.1, CH
66.6, CH2
28.1, CH3
38.7, CH2
23.8, CH2
38.3, CH2
145.5, C
110.3, CH2
18.1, CH3
17.3, CH3
28.5, CH3
32.1, CH3
169.9
Glu3Me4 (1 → 3)
1
2
3
4
5
6
105.4, CH
75.1, CH
88.0, CH
70.8, CH
78.5, CH
62.2, CH
OMe
60.7
21.1
a
Measured at 150 MHz in C5D5N/D2O 4:1.
Table 4
Antifungal activities of compounds (MIC80
,
μ
g⋅mLꢀ 1).
Compoundsa
C. albicans
C. neoformans
C. tropicalis
C. parapsilosis
We confirmed the antitumor activity of coloquadranoside A pre-
liminarily, while the extracorporeal angiogenesis model results also
demonstrated it obviously inhibited the proliferation, migration and
tube formation of HMEC-1 s (Table 7). Angiogenesis is closely related to
the occurrence and development of tumors, as the guarantee of tumor
growth and metastasis.33 The results indicated the antitumor role of
coloquadranoside A through directly inhibition of tumor cell growth and
meanwhile probably suppressing tumor angiogenesis and formation of
microvascular neovascularization. Furthermore, our observation
demonstrated it suppressed the autophosphorylation of VEGFR-2 (IC50
ATTCC76615
ATTCC32609
1
4
16
8
4
2
20
25
30
32
3
4
16
8
4
4
4
16
8
4
5
25
25
25
25
ICZ
TRB
FCZ
<0.125
32
2
1
<0.125
<0.125
0.5
<0.125
2
<0.125
0.25
1
a
Itraconazole (ICZ), Terbinafine (TBNF) and Fluconazole (FCZ) were used as
pos. controls.
of 2.20 μM) and PDGFR-β (IC50 of 4.80 μM), two types of tyrosine kinase
receptors, both of which are closely related to angiogenesis.34–36 In sum,
coloquadranoside A possesses the potential in suppressive activity of
tyrosine kinase receptors and probably inhibits tumor growth and
4

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
Table 5
Cytotoxicity of compounds against human tumor cells in vitro (IC50, μM).
Compounds
A-549
MKN-28
MCF-7
HCT-116
BEL-7402
HO-8910
HL-60
SPC-A4
1
1.27 ± 0.14
1.70 ± 0.99
0.75 ± 0.23
1.47 ± 0.26
1.30 ± 0.13
0.74 ± 0.04
1.83 ± 1.01
2.81 ± 0.50
2.03 ± 0.32
5.53 ± 0.19
1.82 ± 0.09
0.48 ± 0.04
2.03 ± 0.20
2.30 ± 0.51
1.79 ± 0.21
3.03 ± 0.10
1.56 ± 0.22
0.90 ± 0.04
1.36 ± 0.09
1.61 ± 0.42
0.93 ± 0.01
1.01 ± 0.21
1.09 ± 0.02
0.14 ± 0.01
1.45 ± 0.11
2.40 ± 0.12
1.30 ± 0.11
1.75 ± 0.23
1.30 ± 0.21
0.27 ± 0.03
1.94 ± 0.35
2.32 ± 0.50
1.90 ± 0.31
2.14 ± 0.22
1.90 ± 0.41
0.70 ± 0.21
0.60 ± 0.30
1.65 ± 0.32
0.65 ± 0.20
0.82 ± 0.29
1.95 ± 0.20
0.41 ± 0.23
1.50 ± 0.70
3.67 ± 0.72
1.30 ± 0.71
3.65 ± 0.21
1.50 ± 0.71
0.66 ± 0.23
2
3
4
5
HCPa
a
HCP, 10-hydroxycamptothecine (pos. control).
BALB/c-nu mice. As the data of the last day shown, the BEL7402
xenograft mice model was successful (Fig. 5). The increase of average
tumor volume in vehicle group was exponential in the last few days,
while it tended to slow due to coloquadranoside A treatment (Fig. 5).
The ANOVA revealed that the last-day average RTV and average tumor
weight were significantly lower after treatment (P < 0.05; Fig. 5) with
TGI > 50% and T/C < 60% (P < 0.05; Table 8), which suggested their
effectiveness. Taken together, the most obvious finding was that colo-
quadranoside A exhibits an excellent antitumor activity against three
graft tumors in vivo, mainly through reducing the weight and volume of
tumors, and the evaluating indicator like TGI and T/C confirmed our
observation. It may support the hypothesis that coloquadranoside A has
the potential to effectively suppress the growth, aggressiveness and
angiogenesis of a series of tumors, and is expected to become an anti-
tumor lead compound and novel medication just as two positive con-
trols, 5-FU or FT-207.37–39 The future work will carry on toxicology and
Table 6
Effects of compounds against P. oryzae.
Compounds
1
5-FUb
HCPb
MMDC (
a
μ
g/mL)a
≤ 16
≤ 8
≤ 5
μg/ml) are in bold;
Compounds with significant activity (MMDC ≤ 125
MMDC, minimum deformative concentration.
b
5-FU, 5-fluorouracil; HCP, 10-hydroxycamptothecine (pos. control).
aggressiveness by suppressing tumor neovascularization through tyro-
sine kinase autophosphorylation pathway.
2.4. Potential for suppressing tumors in vivo
Because coloquadranoside A displayed potent antitumor effects in
vitro, the activity in vivo was tested in mice next. We established two
homograft tumors in the wild-type mice successfully (Fig. 5), S-180 and
H22 models. An ANOVA revealed that 5-FU significantly inhibited the
weight gaining of tumors in the homograft mice (P < 0.05). The inhi-
bition was also found on the groups of coloquadranoside A (P < 0.01),
suggesting its same ability as 5-FU against the growth of S-180 sarcomas
and H22 tumors in vivo. Meanwhile, TGI of coloquadranoside A with the
dose of 5–50 mg/kg reached more than 35% (P < 0.01, Table 8),
showing valid. Based on its effective inhibition on the growth of two
homograft tumors, the antitumor efficacy of coloquadranoside A in vivo
was proved. These preliminary results revealed coloquadranoside A is
warranted to be further studied on antitumor.
Table 7
IC50 value of 1 on tumor angiogenesis model.
Cell
IC50 (μM) of 1 to different assay
lines
Proliferation
Migration
Tube
VEGFR-
2b
PDGFR-
formationa
βb
HMEC-
1
1.60 ± 0.48
0.05 ±
0.00
–
0.06 ± 0.02
–
–
NIH/
3T3
–
–
2.20 ±
4.80 ±
0.11
0.30
a
Tube-like structure formation of endothelial cells.
For further explanation of the antitumor activity, the xenograft
model of human hepatocarcinoma BEL7402 was established to assay the
effectiveness of coloquadranoside A in the SPF grade immunodeficiency
b
Autophosphorylation level of tyrosine kinase receptors as the evaluating
activity indicator.
Fig. 4. Antitumor assays in vitro of coloquadranoside A (1) against HCT-116 cells. (A). Visual field map of Transwell assay; (B). Hoechst staining on HCT-116 among
groups; (C). Annexin V and propidium iodide double-staining assay determined by flow cytometry; (D). Migration of HCT-116; (E) & (F). Apoptosis of HCT-116
among groups. Note: HCP, 10-hydroxycamptothecine, as pos. control; * P < 0.05, ** P < 0.01, *** P < 0.001, versus vehicle, determined by one-way ANOVA or
Brown-Forsythe and Welch ANOVA tests, n = 3.
5

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
Fig. 5. Therapeutic effect in vivo of coloquadranoside A (1) against three mouse graft tumor models. Average tumor weight in wild type KM mice with the homograft
model of S-180 sarcomas (A) or hepatocarcinoma cells H22 (B) at the last treatment day; the changing trend of tumor volume (C), average RTV (D), and average
tumor weight at the last treatment day (E) in BALB/c-nu mice bearing BEL7402 xenograft tumors; CALD, CAMD and CAHD represent coloquadranoside A with low
dose, middle dose and high dose, respectively; * P < 0.05, ** P < 0.01, *** P < 0.001, versus vehicle; △ P < 0.05, versus CAMD, determined by one-way ANOVA
tests, n = 8.
reflecting the innate immune function, characterized by carbon particle
Table 8
clearance index.42 The clearance and phagocytosis index of immuno-
Effect of coloquadranoside A (1) on the growth of S-180, H22 and BEL7402
suppressed mice was significantly increased by coloquadranoside A,
tumors (n = 8).
which suggests its improvement of mononuclear immune function.
Group
Dosage (mg/kg)
TGI (%)
T/C (%)
P-valuea
These findings may help us to propose that coloquadranoside A en-
hances the innate immune system of immunosuppressed animals.
Furthermore, the hemolysin experiment provided another substantive
evidence of improvement of immune function. The hemolysin level,
positively related to the adaptive immunity ability,43 was significantly
increased by coloquadranoside A in immunosuppressed mice. Its
administration also improved the atrophy of spleen and thymus caused
by CTX,44 showing the potential for immunoenhancement in the im-
mune organs. These evidences suggested that coloquadranoside A
ameliorates the immune organ index, thereby advantageous to the
enhancement of the humoral or adaptive immune function in immu-
nosuppressed condition.
S-180
Vehicle
–
–
–
–
1
0.5
5
21.6
44.2
55.5
39.4
–
–
>0.05
<0.01
<0.001
<0.05
–
1
–
1
50
25
–
–
5-Fu
–
H22
Vehicle
–
1
0.5
5
22.1
40.1
43.3
39.4
–
50.1
58.2
58.6
81.8
–
>0.05
<0.001
<0.001
<0.001
–
<0.05
<0.05
<0.05
<0.01
1
–
1
50
25
–
–
5-Fu
–
BEL7402
Vehicle
–
1
5
45.0
42.2
32.0
20.0
1
50
500
100
1
Together these results provided important insights into coloqua-
dranoside A markedly improving both adaptive immunity and innate
immunity of immunosuppressive mice, while the further researches are
still needed for the specific mechanism of such considerable immuno-
modulatory activity in vivo.
FT-207
a
P-value, compared with vehicle group, determined by one-way ANOVA test,
using for evaluating whether TGI or T/C was valid or not.
pharmacodynamics to characterize its safety and optimal dose in vivo.
3. Conclusions
2.5. Effective immunomodulation in vivo
As extracts of complex mixtures, triterpene glycosides are obtained
in the multistage process of isolation and purification due to their low
content and a large number of congeners and isomers. Ongoing chemical
investigation and bioassay-guided isolation of the sea cucumber
C. quadrangularis led to the characterization of compounds 1–5, among
which new coloquadranoside A (1) was found possessing a 16β-acety-
loxy group and a Δ25 terminal double bond in the holostane aglycone
with a 7(8)–double bond, and a sulfate group linked to C-4 of Xyl1 in the
tetrasaccharide chain. The examined coloquadranoside A displayed the
excellent antifungal, antitumor and immunomodulatory activities, and
we first made a report of the improvement on innate and adaptive im-
mune system under immunosuppression of such glycoside from the sea
cucumber C. quadrangularis. Future work will be required to explore its
exact activity mechanism, toxicology for safety, structure-activity rela-
tionship, and structural optimization. Anyway, the present finding
provides evidence that this marine-derived sulfated triterpene glycoside
may become an attractive candidate for further preclinical testing as a
The immunosuppression of mice established by intraperitoneal in-
jection of cyclophosphamide (CTX) is similar to that of human.40,41 Our
CTX model was successful in mice (P < 0.05, Fig. 6), confirmed by
significantly lower carbon particle clearance index and the atrophy of
spleen and thymus.42–44 Meanwhile, the single most striking observation
to emerge from the data comparison was that coloquadranoside A with
the dose of 50–500 mg/kg significantly increased the clearance index
and adjusted phagocytosis index in the immunosuppressed mice (P <
0.05, Fig. 6); in other words, it improved the clearance and phagocytosis
function of mouse monocyte macrophages in immunosuppressed con-
dition. Similarly, the injection of CTX induced lower thymus and spleen
index and hemolysin level, which were significantly reversed by the
treatment of coloquadranoside A of 50–500 mg/kg (P < 0.001; Fig. 6). It
indicated that coloquadranoside A possesses the immunomodulatory
activity in immunosuppressed mice.
Phagocytosis of mononuclear macrophages is noted as indicators
6

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
Fig. 6. The immunomodulatory activity in vivo of
coloquadranoside
A (1) in immunosuppressed
mouse model. The carbon clearance index (A);
Adjusted phagocytosis index (B); Hemolysin level
(C), shown as OD540nm value of serum; Immune
organ index (D), g/kg; CALD, CAMD and CAHD
represent coloquadranoside
A
with low dose,
#
middle dose and high dose, respectively;
P <
0.05, ## P < 0.01, ### P < 0.001, versus control; *
P < 0.05, ** P < 0.01, *** P < 0.001, versus CTX;
△
△△
P < 0.05,
P < 0.01, versus CTX + CAMD;
determined by one-way ANOVA or Brown-Forsythe
and Welch ANOVA tests, n = 8.
new drug probably.
Fisher Scientific Co., Ltd. DMEM was purchased from GIBCO, Life
Technologies, Grand ISIAND, NY, USA. Sulforoxamine B (SRB) and the
stimulator, VEGF and PDGF-BB, were purchased from Sigma-Aldrich
Corporation, MO, USA. Trichloroacetic acid (TCA) and Tris base buffer
were both analytical pure. Matrigel was purchased from Becton
Dichinson, USA. Tyrosine phosphorylation monoclonal antibody PY-99,
sheep anti mouse biotin labeled IgG (H + L) and Horseradish-
Peroxidase-Streptavidin polymer (HRP-Avidin) were purchased from
Vector, USA. The 10% complement was prepared from serum of guinea
pigs with normal saline. Rabbit erythrocyte was taken from heart blood
with fibrinogen removed, washing with normal saline to make 20%
suspension.
4. Experimental section
4.1. General procedures
Melting points were determined on an XT5-XMT apparatus. Optical
rotations were measured on a Perkin-Elmer-341 polarimeter. IR spectra
were recorded on a Bruker Vector-22 infrared spectrometer. NMR
spectra were recorded in C5D5N on a Bruker Avance-ІІ-600 spectrometer
with TMS as internal standard. ESI-MS was acquired on Q-TOF Micro LC-
MS-MS mass spectrometer. GC–MS were performed on a Finnigan
Voyager apparatus using a DB-5 column (30 m × 0.25 mm i.d., 0.25 μm)
with an initial temperature of 150 ◦C for 2 min and then temperature
programming to 300 ◦C at a rate of 15 ◦C/min. HPLC was carried out on
an Agilent 1100 liquid chromatograph equipped with a refractive index
detector using a Zorba 300 SB-C18 column (250 × 9.4 mm i.d.). Column
chromatographic separations were performed on silica gel H (200–300
4.3. Animal material
Specimens of C. quadrangularis was collected from the South China
Sea near Guangdong province, China in May 2018. Taxonomy of the sea
cucumber was identified by Prof. JR Fang, Fujian Institute of Oceanic
Research, China. A voucher specimen (No. SA201842) was preserved in
School of Medicine, Tongji University, Shanghai, China.
mesh, 10–40
μ
m, Qingdao Marine Chemical Inc.; Qingdao, China),
Lobar Lichroprep RP-C18 (40–63
μm; Merck) and Sephadex LH-20
(Pharmacia). Fractions were monitored by TLC on precoated silica gel
HSGF254 plates (CHCl3-EtOAc-MeOH-H2O, 4: 4: 2.5: 0.5) or RP-C18
(MeOH-H2O, 1: 1), (Qingdao Marine Chemical Inc., Qingdao, China)
and spots were visualized by heating Si gel plates sprayed with 15%
H2SO4 in EtOH.
4.4. Extraction and isolation
The sea cucumber (5 kg, dry weight) was cut into pieces and
extracted with 70% EtOH (20 L, each time for 1 h) under reflux. The
EtOH extracts were combined and then evaporated. The residue was
dissolved in H2O (4 L). The H2O-soluble fraction was passed through a
DA101 resin column (60 × 30 cm; Nankai University, Tianjin, China),
and eluted with dist⋅H2O (until a negative reaction of chloride was
observed) and 50% aq. EtOH. The combined 50% EtOH eluate was
evaporated and subjected to Sepahedex LH-20 column (3 × 50 cm)
equilibrated MeOH/H2O (2:1). The fraction (12 g) containing saponins
was chromatographed over silica gel column (dry column, 500 g, 2 × 50
cm, eluting with CHCl3/MeOH/H2O from 8:2:1 to 6.5:3.5:1, lower phase
stepwise gradient) again. Each subfraction containing saponins was
4.2. Reagents, controls and antibodies
Positive controls for antifungal assay like Itraconazole (ICZ), Terbi-
nafine (TRB) and Fluconazole (FCZ) were supplied by Department of
Pharmacology, School of Pharmacy, Second Military Medical University
(SMMU), China, and 10-hydroxycamptothecin was provided by
Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, China. Hoechst 33,258 and Annexin V-propidium iodide
double-staining flowcytometry reagents were purchased from Sigma-
Aldrich, Shanghai, China. Futraful (FT-207), 5-Fluorouracil (5-FU) and
Fufang Ejiao Jiang (FEJ), as the positive drugs in the intracorporal
screening. Cyclophosphamide (CTX) for injection, dissolved in normal
saline to make 2.5 mg/mL solution immediately before using. Indian
ink, diluted four times with normal saline. Filter and store in refrigerator
at 4 ◦C. Potato medium, including 1.5% sucrose, 20.0% potato and 2.0%
agar. Sabouraud liquid medium, including 1.0% peptone, 0.5% NaCl
and 4.0% glucose. RPMI-1640 culture medium, product by Thermo
purified by semipreparative HPLC (Zorbax SB-C18, 5 μM; 250 mm × 9.4
mm i.d.; 58% aq. MeOH, 1.5 mL/min), and obtained pure 1 (32 mg; tR
35.89 min).
Coloquadranoside A (1). White amorphous powder; mp 223–226 ◦C;
[
α 2D0
]
= -16.7 (c = 0.60, pyridine); IR (KBr): υmax 3419, 1747, 1647, 1233,
1039 cmꢀ 1
;
1H- and 13C NMR: shown in Tables 1 and 2; ESI-MS: m/z
1223 [M+Na]+, m/z 1177 [Mꢀ Na]ꢀ (calc. for C55H85Na2O25S+, 1223).
7

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
4.5. Acid hydrolysis of coloquadranoside A (1)
549, MKN-28, MCF-7, HCT-116, BEL-7402, HO-8910, HL-60, SPC-A4,
was evaluated by SRB assay as described.30 The anticancer agent 10-
hydroxycamptothecin served as a positive control. The activity of
Compound 1 (1 mg) was heated in an ampule with 2 M CF3COOH (1
mL) at 120 ◦C for 2 h. The reaction mixture was evaporated to dryness,
and the residue was partitioned between CH2Cl2 and H2O. The aqueous
phase was evaporated under reduced pressure. Then, pyridine (1 mL)
and NH2OH⋅HCl (2 mg) were added to the dried residue, and the mixture
was stirred at 90 ◦C for 30 min. After that time, 1 mL of Ac2O was added,
and the heating at 90 ◦C was continued for another hour. The soln. was
concentrated, and the resulting aldononitrile peracetates were analyzed
by GC–MS. The carbohydrates were determined by comparing the
retention times and MS behavior with standard aldononitrile peracetates
(Sigma) prepared from authentic sugars by the same procedure per-
formed for the sample. D-xylose, D-quinovose, and 3-O-methyl-D-glucose
were identified in a ratio of 2: 1: 1.
compounds was determined at 100, 10, 1, 0.1, and 0.01 μM (each con-
centration being tested in triplicate). Data were calculated as percent
inhibition (I) according to the formula: I = (ODC - ODT) ⁄ ODC × 100%,
where ODT and ODC are the mean optical densities of the treatment of
compounds and the solvent control, respectively. The concentration
inducing 50% inhibition of cell growth (IC50) was determined graphi-
cally for each experiment by curve fitting using GraphPad Prism (ver.
8.0, GraphPad Software Inc., San Diego, CA, USA), and derived by
DeLean et al.,46 the equation was calculated by Logit method. IC50
values represent the mean of three independent experiments.
4.9. Antitumor and tumor angiogenesis in vitro
4.6. Desulfation of coloquadranoside A(1)
Minimum morphological deformation concentration (MMDC) of
coloquadranoside A, as described,31,32 was tested against P. oryzae.
HCT-116 cells were seeded for antitumor assay, and following colo-
quadranoside A (0, 1/2 IC50, IC50 and two-fold of IC50, respectively) or
HCP as positive control solution were added into the cells and incubated.
Then the migration assay was carried out by routine procedure of
Transwell. Each insert was imaged in for five random fields at 100 ×
magnification, and the analysis was done using ImageJ program (ver.
1.48, National Institutes of Health, Bethesda, MD, USA). As for the
Hoechst assay, briefly, the cells were stained with Hoechst 33,258
staining solution, and the nuclear morphology was examined using an
inverted fluorescence microscope (Leica DMI4000B, Germany, × 400).
Cells were counted from five random fields and the number of apoptotic
cells was expressed as a percentage (%) of the total number of counted
cells. Besides, the cells for apoptosis assay were collected after admin-
Compound 1 (10 mg) was dissolved in pyridine/dioxane (1:1, 3.0
mL) and heated under reflux for 4 h. The mixture was partitioned be-
tween H2O and BuOH. The BuOH extract was evaporated and the res-
idue purified by reversed-phase HPLC (Zorbax-300-SB-C18, 80% MeOH/
H2O) at a rate of 1.5 mL/min to yield the pure 1a (6 mg).
4.7. Strains, cell lines and mice
The strain, Pyricularia oryzae P-2B, was provided by the University of
Tokyo, Japan. The fungus strains Candida albicans ATTCC76615, Cryp-
tococcus neoformans ATTCC32609 were provided by Changzheng hos-
pital, SMMU, Shanghai, China; Candida tropicalis (clinical strain) and
Candida parapsilosis (clinical strain) were provided by Changhai hospi-
tal, SMMU, Shanghai, China. Cancer cell lines, human lung cancer (A-
549), human gastric cancer (MKN-28), human breast cancer (MCF-7),
human colon cancer (HCT-116), human hepatocellular carcinoma (BEL-
7402), human ovarian cancer (HO-8910), human promyelocytic leuke-
mia (HL-60), human lung adenocarcinoma (SPC-A4), mouse sarcoma (S-
180) and mouse hepatocarcinoma (H22), and human microvascular
endothelial cell line, HMEC-1, were provided by Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai, China. Mouse
Fibroblast (NIH/3T3) was purchased from American Type Culture
Collection, Manassas, VA, USA, and its highly expressed lines of VEGFR-
2 and PDGFR-β was obtained by plasmid transfection. BALB/c-nu mice,
SPF grade, provided by Chinese Academy of Medical Sciences, Beijing,
China, while KM mice, wild type, cleaning grade, provided by Shanghai
Laboratory Animal Center, Chinese Academy of Sciences, Shanghai,
China, and all procedures were manipulated by the protocols including
the animal care, and approved by Laboratory Animal Research Center,
Tongji University, Shanghai, China. The cancer lines were maintained in
RPMI-1640 complete medium in a humidified 5% CO2 atmosphere at
37 ◦C. RPMI-1640 complete medium was supplemented with 10% fetal
istration. They were stained with Annexin V-FITC (5
μL) and propidium
iodide (PI; 5
μ
L) afterwards in the dark at 4 ◦C. Immediately examine
with a flow cytometer equipped with FACS Comp software (BD Bio-
sciences, Franklin Lakes, NJ, USA). While HMEC-1 cells were also
seeded as usual, then the drugs were added. The proliferation and
migration assays were carried out by routine procedure of SRB method
or Transwell, then IC50 values were calculated. As for tube-formation
assay, matrigel was quickly added to the plate, allowed to stand to
make the glue solidify. Seed cells and add drugs as above. Culture, then
the total length of tube was measured by Adobe Photoshop to calculate
IC50 values. Additionally, NIH-3 T3 with high VEGFR-2 or PDGFR-β
expression were seeded for phosphorylation assay. The cells were
starved for 24 h before the drugs added. They subsequently were stim-
ulated with VEGF (500 ng/mL) or PDGF-BB (150 ng/mL). Fix with 4%
paraformaldehyde, and seal the culture plates with TPBS. Add PY-99,
IgG (H + L), and HRP-Avidin in turn. The OD was determined at 405
nm after the reaction of ATBS. Calculate IC50 to characterize the phos-
phorylation level of VEGFR-2 or PDGFR-β. Three independent experi-
ments were carried out for each group in these assays in vitro.
bovine serum (FBS), 2 mM L-glutamine, 50
penicillin (100 U/mL) and streptomycin (100
μ
M β-mercaptoethanol,
μ
g/mL).
4.10. Mouse homograft and xenograft models
4.8. Antifungal and cytotoxicity tests in vitro
S-180, H22 and BEL7402 tumor lines were cultured in the usual way.
Select the former two to assay the therapeutic effect of compound 1 in
the homograft tumor mice. The tumor lines were made into cell sus-
pension respectively, inoculated subcutaneously in the right axilla of the
wild-type KM mice, about 2.5 × 106 cells/animal. After 24 h, the mice
were randomly divided into different groups (Table 8), administrated
with different dose drugs intragastrically for 7 days. Sacrifice the mice
24 h after the discontinuation of administration. The average tumor
weight and tumor growth inhibition rate (TGI) would be as evaluation,
the latter calculated by TGI = (WC - WT) / WC × 100%, where WT, the
average tumor weight of the administration groups; WC, the average
tumor weight of the control group; the effective could be proved by TGI
> 30% and P < 0.05. BEL7402 lines were undertaken to the continuing
The antifungal activity of the compounds was tested against four
strains, C. albicans (ATCC76615), C. neoformans (ATCC32609),
C. parapsilosis (clinical strain) and C. tropicalis (clinical strain). The data
of antifungal activity was evaluated by measuring optical density (OD)
at 630 nm using an automatic microplate reader.31,45 MIC80 was defined
as the first well with an approximate 80% reduction in growth compared
with the growth of the drug-free-well. The data represents the means of
three independent experiments in which the compound concentration
was tested in three replicate wells. Itraconazole (ICZ), Terbinafine
(TBNF) and Fluconazole (FCZ) were used as positive controls. The
cytotoxicity of compounds against the eight human cancer cell lines, A-
8

W.-S. Yang et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116188
therapeutic assay. The xenograft model of bearing BEL7402 BALB/c-nu
was established as the above method. After inoculation, the tumor vol-
ume (TV) was measured with vernier caliper at regular intervals,
calculated by TV = 1/2 × a × b2, where a & b represent the long and the
short diameter of tumors. When TV approximately 100–200 mm3, ad-
ministrate intragastrically once a day for 25 days. Measure TV and
weight of each group twice a week. The mice were sacrificed as above.
The tumors were weighed and measured, and TGI, relative tumor vol-
ume (RTV), relative tumor proliferation rate (T/C) and other evaluation
were calculated, while RTV = Vt / V0, and T/C = RTVT / RTVC × 100%,
where V0, the tumor volume measured at the time of grouping (day 0);
Vt, the tumor volume measured at each time (day t); when T/C ≤ 60%
and P < 0.05, valid; otherwise, invalid.
Acknowledgements
We specially thank Prof. Jin-Rui Fang for his helps in taxonomic
identification of the sea cucumber. This work was financially supported
by the National Key Research and Development Program of China
(2019YFC0312502).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116188.
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KM mice were randomly divided into different groups. The treatment
groups were intragastrically administrated with FEJ (10 mL/kg) or
different concentrations of coloquadranoside A (500 mg/kg, 50 mg/kg
and 5 mg/kg), once a day for 14 consecutive days, while the control
group and the model group were administered with 0.5% sodium car-
boxymethyl cellulose with equal volume. On the 7th, 9th and 11th day,
except for the control group, mice in each group were injected with CTX
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with 0.1 mL/10 g one hour after the last administration. Two and ten
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respectively collected and added to 2 mL distilled water. The OD value of
the samples was measured at 680 nm wavelength, and the carbon par-
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ticle clearance index (K) and adjusted phagocytosis index (α) were
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calculated, K = (lgOD1 -lgOD2) / (t2 - t1), and
α
= K1/3 × Wbody / (Wliver
+ Wspleen). Additionally, the mice were also divided into different groups
as above, and after five-time administration, the groups were immu-
nized by intraperitoneal injection of 20% rabbit erythrocyte saline 0.2
mL except for the control. Then, the treatments of drugs were continued
for 8 days. On the 2nd, 4th and 6th day after immunization, the mice
were injected with CTX 30 mg/kg intraperitoneally to establish the
model except for the control. An hour after the last administration, the
blood was taken from eyeballs to prepare serum. At the same time, mice
were dissected, spleen and thymus weighed. The serum was diluted
hundredfold with normal saline. Then, take 1 mL serum to mix with 20%
rabbit erythrocyte suspension 0.5 mL and 10% complement 0.5 mL.
Keep them in 37 ◦C water bath for 30 min. The reaction was terminated
at 0 ◦C. The supernatant’s OD value at 540 nm, having positive corre-
lation with serum hemolysin level, was measured to characterize the
different levels among groups thereby.
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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