In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis
Aim: To confirm the anticancer activity of steroid saponins isolated from the rhizome of Paris polyphylla var. yunnanensis and evaluate the structure-activity relationships of these steroid saponins in vitro and in vivo. Materials and Methods: Eight known steroid saponins were isolated from the rh...
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irk-123456789-1351012018-06-15T03:10:11Z In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis Yan, L.L Zhang, Y.J. Gao, W.Y. Man, S.L. Wang, Y. Original contributions Aim: To confirm the anticancer activity of steroid saponins isolated from the rhizome of Paris polyphylla var. yunnanensis and evaluate the structure-activity relationships of these steroid saponins in vitro and in vivo. Materials and Methods: Eight known steroid saponins were isolated from the rhizome of Paris polyphylla. The LA795 lung adenocarcinoma cell line from mice was chosen to evaluate cytotoxicity by means of MTT assay, and to study apoptosis by means of AnnexinV-FITC/PI flow cytometry. Diosgenin-3-α-L-arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-β-D-glycopyranoside (compound 1), the main steroid saponin of Paris polyphylla, and diosgenin (Dio), the aglycone of major steroid saponins, were evaluated for antitumor activity on LA795 lung adenocarcinoma in T739 inbred mice. Results: The steroid saponins showed remarkable cytotoxicity and caused typical apoptosis in a dose-dependent manner. They were evaluated in vivo by their effect on tumor developed in T739 inbred mice. The oral administration to T739 mice bearing LA795 lung adenocarcinoma of compound 1 and diosgenin significantly inhibited tumor growth, by 29.44% and 33.94%, respectively. HE staining showed that lungs and livers of treated mice underwent various levels of histopathological alterations. It was demonstrated by TUNEL assay that apoptosis rate in tumor cells was increased in comparison to cells in control mice. The 3-O-glycoside moiety and spirostanol structure played an important role in the anticancer activity of steroid saponins, and the number and the variety of glycosides of compounds strongly influenced on their anticancer activity. Conclusion: Rhizoma Paridis saponins showed anticancer activity against lung adenocarcinoma cell line, both in vitro and in vivo, and their effect was dependent on compounds’ structure in a certain degree. 2009 Article In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis / L.L. Yan, Y.J. Zhang, W.Y. Gao, S.L. Man, Y. Wang // Experimental Oncology. — 2009. — Т. 31, № 1. — С. 27-32. — Бібліогр.: 27 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/135101 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Yan, L.L Zhang, Y.J. Gao, W.Y. Man, S.L. Wang, Y. In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis Experimental Oncology |
| description |
Aim: To confirm the anticancer activity of steroid saponins isolated from the rhizome of Paris polyphylla var. yunnanensis and
evaluate the structure-activity relationships of these steroid saponins in vitro and in vivo. Materials and Methods: Eight known
steroid saponins were isolated from the rhizome of Paris polyphylla. The LA795 lung adenocarcinoma cell line from mice was
chosen to evaluate cytotoxicity by means of MTT assay, and to study apoptosis by means of AnnexinV-FITC/PI flow cytometry.
Diosgenin-3-α-L-arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-β-D-glycopyranoside (compound 1), the main steroid
saponin of Paris polyphylla, and diosgenin (Dio), the aglycone of major steroid saponins, were evaluated for antitumor activity on
LA795 lung adenocarcinoma in T739 inbred mice. Results: The steroid saponins showed remarkable cytotoxicity and caused typical
apoptosis in a dose-dependent manner. They were evaluated in vivo by their effect on tumor developed in T739 inbred mice. The
oral administration to T739 mice bearing LA795 lung adenocarcinoma of compound 1 and diosgenin significantly inhibited tumor
growth, by 29.44% and 33.94%, respectively. HE staining showed that lungs and livers of treated mice underwent various levels of
histopathological alterations. It was demonstrated by TUNEL assay that apoptosis rate in tumor cells was increased in comparison
to cells in control mice. The 3-O-glycoside moiety and spirostanol structure played an important role in the anticancer activity
of steroid saponins, and the number and the variety of glycosides of compounds strongly influenced on their anticancer activity.
Conclusion: Rhizoma Paridis saponins showed anticancer activity against lung adenocarcinoma cell line, both in vitro and in vivo,
and their effect was dependent on compounds’ structure in a certain degree. |
| format |
Article |
| author |
Yan, L.L Zhang, Y.J. Gao, W.Y. Man, S.L. Wang, Y. |
| author_facet |
Yan, L.L Zhang, Y.J. Gao, W.Y. Man, S.L. Wang, Y. |
| author_sort |
Yan, L.L |
| title |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| title_short |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| title_full |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| title_fullStr |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| title_full_unstemmed |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| title_sort |
in vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2009 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/135101 |
| citation_txt |
In vitro and in vivo anticancer activity of steroid saponins of paris polyphylla var. yunnanensis / L.L. Yan, Y.J. Zhang, W.Y. Gao, S.L. Man, Y. Wang // Experimental Oncology. — 2009. — Т. 31, № 1. — С. 27-32. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-09T22:42:33Z |
| last_indexed |
2025-07-09T22:42:33Z |
| _version_ |
1837211013373493248 |
| fulltext |
Experimental Oncology 31, 27–32, 2009 (March) 27
Paris polyphylla var. yunnanensis (Fr.) Hand-Mazz.
is a perennial medicinal plant from the Euthyra Franch
group in the Trilliaceae family, mostly distributed in the
southwest of China. The rhizome of Paris polyphylla is
used in traditional Chinese medicine as a haemostatic
and antimicrobial agent [1, 2]. Recently, studies of the
aqueous, ethanolic and methanolic extracts of Paris
polyphylla showed their anticancer activity on several
types of cancer cell lines. More extensive phytochemi-
cal and pharmacological studies further identified
steroid saponins as the main antitumor active com-
ponents [1, 3–6]. Paris saponins are a group of plant
glycosides consisting of a steroid aglycone, to which
one or more sugar chains are attached. They exhibit cell
membrane–permeabilizing properties and, thus, have
been investigated for their therapeutic potential [7].
Steroid saponins from Paris polyphylla are classified in
two main groups: diosgenin (Dio) glycosides and pen-
nogenin glycosides. Furthermore, D-glycopyranoside,
L-rhamnopyranoside and L-arabinofuranoside are
the main glycosides linked in the structure of steroid
saponins [8]. However, the relationship between the
anticancer activity and structure of the steroid saponins
of Paris polyphylla had not yet been reported.
Preliminary screening of Paris polyphylla steroid
saponins anti-proliferative activity revealed inhibitory
effect of these compounds on cell growth of LA795
lung adenocarcinoma cell line. Eight known steroid
saponins were isolated from the rhizome of Paris poly-
phylla [9, 10]. In our experiments, the anticancer activi-
ty of these steroid saponins of was investigated. Their
cytotoxicity was evaluated by MTT assay, and apopto-
sis was studied by AnnexinV-FITC/PI staining. We also
studied the antitumour effects of Diosgenin-3-α-L-
arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-
β-D-glycopyranoside (compound 1), the main steroid
saponin from Paris Polyphylla, and diosgenin (Dio),
the aglycone of compound 1, on inbred strains of
laboratory mice (T739) that carried LA795 metastatic
lung cancer. The relationships between anticancer
activity and structure of Paris polyphylla saponins were
discussed in this paper.
MATERIALS AND METHODS
Plant material and extracts preparation. The
herb Paris polyphylla var. yunnanensis had been
collected from Yunnan Province in the southwestern
part of China, and identified by Dr. Gao. Voucher
specimen (No.057902) was deposited in School of
Pharmaceutical Science and Technology of Tianjin
University. The rhizome of Paris polyphylla was dried
at room temperature, and powdered, and extracted by
organic solvents. After using column chromatography
on Sephadex LH-20, PTLC and PHPLC, eight steroid
saponins, titled compound 1 to 8, were obtained
(Fig. 1) [9, 10]. The purity of compound 4 is > 75%,
IN VITRO AND IN VIVO ANTICANCER ACTIVITY OF STEROID
SAPONINS OF PARIS POLYPHYLLA VAR. YUNNANENSIS
L.L. Yan1, §, Y.J. Zhang2, §, W.Y. Gao1, *, S.L. Man1, Y. Wang1
1School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
2School of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin
300193, China
Aim: To confirm the anticancer activity of steroid saponins isolated from the rhizome of Paris polyphylla var. yunnanensis and
evaluate the structure-activity relationships of these steroid saponins in vitro and in vivo. Materials and Methods: Eight known
steroid saponins were isolated from the rhizome of Paris polyphylla. The LA795 lung adenocarcinoma cell line from mice was
chosen to evaluate cytotoxicity by means of MTT assay, and to study apoptosis by means of AnnexinV-FITC/PI flow cytometry.
Diosgenin-3-α-L-arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-β-D-glycopyranoside (compound 1), the main steroid
saponin of Paris polyphylla, and diosgenin (Dio), the aglycone of major steroid saponins, were evaluated for antitumor activity on
LA795 lung adenocarcinoma in T739 inbred mice. Results: The steroid saponins showed remarkable cytotoxicity and caused typical
apoptosis in a dose-dependent manner. They were evaluated in vivo by their effect on tumor developed in T739 inbred mice. The
oral administration to T739 mice bearing LA795 lung adenocarcinoma of compound 1 and diosgenin significantly inhibited tumor
growth, by 29.44% and 33.94%, respectively. HE staining showed that lungs and livers of treated mice underwent various levels of
histopathological alterations. It was demonstrated by TUNEL assay that apoptosis rate in tumor cells was increased in comparison
to cells in control mice. The 3-O-glycoside moiety and spirostanol structure played an important role in the anticancer activity
of steroid saponins, and the number and the variety of glycosides of compounds strongly influenced on their anticancer activity.
Conclusion: Rhizoma Paridis saponins showed anticancer activity against lung adenocarcinoma cell line, both in vitro and in vivo,
and their effect was dependent on compounds’ structure in a certain degree.
Key Words: Paris polyphylla var. yunnanensis, steroid saponins, diosgenin, anticancer activity.
Received: November 11, 2008.
*Correspondance: E-mail: metayll@hotmail.com
§Authors contributed equally.
Abbreviations used: CPH — cyclophosphamide; Dio — diosgenin;
HE — histological examination; MTT — Methylthiazolyldiphenyl-
tetrazolium bromide; PHPLC — preparative high performance liquid
chromatography; PTLC — preparative thin layer chromatography;
TUNEL — terminal deoxytransferase nick end-labeling.
Exp Oncol 2009
31, 1, 27–32
28 Experimental Oncology 31, 27–32, 2009 (March)
and the purity of other seven compounds is > 90%.
Diosgenin (the purity > 99.9%) was purchased from
Sigma-Aldrich Chemie GmbH.
R’O
O
O
R
R R'
Compound 1 H -α-L-arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-
β-D-glycopyranoside
Compound 2 OH -α-L-arabinofuranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-
β-D-glycopyranoside
Compound 3 H -α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-
β-D-glycopyranoside
Compound 4 OH -α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-
β-D-glycopyranoside
Compound 5 H -α-L-arabinofuranosyl(1→4)-β-D-glycopyranoside
Compound 6 OH -α-L-arabinofuranosyl(1→4)-β-D-glycopyranoside
Compound 7 H -α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl(1→4)]-
[α-L-rhamnopyranosyl(1→2)]-β-D-glycopyranoside
Compound 8 OH
Diosgenin H H
Fig. 1. Structure of diosgenin and compound 1~8 [9, 10]
Cell line and culture. The mice lung adenocarcino-
ma cell line (LA795) was purchased from Peking Union
Medicial College (Beijing, China). The LA795 cells were
maintained in RPMI-1640 medium supplemented with
10% heat-inactivated fetal bovine serum (FBS), penicil-
lin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in
5% CO2. Cells were passaged every 3–4 days.
In vitro assay for cytotoxicity (MTT assay).
LA795 cells (3 × 104 cells/mL) were plated on 96-well
plates (200 μL of medium per well). After 24 h, cells
were synchronized by adding serum-free media. After
another 24 h, the cells were treated with various concen-
trations of DMSO solutions of studied compounds (con-
centration of DMSO < 0.1%). Each solution was added
to eight wells. In another 24 h, 100 μL of 0.5 mg/mL MTT
medium was added to each well for another 4 h. The
supernatant fluid was then removed, 100 μL per well of
DMSO was added, and samples were shaken for 15 min.
Absorbance at 570 nm was measured with an enzyme-
linked immunosorbent assay plate reader. This experi-
ment was carried out in triplicate. Growth inhibition (GI)
was calculated according to the formula: GI = 1 – (A570
of treated cells / A570 of control cells). IC50 values were
calculated using a linear regression of dose-dependent
curves plotted from at least six points.
Annexin V-FITC/PI staining for apoptosis
evalua tion. Apoptosis was quantified using flow
cytometry to measure the levels of detectable
phosphatidylserine on the outer membrane of apop-
totic cells. LA795 cells were seeded on 6-well plates
(3 × 104 cells/mL), and incubated with the compounds
in 0.02% DMSO solutions. The concentration of each
compound was equal to one third from respective IC50
value. After 24 h, cells were harvested by centrifuga-
tion, washed twice with PBS at 4 °C, and re-suspended
in diluted binding buffer from the Annexin V-FITC kit
at a concentration of 1 × 106 cells/mL. 100 μL of this
suspension was added to a 5 mL Flow Cytometry (FC)
tube. 5 μL of AnnexinV-FITC and 10 μL of 20 mg/mL
propidium iodide (PI) were further added to the tube,
followed by 15 min of incubation in the darkness.
400 mL of PBS were then added. Quantitative analysis
of apoptotic cells level was performed using a Flow
Cytometer (Coulter Epics Altra, Beckman Coulter Inc.,
USA). The procedure was carried out three times.
In vivo tumor growth inhibition. T739 inbred mice,
both male and female, weighting 18.0 ± 2.0 g, were
obtained from the Laboratory Animal Breeding and
Research Center, Cancer Hospital, Academy of Medical
Science (Beijing, China). The animals were kept under
pathogen-free conditions, and fed with chow and sterile
water ad lib. This animal study was approved by the
Institutional Animal Care and Use Committee of China,
and Institutional guidelines for animal welfare. LA795
cells (1 × 107/mL) were injected subcutaneously into the
right armpits of the mice. Tumors grew to a volume of
0.5 to 1.0 cm3. After sacrificing the mice, the tumor was
excised, cut into small pieces (volume 0.5–1.0 mm3)
and dipped in 0.9% Sodium Chloride at 4 °C. After filt-
ration and deliquation the pieces dispersed into cells.
Those tumor cells were then injected into recipient mice
(2 × 106/mL, 0.2 mL for each mouse). Thus, T739 mice
(n = 40) bearing LA795 lung adenocarcinoma were
obtained. Five days after injection of LA795 cells, forty
T739 mice bearing LA795 lung adenocarcinoma were
randomly divided into four groups (n = 10 per group):
1) control group (0.1 ml/10 g distilled water, p. o.), 2) cy-
clophosphamide (CPH) treated group (20 mg/kg, i. p.),
3) compound 1-treated group (100 mg/kg, p. o.), and
4) diosgenin-treated group (200 mg/kg., p. o.). Drugs
were administered daily for 14 days, once a day.
At the first and the last days of the experiment, the
body weight of each mouse was measured. The tumor
volume was estimated every other day, on the basis of
two-dimensional tumor measurements performed with
a micrometer, and resorting to the formula suggested by
Kato et al. (1994): Tumor vol. (mm3) = 0.5A × B2, where
A is the longest diameter and B the shortest diameter
of the tumor. The mice were sacrificed at the day 14,
and autopsies were performed. Major organs such
as lung, liver, kidneys, spleen, and tumor mass were
then dissected and their weights were measured. The
spleen index was calculated using the formula: spleen
index = weight of spleen (mg) / body weight (g). The
tumor inhibitory rate was calculated using the formula:
tumor inhibitory rate (%) = 100% × (mean tumor weight
in control mice minus mean tumor weight in treated
mice) / mean tumor weight in control mice.
Histological examination. Dissected organs
(lung, liver, kidneys) were fixed in 10% paraformal-
dehyde (pH 7.5) and then processed for paraffin
embedding according to the standard histological
procedures. 4 μm-thick sections were prepared and
Experimental Oncology 31, 27–32, 2009 (March) 29
stained with HE stain. Histopathological examination
was completed using Olympus microscopy.
TUNEL assay. Apoptotic cells in the tumor sec-
tions were visualized by the terminal deoxyncleotidyl
transferase mediated d-UTP nick and labeling (TUNEL)
technique according to the manufacturer’s instruction
(Chemicon International, Inc, USA). Tissue sections
were treated with 10 μg/mL proteinase K for 10 min at
room temperature. The slides were immersed in a 2%
H2O2 solution to block endogeneous peroxidase activi-
ty. TdT was used to catalyze the addition of biotin-con-
jugated d-UTP to the 3’-OH ends of DNA fragments.
The incorporated biotin was detected by streptavidin
conjugated to horseradish peroxidase. The staining
was then performed using 3, 3’-diaminobenzidine
tetrahydrochloride (DAB) as the chromagen and H2O2
as the substrate. Tissue sections were counterstained
with hematoxylin and then mounted. Cells with deep-
brown stained nuclei were counted as positive.
Statistical analysis. SPSS for Windows (SPSS Inc.)
was used to analyze the data and plot curves. Data was
expressed as the mean ± standard deviation (SD). The
statistical significance of differences observed between
each treatment group and the control was determined
by the Student’s t-test (two-tailed). Differences were
considered statistically significant if p < 0.05.
RESULTS
Inhibition of LA795 cells growth in vitro. Nine
compounds (compounds 1–8 and Diosgenin) were
screened using MTT assay. These compounds showed
remarkable cytotoxicity against LA795 cell in a dose-
dependent manner (Fig. 2). Their median inhibitory
concentration (IC50) are listed in Table 1.
0 1 2 3 4 5 6 7 8 9
0
10
20
30
40
50
60
70
80
90
Compound 1
Compound 2
Compound 3
Compound 4
Compound 7
Compound 8
G
ro
wt
h
in
hi
bi
tio
n
(%
)
Concentration of compound (µmol/L)
0 50 100 150 200 250 300
0
10
20
30
40
50
60
70
80
90
Diosgenin
Compound 5
Compound 6
G
ro
wt
h
in
hi
bi
tio
n
(%
)
Concentration of compound (µmol/L)
Fig. 2. Inhibitory effect of compound 1~8 and diosgenin on
LA795 cell growth. LA795 cells were treated with compounds 1~8
or diosgenin at different concentrations for 24 h, and the growth
inhibition was determined using the MTT assay
Table 1. IC50 of the compounds (x— ± S, n = 3)
Compound IC50 (μmol/L)
Compound 1 1.85 ± 0.11
Compound 2 5.14 ± 0.29
Compound 3 3.06 ± 0.33
Compound 4 2.26 ± 0.47
Compound 5 39.00 ± 3.63
Compound 6 319.53 ± 24.77
Compound 7 1.35 ± 0.19
Compound 8 2.71 ± 0.26
Diosgenin 149.75 ± 10.43
Annexin V-FITC assay of LA795 cells apoptosis.
Many chemical compounds can inhibit the growth of tumor
cells, but not all of them can trigger apoptosis [11]. To
determine whether apoptosis was induced by the com-
pounds mentioned above, we performed flow cytometric
analysis with Annexin V-FITC conjugated to propidium
iodide (PI). Annexin V-FITC-positive, PI-negative (Annexin
V-FITC (+) PI (-)) cells were considered to be in an early
apoptotic stage, while Annexin V-FITC-positive, PI-positive
(Annexin V-FITC (+) PI (+)) cells were considered to be
late apoptotic or necrotic. Apoptosis of LA795 cells was
induced by compounds added at the concentration equal
to one third from their IC50 values, although at different
degree. The rates of apoptosis induced by Paris polyphylla
compounds were significantly higher than apoptotic rate
in the control group (p < 0.01) (Table 2).
Table 2. Flow cytometry analysis of LA795 cells after incubation with drugs
for 24 h (x— ± S, n = 3)
Compound Dosage (μmol·L-1) Annexin (+) PI (-) (%) Annexin (+) PI (+) (%)
Control — 1.1 ± 0.3 4.6 ± 0.7
Compound 1 0.62 12.9 ± 0.5* 36.3 ± 2.6*
Compound 2 1.71 5.4 ± 0.6* 20.0 ± 1.0*
Compound 3 1.02 6.8 ± 0.2* 32.3 ± 3.0*
Compound 4 0.75 8.6 ± 1.0* 34.9 ± 3.1*
Compound 5 13.00 3.7 ± 0.1* 13.5 ± 1.7*
Compound 6 106.51 1.3 ± 0.1 18.2 ± 0.4*
Compound 7 0.45 13.7 ± 0.3* 41.1 ± 2.4*
Compound 8 0.90 10.3 ± 0.9* 39.6 ± 1.9*
Diosgenin 49.92 3.4 ± 0.3* 11.8 ± 1.7*
Notes: Annexin (+) PI (-) (%): the percentage of cells in an early apoptotic stage;
Annexin (+) PI (+) (%): the percentage of cells in a late apoptotic or necrotic
stage. *p < 0.01 scores of drug-administered group vs control group.
Anti-tumor effects of the compounds in vivo.
Although any reduction of the tumor volume was not
observed between the drug treatment groups, tumor
masses increased much slowly than in the control group
during the period of drug administration (Fig. 3). Even-
tual tumor weight in all treated groups was obviously
lower than in the control group (p < 0.01) (Table 3). That
indicated CPH, compound 1 and Dio didn’t make the
solid tumors disappear, but slow down their growth.
6 9 12 15 18
0
1000
2000
3000
4000
5000
6000 Control
CPH
Compound 1
Dio
Days after inoculation
Tu
m
ou
r v
ol
um
e
(m
m
3 )
*
*
Fig. 3. Effect of drug treatment on tumor volume. CPH: Cy-
clophosphamide; Dio: Diosgenin. *p < 0.01 scores of drug-
administered group vs control group.
30 Experimental Oncology 31, 27–32, 2009 (March)
Table 3. Inhibitory effects of drug treatment on LA795 transplanted
tumors in T739 mice
Group Dosage
(mg/kg/d)
Tumour volume
(mm3)
Tumour
weight (g)
Inhibition of growth
of tumour (%)
Control — 5462.5 ± 105.5 6.3 ± 0.6
CPH 20 2912.7 ± 252.9* 2.8 ± 0.4* 56.09
Compound 1 100 4234.0 ± 486.4 4.4 ± 0.5* 29.44
Dio 200 4005.1 ± 762.4 4.2 ± 0.5* 33.94
Notes: CPH: Cyclophosphamide; Dio: Diosgenin. *p < 0.01 scores of drug-
administered group vs control group.
Our original purpose of including the cyclophos-
phamide (CPH) group was to compare the anticancer
activity of compound 1 with the CPH group, because
CPH is generally used for treatment of various types
of cancers. It is a clinically approved anticancer agent
that works by slowing or stopping cell growth. Its use
is becoming more common in anticancer experimental
research, and the dosage (20 mg/kg) of the CPH treat-
ed group was referred to previous reports [12, 13].
The solid tumor in the control group grew well and
the average tumor weight reached 6.3 g. Meanwhile,
CPH exhibited significant anti-tumor activity. In the
CPH group (20 mg/kg, i. p.), the average tumor weight
was only 2.8 g and the inhibitory ratio was 56.09%.
Compound 1 and Diosgenin also exerted a remarkable
inhibitory effect on tumor growth. In the compound
1-treated group (100 mg/kg, p. o.) and the Diosgenin-
treated group (200 mg/kg, p. o.), the tumor weights
were significantly reduced (p < 0.01). The inhibitory
ratio of tumor growth reached 29.44% and 33.94%,
respectively (see Table 3).
The body weight of mice in the treatment groups ap-
peared to be slightly decreased in comparison with the
control group, but these results were not statistically-
valid (p > 0.05) (Table 4). Spleen index in all three treat-
ment groups (CPH, compound 1 and Diosgenin) was
reduced compared with the control group, especially
the spleen index of mice treated with compound 1.
Table 4. Effects of drug treatment on body weight and spleen index of
T739 mice bearing LA795 lung adenocarcinoma
Group Dosage
(mg/kg/d)
Body weight (g)
Spleen indexBefore the
treatment
After the
treatment
Control — 23.7 ± 1.1 23.9 ± 1.0 6.8 ± 1.1
CPH 20 24.4 ± 1.3 23.6 ± 1.3 5.3 ± 0.4
Compound 1 100 24.0 ± 0.8 23.5 ± 1.1 4.5 ± 0.7*
Dio 200 25.0 ± 1.1 24.7 ± 1.1 6.0 ± 0.8
Notes: CPH: Cyclophosphamide; Dio: Diosgenin. *p < 0.05 scores of drug-
administered group vs control group.
Effects of compounds on lung, liver, kidneys,
and tumor mass in mice. In order to investigate the
toxicity of the treated compounds, lung, liver and
kidney sections were prepared and stained by HE.
Lesions of various degree were found in the lungs and
livers of treatment groups. The kidney microstructure
was normal. For the compound 1 group, lungs were
seriously damaged with a large area of hemorrhagic
infarction (Fig. 4, a). For other groups (CPH and
Diosgenin), there were congestion and inflamma-
tory infiltration of lungs. Also, the liver lesions in mice
treated with compound 1 were the most severe among
all three treatment groups. As shown on Fig. 4, b and
4, c, the normal structure of the hepatic lobules was
destroyed, and hydropic degeneration, inflammatory
infiltration and fatty degeneration were observed. For
other treatment groups, slight hydropic degeneration
and inflammatory infiltration was found.
a
b
c
Fig. 4. Pathological changes in lungs and livers of T739 mice
bearing LA795 lung adenocarcinoma upon compound 1 treat-
ment. a, lung tissue, showing hemorrhagic infarction, 100 ×.
b, liver tissue, showing inflammatory infiltration, 400 ×. c, liver
tissue, showing fatty degeneration, 400 ×
TUNEL assay. TUNEL assay of tumor sections
was performed to examine the ability of compound 1
to induce cell apoptosis. The apoptosis index is pre-
sented on Fig. 5. Compared with the control group,
the number of apoptosis cells increased significantly
for all three treatment groups. The apoptosis index of
the compound 1 group was 6-fold higher than that of
the control group, and was almost on the same level
as shown for the CPH group.
Experimental Oncology 31, 27–32, 2009 (March) 31
1 2 3 4
0
20
40
60
80
100
TU
NE
L
po
si
tiv
e
ce
lls
(%
)
*
*
*
Fig. 5. The number of apoptotic cells in tumor sections were detected
by TUNEL assay. Apoptotic cells in tumors increased significantly
in drug-treated groups. CPH: Cyclophosphamide; Dio: Diosgenin.
*p < 0.01 scores of drug-administered group vs control group.
DISCUSSION
Currently chemotherapy is regarded as one of the
most efficient cancer treatment aproach. Although
chemotherapy significantly improves symptoms and
the quality of life of patients with lung cancer, only
modest increase in survival rate can be achieved.
Faced with palliative care, many cancer patients use
alternative medicines, including herbal therapies.
Among these therapies, traditional Chinese medicine
is probably the best established and codified, dating
back several thousand years. Traditionally, Chinese
herbs are used in combinations of single herbs, or
prescriptions — called formulas. Specific herbal ex-
tracts, and combinations, have been designed to treat
specific diseases including cancer [14–16].
Paris polyphylla var. yunnanensis has been tradi-
tionally used for centuries. Some medical formula,
such as Chonglou-Tang, which includes this Chinese
herb as a main ingredient, have been used to treat
cancer. Recent studies pointed to steroid saponins as
the main anti-tumor components of this herb. Previous
studies pointed that Paris polyphylla var. yunnanensis
contained significant amounts of steroid saponins,
including polyphyllin D, diosgeninscin, gracillin, trillin,
methylmotogracillin, and so on [5, 6, 17–21].
In the experiments in vitro, as shown in MTT results,
diosgenin saponins had stronger cytotoxicity than
pennogenin saponins. These compounds had remar-
kable cytotoxicity against LA795 cell, and eight steroid
saponins (except compound 6) had higher inhibition
of cell growth compared with diosgenin.
Compound 7 with four glycosides had shown the
highest rate of cell growth inhibition among other
studied steroid saponins, while compounds 5 and
6, containing two glycosides, exhibited the lowest
inhibition rate both among diosgenin and penno-
genin saponins. This data indicates that the number
of glycosides correlate with the cytotoxicity of steroid
saponins. However, IC50 of compound 4 was lower than
that of compound 3 and compound 8. This phenome-
non could be explained by the fact that the purity of
compound 4 was approximately 75%, and the purity of
other seven compounds was more than 90%.
Compared with compound 3 and compound 2,
compound 1 and 4 showed higher cytotoxicity rate, re-
spectively. Although the number of glycosides in com-
pounds was the same, the varieties of glycosides were
different. Besides glycopyranoside and rhamnopyra-
noside, their main difference was that compounds 1
and compound 2 contain arabinofuranosides, and
that compound 3 and 4 contain rhamnopyranosides.
When there was used aglycone of diosgenin, the
activity of rhamnopyranoside was stronger than of
arabinofuranoside-containing compound. But when
there was aglycone of pennogenin, the effect was op-
posite. This indicated that the variety of glycosides of
steroid saponins also could affect cytotoxicity.
Altogether, the 3-O-glycoside moiety and spiro-
stanol structure were found to be essential for the
proapoptotic activity with regard to anticancer struc-
tural relationship of steroid saponins. Their cytotoxic
activities were strongly influenced by the number and
the variety of steroid saponins.
Compound 1 was the main steroid saponin com-
ponent in Paris polyphylla, and the contents of other
seven compounds were very few. So compound 1 and
diosgenin (Dio), the aglycone of compound 1 were
selected for in vivo experiment. And we hope that the
anti-tumor activities in vivo of other compounds could
be estimated through the results of compound 1. In
in vivo experiment, the molar dose of compound 1 was
set to one-quarter of diosgenin, because of the high
toxicity of compound 1 for mice. The results showed
that compound 1 was as effective as diosgenin with the
4-fold molar dose in inhibition of tumor growth. This
further indicated that glycosides of steroid saponins
promote the anticancer activity of diosgenin. From the
results of tissue sections HE staining, it is seen that
compound 1 caused stronger lung and liver damage
than diosgenin. Probably the 3-O-glycoside structure
may contribute to the toxicity of steroid saponins.
Treatment of tumors is directed not only on inhibition
of cell proliferation, but also on induction of apoptosis
of tumor cells. More and more attention is paid to the
ability of drugs to induce apoptosis in the process of
evaluation of anti-tumor agents’ effectiveness. That
is why we tested the ability of eight steroid saponins
extracted from Paris polyphylla to induce apoptosis by
means of AnnexinV-FITC/PI flow cytometry approach.
The rates of apoptosis in the treatment groups were
significantly higher than that in the control group, which
was shown by AnnexinV-FITC/PI flow cytometry. Addi-
tionally, in TUNEL assay of tumor sections, the number
of apoptotic cells in tumors treated with compound 1
was increased too. This data suggest that one of anti-
tumor mechanisms of steroid saponins is an induction of
tumor cell apoptosis. The main biologic activity ascribed
to saponins is their membrane permeabilizing property.
The main actions are considered changes in membrane
permeability and pore formation [22–24]. In apoptotic
death, an increase of mitochondrial membrane perme-
ability is considered to be one of the key events. A sud-
den increase in permeability of the inner mitochondrial
32 Experimental Oncology 31, 27–32, 2009 (March)
membrane, the so-called mitochondrial permeability
transition, is a common feature of apoptosis and is me-
diated by the mitochondrial permeability transition pore
(mtPTP) [25–27]. So the detailed mechanism of the
eight steroid saponins extracted requires future study.
In conclusion, steroid saponins of Paris polyphylla
var. yunnanensis could be regarded as promising
drugs for cancer therapy, but the mechanisms of their
anti-cancer activity and their toxicity should be further
addressed. In our studies, we demonstrated that the
structure of spirostanol 3-O-glycoside and the number
of glycosides were two main factors responsible for
compounds’ cytotoxicity. Also, we plan to study further
the saponins structure-function relationships, and per-
form structure modifications to design new compounds
with strong anti-tumor activity and low toxicity.
ACKNOWLEDGEMENTS
This work was supported by a grant 07JCZD-
JC05400 from Tianjin Municipal Science and Techno-
logy Commission (Tianjin, China). We also wish to
thank Traditional Chinese Medicine Department at
Tianjin University of Traditional Chinese Medicine for
the use of their equipment.
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