全 文 ：第 21卷第 12期 中国现代医学杂志 Vol. 21 No.12
2011年 4月 China Journal of Modern Medicine Apr. 2011
文章编号： 1005- 8982（2011）12- 1449- 08
·论著·
In vitro screening of effective anti-HBV and anti-tumor compounds
from Rabdosia serra (Masim.) Hara*
HE Ying1, LI Hui-ying2, KANG Li-qun1, ZHANG Xiu-zhen1, XU Hong-juan1, LIU Huan1, CHEN Yu-xiang2
(1.School of Pharmacy, Central South University, Changsha, Hunan 410013, P.R.China; 2.School of Biological
Science and Technology, Central South University, Changsha, Human 410013, P.R.China)
Abstract: 【Objective】To screen and investigate the anti-HBV and anti-tumor activities of separated com－
pounds from Rabdosia serra (Maxim.) Hara for the further isolation of its active entities. 【Methods】Three extracts
from Rabdosia serra by using different solvents (petroleum ether, acetidin or butyl alcohol) were prepared and ana－
lyzed of their anti-HBV activities in HepG2.2.15 cells before further separation. The cytotoxicity of each extract was
tested by MTT assay. The levels of HBsAg, HBeAg and HBV DNA in supernatants from the culture of HepG2.2.15
cells were detected by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain re－
action (PCR). The most effective extract was then separated and the anti-HBV activities of separated compounds
were tested by MTT and ELISA. The anti-tumor effects of three compounds with high cytotoxicity were identified
using MCF-7, BGC-823 and HepG2 cells.【Results】The acetidin extract C2, which was then further separated, had
the most effective anti-HBV activity as shown by significant reductions in the secretion of HBsAg and HBeAg and
the amount of HBV DNA. There were 14 compounds separated from C2, among which A3 and A5 markedly inhibited
the HBsAg secretion while A9 inhibited the HBeAg secretion in a dose-dependent manner. Their TIs were all higher
than that of C2. Moreover, the compounds A6, A7 and A11 had different anti-tumor activities in different tumor cells.
【Conclusions】The acetidin extract from Rabdosia serra and its effective separated compounds have anti-HBV ac－
tivities by strongly inhibiting HBV replication. The further separation and purification can strengthen their anti-HBV
effects. Meanwhile, some compounds have high cytotoxicity in different tumor cells. Our study provides theoretical
evidences for the development of potential and efficient drugs from Rabdosia serra and its clinical use against HBV
and tumor.
Key words: Rabdosia serra (Maxim.) Hara; effective compound; anti-HBV; anti-tumor
CLC number: R965 Document code: A
溪黄草抗乙肝及抗肿瘤活性成分的体外筛选*
何 颖 1，李辉莹 2，康丽群 1，张秀珍 1，胥洪鹃 1，刘 欢 1，陈玉祥 2
（1 .中南大学药学院，湖南 长沙 410013；2.中南大学生物科学与技术学院，湖南 长沙
410013）
摘要：目的 对溪黄草抗乙肝抗肿瘤活性部位进行初步筛选，为进一步分离活性成分奠定基础。方法 首
先在 HepG2.2.15细胞模型上对三种不同方法萃取所得的溪黄草提取物进行抗乙肝活性筛选，以用于进一步分
析提纯。用MTT 实验检测细胞毒性，ELISA和实时定量 PCR 检测细胞上清液中 HBsAg、HBeAg的分泌及乙
肝病毒 DNA含量。然后，用硅胶柱色谱法进一步提取分离活性最大的提取物，用 MTT 和 ELISA检测所分离
Received date: Feb. 21, 2011
*Foundation: Changsha Science and Technology Plan (K0902033- 31) and Hunan Science and Technology Plan (2009FJ3209)
Corresponding author: Chen Yu- xiang, Tel/Fax: +86 731 82650230, E- mail: chenyx@xysm.net
1449· ·
中国现代医学杂志 第 21卷
Rabdosia serra (Maxim.) Hara [Isodon lophan－
thoides (Buch. -Ham.exD.Don) H. Hara], which be－
longs to the Labiatae family, has been widely used in
China for years in the treatment of arthritis, enteritis,
jaundice, hepatitis, acute icteric hepatitis, lepromatous
leprosy and ascariasis[1-3]. The treatment of 68 hepatitis
B patients with decoctions containing Rabdosia serra
obtained a recovery rate of 83.8% [4]. Another clinical
report by Qin Xue-feng[5] also notified the use of Rab－
dosia serra in the treatment of 300 acute icteric he-
patitis patients. The genus Rabdosia is abundant in
diterpenoids, which have diverse bioactivities such as
antibacterial, anti -inflammatory and antitumor activi－
ties[6-9]. In particular, the extract of Rabdosia serra has
a well-documented antioxidant property[10], which may
be the major mechanism underlying its anti -inflam－
matory and anti-tumor effects. Although Rabdosia ser－
ra has been shown to have different pharmacologic ac－
tions, the information regarding the in vitro ant-viral
and anti -tumor capacities of its main active com－
pounds is limited. Thus far, only one report [11] showed
its anti-HBV activity in HepG2.2.15 cells, which only
investigated the secretion of HBsAg and HBeAg. Se-
veral other reports also demonstrated its anti -tumor
activities in Hela cells[12-13] and CaEs-17 cells[14].
Hepatitis B is a serious disease caused by he-
patitis B virus (HBV). Patients infected by HBV will
develop acute or chronic hepatitis, which may result
in cirrhosis, liver failure, liver cancer and even death.
Several anti -viral drugs have been approved for the
treatment of hepatitis B, including interferon IFN -α
and nucleoside analogues Lamivudine, Adefovir, and
Entecavir [15-17]. However, significant unresolved issues
remain with current drugs, such as dose -dependent
side effects, hepatic decompensation, long-term appli－
cation caused viral mutations and drug resistance[17-18],
and rebound phenomenon[19-20]. An imperative medi－
cal need is existing for safe and efficacious new anti-
HBV drugs. It has been widely accepted that natural
products[21], like flavonoids, terpenoids and alkaloids[22-23],
are good sources for promising anti -viral drugs be－
cause of their molecular diversity, multi -functions,
synergistic hepatoprotective effects and low toxicity[24-26].
To explore potential clinical indications of Rabdosia
serra, in this study we extracted and separated effec－
tive compounds from it, investigated their inhibitory
effects on HBsAg and HBeAg secretion and HBV
DNA replication in HepG2.2.15 cells, and identified
their anti-tumor effects in three tumor cells (MCF-7,
BGC -823 and HepG2 cells), which provides evi－
dences for further anti-HBV and anti-tumor studies of
Rabdosia serra.
1 Materials and methods
1.1 Plant and reagents
Rabdosia serra (Maxim.) Hara [Isodon lophan－
thoides (Buch.-Ham.exD.Don) H. Hara] was collected
from Changsha, Hunan Province in China, and au－
thenticated by Professor Chen Yu -xiang (XiangYa
School of Medicine, Central South University, Chang－
sha, China). Ethanol, petroleum ether, acetidin, butyl
alcohol, methanol and chloroform were from Chemical
Plant of Hunan Normal University (Changsha, China).
Lamivudine (3-TC) and fluorouracil (5-Fu) were pro－
各组分的抗乙肝活性。最后，在MCF- 7，BGC- 823和 HepG2细胞中检测细胞毒性最大的三个组分的抗肿瘤
活性。结果 乙酸乙酯萃取分离所得的提取物抗乙肝活性最大，可显著降低 HepG2.2.15细胞的 HBsAg、HBeAg
抗原的分泌，并抑制乙肝病毒 DNA的复制，被用来进一步分离提纯。从乙酸乙酯提取物中分离出 14个组分，
其中 A3和 A5组分对 HBsAg有较好的抑制作用，A9组分对 HBeAg有较好的抑制作用，并均存在明显的量效关
系，且治疗指数均比乙酸乙酯提取物的要高。A6，A7和 A11组分细胞毒性大，对不同的肿瘤细胞有不同的抑制活
性。结论 溪黄草的乙酸乙酯提取物及其分离成分具有很强的抑制乙肝病毒复制的作用，从而具有很好的抗乙
肝病毒活性，进一步提取分离有利于有效成分的纯化而具更强的活性。此外，一些分离成分对不同的肿瘤细胞
还有很高的细胞毒性，具一定的抗肿瘤作用。本实验为溪黄草在临床的使用及其作为潜在的高效抗乙肝和肿瘤
药物的进一步研究开发工作提供了理论基础。
关键词： 溪黄草；活性分离成分；抗乙肝作用；抗肿瘤作用
中图分类号： R965 文献标识码：A
1450· ·
Chloroform Methanol Water
9 1 0.1
8 2 0.1
7 3 0.1
6 4 0.1
4 6 0.1
3 7 0.1
2 8 0.1
1 1 0.1
Table 1 Composition ratio of chloroform-methanol-water
mixture
vided by the Cancer Research Institute of Central
South University. All chemicals were of analytical
grade.
3-(4, 5-di-methylthiazol -2-yl) -2, 5 -diphenyl
tetrazolium (MTT) was purchased from Sigma (St.
Louis, MO, USA). RPMI-1640 medium was purchased
from Invitrogen (USA). Trypsin, fetal bovine serum
(FBS), dimethyl sulfoxide (DMSO) and geneticin
(G418) were acquired from Gibco BRL (Grand Island,
NY, USA). Glutamine, penicillin and streptomycin
were obtained from XiangYa Cell Center of Central
South University (Changsha, China).
1.2 Extraction and separation
The dried Rabdosia serra (Masim.) Hara was
crushed into powder and extracted in 50% ethanol by
refluxing twice each for 3 hours. The ratio of the sol－
vent to raw materials was 20∶1. The primary ethanol
extract was filtered and concentrated to the crude ex－
tracta sicca by a rotary evaporator. 40.2 g extracta
sicca was then dissolved in distilled water in triplicate
and further extracted by petroleum ether, acetidin or
butyl alcohol using a separatory funnel. The final ex－
tracts were collected and concentrated to petroleum
ether extract (C1), acetidin extract (C2) and butyl alco－
hol extraction (C3) respectively. The yield of each ex－
tract was calculated as follow.
Extraction yield (%) = weight of extract / weight
of extracta sicca × 100 %
According to results of the following anti -HBV
study with different extracts, the most effective extract
was further separated by silica gel column chromatog－
raphy. Briefly, 10.2 g of extract was precisely weigh-
ed, dissolved in 20 mL chloroform, and added into the
silica gel column carefully followed by another 50 mL
chloroform. The gradient elution was performed with
chloroform-methanol-water mixtures at different ratios
in sequential order as shown in Table 1. The separat－
ed fractions were identified by thin layer chromatogra－
phy (TLC) and observed by a UV spectrophotometer.
The similar fractions were merged as one compound
and concentrated under negative pressure. The per－
centage (%) of each compound was calculated as fol－
low.
Percentage (%) = weight of compound / weight of
active extract × 100 %
1.3 Sample preparation
The petroleum ether extract C1, acetidin extract
C2, butyl alcohol extract C3, and separated compounds
were dissolved in 0.1% DMSO respectively as stocks.
In subsequent assays, the stock solution was diluted to
concentrations of 0.5, 2, 4, 8 and 16 mg/mL with RP－
MI-1640 medium (supplemented with 10% FBS, 100
U/mL penicillin, 100 U/mL streptomycin, 100 mg/L
G418 and 2 mmol/L glutamine, pH 7.0). All samples
were filtered by syringe filters of 0.22 μm.
1.4 Cell culture
The HepG2.2.15 cell line, human breast carcino－
ma cell line MCF-7, human gastric cancer cell line
BGC-823, and human hepatocellular liver carcinoma
cell line HepG2 were purchased from XiangYa Cell
Center of Central South University (Changsha, China)
and cultured in RPMI -1640 medium supplemented
with 10% FBS, 100 U/mL penicillin, 100 U/mL strep－
tomycin, 100 mg/L G418 and 2 mmol/L glutamine (pH
7.0) in a 5% CO2 incubator at 37℃.
1.5 Cytotoxicity assay
The cytotoxicity of extracts (C1, C2, C3) and sepa－
rated compounds from Rabdosia serra in HepG2.2.15
were evaluated using MTT assay. HepG2.2.15 cells
were seeded in 96-well plates at 2×104 cells/well in
200 μL medium and cultured for 24 h. After cells ad－
hered, the supernatants were replaced carefully with
200 μL samples at different concentrations (0.5, 2, 4,
8 and 16 mg/mL). Cells treated with culture medium
(containing 0.1% DMSO) were set as the negative
第 12期 何 颖，等：溪黄草抗乙肝及抗肿瘤活性成分的体外筛选
1451· ·
中国现代医学杂志 第 21卷
control, while 3-TC (0.1 mg/mL) treated cells were the
positive control. Each group contained 4 parallel
wells. After 6 days treatment, the supernatant was re－
moved and 20 μL MTT (5 mg/mL) was added in each
well, which was then replaced with 200 μL DMSO af－
ter incubation for 4 h to dissolve purple formazan of
MTT. The absorbance (OD value) was detected by a
microplate reader at 490 nm. The cell inhibition rate
was calculated as follow.
Inhibition rate (%) = (ODnegative - ODsample) / (ODnegative
- ODblank) × 100%
The median toxic concentration (TC50), the drug
concentration with which 50% culture cells survived,
was determined by dose -response data from at least
three independent experiments by the Origin software.
1.6 Quantification of HBsAg and HBeAg
The levels of HBsAg and HBeAg were deter－
mined by ELISA. Supernatants were collected on the
sixth day and reserved at -20℃ for analysis. HBsAg
and HBeAg in culture supernatants of HepG2.2.15
cells were quantified using quantitative ELISA test
kits (Rongsheng, Shanghai, China) under manufactur－
ers instructions. The absorbance (OD value) was
measured with a microplate reader at 490 nm. The
antigen inhibition rate was calculated as follow.
Inhibition rate (%) = (ODnegative - ODsample) / (ODnegative
- ODblank) × 100%
The median effect concentration (EC50), the con－
centration at which the drug achieved the inhibition
rate of 50% against antigens, was determined from
dose-response data by the Origin software.
The therapeutic index (TI) of samples was calcu－
lated by TI=TC50 / EC50. When TI ≥ 2, it means that
the drug is effective (positive); when 1 ≤ TI < 2, it
indicates that the drug has a little effect but high cyto－
toxicity (weakly positive); when TI < 1, it suggests that
the drug is noneffective (negative).
1.7 Measurement of HBV DNA by real-time quan-
titative polymerase chain reaction (PCR)
According to TIs of different extracts, the most
effective one was employed for the quantification of
HBV DNA by real-time quantitative PCR. After cells
were incubated with extracts at different concentra－
tions (0.5, 2, 4, 8 and 16 mg/mL) for 6 days, the su－
pernatants were collected. Cells treated with 3 -TC
were set as the positive control. The HBV DNA was
extracted from culture supernatants using DNA Ex－
traction Kits (CASarray, Shanghai, China). Real-time
quantitative PCR was performed in an Applied
Biosystems Prism 7000 instrument (ABI 7000, USA)
using HBV Fluorescent Quantitative PCR Detection
Kits (RongSheng biological technology Co., LTD,
Shanghai, China).
1.8 Anti-tumor activity study
According to the result from cytotoxicity assay of
separated compounds in HepG2.2.15 cells, 3 com－
pounds with high cytotoxicity were selected for the an－
ti-tumor activity study. The growth inhibitory rates of
these compounds in MCF-7, BGC -823 and HepG2
cells were also evaluated by MTT assay. Cells were in－
cubated in 96 -well plates at 1 ×104/well in 200 μL
medium. After cells adhered, the supernatants were
replaced with 200 μL samples at different concentra－
tions (0.5, 2, 4, 8 and 16 mg/mL). Each group con－
tained 4 parallel wells. The following procedures were
the same as described above. The inhibition rate was
calculated as follow.
Inhibition rate (%) = (ODnegitive - ODsample) / (ODcontrol
- ODblank) × 100%
The median inhibitory concentration (IC50), the
concentration at which the drug achieved the inhibi－
tion rate of 50% against culture cells, was determined
by dose-response data from at least three independent
experiments by the Origin software.
1.9 Statistical analysis
Data were expressed as mean ± SD. Statistical
differences between controls and treated groups were
determined by t-test using SPSS 13.0 software. P val－
ues less than 0.05 were considered of statistical sig－
nificance.
2 Results
2.1 Anti -HBV activities of three extracts in
HepG2.2.15 cells
The triplicate suspensions of extracta sicca (40.2
g) were extracted by petroleum ether, acetidin and
1452· ·
Treatment
Concentra-
tion/(mg/mL)
MTT data TC50
/(mg/mL)OD490 IR/%
Negative control 0.724±0.051 0
Positive control 0.1 0.236±0.082 67.352)
16 0.293±0.056 59.522) 10.471
8 0.407±0.048 43.812)
4 0.530±0.051 26.731)
2 0.689±0.089 5.26
0.5 0.749±0.063 - 3.45
16 0.352±0.058 51.342) 14.563
8 0.472±0.054 34.751)
4 0.613±0.037 15.31
2 0.732±0.065 1.13
0.5 0.793±0.056 - 9.57
Acetidin extract
C2
Butyl alcohol
extract C3
butyl alcohol respectively. The weight and yield of
extract C1, C2 or C3 were stated in Table 2, which
showed that the yield of C1 was the lowest while those
of C2 and C3 were almost equal.
HBsAg, HBeAg and HBV DNA are general mark－
ers of HBV replication. The elimination of HBsAg and
HBeAg and the eradication of HBV DNA are general－
ly accepted as epidemiologic indicators of complete
cure of hepatitis B. In this study, we first evaluated
the anti -HBV effects of three extracts by detecting
HBsAg, HBeAg and HBV DNA in the culture super－
natant to find out the most effective one for the further
separation and screening.
2.2 Cytotoxicity of three extracts in HepG2.2.15
cells
The growth inhibitory rates of three extracts in
HepG2.2.15 cells were measured by MTT assay on
day 6. Results showed that C1 had neither cytotoxicity
nor inhibitive effects on the secretion of HBsAg and
HBeAg. Nevertheless, the growth of HepG2.2.15 cells
was inhibited by C2 and C3 in a dose-dependent manner
(Table 3). When treated with C2 and C3 at 16 mg/mL, the
inhibition rates were as high as 59.52 % and 51.34 %
respectively (P <0.01). The TC50 values of C2 and C3
were 10.471 mg/mL and 14.563 mg/mL respectively.
Thus, Rabdosia serra extracts exhibited inhibitory ef－
fects on the proliferation of HepG2.2.15 cells, which
is consistent with its anti-tumor activity as previously
reported.
2.3 Inhibitory effects of different extracts on se－
cretion of HBsAg and HBeAg
HBsAg and HBeAg were quantified by ELISA.
As shown in Table 4 and 5, the treatment of HepG2.2.15
cells with C2 for 6 days resulted in significant reductions
of HBsAg and HBeAg secretion in a dose-dependent
manner, with EC50 value of 2.351 mg/mL for HBsAg and
that of 1.490 mg/mL for HBeAg. Its TIs were 4.453
for HBsAg and 7.028 for HBeAg, which were both
higher than 2, indicating positive effects. Moreover, C2
at 16 mg/mL achieved greatly high IRs of HBsAg and
HBeAg more than 90% , which was more efficacious
than the positive control 3 -TC. C3 was less efficient
than C2, with EC50 value of 8.211 mg/mL for HBsAg
and that of 11.316 mg/mL for HBeAg. The TIs of C3
against HBsAg and HBeAg were both lower than 2,
suggesting weakly positive effects with high toxicity.
MTT and ELISA data showed that the cytotoxici－
ty of C2 was higher than that of C1 or C3, and its in－
hibitory effects on the secretion of HBsAg and HBeAg
were also stronger (TI > 2). These findings (Table 3,
4, 5) demonstrated that C2 had the most efficient anti-
HBV activity. To further confirm the anti-HBV activi－
ty of C2, its effect on the HBV DNA level was evaluat－
ed.
2.4 Effect of acetidin extract C2 on HBV DNA
level
Real-time PCR was performed to determine virus
DNA in supernatants from the HepG2.2.15 cell cul－
tures. In cells treated with C2, the HBV DNA was sig－
nificantly reduced in a dose-dependent manner. Con－
sistent with its inhibitory effects on HBsAg and
HBeAg secretion, C2 at 4, 8 and 16 mg/mL led to a
Extract Weight/g Yield/%
Petroleum ether extract C1 3.4 8.5
Acetidin extract C2 10.2 25.4
Butyl alcohol extract C3 10.7 26.5
Table 2 Weight and yield of different extracts from
Rabdosia serra (Masim.) Hara
Note: 1)P <0.05, 2)P <0.01 when compared with the negative control.
Table 3 Inhibitory effects of extracts C2 and C3 on
HepG2.2.15 cells
第 12期 何 颖，等：溪黄草抗乙肝及抗肿瘤活性成分的体外筛选
1453· ·
中国现代医学杂志 第 21卷
Compound Weight/g Percentage/% TC50/(mg/mL)
A1 0.20 1.961 7.983
A2 1.00 9.80 6.107
A3 0.06 0.59 5.216
A4 0.03 0.29 -
A5 1.21 11.86 5.236
A6 0.93 9.12 0.471
A7 0.61 6.27 1.208
A8 1.23 12.06 5.856
A9 0.02 0.20 8.062
A10 2.10 20.59 6.535
A11 1.78 17.45 0.658
A12 0.32 3.14 13.129
A13 0.03 0.29 -
A14 0.01 0.09 -
Table 6 Weight, weight percentage and TC50 of compounds
from acetidin extract C2
Treatment Concentration/(mg/mL)
ELISA data EC50
/(mg/mL)
TI (TC50/
EC50)OD490 IR/%
Negative control 2.145±0.132 0
Positive control 0.1 0.319±0.083 85.132)
Acetidin extract
C2
16 0.203±0.041 90.522) 2.351 4.453
8 0.449±0.052 79.292)
4 0.921±0.068 57.101)
2 1.360±0.049 36.611)
0.5 1.682±0.052 21.57
Butyl alcohol
extract C3
16 0.810±0.053 62.242) 8.211 1.773
8 1.004±0.092 53.212)
4 1.466±0.085 31.651)
2 1.606±0.112 25.12
0.5 1.965±0.009 8.36
Note: 1)P <0.05, 2)P <0.01 when compared with the negative control.
Table 4 Inhibitory effects of extracts C2 and C3 on HBsAg
secretion from HepG2.2.15 cells
Treatment
Concentra－
tion
/(mg/mL)
ELISA data EC50
/(mg/mL)
TI (TC50/
EC50)OD490 IR/%
Negative control 1.615±0.134 0
Positive control 0.1 0.416±0.016 74.252)
Acetidin extract
C2
16 0.076±0.028 97.322) 1.490 7.028
8 0.266±0.062 87.532)
4 0.571±0.043 64.622)
2 0.882±0.051 47.372)
0.5 1.128±0.079 31.131)
Butyl alcohol
extract C3
16 0.674±0.060 58.252) 11.316 1.287
8 0.995±0.064 38.362)
4 1.249±0.092 22.721)
2 1.343±0.071 16.83
0.5 1.578±0.088 2.32
Note: 1)P <0.05, 2)P <0.01 when compared with the negative control.
Table 5 Inhibitory effects of extracts C2 and C3 on HBeAg
secretion from HepG2.2.15 cells
statistically significant reduction in extracellular HBV
DNA when compared with the negative control (P <
0.05, Figure). This finding indicated that C2 could
markedly inhibit the HBV DNA replication, which
further confirmed its efficient anti-HBV activity.
2.5 Anti -HBV effects of compounds separated
from acetidin extract C2
The most efficient anti-HBV extract C2 was se-
parated and identified by silica gel column chro－
matography and TLC. Total 14 compounds were ob－
tained. Their cytotoxicity in HepG2.2.15 cells was ex－
amined by MTT assay (Table 6). The compounds A4,
A13 and A14 had no cytotoxicity.
Effects of 14 compounds from C2 on the secretion
of HBsAg and HBeAg in HepG2.2.15 cells were in－
vestigated by ELISA. Among them, A3 and A5 had
more satisfactory inhibitory effects on the secretion of
HBsAg with their TIs of 5.934 and 2.655 respectively
(Table 7), while A9 inhibited the secretion of HBeAg
more effectively with the TI of 9.485 (Table 8). Their
inhibitory effects were all in a dose-dependent man－
350
300
250
200
150
100
50
0
Co
nt
rol
C 2
16
mg
/m
L
C 2
8 m
g/m
L
C 2
4 m
g/m
L
C 2
2 m
g/m
L
C 2
0.5
mg
/m
L
ST
C
0.1
mg
/m
L
H
BV
DN
A
(×
10
00
co
pi
es
/m
L)
1) 1)
2)
2)
Figure Inhibitory effect of C2 on HBV DNA level in
supernatants from HepG2.2.15 cell cultures
Note: 1)P <0.05, 2)P <0.01 when compared with control.
1454· ·
Treatment
Concentration
/(mg/mL)
ELISA data EC50 /
(mg/mL)
TI (TC50/
EC50)OD490 IR/%
Negative control 1.947±0.115 0
Positive control 0.1 0.319±0.083 87.132)
A3 16 0.041±0.009 97.862) 0.879 5.934
8 0.083±0.032 96.722)
4 0.285±0.051 89.392)
2 0.735±0.043 62.272)
0.5 1.215±0.052 37.611)
A5 16 0.073±0.021 96.272) 1.972 2.655
8 0.173±0.020 91.112)
4 0.544±0.053 72.052)
2 1.143±0.045 41.321)
0.5 1.683±0.118 13.58
Note: 1)P <0.05, 2)P <0.01 when compared with the negative control.
Table 7 Inhibitory effects of A3 and A5 on HBsAg
secretion from HepG2.2.15 cells
Treatment
Concentration
/(mg/mL)
ELISA data EC50 /
(mg/mL)
TI (TC50/
EC50)OD490 IR/%
Negative control 1.615±0.174 0
Positive control 0.1 0.416±0.046 71.252)
A9 16 0.010±0.013 99.362) 0.850 9.485
8 0.045±0.023 97.212)
4 0.167±0.018 89.652)
2 0.510±0.044 68.412)
0.5 0.958±0.056 40.701)
Note: 1)P <0.05, 2)P <0.01 when compared with the negative control.
Table 8 Inhibitory effect of A9 on HBeAg secretion from
HepG2.2.15 cells
Treatment
Concen－
tration
/(mg/mL)
MCF-7 BGC-823 HepG2
IR/% TC50/(mg/mL)
TC50
/(mg/mL)
TC50
/(mg/mL)
Negative 0 0 0
Positive 0.5 93.352) 99.622) 96.512)
A6 16 32.561) 45.042 33.831) 40.201 1002) 0.597
8 19.35 25.281) 96.352)
4 15.17 18.01 92.512)
2 11.92 10.79 84.632)
0.5 3.27 -5.93 59.392)
A7 16 96.022) 0.902 41.132) 28.151 98.032) 1.156
8 93.322) 30.721) 93.162)
4 75.462) 23.61 76.092)
2 68.912) 13.87 59.632)
0.5 38.641) 7.25 32.76*
A11 16 98.782) 0.795 97.532) 1.368 100.002) 0.744
8 96.032) 93.322) 96.122)
4 81.292) 74.032) 87.262)
2 67.312) 57.762) 78.622)
0.5 47.821) 24.45 53.922)
IR/% IR/%ner. In addition, A3 and A5 at concentrations of 8 and
16 mg/mL even suppressed HBsAg secretion more
powerfully than the positive control 3 -TC did (over
90% vs 87.13% ). Similar findings were observed in
inhibited HBeAg secretion by A9 at 4, 8 and 16 mg/mL
(over 80% vs 71.25% ). When comparing the ELISA
results between extract C2 and its separated com －
pounds, we found that the further separation and pu－
rification had been done, the higher TI was achieved.
Thus, A3, A5 and A9 were the essential effective anti-
HBV compounds from Rabdosia serra with high effi －
ciency and low toxicity. However, after separation the
compounds could only impact on one antigen, either
HBsAg or HBeAg, while before that C2 could inhibit
both of them. Though further separation and purifica－
tion made inhibitory effects less extensively, they
could purify out the most effective compounds for
more specific targeting and better controlling. By re－
combining the active separated compounds, we could
also make the optimal effects realized, which is in ac－
cordance with the theory of effective compounds
group[27-28].
2.6 Anti -tumor effects of compounds separated
from acetidin extract C2
Among 14 compounds separated from C2, A6, A7
and A11 had high cytotoxicity in HepG 2.2.15 cells
with the TC50 as low as 0.471, 1.208 and 0.658 mg/mL
respectively, which were then selected for the further
anti-tumor study in three tumor cells (MCF-7, BGC-
823 and HepG2 cells) for the development of new an－
ti-tumor drugs from Rabdosia serra.
As shown in Table 9, the growth of HepG2 cells
was significantly inhibited in a dose-dependent man－
ner by all three compounds, and the most effective
one was A6 with the TC50 of 0.597. However, A6 had
little effect on other two tumor cells with the TC50 over
40 mg/mL. A7 could inhibit MCF-7 and HepG2 cells
with the TC50 of 0.902 and 1.156 mg/mL respectively,
but only had a slight effect on BGC-823 cells with
Table 9 Effects of A6, A7 and A11 on three cells viability
(%, x±s)
第 12期 何 颖，等：溪黄草抗乙肝及抗肿瘤活性成分的体外筛选
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the TC50 of 28.151 mg/mL. A11 had a markedly in －
hibitory effect on all three cells with the TC50 of
0.795, 1.368 and 0.744 mg/mL respectively. Moreover,
obvious enhancements of cytotoxicity were observed in
these compounds when compared with acetidin extract
C2, and they showed different cytotoxicity in different
tumor cells. These findings indicated that further iso－
lation increased the anti -tumor activities as well as
targeting abilities of separated compounds. Thus these
active compounds derived from C2 could be applied to
the prevention of different cancers based on their spe－
cific targeting on different tumor cells.
3 Discussion
In conclusion, our study demonstrated that the
acetidin extract from Rabdosia serra and its separated
compounds had potent anti-HBV activities in vitro. A－
mong the purified compounds, there were three with
high anti -tumor activities in different tumor cells in
vitro. Further separation and purification could appa-
rently increase the effectivenesses. The in vitro potency
and efficacy of active compounds from Rabdosia serra
present the possibility and feasibility to develop a po－
tential therapeutic agent from this natural product for
better management of hepatitis B and cancers. Our
study fills gaps in the in vitro research of anti-HBV
and anti -tumor effective compounds from Rabdosia
serra. The activity screening in this study also pro－
vides evidences for the further development and clini－
cal use of prospective anti -tumor and anti -HBV
medicines from Rabdosia serra. Nevertheless, further
in vivo studies are necessary to reconfirm the activity
and safety of Rabdosia serra before its clinical appli－
cation.
Acknowledgements
We thank for the support from Changsha Science
and Technology Plan (K0902033-31) and Hunan Sci－
ence and Technology Plan (2009FJ3209).
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