Inhibitory Effect of Buddlejasaponin IV on Hepatocarcinoma 22(H22)in Mice

Yueyuan CHEN, Yonglin HUANG, Jiejing CHEN, Fenglai LU, Dianpeng LI*

1. Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin 541006, China;

2. Guangxi Key Laboratory of Metabolic Diseases Research, Guilin 541002, China

Buddlejasaponin IV (BJ-IV) is a triterpenoid saponin containing Δ11-13, 28-epoxy structure.Studies have shown that buddlejasaponin IV has significant anti-inflammatory and analgesic effects, and the mechanisms may be related to inhib ited NO, PGE2 and TNF-α[1]. Buddlejasaponin IV also has anti-hyperchlesterolemia and anti-hyperlipidemia effects, which are equivalent to those of probucol[2].Moreover,buddlejasaponin IV also has anti-liver fibrosis activity in vitro[3]. There are rare reports on inhibitory effect of buddlejasaponin IV on H22tumor in vivo. In this study, the inhibitory effect of buddlejasaponin IV on growth of transplanted H22tumor in mice was investigated. The buddlejasaponin IV was injected into abdominal cavities of mice, and the H22tumorbearing mice were treated as the pharmacodynamic models. The tumor inhibition rate, as well as antioxidant enzyme activities in mice serum, was determined. The possible mechanism of inhibitory effect of buddlejasaponin IV on growth of tumor in vivo was studied,providing pharmacological basis for development of buddlejasaponin IV.

Materials and Methods

Experimental animal, tumor strain and reagents

Total 50 4-6-week-old Kunming mice (SPF,half male and half female)with weight of 18-22 g were randomly divided into five groups,and they were provided by the Animal Experiment Center of Guangxi Medical University(SCXK (G)2003-0003).The H22tumor strain was purchased from the China Center for Type Culture Collection(preservation center of Wuhan University),and its subculture was performed by the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization.The cyclophosphamide(CTX) for injection (Batch No.03110121) was produced by the Zhangjiagang City Hengrui Pharmaceutical Machinery Co., Ltd. The superoxide dismutase (SOD), malondialdehyde (MDA), gamma glutamyl transpeptidase (GGT) and alkaline phosphatase (AKP) detection kits were purchased from the Nanjing Jiancheng Bioengineering Institute(Batch No. 20101228). The buddlejasaponin IV with purity higher than 99.8% was extracted from Clinopodium by the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization[4].

Establishment of H22 tumor-bearing mice model

The H22strain was injected into abdominal cavities of mice, and 7 d after the inoculation, appropriate amounts of ascites were sampled from the mice under sterile conditions. The sampled ascites samples were diluted with sterile saline,and then centrifuged at 1 000 r/min for 5 min. The H22cells were washed 3 times and diluted to 5×106cells/ml (percentage of viable cells was higher than 95%). The isolated H22cells were injected to right axillary skins of the mice with inoculation amount of 1×106cells/mouse.

Grouping and treatment

The mice were randomly divided into five groups 24 h after the inoculation, and there were 10 mice in each group. In the model control group,distilled water was given to mice by gavage. In the CTX group, the mice were injected with CTX according to the amount of 20.0 mg/kg.In the high-,middle- and low-dosage BJ-IV treatment groups, the mice were injected with BJ-IV according to the amounts of 1.00, 0.50 and 0.25 mg/kg, respectively once a day. For various groups,the injection was all performed once a day. The injection lasted for 10 consecutive days, and all the mice were sacrificed the next day.The blood was sampled from their eyeballs. Subsequently, the blood samples were centrifuged at 3 000 r/min for 15 min at 4℃, and the serum samples were preserved at -20 ℃for use. The tumor tissues, spleens and thymuses were stripped and weighted by electronic scale. The tumor inhibition rate, thymus index and spleen index were calculated as follows:

Tumor inhibition rate = [(Tumor weight in the control group - Tumor weight in the BJ-IV treatment group) /Tumor weight in the control group]×100%;

Thymus index = Thymus weight(mg)/Mouse body weight(g);

Spleen index = Spleen weight(mg)/Mouse body weight(g).

SOD activity, MDA content, GGT activity and AKP activity in serum of H22 tumor-bearing mice

The SOD activity was determined with xanthine oxidase (hydroxylamine)method; the DMA content was determined with thiobarbituric acid reactive substances assay; the GGT and AKP activities were all determined with chemical colorimetric method.The determination above was all performed in accordance with the instructions of kits produced by the Nanjing Jiancheng Bioengineering Institute. In the determination of SOD activity, MDA content, GGT activity and AKP activity, a UV spectrophotometer (OPTIZEN2120)was used.

Table 1 Inhibitory effect of buddlejasaponin IV on hepatocarcinoma 22(H22)in mice

Data processing and statistics

The results about tumor weight,SOD activity, MDA content, GGT activity and AKP activity were all expressed as mean ± standard deviation. With the ANOVA model of SPSS 13.0, the differences among groups were compared, and the multiple comparisons were performed with the LSD model.

Results and Analysis

Growth situations of tumor and mice

On day 2, grain-sized, irregular and unsmooth tumor nodules were shown in the right armpits of mice.The growth of tumors in mice in the model control group was the fastest,followed by that in the low-dosage buddlejasaponin IV treatment group, and the growth of tumors in mice in the CTX group was the slowest. In terms of feed intake, activity and hair color, the mice in the BJ-IV treatment groups were better than those in the CTX group.

Inhibitory effect of buddlejasaponin IV on H22 tumor in mice

The results of antitumor experiment in vivo showed that the high-,middle- and low-dosage buddlejasaponin IV all showed inhibitory effect on H22tumor.As shown in Table 1,the tumor inhibition rates in the high-,middle- and low-dosage buddlejasaponin IV treatment groups were 56.96% ,50.63% and 35.44%, respectively. In the CTX group, the tumor inhibition rate was 69.49%. There were significant differences in tumor inhibition rate between model control group and the other groups (P＜0.01).During the experiment, the weights of mice in various groups were increased in varying degrees. Among the groups, the weight of mice in the CTX group was increased slowest. The thymus index of mice in the CTX group was de-creased (P＜0.01), indicating that CTX inhibited the growth of thymuses of mice. Compared with the control group, buddlejasaponin IV showed no significant effects on spleen and thymus weights of mice. It indicated that buddlejasaponin IV had no significant inhibitory effects on spleens and thymuses of mice.

Table 2 SOD activities,MDA contents,GGT activities and AKP activities in serums of mice in all the groups

Effects of buddlejasaponin IV on SOD activity, MDA content, GGT activity and AKP activity in serum of H22 tumor-bearing mice

Compared with those in the model control group, the serum SOD activities in the high- and middle-dosage buddlejasaponin IV treatment groups were significantly increased (P＜0.05),and the serum MDA contents in the three buddlejasaponin IV treatment groups were all decreased (P＜0.01).Significant differences were observed in serum GGT and AKP activities between the buddlejasaponin IV treatment groups and the model control group(P＜0.01).There were no significant differences between the CTX and model control groups except in SOD activity. It suggests that buddlejasaponin IV can scavenge free radicals in tumor-bearing mice, and it can upregulate the tolerance of body to oxidative stress. Moreover, buddlejasaponin IV can also inhibit the sustained damage to liver functions(Table 2).

Discussion

Buddlejasaponin IV (BJ-IV) is a triterpenoid saponin containing Δ11-13,28-epoxy structure. Studies have shown that triterpenoid saponins that have similar structures with buddlejasaponin IV have some anti-tumor activity[5-6], and they can also improve immunomodulatory capacity to some extent[7-8].Cyclophosphamide is a broadspectrum anti-tumor drug, and it can be used to establish immunosuppressed mice models[9]. Therefore, in this study, CTX was selected as the positive control to investigate the inhibitory effect of buddlejasaponin IV on H22tumor in mice.

The guiding principle for antineoplastic pharmacodynamics was as follows: the results of anti-tumor experiment in vivo are an important indicator for evaluating the effectiveness of anticancer drugs[10].Li et al.[11]and Chen et al.[12]considered that the axillarilytransplanted H22tumor-bearing mice are a class of recognized models for studying anti-tumor effects and related mechanisms. Oxidative stress is an important mechanism of tumorigenesis[13]. Currently, it has become a new direction to look for effective antioxidants in vivo in anticancer research.SOD is an important antioxidant enzyme in animal bodies, and it can protect the body from free radical damage. SOD activity can be used to reflect the free radical-scavenging ability of the body.MDA is the end product of cellular lipid peroxides. MDA content can reflect the degree of lipid peroxidation in the body,thus the degree of cell damage can be inferred indirectly.The results of this study showed that the serum SOD activities of mice in the normal control and middle- and highdosage buddlejasaponin IV treatment groups were significantly higher than that of mice in the model control group(P ＜0.05). Buddlejasaponin IV could significantly up-regulate the SOD activity reduced by growth of H22tumor,improving the immunity of body. The MDA contents in the buddlejasaponin IV treatment groups were significantly lower than that in the model control group (P＜0.01),but there was no significant difference in serum MDA content between the normal and model control groups. The reduced serum MDA content meant reduced free radicals and liver damage. Alkaline phosphatase (AKP)is widely distributed in various body organs, but its content is highest in liver.Liver damage and lipid peroxidation caused by free radicals will cause damage to the structure of biofilms and lead to functional inactivation,thereby increasing permeability of cell membrane. Then, the liver intracellular enzymes, such as AKP, will enter blood circulation, resulting in higher AKP level in blood[14-15]. Serum GGT is mainly from the hepatobiliary system.In the onset of liver tumor,intra-hepatic obstruction will occur, inducing the generation of GGT by liver cells;at the same time,tumor cells can also synthesize GGT, resulting in higher GGT content in serum.The results of this study showed that the AKP and GGT contents in the model control group were all significantly higher than those in the normal control group,indicating that the H22tumor-bearing mouse models were successfully established, and the invasion of tumor cells caused damage to cell membrane structure and functional inactivation, thereby resulting in higher AKP content in serum of mice. There were significant differences in serum AKP and GGT contents between the model control and buddlejasaponin IV treatment groups,but there were no significant differences in serum indexes between the model control and CTX groups, indicating that buddlejasaponin IV has certain protective effect on liver cells.

In short,buddlejasaponin IV has a certain anti-hepatoma effect in vivo,and the related mechanism may be associated with regulating the body’s antioxidant capacity. However, where buddlejasaponin IV has inhibitory effects on other kinds of tumors, as well as the related mechanisms, needs tobe studied in depth.

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