| Ginseng(Panax ginseng C.A. Meyer) is a traditional Chinese herbal medicine, anticancer is one of the major biological functions. Ginsenosides is the major active component which processed anticancer activity. They are classified into three types: protopanaxadiol(PPD)-type, protopanaxatriol(PPT)-type and oleanolic acid(OA)-type. The application of ginseng is usually as the form of mix ginsenosides, previous work has found that a variety of ginsenosieds(ginsenoside Re, Rg1 and Ro) can promote growth of cancer cells and vascular endothelial cells. Cancer patients should not taking ginseng overall in traditional view, instead that they need to take the secondary ginsenosides(Rg3, Rh2, etc.). Therefore, the research on anti-tumor activity of ginsenoside is helpful for understand the overall efficacy of ginseng and maybe the guidance of ginseng in anti-tumor clinical application.This paper consists of four parts:Part 1: Effects of dried ginseng, lyophilized ginseng and red ginseng on tumor metastasis.Dried ginseng, lyophilized ginseng and red ginseng were producted from the same year and the same variety of ginseng, respectively. Experimental lung metastasis model was used to investigate the effect of three different processing methods of ginseng on tumor metastasis. B16F10 melanoma high metastatic cells were injected via tail vein to the C57BL/6J mice. The experimental mice were divided into six groups, blank control(did not received cancer cells), model control, positive control and administration of dried ginseng, lyophilized ginseng or red ginseng groups. Normal and model mice were intragastric administered with normal saline. The positive mice were intraperitoneal injected cyclophosphamide(CTX) for 20 mg /kg. The intragastric administered dosages of dried ginseng, lyophilized ginseng and red ginseng were all 900 mg/kg. After two weeks of successively administration, the mice were sacrificed and the number of B16F10 metastasis loci on lungs was determined. Then, the inhibition ratio was calculated. The experiment in vivo and pathological section results showed that dried ginseng, lyophilized ginseng and red ginseng were all possesses anti-tumor metastasis activity in mice, the inhibition ratio were 68.67, 66.08 and 35.42%, respectively. The major ginsenosides of the ginseng were Rb1, Rb2, Rc, Rd, Re, Rg1 and Ro by HPLC. The dissolution of red ginseng was the lowest among the three samples.Part 2: Effects of PPD-type ginsenosides on tumor growth and angiogenesis.Effects of PPD-type ginsenosides: Rb1,(S)-Rg3,(R)-Rg3, Rg5, CK and PPD on tumor growth and angiogenesis were investigated by B16F10 and HUVEC cytotoxicity test, tube formation assay, transwell assay, CAM assay. Tumor xenograft model and tumor angiogenesis model were used to investigate the effect of ginsenoside Rb1 and(S)-Rg3 on tumor growth and tumor angiogenesis in mice. The results showed that ginsenoside Rb1(1-30 g/m L) and(R)-Rg3(1-30 g/m L) has no significant effect on the growth of B16F10 cells, high concentration(100 g/m L) exhibit weak cytotoxic activity, Rb1(1-100 g/m L) and(R)-Rg3(1-100 g/m L) had no significant influence on the growth of HUVEC cells. Ginsenoside Rg5,(S)-Rg3, CK and PPD have strong cytotoxicity on B16F10 and HUVEC cells, the IC50 value was 12.89, 8.48, 7.51, 5.35μg/m L for B16F10 cells and 13.90, 19.68, 8.36, 11.61 μg/m L for HUVECs, respectively. In addition, the result of tube formation assay showed that treatment with Rb1(100 μg/m L),(S)-Rg3(30 μg/m L), Rg5(25 μg/m L) resulted in 9.04, 71.12 and 34.17% inhibition of HUVECs tube formation, respectively. Administeration of(R)-Rg3(100 μg/ml) and PPD(25 μg/ml) had no significant inhibitory effect on tube formation;The inhibiting effect of CK for HUVEC cells tube formation was the strongest among the tested drugs, none tube was visible after the cells were administered CK at 15 μg/m L. Transwell assay results showed that CK(1.25-15 μg/m L), PPD(5-25 μg/m L) and Rb1(1-30 μg/m L) had no significant effect on the migration and invasion ability of HUVEC cells. The promotion rate was 5.7% and 6.1% on HUVECs migration and invasion when the concentration of Rb1 was 100 μg/m L(p>0.05), respectively. Ginsenoside(S)-Rg3(10-30 μg/m L) and Rg5(5-25 μg/m L) inhibit HUVEC cells migrate and invade in a concentration-dependent manner. When the concentrations of(S)-Rg3 were 20, 25 and 30 g/m L, the migration inhibition rate of HUVECs were 23.3, 27.8 and 37.8%, as well as the cell invasion inhibition rate was 22, 18.3 and 26.8%, respectively. When the concentrations of Rg5 were 15, 20 and 25 μg/m L, the migration inhibition rate of HUVECs were 17.6, 20.6 and 40.2%, as well as the cell invasion inhibition rate was 25.5, 29.6 and 51.0%, respectively. CAM assay result showed that ginsenoside Rb1 significantly promoted angiogenesis of the first and second grade blood vessels(p < 0.05), and(S)-Rg3,(R)-Rg3, Rg5, CK significantly inhibited firstly vascular and secondary angiogenesis(p < 0.05), PPD inhibited CAM angiogenesis on third branch of blood vessels(p > 0.05). In vivo, after intraperitoneal injection of ginsenoside Rb1(30 mg/kg bodyweight), promotion rate was 14.2% on tumor volume while thepromotion rate was 53.3% on angiogenesis(p < 0.05). Intraperitoneal injection of(S)-Rg3(30 mg/kg) inhibit the tumor growth and angiogenesis, the inhibition rate was 75.3% and 34.2%(p < 0.01). However, the two ginsenosides has the immunity enhancement of tumor-bearing mice.Part 3: Effects of PPT-type ginsenosides on tumor growth and angiogenesis.PPT-type ginsenosides: Re, Rg1,(S)-Rh1,(R)-Rh1 and PPT on tumor growth and angiogenesis were investigate by the methods which in accordance with part 2. Tumor xenograft model and tumor angiogenesis model were used to investigate the effect of ginsenoside Re and Rg1 on tumor growth and tumor angiogenesis in mice. The results showed that ginsenoside Re(1-100 μg/m L) and Rg1(1-100 μg/m L) showed no significant effect on B16F10 and HUVEC cells. The inhibition ratio of(S)-Rh1(30 μg/m L) and(R)-Rh1(30 μg/m L) on B16F10 was 10.42(p >0.05) and 21.59%(p < 0.01), respectively.(S)-Rh1(100 μg/m L) significantly enhance the proliferation of HUVEC cells, the promotion rate was 72.4%(p < 0.01), but(R)-Rh1(1-100 μg/m L) showed no significant effect on HUVEC cells growth. PPT(5-25 μg/m L)inhibited B16F10 cells viability in a concentration-dependent manner, the IC50 value was 14.8 μg/m L, the inhibition rate of PPT on HUVEC was 42.4%(p < 0.01). PPT(80 μg/m L) inhibited HUVECs tube formation, the inhibition was 44.97%. Moreover, ginsenoside Re(1-100 μg/m L), Rg1(1-100 μg/m L),(S)-Rh1(1-100 μg/m L) and(R)-Rh1(1-100 μg/m L) showed no significant effect on the HUVEC cells tube formatted. Ginsenoside Re(100 μg/m L) and Rg1(100 μg/m L) had weakly enhance ability on HUVEC cell proliferation(p >0.05).(S)-Rh1(100 μg/m L) inhibited the migration and invasion of HUVEC cells weakly(p >0.05);(R)-Rh1 showd no significant effect on HUVEC cells migration, the inhibition on invasion of HUVEC cells was 5.0%(p >0.05). High concentration of PPT(80 μg/m L) resulted in 5.89%(p >0.05) inhibition of migration and 9.11%(p >0.05) inhibition of invasion on HUVECs. Ginsenoside Rg1,(S)-Rh1 and(R)-Rh1 significantly promoted the first grade vessel of CAM, and they showed different degrees of promoting effect on secondary blood vessels. PPT showed no significant effect on the angiogenesis in CAM assay. In vivo, intraperitoneal injection of ginsenoside Re(30 mg/kg) promoted tumor growth, the tumor volume was 1.49 times as much as model mice, the promotion rate on angiogenesis was 57.7%(p < 0.01). Intraperitoneal injection of ginsenoside Rg1(30 mg/kg) showed weak anticancer activity in tumor growth. The inhibition rate on tumor volume was 13.8%, and the promotion rate on angiogenesis was 18.1%(p >0.05). However, ginsenoside Re and Rg1 showed immunity enhancement on tumor-bearing mice.Part 4: Effects of OA-type ginsenosides on tumor growth and angiogenesis.In part 4, effects of OA-type ginsenosides: ginsenoside Ro(Ro), zingibroside R1(R1), chikusetsusaponin Iva(IVa), calendulaoside E(E), songoroside A(S-A), oleanolic acid(OA) and oleanonic acid(oxo-OA) on tumor growth was investigate in vitro and in vivo. The effect of these tested drugs on angiogenesis was investigated in vitro. In addition, hemolysis test was carried out to determine if the cell toxicities of the OA-type ginsenosides are due to cholesterol related membrane disruption. Ginsenoside Ro and IVa were not show significant effects on the cell viability of B16F10 cells at low concentration(1.0-30 μg/m L). Both Ro and Iva caused a slight decrease in cell viability(5% of Ro and 13% of IVa) at high concentration(100 μg/m L). However, Ro and Iva were enhanced the proliferation of HUVEC cells. OA inhibited B16F10 cells viability in a concentration-dependent manner, the inhibition was 10% with a 1 μg/m L treatment and 39% with a 100μg/m L treatment, respectively. Moreover, OA(100 μg/m L) was observed toxic to HUVEC cells with the inhibition rate of 28%. R1, S-A, oxo-OA and E remarkably inhibited B16F10 and HUVEC cell viability, the IC50 value was 25.8, 8.5, 3.1, 2.2 μg/m L for B16F10 cells and 24.0, 2.7, 15.0, 6.8 μg/m L for HUVECs,respectively. Tube formation results showed that the length(%) of tube formatted of Ro(100 μg/m L)、Iva(100 μg/m L), OA(100 μg/m L), R1(40 μg/m L), oxo-OA(30 μg/m L) were 117.0, 72.0, 53.0, 39.0 and 34.2%, respectively. S-A(12 μg/m L) and E(10 μg/m L) remarkably inhibited HUVECs tube formation, none tube was visible with the concentration increased. The results of the haemolysis test demonstrated that the haemolysis rate of R1 at different concentrations(20, 30 μg/m L) was 2.99 and 83.11%, respectively. E coursed complete hemolysis at 4 μg/m L. Other OA-type ginsenosides did not course hemolysis. All the OA-type ginsenosides exhibited anti-tumor activities in vivo. Intraperitoneal injection of E(5 mg/kg) displayed almost the same inhibition as oleanolic acid(25 mg/kg), the the inhibition was 85.4 and 84.9%, respectively. The inhibitory effects of Iva(25 mg/kg), Ro(25 mg/kg) and OA(25 mg/kg) were observed to be similar, approximately 75%. S-A(25 mg/kg) and R1(25 mg/kg) exhibited weaker anti-tumor activity in vivo.The conclusions are as follows:In this paper, the traditional view that "the cancer patients can not take ginseng, they should take the secondary ginsenoside" was clarified. It was proved that the whole application of ginseng has anti-tumor effect. The effect of the representative ginsenosides on the melanoma and angiogenesis was investigated. Finally this paper drawed the following conclusions1. Dried ginseng, lyophilized ginseng, red ginseng are all process anti tumor metastasis activity.2. Ginseng with the same year and the same variety but processed by different methods exist difference in anti tumor metastasis activity. Dried ginseng and lyophilized ginseng has good anti tumor metastasis activity, there was no difference between them, red ginseng own weakest activity among the three ginseng products. 3. Ginsenoside(S)-Rg3、Rg5、CK and PPD process anti tumor and anti angiogenesis activity. 4.(S)-Rg3 possessed stronger anit tumor and angiogenesis activities than(R)-Rg3. 5. The structure-cytotoxicity relationship of OA-type ginsenosides are as follows: OA-type ginsenosides with a glycosyl group at C-3 and a carboxyl group at C-28 exhibited higher toxicity against B16F10 cells than those with two glycosyl groups respectively distributed at C-3 and C-28; OA-type ginsenosides with a glycosyl group at C-3 and without a glycosyl group at C-28 exhibited toxicity against HUVECs, and the OA-type ginsenosides with two glycosyl groups at C-3 and C-28 improve the proliferation of HUVECs.6. The antitumor activity of OA type ginsenosides in mice did not follow the structure activity relationship in vitro. The anti-tumor activity of ginsenoside Ro was observed to be a synergistic effect of its metabolites produced in vivo. |
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