Ginsenoside Rh2 Enhances Antitumour Activity and Decreases Genotoxic Effect of Cyclophosphamide


Author for correspondence: Rongliang Zheng, School of Life Sciences, Lanzhou University, Lanzhou 730000, People's Republic of China (fax +86 535 6713858, e-mail


Abstract: Ginsenoside Rh2, a panoxadiol saponins, possesses various antitumour properties. Cyclophosphamide, an alkylating agent, has been shown to possess various genotoxic and carcinogenic effects, however, it is still used extensively as an antitumour agent and immunosuppressant in the clinic. Previous reports reveal that cyclophosphamide is involved in some secondary neoplasmas. In this study, the antitumour activity and genotoxic effect of oral intake of ginsenoside Rh2 combined with intraperitoneal injection of cyclophosphamide was investigated. Meanwhile, C57BL/6 mice bearing B16 melanoma and Lewis lung carcinoma cells were respectively used to estimate the antitumour activity in vivo. The clastogenic activity in bone marrow polychromatic erythrocytes was assayed by frequency of micronucleus. The DNA damage in peripheral white blood cells was assayed by single cell gel electrophoresis as well. The results indicated that oral administration of Rh2 (5, 10 and 20 mg/kg body weight) alone has no obvious antitumour activity and genotoxic effect in mice, while Rh2 synergistically enhanced the antitumour activity of cyclophosphamide (40 mg/kg body weight) in a dose-dependent manner. Rh2 decreased the micronucleus formation in polychromatic erythrocytes and DNA strand breaks in white blood cells in a dose-dependent way. Our results suggest that ginsenoside Rh2 is able to enhance the antitumour activity and decrease the genotoxic effect of cyclophosphamide.

Panax ginseng has been used worldwide for thousands of years as a traditional herb medicine. Ginseng is considered to promote longevity (Attele et al. 1999; Liu et al. 2000), enhance resistance to many diseases (Keum et al. 2003), and help maintain the equilibrium of the human body under stress known as the adaptogenic effect (Ben-Hur & Fulder 1981; Ong & Yong 2000). The major effective components of ginseng are ginsenosides. Ginsenoside Rh2 belongs to the protopanaxadiol family and has drawn some attention in Asian laboratories due to its potential tumour-inhibitory effect. Rh2 could induce the differentiation and apoptosis in some malignant cell lines in vitro (Kim et al. 1998; Zeng & Tu 2003; Kim & Jin 2004; Cheng et al. 2005). Studies have demonstrated that Rh2 synergistically enhances the cytotoxic effects of paclitaxel and mitoxantrone in vitro (Jia et al. 2004). The orally administered Rh2 could inhibit the growth of human ovarian cancer in nude mice and prolong the survival period significantly when combined with cisplatin (Nakata et al. 1998). However, the useful information about the pharmacological and genotoxic effects of Rh2 combined with chemotherapeutic agents is scarce. The present study aimed at investigating the influence of Rh2 on the antitumour acitivity and genotoxic effect of the alkylating agent cyclophosphamide used extensively as antitumour agent and immunosuppressant.

Materials and Methods

Chemical reagents. 20(S)-ginsenoside Rh2 (Rh2) with more than 99.1% purity was prepared and identified according to the method reported by Chen et al. (1997). The chemical structure of Rh2 is shown in fig. 1. Cyclophosphamide was purchased from Jiangsu Hengrui Medicine Co., Ltd. Ethidium bromide (EtBr) and Triton X-100 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Low melting point (LMP) agarose and normal agarose (electrophoresis grade) were obtained from Gibco-BRL (Grand Island, NY, USA). Heparin sodium was bought from Roche (Brazil) under the commercial name Liquemine®.

Figure 1.

Structure of ginsenoside Rh2.

Animals. SPF C57BL/6 male mice, aged 5–7 weeks and weighing from 18 to 22 g, were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China. The mice were acclimatized to laboratory conditions (22±3 ° and 60% humidity) for 7 days, with a commercial standard mouse cube diet (Shandong Province Laboratory Animal Center, Jinan, China) and water ad libitum. After acclimatization, the mice were randomly divided into control and treated groups. All animal experiments were conducted in compliance with the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” and “Guiding Principles in the Use of Animals in Toxicology”.

Cell lines. Lewis lung carcinoma (LLC) cells and B16 melanoma (B16) cells were obtained from China Center for Type Culture Collection (CCTCC), Wuhan, China.

In vivo antitumour activity. LLC cells or B16 cells were cultured in DMEM medium with 10% heat-inactivated FBS. C57BL/6 mice were implanted subcutaneously with 0.2 ml (1×107 cells/ml in sodium chloride) LLC cells or B16 cells. After implantation for 24 hr, mice bearing LLC or B16 were randomly assigned to eight groups as follows: 1) control, 2) 40 mg/kg cyclophosphamide alone, 3) 5 mg/kg Rh2, 4) 10 mg/kg Rh2, 5) 20 mg/kg Rh2, 6) 5 mg/kg Rh2 combined with cyclophosphamide, 7) 10 mg/kg Rh2 combined with cyclophosphamide and 8) 20 mg/kg Rh2 combined with cyclophosphamide. Each group consisted of 10 mice. Rh2, suspended in saline, was orally administered once a day for 10 consecutive days. Cyclophosphamide, dissolved in saline, was injected by intraperitoneally as a single dose of 40 mg/kg body weight at the first day. Controls received the vehicle alone (20 ml/kg). Mice were sacrificed by cervical dislocation on day 11. Implanted sarcomas were separated and weighed.

Micronucleus formation. After acclimation, eighty female mice were randomly assigned to the eight groups. Rh2 was administered orally for 3 consecutive days. After the last administration, a single dose of cyclophosphamide 40 mg/kg body weight was injected intraperitoneally while the vehicle alone (20 ml/kg) was injected as the control. Blood (about 50 μl) was obtained after cyclophosphamide treatment for 24 hr from murine tail tips by a small incision and immediately mixed with heparin sodium (20 μl). The animals were then sacrificed by cervical dislocation. Bone marrow smears and staining were done following the procedure described by Schmid (1976). Femoral bone marrow was flushed out by using 1% sodium citrate solution at 37 °. The marrow was homogenized and centrifuged at 1000×g for 5 min. The supernatant was decanted and the pellet was homogenized with the residual fluid and smeared over the slide, then fixed by methanol for 5 min. Staining was carried out with May and Grunwald reagent followed by 10% buffered Giemsa. Slides were rinsed with distilled water, dried in air and scored. For each animal, 1000 polychromatic erythrocytes were examined under an oil immersion objective by utilizing a number of fields, and the number of micronucleated polychromatic erythrocytes were recorded. Every individual experiment was performed in triplicate.

Comet assay. The alkaline version of the comet assay (single cell gel electrophoresis, SCGE) was performed according to the protocol developed by Franke et al. (2005). Seven μl blood cells/heparin mixture were embedded in 93 μl LMP agarose (0.75 g/100 ml PBS) and the resulting mixture was spread over a precoated microscope slide (1.5 g/100 ml agarose). A cover glass was then gently placed over the slide and put at 4 ° for 5 min. to allow gel solidification. After removal of the coverslips, the cells were lysed at 4 ° for at least 1 hr in a freshly prepared, ice-cold solution of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma Base, 1% Na-lauryl sarcosinate, pH 10, and 1% Triton X-100, and 10% fresh DMSO added. Shortly after lysis, the slides were placed on a horizontal electrophoresis unit. Then they were exposed to alkali (300 mM NaOH, 1 mM Na2EDTA, pH>13) at 4 ° for 20 min. to allow DNA unwinding. Electrophoresis was performed at 300 mA and 25 V (0.90 V/cm) at 4 ° for 15 min. The slides were then neutralized (Tris 0.4 M, pH 7.5), stained with ethidium bromide (20 μg/ml), and were observed and photographed at 400× magnification using a Nikon TE2000 reverse fluorescence microscope equipped with an excitation filter (BP 546/12 nm) and a barrier filter (590 nm). The DNA damage degree was assessed with CASP, a free SCGE analysis system published by Konca et al. (2003). The images of one hundred cells were randomly selected from each slide and the tail moment was measured. The tail moment is positively correlated with the level of DNA breakage in a cell. The mean value of the tail moment in a particular sample was taken as an index of DNA damage in this sample.

Statistical analysis. The results were expressed as mean±S.D. and analyzed by one-way analysis of variance (ANOVA). The P value less than 0.05 was accepted as statistically significant.


Rh2with cyclophosphamide decreases the tumour growth synergestically. To explore the possible synergistic effects of Rh2 with conventional chemotherapeutic drug, cyclophosphamide, B16 melanoma cells and Lewis lung carcinoma cells were transplanted subcutaneously into C57BL/6 mice. As shown in fig. 2, Rh2 alone had little inhibitory effects on the growth of both tumours. At 20 mg/kg, the inhibition ratio was 18.6% on B16 melanoma and 20.0% on Lewis lung carcinoma. The inhibition ratio of cyclophosphamide alone was 32.6% on B16 melanoma and 21.1% on Lewis lung carcinoma. However, the combination of Rh2 and cyclophosphamide significantly inhibited the tumour growth. The inhibition ratio reached 64.1% on B16 and 61.8% on LLC after treatment with 20 mg/kg Rh2 combined with cyclophosphamide 40 mg/kg (fig. 2).

Figure 2.

Synergestic inhibition of ginsenoside Rh2 and cyclophosphamide on tumour growth in vivo. A. C57BL/6 mice bearing B16 melanoma cells were administrated with Rh2 (5∼20 mg/kg) or cyclophosphamide (CP, 40 mg/kg) as described in Materials and Methods. The implanted sarcomas were separated and weighed on day 11. B. C57BL/6 mice bearing Lewis lung carcinoma cells were administrated with Rh2 (5∼20 mg/kg) or CP (40 mg/kg) as described in Materials and Methods. The implanted sarcomas were separated and weighed on day 11. Data are the mean±S.D. from 10 individual treatments. *P<0.05; **P<0.01 versus control (Ctr); #P<0.05; ##P<0.01 versus CP-treated group.

Rh2 inhibits the micronuclei yield induced by cyclophosphamide. Cyclophosphamide alone significantly increases micronucleus in polychromatic erythrocytes (fig. 3), approximately 13 times higher than that in the control group. Pretreatment with Rh2 partially blocked the micronucleus in a dose-dependent way. The inhibition of Rh2 20 mg/kg on micronucleus reached 44.4% (fig. 3).

Figure 3.

Cyclophosphamide-induced micronucleus formation can be inhibited by co-treatment with Rh2, while Rh2 alone has no any influence on micronucleus. After cyclophosphamide (CP, 40 mg/kg) treatment for 24 hr, mice were sacrificed and the marrow cells in femoral bone were collected. Smear sliders of bone marrow cells were stained with Gimsa and the micronucleus (MN) in 1000 polychromatic erythrocytes (PCEs) were counted under an oil immersion objective. Data are the mean±S.D. from 10 individual treatments; **P<0.01 versus CP alone group.

Rh2 inhibits the DNA damage induced by cyclophosphamide. Cyclophosphamide alone caused serious DNA damage detected by SCGE. A significant increase of olive tail moment was observed. In our study, we observed that Rh2 alone had no influence on DNA. While combined with cyclophosphamide, Rh2 decreased the olive tail moment in a dose-dependent manner (fig. 4).

Figure 4.

Cyclophosphamide induced DNA-damage can be inhibited by Rh2, while Rh2 alone has no any influence on DNA. After cyclophosphamide (CP, 40 mg/kg) treatment for 24 hr, blood was obtained from murine tail tips. Commet assay was performed as described in Materials and Methods. The tail moment was measured by CASP software. The mean value of the tail moment in a particular sample was taken as an index of DNA damage in this sample. Data are the mean±S.D. from 10 individual treatments. *P<0.05, ***P<0.001 versus CP-treated group.


The present study demonstrated that oral administration of ginsenoside Rh2 showed a synergistic enhancement on antitumour activity of cyclophosphamide in a dose-dependent way. As well as Rh2 decreased the genotoxic effects induced by cyclophosphamide in vivo proved by micronuclei formation and DNA damage.

Improvements in the therapy of malignant disease during the past two decades have allowed long-term survical and cure in many patients. However, in recent years secondary malignancies have been recognized increasingly as an important late complication after chemo- and radiotherapy (Kaldor et al. 1990; Levine & Bloomfield 1992). Some reports indicate that a few secondary malignant cases are related to cyclophosphamide (Yokoyama et al. 2000; Agarwala et al. 2001; Heller et al. 2003) and that cyclophosphamide could induce the formation of urinary bladder tumours in rats (Habs & Schmahl 1983), while still being used extensively for its efficacy on primary tumours, as the first-line chemotherapy agent against breast cancer, small cell lung cancer, cervical cancer and non-Hodgkin's lymphoma. It has been shown that cyclophosphamide could produce chromosome damage, micronuclei, sister chromatid exchanges and DNA strand breaks in many kinds of mouse cells (Jenderny et al. 1988; Agrawal & Kuma 1998; Franke et al. 2005). It is obvious that all above indices are related with carcinogenesis. Therefore, it is necessary to find a compound that can decrease the genotoxic effects without having any negative effect on the antitumour activity of cyclophosphamide.

Panax ginseng has been used as a medicinal plant in China for thousands of years. The current use has been focused on cancer therapy. Epidemiological and experimental studies have shown that Panax ginseng has chemopreventive and antitumour effects (Chang et al. 2003; Helms 2004). Native ginseng contain trace amount of ginsenoside Rh2 (about 0.01%). Previous studies demonstrated that some natural ginsenosides, such as Rg3, Rb1, Rb2 and Rc could be metabolised to Rh2 by human intestinal bacteria (Bae et al. 2004), which suggests that Rh2 may contribute to ginseng's chemoprevent effects. Zhu et al. (1995) found that Rh2 could inhibit sister chromatid exchange formation induced by mitomycin C. Ginseng (Lee et al. 2004) and its extracts (Panwar et al. 2005) could suppressed micronuclei formation and DNA strand breaks induced by irradiation or a typical carcinogen, 7,12-dimethylben (a) anthracene. Tatsuka et al. (2001) also found that Rh2 could block the initiating activity on carcinogenic effects of 3-methylcholanthrene in mammalian cells in vitro. All the above studies indicate that ginsenoside Rh2 possesses antigenotoxic effects. Lee et al. (2005) also found that 20(S)-ginsenoside Rh2 inhibited t-butylhydroperoxide-induced liver damage. t-Butylhydroperoxide, an oxidizing agent, is known to induce oxidative stress and generate lipid peroxidation (Rohn et al. 1993), which indicates that ginsenoside Rh2 exerts its protective effects against oxidative stress damage. Numerous studies have shown that cyclophosphamide exposure enhances intracellular reactive oxygen species production, suggesting that biochemical and physiological disturbances may result from oxidative stress (Salvia et al. 1999; Manda & Bhatia 2003). Ginsenoside Rh2 may protect the cells from genotoxic damage induced by cyclophosphamide through modulating the oxidative stress status.

In conclusion, our studies indicate that Rh2 significantly inhibits the genotoxic effects induced by cyclophosphamide in vivo, and moreover, enhances the antitumour activity of cyclophosphamide. The results support Rh2 as a powerful candidate drug for enhancing the therapeutic effects and decreasing the toxic effects of chemotherapy.