Anti-proliferative effects of raw and steamed extracts of Panax notoginseng and its ginsenoside constituents on human liver cancer cells
© Toh et al; licensee BioMed Central Ltd. 2011
Received: 17 August 2010
Accepted: 24 January 2011
Published: 24 January 2011
Panax notoginseng is a potential source of anticancer compounds. This study aims to investigate the effects of steaming on the chemical profile of P. notoginseng and the anti-proliferative effects of P. notoginseng on liver cancer cells.
Samples of powdered raw P. notoginseng roots were steamed for various durations. Extracts of the raw and steamed samples were subjected to ultra-high pressure liquid chromatography/mass spectrometry (UHPLC-MS) analysis for chemical profiling. The anti-proliferative effects on three human liver cancer cells, namely SNU449, SNU182 and HepG2, were evaluated using colorimetric WST-1 assay.
Steaming changed chromatographic and pharmacological profiles of P. notoginseng, causing differences in activities such as inhibition of cancer growth. Steamed P. notoginseng exhibited greater anti-proliferative effects against liver cancer cells (SNU449, SNU182 and HepG2) than its raw form; steaming up to 24 hours increased bioactivities. Steaming increased the concentrations of ginsenoside Rh2, Rk1, Rk3 and 20S-Rg3 and enhanced growth inhibition of liver cancer cells.
Steaming changes the chemical profile as well as anti-cancer biological activities of P. notoginseng. Steamed P. notoginseng contains potential compounds for the treatment of liver cancer.
Some Chinese medicinal herbs exhibit anti-tumour activities [1, 2]. The raw form of Panax notoginseng (Burk.) F.H. Chen (Sanqi) is used in Chinese medicine to arrest internal and external haemorrhages, eliminate blood stasis, improve blood circulation, disperse bruises, reduce swelling and pain . The steamed form, on the other hand, is used as a tonic to nourish blood by increasing the production of various blood cells to treat anaemia . The roots of P. notoginseng which exhibited anticancer activities [4–6] were effective against colorectal [5, 6], lung , gastric [8, 9], skin , prostate  and liver  cancer. The ethanol extracts of P. notoginseng inhibited spleen tumour growth and liver metastasis in vivo; however, the in vitro anti-proliferative effects of P. notoginseng on liver cancer have yet to be evaluated.
Ginsenosides or dammarane-type triterpenoidal saponins, the main bioactive constituents of P. notoginseng[12, 13], are effective in preventing and treating cardiovascular and cerebrovascular diseases. These compounds are also immunoregulatory, possessing properties such as hepatoprotection and anti-carcinogenesis . Ginsenoside Rd was effective against human cervical cancer  and 20S-25-methoxyl-dammarane-3β, 12β, 20-triol was effective against several other types of cancer in vitro. Ginsenoside Rh2 inhibited the growth of human hepatoma cell SK-Hep-1 . Ginsenoside Rk1 controlled human hepatocellular carcinoma cell HepG2  proliferation. Ginsenosides Rg3, Rg5, Rk1, Rs5 and Rs4 are 50% more effective than cisplatin in inhibiting growth in human hepatoma cell SK-Hep-1 .
Processing of P. notoginseng (eg steaming) may change its composition [20–23] and alter its biological activities [6, 24]. While a study showed that steaming of P. notoginseng increased its anticancer activities , the correlation between altered composition of P. notoginseng and growth inhibition has not been established. Moreover, the relations between compositional changes in steamed P. notoginseng and changes in biological activities (eg antiproliferation on liver cancer cells) have yet to be investigated.
This study investigates the effects of steaming on the chemical profile of raw P. notoginseng and the effects of steaming duration on anti-proliferative activities of P. notoginseng in three liver cancer cell lines.
Leucine-enkephalin and formic acid were purchased from Sigma-Aldrich (USA). Acetonitrile (HPLC grade) was purchased from Merck (USA). Methanol (HPLC grade) was purchased from Fisher Scientific (USA). Distilled water was prepared 'in-house' using a MilliQ system (Millipore, USA). Ginsenosides Rg1, Rb1, Rc, Rb1, Rd and Re were purchased from Indofine Chemical Company (USA). Dimethyl sulfoxide (DMSO) was purchased from MP Biomedicals (USA). Notoginsenoside R1 was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). Rg3 was purchased from ChromaDex, Inc (USA). Rh1, Rh2 and Rg2 were purchased from Delta Information Centre for Natural Organic Compounds (China). P. notoginseng roots were obtained from Wenshan, Yunnan Province, China. The materials were identified to be P. notoginseng through morphological characteristics as well as qualitative and quantitative analyses with comparisons to the authenticated herb obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) [20–23]. A voucher sample was kept at the Department of Pharmacy Herbarium, National University of Singapore.
Human liver cancer cell lines SNU449 (CRL-2234), SNU182 (CRL-2235) and HepG2 (HB-8065) were purchased from American Type Culture Collection (ATCC, USA) and cultured in RPMI 1640 (for SNU449 and SNU182 cells) and Dulbecco's Modified Eagle Medium DMEM (for HepG2 cells) media supplemented with 10% fetal bovine serum FBS in a humidified atmosphere of 5% CO2 at 37°C. Cell proliferation was evaluated using the WST-1 assay (Roche, Germany) according to the manufacturer's instructions.
Steaming and extraction of raw P. notoginseng
Samples of the powdered raw P. notoginseng root were steamed at 120°C in an Hirayama Hiclave™ HV-50 autoclave (Hirayama (HMC), Japan) for 2, 6, 9, 15 or 24 hours. The powder was then vacuum dried at 80°C until constant weight and extracted with a Branson ultrasonicator (model 5510, USA) as described by Chan et al.. Six individual extractions were performed on the raw and steamed samples to generate six replicates each of the raw and steamed extracts .
Ultra-high pressure liquid chromatography (UHPLC) and mass spectrometry (MS)
Ultra-high pressure liquid chromatography (UHPLC) was performed using a Waters ACQUITY UPLC™ system (USA), equipped with a binary solvent delivery system (Waters, USA) and an auto-sampler (Waters, USA). The chromatography was performed on a 100 × 2.1 mm Waters ACQUITY C18 1.7 μm column. The mobile phase consisted of (A) 0.1% formic acid in distilled water and (B) acetonitrile containing 0.1% formic acid.
Mass spectrometry (MS) was performed using a QTOF premier™, a quadruple TOF mass spectrometer (Waters, USA). The system was tuned for optimal sensitivity and resolution using leucine-enkephalin (2 ng/μL) and syringe pump infused at 3 μl/min in negative electrospray (ES-) ionization mode. The TOF mass spectrometer was operated in the 'W' mode and tuned using the standard compound ginsenoside Rg1. Data were centroided during acquisition using independent reference lock-mass ions via the LockSpray™ (Waters, USA) interface to ensure mass accuracy and reproducibility. Leucine-enkephalin was used as the reference compound (2 ng/μL) at an infusion flow rate of 3 μL/min. Sodium formate was used for calibration for accurate mass. The conditions for the UHPLC analysis with MS detection were as previously reported .
A standard mixture containing ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2 and notoginsenoside R1 was prepared in 50% (v/v) methanol. A volume of 2 μl of a standard mixture was used for the validation of retention time reproducibility and mass accuracy. A blank sample consisting of 50% methanol (2 μl) was injected between analyses to validate inter-sample cross-talking effect.
Cell proliferation analysis
Raw and steamed P. notoginseng extracts were dissolved in 100% DMSO. Ginsenosides were dissolved in water. Cells were seeded in 96-well plates with the optimized cell density for the three cell lines, namely SNU449 (4 × 103 cells/well), SNU182 (4 × 103 cells/well), HepG2 (1.1 × 104 cells/well). After 24 hours of incubation, various concentrations of extracts/ginsenosides were added to the wells. The final concentration of DMSO for the extracts was 0.5% (v/v). A total of 100 μl of sample was added to each well. DMSO was used as control for the extracts and water was used as control for the ginsenosides. Controls were exposed to culture medium containing 0.5% DMSO or 10% water without drugs. All experiments were performed in quadruplicates and repeated three times. Three extracts obtained separately on three independent occasions were tested in the experiments. The SNU449 cell line was treated for 48 hours while SNU182 and HepG2 cell lines were treated for 72 hours. Cell proliferation was evaluated using WST-1 assay. At the end of the drug exposure period, the medium was replaced with 100 μl of the (10% v/v) WST-1 in fresh medium in each well and incubated for one hour. Absorbance was read at 440 nm with reference at 650 nm. The effect of herbal extracts or ginsenosides on cell proliferation was calculated as percentage of cell viability measured in the presence of 0.5% (v/v) DMSO. Results are presented as a percentage of the vehicle with values being mean ± standard deviation of three individual experiments conducted in triplicates each.
Differences in anti-proliferative activities between P. notoginseng samples and ginsenosides were compared against the control using one-way analysis of variance (ANOVA) and Tukey's multiple comparison post-tests (GraphPad Prism 4, USA). Spearman correlation method is used for the correlation studies of duration of steaming of P. notoginseng samples and its anti-proliferative activities. Results were considered statistically significant when P < 0.05. Inhibition concentration (IC50) was determined using Calcusyn software (Biosoft, UK).
Results and Discussion
Anti-proliferative activities of P. notoginseng extracts in liver cancer cells
The anti-proliferative effects of extracts of the raw and steamed P. notoginseng root were evaluated on three liver cancer cell lines. Due to the inherent different cell doubling times of the respective cell lines, the duration of treatment was optimized to 48 hours for SNU449 and SNU182, 72 hours for HepG2 respectively.
Anti-proliferative effects (percentage viabilities) of raw and steamed P. notoginsen g extracts on the SNU449, SNU182 and HepG2 liver cancer cells.
Steamed 2 hours
Steamed 6 hours
Steamed 9 hours
Steamed 15 hours
Steamed 24 hours
Correlation between duration of steaming of P. notoginsen g and anti-proliferative activities (percentage inhibition on proliferation of cells) on SNU449, SNU182 and HepG2 liver cancer cells
Inhibition of proliferation of SNU449, SNU182 and HepG2 human liver cancer cells by raw and steamed P. notoginsen g extracts
Steamed 2 hours
Steamed 6 hours
Steamed 9 hours
Steamed 15 hours
Steamed 24 hours
Anti-proliferative activities of ginsenosides in liver cancer cells
Ginsenosides Rk3, Rh2, 20S-Rg3 and Rk1 enriched in steamed P. notoginseng all significantly inhibited liver cancer cell growth (P < 0.001) at 0.25 mg/ml (Figure 4B). Ginsenoside Rk3 reduced cell viabilities of HepG2, SNU449 and SNU182 to 0.04 ± 0.3% (P < 0.001), 1.0 ± 1.2% (P < 0.001) and 34.4 ± 9.3% (P < 0.001) respectively. Ginsenosides Rh2 and Rk1 were the most potent among the four ginsenosides examined as exposure to 0.25 mg/ml of Rh2 resulted in minimal cell viability in all three liver cancer cells (0.4-1.4%; Figure 4B). Ginsenoside 20S-Rg3 effectively inhibited cell growth of SNU449 (P < 0.001) but was comparatively less effective on SNU182 and HepG2 cells whereas ginsenoside Rk3 inhibited the growth of SNU449 (P < 0.001) and HepG2 (P < 0.001), and to a lesser extent SNU182 cells (P < 0.001). Lastly, ginsenoside 20R-Rh1 significantly inhibited the growth of SNU449 (P = 0.003) and HepG2 (P < 0.001) while ginsenoside 20S-Rh1 inhibited the growth of SNU449 cells only (P = 0.005).
The differences in the anti-proliferative activities among the ginsenosides in the raw and steamed P. notoginseng are consistent with the findings that extracts of raw and steamed P. notoginseng show differential anti-proliferative activity on liver cancer cell lines.
The present study reports for the first time the in vitro anti-proliferative activities of ginsenoside Rk3.
Dose-response of selected ginsenosides on the SNU449 cell line
Inhibitory concentration of different ginsenosides on proliferation of SNU449 Human Liver Cancer Cells
Steaming selectively enriched growth-inhibiting ginsenosides
The present study demonstrated distinctive chemical profiles between the raw and steamed P. notoginseng. The steamed herb was significantly more effective in inhibiting the growth of liver cancer cells. The anti-proliferative activities of P. notoginseng increased with progressive steaming up to 24 hours as this process enriched the bioactive components such as ginsenosides Rh2, Rk1, Rk3 and 20S-Rg3.
Sun et al. also reported that steaming the root of P. notoginseng affected its chemical composition and anticancer and anti-proliferative activities in SW-480 human colorectal cancer cells. However, the durations of steaming in their study were shorter (120°C for 1, 2, 4 and 6 hours) and the cancer cell line was different from what was used in our study. Furthermore they only studied three ginsenosides (Rb1, Rg1 and Rg3), of which only 20S-Rg3 showed significant anti-proliferative effects against SW-480. In the present study, the durations of steaming were longer (120°C for 2, 6, 9, 15 and 24 hours) and three human liver cancer cell lines (HepG2, SNU449 and SNU182) were used. In addition, more saponins were investigated for their anti-proliferative activities. In particular, Rk3, Rh2, Rk1 and 20S-Rg3 were the most anti-proliferative and ginsenoside Rk3 was reported for the first time to possess anti-proliferative activities.
It would be of future interest to investigate the in vivo anti-proliferative effects of raw and steamed P. notoginseng and the saponins enriched by the steaming process.
Steaming changes the chemical profile as well as anti-proliferative biological activities of P. notoginseng. Steamed P. notoginseng contains potential compounds for the treatment of liver cancer.
- The abbreviations used include the following:
(ANOVA): analysis of variance
ultra-high pressure liquid chromatography
ultra-high pressure liquid chromatography - time-of-flight mass spectrometry and 50% inhibitory concentration (IC50).
The project was supported by the National University of Singapore academic research grants (R-148-000-079-112 to HLK and R-148-000-100-112 to ECYC) and the graduate research scholarships to DFT and PDN.
- Konkimalla VB, Efferth T: Anti-cancer natural product library from traditional Chinese medicine. Comb Chem High Throughput Screen. 2008, 11: 7-15. 10.2174/138620708783398368.View ArticlePubMedGoogle Scholar
- Efferth T, Kahl S, Paulus K, Adams M, Rauh R, Boechzelt H, Hao X, Kaina B, Bauer R: Phytochemistry and pharmacogenomics of natural products derived from traditional Chinese medicine and Chinese materia medica with activity against tumor cells. Mol Cancer Ther. 2008, 7: 152-161. 10.1158/1535-7163.MCT-07-0073.View ArticlePubMedGoogle Scholar
- The State Pharmacopoeia Commission of People Republic of China: Pharmacopoeia of the People's Republic of China. 2005, Beijing: Chemical Industry Press, 1: 10-11.Google Scholar
- Konoshima T, Takasaki M, Tokuda H: Anti-carcinogenic activity of the roots of Panax notoginseng. II. Biol Pharm Bull. 1999, 22: 1150-1152.View ArticlePubMedGoogle Scholar
- Wang CZ, Xie JT, Zhang B, Ni M, Fishbein A, Aung HH, Mehendale SR, Du W, He TC, Yuan CS: Chemopreventive effects of Panax notoginseng and its major constituents on SW480 human colorectal cancer cells. Int J Oncol. 2007, 31: 1149-1156.PubMed CentralPubMedGoogle Scholar
- Sun S, Wang CZ, Tong R, Li X, Fishbein A, Wang Q, He T, Du W, Yuan C: Effects of steaming the root of Panax notoginseng on chemical composition and anticancer activities. Food Chem. 2010, 118: 307-314. 10.1016/j.foodchem.2009.04.122.View ArticleGoogle Scholar
- Park SC, Yoo HS, Park C, Cho CK, Kim GY, Kim WJ, Lee YW, Choi YH: Induction of apoptosis in human lung carcinoma cells by the water extract of Panax notoginseng is associated with the activation of caspase-3 through downregulation of Akt. Int J Oncol. 2009, 35: 121-127. 10.3892/ijo_00000371.View ArticlePubMedGoogle Scholar
- Wang ZB, Li JX, Zhu LQ, Niu FL, Cui W: Inhibiting effects of Panax notoginseng extracts on proliferation of GES-1 cells and MNNG-transformed GES-1 cells. Zhong Xi Yi Jie He Xue Bao. 2004, 2: 445-449. 10.3736/jcim20040613.View ArticlePubMedGoogle Scholar
- Li JX, Wang ZB, Zhu LQ, Niu FL, Cui W: Apoptosis-promoting effect of Panax notoginseng extracts on MNNG-transformed GES-1 cells. Zhong Xi Yi Jie He Xue Bao. 2005, 3: 123-127. 10.3736/jcim20050212.View ArticlePubMedGoogle Scholar
- Wang W, Wang H, Rayburn ER, Zhao Y, Hill DL, Zhang R: 20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol, a novel natural product for prostate cancer therapy: activity in vitro and in vivo and mechanisms of action. Br J Cancer. 2008, 98: 792-802. 10.1038/sj.bjc.6604227.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen PF, Liu LM, Chen Z, Lin SY, Song WX, Xu YF: Effects of ethanol extracts of Panax notoginseng on liver metastasis of B16 melanoma grafted in mice. Zhong Xi Yi Jie He Xue Bao. 2006, 4: 500-503. 10.3736/jcim20060512.View ArticlePubMedGoogle Scholar
- Wang CZME, W S, W JA, Y CS: Phytochemical and analytical studies of Panax notoginseng (Burk.) F.H. Chen. J Nat Med. 2006, 60: 97-106. 10.1007/s11418-005-0027-x.View ArticleGoogle Scholar
- Zhu YP: Chinese Material Medica. 1998, Australia: Association HMGoogle Scholar
- Ng TB: Pharmacological activity of sanchi ginseng (Panax notoginseng). J Pharm Pharmacol. 2006, 58: 1007-1019. 10.1211/jpp.58.8.0001.View ArticlePubMedGoogle Scholar
- Yang ZG, Sun HX, Ye YP: Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis. Chem Biodivers. 2006, 3: 187-197. 10.1002/cbdv.200690022.View ArticlePubMedGoogle Scholar
- Zhao Y, Wang W, Han L, Rayburn ER, Hill DL, Wang H, Zhang R: Isolation, structural determination, and evaluation of the biological activity of 20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol [20(S)-25-OCH3-PPD], a novel natural product from Panax notoginseng. Med Chem. 2007, 3: 51-60. 10.2174/157340607779317508.View ArticlePubMedGoogle Scholar
- Lee KY, Park JA, Chung E, Lee YH, Kim SI, Lee SK: Ginsenoside-Rh2 blocks the cell cycle of SK-HEP-1 cells at the G1/S boundary by selectively inducing the protein expression of p27kip1. Cancer Lett. 1996, 110: 193-200. 10.1016/S0304-3835(96)04502-8.View ArticlePubMedGoogle Scholar
- Kim YJ, Kwon HC, Ko H, Park JH, Kim HY, Yoo JH, Yang HO: Anti-tumor activity of the ginsenoside Rk1 in human hepatocellular carcinoma cells through inhibition of telomerase activity and induction of apoptosis. Biol Pharm Bull. 2008, 31: 826-830. 10.1248/bpb.31.826.View ArticlePubMedGoogle Scholar
- Park IH, Piao LZ, Kwon SW, Lee YJ, Cho SY, Park MK, Park JH: Cytotoxic dammarane glycosides from processed ginseng. Chem Pharm Bull (Tokyo). 2002, 50: 538-540. 10.1248/cpb.50.538.View ArticleGoogle Scholar
- Lau AJ, Woo SO, Koh HL: Analysis of saponins in raw and steamed Panax notoginseng using high-performance liquid chromatography with diode array detection. J Chromatogr A. 2003, 1011: 77-87. 10.1016/S0021-9673(03)01135-X.View ArticlePubMedGoogle Scholar
- Chan EC, Yap SL, Lau AJ, Leow PC, Toh DF, Koh HL: Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng. Rapid Commun Mass Spectrom. 2007, 21: 519-528. 10.1002/rcm.2864.View ArticlePubMedGoogle Scholar
- Lau AJ, Seo BH, Woo SO, Koh HL: High-performance liquid chromatographic method with quantitative comparisons of whole chromatograms of raw and steamed Panax notoginseng. J Chromatogr A. 2004, 1057: 141-149. 10.1016/j.chroma.2004.09.069.View ArticlePubMedGoogle Scholar
- Toh DF, New LS, Koh HL, Chan EC: Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng. J Pharm Biomed Anal. 2010, 52: 43-50. 10.1016/j.jpba.2009.12.005.View ArticlePubMedGoogle Scholar
- Lau AJ, Toh DF, Chua TK, Pang YK, Woo SO, Koh HL: Antiplatelet and anticoagulant effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with Panax ginseng and Panax quinquefolium. J Ethnopharmacol. 2009, 125: 380-386. 10.1016/j.jep.2009.07.038.View ArticlePubMedGoogle Scholar
- Yue PY, Mak NK, Cheng YK, Leung KW, Ng TB, Fan DT, Yeung HW, Wong RN: Pharmacogenomics and the Yin/Yang actions of ginseng: anti-tumor, angiomodulating and steroid-like activities of ginsenosides. Chin Med. 2007, 2: 6-26. 10.1186/1749-8546-2-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Christensen LP: Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res. 2009, 55: 1-99. full_text.View ArticlePubMedGoogle Scholar
- Toh DF: Chemical and pharmacological evaluations of Panax notoginseng and Swietenia macrophylla. PhD thesis. 2010, National University of Singapore, Department of PharmacyGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.