Anti-proliferative effects of raw and steamed extracts of Panax notoginseng and its ginsenoside constituents on human liver cancer cells

Background 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. Methods 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. Results 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. Conclusion 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.


Background
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 [3]. 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 [3]. The roots of P. notoginseng which exhibited anticancer activities [4][5][6] were effective against colorectal [5,6], lung [7], gastric [8,9], skin [4], prostate [10] and liver [11] cancer. The ethanol extracts of P. notoginseng inhibited spleen tumour growth and liver metastasis in vivo [11]; however, the in vitro anti-proliferative effects of P. notoginseng on liver cancer have yet to be evaluated.
While a study showed that steaming of P. notoginseng increased its anticancer activities [6], 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.

Materials
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 Rg 1 , Rb 1 , Rc, Rb 1 , Rd and Re were purchased from Indofine Chemical Company (USA). Dimethyl sulfoxide (DMSO) was purchased from MP Biomedicals (USA). Notoginsenoside R 1 was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). Rg 3 was purchased from ChromaDex, Inc (USA). Rh 1 , Rh 2 and Rg 2 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][21][22][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% CO 2 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. [21]. Six individual extractions were performed on the raw and steamed samples to generate six replicates each of the raw and steamed extracts [21].
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 Rg 1 . 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 [23].

Method validation
A standard mixture containing ginsenosides Rb 1 , Rb 2 , Rc, Rd, Re, Rg 1 , Rg 2 , Rg 3 , Rh 1 , Rh 2 and notoginsenoside R 1 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 crosstalking 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 × 10 3 cells/well), SNU182 (4 × 10 3 cells/well), HepG2 (1.1 × 10 4 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.

Statistical analysis
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 (IC 50 ) was determined using Calcusyn software (Biosoft, UK). Figure 1 shows the morphology of the raw P. notoginseng, powdered P. notoginseng and steamed P. notoginseng (15 hours). The raw and steamed P. notoginseng showed distinct chromatographic profiles, indicating that steaming altered the composition of ginsenosides by increasing the generation of non-polar ginsenosides which eluted later from the column (Figure 2). Chromatographic analyses of raw and steamed P. notoginseng were consistent with those reported by Lau et al. [22], who noted that the quantitative differences were correlated to the duration of steaming. Furthermore, Lau et al reported that the concentrations of less polar saponins such as ginsenosides 20S-Rh 1 , 20R-Rh 1 , Rk 3 , Rh 4 , 20S-Rg 3 , 20R-Rg 3 , Rk 1 and Rg 5 increased in steamed P. notoginseng [20,22]. Ginsenosides in the raw P. notoginseng underwent hydrolysis to form other ginsenosides upon steaming. For example, ginsenosides Rh 1 , Rh 2 and Rg 3 were produced from ginsenoside Rb 1 through deglycosylation where the glycosyl moiety at C-20 was partially detached [25]. The ginsenosides Rk 1 , Rk 3 and Rg 5 were the examples of ginsenosides generated by the loss of water from the corresponding ginsenosides with a free hydroxyl group at C-20 [26]. One of the major saponin in raw P. notoginseng was ginsenoside Rg 1 . This study found that the concentration of ginsenoside Rg 1 in the raw P. notoginseng was reduced from 38.9 to 1.0 mg/g upon steaming for 15 hours [27]. Taken together, these findings indicate that steaming changed the compositional profiles of the Panax species and altered their biological activities.

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.
The anti-proliferative effects of P. notoginseng extracts on the three cell lines are in Table 1. Raw P. notoginseng at 0.25 mg/ml did not show any inhibition of cell proliferation in SNU182 and HepG2 whereas the growth of SNU449 cells was inhibited by about 20% (P = 0.018).
In contrast, steamed P. notoginseng significantly inhibited the proliferation of all three liver cancer cell lines in vitro. The actual P values and percentage viabilities of the raw and steamed P. notoginseng extracts at 250 μg/ml are shown in Table 1. Spearman correlation study was conducted to study the correlation between the antiproliferative activities of P. notoginseng with duration of steaming. The increased duration of steaming on P. notoginseng was directly correlated with the higher inhibitory effect of the steamed extracts on cell growth. The P values and the correlation factor were presented in Table 2. The results of Spearman correlation study indicates that the correlation of the duration of steaming of P. notoginseng and its anti-proliferative effects on the three liver cancer cells were statistically significant. In addition, the steamed P. notoginseng demonstrated a dose-dependent growth inhibition in all three cell lines.
The raw P. notoginseng has no effects on cell growth up to a concentration of 1 mg/ml. Thus, the IC 50 for raw P. notoginseng were not determined for all three cell lines. The IC 50 value decreased as the duration of steaming increased, indicating that longer steaming led to greater anti-proliferative effects in all three cell lines (Table 3).

Anti-proliferative activities of ginsenosides in liver cancer cells
To identify the active components responsible for the anti-proliferative activities of P. notoginseng, we screened ginsenosides enriched in steamed P. notoginseng, namely 20S-Rh 1 , Rk 3 , Rk 1 , 20S-Rg 3 , Rh 2 and 20R-Rh 1 . Ginsenosides Rg 1 , Rb 1 , Rd, Re and notoginsenoside R 1 which predominate in raw P. notoginseng were also assessed for comparison. The structures of these ginsenosides and notoginsenoside are shown in Figure 3.
The ginsenosides in the raw P. notoginseng, namely Rg 1 , Rb 1 , Re, Rd and notoginsenoside R 1 , exerted different growth responses in all three cell lines ( Figure 4A). At 0.25 mg/ml, all of them reduced cell growth by 20-30% in SNU449 cell ( Figure 4A). In SNU182 cells, cell viability was not significantly affected by any of the ginsenosides. Ginsenoside Rg 1 , Re and notoginsenoside R 1 exerted significant anti-proliferative effects, resulting in lowered cell viabilities of 65-85% in the HepG2 cell line.
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 antiproliferative activity on liver cancer cell lines.
The present study reports for the first time the in vitro anti-proliferative activities of ginsenoside Rk 3 .

Dose-response of selected ginsenosides on the SNU449 cell line
The dose-response of four constituent ginsenosides, namely Rh 2 , Rk 1 , Rk 3 and 20S-Rg 3 , were further assessed for efficacy for inhibiting growth in the SNU449 cell line. The IC 50 values for ginsenosides Rh 2 . Rk 1 , Rk 3 and 20S-Rg 3 are listed in Table 4. As expected, ginsenoside Rh 2 was the most potent, followed by Rk 1 , Rk 3 and 20S-Rg 3 . The IC 50 values of ginsenosides Rh 2 , Rk 1 and Rk 3 were lower than the IC 50 of the most potent steamed P. notoginseng extract which is the 24 hour steamed sample.

Steaming selectively enriched growth-inhibiting ginsenosides
The anti-proliferative effects of the steamed P. notoginseng were positively correlated with the duration of steaming ( Figure 5). The longer the steaming duration is, the greater the anti-proliferative effects. Increasing the steaming duration resulted in the formation of more ginsenosides Rk 3 , Rk 1 , 20S-Rg 3 and Rh 2 as indicated by the increased peak area of the four ginsenosides ( Figure 6). The duration of steaming P. notoginseng powder correlated with increasingly enriched ginsenoside Rk 3 , Rk 1 , 20S-Rg 3 and Rh 2 in the extracts. Cellular exposure to these extracts resulted in a dose-dependent reciprocal inhibition of cell proliferation and cell viability. These indicate that active components were increasingly generated upon steaming, thereby increasing the anti-proliferative activities. The varying proportions of the active components in the P. notoginseng extracts resulted in differences in their anti-proliferative activities. 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 Rh 2 , Rk 1 , Rk 3 and 20S-Rg 3 .
Sun et al. [6] also reported that steaming the root of P. notoginseng affected its chemical composition and anticancer and anti-proliferative activities in SW-480    Figure 4 Anti-proliferative effects of the ginsenosides from P. notoginseng. In vitro anti-proliferative effects of ginsenosides in the raw (A) and steamed (B) Panax notoginseng extracts in SNU449 (black square), SNU182 (white square) and HepG2 (grey square) human liver cancer cells. The cells were exposed to these ginsenosides at 250 μg/ml for 48 hours (SNU449 and SNU182) or 72 hours (HepG2) and assayed by WST-1. Plot shows the average percentage cell viability ± standard deviation as compared to vehicle control (100 ± 4.5% viability) of three independent experiments conducted in triplicates each. Statistical significance was considered when P < 0.05 (*) and P < 0.001 (**). (A) Ginsenosides Rg 1 , Rb 1 , Re, Rd and notoginsenoside R 1 in the raw P. notoginseng were screened. Most of the ginsenosides in the raw P. notoginseng showed significant anti-proliferative activities against SNU449 and HepG2 but not SNU182.   ), 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, Rk 3 , Rh 2 , Rk 1 and 20S-Rg 3 were the most anti-proliferative and ginsenoside Rk 3 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.

Conclusion
Steaming changes the chemical profile as well as antiproliferative biological activities of P. notoginseng.
Steamed P. notoginseng contains potential compounds for the treatment of liver cancer.   Duration of steaming (hours) Figure 6 The changes of the total peak area (plus sign) and individual peak area of ginsenoside Rk 3 (triangle), Rk 1 (cross), 20S-Rg 3 (square) and Rh 2 (circle) in P. notoginseng extracts with the duration of steaming.