MATERIALS AND METHODS

Cell lines and chemicals: The human esophageal cancer cell line (EC109) and human liver cancer cell line (HepG2) were purchased from Beinglay Biotech (Wuhan, Hubei, China). Cells were grown in DMEM medium supplemented with 10% fetal bovine serum, 100 μg mL^–1 penicillin and 100 μg mL^–1 streptomycin in a 5% carbon dioxide humidified incubator at 37°C. Experiments were performed when cells were approximately 80% confluent. The 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), butylated hydroxytoluene (BHT), cisplatin and camptothecin were purchased from Sigma (Sigma-Aldrich, Sternhein, Germany).

Sample preparation: Scutellaria barbata was purchased from Hao Yi Sheng Chinese herbal medicine store, Jinghua Street, Luoyang. The original plant was collected in Shandong province, China (July, 2015) and the identity kindly confirmed as S. barbata D. Don by Professor Wenliang Li at the Department of Entomology, Henan University of Science and Technology. A voucher specimen has been deposited in natural products research laboratory, School of Chemical Engineering and Pharmaceutics (access number: JGS2015-039A).

The dry herb of S. barbata was minced and extracted with light petroleum twice for 24 h at room temperature. The solvent were completely removed at 35°C under reduced pressure and the residue was dried in the fume cupboard. The dried herbal residues were then extract with distilled water at proposed ratio (1:6, 1:10, 1:14, 1:18 and 1:24) in the ultrasonic cleaning machine for proposed minutes (10, 20, 30, 40 and 50 min) filtered and collected supernatants. Added the same amount of distilled water as before and repeated proposed times (1, 2, 3, 4 and 5). The collected supernatants were then combined and centrifuged at 1500 rpm for 8 min. The solvent were reduced to 1/4 volume at 35°C under reduced pressure. The condensed extracts were mixed with chloroform/butanol (5:2) at volume ratio of 1:1. The mixture was standstill for 50 min. The water layer (upper) was then separated using separating funnel. The water extracts were mixed with absolute ethanol to achieve the final ethanol concentration at proposed percentage (50, 60, 70, 80 and 90%). The mixture was standstill for 4 h and centrifuged to precipitate the polysaccharides. The isolated polysaccharides were then washed by distilled water and dried at -30°C under reduced pressure for 2 h.

Cell proliferation assay: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed as follows: briefly, 200 μL cell suspension was seeded at a concentration of 1.5×10⁴ cells mL^–1 in a 96-well plate. After overnight incubation, serial doubling dilutions of PSB (100-3.1 μg mL^–1) were added in 10 μL aliquots. The negative control was 10 μL 0.01% DMSO in Hank’s solution. Cisplatin (100-3.1 μg mL^–1) was used as positive control. Each concentration was tested three times. After 24 h incubation, 20 μL MTT solution (5 mg mL^–1, Sigma-Aldrich Ltd, Gillingham, Dorset, UK) was added and incubated at 37°C for 4 h to develop color. The Optical Density (OD) was measured at 590 nm using a Multiskan Spectrum Reader. The growth inhibition was determined using cytotoxicity:

Apoptosis assay: The cell suspension (800 μL) was seeded at a concentration of 1.5×10⁴ cells mL^–1 in a 24-well plate with 30 mm cover slide plated in the bottom of the well. After overnight incubation, 50 μg mL^–1 (Value close to the IC₅₀ value) PSB were added in 40 μL aliquots. The negative control was 40 μL 0.01% DMSO in Hank’s solution. Camptothecin (50 μg mL^–1) was used as positive control. The cell-seeded cover slides were removed from 24-well plates at 24 h after treatment. The cells were then stained using the Annexin-V-FLUOS staining kit (Roche Applied Science, Mannheim, Germany). Labelling solution (20 μL Annexin-V-FLUOS-labelling reagent in 1 mL HEPES buffer with 20 μL propidium iodide reagent) was used to stain apoptotic cells which were then visualized by green fluorescence microscopy with excitation wavelength at 488 nm.

Total antioxidant activity test: Prepared PSB and BHT at concentration of 0.24, 0.48, 0.72, 0.96, 1.20 mg mL^–1. Prepared the stock solution of phosphorus molybdenum reagent (0.6 mol L^–1 sulfuric acid, 28 mmol L^–1 sodium phosphate and 4 mmol L^–1 ammonium molybdate). The 1.0 mL of each samples were mixed with 3.0 mL phosphorus molybdenum reagent solution. The mixtures were placed in 95 water bath for 90 min. The samples were cooled down until room temperature and then measured the absorbance at 625 nm. The distilled water was used as a blank and BHT was positive control.

ABTS free radical scavenging assay: Prepared PSB and BHT at concentration of 0.24, 0.48, 0.72, 0.96, 1.20 mg mL^–1. The 0.8 mL of 7.4 mmol L^–1 ABTS solution and 0.8 mL of 2.6 mmol L^–1 K₂S₂O₈ were mixed and reacted in dark for 12 h. The prepared mixture was then diluted 100 times using absolute ethanol to produced ABTS⁺ working solution. The 4 mL ABTS⁺ working solution was mixed with 1 mL sample and 1 mL absolute ethanol. The mixtures were then shake for 1 min and measure the absorbance at 734 nm as follows:

where, A₀ is absorbance of ABTS⁺ working solution+ absorbance of absolute ethanol and A is absorbance of ABTS⁺ working solution+ absorbance of samples.

Statistical analysis: The data were shown as Mean±Standard Deviation (SD). Statistical comparison among treatments was carried out using one-way analysis of variance (ANOVA). The statistical significances between control and sample groups were calculated by the Student’s t-test. Data were taken as significant where p<0.05.

RESULTS AND DISCUSSION

Optimization of PSB isolation: The water extraction following alcohol precipitation is the regular principle used for herbal polysaccharides isolation. In this study, optimized the PSB isolation method through adjusting the material/water ratio, ultrasonic parameters and ethanol percentage. In the single factor experiment, the content of PSB was found to decrease along the S. barbata (g)/water (mL) (1:6, 1:10, 1:14, 1:18 and 1:24) decrease. The isolation with shorter ultrasonic time (10, 20 and 30 min) didn’t produce less PSB than that of longer time (40 and 50 min) but repeat ultrasonic times (2, 4 and 5 times) could lead to a higher polysaccharides content. About 60, 80 and 90% ethanol resulted in the higher amount precipation of PSB than 50 and 70% ethanol. Based on these, a L9 (3⁴) orthogonal experiment was designed to optimized the ultrasonic-assisted PSB isolation. Four factors including material/water ratio; (A) Ultrasonic time, (B) Repeat times, (C) Ethanol percentage and (D) Under three conditions (A:1:6, 1:10 and 1:14, B: 10, 20 and 30 min, C: 2, 4 and 5 and D: 60, 80 and 90%) were used as parameters. The analytical results indicated that the best isolation condition was A₃B₁C₃D₂. The optimized method parameters for PSB isoaltion were thus determined as material/water ratio at 1:14, 10 min ultrasonic, repeat 5 times and 80% ethanol usage. This set of parameters was further used to validate experimentally. A mean value of PSB yield rate was at 2.96±0.24% (n = 4) indicating that the optimized extraction process was adequate for the PSB isolation. Compared to the traditionally used PSB isolation, this ultrasonic-assisted PSB isolation method is characterized with higher yield rate and less time and resources consuming¹⁰. Moreover, the ultrasonic employed in this method is characterized of fast, clean and efficiency. As the whole isolation was performed in the room temperature and the bioactive compounds were remained in the maximum amounts of their original structure without heat degradation. The significant of this development is to greatly facilitate the gathering of large amounts of PSB which would promote the current and future study and finally transform PSB to the valuable medicinal products.

Anticancer activities of PSB in vitro: The anticancer activities of S. barbata have been widely reported in both of the in vitro and in vivo models^11-13. Previously, the flavonoids and diterpenoid alkaloids were recognized as the major active anticancer components of S. barbata^3,14. Recently, the inhibitory effects of PSB were found in 95-D cancer cell lines and xenograft tumours^5-7. However, the current studies were only limited in just lung cancer cells or animal models. In this study, the anticancer activities of PSB were further determined in human esophageal cancer cell EC109 and human liver cancer cell HepG2 in vitro. As shown in Fig. 1, PSB inhibited the proliferation of both of the cancer cell lines in vitro on a dose-dependent manner. The IC50 values of PSB on EC109 and HepG2 were 43.62±1.51 μg mL^–1 and 60.44±2.31 μg mL^–1, respectively. Moreover, the PSB (50 μg mL^–1) could induce the apoptosis of EC109 and HepG2 cells under 24 h treatment. As these results were highly in agreement with the data reported in previous study, PSB could be confirmed as an effective inhibitor for the growth of cancer cells through apoptosis-inducing mechanisms^6,7.

Fig. 1(a-d):	(a) Growth inhibition of PSB on EC109, (b) HepG2, (c) Apoptosis induced by PSB on EC109 and (d) HepG2

Fig. 2(a-b):	(a) Total antioxidant activity of PSB and BHT (Left) and (b) ABTS free radical scavenging rate of PSB (Right)

The more detailed mechanisms may include regulation the CDK/cyclin cascade and apoptotic proteins such as Bcl-2, Bax, Cyclin D1, CDK4 and p21^11,15. As reported by Lin et al.¹¹ the ethanol extract of S. barbata could significantly suppress the activation of STAT3, Erk and p38 signalings in color cancer tissues through regulation of multiple critical genes expression such as Bcl-2, Bax, Cyclin D1, CDK4 and p21. The chloroform fraction of S. barbata was reported to increase the ratio of the pro-apoptotic Bax/Bcl-2 and decreased the expression of the pro proliferative cyclin D1 and cyclin-dependent kinase 4 in human colon cancer cells¹⁵. According to a previous genomics screening study reported that the S. barbata induced apoptotic death of cancer cells need multiple genes involved in mechanisms including control of DNA damage, cell cycle, nucleic acid binding and protein phosphorylation¹². The PSB may also involve in this complex regulation network. Taken together, this significant of current results is to firstly evident the anticancer activities and apoptosis-inducing abilities of PSB on human esophageal and liver cancer cells in vitro which expand the fundamental understanding of the anticancer activity of S. barbata and support its clinical use. However, the detailed underlying mechanisms of anticancer effects of PSB still remain as an ongoing challenge and thus are highly suggested to be further studied.

Antioxidant activities of PSB: Oxidative stress has been known as a causative factor for progression of diseases as well as cancer. In this study, the antioxidant activities of isolated PSB were examined since, the wide use of S. barbata in medicine. Characterization of antioxidant effects of PSB was determined and compared to standard butylated hydroxytoluene (BHT). Two different assays including total antioxidant activity test and ABTS free radical scavenging assay were employed for the comparison. As shown in Fig. 2, PSB presented a dose-dependent antioxidant activity and this activity could achieve similar effects as BHT did at lower concentration level (0.24 and 0.48 mg mL^–1). In the ABTS free radical scavenging assay, the different concentration of BHT demonstrated a constant high scavenging rate (over 95%) while, PSB’s effects increased gradually and reach BHT’s value at concentration of 1.2 mg mL^–1. Taken together, the isolated PSB presented a great antioxidant and free radical scavenging activity which was in good agreement with the research reports in previous study⁸. The results above could confirmed PSB as the major active group constitutes the antioxidant activities of herbal S. barbata thus, providing a valuable natural antioxidant for further investigation and development. Moreover, current study of antioxidant activities of PSB were all conducted to test in vitro assays and no in vivo experiments reported so far. Therefore, in vivo efficacy, side effects and safety of PSB remain unknown. The proper designed in vivo models, comprehensive well-controlled and double-blind clinical trials are urgently needed.

CONCLUSION

Overall, the herb S. barbata is widely used in TCM hospitals of China and its medicinal values are gradually accepted worldwide. Therefore, the exploration of bioactivities of its major components is important to underpin the fundamental understanding of the medicinal activities of S. barbata and support its clinical use. In this study, this study has optimized an ultrasonic-assisted method to extract the polysaccharides from S. barbata. The PSB’s anticancer and antioxidant activities which are two of most used medicinal properties of S. barbata were then determined in in vitro experimental models. The significant of this results to current knowledge of the research field include; (1) The developed PSB isolation method provides higher yield rate and consumes less time/resources compared to the method already published which would promote the current and future research in relevant fields, (2) The growth inhibitory and apoptotic effects of PSB are firstly determine in those two cancer cell lines (EC109 and HepG2) in vitro. It determined that PSB was a significant bioactive group of components constitute the anticancer and antioxidant effects of S. barbata providing a potential natural medicinal resource for further investigation and development. The further studies are strongly suggested to explore its underlying mechanisms of bioactivities and develop the PSB-based drugs and health products or addictives.

ACKNOWLEDGMENT

The authors gratefully acknowledge the University Scientific Research Funding (2014QN012) from Henan University of Science and Technology and the Research Fund of Returned Overseas Personnel (13530001) from Ministry of Education of the People's Republic of China.