Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo

Journal of Ethnopharmacology 137 (2011) 64–69

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Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo Judy Yuet-Wa Chan a,b , Johnny Chi-Man Koon b , Xiaozhou Liu a , Michael Detmar c , Biao Yu d , Siu-Kai Kong e , Kwok-Pui Fung a,b,∗ a

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China c Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland d State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China e Department of Biochemistry, Faculty of Science, The Chinese University of Hong Kong, Hong Kong, China b

a r t i c l e

i n f o

Article history: Received 21 October 2010 Received in revised form 8 February 2011 Accepted 11 April 2011 Available online 31 May 2011 Keywords: Anti-angiogenesis Human microvascular endothelial cells Zebrafish Vascular endothelial growth factor

a b s t r a c t Ethnopharmacological relevance: Angiogenesis, the process of blood vessel formation, is critical to tumour growth. The importance of angiogenesis in tumour development has lead to the development of antiangiogenic strategies to inhibit tumour growth. In this study, polyphyllin D (PD), an active component in Chinese herb, Paris polyphylla, was evaluated for its potential anti-angiogenic effects. Materials and methods: The inhibitory effects of PD on three important processes involved in angiogenesis, i.e. proliferation, migration and differentiation were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using zebrafish embryos as an animal model of angiogenesis, the anti-angiogenic effect of PD was further verified in vivo. Results: PD suppressed the growth of HMEC-1 cells at 0.1–0.4 ␮M without toxic effects. At 0.3 ␮M and 0.4 ␮M, PD significantly inhibited endothelial cell migration and capillary tube formation. About 70% of the zebrafish embryos showed defects in intersegmental vessel formation upon treatment with PD at concentrations of 0.156 ␮M and 0.313 ␮M. Conclusion: The anti-angiogenic effects of PD have been explored in the study which implied a potential therapeutic development of PD in cancer treatment. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Polyphyllin D (PD) is an active component in the Liliaceae family plant Paris polyphylla which was used as prescribed medicine in China. The rhizome of the plant is known as Chong-lou and was used to treat tumours of liver, digestive tract, leukemia and symptoms such as sore throat and convulsion [Guo et al., 2008; Yan et al., 2009]. One of its major components, PD, is classified as a steroidal saponin. The systemic name of PD is diosgenyl ␣-l-rhamnopyranosyl-(1 → 2)-[(␣-l-arabinofuranosyl(1 → 4)-␤-d-glucopyranoside)] [Deng et al., 1999]. Our previous findings showed that PD exerted anti-proliferative effects on human breast tumour cells (MCF-7 and MDA-MB-231), human

Abbreviations: PD, polyphyllin D; VEGF, vascular endothelial growth factor; HMEC, human microvascular endothelial cell; ISV, intersegmental vessel. ∗ Corresponding author at: Room 603, Mong Man Wai Building, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. Tel.: +852 26096873; fax: +852 26037732. E-mail address: [email protected] (K.-P. Fung). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.04.021

hepatocellular carcinoma cells (HepG2) as well as its multi-drug resistant counterpart R-HepG2 through the induction of apoptosis [Cheung et al., 2005; Lee et al., 2005; Ong et al., 2008]. In vivo studies also indicated that PD is effective in reducing the tumour size of human breast tumour xenografts in nude mice with no apparent toxicity to the host [Lee et al., 2005]. Anti-apoptotic effects of PD have been demonstrated previously. In this study, the potential anti-angiogenic effect of PD will be examined. Angiogenesis, the process of new blood vessel formation, is important in the normal development of the embryo and fetus as well as in normal physiological processes including wound healing and reproductive functions [Bussolino et al., 1997; Dvorak, 2005]. Angiogenesis also plays a crucial role in the development of cancer. Small solid tumours at the initial stage are not vascularized. New blood vessel formation might favor the transition from hyperplasia to neoplasia which marks the onset of uncontrolled tumour growth [Gordon et al., 2010]. Vascular endothelial growth factor (VEGF), one of the most specific and potent mitogens for endothelial cells, is known to induce neovascularization of tumours. VEGF is over-expressed in tumour cells and it can activate endothelial and, possibly, also tumour cells to migrate and invade adjacent

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2006]. HMEC-1 cells (1.5 × 104 cells/well) were seeded in 96well culture plates. After treatment with various concentrations of PD (0.1–0.9 ␮M) or vehicle (0.0008% DMSO) for 24 h, the medium was replaced by 40 ␮L of MTT solution (5 mg/mL in PBS). The cells were then incubated for 2 h at 37 ◦ C. Subsequently, MTT solution was removed and 100 ␮L of DMSO was added. Absorbance was measured at 540 nm using a microplate reader. The percentage of cell viability was calculated against vehicle control. 2.3. Measurement of cell proliferation by direct cell counting

Fig. 1. The chemical structure of PD.

tissue [Murukesh et al., 2010]. Various inhibitors of angiogenesis have been developed to inhibit or slow down the growth of tumours by blocking blood vessel formation, mostly through suppressing VEGF signaling pathways. Bevacizumab (Avastin® ) is the first clinically used angiogenesis inhibitor approved by the U.S. Food and Drug Administration (FDA) in 2004 [Shih and Lindley, 2006; Lien and Lowman, 2008]. It is a humanized monoclonal antibody against VEGF which neutralizes and inhibits all active isoforms of VEGF from binding to the VEGF receptors VEGFR-1 (flt-1) and VEGFR2 (KDR/flk-1). Both receptors are membrane-associated tyrosine kinase receptors responsible for downstream survival and proliferation pathways. Neutralization of VEGF leads to prevention of neovasculature formation and hence limitation of blood supply which might allow a greater capacity for oxygen and chemotherapeutic drugs to reach specific target cells. In this study, the anti-angiogenic potential of PD was explored, using the human microvascular endothelial cell line HMEC-1 for in vitro studies of cell proliferation, migration and tube formation. Moreover, the potential inhibition of angiogenesis by PD was also studied in vivo using a zebrafish model. 2. Materials and methods 2.1. Materials Diosgenyl ␣-l-rhamnopyranosyl-(1 → 2)-[(␣-larabinofuranosyl-(1 → 4)-␤-d-glucopyranoside)] (PD) was synthesized from diosgenyl-␤-d-glucopyranoside as described previously [Li et al., 2001]. The chemical structure of PD is shown in Fig. 1. The method for synthesizing PD was illustrated in our previous publication [Li et al., 2001] in which Nuclear Magnetic Resonance (NMR) was applied to confirm the structure of the final product. PD was dissolved in DMSO at 50 mM as stock solution. Solvent control with 0.0008% DMSO was used in the assays of this study as 0.4 ␮M of PD was the highest concentration applied. Other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise specified. Human microvascular endothelial cell line HMEC-1 was purchased from the American Type Culture Collection (Manassas, USA). The cells were maintained in MCDB131 medium supplemented with 10% fetal bovine serum (Invitrogen, CA, USA), 10 ng/mL epidermal growth factor, 1 ␮g/mL hydrocortisone, penicillin (100 IU/mL) and streptomycin (100 ␮g/mL) in a humidified incubator with 5% CO2 at 37 ◦ C. 2.2. Measurement of cell viability by MTT assay Cell viability was determined by 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [Chan et al.,

HMEC-1 cells (1.5 × 104 cells/well) were seeded into 24-well plates. After overnight incubation, various concentrations of PD or vehicle were added to the wells. Cells were incubated for 24 h, 48 h or 72 h before harvest by trypsinization. The number of viable cells in each well was determined by trypan blue dye exclusion method. 2.4. Measurement of DNA synthesis HMEC-1 cells (5 × 103 cells/well) were seeded into 96-well culture plates and synchronized with 1% FBS for 24 h [Noguer et al., 2009]. After treatment with various concentrations of PD or vehicle for 24 h, 0.5 ␮Ci of 3 H-thymidine in PBS was added per well and the cells were incubated at 37 ◦ C for 6 h. Then, DNA was harvested on microfilters with a cell harvester (Beckman Coulter, Brea, USA). The amount of DNA synthesized was determined by measuring the radioactivity of the filter using a microplate scintillation counter (Beckman Coulter, Brea, USA). 2.5. Cell cycle analysis HMEC-1 cells (3 × 105 cells/well) were seeded into 6-well culture dishes. After synchronization, the cells were treated with various concentrations of PD or vehicle for 24 h. Then, the cells were harvested and fixed in 70% ethanol. Before performing flow cytometry, ethanol was removed and the cells were incubated with RNase (8 ␮g/mL) and PI (10 ␮g/mL) for 30 min. Cell cycle distribution was then detected using a flow cytometer (BD FACSCanto, BD BioSciences, CA, USA), and the results were analyzed using ModfitLT version 3.0 software. 2.6. Wound healing assay Wound healing assay, also known as scratch assay, was carried out as described previously [Gebäck et al., 2009]. HMEC-1 cells were grown to full confluence in the wells of 24-well plates. Then, two crosses on cell cultures were scratched with a pipette tip and the wells were washed with PBS to remove detached cells. Cells were then incubated with various concentrations of PD or vehicle for 15 h. Photographs were taken before and after PD or vehicle treatment at 40× magnification by an inverted microscope (Nikon Eclipse TS100). Data set was analyzed with the TScratch software using the default parameter settings [Gebäck et al., 2009]. Four replicates were done in each individual experiment and the presented images are representatives of triplicate experiments with similar outcome. 2.7. Tube formation assay Each well of 48-well culture plates was coated with 150 ␮L of Matrigel and allowed to solidify. HMEC-1 cells (5 × 104 cells/well) were then seeded into the wells with the addition of various concentrations of PD or vehicle, and incubated for 24 h. The network of

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Fig. 3. PD inhibits DNA synthesis, as assessed by reduced incorporation of 0.5 ␮Ci of 3 H-thymidine. HMEC-1 cells upon treatment with or without PD were incubated with 0.5 ␮Ci of 3 H-thymidine for 6 h. Then, DNA incorporated with 3 H-thymidine was harvested on microfilters and the amount of radioactivity was measured by scintillation counter. Data are expressed as mean ± SD for 3 independent trials with 6 replicates in each trial (n = 18). **p < 0.01 (One Way ANOVA).

3. Results 3.1. Inhibition of HMEC-1 cell growth by PD

Fig. 2. PD inhibits the proliferation of HMEC-1 cells. The percentage of viable cells was evaluated using the MTT assay. Cells without PD treatment were set as 100% survival. Cell proliferation was inhibited with increasing concentrations of PD at the range of 0.1–0.9 ␮M (A). At the same concentration range of PD, the growth of HMEC-1 cells was examined for 3 days by direct cell counting. At concentrations of 0.4 ␮M or below, the cell numbers increased until day 3 but the proliferation rate was reduced when PD concentration was increased from 0.1 to 0.4 ␮M (B). Data are expressed as mean ± SD for 8 replicates (n = 8). *p < 0.05, ***p < 0.0001 (One Way ANOVA).

tubes formed in each well was photographed in 3 random regions. The total length of tubes per image was analyzed using Image-Pro Plus 6.0 software. 2.8. In vivo study The transgenic zebrafish line TG (flil:EGFP), whose endothelial cells express eGFP, was purchased from Zebrafish International Resource Center in University of Oregon. Embryos at 1–4 cell stage were distributed into 6-well plates. After 20 h, different concentrations of PD or vehicle were added to each well for 2 h before the drug was replaced by fresh clean water. After further incubation for 26 h, intersegmental vessel (ISV) formation was studied under a fluorescence microscope. The number of absent ISV in each zebrafish embryo was counted. Inhibition of angiogenesis was recorded when there were one or more defects in ISV formation. The extent of inhibition of ISV formation was defined as no inhibition (no missing vessel), mild inhibition (1–3 defective ISVs) or severe inhibition (4 or more defective ISVs). 2.9. Statistical analyses

The effect of PD on the viability of HMEC-1 cells was examined by MTT assay. We found that PD at all selected concentrations between 0.1 and 0.9 ␮M caused a significant decrease in cell viability after 24 h incubation (p < 0.05, Fig. 2A). By direct cell counting, PD was shown to inhibit cell proliferation at 0.1 ␮M and 0.2 ␮M after 3 days of treatment (p < 0.05). At concentrations of 0.3 ␮M and 0.4 ␮M, PD suppressed HMEC-1 cell proliferation after 2 and 3 days (p < 0.001) (Fig. 1B). At PD concentrations higher than 0.4 ␮M, a cytotoxic effect was seen since the number of cells decreased from day 2 to day 3 (Fig. 2B). Therefore, non-toxic concentrations of PD (0.1–0.4 ␮M) were selected for the additional in vitro studies. 3.2. Effects of PD on DNA synthesis and cell cycle distribution of HMEC-1 cells PD at concentrations of 0.2 ␮M, 0.3 ␮M and 0.4 ␮M significantly inhibited thymidine incorporation by about 38% (p < 0.05, Fig. 3). The effect of PD on cell cycle distribution was evaluated by PI staining. There was no significant change in the percentage of cell population in any specific phase of the cell cycle upon PD treatment. However, there was a tendency of a decreased population in G0 /G1 phase (from 56.03% to 48.71%) and an increased population in G2 /M phase (from 18.63% to 26.53%) when HMEC-1 cells were treated with increasing concentrations of PD from 0.1 ␮M to 0.4 ␮M (Table 1).

Table 1 HMEC-1 cells were stained with propidium iodide and the percentage population at different cell cycle phases was quantified using flow cytometry. The percentage of cell population of G0 /G1 , S and G2 /M phase in control and PD-treated cells of three independent trials is summarized. Data are expressed as mean ± SD for 3 trials (n = 3). [PD] (␮M)

One Way ANOVA with Dunnett’s Multiple Comparison Test was used to analyze the statistical significance of the results. All experimental results were expressed as mean ± standard deviation (SD). For all assays, the significance of difference was calculated between PD-treated samples and vehicle-treated samples. The results were considered as statistically significant when p-values were ≤0.05.

Percentage of cell population G0 /G1

0 0.1 0.2 0.3 0.4

56.03 56.98 52.19 51.07 48.71

S ± ± ± ± ±

2.93 1.90 4.62 5.75 2.21

25.35 25.56 25.39 25.65 24.75

G2 /M ± ± ± ± ±

1.22 0.83 0.89 0.91 0.82

18.63 17.47 22.41 23.28 26.53

± ± ± ± ±

1.94 2.12 4.63 5.05 1.49

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Fig. 4. PD inhibits endothelial cell migration. Cell migration was evaluated by measuring the percentage of open wound area before and after 15 h incubation. A representative figure shows the image of one of three trials. An increased open wound area was observed in 0.4 ␮M PD-treated cells when compared to vehicle control cells (A). The results of three independent trials are summarized. For vehicle control cells, about 50% of wound area was closed after 15 h. When cells were treated with 0.3 or 0.4 ␮M of PD, the percentage of open wound area was significantly increased (B). Data are expressed as mean ± SD for 12 replicates (4 replicates in each trial, three trials performed). ***p < 0.001 (One Way ANOVA).

3.3. Inhibition of endothelial cell migration by PD In vehicle-treated HMEC-1 cells, about 50% of wound area was closed after 15 h incubation. Upon treatment with 0.3 ␮M and 0.4 ␮M of PD, the percentage of open wound area was significantly increased to 70–80% (Fig. 4A and B), indicating that endothelial cell migration was inhibited.

3.4. Suppression of tube formation by PD HMEC-1 cells without PD treatment formed a more or less complete tubular network after 24 h incubation. Upon treatment with various concentrations of PD, the network formed was less complete with shorter tube length (Fig. 5A). The total length of all tubes formed in each sample was measured and summarized in Fig. 5B. PD inhibited the tube formation of HMEC-1 cells in a dose-dependent manner with a significant suppression at concentrations of 0.2 ␮M, 0.3 ␮M and 0.4 ␮M.

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Fig. 5. PD inhibits capillary tube formation. For control cells without PD treatment (0 ␮M), a complete tubular network of HMEC-1 was found. When the cells were treated with various concentrations of PD (0.1–0.4 ␮M), a less complete network with shorter tube length was observed (A). The length of all tubes was added up and plotted as total tube length. At concentrations of 0.2, 0.3 and 0.4 ␮M, PD significantly inhibited tube formation (B). Data are expressed as mean ± SD for 12 replicates (4 replicates in each trial, three trials performed). *p < 0.05 or ***p < 0.001 (One Way ANOVA).

3.5. Anti-angiogenic effects of PD in zebrafish embryos In negative controls (vehicle only), complete ISV formation was observed in most treated embryos and less than 2% of the embryos showed severe inhibition (Fig. 6A). However, when the embryos were treated with PD, nearly 70% of the treated embryos showed inhibition of ISV formation, ranging from mild to severe, at concentrations of 0.156 ␮M and 0.313 ␮M (Fig. 6D). 4. Discussion Polyphyllin D (PD) is a steroidal saponin found in the traditional Chinese medicine Paris polyphylla, which has been used in China to treat various diseases [Pharmacopoeia Commission of the People’s Republic of China, 1990]. PD was chemically synthesized by our group [Deng et al., 1999; Li et al., 2001], and the anti-proliferative activities and associated mechanisms of PD in tumour cells have

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Fig. 6. PD impairs the formation of intersegmental vessels (ISVs) in zebrafish embryos. Representative images of zebrafish embryos with normal ISV formation after incubation in vehicle (0.01% DMSO), and with impaired ISV formation upon PD treatment (0.313 ␮M) (A and B). Defective ISV formed in the PD-treated group is enlarged. White arrows indicate the missing vessels (C). Inhibition of ISV formation was assessed by counting the number of missing vessel in each zebrafish embryo. The inhibition of ISV formation was scored as described in Section 2. The total number of zebrafish counted in each treatment group was more than 40 (n > 40). In the vehicle control group, about 60% of zebrafish showed no inhibition of formation. Upon treatment with SU5416 (2 ␮M), there was a strong inhibition (∼20%). When zebrafish were treated with PD at concentrations of 0.156 or 0.313 ␮M, ISV formation was impaired (D).

been investigated [Cheung et al., 2005; Lee et al., 2005; Ong et al., 2008]. It was also reported that PD inhibits P-glycoproteinmediated anti-tumour drug efflux in multidrug resistant human leukemia cells [Nguyen et al., 2009]. In a proteomic study, PD was found to upregulate typical endoplasmic reticulum stress-related proteins/genes including glucose-regulated protein 78 and protein disulfide isomerase [Siu et al., 2008]. In the present study, we found that PD also exerts potent anti-angiogenic effects in vitro and in vivo. Angiogenesis, a process involving the formation of new blood vessels, is important for many physiological processes such as wound healing, tissue regeneration and fetal development. The significance of angiogenesis in tumourigenesis has been well demonstrated, leading to anti-angiogenesis as an important strategy in cancer therapy. Avastin (bevacizumab) is the first angiogenic inhibitor approved by FDA. It has been clinically applied since 2004. In the past decade, many other anti-angiogenic agents have been developed and have sometimes shown potent effects in inhibiting tumour growth. Some of them, such as epigallocatechin gallate, berberine, celastrol and gambogic acid [Huang et al., 2008; Liu et al., 2008; Yi et al., 2008; Tang et al., 2009] are natural products isolated from herbal materials. To investigate the anti-angiogenic proper-

ties of PD, the human microvascular endothelial cell line HMEC-1 was used in this study. When compared to human umbilical vein endothelial cells (HUVEC) which are primary cells with limited life-span and various characteristics due to multi-donor origin and derivation from large vessels, HMEC-1 are generally better characterized and are more stable [Nanobashvili et al., 2003]. Moreover they are derived from the microvasculature which is the immediate target of angiogenic factors. As angiogenesis involves coordinated migration, proliferation and differentiation of endothelial cells, the in vitro HMEC-1 model has been used in this study to investigate the effects of PD on distinct phases of blood vessel formation. We found that PD inhibited the proliferation of HMEC-1 cells at a non-toxic concentration range of 0.1–0.4 ␮M. At PD concentrations higher than 0.4 ␮M, a cytotoxic effect was seen since the number of cells decreased from day 2 to day 3. The anti-proliferative property of PD was further verified by inhibition of thymidine incorporation during DNA synthesis and a tendency of reduced cell numbers in the G0 /G1 phase of the cell cycle, which is the growth phase with a high rate of biosynthetic activity, especially regarding DNA replication. To examine the effect of PD on endothelial cell migration, we used a scratch assay which is considered

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as an in vitro wound healing assay [Liang et al., 2007]. The most commonly used cell migration models are the scratch assay and the Boyden chamber assay. The scratch assay is advantageous in studying the regulation of cell migration by cell–cell interactions or cell interactions with the extracellular matrix because the cells involved are not prepared in suspension [Liang et al., 2007]. For the cells treated with PD at 0.3 ␮M and 0.4 ␮M, the open wound area was significantly increased when compared to the control. In the process of angiogenesis, endothelial cells migrate and proliferate towards angiogenic signals (e.g. angiogenic factors), resulting in the formation of capillary sprouts and tubes with a new basement membrane [Folkman and D’Amore, 1996; Hanahan and Folkman, 1996; Risau, 1997]. It has been reported that the differentiation process involves several steps in blood vessel formation, including cell adhesion, migration, alignment, protease secretion, and tube formation [Arnaoutova et al., 2009]. Thus, the length of capillary tubes formed on Matrigel layer was evaluated which could partly explain the effect on endothelial cell differentiation. It was found that PD at concentrations 0.2–0.4 ␮M was effective in inhibiting the tube formation of HMEC-1 cells. Summarizing the results of the various in vitro experiments, PD was found to exhibit antiangiogenic effects by suppressing the proliferation, migration and at least partly differentiation of endothelial cells. These anti-angiogenic effects of PD were confirmed in an in vivo zebrafish model. Zebrafish development is a useful system for chemical screens. Large numbers of transparent embryos can be produced for rapid screening with easy manipulation. Furthermore, the embryos exhibit several features of tumour biology, including rapidly dividing cells, apoptosis and angiogenesis [Serbedzija et al., 1999; Tran et al., 2007]. Thus, the zebrafish model is well suited for studying the properties of potential anti-cancer drugs. In our study, the established anti-angiogenic compound SU5416 was applied as positive control drug. It is a small molecule tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 [Katanasaka et al., 2008]. When either SU5416 or PD was added, defective ISV formation was found. The severity of ISV defects was increased with increasing concentrations of PD. No difference in survival rate was observed between PD-treated group and control group, indicating that the overall development was unaffected and that the inhibition of blood vessel formation was specific. In conclusion, it was found that PD inhibited angiogenesis by suppressing cell proliferation, migration and tube formation in vitro. In vivo, PD impaired the formation of intersegmental vessels in zebrafish embryos. Together, these effects indicated that PD might also have antitumoural effects via inhibition of tumour angiogenesis. Acknowledgements This study was supported by Ming Lai Foundation, The International Association of Lions Clubs District 303 – Hong Kong and Macau Tam Wah Ching Chinese Medicine Resource Centre in Institute of Chinese Medicine, The Chinese University of Hong Kong. Dr. Johnny Koon was supported by a Hop Wai Short-term Research Grant to visit the Swiss Federal Institute of Technology, ETH Zurich, Switzerland. References Arnaoutova, I., George, J., Kleinman, H.K., Benton, G., 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis 12, 267–274. Bussolino, F., Mantovani, A., Persico, G., 1997. Molecular mechanisms of blood vessel formation. Trends in Biochemical Sciences 22, 251–256. Chan, J.Y., Tang, P.M., Hon, P.M., Au, S.W., Tsui, S.K., Waye, M.M., Kong, S.K., Mak, T.C.W., Fung, K.P., 2006. Pheophorbide a, a major antitumor component purified from Scutellaria barbata, induces apoptosis in human hepatocellular carcinoma cells. Planta Medica 72, 28–33.

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