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Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: involvement of cytochrome P450 CYP2E1.

Abstract

Acetaminophen is a commonly used drug for the treatment of patients with common cold and influenza. However, an overdose of acetaminophen may be fatal. In this study we investigated whether mice, administered intraperitoneally with a lethal dose of acetaminophen, when followed by oral administration of Phyllanthus urinaria extract, may be prevented from death. Histopalhological analysis of mouse liver sections showed that Phyllanthus urinaria extract may protect the hepatocytes from acetaminophen-induced necrosis. Therapeutic dose of Phyllanthus urinaria extract did not show any toxicological phenomenon on mice. Immunohistochemical staining with the cytochrome P450 CYP2E1 antibody revealed that Phyllanthus urinaria extract reduced the cytochrome P450 CYP2E1 protein level in mice pre-treated with a lethal dose of acetaminophen. Phyllanthus urinaria extract also inhibited the cytochrome P450 CYP2E1 enzymatic activity in vitro. Heavy metals, including arsenic, cadmium, mercury and lead, as well as herbicide residues were not found above their detection limits. High performance liquid chromatography identified corilagin and gallic acid as the major components of the Phyllanthus urinaria extract. We conclude that Phyllanthus urinaria extract is effective in attenuating the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme may be an important factor for its therapeutic mechanism.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Acetaminophen; Cytochrome 450 CYP2E1; Hepatoprotection; Hepatotoxicity; Phyllanthus urinaria

Introduction

Acetaminophen (APAP) has been widely used as a medicine for pain and fever relief (Whitcomb 1994). Since APAP can be purchased easily from any pharmaceutical outlet and even from supermarkets, without prescriptions from clinicians, it is commonly considered as a "safe drug" when taken within the suggested therapeutic dose. However, APAP can be hepatotoxic when an overdose is administered and, warning messages are present in the package. Clinically, APAP has been demonstrated to be nephrotoxic and hepatotoxic from animal experiments and in human beings (Curry et al. 1982; Keaton 1988; Vermeulen et al. 1992; Bonkovsky et al. 1994).

The use of herbal extracts in the treatment of human diseases is becoming very popular worldwide. Scientific approaches further magnify the reliability of the use of herbal extracts as complimentary medicine. Extracts and molecules from medicinal plants have been demonstrated to be important in the development of agents against human viruses, such as herpes simplex viruses 1 and II (Khan et al. 2005) and antitumor drugs (Lampronti et al. 2003). They are also compounds of interest in the treatment of genetic diseases, including thalassemia (Bianchi et al. 2008). The ethanol extract from anomalous fruit of Gleditsia sinensis has been well documented for its anticancer properties on human cancer cell lines (Chui et al. 2005; Tang et al. 2007).

In this respect, Phyllanthus urinaria (P. urinaria) has been extensively investigated for its possible anticancer activity. The boiled water extract from the whole plant of P. urinaria has been reported to induce apoptosis in a number of human cancer cell lines, including leukaemia, hepatoblastoma, nasopharyngeal carcinoma and fibrosarcoma but not the normal human endothelial cells and liver cells (Huang et al. 2004a). The bcl-2 anti-apoptotic protein was further shown to be down-regulated after treatment of Lewis lung carcinoma cells with this boiled water extract of P. urinaria (Huang et al. 2003). Further mechanistic investigation using HL-60 human acute promyelocytic leukaemia cell line suggested that induction of apoptosis by the boiled water extracts of P. urinaria is associated with the activation of the CD95 Fas receptor/ ligand expression and ceramide-mediated pathways (Huang ct al. 2004b). This boiled water extract of P. urinaria was further shown to exhibit anti-tumor and anti-angiogenic effects in mice bearing Lewis lung carcinoma. P. urinaria can reduce the blood vessel density and decrease the matrix induced tube formation of human umbilical cord endothelial cells as well as the their migration (Huang et al. 2006). The medicinal use of P. urinaria, however, is not restricted to the treatment of neoplastic diseases. The use of P. urinaria as hepatopro-tective agent in tetrachloromethanc induced hepatic injury has been previously documented (Lee et al. 2006).

In the present study, we conducted experiments designed to explore the hepatoprotective activity of P. urinaria in a mice model twenty four hours after the administration of a lethal dose of APAP (550mg/kg). This APAP dosage causes, in C57BI6 mice, a very low survival rate, since the majority of them succumb within two weeks (Wong et al., personal communication). The employed experimental model system simulates a clinical condition similar to those of patients admitted for acute liver injury to the emergency departments of hospitals. Our studies were designed to determine whether P. urinaria extracts may be effective in attenuating the APAP induced hepatotoxicity; cytochrome P450 CYP2E1 enzyme was chosen as a biochemical marker and may be an important factor by which to further explore the mechanisms of potential therapeutic relevance in our animal experimental model system.

Materials and methods

Chemicals and reagents

Unless otherwise stated, all the reagents, including APAP. were purchased from Sigma chemicals. The physiological saline for APAP injection was obtained from Baxter. Silymarin was purchased from Sigma chemical. Primary antibody conjugated with biotin against mouse cytochrome P450 CYP2E1 and substrate for peroxidase were purchased from US Biologicals, while secondary antibody and the subsequent signal detection reagents were purchased from Dako. The in vitro cytochrome P450 CYP2EI kit was purchased from In Vitrogen.

Preparation of the P. urinaria extract

P. urinaria in powdered form was kindly provided by the Bioactive Technologies Ltd. (Hong Kong). Briefly, whole plants of P. urinaria were identified, confirmed and a voucher sample was kept by the company. The plants were then excised and 5 kilograms (dry weight) was extracted with 30 litres of 80% ethanol for 90min. The percentage of yield was 11%. Afterwards, the dried powder was dissolved completely in distilled water and adjusted to a final concentration of l0mg/ml. The greenish yellow mixture was sterile filtered and stored at --20 CC until future use.

Animal care

Eight weeks old C57B16 mice, weighing approximately 20-25 g, were purchased from the animal unit of The Chinese University of Hong Kong and maintained in a conventional sanitary facility, in accordance with the institutional guidelines on animal care, with the required consistent temperature and relative humidity. All the procedures were approved by the Animal Research Ethics Committee.

APAP treatment on mice

APAP was dissolved in physiological saline. A total of 37 mice were included in the study. On day one, acute liver injury was induced by intraperitoneally (i.p.) administered APAP at a dose of 550 mg/kg of body weight. From day two to day four, treatment groups received various doses of P. urinaria extract or silymarin (positive reference) once daily while APAP group received water. Two additional groups consisted of mice treated with (i) buffer i.p. at day 1 and P. urinaria extract at the dose of 200 mg/kg daily for three days, and (ii) buffer i.p. at day 1 and water for three days. The mortality rate and change of body weight in the animals were monitored and recorded. On day 5, all the mice were sacrificed and autopsies was performed to collect vital organs.

Haematoxylin and eosin (H and E) histochemistry staining

Sections of mouse liver from autopsy samples were dewaxed, washed with phosphate buffered saline (PBS) and then stained with H and E for nucleus and cytoplasm staining using the conventional protocol reported elsewhere. Slides were then premounted and inspected under a light microscope.

Immunohistochemistry analysis of cytochrome 450 CYP2E1

Sections of mouse liver from autopsy samples were dewaxed with xylene and gradient concentrations of ethanol. Possible endogenous peroxidase was blocked and slides were washed with PBS. Slides were then blocked again and treated with diluted primary antibody (rabbit anti-rat cytochrome P450 CYP2E1) in PBS. Slides were washed with PBS and then treated with the secondary antibody CSA II rabbit link. After washing, slides were further treated with amplification reagent and anti-fluorescein-HRP. Afterwards, slides were incubated with DAB substrate. Nuclei were stained with haematoxylin and finally slides were inspected under a light microscope.

In vitro cytochrome P450 CYP2E1 enzyme assay

Detailed procedures can be found in the user guide manual supplied with the reagent kit. Briefly, various concentrations of P. urinaria extract were mixed with the reagent buffer, the cytochrome P450 CYP2E1 enzyme and the regeneration system. Before the final addition of the substrate for the cytochrome P450 CYP2E1 enzyme, an excitation emission reading was recorded for any possible background fluorescence. The substrate was added, and after 30 min incubation, diethyldithiocarbamate, at a final concentration of 100 [mu]M, was added to terminate the reaction. A second excitation emission reading was recorded, and results were analysed.

Analytical chemistry analysis for P. urinaria extract

Any possible contaminations of heavy metal and herbicide from P. urinaria extract were examined. The herbicide list included aldrin, cis-chlordane, trans-chlordane, oxychlordane, p,p'-DDD (4,4'-DDD), p,p'-DDE, (4,4'-DDE), o,p'-DDT (2,4'-DDT), p,p'-DDT (4,4'-DDT), dieldrin, endrin, heptachlor, heptachlor epoxide isomer B, hexachlorobenzene, hexachlorocyclo-hexane (BHC) [alpha]-isomer, hexachlorocyclohexane (BHC) [beta]-isomer, hexachlorocyclohexane (BHC) [gamma]-isomer (lindane), hexachlorocyclohexane (BHC) [sigma]-isomer, methyl pentachlorophenyl sulphide (MPCPS), pentachloroani-line and pentachloronitrobenzene (quintozene). Three independent tests were performed and results were expressed as mean [+.or.-] standard deviations from three independent experiments.

Method for heavy metals analysis

Individual stock standard solution of arsenic (As), cadmium (Cd), mercury (Hg) and lead (Pb; 1000 mg/1) were purchased from The National Institute of Metrology, PR China, and were of purity [greater than or equal to]99.99%. Stock solutions containing As, Cd and Pb each at 200 [mu]g/l and Hg at 100 [mu]g/l were prepared in 2% v/v nitric acid solution. The working solutions ranged from 0.2 to 20 [mu]g/1 for As, Cd and Pb and from 0.2 to 20 [mu]g/1 for Hg and were obtained daily by appropriate dilutions with 2% v/v nitric acid solution. Indium (In) and bismuth (Bi) at 2 mg/1 and germanium (Ge) at 20 mg/1 were added to the working solutions as an internal standard. Water (18 M[omega]) was prepared with a Milli-Q system (Millipore, US). It was further analyzed by ICP-MS (PerkinElmer Sciex Elan 6100 inductive couple plasma mass spectrometer equipped with a concentric quartz nebulizer).

Method for pesticide residues analysis

Individual stock standard solutions of organochlorine pesticide (500 mg/1) were purchased from Supercol and Chem Service, and were of > 98% purity. Stock solutions containing 20 pesticides each at concentrations ranging from 2 to 10 mg/l were prepared in iso-octane and stored at ~4[degrees]C in amber glass bottles. The working solutions ranging from 0.02 to 0.15 mg/l were obtained daily by appropriate dilutions with iso-octane. 1-Bromo-2-nitrobenzene at 0.2 mg/l was added to the working solutions as an internal standard. All the solvents (acetone, dichloromethane, ethyl acetate, n-hexane and iso-octane (LabScan, Thailand)) were of pesticide grade. Anhydrous sodium sulphate (Sigma-Aldrich, US) was of analytical grade. Analytical regent grade chemicals and water (18 M[omega]) were prepared with a Milli-Q system (Millipore, US).

GC-ECD condition

Gas chromatographic analyses were performed on an Ahgent 6890 gas chromatograph equipped with an electron capture detector. A DB-17MS fused silica capillary column of 30 m x 0.25 mm i.d. and 0.25 m film thickness from J&W Scientific was used. Helium (purity [greater than or equal to]99.999%) was used as a carrier gas at a flow rate of 1 ml/min. A one liter extract was injected in splitless mode. The injection temperature was 210 [degrees]C. The oven temperature was programmed from initial temperature 100[degrees]C (held for 2 min) to 165 [degrees]C at 20[degrees]C/min, followed by 200 [degrees]C at 10[degrees]C/min, 230 [degrees]C at 2 [degrees]C/min, 265 [degrees]C at 3 [degrees]C/min and finally at 280 [degrees]C (held for l0 min.). The detector was 300 [degrees]C. Results were further confirmed by GC-MS.

Pesticide residues sample preparation

Sample was mixed with 100 ml ethyl acetate and 4 g anhydrous sodium chloride. The mixture was sonicated for 15 min and the solids were allowed to settle. After double extraction of residue each with 50 ml ethyl acetate, the supernatant was combined and evaporated to near dryness. The residual extract was diluted to 10 ml with dichloromethane for subsequent purification in GPC.

HPLC analysis for P. urinaria extract

To identify major components from the P. urinaria, HPLC analysis was performed. Corilagin and gallic acid were used as markers and serial combinations were prepared. Agilent 1100 series HPLC and Symmetry C18 (5 urn, 4.6 mm x 250 mm) column was used and detected with DAD detection using wavelength of 270 nm.

Mobile phase consisted of acetonitrile and trifluor-oacetic acid (0.1%) while the gradient is shown in Table 1. Flow rate was adjusted to 0.8 ml per minute and injection volume was 10[mu]l. Then calibration curves for both markers were set where peak areas were correlated to the corresponding concentrations. According to the retention time, markers were identified from the P. urinaria extract again and the relative concentrations were further estimated according to their corresponding peak areas. The calculated results for the concentration of major components were expressed as mean [+ or -] standard derivation obtained from three independent experiments.
Table 1. HPLC mobile phase condition for the ingredients
identification in P. urinaria extract.

Time (min)  A (%)  B (%)

0           3      97

10          3      97

15          10     90

40          20     80

45          30     70

50          3      97

60          3      97

A: Acetonitrile. B: 0.1% trifluoroacetic acid. The percentage is in
terms of total volume.


Results

P. urinaria extract inhibits APAP induced hep atotoxicity in vivo

Table 2 shows that when APAP was administrated intraperitoneally to mice at a dose of 550 mg/kg without further treatments, a high mortality rate and rapid drop in body weight (Fig. 1) were observed. Hematoxylin (H) and Eosin (E) staining of the liver autopsy samples showed extensive necrotic features (Fig. 3A). When mice were treated with APAP together with oral administration of P. urinaria extract, an improvement of survival rate (Table 2) as well as body weight variation (Fig. 1) was observed and noticeably, its improvement of survival rate is similar to the positive reference, silymarin (Table 2). After increasing the dose of P. urinaria extracts from 20 to 200 mg/kg per day for a continuous treatment of three days, liver autopsies also showed significant improvement in cytoplasm integrity (Figs. 3B to D). Each of the five control mice only treated with 200 mg/kg of P. urinaria for 3 days exhibited no evidence of necrotic feature with high integrity of cytoplasm, as was evident from the H and E staining of liver autopsy sections (Fig. 4). The body weight of animals which have been treated with only vehicle or P. urinaria were found to be increased (data not shown).
Table 2. Survival percentage of mice treated with various
combinations of APAP and P. urinaria extract.

                               Number of day

                               1    2    3    4    5

                               Percentage of mice survival

0                     (n = 5)  100  100  100  100  100

APAP only             (n = 5)  100  80   0    0    0

APAP + 20 mg/kg (a)   (n = 5)  100  100  100  100  100

APAP + 40 mg/kg (a)   (n = 5)  100  100  100  100  80

APAP + 80 mg/kg (a)   (n = 7)  100  100  100  100  85

APAP + 200 mg/kg (a)  (n = 5)  100  100  100  100  100

APAP + 100 mg/kg (b)  (n = 7)  100  100  100  100  100

APAP + 200 mg/kg (b)  (n = 7)  100  100  100  100  100

200 mg/kg (a)         (n = 5)  100  100  100  100  100

APAP (single dose at 550 mg/kg on day 1).

"n"--the number of mice involved in the corresponding
experimental group.

(a) P. urinaria extract (single dose daily for 3 consecutive
days from day 20 to 4 after a single dose of APAP at 550 mg/kg
on day 1).

(b) Silymarin (single dose daily for 3 consecutive days from
day 2 to 4 after a single dose of APAP at 550 mg/kg on day 1).


[FIGURE 1 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Cytochrome P450 CYP2E1 is involved in the protective activity of P. urinaria extract against APAP induced hepatotoxicity

Sections of liver autopsy samples from treated mice were further investigated for any possible changes in the protein level of cytochrome P450 CYP2E1 by using a specific antibody. As shown in Fig. 5, samples from vehicle treated control mice showed relatively higher protein level of cytochrome P450 CYP2E1 when compared with samples obtained from mice treated with only P. urinaria extract (Figs. 5A and B). The inhibitory effects of P. urinaria extract were confirmed in the liver autopsy sections from mice treated with APAP followed by P. urinaria extract; in this case an even higher decrease in the protein level of cytochrome P450 CYP2E1 was observed (Fig. 5C). In agreement with the data shown in Fig. 5, the in vitro enzymatic assay for the effect of P. urinaria extract on cytochrome P450 CYP2E1 showed a dose dependent inhibition (Fig. 6). The 50% inhibitory concentration was about 500 [mu]g/ml under this experimental system.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Analytical chemistry for the detection of heavy metals and herbicides

Analytical chemistry assays of P. urinaria extract showed that heavy metals, including arsenic, cadmium, mercury and lead, were not present above the detection limit (0.05 mg/kg). With respect to herbicide residues, none of the tested targets were detected above the detection limit (0.02 mg/kg; data not shown).

HPLC analysis for the detection of major ingredients

HPLC analysis of P. urinaria extract allowed the identification of two distinct peaks, which were superimposed on the marker sample HPLC profile according to their retention times. The calculated concentration of gallic acid was found to be 72.34[+ or -]0.86 mg/1, while corilagin was found to be 956.38[+ or -]13.52 mg/1. In addition, unidentified minor peaks were also detected within the HPLC profile of the P. urinaria extract (Fig. 2).

[FIGURE 2 OMITTED]

Discussion

APAP is commonly used as an analgesic and antipyretic medicine (Whitcomb 1994). It has been suggested that the mechanism of action of APAP involves cytochrome P450 CYP2E1 to produce a toxic product, the N-acetyl-p-benzoquinone imine (NAPQI). This NAPQI can further interact with the glutathione (GSH) in the liver (Dahlin et al. 1984). If an overdose of APAP is administrated, it reduces hepatic GSH, and NAPQI thus binds covalently to cysteine residues on proteins, causing the production of 3-(cysteine-S-yl) APAP adducts (Mitchell et al. 1973). APAP induced hepatotoxicity is pathologically characterized by its centrilobular hepatic necrotic features. Since cyto-chrome 450 CYP2EI plays a crucial role in bio-transformation of toxic chemicals, it appears that any reagent which can down-regulate the cytochrome 450 CYP2E1 activity would be a potential hepatoprotective regimen candidate to protect the hepatocytes from APAP induced toxicity.

The Phyllanthus spp. family has been widely used for medicinal purposes. P. amarus was demonstrated to possess anti-inflammatory qualities by inhibiting iNOs, COX-2 and cytokines through the NF-kB pathway (Kiemer et al. 2003). In respect to the possible hepatoprotective activity, the whole plant extract of P. maderaspatensis from India was reported to have remarkable hepatoprotective function against APAP induced hepatotoxicity as judged from the serum marker enzymes (Asha et al. 2004). Bhattacharjee and Sil (2006) further demonstrated in India, that the partially purified protein fraction of P. niruri protects experimental mice against APAP induced hepatotoxicity. These results demonstrated that P. maderaspatensis and P. niruri protect hepatocytes against oxidative stress in mice, probably by increasing oxidative defence. These studies have brought attention to the effects of sub-lethal doses of APAP.

In the present study, we attempted to develop an experimental design mimicking the actual clinical situation of patients accidentally consuming lethal doses of APAP. Under such circumstances, acute liver toxicity was observed with extensive pathological necrotic features, leading to death within 3 days. A twenty-four hours post-treatment with P. urinaria extract, however, significantly improved survival, inhibiting toxicity of APAP treated mice. The relative changes in body weight of mice indicated that P. urinaria extract may gradually decrease the toxic effects of APAP. End point autopsy analysis further demonstrated that P. urinaria extract can significantly protect the mice liver from APAP induced necrosis and that this phenomenon was dose dependent. Pathological studies revealed that the maximum dose of P. urinaria extract tested for post APAP treatment was non-toxic as high cytoplasmic integrity of hepatocytes was determined in samples from each of the five employed subjects.

To propose possible mechanisms of action, the effects of P. urinaria extract against the target enzyme cytochrome P450 CYP2E1 was investigated. Immuno-histochemistry staining using the cytochrome P450 CYP2E1 mouse specific antibody clearly demonstrated that P. urinaria extract induces a sharp decrease in the hepatic cytochrome P450 CYP2E1 protein level in mice which were pre-treated with APAP. Notably, P. urinaria extract treated groups also showed lower level of hepatic cytochrome P450 CYP2E1 protein when compared with the vehicle group. Due to the strong necrotic features of the liver sections from mice treated with only APAP, we were unable to perform the immunohistochemistry staining on those sections. As APAP could induce the mRNA of cytochrome P450 CYP2E1 (Simpson et al. 2003), we speculate that P. urinaria extract acts by depressing the liver cytochrome P450 CYP2E1 protein levels stimulated by APAP. Since we further demonstrated that P. urinaria extract inhibits the cytochrome P450 CYP2E1 enzymatic activity in vitro, both findings imply that this extract reasonably reduces the conversion of APAP into 3-(cysteine-S-y1) APAP adducts in the liver.

The acceptability and reliability of herbal extracts used for medicinal purpose always face difficulties of heavy metal and herbicides contamination. In addition, the presence of unwanted contaminants within plant extracts may cause errors in the interpretation of the results on biological activity; in the case when contaminants retain biological effects. In order to address this issue, panels of heavy metals and herbicides were screened, but none of the targeted heavy metals or herbicides was found to be present above the corresponding detection limits.

It is always argued that the study of herbal extracts which makes definitive conclusions about specific compounds is impossible. However, such extracts have been used in traditional medicine. Therefore, we were interested in determining the presence of characterized molecules within the P. urinaria extract. To this aim, HPLC was performed, and the obtained spectrum was compared with two control markers, corilagin and gallic acid. These molecules were chosen as markers because they are expected to be present in P. urinaria. As anticipation, both corilagin and gallic acid were identified as major peaks within the P. urinaria extract. Notably, gallic acid is the major component of the tannic acid. Previous research has shown that tannic acid reduces the hepatic cytochrome P450 CYP2E1 protein twenty four hours after a single dose i.p. injection from 20 to 80 mg/kg (Krajka-Kuzniak and Baer-Dubowska 2003). Is gallic acid also an inhibitor of cytochrome P450 CYP2E1 enzyme as tannic acid? Park et al. (2005) reported that gallic acid isolated from Orostachys japonicus may also attenuate the hepatic toxicity from mice induced by an i.p. injection of bromobenzene. Orally administrated gallic acid at a dose of 20 mg/kg/day may reduce the aniline hydroxylase activity (cytochrome P450 CYP 2E1 activity). Gallic acid may further restore the activity of epoxide hydrolase which was decreased by bromobenzene. Furthermore, the hepatic lipid peroxidation induced by bromobenzene was prevented with gallic acid. Their results suggest that gallic acid of O. japonicus may protect liver from bromobenzene toxicity by, at least in part, inhibiting the cytochrome P450-dependent mono-oxygenase activities and by enhancing the epoxide hydrolase activity. Since the theoretically calculated amount of gallic acid received by the mice from our P. urinaria extract was only approximately equal to 1.5 mg/kg/day, we speculate that gallic acid from our P. urinaria might only be in part responsible for the mechanisms involved in the hepatoprotective function as well. Further experimental work is still on-going to elucidate whether other components from our P. urinaria also participate in hepatoprotection.

Recently, the protective effects of Pycnogenol on carbon tetrachloride-induced hepatotoxicity in Spraguc-Dawley rats was reported (Yang et al. 2008). Here, our results demonstrate that P. urinaria extract is effective in allowing survival of mice after receiving an overdose of APAP by protecting the hepatocytes from necrosis. The underlying mechanism involves the down-regulation of hepatic cytochrome P450 CYP2E1 protein after stimulation from a lethal dose of APAP. Chemical composition analysis showed that corilagin and gallic acid are the major components where gallic acid may be partly responsible for the therapeutic action of P. urinaria extract. We assume that P. urinaria extract can be potentially used as a complementary medicine in emergency treatment for the overdose of APAP in the future provided that more favourable pre-clinical and clinical data are available to support our hypothesis.

Acknowledgements

We acknowledge a Niche area grant offered by the Hong Kong Polytechnic University to Dr. C.H. Chui (HK$200,000; BB8Q) and a postgraduate research fund to Mr. D.K.P. Hau from The Baptist University of Hong Kong (40-40-173 RDD Development Fund). Professor R. Gambari is sponsored by AIRC (Italian Association for Cancer Research). Lastly, Mr. D.K.P. Hau would like to thank the supervision from Professor W.F. Fong and Bioactive Technologies Limited (Hong Kong) for the supply of P. urinaria extract.

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Desmond K wok Po Hau (a), Roberto Gambari (b), Raymond Siu Ming Wong (c), Marcus Chun Wah Yuen (d), Gregory Yin Ming Cheng (c), Cindy Sze Wai Tong (c), Guo Yuan Zhu (a), Alexander Kai Man Leung (a), Paul Bo San Lai (e), Fung Yi Lau (c), Andrew Kit Wah Chan (c), Wai Yeung Wong (f), Stanton Hon Lung Kok (d), Chor Hing Cheng (d), Chi Wai Kan (d), Albert Sun Chi Chan (d), Chung Hin Chui (c),(*), Johnny Cheuk On Tang (d),(*), David Wang Fun Fong (a),(*)

(a) Research and Development Division, School of Chinese Medicine, Horn Kong Baptist University, Hong Kong, China

(b) Bio PharmaNet, Department of Biochemistry and Molecular Biology, The University of Ferrara, Ferrara, Italy

(c) Department of Medicine and Therapeutics, Li Ka Slung Medical Sciences Building, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

(d) Institute of Textiles and Clothing and Applied Biology, The Hong Kong Polytechnic University, Hong Kong, China

(e) Department of Surgery, Li. Ka Shing Medical Sciences Building, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

(f) Department of Chemistry, Hong Kong Baptist University, Hong Kong, China

* Corresponding author. Tel.: +852 26323120; fax: +8522637 5396 (C.H. Chui).

* Corresponding author. Tel.: +852 34008727; fax: +852 23649932 (J.C.O. Tang).

* Corresponding author. Tel.: +852 34115308; fax: +852 3411 2902 (D.W.F. Fong).

E-mail addresses: chchui@graduate.hku.hk (C.H. Chui), bccotang@inet.polyu.edu.hk (J.C.O. Tang), wfforg@hkbu.edu.hk. (D.W.F. Fong).

0944-7113/$ - see front matter[C] 2009 Elsevier GmbH. All rights reserved.

doi:10.1016/j. phymed. 2009.01.008
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Author:Hau, Desmond Kwok Po; Gambari, Roberto; Wong, Raymond Siu Ming; Yuen, Marcus Chun Wah; Cheng, Gregor
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Aug 1, 2009
Words:5369
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