Scholarly article on topic 'Hepatoprotective activity of Chhit-Chan-Than extract powder against carbon tetrachloride-induced liver injury in rats'

Hepatoprotective activity of Chhit-Chan-Than extract powder against carbon tetrachloride-induced liver injury in rats Academic research paper on "Chemical sciences"

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{Antioxidants / "Carbon tetrachloride" / Hepatoprotection / " Ocimum gratissimum " / "Oxidative stress"}

Abstract of research paper on Chemical sciences, author of scientific article — Yi-Chun Lin, Kuei-Mei Cheng, Hsin-Yu Huang, Pei-Yu Chao, Jin-Ming Hwang, et al.

Abstract The capability of Chhit-Chan-Than extract powder (CCTEP, 10% aqueous Ocimum gratissimum L. extract) to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity in vivo was investigated. Wistar rats were divided into five groups. Group A was a normal control group given only vehicle; Group B, the hepatotoxic group, was injected intraperitoneally twice a week with repeated 8% CCl4/olive oil (0.1 mL/100 g of body weight); Groups C–E, extract-treated groups received CCl4 and different doses of CCTEP (100 mg/kg and 200 mg/kg) or silymarin (200 mg/kg of body weight) daily by gavage for 8 weeks, respectively. The results showed that the CCl4-induced histopathogical changes may be prevented by CCTEP through reducing the intercellular collogen stack, dropping blood serum alanine aminotransferase and aspartate aminotransferase levels, and restoring the catalase activity and glutathione content. The hepatoprotective properties were further confirmed by the marked improvement in histopathological examination and by quantitative steatosis-fibrosis scoring. The above results suggest that CCTEP is able to prevent the liver inflammation and fibrosis induced by repeated CCl4 administration, and the hepatoprotective effects might be correlated partly with its antioxidant and free radical scavenging effects.

Academic research paper on topic "Hepatoprotective activity of Chhit-Chan-Than extract powder against carbon tetrachloride-induced liver injury in rats"

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

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Research Article

Hepatoprotective activity of Chhit-Chan-Than extract powder against carbon tetrachloride-induced liver injury in rats

Yi-Chun Lin a'1, Kuei-Mei Cheng b1, Hsin-Yu Huang c,d, Pei-Yu Chaoef, Jin-Ming Hwangg, Hsueh-Hui Leeh,i, Cheng-You Luj, Yung-Wei Chiua,j'*, Jer-Yuh Liu k'1'**

a Emergency Department, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan, ROC b Department of Sports Management, National Taiwan University of Physical Education and Sport, Taichung, Taiwan, ROC

c Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC d Department of Hospitality Management, Wu Feng University, Chiayi, Taiwan, ROC

e Department of Leisure Industry Management, National Chin-Yi University of Technology, Taichung, Taiwan, ROC f Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC g School of Applied Chemistry, Health Care and Management College, Chung Shan Medical University, Taichung, Taiwan, ROC

h Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC i Department of Clinical Laboratory, Kuang Tien General Hospital, Taichung, Taiwan, ROC j Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC k Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan, ROC l Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan, ROC

ELSEVIER

ARTICLE INFO

ABSTRACT

Article history: Received 29 April 2013 Received in revised form 9 July 2013

Accepted 2 September 2013 Available online xxx

Keywords: Antioxidants Carbon tetrachloride Hepatoprotection

The capability of Chhit-Chan-Than extract powder (CCTEP, 10% aqueous Ocimum gratissimum L. extract) to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity in vivo was investigated. Wistar rats were divided into five groups. Group A was a normal control group given only vehicle; Group B, the hepatotoxic group, was injected intraperitoneally twice a week with repeated 8% CCVolive oil (0.1 mL/100 g of body weight); Groups C—E, extract-treated groups received CCl4 and different doses of CCTEP (100 mg/kg and 200 mg/kg) or silymarin (200 mg/kg of body weight) daily by gavage for 8 weeks, respectively. The results showed that the CCl4-induced histopathogical changes may be prevented by CCTEP through reducing the intercellular collogen stack, dropping blood serum alanine aminotransferase and aspartate aminotransferase levels, and restoring the catalase activity and glutathione content. The hepatoprotective properties were further confirmed by the marked improvement in histopathological examination

* Corresponding author. Emergency Department, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan, ROC.

** Corresponding author. Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan, ROC.

E-mail addresses: hboxygen@gmail.com (Y.-W. Chiu), jyl@mail.cmu.edu.tw (J.-Y. Liu). 1 These authors share equal contribution to this article. 1021-9498/$ — see front matter Copyright © 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/jjfda.2013.09.012

2 JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

Ocimum gratissimum and by quantitative steatosis-fibrosis scoring. The above results suggest that CCTEP is able

Oxidative stress to prevent the liver inflammation and fibrosis induced by repeated CCl4 administration,

and the hepatoprotective effects might be correlated partly with its antioxidant and free radical scavenging effects. Copyright © 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan

LLC. All rights reserved.

1. Introduction

Chronic liver diseases are common worldwide and are caused by long-term exposure to toxic chemicals, drugs, alcohol, and/ or viral infection, which are characterized by a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [1,2]. Iterative hepatic injury induces chronic inflammation associated with dynamic healing response, ultimately leading to cirrhosis and its life-threatening complications [3]. In the absence of an effective pharmacological therapy for such pathological processes, the research of herbal drugs or plant extracts for the treatment of liver diseases has increased all over the world [4]. For example, silymarin, a flavonolignan mixture of milk thistle (Silybum marianum), is one such popular hepatoprotective herbal drug. Silymarin exhibits significant hepatoprotective effects, which have been well demonstrated using several in vitro and in vivo models [5,6], and is thus often used as a standard reference in this field. Clearly, there exists a critical need to validate the efficacy of traditional herb medicine and plant extracts using scientific evidence and experimental models in order to explore novel or alternative approaches for liver diseases.

The Ocimum species are widely distributed in tropical and subtropical regions and commonly used as a food spice and traditional herb in many countries. Among the Ocimum species, Ocimum gratissimum L. is a well-known medicinal plant since ancient times. It is popularly known as Vana Tulsi in India [7,8], Chhit-Chan-Than in Taiwan [9,10] and clove basil in most Western countries [11]. The leaf of O. gratissimum has a strong-smelling aromatic flavor and is rich in antioxidants and phytoconstituents. It has been prepared in a variety of forms for consumption and is known for its multiple pharmacological properties including anti-inflammation [10,12], analgesic and spasmolytic activities [13], antidiarrheal activity [14], antitumor activity [15,16], antiviral activity [17], anti-hyperglycemic activity [18], and improvement of phagocytic function [19]. Our research group demonstrated the beneficial effects of the aqueous O. gratissimum extract (OGE), which exerts potent antioxidant action in in vitro and in vivo studies [12,20]. Using an in vitro model, OGE also exerts hep-atoprotective effects against oxidative stress [21]. OGE pre-treatment, in a dose-dependent manner, restored the decrease in cell viability in H2O2-treated human hepatoma HepG2 cells. Herbal extracts of Ocimum species have also been shown to inhibit liver cirrhosis using the animal models [22,23]. Nevertheless, the in vivo hepatoprotection of OGE underlying its antioxidant property remains sketchy.

Carbon tetrachloride (CCl4) is an extensively used hepato-toxic agent in preclinical animal studies [24]. It is the best-characterized chemical for study of xenobiotic-induced free

radical mediated acute liver injury in rats [25]. CCl4-induced liver damage results in increased necrosis, steatosis, and foamy degeneration followed by the progression of fibrosis [26]. During hepatocellular damage, varieties of enzymes normally located in the cytosol are released into the blood flow, where their quantification in plasma is a useful biomarker. The metabolism of CCl4, catalyzed by liver micro-somal cytochrome P450, rapidly overproduces free radicals that deplete hepatic glutathione (GSH) and initiates a chain lipid peroxidation of the hepatocyte membrane. CCl4-induced generation of peroxy and superoxide radicals result in the inactivation of catalase (CAT) and superoxide dismutase. These phenomena ultimately result in oxidative stress and hepatocyte injuries. Liver injury induced by CCl4 is a classical system of xenobiotic-induced hepatotoxicity and has been used extensively for decades for the screening of anti-hepatotoxic/hepatoprotective activities of different drugs [27].

Blocking the CCl4-induced peroxidation chain reaction that results in hepatotoxicity is one of the potential strategies by which functional food could exert a hepatoprotective effect. The most remarkable pathological characteristics in the liver of CCl4-treated animals resemble closely the processes of chronic hepatitis and cirrhosis in human [28]. Thus, male Wistar rats challenged by CCl4 were used as the animal model of chemical-induced hepatotoxicity in our recent studies [20] and also in the present study. The purpose of this project was to evaluate the hepatoprotective effect of Chhit-Chan-Than extract powder (CCTEP, containing 10% OGE) against acute CCl4-induced hepatic damage.

2. Methods

2.1. Preparation of extract powder and composition analysis

The CCTEP used in this study was made with 10% aqueous OGE and 90% dextrin as an excipient. Aqueous extract of O. gratissimum leaf was prepared as described elsewhere [12,16,20,21,29]. Briefly, leaves of O. gratissimum were harvested and washed with distilled water, followed by homog-enization with distilled water using polytron. The homogenate was incubated at 95 °C for 1 hour and then filtered through two layers of gauze. The filtrate was centri-fuged at 20,000 x g at 4 °C for 15 minutes to remove insoluble pellets and the supernatant was thereafter collected as OGE solution. Subsequently, the aqueous solution was mixed with water-soluble dextrin as an excipient. The powders obtained by spray drying were packaged in sealed metallized bioriented polypropylene bags at 24 ± 2 °C prior to the subsequent

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10 3

analyses. The total polyphenol and flavonoid contents were determined by the Folin-Ciocalteau method [30,31] and Lamaison method [32], respectively. The polyphenol content of OGE was analyzed as previously described [12,16,20,21,29] and revealed that the final product of CCTEP contained 1.77% polyphenolic acid and 0.69% flavonoids.

2.2. Treatment of animals

Male Wistar rats weighing 200-240 g were housed in conventional cages with free access to water and rodent chow at 20-22 °C with a 12-hour light-dark cycle. All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Chung Shan Medical University (Taichung, Taiwan, R.O.C.) for the care and use of laboratory animals. The experimental setting is Group A were treated with olive oil (1.0 mL/kg) by intraperitoneal injection, and Groups B-E were treated with 8% CCVolive oil (1.0 mL/kg) by intraperitoneal injection, twice every week (Monday and Thursday) for 8 weeks, as described by Hernandez-Munoz et al [33] with some modifications. On Tuesday, Wednesday, Friday, and Saturday, Groups A and B were treated with water, Groups C and D were treated with low dose (100 mg/kg) and high dose (200 mg/kg) of CCTEP, respectively, and Group E was treated with silymarin (200 mg/ kg); all by oral gavage. The mortality rate and body weight were recorded daily. At the end of the experiment, blood and organs were immediately collected after the animals were sacrificed. Liver tissue samples were taken from the left liver lobe and cut into two pieces. One piece was fixed in formalin for pathological examination. The other piece was subjected to biological analyses. The blood samples and organ tissues were all collected and stored at -80 °C until use.

2.3. Biochemical assays of serum

All biochemical assays were conducted in the clinical laboratories of the Kuang Tien General Hospital, Taichung, Taiwan. The levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured on a Beckman Coulter Synchron Clinical Systems Analyzer (LX20PRO Autoanalyzer; Beckman Coulter Inc., Taipei, Taiwan). Specifically, AST was measured by an oxaloacetate/malate dehydrogenase method, whereas ALT was measured by a pyruvate/lactate dehydro-genase method. Serum total cholesterol and triglyceride were measured on a Hitachi 7170 autoanalyzer (Hitachi, Tokyo, Japan).

2.4. Preparation of liver homogenate and assay of protein content

A 1 g sample of liver tissue in 10 mL ice-cold potassium phosphate buffer (pH 7.0) was homogenized in an IKA-WEAR homogenizer (RW 20 DZM; IKA, Staufen, Germany). The homogenate solution was centrifuged at 12,000 x g for 30 minutes. The supernatant was collected and stored at -80 °C for protein content assay, GSH and the antioxidant enzyme activities in the liver. The supernatant was collected and assayed for protein content by the Bradford method, using a

protein assay kit supplied by Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA), with bovine serum albumin as a standard.

2.5. Determination of GSH in the liver

GSH was determined by titration with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) [34] as described previously [35], with some modifications. Proteins of 0.4 mL liver homogenates were precipitated by the addition of 0.4 mL a metaphosphoric acid solution (1.67 g metaphosphoric acid, 0.20 g EDTA, and 30.0 g NaCl in 100 mL H2O). After 40 minutes, the protein precipitate was recovered by centrifugation at 12,000 x g in a microcentrifuge (Microfuge; Beckman) at 4 °C for 30 minutes. A 100 mL aliquot of the supernatant was combined with 0.7 mL 300 mM Na2HPO4, and the absorbance at 412 nm was read against a blank consisting of 0.1 mL supernatant plus 0.7 mL H2O. A100 mL aliquot of DTNB (0.02%, w/ v 1% sodium citrate) was then added to the blank and sample. Absorbance of the sample was read against the blank at 412 nm. The GSH content was determined using a calibration curve prepared with an authentic sample. An aliquot of lysate was used for the determination of protein content. GSH values are expressed as nmol/mg protein.

2.6. CAT assay

CAT activity was measured by the method of Kawamura et al [36]: 0.1 mL supernatant was added to cuvette containing 1.9 mL 50 mM phosphate buffer (pH 7.0). Reaction was started by adding 1.0 mL freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. Activity of CAT was expressed as U/mg of protein.

2.7. Histopathologic study

A portion of the liver was fixed in 10% formalin, processed using routine histology procedures, embedded in paraffin, cut in 5 mm sections, and mounted on a slide. The sections were then stained with hematoxylin-eosin dye and studied for histopathological changes (40x), i.e., necrosis, fatty changes, ballooning degeneration, and lymphocyte infiltration. Masson stain was used for liver steatosis and fibrosis scoring. Liver steatosis was graded on a 3-point scale: 1+, hepatocytes in the area of one-third of the lobules showing fatty accumulation; 2+, for two-thirds; and 3+, for all hepatocytes. The criteria used for scoring fibrosis severity were as follows: 0, normal; 1+, fibrosis present (collagen fiber present that extends from portal triad or central vein to peripheral region); 2+, mild fibrosis (collagen fiber present with extension without compartment formation); 3+, moderate fibrosis (collagen fiber present with some pseudo lobe formation); and 4+, severe fibrosis (collagen fiber present with thickening of the partial compartments and frequent pseudo lobe formation).

2.8. Statistical analysis

The experimental results are expressed as the mean ± standard deviation. Data were assessed using analysis of variance (ANOVA). Student t test was used in the

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

comparison between groups. A p value <0.05 was considered statistically significant.

Table 1 — The effects of Chhit-Chan-Than extract powder on the ratio of organ to body weight (BW) in CCU-induced liver injury rats.

Results

3.1. Clinic observation, body weight, and organ weight change

No dead rat was observed during the 8-week test of hep-aprotective effect of CCTEP. However, in the CCl4-treated only group the rats look emaciated at Week 7 or Week 8, possibly due to the liver damage.

The change of body weight may be in response to the rat growth rate and physiologic conditions. Fig. 1 shows the change of body weight during the 8-week test of hep-atoprotective effect of CCTEP. The CCl4-treated control group rats (B, average weight 382.1 ± 53.9 g) had a reduced mean body weight (p < 0.01) as compared with those of normal control group (A, weight 440.8 ± 35.0 g). The weight gain in Group A (240.8 ± 25.8 g) administrated with olive oil and water was the most obvious, but is small in Group B (average 180.0 ± 37.1 g) administrated with CCl4. In the interventional groups, supplement of silymarin or CCTEP with CCl4 did not adequately resume the lost weight caused by CCl 4. In Group C (440.0 ± 12.9 g), Group D (392.1 ± 19.1 9), and Group E (398.3 ± 41.3 g) administrated with different dose of CCTEP and silymarin, the scope of weight gain has restored, but was not statistically significant.

Fig. 1 — Effects of Chhit-Chan-Than extract powder (CCTEP) on body weight after CCU treatment in rats. CCTEP and CCI4 were given as described in "Methods". (A) Normal control group; (B) CCU-treated control group; (C) CCI4- and 100 mg/kg CCTEP-treated group; (D) CCI4- and 200 mg/kg CCTEP-treated group; and (E) CCl4- and 200 mg/kg silymarin-treated group. Data are presented as mean ± standard deviation, n = 8. *p < 0.05 as compared with the normal control group.

Group Ratio of Ratio of spleen Ratio of

liver to BW to BW kidney to BW

A 2.63 ± 0.04 0.20 ± 0.02 0.67 ± 0.02

B 3.79 ± 0.12* 0.25 ± 0.02 0.85 ± 0.09**

C 3.33 ± 0.21** 0.22 ± 0.03 0.68 ± 0.01***

D 3.09 ± 0.11*** 0.21 ± 0.01 0.70 ± 0.03***

E 3.39 ± 0.31** 0.21 ± 0.01 0.74 ± 0.00

The ratios of organ to body weight are expressed as mean ± SE, n = 8.

*p < 0.01 as compared with the control group.

**p < 0.05 as compared with the control group.

***p < 0.05 as compared with the CCl4 only group.

A = control group; B = CCl4-treated group; C = CCl4- and 100 mg/kg

Chhit-Chan-Than extract powder-treated group; D = CCl4- and

200 mg/kg Chhit-Chan-Than extract powder-treated group; E = CCl4-

and 200 mg/kg silymarin-treated group.

After the final experiment, the rats were sacrificed, and the liver, kidney, and spleen were weighed. Table 1 shows that the ratio of organ weight in Group B increased, but has restored in Groups C—E. The ratio of liver weight to body weight in Group B significantly increased as compared with Group A (p < 0.01), whereas that in Groups C and D also slightly increased (p < 0.05). However, in Group D, the ratio of liver weight to body weight restored as compared with Group B (p < 0.05). The ratio of kidney weight to body weight in Group B significantly increased as compared with Group A (p < 0.05), and restored in Groups C and D (p < 0.05). The ratio of spleen weight to body weight did not show any remarkable difference in each group.

3.2. Serum biochemistry examination

Measurements of serum biochemical markers were taken at the end of the experiment. Fig. 2 shows that the levels of tri-glycerine and cholesterol did not alter in all groups at 8 weeks. AST and ALT in the CCl4-treated group increased markedly to 761 ± 149 U/L and 788 ± 148 U/L as compared with the control group at 46 ± 10 U/L and 151 ± 45 U/L, respectively. When cotreated with 100 mg/kg CCTEP, 200 mg/kg CCTEP (Fig. 2), or 200 mg/kg silymarin, the AST levels were restored to 583 ± 128 U/L, 440 ± 50 U/L, and 522 ± 267 U/L, respectively, whereas the corresponding ALT levels were restored to 504 ± 98 U/L, 525 ± 76 U/L, and 500 ± 196 U/L, respectively.

3.3. Anti-oxidative enzyme activity

Effects of CCTEP on hepatic CAT and GSH levels for all experimental groups are shown in Table 2. CCl4 treatment caused a significant decrease of GSH level in liver tissue ho-mogenates compared with the normal group. Pretreatment of 100 mg/kg CCTEP (C) enhanced the level of GSH compared with CCl4-treated group. Treatment with CCTEP (200 mg/kg, D) prior to the CCl4 treatment elevated the GSH level. Similar results were obtained with silymarin (200 mg/kg, E).

The activity of liver CAT in the CCl4 treated rats (Group B) decreased significantly as compared with the control (Group

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

Fig. 2 - The effects of Chhit-Chan-Than extract powder on serum AST, ALP, TG, and cholesterol in rats treated with CCI4 for 8 weeks. Chhit-Chan-Than extract powder and CCI4 were given as described in "Methods". Data are presented as mean ± standard deviation, n = 8. *p < 0.05 as compared with the normal control group. ALT = alanine amino transferase; AST = aspartamine amino transferase; Cho = cholesterol; TG = triglyceride.

A; p < 0.01; Table 2). The activity of liver CAT was restored in rats treated with CCTEP (100 mg/kg, Group C), CCTEP (200 mg/ kg, Group D), and the silymarin (200 mg/kg, Group E).

3.4. Histopathologic study

The general liver morphological changes and fibrosis induced by CCl4 administration were evidenced by both qualitative and quantitative histopathological examination. As compared with the normal rat liver, the tissue section in Figs. 3 and 4 show that the CCl4-induced chronic liver injury was concave on the liver surface and had lymphocyte infiltration in the

Table 2 - Effects of Chhit-Chan-Than extract powder on the liver glutathione levels and catalase activity after CCU-treated in rats.a

Group Glutathione

(nmol/mg protein)

Catalase activity (IU/mg protein)

B C D E

7.7 ± 0.4

5.8 ± 0.4*"

8.7 ± 0.9*" 9.1 ± 0.4*"

8.8 ± 0.9*"

8.2 ± 1.6 4.6 ± 0.1* 5.4 ± 0.3**' 6.1 ± 0.3*** 5.3 0.2**'

The liver GSH value and catalase activity are expressed as mean ± SE, n = 8.

*p < 0.01 as compared with the control group.

**p < 0.05 as compared with the control group.

***p < 0.01 as compared with the CCl4-treated group.

****p < 0.05 as compared with the CCl4-treated group.

A = control group; B = CCl4-treated group; C = CCl4- and 100 mg/kg

Chhit-Chan-Than extract powder-treated group; D = CCl4- and

200 mg/kg Chhit-Chan-Than extract powder-treated group; E = CCl4-

and 200 mg/kg silymarin-treated group.

a Chhit-Chan-Than extract powder and CCl4 are given as described in "Methods" in the main text.

central vein. The hepatic cells were found to be cloudy, swelling, and necrotic, cytoplasmic vacuolization and fatty degeneration were observed in the centrilobular zone and mid zone. Masson trichrome stain clearly demonstrated that the CCl4-intoxicated liver became fibrotic with fiber extension and collagen accumulation (Fig. 5). These pathological changes (such as fibrosis and inflammation) were ameliorated by cotreatment of silymarin or CCTEP, which showed a dose-dependent effect (Figs. 4 and 5). The severity of the liver morphological changes and fibrosis induced by CCl 4 treatment were scored and summarized in Table 3. As shown, four out of eight CCl4-exposed rats had liver morphological change greater than Grade 3+, and five out of eight had liver fibrosis and necrosis greater than Grade 2. The CCTEP exposure greatly improved liver morphological changes, fibrosis, and necrosis. The average severity scores of the CCTEP-treated rats were markedly reduced in a dose-dependent manner.

Discussion

CCl4 has been widely used in animal models to investigate chemical toxin-induced liver damage and as an excellent model to evaluate the efficacy of hepatoprotectants [37-39]. The ability of a hepatoprotective drug to reduce the injurious effects or to preserve the normal hepatic physiological mechanisms that have been disturbed by a hepatotoxin, is the index of its protective effects [40]. The purpose of this research program is to evaluate the hepaprotective effects of CCTEP in CCl4-induced liver damage in Wistar rats. In this study, rats treated with CCl4 developed significant hepatic damage as manifested by a significant increase in plasma activities of AST and ALT, which are indicators of hepatocyte damage and loss of functional integrity. The CCTEP treatment stimulated hepatoprotective effects that were proven by attenuating

6 JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

Fig. 3 - Livers from CCI4- and/or Chhit-Chan-Than extract powder (CCTEP)-treated rats. CCTEP and CCI4 were given as described in "Methods". (A) Normal control group; (B) CCU-treated control group; (C) CCI4- and 100 mg/kg CCTEP-treated group; (D) CCl4- and 200 mg/kg CCTEP-treated group; and (E) CCI4- and 200 mg/kg silymarin-treated group.

serum AST and ALT activities. The antioxidant activities of the CCTEP can ameliorate oxidative stress contributing to the amount of intracellular antioxidant enzymes, which was confirmed by GSH and CAT assay. Centrilobular necrosis, lymphocytes infiltration and steatosis were apparent in CCl4-treated group, whereas administration of the CCTEP significantly reverses these abnormal indexes. This indicates that CCTEP can ameliorate oxidative stress to preserve hepatic function and hepatic injury induced by CCl4.

Free radicals/reactive oxygen species (ROS) and oxidative stress play a central role in liver disease pathology and progression [1,2]. Cellular injury occurs when ROS generation exceeds the cellular capacity of removal [41]. The cleavage of CCl4 leads to the formation of highly unstable free radicals (CCl3» or CCl3O2»), to initiate peroxidation [26]. The oxidant species may in turn trigger the signal transduction pathways

involved in the antioxidant responses. Antioxidant enzymes (CAT, superoxide dismutase, glutathione peroxidase, etc.) represent one protection system against oxidative tissue-damage [42]. CAT is known to break H2O2 down to H2O and O2 and can be found in the peroxisome and mitochondria, especially in liver. Superoxide dismutase removes superoxide radicals by converting them into H2O2, which, in turn, can be rapidly converted into water by catalase and glutathione peroxidase. Thus, the inhibition of free radicals generation retards CCl4-induced lipid peroxidation. In the present study, antioxidant enzymes activities in the liver of CCl4-treated group rats were significantly lower than those in the normal control group. Previous studies have indicated that CCl4 reduces the activities of antioxidant enzymes and causes hep-atopathy [26]. Interestingly, impaired hepatic antioxidant enzymes activities were brought back via the treatment of

Fig. 4 - Effects of Chhit-Chan-Than extract powder (CCTEP) on liver pathologic analysis by hematoxylin-eosin stain after CCU treatment in rats. CCTEP and CCl4 were given as described in "Methods". (A) Normal control group; (B) CCl4-treated control group; (C) CCl4- and 100 mg/kg CCTEP-treated group; (D) CCl4- and 200 mg/kg CCTEP-treated group; and (E) CCl4- and 200 mg/kg silymarin-treated group (original magnification: 40 x).

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

Fig. 5 - Effects of Chhit-Chan-Than extract powder (CCTEP) on liver pathologic analysis by Masson's stain after CCl4 treatment in rats. CCTEP and CCl4 were given as described in "Methods". (A) Normal control group; (B) CCl4-treated control group; (C) CCl4- and 100 mg/kg CCTEP-treated group; (D) CCl4- and 200 mg/kg CCTEP-treated group; and (E) CCl4- and 200 mg/ kg silymarin-treated group.

CCTEP to CCl4-treated rats. Our present works clearly showed an elevation of CAT level in liver, suggesting that it can restore these antioxidant enzymes and/or activate enzyme activity in the damage caused by CCl4.

GSH is believed to be a highly effective extra- and intra-cellular antioxidant compound that neutralizes H2O2 and hydroperoxides by its scavenging and antioxidant properties [43]. In this study, GSH content in the liver of CCl 4-intoxi-cated rats was declined by CCl4 treatment and its subsequent enhancement revealed antioxidant effects of CCTEP. A number of studies have revealed that GSH conjugates play a major role in eliminating the CCl4-induced toxic metabolites that are the main cause of liver injuries. The maintenance of sufficient glutathione level is important for the prevention of CCl4-induced damages. Toxicants that deplete GSH or influence the

activity of GSH-dependent enzyme(s) may result in toxic responses. The study herein presented was instigated by other reports [44] where CCl4 administration caused depletion in GSH contents. The mechanism of hepatoprotection by CCTEP against the CCl4 toxicity might be partially due to the restoration of GSH concentration in liver.

CCl4 is metabolized by cytochrome P450 2E1 to the tri-chloromethyl radical (CCl3»), which is assumed to initiate free radical-mediated lipid peroxidation leading to the accumulation of lipid-derived oxidation products that cause liver injury [45]. Alternatively, oxidative stress generated from cytochrome P450-catalyzed metabolism of xenobiotics can result in protective cellular responses, leading to the enhancement of cell survival upon noxious stimuli [46]. Studies have indicated that some phytochemicals are found to regulate

Table 3 - The effects of Chhit-Chan-Than extract powder extract powder on histopathological evidence of CCl4-induced liver fibrosis rats.a

Group Severity score of fatty metamorphosis (incidence) Severity score of fatty hepatic fibrosis (incidence)

N 0 1 2 3 Mean ± SE 01 2 3 4 Mean ± SE

A 8 8 0 0 0 0.0 ± 0.0 8 0 0 0 0 0.0 ± 0.0

B 8 1 2 1 4 2.0 ± 0.4* 0 1 4 3 0 2.3 ± 0.3*'****

C 8 1 1 3 3 2.0 ± 0.4* 2 4 2 0 0 1.0 ± 0.3*'****

D 8 3 3 2 0 2.0 ± 0.4* 5 2 1 0 0 0.5 ± 0.3***

E 8 2 4 2 0 1.0 ± 0.3* 4 3 1 0 0 0.6 ± 0.3**'***

Data represent the number of rats rated with a given level of fatty metamorphosis or hepatic fibrosis; see "Methods" in the main text for additional details.

*p < 0.01 as compared with the control group. **p < 0.05.

***p < 0.01 as compared with the CCl4 only group by the Student t test.

****p < 0.05.

SE = standard error.

a Chhit-Chan-Than extract powder and CCl4 are given as described in "Methods" in the main text.

8 JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

cytochrome P450 enzymes and demonstrate hepatoprotective effects, especially on the specific protein activity by mRNA expression [47,48]. The present study has found that CCTEP contributes to the amount of catalase and GSH inside the liver after chronic CCl4 treatment (Table 2). Catalase is known to breakdown H2O2, the downstream product of free radical metabolic cascade, and thus protect liver from oxidative stress. In a previous study, we clearly demonstrated in vitro OGE's powerful antioxidant activity with 80% scavenging effect, which is as effective as a-tocopherol, as well as lowing thiobarituric acid-reactive substances (TBARS) in H2O2-treated HepG2 cells [21]. Taken together with the present study, it can therefore be claimed that CCTEP lowers oxidative stress-induced liver injury by increasing antioxidants inside the liver. Induction of GSH levels may be due to the activation of GSH synthesizing enzymes such as g-glutamylcysteine syn-thetase and GSH synthetase, which are key enzymes in the biosynthesis of GSH [49]. These results could indicate that CCTEP either inhibits ROS induced lipid peroxidation, or directly lowers ROS production, possibly through the inhibition of CYP2E1 activity. Some antioxidants, such as Vitamin C and E or garlic acid, are shown to possess similar mechanisms [50,51].

For the therapeutic strategies of liver injury and disease, it is important to find antioxidant compounds that are able to block liver injuries through free radicals generated by toxic chemicals. Therefore, the present study speculated that the aqueous O. gratissimum extract protects against diseases that are caused by oxidative stress because it is rich in polyphenols and has excellent radical scavenging ability. Our preliminary phytochemical screening discovered a significant quantity of simple phenols and flavonoids inside OGE, including catechin, caffeic acid, epicatechin, and rutin [12,16,20,29]. Other researchers have reported that green tea catechin is capable of inhibiting the activation of hepatic stellate cells (HSCs), which leads to liver fibrosis by lowering the secretion of liver fibril-forming collagen [52], rutin is capable of lowering liver oxidative damage [53-55], and caffeic acid is capable of treating liver cirrhosis by suppressing H2O2 formation [56]. Primarily, the hepatoprotective effects of these phytocon-stituents may inhibit lipid peroxidation in the liver, which suggests that it may prevent free radical damage and thus protect the liver from oxidative stress [57].

In the present study, although CCTEP could not completely repair the hepatic injury induced by CCl4, it is possible that the CCl4 dose at the late stage was too high so that the liver damage could not be restored. The trend of protective effects on AST and ALT levels between CCl4-control group (Group B) and interventional groups (Groups C-E) is clearly demonstrated in Fig. 2 and described in the Section 3.2. "Serum biochemistry examination". However, the evident reduction of values cannot reach significance by ANOVA analysis (due to the small experimental animal number and data variance). Thus, the transaminase-restored effects of CCTEP and sily-marin were hard to clarify completely in this study. Fortunately, the diagnostic value of histopathologic morphology in long-term hepatic injury is more meaningful than that of AST or ALT in the acute stage. In a chronic CCl4-treated rat model, progressive scarring of the liver (fibrogenesis) is characterized by an excessive deposition of extracellular matrix (ECM)

proteins (predominantly types I and III collagen) due to the activation of HSCs. Our laboratory also demonstrated that OGE possesses an antiliver fibrosis property by inhibiting in vitro HSCs activation, as well as effectively suppressing the expression of a-smooth muscle actin and type I collagen using an in vivo model (unpublished data).

CCTEP used in this study was developed from aqueous OGE for further commercial products such as oral capsules or beverage power package. The powder was made with 10% OGE and 90% dextrin as an excipient. Several products and phy-toconstituents deriving from O. gratissimum have been investigated by different models and protocols in vitro or in vivo in our laboratory [12,16,20,21,29,58]. The hepatoprotective efficacy of OGE has already been reported by our research group [21,29], as well as the proposal molecular mechanisms [29]. The motive of the present study was to explore the newly-developed product with similar protective property, but less molecular mechanism is discussed. The hepatoprotective effects of O. gratissimum extract were associated not only with antioxidative mechanisms but also with inflammatory pathways, cell death pathways, and activation of HSCs, which are discussed elsewhere. Our previous works [29] demonstrated the beneficial effects of OGE in acute hepatic injury in vivo and found that significantly decreased stress proteins including HSP70 and iNOS were observed. The antioxidant activities of OGE can directly ameliorate oxidative stress by inhibiting lipid peroxidation and contributing to the levels of intracellular antioxidant enzymes, which were confirmed by TBARS assay and antioxidant assay (unpublished data). The cytoprotective property may also be achieved by regulating Bcl-2 expression and Akt phosphorylation, and slightly affecting the phos-phorylation of mitogen-activated protein kinases including p38MAPK and JNK that reduce proinflammatory mechanisms [12,21].

Studies indicate that different doses of drugs may have different physiologic or pharmacologic effects [59], and low doses may be ineffective due to low absorption in the gastrointestinal tract [60]. Previous studies have established that silymarin, the current leading hepatoprotective herbal extract, works best at 0.2 g/kg body weight [6,61]. By contrast, another study has shown that silymarin failed to reverse fibrosis significantly, which was previously established by prolonged chronic CCl4 administration in rats when compared with an appropriate vehicle control [62]. This difference in results may be due to the diverse doses of herbal extract used. Previous studies reported that dried flower Hibiscus sabdariffa L. extract treatment works ideally by being mixed in with a regular diet at 5% of the diet [63], aqueous extract of Anoecto-chilus formosanus has been reported to work at 2.0 g/kg body weight [64], and Ginkgo biloba extract has shown promising results at 0.5 g/kg body weight [65]. In the present study, the suppression of CCTEP at 200 mg/kg body weight was similar to that of silymarin at the same dose. This demonstrates the potency of CCTEP and suggests that O. gratissimum may have the potential to be an herb choice in the treatment of chronic liver disease.

In conclusion, the present study showed that CCTEP (10% aqueous O. gratissimum L. extract) has a remarkable protective effect against CCl4-induced liver damage. The micronutrient functions of O. gratissimum may be as an effecter of important

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

cellular stress response pathways that ultimately influence endogenous cellular antioxidant levels. The inhibitory effects of CCTEP may have therapeutic achievements in chemical-induced hepatic injury in vivo, and thus dietary CCTEP may be useful as a hepatoprotective agent against chronic liver disease in humans.

Acknowledgments

We are grateful for the support of the National Science Council, Taiwan, R.O.C. (NSC 98-2320-B-039-042-MY3 and NSC 99-2632-B-039 -001-MY3) and in part by the Taiwan Department of Health Cancer Research Center of Excellence (D0H100-TD-C-111-005), Taiwan, R.O.C.

REFERENCES

[1] Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free Radic Biol Med 2003;34:1—10.

[2] Vitaglione P, Morisco F, Caporaso N, et al. Dietary antioxidant compounds and liver health. Crit Rev Food Sci Nutr 2004;44:575—86.

[3] Lotersztajn S, Julien B, Teixeira-Clerc F, et al. Hepatic fibrosis: molecular mechanisms and drug targets. Annu Rev Pharmacol Toxicol 2005;45:605—28.

[4] Girish C, Pradhan SC. Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin. Fundam Clin Pharmacol 2008;22:623—32.

[5] Li CC, Hsiang CY, Wu SL, et al. Identification of novel mechanisms of silymarin on the carbon tetrachloride-induced liver fibrosis in mice by nuclear factor-kappaB bioluminescent imaging-guided transcriptomic analysis. Food Chem Toxicol 2012;50:1568—75.

[6] Tsai JH, Liu JY, Wu TT, et al. Effects of silymarin on the resolution of liver fibrosis induced by carbon tetrachloride in rats. J Viral Hepat 2008;15:508—14.

[7] De Mattia F, Bruni I, Galimberti A, et al. A comparative study of different DNA barcoding markers for the identification of some members of Lamiacaea. Food Res Int 2011;44:693—702.

[8] Mahapatra SK, Chakraborty SP, Das S, et al. Methanol extract of Ocimum gratissimum protects murine peritoneal macrophages from nicotine toxicity by decreasing free radical generation, lipid and protein damage and enhances antioxidant protection. Oxid Med Cell Longev 2009;2:222—30.

[9] Lin JK, Chang CH. The pharmacognostical studies on Taiwan folk medicine "Chhit-Chan-Than". Chia-Nan Annual Bulletin 1992;18:28—43.

[10] Lin CC, Lin JK, Chang CH. Evaluation of hepatoprotective effects of "Chhit-Chan-Than" from Taiwan. Int J Pharmacogn 1995;33:139—43.

[11] Ouyang X, Wei L, Pan Y, et al. Antioxidant properties and chemical constituents of ethanolic extract and its fractions of Ocimum gratissimum. Med Chem Res 2013;22:1124—30.

[12] Lee MJ, Chen HM, Tzang BS, et al. Ocimum gratissimum aqueous extract protects H9c2 myocardiac cells from H2O2-induced cell apoptosis through Akt signalling. Evid Based Complement Alternat Med 2011;2011:578060.

[13] Aziba PI, Bass D, Elegbe Y. Pharmacological investigation of Ocimum gratissimum in rodents. Phytother Res 1999;13:427—9.

[14] Ilori M, Sheteolu AO, Omonigbehin EA, et al. Antidiarrhoeal activities of Ocimum gratissimum (Lamiaceae). J Diarrhoeal Dis Res 1996;14:283—5.

[15] Nangia-Makker P, Tait L, Shekhar MP, et al. Inhibition of breast tumor growth and angiogenesis by a medicinal herb: Ocimum gratissimum. Int J Cancer 2007;121:884-94.

[16] Chen HM, Lee MJ, Kuo CY, et al. Ocimum gratissimum aqueous extract induces apoptotic signalling in lung adenocarcinoma cell A549. Evid Based Complement Alternat Med 2011;2011:739093.

[17] Ayisi NK, Nyadedzor C. Comparative in vitro effects of AZT and extracts of Ocimum gratissimum, Ficus polita, Clausena anisata, Alchornea cordifolia, and Elaeophorbia drupifera against HIV-1 and HIV-2 infections. Antiviral Res 2003;58:25-33.

[18] Aguiyi JC, Obi CI, Gang SS, et al. Hypoglycaemic activity of Ocimum gratissimum in rats. Fitoterapia 2000;71:444-6.

[19] Atal CK, Sharma ML, Kaul A, et al. Immunomodulating agents of plant origin. I: preliminary screening. J Ethnopharmacol 1986;18:133-41.

[20] Li PC, Chiu YW, Lin YM, et al. Herbal supplement ameliorates cardiac hypertrophy in rats with CCl4-induced liver cirrhosis. Evid Based Complement Alternat Med 2012;2012:139045.

[21] Chiu YW, Lo HJ, Huang HY, et al. Antioxidant and cytoprotective activities of Ocimum gratissimum extracts against hydrogen peroxide induced toxicity in human HepG2 Cells. J Food Drug Anal 2013;21:253-60.

[22] Chaturvedi R, George S, John A. Preventive and protective effects of wild basil in ethanol-induced liver toxicity in rats. Br J Biomed Sci 2007;64:10-2.

[23] George S, Chaturvedi P. A comparative study of the antioxidant properties of two different species of Ocimum of southern Africa on alcohol-induced oxidative stress. J Med Food 2009;12:1154-8.

[24] Matkowski A. Plant in vitro culture for the production of antioxidants - a review. Biotechnol Adv 2008;26:548-60.

[25] Rechnagel RO, Glende Jr EA. Carbon tetrachloride hepatotoxicity: an example of lethal cleavage. CRC Crit Rev Toxicol 1973;2:263-97.

[26] Recknagel RO, Glende Jr EA, Dolak JA, et al. Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 1989;43:139-54.

[27] Alqasoumi S. Carbon tetrachloride-induced hepatotoxicity: protective effect of 'Rocket' Eruca sativa L. in rats. Am J Chin Med 2010;38:75-88.

[28] Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver injury. Gut 2005;54:1024-33.

[29] Chiu CC, Huang CY, Chen TY, et al. Beneficial effects of Ocimum gratissimum aqueous extract on rats with CCl4-induced acute liver injury. Evid Based Complement Alternat Med 2012;2012:736752.

[30] Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 1965;16:144-58.

[31] Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat Protoc 2007;2:875-7.

[32] Lamaison JL, Carnat A, Petitjean-Freytet C. Tannin content and inhibiting activity of elastase in Rosaceae. Ann Pharm Fr 1990;48:335-40 [Article in French].

[33] Hernandez-Murioz R, Diaz-Murioz M, Su^rez-Cuenca JA, et al. Adenosine reverses a preestablished CCl4-induced micronodular cirrhosis through enhancing collagenolytic activity and stimulating hepatocyte cell proliferation in rats. Hepatology 2001;34:677-87.

[34] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.

[35] Hsiao G, Lin YH, Lin CH, et al. The protective effects of PMC against chronic carbon tetrachloride-induced hepatotoxicity in vivo. Biol Pharm Bull 2001;24:1271-6.

JOURNAL OF FOOD AND DRUG ANALYSIS XXX (2013) 1—10

[36] Kawamura N, Taniguchi N, Gutteridge JMC. Experimental protocols for reactive oxygen and nitrogen species. , New York: Oxford University Press Inc; 2000.

[37] Lee KJ, Choi JH, Jeong HG. Hepatoprotective and antioxidant effects of the coffee diterpenes kahweol and cafestol on carbon tetrachloride-induced liver damage in mice. Food Chem Toxicol 2007;45:2118-25.

[38] Rudnicki M, Silveira MM, Pereira TV, et al. Protective effects of Passiflora alata extract pretreatment on carbon tetrachloride induced oxidative damage in rats. Food Chem Toxicol 2007;45:656-61.

[39] Desai SN, Patel DK, Devkar RV, et al. Hepatoprotective potential of polyphenol rich extract of Murraya koenigii L.: an in vivo study. Food Chem Toxicol 2012;50:310-4.

[40] Yadav NP, Dixit VK. Hepatoprotective activity of leaves of Kalanchoe pinnata Pers. J Ethnopharmacol 2003;86:197-202.

[41] Tang NY, Liu CH, Su SY, et al. Uncaria rhynchophylla (miq) Jack plays a role in neuronal protection in kainic acid-treated rats. Am J Chin Med 2010;38:251-63.

[42] Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 1990;186:1-85.

[43] Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol 2005;45:51-88.

[44] Khan MR, Ahmed D. Protective effects of Digera muricata (L.) Mart. on testis against oxidative stress of carbon tetrachloride in rat. Food Chem Toxicol 2009;47:1393-9.

[45] Poli G, Albano E, Dianzani MU. The role of lipid peroxidation in liver damage. Chem Phys Lipids 1987;45:117-42.

[46] Owuor ED, Kong AN. Antioxidants and oxidants regulated signal transduction pathways. Biochem Pharmacol 2002;64:765-70.

[47] Chiu PY, Leung HY, Poon MK, et al. Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver. Mol Cell Biochem 2006;293:87-92.

[48] Bhadauria M, Nirala SK, Shukla S. Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride. Mol Cell Biochem 2007;302:215-24.

[49] Griffith OW. Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. J Biol Chem 1982;257:13704-12.

[50] Sheweita SA, Abd El-Gabar M, Bastawy M. Carbon tetrachloride-induced changes in the activity of phase II drug-metabolizing enzyme in the liver of male rats: role of antioxidants. Toxicology 2001;165:217-24.

[51] Sheweita SA, El-Gabar MA, Bastawy M. Carbon tetrachloride changes the activity of cytochrome P450 system in the liver of male rats: role of antioxidants. Toxicology 2001;169:83-92.

[52] Nakamuta M, Higashi N, Kohjima M, et al. Epigallocatechin-3-gallate, a polyphenol component of green tea, suppresses both collagen production and collagenase activity in hepatic stellate cells. Int J Mol Med 2005;16:677—81.

[53] Funabiki R, Takeshita K, Miura Y, et al. Dietary supplement of G-rutin reduces oxidative damage in the rodent model. J Agric Food Chem 1999;47:1078—82.

[54] Afanas'ev IB, Ostrachovich EA, Korkina LG. Effect of rutin and its copper complex on superoxide formation and lipid peroxidation in rat liver microsomes. FEBS Lett 1998;425:256—8.

[55] Paduraru I, Saramet A, Danila G, et al. Antioxidant action of a new flavonic derivative in acute carbon tetrachloride intoxication. Eur J Drug Metab Pharmacokinet 1996;21:1—6.

[56] Hatano T, Miyatake H, Natsume M, et al. Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects. Phytochemistry 2002;59:749—58.

[57] Wu J, Zern MA. Hepatic stellate cells: a target for the treatment of liver fibrosis. J Gastroenterol 2000;35:665—72.

[58] Ye JC, Hsiao MW, Hsieh CH, et al. Analysis of caffeic acid extraction from Ocimum gratissimum Linn. by high performance liquid chromatography and its effects on a cervical cancer cell line. Taiwan J Obstet Gynecol 2010;49:266—71.

[59] Dehmlow C, Murawski N, de Groot H. Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cells. Life Sci 1996;58:1591—600.

[60] Pepping J. Milk thistle: Silybum marianum. Am J Health Syst Pharm 1999;56:1195—7.

[61] Zhao J, Agarwal R. Tissue distribution of silibinin, the major active constituent of silymarin, in mice and its association with enhancement of phase II enzymes: implications in cancer chemoprevention. Carcinogenesis 1999;20:2101—8.

[62] Muriel P, Moreno MG, Hernandez Mdel C, et al. Resolution of liver fibrosis in chronic CCl4 administration in the rat after discontinuation of treatment: effect of silymarin, silibinin, colchicine and trimethylcolchicinic acid. Basic Clin Pharmacol Toxicol 2005;96:375—80.

[63] Liu JY, Chen CC, Wang WH, et al. The protective effects of Hibiscus sabdariffa extract on CCl4-induced liver fibrosis in rats. Food Chem Toxicol 2006;44:336—43.

[64] Shih CC, Wu YW, Lin WC. Aqueous extract of Anoectochilus formosanus attenuate hepatic fibrosis induced by carbon tetrachloride in rats. Phytomedicine 2005;12:453—60.

[65] Liu SQ, Yu JP, Chen HL, et al. Therapeutic effects and molecular mechanisms of Ginkgo biloba extract on liver fibrosis in rats. Am J Chin Med 2006;34:99—114.