ETM Experimental and Therapeutic Medicine 1792-0981 1792-1015 D.A. Spandidos 10.3892/etm.2013.1189 etm-06-03-0796 Articles Synthesis and hepatoprotective properties of Acanthus ilicifolius alkaloid A and its derivatives LIULIN1 FANHUI2 QIPING1 MEIYAN3 ZHOULIJUAN1 CAILIPING1 LINXING1 LINJUN1 Guangxi Medical University, Nanning, Guangxi 530021, P.R. China Shanghai Jiaotong University, Shanghai 200240, P.R. China The First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China Correspondence to: Professor Jun Lin, Pharmacy Department, Guangxi Medical University, 22 Shuangyong Road, Nanning, Guangxi 530021, P.R. China, E-mail: junlin989@sina.com 9 2013 28 06 2013 6 3 796 802 01 03 2013 13 06 2013 Copyright © 2013, Spandidos Publications 2013 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Acanthus ilicifolius alkaloid A (4-hydroxy-2(3H)benzoxazolone, HBOA) is a naturally occurring compound that has been separated from Acanthus ilicifolius. Previous studies have reported the beneficial effects of HBOA on HSC-T6 cells. This study was undertaken in order to synthesize HBOA and two of its derivatives, specifically, 4-acetoxy-2(3H)-benzoxazolone (AcO-BOA) and 3-acetyl-4-acetoxy-2-benzoxazolone (TC-3), and to investigate the hepatoprotective potentials of these three compounds on CCl4-induced liver injury in mice. HBOA was prepared from 2-nitroresorcinol by a 'one pot' reduction and subsequent cyclization with urea. The acyl derivatives, AcO-BOA and TC-3, were prepared from HBOA using a substitution reaction. The compounds were synthesized with good yields (63.08–68.22%). An acute liver injury model was established by administering CCl4 intraperitoneally to Kunming mice. The mice were then intragastrically administered bifendate (150 mg/kg) or the synthesized compounds at three different doses (200, 100 and 50 mg/kg). The treatment with CCl4 was observed to increase the levels of aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH) and malondialdehyde (MDA) and decrease the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (Gpx) in the liver tissues of the mice. Furthermore, treatment with CCl4 elevated the expression level of the proinflammatory mediator TNF-α. However, HBOA and its derivatives attenuated the changes induced by CCl4. Furthermore, CCl4-induced histopathological changes were reduced by treatment with these compounds. These results suggest that HBOA and its acyl derivatives are able to significantly alleviate the hepatotoxicity induced by CCl4 in mice.

 Acauthus ilicifolius alkaloid A synthesis hepatoprotective potential acute liver injury Introduction

The liver is a vital organ in mammals, including rodents. Hepatotoxic agents may cause severe damage to hepatocytes; not only do they potentially catabolize radical-induced lipid peroxidation, damage the membranes of liver cells and organelles and cause swelling and necrosis of hepatocytes, but they may also induce an inflammatory response and release mediators of inflammation (1). CCl4 is used as a model agent for inducing acute liver injury in mice. CCl4-induced liver injury is associated with the aggravation of lipid peroxidation and the attenuation of antioxidant enzyme function (2). These changes result from the formation of reactive intermediates such as trichloromethyl (•CCl3) free radicals and reactive oxygen species (ROS) (3). Furthermore, CCl4-induced liver injury has been reported to cause significant increases in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH) and malondialdehyde (MDA) and reductions in the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (Gpx) (4). Therefore, reducing lipid peroxidation or oxidative stress may be an effective therapeutic strategy for preventing and treating CCl4-induced liver injury.

Acanthus ilicifolius L. (Acanthaceae) has spinous edges to its evergreen leaves and stipular spines at the stem nodes. It is a plant found in marshy habitats throughout the mangroves of China, including those of the Guangxi region and Guangdong and Fujian provinces, and in various Asian countries such as India, Burma and Thailand. In traditional Chinese medicine, A. ilicifolius is used to treat inflammation, hepatitis, swollen spleens, asthma, gastralgia and malignant tumors. Previous studies have reported that the crude alcoholic extract of A. ilicifolius exhibits antioxidant, hepatoprotective (5), antitumor (6) and antimicrobial activities (7).

Acanthus ilicifolius is protected in China. Acanthus ilicifolius alkaloid A (4-hydroxy-2(3H)-benzoxazolone, HBOA) has been separated from the Acanthus ilicifolius plant in the pharmaceutical chemistry laboratory of Guangxi Medical University. Moreover, in order to avoid the exploitation of the mangroves, we attempted to synthesize HBOA and successfully synthesized the core heterocyclic nucleus, 2(3H)-benzoxazolone (BOA) of HBOA. Benzoxazolone heterocycles are considered to be ‘privileged scaffolds’ in the design of pharmacological probes. Medicinal chemists have paid considerable attention to these units due to their capacity for mimicking phenol or catechol moieties in a metabolically stable template (8). Furthermore, 3-substituted BOA derivatives with more potent anti-inflammatory activity than aspirin have been reported (9). Therefore, HBOA, as a lead compound, may be modified to synthesize novel drugs with pharmacological activities.

There are a number of methods that may be used for the synthesis of BOA, including reactions between 2-aminophenol and urea phosgene or carbon monoxide; the reaction with urea is normally used in industry (10). A method of synthesizing HBOA from 2-aminoresorcinol hydrochloride and carbonyldiimidazole (CDI) by cyclization in dichloromethane, and a method for synthesizing 4-acetoxy-2(3H)-benzoxazolone (AcO-BOA) have been reported (11). The reaction scheme for HBOA is presented in Fig. 1A. Pharmacological studies of HBOA and its derivatives have been reported, and describe its use as an anti-inflammatory agent (12), analgesic (13,14), antioxidant, anti-convulsant and as a Type 2 diabetes agent (15–17). Our previous in vitro study demonstrated that HBOA and its derivatives possess hepatoprotective activity against the induction of apoptosis in hepatic stellate cells (HSC-T6), and the two acyl derivatives were shown to be more potent than HBOA (18). These findings prompted us to conduct the current study to investigate the hepatoprotective activity of HBOA and its acetylated derivatives in mice.

 Materials and methods Materials

Melting points (M.p.) were determined using an electrothermal melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and are uncorrected. The IR spectra of the compounds were recorded using a Spectrum One FT-IR spectrometer (Perkin-Elmer, Waltham, MA, USA). NMR (1H and 13C) spectra were recorded on a Bruker AV-500 MHz NMR spectrometer (Bruker BioSpin, Shanghai, China), and chemical shifts are expressed in parts per million (ppm, for δ) relative to tetramethylsilane (TMS) as an internal standard with CDCl3 as the solvent. Spin multiplets are provided as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). All compounds reported in the present study had IR, 1H and 13C-NMR data consistent with their structures. TLC analyses were performed on TLC plates (silica gel, GF254; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China).

Bifendate (Bif) was purchased from Guangzhou Xingqun Pharmaceutical Co., Ltd. (Guangzhou, China); ALT, AST, LDH, MDA, SOD, CAT, GSH and Gpx testing kits were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The tumor necrosis factor-α (TNF-α) kit was obtained from Wuhan Boster Bio-engineering Co., Ltd (Wuhan, China).

 Methods Synthesis of 2-nitroresorcinol

The intermediate product, 2-nitroresorcinol was synthesized by the reaction of resorcinol with a mixed acid consisting of concentrated sulfuric acid and nitric acid according to a method reported previously (19) (yield, 30.12%; M.p., 83–84°C).

 Synthesis of HBOA

Activated carbon (18 g) was added to a solution of 8 g hexahydrated ferric chloride dissolved in 480 ml methanol and refluxed for ~30 min. To this mixture, 62 g 2-nitroresorcinol was added and the mixture was refluxed with stirring. Subsequently, 100 ml (75%) hydrazine hydrate was added dropwise to the mixture which was then refluxed for 4–5 h. The mixture was filtered to remove the activated carbon, and the solvent was evaporated under reduced pressure. Upon obtaining a homogenous paste, 620 ml ethyl acetate was added and the mixture was heated to 90°C. To the stirred mixture, 144 g urea was added and refluxing was conducted for 4–5 h. Following the removal of solvent under reduced pressure, 500 ml water was added and the mixture was refluxed twice for 30 min each. A crude product was obtained through filtration and subsequent drying. The product was extracted with n-butyl alcohol (3 × 800 ml). The extract was evaporated to dryness under reduced pressure and the resulting light brown crystals were filtered and recrystallized from ethanol (C7H5NO3; yield, 63.08%; M.p., 293-29°C). IR (KBr, cm−1), 3,289 (-OH, phenolic hydroxy), 1,741 (C=O, lactam) and 1,659 (C=O, amide); 1H-NMR (500 MHz; CDCl3) δ (ppm), 6.76 (1H, dd, H5), 6.74 (1H, dd, H7) and 6.94 (1H, m, H6).

 Synthesis of AcO-BOA (TC-2)

To a stirred and cooled solution of 10.7 g HBOA in 750 ml acetone, at <5°C, was added 11.9 g triethylamine. Subsequently, a solution of 6.9 g acetyl chloride in 20 ml of acetone was added dropwise and the mixture was stirred for 5 h; the temperature did not rise above 5°C. Stirring proceeded for a further 5 h at room temperature and the mixture was left for a further 24 h. Following evaporation to dryness, the white precipitate of AcO-BOA was collected by filtration, washed with water, dried and recrystallized from water (C9H7NO4; yield, 68.22%; M.p., 191–193°C). IR (KBr, cm−1), 3,127 (-OH, phenolic hydroxy), 1,761 (C=O, lactam) and 1,639 (C=O, amide); 1H-NMR (500 MHz; CDCl3) δ (ppm), 7.15 (1H, dd, H5), 6.99 (1H, dd, H7), 7.17 (1H, m, H6) and 2.07 (3H, s, -CH3).

 Synthesis of TC-3

To a solution of 11.0 g AcO-BOA in 750 ml acetone, which was stirred and cooled at <5°C, was added 11.9 g triethylamine. Then, 11.36 g acetyl chloride in 20 ml acetone was added dropwise. The reaction mixture was stirred for 2 h at <5°C. After stirring at room temperature for another day, the solvent was removed under reduced pressure and the residue was obtained in water and extracted with methenyl trichloride. Trichloromethane was recovered and white crystals of TC-3 were collected by filtration and recrystallized from ethanol (C11H9NO5; yield, 64.62%; M.p., 91–93°C). IR (KBr, cm−1), 1,787 (C=O, lactam) and 1,602 (C=O, amide); 1H-NMR (500 MHz; CDCl3) δ (ppm), 7.12 (1H, dd, H5), 6.96 (1H, dd, H7), 7.25 (1H, m, H6), 2.71 (3H, s, phenyl-OCOCH3) and 2.32 (3H, s, -NCOCH3).

 Determination of compound purity

A Shimadzu HPLC system (Shimadzu, Tokyo, Japan) was utilized for the experiments and consisted of the following components: a 3500 pump, AS 3000 auto sampler, and PDAD 1000 detector. A welchrom-C18, 250×4.6 mm, 5 μm analytical column (Welch Materials, Inc., Potomac, MD, USA) was used. The instrumental settings were: flow rate, 1 ml/min; column oven temperature, 30°C; detector wavelength, 215 nm, 215 nm and 210 nm, respectively; and injection volume, 10 μl. Data acquisition was made using LCsolution version 1.2 (Shimadzu). Peak purities were checked using a photodiode array detector. The mobile phase consisted of methanol and water (50:50, v/v). The mobile phases were filtered through a 0.45-μm nylon filter and degassed. The three compounds were dissolved in the mobile phase.

 Animals and treatment

Local Kunming mice of both genders, ranging from 18–22 g in weight, were provided by the Experimental Animal Center of Guangxi Medical University (Guangxi, China). This study was conducted in accordance with protocols approved by the Institutional Ethics Committee of Guangxi Medical University.

The mice were divided randomly into various groups containing ten mice in each group: the normal control group, the sodium carboxymethyl cellulose (CMC-Na) group, the CCl4 model group, the Bif group and the HBOA, AcO-BOA and TC-3 (high dose, middle dose and low dose) groups. The acute liver injury model was established through the intraperitoneal injection of 0.1 ml/10 g body weight of 0.15% (v/v) CCl4 solution in peanut oil to the mice, with the exception of the control and CMC-Na groups. The synthesized compounds were administered orally (at 200, 100 and 50 mg/kg) in 6% CMC solution and the Bif group mice were treated with Bif (150 mg/kg) 8 h later. The mice in the control group received the same volume of normal saline solution, and the CMC-Na and model groups received the same volume of 6% CMC. An additional dose of the treatment was administered after a further 16 h. At 8 h after the last treatment the mice were sacrificed by cervical dislocation. Serum samples were obtained from the eyeballs of the mice and stored at −20°C. The livers were removed from the mice and weighed. Liver tissue (0.2 mg) was accurately weighed, homogenized and centrifuged (1,575 × g for 10 min). The supernatant was separated and stored at −20°C. The remaining liver samples were fixed in 10% formaldehyde for histological analysis.

 Liver function and biochemical assays

The liver index was calculated for each group using the following formula: Liver index = [mouse liver weight (g)/mouse weight (g)] × 100. The serum levels of ALT and AST were determined using commercially available kits according to the manufacturer's instructions. Levels of LDH in the serum and levels of MDA, SOD, CAT, GSH and Gpx in the liver tissue were measured in accordance with the kit manufacturer's instructions. The inhibition rate of MDA was calculated as follows: (level in the model group - level in the treatment group)/level in the model group × 100. The induction rates of SOD and GSH were calculated as follows: (level in the treatment group - level in the model group)/level in the model group × 100.

 Histopathology and immunohistochemistry

Liver tissue sections fixed in 10% formaldehyde were embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E). The TNF-α levels in the liver tissues were measured using an immunohistochemical assay. The degree of liver damage was examined blindly using an Olympus CX41 biological microscope (Tokyo, Japan).

 Statistical analysis

Statistical analysis was performed using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). One way analysis of variance (ANOVA) was used to compare the means among the groups. Data are presented as the means ± SE. P<0.05 was considered to indicate a statistically significant difference.

 Results Chemistry

HBOA and its derivatives were synthesized and the purities of the products were determined. The chemical structures of the products are presented in Fig. 1B. The purities of HBOA, AcO-BOA and TC-3 were 99.26, 94.42 and 93.73%, respectively.

 Pharmacology Liver index

The liver index in the model group was significantly higher compared with that of the normal control group. However, the liver indices in the high-dose HBOA and AcO-BOA and mid- and high-dose TC-3 groups were markedly reduced compared with that of the model group (Table I).

 Effect of HBOA

The levels of serological indicators (AST, ALT, MDA) were significantly higher in the model group compared with the normal control group. Compared with the values in the model group, high-dose HBOA induced significant reductions in the activities of ALT and AST and increases in the activities of SOD, Gpx and CAT, and mid- and high-dose HBOA induced significant reductions in the levels of MDA and LDH and increases in the activity of GSH (Fig. 2Aa; Table II). The expression of TNF-α was significantly reduced by high-dose HBOA (Fig. 3).

 Effect of AcO-BOA

Acute liver injury induced by CCl4 provoked significant reductions in the activities of liver SOD and GSH, and increased the liver MDA and serum ALT and AST levels. The results demonstrated that the activities of liver SOD and GSH were increased by treatment with high-dose AcO-BOA, whereas the liver MDA and serum ALT and AST levels were markedly reduced (Fig. 2Ab; Table III).

 Effect of TC-3

CCl4 intoxication reduced the activities of SOD and GSH and raised the levels of ALT, AST and MDA. Conversely, in mice treated with TC-3, the levels of AST and MDA were reduced and SOD and GSH levels were elevated (Fig. 2Ac; Table IV).

 Inhibition rate of MDA and induction rate of SOD and GSH

A high-dose of each of the three compounds demonstrated the highest MDA inhibition rates and the highest SOD and GSH induction rates (Fig. 2B).

 Histopathological observation

The degree of liver injury observed in mice was evaluated by H&E staining, which demonstrated that the livers from the normal control and CMC-Na control mice exhibited a normal liver lobular architecture with a central vein and radiating hepatic cords (Fig. 4A and B). In contrast to normal mouse liver morphology, CCl4-induced injury was evidenced by disruption of the liver lobular architecture, liver cell edema, fatty degeneration of the liver, inflammation and granuloma (Fig. 4C). These alterations were reduced in the liver sections of mice that received the three compounds in high and medium doses (Fig. 4E–G). Concurrent administration of bifendate attenuated the CCl4-induced changes in the liver architecture (Fig. 4D).

 Discussion

Regarding the chemical structure of HBOA, it contains hydroxyl and imine bases that may act as either hydrogen bond donors or acceptors and play a role in the action of the drug and in the process of receptor binding. Calış et al observed that the substitution of the benzene ring of benzoxazolinone derivatives by an acyl moiety resulted in enhanced anti-inflammatory and analgesic activities (20). Therefore, we hypothesize that 3-substituted or 3,4-disubstituted derivatives of HBOA may possess a greater protective effect on the liver than HBOA.

Products were carbonized by concentrated sulfuric acid. In order to avoid carbonization and maintain the concentration of sulfuric acid, we added distilled water or filtrate when distilling. We obtained 2-nitroresorcinol by drip type water vapor distillation in order to avoid carbonising the product. HBOA was obtained from 2-nitroresorcinol by a ‘one-pot’ reduction reaction and subsequent cyclization with urea. In this reaction, hexahydrated ferric chloride-activated carbon was used as a catalytic agent and hydrazine hydrate was used as a reducing agent. We have previously compared the applicability of reduction with hydrazine hydrate with that of stannous chloride dissolved in hydrochloric acid; reduction by hydrazine hydrate was observed to be simpler and more convenient (21). However, hydrazine hydrate must be evaporated to dryness to prevent it from reacting with urea. AcO-BOA and TC-3 were subsequently obtained by the reaction of HBOA with acetyl chloride, an acylating agent, in a basic triethylamine solution.

HPLC was used to determine the purity of the compounds. The method provides excellent separation of target compounds from unknown impurities with a resolution of >1.5 and a tailing factor range of 0.95–1.5. We observed that AcO-BOA and TC-3 are readily hydrolyzed in aqueous solution. Therefore, we employed a solution of CMC as required in order to avoid hydrolysis.

CCl4-induced liver injury is dependent upon reductive dehalogenation catalyzed by cytochrome p450 in the endoplasmic reticulum of hepatic cells leading to the generation of an unstable complex trichloromethyl radical (•CCl3). The superoxide anion O2−, H2O2 and the hydroxyl radical (•OH) are reactive oxygen species (ROS) produced in mitochondria (4). Lipid peroxidation is an important consequence of the metabolism of CCl4(22). These oxygen radicals contribute to the process of lipid peroxidation. However, cells have various mechanisms for protecting themselves from the toxic effects of ROS, including free radical scavengers and chain reaction terminators such as SOD, CAT, GSH and Gpx systems (23). Liver injury via the ROS pathway causes increases in the levels of ALT, AST, MDA and LDH, the syntheses of which are increased due to the extensive damage of the liver cells (4,24).

The current study demonstrates that HBOA and its derivatives exhibit protective effects against acute liver injury, confirmed by the serum ALT and AST levels and H&E staining. Our investigation revealed that HBOA and its derivatives exhibit potent hepatoprotective effects against CCl4-induced liver damage in mice. This may be the result of increasing the activity of the antioxidant-defense system and the inhibition of lipid peroxidation.

Many hepatoprotective drugs have been of interest due to their antioxidant activity (25). In the current study, the induction of acute liver injury by CCl4 increased the levels of MDA in liver tissue. MDA is a product of lipid peroxidation; elevated levels of MDA may reflect the degree of lipid peroxidation injury in liver cells (2). High doses of HBOA and its derivatives are able to reduce the levels of hepatic MDA. Compared with the CCl4 model group, treatment with HBOA increased the activity or level of SOD, CAT, Gpx and GSH, which scavenge free radicals and simultaneously reduce lipid peroxidation, thus alleviating the oxidative damage caused by CCl4. The results suggest that HBOA protects against the damage caused by the oxidation of hepatic cellular membranes by free radical scavenging. Treatment of the mice with the two derivatives of HBOA attenuated the hepatic SOD and GSH depletion induced by the intraperitoneal administration of CCl4. Therefore, we hypothesize that the protective actions of the two derivatives are related to their ability to scavenge free radicals.

TNF-α activates various intracellular pathways which regulate inflammation, cell death and proliferation. In the liver, TNF-α not only mediates hepatotoxicity but also contributes to the restoration of functional liver mass by promoting hepatocyte proliferation and liver regeneration (26). TNF-α expression was observed to be lower in the group treated with HBOA than in the model group, which demonstrates that the hepatoprotective effect of HBOA involved an anti-inflammatory mechanism and inhibiting the activity of secreted TNF-α on Kupffer cells (KCs).

In conclusion, these preliminary studies indicate that HBOA and its two acyl derivatives elicit a protective effect on CCl4-induced liver injury in vivo. This study supports the traditional use of Acanthus ilicifolius as an herbal remedy for various liver diseases. However, further studies are required in order to evaluate the mechanism of action these compounds.

 Acknowledgements

The authors acknowledge financial support from the Science and Technology Research Development of Guangxi Province, Guangxi Natural Science Foundation (2009AM4019) and the Department of Education, Guangxi Province.

 Abbreviations CCl4

carbon tetrachloride

 ALT

alanine aminotransferase

 AST

aspartate aminotransferase

 LDH

lactic dehydrogenase

 MDA

malondialdehyde

 SOD

superoxide dismutase

 CAT

catalase

 GSH

glutathione

 Gpx

glutathione peroxidase

 References SpitellerGAre lipid peroxidation processes induced by changes in the cell wall structure and how are these processes connected with diseases?Med Hypotheses6069832003 AminAMahmoud-GhoneimDZizyphus spina-christi protects against carbon tetrachloride-induced liver fibrosis in ratsFood Chem Toxicol47211121192009 LinHMTsengHCWangCJLinJJLoCWChouFPHepatoprotective effects of Solanum nigrum Linn extract against CCl4-induced oxidative damage in ratsChem Biol Interact1712832932008 WangCYMaFLLiuJTTianJWFuFHProtective effect of salvianic acid A on acute liver injury induced by carbon tetrachloride in ratsBiol Pharm Bull3044472007 BabuBHShyleshBSPadikkalaJAntioxidant and hepatoprotective effect of Acanthus ilicifoliusFitoterapia722722772001 BabuBHShyleshBSPadikkalaJTumour reducing and anticarcinogenic activity of Acanthus ilicifolius in miceJ Ethnopharmacol7927332002 GaneshSVennilaJJScreening for antimicrobial activity in Acanthus ilicifoliusArch Appl Sci Res23113152010 PoupaertJCaratoPColacinoEYousS2(3H)-Benzoxazolone and bioisosters as ‘privileged scaffold’ in the design of pharmacological probesCurr Med Chem128778852005 GulcanHOKupeliEUnluSYesiladaESahinMF4-(5-chloro-2(3H)-benzoxazolon-3-yl) butanoic acid derivatives: synthesis, antinociceptive and anti-inflammatory propertiesArch Pharm (Weinheim)3364774822003 TangLBanSRFengXELinWHLiQSSynthesis and activities of new 4-hydroxy benzoxazolone derivativesChin Chem Lett2163662010 GülcanHOÜnlüSBanoğluEŞahinMFKüpeliEYeşiladaESynthesis of new 4-(5-chloro-2-oxo-3Hbenzoxazol-3-yl) butanamide derivatives and their analgesic and anti-inflammatory propertiesTurk J Chem274674762003 CharlierCMichauxCDual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-inflammatory drugsEur J Med Chem386456592003 ÖnkolTSahinMFYildirimEErolKItoSSynthesis and antinociceptive activity of (5-chloro-2(3H)-benzoxazolon-3-yl) propanamide derivativesArch Pharm Res27108610922004 WangZYChenYHZhengGJYangCMLongSJStudy on the anti-inflammatory and analgesic effects of ilicifolius alkaloids A and its derivatives acetyl ilicifolius alkaloids AChin Hosp Pharm J318078102011 SoyerZBasMPabuccuogluAPabuccuogluVSynthesis of some 2(3H)-benzoxazolone derivatives and their in-vitro effects on human leukocyte myeloperoxidase activityArch Pharm (Weinheim)3384054102005 UcarHVan derpoortenKCacciaguerraSSpampinatoSStablesJPDepoverePIsaMMasereelBDelargeJPoupaertJHSynthesis and anticonvulsant activity of 2(3 H)-benzoxazolone and 2(3H)-benzothiazolone derivativesJ Med Chem41113811451998 BellMCrowellTMatthewsDMcdonaldJNeelDShukerASelective β3 adrenergic agonistsUS Patent5,786,356Filed June 25, 1997; issued July 28, 1998 LinJFanHEffect of 4-hydroxy-2-benzoxazolone on the proliferation of HSC-T6 cellsLishizhen Med Materia Medica Res10237123722011 WeiCMOptimizing the preparation process for 2-nitroresorcinolJ Huaiyin Teach (Nat Sci)31351382004 CalışÜGökhanNErdoğanHSynthesis of some novel 3-methyl-6-(2-substituted propanoyl/propyl)-2-benzoxazolinone derivatives and anti-nociceptive activityFarmaco567197242001 WeiXChenYHLongSJSynthesis of 2-aminoresorcinol hydrochlorideNorthwest Pharmaceutical Journal31681692008 RecknagelROGlendeEAJrDolakJAWallerRLMechanisms of carbon tetrachloride toxicityPharmacol Ther431391541989 YuJWangYQianHZhaoYLiuBFuCPolyprenols from Taxus chinensis var. mairei prevent the development of CCl4-induced liver fibrosis in ratsJ Ethnopharmacol1421511602012 Al-DbassAMAl-DaihanSKBhatRSAgaricus blazei Murill as an efficient hepatoprotective and antioxidant agent against CCl4-induced liver injury in ratsSaudi J Biol Sci193033092012 DomitrovićRJakovacHBlagojevićGHepatoprotective activity of berberine is mediated by inhibition of TNF-α, COX-2, and iNOS expression in CCl4-intoxicated miceToxicology28033432011 SchwabeRFBrennerDAMechanisms of liver injury. I TNF-alpha-induced liver injury: role of IKK, JNK and ROS pathwaysAm J Physiol Gastrointest Liver Physiol290G583G5892006

(A) Reaction scheme for 4-hydroxy-2(3H)-benzoxazolone (HBOA): 2-itroresorcinol is reduced and subsequently cyclized with urea in a ‘one-pot’ synthesis. (B) The chemical structure of HBOA and its derivatives. 4-Acetoxy-2(3H)-benzoxazolone (AcO-BOA) and 3-acetyl-4-acetoxy-2-benzoxazolone (TC-3) were prepared by HBOA acetylation.

(A) Effects of (a) HBOA, (b) AcO-BOA and (c) TC-3 on serum ALT and AST levels. Data are presented as the means ± SE (n=10). **P<0.01 compared with the normal control group; #P<0.05 and ##P<0.01 compared with the CCl4 group. (B) Comparison of (a) the MDA inhibition rate and the induction rates of (b) SOD and (c) GSH following treatment with HBOA, AcO-BOA or TC-3 in mice with acute liver injury. Results are presented as: (level in the model group - level in the treatment group)/level in the model group × 100 and (level in the treatment group - level in the model group)/level in the model group × 100, respectively. HBOA, 4-hydroxy-2(3H)-benzoxazolone; AcO-BOA, 4-acetoxy-2(3H)-benzoxazolone; TC-3, 3-acetyl-4-acetoxy-2-benzoxazolone; ALT; alanine aminotransferase; AST, aspartate aminotransferase; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione.

Effects of HBOA on the expression of TNF-α in the liver tissues of mice with acute liver injury (magnification, ×400; Immunohistochemical stain for TNF-α diluted 1:150).(A) NC group; (B) CMC-Na group; (C) CCl4 model group; (D) Bif group; (E) high-dose HBOA group; (F) mid-dose HBOA group; (G) low-dose HBOA group. (H) The quantification of TNF-α staining. Data are presented as the means ± SE (n=10). **P<0.01 compared with the NC group; #P<0.05 compared with the CCl4 group. HBOA, 4-hydroxy-2(3H)-benzoxazolone; TNF-α, tumor necrosis factor-α; NC, normal control; CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.

Effects of HBOA and its derivatives on histopathological changes induced by CCl4 in the mouse liver. Liver sections were H&E stained (magnification, ×100): (A) normal control group; (B) CMC-Na group; (C) CCl4 model group; (D) CCl4 + Bif 150 mg/kg group; (EH) CCl4 + HBOA 200 mg/kg group; (EM) CCl4 + HBOA 100 mg/kg group; (EL) CCl4 + HBOA 50 mg/kg group; (FH) CCl4 + AcO-BOA 200 mg/kg group; (FM) CCl4 + AcO-BOA 100 mg/kg group; (FL) CCl4 + AcO-BOA 50 mg/kg group; (GH) CCl4 + TC-3 200 mg/kg group; (GM) CCl4 + TC-3 100 mg/kg group; (GL) CCl4 + TC-3 50 mg/kg group. HBOA, 4-hydroxy-2(3H)-benzoxazolone; H&E, hematoxylin and eosin; AcO-BOA, 4-acetoxy-2(3H)-benzoxazolone; TC-3, 3-acetyl-4-acetoxy-2-benzoxazolone; CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.

Effect of HBOA, AcO-BOA and TC-3 on the liver index in mice (%).

Group HBOA AcO-BOA TC-3
Control 4.56±0.51 4.93±0.41 5.59±0.34
CMC-Na 4.60±0.36 5.17±0.59 5.35±0.49
CCl4 5.28±0.45a 5.66±0.75b 6.06±0.46a
Bif 4.61±0.35c 4.83±0.30c 5.29±0.18d
High-dose 4.73±0.44d 4.88±0.49d 5.43±0.38d
Med-dose 5.38±0.79 5.23±0.59 5.44±0.20d
Low-dose 5.77±0.86 5.84±0.98 5.52±0.47c

Data are presented as the means ± SE. n=10 per group.

P<0.01 and

P<0.05 compared with the normal control group;

P<0.01 and

P<0.05 compared with the CCl4 group.

HBOA, 4-hydroxy-2(3H)-benzoxazolone; AcO-BOA, 4-acetoxy-2(3H)-benzoxazolone; TC-3, 3-acetyl-4-acetoxy-2-benzoxazolone; CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.

Effect of HBOA on SOD, MDA, GSH, Gpx, CAT and LDH levels (n=10).

Group SOD (U/mgprot) MDA (nmol/mgprot) GSH (nmol/mgprot) Gpx (U/mgprot) CAT (U/mgprot) LDH (U/l)
Control 200.84±41.95 2.20±0.60 2.20±0.83 368.74±101.27 12.74±3.29 5423.47±1054.40
CMC-Na 215.18±43.38 2.36±0.93 2.95±1.41 374.33±103.56 11.30±5.53 5942.01±1074.70
CCl4 139.49±30.52a 3.79±1.74a 1.02±0.36a 269.06±61.43a 8.18±2.82a 7907.96±2420.00a
Bif 191.61±36.57c 1.88±0.78c 1.63±0.89b 344.46±72.22b 11.41±1.81c 5967.66±1305.49b
High-dose HBOA 196.81±27.16c 1.90±0.91c 1.66±1.00b 341.57±76.65b 12.92±5.41b 5288.04±1151.85c
Med-dose HBOA 168.85±42.47 2.08±0.49c 1.42±0.53b 263.04±81.16 11.38±6.18 5764.20±865.38b
Low-dose HBOA 160.99±44.77 2.81±1.89 1.31±0.91 278.41±68.83 7.25±3.25 7172.09±370.51

Data are presented as the means ± SE.

P<0.01 compared with the normal control group;

P<0.05 and

P<0.01 compared with the CCl4 group.

HBOA, 4-hydroxy-2(3H)-benzoxazolone; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; Gpx, glutathione peroxidase; CAT, catalase; LDH, lactic dehydrogenase, CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.

Effect of high-, mid- and low-dose AcO-BOA on SOD, MDA and GSH levels (n=10).

Group SOD (U/mgprot) MDA (nmol/mgprot) GSH (nmol/mgprot)
Control 291.51±92.07 1.59±0.48 4.42±2.32
CMC-Na 305.37±84.18 1.89±0.59 3.68±1.59
CCl4 203.72±49.49a 2.58±0.85b 2.16±0.78b
Bif 269.34±82.90c 1.32±0.47d 3.27±0.86d
High-dose 288.98±102.00c 1.79±0.70c 3.33±0.95d
Mid-dose 217.58±74.44 2.65±0.67 1.11±0.56
Low-dose 227.66±56.68 2.80±1.04 2.77±0.15

Data are presented as the means ± SE.

P<0.05 and

P<0.01 compared with the normal control group;

P<0.05 and

P<0.01 compared with the CCl4 group.

AcO-BOA, 4-acetoxy-2(3H)-benzoxazolone; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.

Effect of high-, mid- and low-dose TC-3 on SOD, MDA and GSH levels (n=10).

Group SOD (U/mgprot) MDA (nmol/mgprot) GSH (nmol/mgprot)
Control 246.81±86.41 2.81±0.62 4.57±2.30
CMC-Na 262.09±33.99 2.94±0.23 4.96±1.07
CCl4 193.37±22.36a 3.62±0.92a 2.57±0.84a
Bif 290.39±63.25b 2.74±0.90b 4.81±1.43c
High-dose 288.45±42.52c 2.32±0.65c 3.94±1.14c
Mid-dose 216.15±20.75c 2.33±0.45c 2.32±1.05c
Low-dose 208.76±54.45 4.30±1.35 2.19±1.15c

Data are presented as the means ± SE.

P<0.05 compared with the normal control group;

P<0.05 and

P<0.01 compared with the CCl4 group.

TC-3, 3-acetyl-4-acetoxy-2-benzoxazolone; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione; CMC-Na, sodium carboxymethyl cellulose; Bif, bifendate.