This article has Open Peer Review reports available.
San Huang Shel Shin Tang beta-cyclodextrin complex augmented the hepatoprotective effects against carbon tetrachloride-induced acute hepatotoxicity in rats
- Yu-Lan Yeh1, 2,
- Wei-Jen Ting3,
- Wei-Wen Kuo4,
- Hsi-Hsien Hsu5,
- Yueh-Min Lin1, 2,
- Chia-Yao Shen6,
- Chung-Ho Chang7,
- Viswanadha Vijaya Padma8,
- Yuhsin Tsai†9Email author and
- Chih-Yang Huang†3, 9, 10Email author
© Yeh et al. 2016
Received: 14 April 2015
Accepted: 15 May 2016
Published: 27 May 2016
San Huang Shel Shin Tang (SHSST) is a traditional herbal decoction used as a hepato-protective agent and is composed of Rheum officinale Baill, Scutellaria baicalnsis Geprgi and Coptis chinensis Franch (2:1:1 w/w). Beta-cyclodextrin (β-CD) modification may potentially increase the solubility and spectral properties of SHSST.
In this research, the hepato-protective effects of unmodified SHSST, β-CD modified SHSST complex (SHSSTc) and silymarin were evaluated in carbon tetrachloride (CCl4) induced acute hepatotoxicity in rats.
SHHSTc (40 mg/kg/day) and silymarin (100 mg/kg/day) both decreased the CCl4-induced cirrhosis pathway-related transforming growth factor beta (TGF-β) and apoptosis pathway-related caspase-8 protein expressions, but SHSST (40 mg/kg/day) did not reduce TGF-β and caspase-8 significantly . Moreover, SHHSTc (40 mg/kg/day) enhanced the activation of insulin-like growth factor 1 receptor (IGF1R) mediated survival pathway than the silymarin (100 mg/kg/day) to protect the liver from damage induced by CCl4.
β-CD modification promotes hepato-protective effects of SHSST and reduces the required-dosage of the SHSST.
Hepatic fibrosis is one of the most critical pathological features of chronic liver disease. Liver fibrosis is associated with the inflammatory and reparative phase of hepatic fibrosis by activated hepatic stellate cells (HSC) and can be characterized by an excessive deposition of extracellular matrix (ECM) components in the liver parenchyma . Liver fibrosis is known to be a serious chronic disease and it is difficult to treat with an aggressive treatment due to possible cirrhosis outcomes. This makes it necessary to develop a new complex or cocktail of more efficient drugs with sufficiently low dosages to prevent side effects.
SHSST is a cocktail of traditional herbal decoction, with reported bioactivities such as hypotension, antioxidation, anti-inflammation and cardio-protective effects . Rheum was discovered to have a hepato-protective effect and can be used to treat carbon tetrachloride (CCl4)-induced liver injury in rats [3, 4]. Scutellaria and Coptis were also reported to elicit similar liver protection against acute hepatotoxicity [5–7]. The similar liver protection effects of Rheum officinale, Scutellaria baicalnsis and Coptis chinensis are due to the similarity in the constituent bioactive compounds such as baicalein, which is a flavonoid [8–10].
In our previous study, the tumor necrosis factor ligand superfamily member 6 (FAS) was activated in the course of CCl4-induced liver failure, with down stream apoptotic protein caspase-8 on Fas-associated protein with death domain (FADD) released to cytosol, causing cleavage of caspase 3 and progression of cellular apoptosis [11, 12]. CCl4 induced liver failure occurs through an oxidation process when CCl4 is transported through the vascular system to the liver and by catalysis by mixed function oxidase (MFO) such as P450, changes into methane chloride or radicals. These radicals cause protein metabolic obstruction and inflammation in the liver, producing CCl4 induced acute liver injury [13, 14]. Silymarin can provide an anti-oxidation function to block the CCl4 catalyzing process and protect the liver from CCl4 induced acute liver injury but not through the P450 inhibition . Many flavonoid compounds play the same role with their anti-oxidative function. Baicalein in SHSST can also enhance cell survival ability through PI3K-Akt pathway activation. The active Akt can keep the Bcl-2-associated death promoter (Bad) protein in phosphorylated type and prevent cell apoptosis . Here, the liver protection function of SHSST was tested and compared with silymarin.
In our previous research, beta- cyclodextrin (β-CD) increased the solubility and spectral properties of guest molecules, especially the hydrophobic drugs, without changing their intrinsic property to permeate the cell membranes [17–19]. Thus, β-CD complex synthesis with the indicated compound or herbal decoction can enhance the solubility, stability and bioavailability of drugs [20, 21]. This research evaluated the liver protection effects of the β-CD modified SHSST complex (SHSSTc), SHSST and silymarin in CCl4 induced acute hepato-toxicity in rats.
Preparation of SHSST and SHSST-β-CD complex
The SHSST was purchased from PaiAn pharmacy (Taichung, Taiwan). The SHSST-β-CD complex was prepared by coprecipitation. β-CD (70.0 g) was dissolved in distilled water (85 ml) at 70 °C in a water bath for 1 h. SHSST (10.0 g) in ethanol (15 mL) was slowly added to the β-CD solution with continuous agitation and stirred continuously for 6 h. Following that, 40 mL of ethanol was added drop wise to regulate the solubility of the hydrophobic solute in β-CD solution. The solution was then refrigerated overnight at 4 °C. The precipitated SHSSTc (SHSST-β-CD complex, SHSST: β-CD = 1: 9 in weight) was recovered by filtration and washed with ethanol to remove unencapsulated SHSST. This residue was dried in a vacuum oven at −20 °C for 48 h. The final powder was stored at 4 °C until use.
The animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (No.100-3-B, date 2010-9-1). There were 30 SD rats (300 g in body weight, aged 10 weeks) purchased from BioLASCO Taiwan Co., Ltd and were divided into 5 groups (n = 6 each). The groups were designated as control, CCl4 (Sgma-Aldrich, Taipei, Taiwan) intraperitoneal injection treatment, CCl4 intraperitoneal injection combined with SHSSTc (40 mg/kg/day) oral treatment, CCl4 intraperitoneal injection combined with SHSST oral treatment (40 mg/kg/day), CCl4 intraperitoneal injection combined with silymarin (Sigma-Aldrich, Taipei, Taiwan) oral treatment. CCl4 intraperitoneal injection (100 mg/kg/day) treatment was performed on the 7th day following 6 days of pretreatment with the test materials. After 24 h CCl4 intraperitoneal injection, all the rats were killed by decapitation and samples were collected immediately.
Blood biochemical analysis
Blood was collected from rat in each group during the while decapitation and measured using the blood routine examination protocol at China Medical University Hospital. The following parameters were analyzed: TC (total cholesterol), cholesterol, AST (aspartate transaminase), ALT (alanine transaminase), BUN (blood urea nitrogen), CK (creatine kinase).
Hemotoxyline and eosin staining
Livers from rats in each group were soaked in 10 % formalin, dehydrated through graded alcohols and embedded in paraffin wax. Following that, 2 μm thick paraffin sections were cut from these paraffin-embedded tissue blocks. The tissue sections were deparaffinized by immersion in xylene and rehydrated. Sections were stained with hematoxylin and eosin (H&E), immersed in graded alcohols followed by xylene and mounted in mounting medium kit (Surgipath, Leica Biosystems, Instrument Co., Richmond, USA). Photomicrographs were obtained using Zeiss Axiophot microscopes (Taiwan Instrument Co., Taipei, Taiwan).
Masson’s trichrome staining
Rat livers from each group were soaked in 10 % formalin, dehydrated through graded alcohols and embedded in paraffin wax. Following that, 2 μm thick paraffin sections were cut from these paraffin-embedded tissue blocks. The tissue sections were deparaffinized by immersion in xylene and rehydrated. Samples were then stained with Masson’s trichrome (MT) using HT15 Sigma Trichrome stain (Masson) kit (HT1079, Sigma-Aldrich, Taipei, Taiwan) and the procedure was according to the protocol of the kit. MT satin was used to investigate liver histological and fibrotic changes and photomicrographs were obtained using Zeiss Axiophot microscopes (Taiwan Instrument Co., Taipei, Taiwan).
Tissue protein extraction
Liver tissue extracts from 6 rats in each group were obtained by homogenizing in a lysis buffer (0.05 M Tris–HCl, pH 7.4, 0.15 M NaCl, 0.25 % deoxycholic acid, 1 % NP-40, 1 mM EDTA) at a ratio of 100 mg tissue/1 ml buffer. The homogenates were placed on ice and then centrifuged at 13,000 rpm for 40 min. The supernatants were collected and stored at −80 °C for further experiments.
Western blot assay
Liver tissue extract protein concentrations were determined using the Lowry protein assay. Protein samples were separated in a 12 % SDS polyacrylamide gel electrophoresis (SDS-PAGE) with a constant voltage of 75 V for 120 min. Proteins were then transferred to Hybond-C membranes (GE healthcare UK limited., Buckinghamshire, UK) using 50 volt for 3 h. PVDF membranes were incubated in 3 % bovine serum albumin (BSA) in Tris-buffered solution (TBS). Primary antibodies (Santa Cruz Biotechnology, CA, USA) were added into the membranes to recognize the respective proteins. After washing 3 times, horseradish peroxidase-labeled antibodies were then used. Last, after 3 times washed and pictures were taken with Fujifilm LAS-3000 (GE healthcare UK limited, Buckinghamshire, UK).
The results shown are the means ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variants. The Student’s t-test was used for paired samples.
Blood Biochemical analysis of the experimental rats
28.0 ± 2.5
42.9 ± 9.7***
25.9 ± 5.1##
38.6 ± 11.6
35.1 ± 3.9
27 ± 14
195 ± 20***
93 ± 15###
116 ± 19##
124 ± 25##
50 ± 15
57 ± 14
50 ± 15
56 ± 17
50 ± 18
45 ± 20
54 ± 19
46 ± 43
47 ± 13
46 ± 13
483 ± 56
1399 ± 147***
998 ± 63#
1137 ± 85
1024 ± 72#
29 ± 7
35 ± 7
31 ± 6
33 ± 7
32 ± 7
CCl4 can be changed into CHCl3 through oxidation in the liver . However, the products after CCl4 oxidation also include free radicals, such as trichloromethyl free radical (CCl3•) and trichloromethylperoxyl free radical (CCl3OO•), these free radical compounds will react with the lipoprotein in hepatocytes causing cholesterol accumulation because of the problems in lipid metabolism . Silymarin can provide an anti-oxidant function and reduce the amount of free radicals in the liver, providing liver protection . The flavonoids rich SHSST might also present a similar function, but the result shown the liver protection effect of SHSST was still very week (Table 1, Fig. 1). SHSSTc treatment acts better than SHSST even silymarin owing to the modulations in its bioavailability for being modified with the β-CD complex.
Silymarin is reported to provide protection against liver cirrhosis and it reduces the levels of hepatic fibrosis markers including serum TGF-β1, especially in the CCl4 induced liver injury model . TGF-β1/p-Smad signaling expression can enhance collagen synthesis in fibroblasts . The MT staining assay showed collagen accumulation in the CCl4 induced liver fibrosis group and TGF-β1/p-Smad signaling over expression within 24 h (Figs. 1, 2). In 1 week pretreatment with silymarin and SHSSTc, TGF-β1/p-Smad signaling and collagen accumulation were blocked (Fig. 1, 3). The experimental data suggests that SHSSTc and silymarin have similar anti- cirrhosis effects.
SHSSTc treatment efficiently protects the hepatocytes in CCl4 induced liver injury. The possible protection mechanism is through FAS/FADD/caspase-8 apoptosis pathway inhibition and IGF1R/PI3K/Akt survival pathway activation [27, 28] (Fig. 4). Here, silymarin could only inhibit apoptosis through caspase-8 suppression but not through IGF1R pathway activation. This may be the difference between SHSSTc and silymarin in liver protection which makes SHSSTc protect the liver better than silymarin in this CCl4 induced liver injury animal model.
SHSST modification with β-CD complex to SHSSTc enhances the liver protection effects of SHSST. SHSSTc shows similar inhibitory effects with silymarin in the FAS/FADD/caspase-8 apoptosis pathway and TGF-β fibrosis pathway. However, SHSSTc augmented the IGF1R/PI3K/Akt survival pathway more than silymarin and displayed stronger protection against CCl4 induced liver injury.
ALT, alanine transaminase; AST, aspartate transaminase; Bad: Bcl-2-associated death promoter; BUN, blood urea nitrogen; CCl4, carbon tetrachloride; CK, creatine kinase; ECM, extracellular matrix; FADD, Fas-associated protein with death domain; FAS, tumor necrosis factor ligand superfamily member 6; HSC, hepatic stellate cells; MFO, mixed function oxidase; SHSST, San Huang Shel Shin Tang; SHSSTc, β-CD modified SHSST complex; TC, total cholesterol; TGF-β, transforming growth factor beta; β-CD, Beta-cyclodextrin
This study is supported in part by Taiwan and from Taiwan Ministry of Health and Welfare Clinical Trial and Research Centre of Excellence (MOHW105-TDU-B-212-133019).
Availability of data and materials
We have presented all our data in the form of figures and tables. The datasets supporting the conclusions of this article are included within the article.
YLY, WJT, WWK, HHH and CYS contributed conception, design and acquisition of data. YML, CHC and VVP contributed the analysis and interpretation of data. YT drafted the manuscript and CYH revised and gave final approval of the version to be published. All authors were read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The animal experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (No.100-3-B, date 2010-9-1).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibro genesis. J Gastroenterol Hepatol. 2007;22:S79–84.View ArticlePubMedGoogle Scholar
- Cheng WY, Wu SL, Hsiang CY, Li CC, Lai TY, Lo HY, Shen WS, Lee CH, Chen JC, Wu HC, Ho TY. San-Huang-Xie-Xin-Tang and its herbal components on the gene expression profiles in HepG2 cells. Am J Chin Med. 2008;36:783–97.View ArticlePubMedGoogle Scholar
- Wang JB, Zhao HP, Zhao YL, Jin C, Liu DJ, Kong WJ, Fang F, Zhang L, Wang HJ, Xiao XH. Hepatotoxicity or hepatoprotection? Pattern recognition for the paradoxical effect of the Chinese herb Rheum palmatum L. in treating rat liver injury. PLoS One. 2011;6:e24498.View ArticlePubMedPubMed CentralGoogle Scholar
- Fang F, Wang JB, Zhao YL, Jin C, Kong WJ, Zhao HP, Wang HJ, Xiao XH. A comparative study on the tissue distributions of rhubarb anthraquinones in normal and CCl4-injured rats orally administered rhubarb extract. J Ethnopharmacol. 2011;137:1492–7.View ArticlePubMedGoogle Scholar
- Chien CF, Wu YT, Tsai TH. Biological analysis of herbal medicines used for the treatment of liver diseases. Biomed Chromatogr. 2011;25:21–38.View ArticlePubMedGoogle Scholar
- Nan JX, Park EJ, Kim YC, Ko G, Sohn DH. Scutellaria baicalensis inhibits liver fibrosis induced by bile duct ligation or carbon tetrachloride in rats. J Pharm Pharmacol. 2002;54:555–63.View ArticlePubMedGoogle Scholar
- Ye X, Feng Y, Tong Y, Ng KM, Tsao S, Lau GK, Sze C, Zhang Y, Tang J, Shen J, Kobayashi S. Hepatoprotective effects of Coptidis rhizoma aqueous extract on carbon tetrachloride-induced acute liver hepatotoxicity in rats. J Ethnopharmacol. 2009;124:130–6.View ArticlePubMedGoogle Scholar
- Püssa T, Raudsepp P, Kuzina K, Raal A. Polyphenolic composition of roots and petioles of Rheum rhaponticum L. Phytochem Anal. 2009;20:98–103.View ArticlePubMedGoogle Scholar
- Wang CZ, Calway TD, Wen XD, Smith J, Yu C, Wang Y, Mehendale SR, Yuan CS. Hydrophobic flavonoids from Scutellaria baicalensis induce colorectal cancer cell apoptosis through a mitochondrial-mediated pathway. Int J Oncol. 2013;42:1018–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu L, Chen Z. Analysis of four alkaloids of Coptis chinensis in rat plasma by high performance liquid chromatography with electrochemical detection. Anal Chim Acta. 2012;737:99–104.View ArticlePubMedGoogle Scholar
- Huang CY, Yang AL, Lin YM, Wu FN, Lin JA, Chan YS, Tsai FJ, Tsai CH, Kuo CH, Lee SD. Anti-apoptotic and pro-survival effects of exercise training on hypertensive hearts. J Appl Physiol (1985). 2012;112:883–91.View ArticleGoogle Scholar
- Lee SD, Shyu WC, Cheng IS, Kuo CH, Chan YS, Lin YM, Tasi CY, Tsai CH, Ho TJ, Huang CY. Effects of exercise training on cardiac apoptosis in obese rats. Nutr Metab Cardiovasc Dis. 2013;23:566–73.View ArticlePubMedGoogle Scholar
- Chaudhury S, Mehendale HM. Amplification of CCl4 toxicity by chlordecone: destruction of rat hepatic microsomal cytochrome P-450 subpopulation. J Toxicol Environ Health. 1991;32:277–94.View ArticlePubMedGoogle Scholar
- Klingensmith JS, Mehendale HM. Destruction of hepatic mixed-function oxygenase parameters by CCl4 in rats following acute treatment with chlordecone, Mirex, and phenobarbital. Life Sci. 1983;33:2339–48.View ArticlePubMedGoogle Scholar
- Kim HJ, Chun YJ, Park JD, Kim SI, Roh JK, Jeong TC. Protection of rat liver microsomes against carbon tetrachloride-induced lipid peroxidation by red ginseng saponin through cytochrome P450 inhibition. Planta Med. 1997;63:415–8.View ArticlePubMedGoogle Scholar
- Zhang HB, Lu P, Guo QY, Zhang ZH, Meng XY. Baicalein induces apoptosis in esophageal squamous cell carcinoma cells through modulation of the PI3K/Akt pathway. Oncol Lett. 2013;5:722–8.PubMedGoogle Scholar
- Tsai Y, Tsai HH, Wu CP, Tsai FJ. Preparation, characterisation and activity of the inclusion complex of paeonol with b-cyclodextrin. Food Chem. 2010;120:837–41.View ArticleGoogle Scholar
- Yuan C, Jin Z, Xu X, Zhuang H, Shen W. Preparation and stability of the inclusion complex of astaxanthin with hydroxypropyl-b-cyclodextrin. Food Chem. 2008;109:264–8.View ArticlePubMedGoogle Scholar
- Vyas A, Saraf S, Saraf S. Cyclodextrin based novel drug delivery systems. J Incl Phenom Macro. 2008;62:23–42.View ArticleGoogle Scholar
- Uekama K, Hirayama F, Irie T. Cyclodextrin Drug Carrier Systems. Chem Rev. 1998;98:2045–76.View ArticlePubMedGoogle Scholar
- Kang J, Kumar V, Yang D, Chowdhury PR, Hohl RJ. Cyclodextrin complexation: Influence on the solubility, stability, and cytotoxicity of camptothecin, an antineoplastic agent. Eur J Pharm Sci. 2002;15:163–70.View ArticlePubMedGoogle Scholar
- Raymond P, Plaa GL. Effect of dosing vehicle on the hepatotoxicity of CCl4 and nephrotoxicity of CHCl3 in rats. J Toxicol Environ Health. 1997;51:463–76.PubMedGoogle Scholar
- Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol. 2003;33:105–36.View ArticlePubMedGoogle Scholar
- Velussi M, Cernigoi AM, De Monte A, Dapas F, Caffau C, Zilli M. Long-term (12 months) treatment with an anti-oxidant drug (silymarin) is effective on hyperinsulinemia, exogenous insulin need and malondialdehyde levels in cirrhotic diabetic patients. J Hepatol. 1997;26:871–9.View ArticlePubMedGoogle Scholar
- Wei F, Liu SK, Liu XY, Li ZJ, Li B, Zhou YL, Zhang HY, Li YW. Meta-analysis: silymarin and its combination therapy for the treatment of chronic hepatitis B. Eur J Clin Microbiol Infect Dis. 2013;32:657–69.View ArticlePubMedGoogle Scholar
- Xue W, Yunliang Q, Rong J, Yan W, Jun C, Chen W, Danru W. Effects of TRAP-1-Like Protein (TLP) Gene on Collagen Synthesis Induced by TGF-b/Smad Signaling in Human Dermal Fibroblasts. Plos One. 2013;8:e55899.View ArticleGoogle Scholar
- Hsieh SR, Cheng WC, Su YM, Chiu CH, Liou YM. Molecular targets for anti-oxidative protection of green tea polyphenols against myocardial ischemic injury. BioMedicine. 2014;4:7–16.View ArticleGoogle Scholar
- Wang CH, Lin WD, Bau DT, Chou IC, Tsai CH, Tsai FJ. Appearance of acanthosis nigricans may precede obesity: An involvement of the insulin/IGF receptor signaling pathway. BioMedicine. 2013;3:82–7.View ArticleGoogle Scholar
- Li CY, Hou YC, Lee Chao PD, Shia CS, Hsu IC, Fang SH. Potential ex vivo immunomodulatory effects of San-Huang-Xie-Xin-Tang and its component herbs on mice and humans. J Ethnopharmacol. 2010;127:292–8.View ArticlePubMedGoogle Scholar
- Parveen R, Baboota S, Ali J, Ahuja A, Vasudev SS, Ahmad S. Effects of silymarin nanoemulsion against carbon tetrachloride-induced hepatic damage. Arch Pharm Res. 2011;34:767–74.View ArticlePubMedGoogle Scholar