Protective effect of the aqueous extract from the root of Platycodon grandiflorum on cholestasis-induced hepatic injury in mice

Abstract

Context: The root of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae) has been widely studied for its hepatoprotective effects against various hepatotoxicants.

Objective: The present study evaluated the protective effect of the standardized aqueous extract of P. grandiflorum (BC703) on cholestasis-induced hepatic injury in mice.

Materials and methods: BC703 is a standardized aqueous extract of P. grandiflorum in reference to platycodin D (at least 0.8%). The mice were allocated into five groups as follows: Sham-operated, bile duct ligation (BDL) alone, and BDL with BC703 (1, 5, and 10 mg/kg BW) treated group. BC703 was given for 3 consecutive days before BDL operation. The animals were sacrificed by CO₂ anesthesia post-24 h of BDL operations.

Results and discussion: Serum alanine aminotransferase and serum aspartate aminotransferase increased to 395.2 ± 90.0 and 266.0 ± 45.6 Unit/L in the BDL alone group and decreased with BC703 in a dose-dependent manner. Especially the 10 mg/kg of BC703-treated mice showed a 77% decrease of serum alanine aminotransferase and 56% of aspartate aminotransferase as compared with BDL alone. Decreased antioxidant enzyme levels in BDL alone group were elevated in BC703-treated groups ranging from 7 to 29% for glutathione and from 13 to 25% for superoxide dismutase. BC703 treatment also attenuated malondialdehyde (from 3 to 32%) and nitric oxide levels (from 32 to 50%) as compared with BDL alone. Histopathological studies further confirmed the hepatoprotective effect of BC703 in BDL-induced cholestesis.

Conclusion: BC703 could attenuate liver injury by BDL in mice, and test results indicate that BC703 might be useful in cholestatic liver injury.

Keywords::

• Bile duct ligation
• cholestasis
• hepatotoxicity
• platycodin D
• Platycodon grandilflorum
• standardized aqueous extract

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Introduction

Cholestasis is a condition caused by either a functional defect in bile formation or impairment in its secretion and flow (Arrese & Trauner, 2003). Long-standing cholestasis could lead to portal hypertension, hepatic dysfunction, and eventually hepatic cirrhosis (Parola et al., 1996). The pathogenesis is uncertain, but several studies reported that free radicals may play a role in cholestatic liver injury (Montilla et al., 2001; Pastor et al., 1997). Bile acids enhanced the release of free radicals from activated polymorphonuclear and inflammatory cells in cholestatic liver lesions (Dahm et al., 1988). Furthermore, accumulated lipophilic bile acid during cholestasis impairs the activity of the electron transport chain in the liver mitochondria and reduces its antioxidant capacity (Dueland et al., 1991).

The bile duct ligation (BDL) model is a well-known experimental model for research in cholestasis (Kountouras et al., 1984). Moreover, it is an important model for hepatocellular injury, producing changes analogous to primary sclerosing cholangitis, and other liver diseases in humans (Vierling, 2001).

Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae) has been traditionally used as medicine for various respiratory diseases including bronchitis, asthma, and pulmonary tuberculosis in East Asian countries. It contains at least 90% of carbohydrate, 2.4% of protein, 0.1% of lipid, and 1.5% of ash (Lee, 1973). It has been reported that the polysaccharides from the aqueous extract of P. grandiflorum root prevent hypercholesterolemia, hyperlipidemia, and diabetes (Kim et al., 2000). Moreover, the inulin-type polysaccharides, (1→2)-β-d-fructan, isolated from the roots of P. grandiflorum demonstrated an selective immunological effect on B cells and macrophages, but not on T cells (Han et al., 2001). Triterpenoid saponins were also found in the roots of P. grandiflorum such as platycodins (A, D, D₂, and D₃), 2″- and 3″-O-acetyl polygalacin D₂, platyconic acid, and platycosides (A, B, C, D, E, and F) (Kim et al., 2007). These saponins exhibit a variety of pharmacological activities, such as anti-inflammatory, anticancer, and immune enhancing effects (Kim et al., 2001; Shin et al., 2009; Xie et al., 2009). These saponins are believed to have protective effects against some chemical-induced hepatotoxicity (Khanal et al., 2009; Lee et al., 2001). However, the protective effect of the aqueous extract from P. grandiflorum has not been reported against biliary obstruction–induced injury in the liver.

The present study investigated the hepatoprotective effect of the standardized aqueous extract from P. grandiflorum (BC703) on the cholestasis-induced hepatic injury in mice.

Materials and methods

Chemicals

A superoxide dismutase (SOD) assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Thiobarbituric acid reactive substance (TBARS), glutathione (GSH), nitric oxide (NO) assay kits were from BioAssay Systems (Hayward, CA, USA). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) test kits were obtained from IDEXX (Westbrook, ME, USA). All other reagents were purchased from Sigma Chemicals (St. Louis, MO, USA). The standardized P. grandiflorum aqueous extract, BC703, was manufactured by SkyHerb Pharmaceuticals (Zhejiang, China) according to full good manufacturing practice. BC703 was standardized in reference to platycodin D (at least 0.8%) using validated high performance liquid chromatography (HPLC) assay method. Shortly, the root of P. grandiflorum (100 g) was cut into slices and extracted in distilled water with occasional shaking at 60–90°C for 6–10 h. The aqueous extract was then filtered, concentrated under reduced pressure in a rotary evaporator, and spray-dried into powder (36 g).

HPLC–ELSD analysis

HPLC fingerprint analysis of BC703 was carried out as previously described, with some modification (Kim et al., 2007). In brief, HPLC analysis of BC703 was performed on a Younglin ACME 9000 (Seoul, Korea) equipped with a Sedex 55 evaporative light scattering detector (ELSD; SEDERE, Alfortville, France). Sample separation was achieved in a Gemini C₁₈ column (100 × 4.6 mm, 3-µm particle size; Phenomenex, Torrance, CA, USA) with a precolum (C₁₈, 3.5 µm, 2 × 20 mm) at room temperature. The mobile phase consisted of 0.1% formic acid/methanol/acetonitrile (75:5:20, v/v/v; A) and 0.1% formic acid/methanol/acetonitrile (70:5:25, v/v/v; B), and gradient runs programmed as follows: 0–10 min (0% B), 10–17 min (0–50% B), 25–34 min (50–80% B), 42–52 min (100% B), and then equilibration with 0% B for 10 min at a flow rate of 1 mL/min. The injection volume was 20 µL. The ELSD was set to a probe temperature of 70°C, a gain of 7, and the nebulizer gas nitrogen adjusted to 2.5 bar.

Animals

Male Crl:CD1(ICR) mice (5–6 weeks; 32–36 g) were used for this study. They were obtained from Orient Bio (Seongnam, Korea) and acclimated for 1 week before experiments. They were housed under standard laboratory conditions (12 h light/dark cycle, 22–23°C) with standard mouse food pellet and water ad libitum. The mice were randomly allocated into five groups as follows: Group 1 (sham-operated, n = 5), Group 2 (BDL alone, n = 5), Group 3 (BDL and 1 mg/kg of BC703 treated, n = 5), Group 4 (BDL and 5 mg/kg of BC703 treated, n = 5), and Group 5 (BDL and 10 mg/kg of BC703 treated, n = 5). The sham-operated group and BDL alone group were orally administered distilled water, and the others were orally administered with BC703 (1, 5, and 10 mg/kg) for 3 consecutive days before BDL operation. BC703 were dissolved in distilled water, and dosing volumes were 5 mL/kg of body weight. At 3 h after the last treatment, common BDL was performed under anesthesia with intraperitoneal injection of zolazepam and tiletamine combination (Zoletil 50^®, Virbac, Carros, France). After midline laparotomy, the duodenum was retracted, and common bile duct was carefully double ligated with a 6-0 nylon suture and dissected. Sham-operated mice received an identical laparotomy and isolation of the common bile duct without ligation. After 24 h of BDL, survived mice were sacrificed by cardiac puncture under CO₂ anesthesia. Blood samples were collected and serum was separated by centrifuging at 800 g for 15 min, and the serum samples were subjected to biochemical investigations. Liver samples from each mouse were taken for histopathological and biochemical examinations. Left lobes of liver were fixed immediately with 10% buffered formalin phosphate solution. Serum samples and other parts of livers were stored at −70°C until analysis. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Chungnam National University (Daejeon, Korea).

Serum biochemical examination

The serum ALT, AST activities were determined on a dry chemistry system, the Vettest 8008 blood chemistry analyzer (IDEXX Laboratories, Westbrook, ME, USA).

Oxidant and antioxidant parameters

The level of SOD was measured by using the tetrazolium salt assay (Superoxide Dismutase Assay Kit, Cayman Chemical Company, Ann Arbor, MI, USA). The content of malondialdehyde, a terminal product of lipid peroxidation, was measured with the thiobarbituric acid reduction method using a commercially available kit (QuatiChrom TBARS Assay kit, BioAssay Systems, Hayward, CA, USA). The hepatic GSH level was determined using the improved DTNB (5,5′-dithiobis-[2-nitrobenzoic acid]) method (QuantiChrom GSH Assay kit, BioAssay Systems, Hayward, CA, USA). NO was measured by improved Griess method (QuantiChrom NO Assay kit, BioAssay Systems, Hayward, CA, USA).

Histopathological examination

The liver slices were taken from a part of the left lobe in each groups and fixed in a 10% buffered formalin phosphate solution and embedded in paraffin. The block was cut into 5 µm sections and stained with hematoxylin and eosin (H & E). Randomly selected slides from each group were analyzed under a light microscope.

Statistical analysis

Results were expressed as mean ± standard error (SEM). The significance of differences among experimental groups were determined using the one way analysis of variance (ANOVA) test. Where significant effects were found, post hoc analysis using the Dunnett test or Least Significant Difference test was performed and p < 0.05 was considered to be statistically significant.

Results

HPLC fingerprint analysis of BC703

In HPLC/ELSD analysis, the peak of platycodin D was found at 37.8 min, and the percentage of platycodin D in the BC703 was approximately 0.88% (Figure 1). It also includes deapio-platycoside E, platycoside E, deapio-platycodin D₃, platycodin D₃, deapio-platycodin D, platycodin D, polygalacin D, 3″-O-acetyl polygalacin D, platycodin A, and 2″-O-acetyl polygalacin D. In addition, it was reported that the aqueous extract of P. grandiflorum contained a polysaccharide, an inulin-type polyfructose, (1→2)-β-d-fructan (Han et al., 2001; Oka et al., 1992).

Figure 1.  Representative HPLC chromatograms of triterpenoids in BC703. The numbers indicate each platycoside: 1, deapio-platycoside E; 2, platycoside E; 3, platycodin D₃; 4, deapio-platycodin D; 5, platycodin D; 6, 3″-O-acetyl platycodin D; 7, polygalacin D; 8, 2″-O-acetyl platycodin D; 9, an unknown compound.

Assessment of serum hepatic enzyme activities

Effects of BC703 on serum hepatic enzyme activities were shown in Figure 2. The levels of serum ALT and AST in the BDL alone group significantly increased to 395.2 ± 90.0 and 266.0 ± 45.6 Unit/L compared with the sham-operated group (p < 0.05). However, BC703 (1, 5, and 10 mg/kg) pretreatment dose-dependently attenuated these BDL-induced elevations in serum ALT and AST levels up to 77 and 56%, respectively.

Figure 2.  Effect of BC703 on liver function in the bile duct ligation (BDL)-induced acute hepatic injury. Mice were given orally BC703 (0, 1, 5, and 10 mg/kg) once daily for 3 consecutive days prior to BDL. Values are expressed as means ± standard error (SEM). *p < 0.05, a significant difference in comparison with the positive control group.

Effect of BC703 on GSH, SOD, TBARS, NO values

Hepatoprotective effects of BC703 were evaluated by GSH, SOD, TBARS, and NO levels in liver homogenates (Figure 3). In the BDL alone group, significantly reduced activities of GSH and SOD were shown, whereas the levels of NO and TBARS were increased as compared with the sham-operated group (p < 0.05). Decreased GSH values by BDL were increased to 5.6 ± 0.94, 5.9 ± 1.36, and 6.7 ± 0.17 nmole/mg of protein in the BC703-treated group at 1, 5, and 10 mg/kg, respectively. Similarly, SOD levels were also increased in the BC703-treated group in a dose-dependent fashion. The level of hepatic NO was remarkably decreased, ranging from 32 to 50%, by BC703 pretreatment. BC703 also attenuated the increased TBARS level in BC703-treated group.

Figure 3.  Effect of BC703 on the hepatic GSH (A), SOD (B), lipid peroxidation (C) and NO (D) content in the bile duct ligation (BDL)-induced acute hepatic injury. Mice were given orally BC703 (0, 1, 5, and 10 mg/kg) once daily for 3 consecutive days prior to BDL. Values are expressed as means ± standard error (SEM). *p < 0.05, a significant difference in comparison with the positive control group.

Histopathological findings

In sham-operated animals, there were no microscopic structural changes of liver from normal hepatic architecture (Figure 4). The liver from the BDL alone group showed significant histopathological alteration of liver, such as mild ductal proliferation, polymorphic nucleus, hepatocyte necrosis, and inflammatory cell infiltration. In the low dose, BC703 (1 mg/kg) did not reduce the BDL-induced histopathlogical alteration, with mild ductal proliferation and inflammatory cell infiltration still present. The higher dose BC703 (5, 10 mg/kg) treated groups showed much less necrosis of hepatocyte and infiltration of inflammatory cell than the BDL alone group.

Figure 4.  Histopathological changes of the liver stained with hematoxyline and eosin on the bile duct ligation (BDL)-induced liver injury (×100). Mice were given orally BC703 (0, 1, 5, and 10 mg/kg) once daily for 3 consecutive days prior to BDL. (A) nontreated sham-operated group; (B) nontreated BDL group; (C) BC703 (1 mg/kg) treated BDL group; (D) BC703 (5 mg/kg) treated BDL group; (E) BC703 (10 mg/kg) treated BDL group. Arrows indicate the bile duct proliferation and hepatocyte necrosis.

Discussion

BDL is a good model to evaluate for the protective or curative effect of candidates against liver injury by cholestasis. In the early stage of BDL, increased activities of serum enzyme levels of ALP and ALT following hepatocyte injury were shown. In addition, even 8 h after BDL, the liver presented clusters of injured hepatocytes and initiation of cholangiocellular proliferation in the large bile duct (Georgiev et al., 2008; Portincasa et al., 2007). In the present study, levels of serum liver enzymes in the BDL alone group were significantly increased by BDL in comparison with the sham-operated group, and these elevated enzyme levels were decreased by pretreatment of BC703.

GSH is a major antioxidant that prevents the damage to cellular components caused by reactive oxygen species (Pastor et al., 1997). In cholestasis, the retention of hydrophobic bile salts may affect the pathways that could decrease GSH levels and stimulate GSH efflux from hepatocytes and decrease SOD levels, which plays an important role in the antioxidant defense system in the liver by eliminating superoxide anion radicals (Gumpricht et al., 2000). BDL also resulted in an increase of lipid peroxidation levels followed by oxidative stress. In this study, the pretreatment of BC703 significantly attenuated BDL-induced hepatic injury and prevented the GSH, SOD depletion. In addition, the index of oxidative stress, such as NO and TBARS values, in BDL alone group was significantly increased, but ameliorated in BC703-treated groups. These results were consistent with our previous study which the aqueous extract from the root of P. grandiflorum ameliorated the thioacetamide-induced fulminant hepatic failure in mice (Lim et al., 2011). BC703 may reduce oxidative stress by recovery of antioxidative enzymes, together with decrease in lipid peroxidation and oxidative stress. However, TNF-α in BDL-induced cholestasis mediates acute and chronic liver injury and plays an important role in hepatic fibrosis and cirrhosis (Gäbele et al., 2009). Khanal et al. (2009) reported that saponins isolated from the root of P. grandiflorum decreased the TNF-α level on acute ethanol-induced hepatic injury in mice.

In histopathological findings, the BDL alone group showed the proliferation of the bile duct and hepatocyte necrosis. Although the BC703-treated group (10 mg/kg) showed reduced ductal proliferation and necrosis of hepatocytes in periductal area compared with the positive control group, severe hepatic injury including neoductulogenesis and broad biliary infarcts and hepatic necrosis was not shown in both the BDL alone group and BC703-treated groups. Several studies have assessed hepatocellular injury on Day 3 or 7 after BDL (Canbay et al., 2002; Miyoshi et al., 1999). Georgiev et al. (2008) reported that Day 3 after BDL is a valid time for histological quantification with proliferation of hepatocytes and biliary epithelial cells, but serum hepatic enzymes such as ALT and AST should be measured after 24–48 h to determine the degree of acute cholestatic liver injury. Consistently, a definite histopathological change among the experimental groups was not shown at 24 hr after BDL. However, an evident change in the serum enzyme and hepatic antioxidant index of the BDL operated groups was found in the present study.

Conclusion

The administration of BC703 protects against acute hepatic injury in the early stage of cholestatic mice. Further studies are needed to assess its hepatoprotective effect against the long-term BDL-induced fibrosis.

Declaration of interest

This research was supported by Industrialization Support Program for Bio-technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.