Ginseng essence, a medicinal and edible herbal formulation, ameliorates carbon tetrachloride-induced oxidative stress and liver injury in rats.

BACKGROUND: Ginseng essence (GE) is a formulation comprising four medicinal and edible herbs including ginseng (Panax ginseng), American ginseng (Panax quinquefolius), lotus seed (Nelumbo nucifera), and lily bulb (Lilium longiflorum). This study was aimed at investigating the hepatoprotective effect of GE against carbon tetrachloride (CCl4)-induced liver injury in rats.
METHODS: We treated Wistar rats daily with low, medium, and high [0.625 g/kg body weight (bw), 1.25 g/kg bw, and 3.125 g/kg bw, respectively] doses of GE for 9 wk. After the 1st wk of treatment, rats were administered 20% CCl4 (1.5 mL/kg bw) two times a week to induce liver damage until the treatment ended.
RESULTS: Serum biochemical analysis indicated that GE ameliorated the elevation of aspartate aminotransferase and alanine aminotransferase and albumin decline in CCl4-treated rats. Moreover, CCl4-induced accumulation of hepatic total cholesterol and triglyceride was inhibited. The hepatoprotective effects of GE involved enhancing the hepatic antioxidant defense system including glutathione, glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase, and catalase. In addition, histological analysis using hematoxylin and eosin and Masson's trichrome staining showed that GE inhibited CCl4-induced hepatic inflammation and fibrosis. Furthermore, immunohistochemical staining of alpha-smooth muscle actin indicated that CCl4-triggered activation of hepatic stellate cells was reduced.
CONCLUSION: These findings demonstrate that GE improves CCl4-induced liver inflammation and fibrosis by attenuating oxidative stress. Therefore, GE could be a promising hepatoprotective herbal formulation for future development of phytotherapy.

Introduction

Chronic liver disease and cirrhosis are the leading causes of death in Taiwan and have been responsible for an increasing number of fatalities in recent years [1]. Prevention of liver disease has become an important task for public health authorities in the absence of the discovery of an actual curative therapeutic agent. Numerous studies have demonstrated that oxidative stress is a mediator of acute and chronic liver injuries [2], [3], [4]. In addition, loss of balance between the antioxidant defense system and free radicals in the body can trigger inflammation and may lead to chronic diseases such as liver and cardiovascular diseases as well as diabetes and cancer [5], [6]. Therefore, the use of antioxidants from herbal medicines or functional foods is a reasonable treatment strategy for inhibiting inflammation and oxidative damage to reduce the incidences of such diseases.

Medicine food homology (藥食同源 yào shí tong yuán) means that food and traditional Chinese medicine originated at the same time in ancient China. Based on this concept, medicine food homology materials are considered a treasure house of functional factors for current functional foods [7]. Ginseng essence (GE) is an herbal formulation comprising four Chinese Materia Medica (中藥 zhōng yào) plants including ginseng (人參 rén shēn, Panax ginseng), American ginseng (西洋參 xī yáng shēn, Panax quinquefolius), lotus seed (蓮子 lián zǐ, Nelumbo nucifera), and the lily bulb (百合 bǎi hé, Lilium longiflorum). They are allowed to be used not only as traditional Chinese medicine but also as food ingredients in Taiwan. Previous studies have indicated that ginseng and American ginseng as well as their main active compounds the ginsenosides have a number of biological benefits including hepatoprotective [8], anti-inflammatory [9], antidiabetic [10], and tumor growth reduction [11]. Lotus seeds have been found to have hepatoprotective [12], blood sugar lowering [13], anti-inflammatory, and antioxidative activities, and the ability to prevent diabetes [14]. In addition, a few studies have shown that lily reduces inflammation [15], prevents cancer [16], inhibits fungal growth [17], and inhibits oxidative reactions [18]. Therefore, we hypothesized that the herbal formulation GE may have therapeutic potential for the prevention of liver injury via free radical scavenging as well as anti-inflammatory activities.

Carbon tetrachloride (CCl4) is a well-known hepatotoxin, which is widely used to induce acute toxic liver injury in animals. Numerous studies have shown that CCl4 is metabolized by the cytochrome P450 enzyme system to yield reactive metabolic products including trichloromethyl free radicals, which can initiate the process of lipid peroxidation and ultimately result in the overproduction of reactive oxygen species (ROS) and hepatocyte injuries [19], [20]. The rat model of CCl4-induced liver injury is well established and is one of the methods for the evaluation of hepatoprotective agents recommended by the Ministry of Health and Welfare, Taiwan. In addition, silymarin (Silybum marianum) is an herbal product containing a mixture of flavonolignan isomers. Silymarin is used as a positive control in the animal model because numerous studies have shown that it can prevent CCl4-induced lipid peroxidation and hepatotoxicity by decreasing the metabolic activation of CCl4 and acting as a chain-breaking antioxidant [21], [22], [23]. Therefore, the aim of this study was to investigate whether GE can protect the rat liver against CCl4-induced oxidative damage and inflammation. In this study, male Wistar rats were treated with GE [0.625 g/kg body weight (bw)/d, 1.25 g/kg bw/d, and 3.125 g/kg bw/d] or silymarin (positive control, 0.5 g/kg bw/d) for 9 wk. After the 1st wk of treatment, rats were gavaged with 20% CCl4 at 1.5 mL/kg bw two times/wk to induce liver injury. After treating the animals, serum biochemical and antioxidant enzyme levels were determined, and histopathological observation of hepatic inflammation and fibrosis was performed to assess the hepatoprotective effect of GE against CCl4-induced liver injury in rats.

Materials and methods

Preparation of GE

GE was obtained from Quaker Co., Ltd. (Taoyuan, Taiwan) and contained a mixture of P. quinquefolius, P. ginseng, N. nucifera, and L. longiflorum at a ratio of 1.66:1:1:1 (dry weight). The mixture was extracted with steam at 105°C for 30 min, cooled at 8°C for 12 h, and then filtered two times at 50°C. The filtrate was then freeze-dried to a powder, which was used to prepare different doses of GE in 0.5% carboxymethyl cellulose for the animal experiments.

Treatment of animals

Seventy-two male Wistar rats (weight: 240–260 g; age: 7 wk old) were obtained from BioLASCO Co., Ltd. (Yilan, Taiwan). All animals were handled in accordance with the guidelines of the National Taiwan University Animal Care Committee, which approved the study (Approval Number: NTU-99-EL-98). Standard experimental conditions were as follows: temperature, 22 ± 3°C; humidity, 50–70%; and a 12-h light/dark cycle. After 1 wk of acclimatization, the rats were randomly divided into six groups of 12 rats each including the control (normal control); CCl4 (negative control); CCl4 with silymarin (CCl4 + silymarin); and CCl4 with low-, medium-, and high-dose GE (CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE, respectively). The CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups were orally treated with silymarin (0.5 g/kg bw/d), LGE, MGE, and HGE (0.625 g/kg bw/d, 1.25 g/kg bw/d, and 3.125 g/kg bw/d), respectively, whereas the control and CCl4 groups were orally treated with equal volumes of the vehicle (0.5% carboxymethyl cellulose). After 1 wk of treatment, rats in the CCl4, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups were further administered 20% CCl4 (1.5 mL/kg bw, two times a week) for 8 wk to induce hepatic fibrosis, whereas rats in the control group were administered equal volumes of the vehicle (olive oil). Blood samples were then collected from the inferior vena cava of the rats, and each liver was isolated and stored at −80°C until further analysis. Schematic diagrams are shown in Fig. 1, which presents the design for the control, negative control (CCl4), and treatment groups (CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE).

Fig. 1

Schematic diagrams showing the design for studying protective activity of ginseng essence on carbon tetrachloride (CCl4)-induced liver injury in rats. Treatments of animals are detailed in the “Materials and Methods” section. Ac, acclimatization; CMC, carboxymethyl cellulose; HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Phytochemical analysis

First, 1 g of freeze-dried GE powder was sonicated in 2 mL of 70% methanol for 1 h to obtain the extract, which was centrifuged at 6,000 rpm at 4°C for 30 min. The supernatant was then collected, filtered using a 0.22-μm syringe filter, and the filtrate was analyzed using high-performance liquid chromatography (HPLC). Qualitative analysis of the major active components (i.e., ginsenosides) in GE was further performed using HPLC (Jasco LC-Net II/ADC and Jasco PU-2089 Plus Quaternary gradient pump, Tokyo, Japan). The HPLC chromatographic conditions were maintained according to previous reports, but with slight modifications [24], [25]. The HPLC procedure was carried out on a Luna C18 column (5-μm pore size, 250 × 4.6 mm inner diameter; Scientific Hightek Co., Taipei, Taiwan) using a gradient solvent system consisting of phosphate buffer (Solvent A, pH 5.82) and acetonitrile (Solvent B). The two-solvent system was run as follows: 20% B (0.01 min), 20.3% B (25 min), 26.8% B (28 min), 26.8% B (38 min), 31% B (48 min), 31% B (58 min), 35.6% B (68 min), 50% B (78 min), 95% B (83 min), 95% (88 min), 20.3% B (90 min), and 20.3% B (95 min). The peaks were recorded using a UV/Visible detector (Jasco UV-2075 Plus) at 202 nm, and the solvent flow rate was maintained at 1.0 mL/min.

Serum biochemistry

To assess the liver damage in the rats, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and albumin, total cholesterol (TC), and triglyceride (TG) levels were determined using SPOTCHEM EZ reagent strips (Arkray, Inc., Kyoto, Japan).

Histological analysis

For the histological examination, the anterior portions of the left lateral lobe of the rat livers were sectioned, fixed in 10% neutral-buffered formalin, embedded in paraffin, and sliced into 5-μm sections. The sections were then hematoxylin and eosin or Masson's trichrome stained. A blinded histological assessment of the liver sections was then performed by a veterinary pathologist at the Graduate Institute of Veterinary Pathobiology of the National Chung Hsing University, Taiwan. Histological changes were evaluated in nonconsecutive histological fields, randomly chosen at a magnification of 100×.

Hepatic antioxidant enzyme activities and total glutathione content

The frozen liver tissue was homogenized, centrifuged, and collected as previously described [26]. The resulting supernatant was then used to determine total glutathione content and antioxidant enzymatic activities including glutathione peroxidase (GPx), glutathione reductase (GRd), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) using Cayman assay kits (Cayman, MI, USA).

Immunohistochemistry

Immunohistochemical analysis of the rat livers was performed as previously described with slight modifications [27]. In brief, the rats were killed, and their livers were trimmed into a strip, soaked in 10% formalin, embedded in paraffin, and then sectioned. The liver sections were dewaxed, hydrated, subjected to heat-induced antigen retrieval, blocked in blocking buffer, and then incubated overnight at 4°C with an anti-alpha-smooth muscle actin (α-SMA) antibody (1:100; Dako, Denmark, Europe). The sections were then washed and further incubated with Super Enhancer and a poly-horseradish-conjugated reagent. The color was developed by incubating the sections with the 3,3′-diaminobenzidine and substrate reaction mixtures (1:38) as well as with hematoxylin. After washing the sections with water, the specific staining was visualized using light microscopy.

Statistical analysis

All the experimental data were represented as the mean ± standard deviation. The statistically significant differences in the data were analyzed using a one-way analysis of variance followed by Duncan multiple comparison test using statistical analysis software (SAS) version 9.2 (Cary, NC, USA). Differences were considered significant when p values are less than 0.05.

Results and discussion

Ginsenoside content of GE

Previous studies have indicated that ginsenosides Rb1 [28], Rg1 [8], and Rg3 [29] are the active components of ginseng with hepatoprotective effect. Therefore, in this study, we performed HPLC analysis to determine the composition of ginsenosides in the GE by comparing its retention time peaks with those of the reference ginsenoside standards (Fig. 2). The quantitative results of the chromatographic analysis revealed that GE contained ginsenosides Rg1, Re, Rb1, Rc, Rd, and Rg3 at levels of 31.74 ppm, 15.57 ppm, 52.79 ppm, 11.24 ppm, 11.69 ppm, and 3.24 ppm, respectively. Therefore, we reasonably presumed that these ginsenosides are the main active ingredients in the GEs that exhibit liver protection.

Fig. 2

The chromatogram of HPLC showing ginsenosides in ginseng essence including (1) ginsenoside Rg1; (2) ginsenoside Re; (3) ginsenoside Rb1; (4) ginsenoside Rc; (5) ginsenoside Rd; (6) ginsenoside Rg3. HPLC chromatographic conditions are detailed in the “Materials and Methods” section.

Changes in rat body and organ weights

A decrease in body weight is usually regarded as direct evidence of toxic injury in rodents. Administration of CCl4 induces physiological changes in the body of organisms that may slow body weight gain and even cause a decrease [30]. Table 1 shows that the final body weight of the CCl4 group was significantly lower than that of the control group. By contrast, the final body weight of the CCl4 + silymarin, CCl4 + MGE, and CCl4 + HGE groups was significantly higher than that of the CCl4 group (p < 0.05). The result indicates that changes in body weight were induced in rats administered CCl4. However, supplementation with MGE and HGE inhibited the decrease in body weight, and the result was similar to that of the silymarin group. Therefore, GE might inhibit the weight loss caused by CCl4 in rats. The results of the organ weight determinations shown in Table 1 revealed that the absolute liver weight of all CCl4-treated groups including the CCl4, CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups was significantly higher than that of the control group (p < 0.05). By contrast, the relative liver weight of the CCl4 + silymarin and CCl4 + HGE groups was significantly lower than that of the CCl4 group (p < 0.05). There was no significant difference in absolute spleen weight between all six groups, whereas the relative spleen weight of the CCl4 group was significantly higher than that of the other groups (p < 0.05). The absolute kidney weight of all the CCl4-treated groups was significantly higher than that of the control group (p < 0.05). However, the relative kidney weight of the CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups was significantly lower than that of the CCl4 group (p < 0.05). Evidence of change during the assessment of organ weight is often considered an important indicator of organ damage, and therefore can be used to evaluate responses to toxicants [31]. Damage to the liver could cause liver cell microvilli and sinusoidal endothelial cell fenestrae to disappear, alter the structure of the smooth endoplasmic reticulum, inactivate hepatic stellate cells (HSCs) and Kupffer cells, increase collagen content, result in accumulation of fibers in the liver, and then cause changes in liver weight [32]. Liver fibrosis can result in the blockage of blood flow to the liver and increase in portal pressure, which could lead to the retention of blood by the spleen, and thereby cause splenomegaly [33].

Table 1

Effects of ginseng essence on body and organ weights of rats with carbon tetrachloride-induced liver injury

Group1)	Initial weight	Final weight	Liver weight	Relative liver weight	Spleen weight	Relative spleen weight	Kidney weight	Relative kidney weight
Control	291 ± 9a	448 ± 29a	14.43 ± 2.45c	3.27 ± 0.44c	0.93 ± 0.15a	0.22 ± 0.03b	2.62 ± 0.23b	0.59 ± 0.03c
CCl4	295 ± 10a	398 ± 7c	17.84 ± 1.82a	4.06 ± 0.33a	1.02 ± 0.14a	0.30 ± 0.04a	2.90 ± 0.19a	0.94 ± 0.11a
CCl4 + silymarin	287 ± 13a	423 ± 17b	16.42 ± 1.42ab	3.53 ± 0.66bc	1.07 ± 0.25a	0.24 ± 0.03b	2.69 ± 0.42ab	0.68 ± 0.07b
CCl4 + LGE	286 ± 6a	415 ± 30bc	16.55 ± 1.05ab	3.84 ± 0.26ab	0.98 ± 0.08a	0.24 ± 0.03b	2.74 ± 0.09ab	0.66 ± 0.05b
CCl4 + MGE	292 ± 7a	424 ± 23b	16.76 ± 1.36ab	3.87 ± 0.37ab	1.07 ± 0.05a	0.24 ± 0.01b	2.79 ± 0.13ab	0.65 ± 0.02b
CCl4 + HGE	291 ± 6a	416 ± 19bc	15.99 ± 1.08b	3.59 ± 0.24bc	0.96 ± 0.11a	0.22 ± 0.02b	2.69 ± 0.28ab	0.65 ± 0.04b

Data are represented as the mean ± standard deviation (n = 12). Values with different superscripts within the same column are significantly different among groups according to a one-way analysis of variance coupled with Duncan multiple test (p < 0.05).

Control, vehicle (0.5% CMC + olive oil); CCl4, 20% CCl4; CCl4 + silymarin, 20% CCl4 + silymarin 0.5 g/kg bw/d; CCl4 + LGE, 20% CCl4 + GE 0.625 g/kg bw/d; CCl4 + MGE, 20% CCl4 + GE 1.25 g/kg bw/d; CCl4 + HGE, 20% CCl4 + GE 3.125 g/kg bw/d. bw, body weight; CMC, carboxymethyl cellulose; CCl4, carbon tetrachloride; HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Our study results show that after chronic administration of CCl4, the absolute and relative weights of the liver, spleen, and kidney were significantly higher than those of the normal control animals. However, after 8-wk supplementation with GE, the relative liver weight of the high-dose group (CCl4 + HGE) was significantly lower than that of the negative control group (CCl4) and was close to that of the normal control. Furthermore, the relative spleen and kidney weights in all three GE-treated groups (CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE) were significantly lower than those of the negative control group. These results imply that supplementation with GE may significantly improve the swelling and inflammation induced by CCl4 in the liver, spleen, and kidney of rats.

Serum biochemical analysis

Previous studies have demonstrated that once the liver is exposed to CCl4, ALT and AST are released and flow into the bloodstream. The notable increase in serum ALT and AST levels is considered an indicator of liver injury [34]. Figs. 3A and 3B show that compared with the control, administration of CCl4 strongly elevated both serum ALT and AST levels of rats (control and CCl4, 41 ± 5 IU/L and 1,387 ± 216 IU/L, and 110 ± 15 IU/L and 1,511 ± 459 IU/L, respectively). However, the levels of serum ALT and AST of the silymarin- and GE-treated groups were significantly lower than those of the control group (ALT: CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups, 766 ± 295 IU/L, 857 ± 314 IU/L, 784 ± 201 IU/L, and 844 ± 379 IU/L, respectively; AST: CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups, 1061 ± 421 IU/L, 961 ± 415 IU/L, 899 ± 139 IU/L, and 980 ± 531 IU/L, respectively, p < 0.05). Therefore, the results suggest that GE has hepatoprotective effects against CCl4-induced liver injury in rats.

Fig. 3

Effects of ginseng essence on serum (A) alanine aminotransferase (ALT), (B) aspartate transferase (AST), and (C) albumin levels of rats with carbon tetrachloride (CCl4)-induced liver injury. Data are represented as the mean ± standard deviation (n = 12). Values with different superscripts are significantly different among groups according to a one-way analysis of variance coupled with Duncan multiple test (p < 0.05). HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

In addition, Fig. 3C shows that the serum albumin level of CCl4-treated rats obviously decreased compared with that of the controls (23.5 ± 2.6 g/L and 33.2 ± 1.2 g/L, respectively). Furthermore, supplementation with GE induced serum albumin levels in the CCl4 + MGE and CCl4 + HGE groups that were close to those of the CCl4 + silymarin group (26.6 ± 2.4 g/L and 26.7 ± 3.1 g/L vs. 27.1 ± 2.2 g/L, respectively) and significantly higher than those of the CCl4 group (p < 0.05). Previous studies have found that CCl4 decreased serum total protein and albumin in carps [35], inhibited protein synthesis, and reduced ribosomal RNA methylation [36]. Administration of CCl4 could inhibit the synthesis of albumin and other proteins in the liver, and result in significantly lower serum albumin level [37]. Moreover, administration of silymarin (0.5 g/kg bw and 1.0 g/kg bw) in the diet significantly reduced the induction of CCl4-induced liver injury, and this effect may be mediated by an increase in ribosome formation and stimulation of DNA and protein synthesis [38], [39]. Accordingly, the results shown in Fig. 3 suggest that GE exerted hepatoprotective effects by inhibiting the CCL4-induced increase in serum ALT and AST levels and a decrease in serum albumin content.

The effects of GE administration on serum and liver TG and TC levels in CCl4-treated rats are shown in Fig. 4. The result shows that the serum TG level of the CCl4 group (51 ± 5 mg/dL) was significantly lower than those of the CCl4 + silymarin, GE-treated CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups (74 ± 25 mg/dL, 70 ± 19 mg/dL, 73 ± 22 mg/dL, and 71 ± 17 mg/dL, respectively, p < 0.05). Furthermore, the serum TC and liver TC and TG levels of the CCl4 group were significantly higher than those of the silymarin- and GE-treated groups (serum TC: CCl4, CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups, 85 ± 15 mg/dL, 62 ± 14 mg/dL, 60 ± 20 mg/dL, 59 ± 16 mg/dL, and 59 ± 15 mg/dL, respectively; liver TC: CCl4, CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups, 62 ± 15 mg/dL, 33 ± 8 mg/dL, 39 ± 10 mg/dL, 41 ± 17 mg/dL, 37 ± 13 mg/dL, respectively; liver TG: CCl4, CCl4 + silymarin, CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups, 28 ± 7 mg/dL, 20 ± 4 mg/dL, 22 ± 5 mg/dL, 22 ± 6 mg/dL, and 22 ± 7 mg/dL, respectively, p < 0.05). These results indicate that CCl4 inhibited the secretion of TG, and thereby enhanced fat accumulation. CCl4 induces an increase in hepatic TG levels, which causes its accumulation in the liver, leading to hepatomegaly. Previous studies have proposed various hypotheses to explain the mechanism of fatty liver induction by CCl4. Recknagel et al [40] demonstrated that CCl4 affected liver ribosomal division and protein synthesis, as well as inhibited the secretion of lipoproteins. In addition, CCl4-triggered lipid accumulation and inactivation of metabolism-related enzymes have been found to reduce liver cytochrome P450 content in vivo and in vitro [41], [42]. Administration of CCl4 in rats for 6 wk increased liver TG but decreased it in the serum [43], and increased cholesterol in the liver and serum [44]. Supplementation with GE inhibits CCl4-induced lipid accumulation in the liver, which implies that GE might play an important role in lipoprotein synthesis and lipid transport by attenuating the inactivation of metabolizing enzymes.

Fig. 4

Effects of ginseng essence on (A) serum total cholesterol (TC), (B) serum triglyceride (TG), (C) liver TC, and (D) liver TG following carbon tetrachloride (CCl4)-induced liver injury in rats. Data are represented as the mean ± standard deviation (n = 12). Values with different superscripts are significantly different among groups according to a one-way analysis of variance coupled with Duncan multiple test (p < 0.05). HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

In vivo antioxidant defense system

All aerobic organisms possess similar inherent and effective in vivo antioxidant defense systems, which include the antioxidant enzymes (SOD, GPx, and CAT) and nonenzymatic antioxidants such as glutathione. Antioxidant enzymes play an important role in the prevention of free radical-induced oxidative damage as well as reduction of antioxidant activities and capacity in vulnerable organisms [45]. Administration of CCl4 is known to lead to the generation of trichloromethyl free radicals and overproduction of ROS, leading to hepatic injuries in rats. The relationship between hepatoprotective effect and antioxidant scavenging activity is highly correlated. Table 2 shows the effects of GE on liver glutathione levels and antioxidant enzyme activities in CCl4-treated rats. Supplementation with GE resulted in significantly higher glutathione, GPx, GRd, GST, SOD, and CAT levels in the extract-treated groups than in the CCl4 group (p < 0.05). The enzyme levels of the GE-treated groups were close to those of the silymarin-treated group.

Table 2

Effects of ginseng essence on liver glutathione and antioxidant enzyme activities of rats with carbon tetrachloride-induced liver injury

Group1)	Glutathione (nmoL/mg protein)	GPx (nmoL/min/mg protein)	GRd (nmoL/min/mg protein)	GST (nmoL/min/mg protein)	SOD (U/mg protein)	CAT (U/mg protein)
Control	71 ± 8b	54 ± 8a	40 ± 8bc	493 ± 29a	1,446 ± 323a	129 ± 16a
CCl4	43 ± 9c	35 ± 11b	28 ± 6c	354 ± 11b	535 ± 87b	53 ± 13b
CCl4 + silymarin	97 ± 8a	61 ± 13a	54 ± 12ab	524 ± 18a	1,292 ± 307a	129 ± 24a
CCl4 + LGE	98 ± 5a	51 ± 18a	50 ± 20ab	460 ± 58a	1,368 ± 252a	136 ± 39a
CCl4 + MGE	102 ± 14a	52 ± 8a	64 ± 27a	528 ± 50a	1,401 ± 167a	152 ± 30a
CCl4 + HGE	99 ± 6a	51 ± 6a	52 ± 14ab	570 ± 33a	1,247 ± 244a	131 ± 31a

Data are represented as the mean ± standard deviation (n = 12). Values with different superscripts within the same column are significantly different among groups according to a one-way analysis of variance coupled with Duncan multiple test (p < 0.05).

Control, vehicle (0.5% CMC + olive oil); CCl4, 20% CCl4; CCl4 + silymarin, 20% CCl4 + silymarin 0.5 g/kg bw/d; CCl4 + LGE, 20% CCl4 + GE 0.625 g/kg bw/d; CCl4 + MGE, 20% CCl4 + GE 1.25 g/kg bw/d; CCl4 + HGE, 20% CCl4 + GE 3.125 g/kg bw/d. bw, body weight; CAT, catalase; CCl4, carbon tetrachloride; CMC, carboxymethyl cellulose; GPx, glutathione peroxidase; GRd, glutathione reductase; GST, glutathione S-transferase; HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence; SOD, superoxide dismutase.

In addition, glutathione is the major noncellular enzymatic antioxidant, which directly or indirectly scavenges free radicals effectively via enzymatic reactions. Previous studies have found that acute administration of CCl4 depletes glutathione contents in mammals. The mechanism of CCl4-induced liver injury showed that conjugation with glutathione plays a critical role in reducing the metabolism of toxins [46]. In this study, treatment with GE significantly inhibited the reduction of glutathione levels. These results indicate that the hepatoprotective effects of GE may be exerted by reducing ROS generation. Furthermore, the attenuation of oxidative stress by GE was similar to that shown by silymarin, which is consistent with previous studies [22], [23].

Histopathological analysis

Hepatic inflammation and fibrosis are common outcomes of liver damage. Hematoxylin and eosin staining is performed to observe CCl4-induced physiological changes in the rat liver, whereas Masson's trichrome staining is a commonly used collagen staining method for liver fibrosis detection [47]. Fig. 5, Fig. 6 show the histological analyses of liver inflammation and fibrosis, respectively. The liver portal peripheral inflammation (vacuoles) and fibrosis were evaluated and scored by a blinded veterinary pathologist. As shown in Table 3, scores of the liver portal peripheral inflammation in the CCl4 + LGE and CCl4 + HGE groups (2.60 ± 1.07 and 2.40 ± 0.97, respectively) were slightly lower than those of the CCl4-treated group. In addition, the CCl4 + MGE group showed significantly lower scores than the CCl4 group (2.10 ± 1.29 and 3.20 ± 0.79, respectively, p < 0.05). The liver fibrosis scores of the GE-treated CCl4 + LGE, CCl4 + MGE, and CCl4 + HGE groups (1.70 ± 0.95, 1.10 ± 0.74, and 1.60 ± 0.84, respectively) were close to those of silymarin + CCl4 and significantly lower than those of the control (1.00 ± 0.82 and 2.40 ± 0.70, respectively, p < 0.05). Therefore, GE obviously inhibited CCl4-induced hepatic inflammation and fibrosis in rats.

Fig. 5

Pathological examination of effects of ginseng essence on liver inflammation in rats with carbon tetrachloride (CCl4)-induced liver injury after 8-week treatment. Livers were stained with hematoxylin and eosin and visualized at 100× magnification. HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Fig. 6

Pathological examination of effects of ginseng essence on liver fibrosis in rats with carbon tetrachloride (CCl4)-induced liver injury after 8-week treatment. Livers were stained with Masson's trichrome and visualized at 100× magnification. HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Table 3

Scoring of the effect of ginseng essence on liver inflammation and fibrosis of carbon tetrachloride-induced liver injury in rats1)

Group2)	Histopathological score of liver
	Inflammation score	Fibrosis score
Control	0.00 ± 0.00c	0.00 ± 0.00c
CCl4	3.20 ± 0.79a	2.40 ± 0.70a
CCl4 + silymarin	2.50 ± 0.53a	1.00 ± 0.82b
CCl4 + LGE	2.60 ± 1.07ab	1.70 ± 0.95b
CCl4 + MGE	2.10 ± 1.29b	1.10 ± 0.74b
CCl4 + HGE	2.40 ± 0.97ab	1.60 ± 0.84b

Histological indices of hepatic vacuolization and necrosis were quantified by a blinded veterinary pathologist based on numerical scoring of liver biopsy specimens. Liver damage was graded on a scale of 0–4 as follows: 0 = none, 1 = slight, 2 = mild, 3 = moderate, and 4 = remarkable. Data are represented as the mean ± standard deviation (n = 12). Values with different superscripts within the same column are significantly different among groups according to a one-way analysis of variance coupled with Duncan multiple test (p < 0.05).

Control, vehicle (0.5% CMC + olive oil); CCl4, 20% CCl4; CCl4 + silymarin, 20% CCl4 + silymarin 0.5 g/kg bw/d; CCl4 + LGE, 20% CCl4 + GE 0.625 g/kg bw/d; CCl4 + MGE, 20% CCl4 + GE 1.25 g/kg bw/d; CCl4 + HGE, 20% CCl4 + GE 3.125 g/kg bw/d. bw, body weight; CMC, carboxymethyl cellulose; CCl4, carbon tetrachloride; HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Immunohistochemical staining of α-SMA

α-SMA can be used as a specific marker to assess the activation of HSCs, which were stained red when the reaction was positive. As shown in Fig. 7, no visible positive reaction was observed in normal controls, indicating that no HSCs were activated. Compared with the control group, the other CCl4-treated group showed a significant increase in the number of activated HSCs. The CCl4 group showed the highest positive response, indicating that the number of activated HSCs was significantly higher than it was in the GE-treated groups. The results showed that treatment with silymarin as well as LGE, MGE, and HGE reduced the number of HSCs activated by CCl4.

Fig. 7

Effects of ginseng essence on activation of hepatic stellate cells of rats with carbon tetrachloride (CCl4)-induced liver injury after 8-week treatment. Immunohistochemical staining of alpha-smooth muscle actin was performed on liver sections and visualized at 100× magnification. HGE, high-dose ginseng essence; LGE, low-dose ginseng essence; MGE, medium-dose ginseng essence.

Fibrosis and cirrhosis of the liver can be observed following the accumulation of extracellular matrix, which leads to the formation of excessive collagen by activation of cells such as HSCs and fibroblasts [48]. In pathological progress, the activation of HSCs could increase the synthesis of extracellular matrix proteins, which changes the structure of liver sinusoid endothelial cells causing necrosis [49]. Although the mechanism mediating hepatic fibrosis is still not fully understood, maintaining the shape of HSCs may prevent or mitigate the development of liver fibrosis. It has been shown that treatment of rats with CCl4 can cause collagen accumulation in the liver and increase the expression of α-SMA, which implies that HSCs have a tendency to induce liver fibrosis. Therefore, a decrease in the positive reaction of α-SMA could indicate the inactivation of HSCs and mitigation of fibrosis. In summary, the results showed that GE might have effectively inhibited liver fibrosis by reducing the activation of HSCs in CCl4-treated rats.

In conclusion, we found that GE contains ginsenosides including Rg1, Re, Rb1, Rc, Rd, and Rg3, which could exert hepatoprotective effects. Furthermore, the results of the animal experiments demonstrate that GE significantly reduced the liver injury induced by CCl4 in rats by ameliorating the oxidative stress, reducing inflammation, and inhibiting the activation of HSCs. Therefore, GE could be a promising hepatoprotective herbal formulation for future development of phytotherapy.

Conflicts of interest

All authors have no conflicts of interest to declare.