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Revista Brasileira de Farmacognosia

Print version ISSN 0102-695XOn-line version ISSN 1981-528X

Rev. bras. farmacogn. vol.26 no.3 Curitiba May/June 2016

http://dx.doi.org/10.1016/j.bjp.2016.01.001 

Original articles

The essential oil of Artemisia capillaris protects against CCl4-induced liver injury in vivo

Qinghan Gaoa  b  1

Xin Zhaoa  1

Lei Yina  1

Yuanbin Zhanga 

Bing Wanga 

Xiuli Wua 

Xinhui Zhanga 

Xueyan Fua  c  * 

Weihong Sund  * 

aSchool of Pharmacy, Ningxia Medical University, Yinchuan, China

bSchool of Public Health, Ningxia Medical University, Yinchuan, China

cKey Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education (Ningxia Medical University), Yinchuan, China

dGeneral Hospital of Ningxia Medical University, Yinchuan, China

Abstract

To study the hepatoprotective effect of the essential oil of Artemisia capillaris Thunb., Asteraceae, on CCl4-induced liver injury in mice, the levels of serum aspartate aminotransferase and alanine aminotransferase, hepatic levels of reduced glutathione, activity of glutathione peroxidase, and the activities of superoxide dismutase and malondialdehyde were assayed. Administration of the essential oil of A. capillaris at 100 and 50 mg/kg to mice prior to CCl4 injection was shown to confer stronger in vivo protective effects and could observably antagonize the CCl4-induced increase in the serum alanine aminotransferase and aspartate aminotransferase activities and malondialdehyde levels as well as prevent CCl4-induced decrease in the antioxidant superoxide dismutase activity, glutathione level and glutathione peroxidase activity (p < 0.01). The oil mainly contained β-citronellol, 1,8-cineole, camphor, linalool, α-pinene, β-pinene, thymol and myrcene. This finding demonstrates that the essential oil of A. capillaris can protect hepatic function against CCl4-induced liver injury in mice.

Keywords Essential oil; Carbon tetrachloride; Hepatoprotective effects; In vivo; H&E; GC–MS

Introduction

Liver disease, a common disorder caused by viral hepatitis, alcoholism, liver-toxic chemicals, unhealthy dietary habits and environmental pollution, is a global concern (Papay et al., 2009). However, medical treatment for this disease is often hard to administer and has a confined effect. Traditional Chinese herbal medicines, which underlie numerous prescriptions used to treat liver diseases, are still widely used by the Chinese (Zhao et al., 2014). Artemisia capillaris Thunb., Asteraceae, according to the Bencao Gangmu, the most famous records of Chinese Traditional Medicine, has been widely used as a medicine to clear heat, promote diuresis and remove jaundice and has also been used as a flavor in beverages, vegetables, and pastries because of its particular fragrance. A. capillaris has been regarded as a type of Chinese folk medicine and food by a growing number of people. Therefore, there have been considerable efforts to develop useful herbal medicines, such as A. capillaris, for the treatment of liver disease.

In recent years, herbal medicines have gained more attention and popularity for the treatment of liver disease because of their safety and efficacy (Ding et al., 2012). A. capillaris has been proven to possess good hepatoprotective activity based on modern pharmacological methods (Han et al., 2006). It is also an important medicinal material in China and is a popular anti-inflammatory (Cha et al., 2009a), choleretic (Yoon and Kim, 2011), and anti-tumor (Feng et al., 2013) herbal remedy.

Phytochemical studies have revealed a number of volatile essential oils, coumarins, and flavonol glycosides as well as a group of unidentified aglycones from A. capillaris (Komiya et al., 1976; Yamahara et al., 1989). The essential oil of A. capillaris (AEO) is one of the main pharmacological active compounds and confers anti-inflammatory (Cha et al., 2009a) and anti-apoptotic properties (Cha et al., 2009b). However, as AEO is one of the main compounds of A. capillaris, the potential hepatoprotective activities of the major constituents from A. capillaris should be explored.

In this study, the protective effect of AEO on carbon tetrachloride (CCl4)-induced hepatotoxicity was evaluated by biochemical methods, such as hepatic reduced glutathione (GSH), malondialdehyde (MDA) levels, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity, as well as the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum. The extent of CCl4-induced liver injury was also analyzed through histopathological observations, accompanied with phytochemical analysis by GC–MS to identify the constituents of AEO.

Materials and methods

Drugs and chemicals

The essential oil of Artemisia capillaris Thunb., Asteraceae, was obtained from Kangshen Natural oils Co., Ltd (Jishui, Jiangxi Province, China). CCl4 was purchased form Damao Chemical Company (Tianjin, China). Bifendate tablets were purchased from Yunpeng, Shanxi Pharmaceutical Co., Ltd. (no. A130602). The diagnostic kits used for the determination of ALT, AST, SOD, MDA, GSH and GSH-Px were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China).

AEO analysis

AEO was analyzed by gas chromatography–mass spectrometry (GC/MS) using a Shimadzu QP2010 plus GC with Rxi-5Sil MS (30 m × 0.25 mm; 0.25 µm film thickness). The GC/MS was run under the following conditions: a fused-silica capillary column with helium at 1.60 ml/min and an injector at 250 °C. The GC oven temperature was initially held at 40 °C for 5 min. Thereafter, the temperature was raised with a gradient of 3 °C min−1 until it reached 230 °C. This temperature was held for 3 min. Finally, the temperature was then raised with a gradient of 1 °C min−1 until 260 °C and again held for 10 min. Mass spectra were taken at 70 eV from 33 to 500 Da. Identification of the compounds was based on the comparison of the mass spectral data on a computer matched with NIST (similarity index >85%) and those described in the literature (Sylvestre et al., 2006; Cheng et al., 2008; Ait-Ouazzou et al., 2012; Argyropoulou and Skaltsa, 2012; SĆavar et al., 2012; Ben Mansour et al., 2013; Chen et al., 2013; Gouveia and Castilho, 2013; Murugan and Mallavarapu, 2013; Singh et al., 2013; Bagheri et al., 2014; Da Silva et al., 2014; Desai et al., 2014; Pandey et al., 2014; Qi et al., 2014; Rather et al., 2014; Sadgrove et al., 2014; Sereshti et al., 2014; Tao et al., 2014; Tian et al., 2014). The identification of compounds was performed according to their retention indices relative to C8–C24 n-alkanes and MS.

Animals

Male ICR mice at 6–8 weeks of age (18–22 g) were purchased from the Medicine Experiment Animal Center of Ningxia Medicine University. The animals were allowed to acclimatize for two days to animal room conditions and were maintained on a standard pellet diet and water ad libitum. The food was withdrawn on the day before the experiment, but the animals were allowed free access to water. All animals were cared for in compliance with the Guide for the Care and Use of Laboratory Animals (1996). The experimental procedures were approved by our institutional animal research ethics committee (approval number: SYXK (NING) 2011-0001).

Rodent model design

The animals were randomly divided into five groups of fifteen mice each. The control group and model group mice were gavaged with sesame oil for 6 days. Positive control group mice were gavaged with bifendate tablets (BT, 10 mg/kg) for 6 days. The experimental groups were treated with 100 mg/kg and 50 mg/kg AEO dissolved in sesame oil for 6 days. On day 6, the control group was treated with sesame oil, and all of the other groups were treated with a single dose of 0.2% CCl4 in sesame oil (10 ml/kg) by intraperitoneal injection. The mice were then fasted free of water, and blood samples were collected from the retrobulbar vessels; collected blood was centrifuged at 3000 × g for 10 min to separate the serum. Cervical dislocation was performed immediately after withdrawal of blood, and liver samples were promptly removed. One part of the liver sample was immediately stored at −20 °C until analysis, and another part was excised and fixed in a 10% formalin solution; the remaining tissues were stored at −80 °C for histopathological analysis (Wang et al., 2008; Hsu et al., 2009; Nie et al., 2015).

Measurement of the biochemical parameters in the serum

Liver injury was assessed by estimating the enzymatic activities of serum ALT and AST using the corresponding commercial kits according to the instructions for the kits (Nanjing, Jiangsu Province, China). The enzymatic activities were expressed as units per liter (U/l).

Measurement of MDA, SOD, GSH and GSH-Px in liver homogenates

Liver tissues were homogenized with cold physiological saline at a 1:9 ratio (w/v, liver:saline). The homogenates were centrifuged (2500 × g for 10 min) to collect the supernatants for the subsequent determinations. Liver damage was assessed according to the hepatic measurements of the MDA and GSH levels as well as the SOD and GSH-Px activities. All of these were determined following the instructions on the kit (Nanjing, Jiangsu Province, China). The results for MDA and GSH were expressed as nmol per mg protein (nmol/mg prot), and the activities of SOD and GSH-Px were expressed as U per mg protein (U/mg prot).

Histopathological analysis

Portions of freshly obtained liver were fixed in a 10% buffered paraformaldehyde phosphate solution. The sample was then embedded in paraffin, sliced into 3–5 µm sections, stained with hematoxylin and eosin (H&E) according to a standard procedure, and finally analyzed by light microscopy (Tian et al., 2012).

Statistical analysis

The results were expressed as the mean ± standard deviation (SD). The results were analyzed using the statistical program SPSS Statistics, version 19.0. The data were subjected to an analysis of variance (ANOVA, p < 0.05) followed by Dunnett's test and Dunnett's T3 test to determine the statistically significant differences between the values of various experimental groups. A significant difference was considered at a level of p < 0.05.

Results and discussion

Constituents of AEO

Upon GC/MS analysis, the AEO was found to contain 25 constituents eluted from 10 to 35 min, and 21 constituents accounting for 84% of the essential oil were identified (Table 1). The volatile oil contained monoterpenoids (80.9%), sesquiterpenoids (9.5%), saturated unbranched hydrocarbons (4.86%) and miscellaneous acetylene (4.86%). Compared with other studies (Guo et al., 2004), we found abundant monoterpenoids (80.90%) in the AEO. The results showed that the most abundant constituent of AEO is β-citronellol (16.23%). Other major components of AEO include 1,8-cineole (13.9%), camphor (12.59%), linalool (11.33%), α-pinene (7.21%), β-pinene (3.99%), thymol (3.22%), and myrcene (2.02%). The variation in the chemical composition may be related to the environmental conditions that the plant was exposed to, such as mineral water, sunlight, the stage of development and nutrition.

Table 1 Chemical composition of essential oil from Artemisia capillaris Thun. (AEO). 

Compound Rt a RI calc b RI lit c %d
α-Pinene 11.133 928 933 7.21
Camphene 11.340 943 946 1.52
Sabinene 13.269 968 969 1.09
β-Pinene 13.640 975 974 3.99
Myrcene 14.313 987 988 2.02
Iso-cineole 15.612 1012 1012 1.27
p-Cymene 16.025 1020 1020 2.28
Limonene 16.273 1025 1024 2.00
1,8-Cineole 16.424 1028 1026 13.09
Artemisia ketone 17.965 1057 1056 1.44
Terpinolene 19.226 1081 1086 1.30
Linalool 20.112 1098 1095 11.33
trans-Pinocarveol 17.965 1135 1135 1.44
Camphor 22.215 1140 1141 12.59
Terpinen-4-ol 23.998 1176 1174 1.21
α-Terpineol 24.744 1191 1186 1.89
Dodecane 25.173 1199 1200 2.44
β-Citronellol 26.407 1226 1211 16.23
Thymol 29.356 1288 1289 3.22
Longipinene 34.330 1401 1402 2.16
(E)-Caryophyllene 34.813 1413 1417 1.73

aRt – retention time (min) form a linear temperature program.

bRIcalc – retention index calculated for each compound.

cRIlit – retention index obtain from published literatures.

d% – relative abundances from the peak area integration.

As previously suggested, few studies have reported the hepatoprotective effects of some of these major compounds. However, there are findings that indicate that major compounds possess an anti-inflammatory effect, such as thymol (Braga et al., 2006), eucalyptol, limonene, and linalool (Ku and Lin, 2013). β-Myrcene and 1,8-cineole were also found to eliminate TCDD-induced oxidative stress in rats (Ciftci et al., 2011).

Measurement of the biochemical parameters in the serum

CCl4-induced hepatic injury is a commonly used experimental animal model (Johnston and Kroening, 1998). A previous study reported that CCl4 administration is associated with activation by the cytochrome system (e.g., CYP2E1) to form trichloromethyl radicals (CCl3 === Inserir caracter correspondente ao PDF === ). It is generally thought that CCl4 toxicity is due to a reactive intermediate that is generated by its reductive metabolism. This highly reactive intermediate is known to induce lipid peroxidation, oxidative stress, hepatic necrosis and apoptosis (Weber et al., 2003).

In this assay, the results of the hepatoprotective effects of AEO on the enzymatic activities of serum ALT and AST are shown in Fig. 1A and B, respectively. An intraperitoneal injection of CCl4 induced a notable increase in ALT and AST activities in comparison with untreated control mice (p < 0.01); the increased serum activities of ALT and AST enzymes in CCl4 treated mice confirmed the hepatic damage. Conversely, pretreatment with AEO at doses of 100 mg/kg and 50 mg/kg, as well as bifendate tablets, obviously reversed the toxin-induced increase in the ALT and AST levels compared with toxin model group 2 (p < 0.01). When the dose reached 100 mg/kg, the results were as good as BT.

Fig. 1 Effects of oral pretreatment with AEO on CCl4-induced hepatic injury. The results of the biochemical parameters (ALT, AST) and hepatic levels (MDA, SOD, GSH and GSH-Px) are represented as the mean ± SD (n = 15). The mice were pretreated with AEO (100, 50 mg/kg) once daily for 6 consecutive days. The control mice were given sesame oil. Differences between the groups were determined by ANOVA followed by Dunnett's test. *p < 0.05, **p < 0.01 compared to the model group, and # p < 0.05, ## p < 0.01 compared to the control group. 

ALT and AST are important metabolic enzymes of the liver. After CCl4 administration, the serum ALT and AST activities in mice were evaluated. These enzymes normally exist in the cytoplasm, but upon liver injury, they can enter the circulatory system due to toxicity-mediated altered permeability of the cellular membrane (Wills and Asha, 2006). In a previous study, it was reported that administration of CCl4 in mice caused increased ALT and AST activities.

Our data showed that the liver damage induced by CCl4 elevates liver marker enzymes. Elevated activities of serum enzymes, ALT and AST are indicative of cellular leakage and the loss of the functional integrity of the cell membrane in the liver, while pretreatment with AEO effectively decreased the amount of AST and ALT leakage.

Measurement of MDA, SOD, GSH and GSH-Px in liver homogenates

The levels of MDA suggest enhanced peroxidation leading to tissue damage and failure of the antioxidant-defense mechanisms to prevent the formation of excessive free radicals (Cheng et al., 2013; Pareek et al., 2013). However, AEO at 50 and 100 mg/kg could markedly prevent the increase in MDA formation (Fig. 1C), which clearly demonstrated the ability of AEO to relieve lipid peroxidation. MDA is well known to be the most abundant individual aldehyde resulting from lipid peroxidation and is commonly used as an indicator of liver tissue damage involving a series of oxidative chain reactions (Huang et al., 2012). In the present research, mice treated with CCl4 showed a striking increase in MDA levels compared to untreated normal mice (p < 0.01). SOD and GSH-Px are the major enzymes that play an important role in the elimination of toxic metabolites, which are the major cause of the liver pathology caused by CCl4 (Cengiz et al., 2013; Xia et al., 2013). Here, administration of CCl4 to mice sharply decreased the SOD and GSH-Px activities in mouse liver tissues, as evidenced by the inhibition of their enzymatic activities. However, the decrease in these enzymatic activities was significantly elevated by pretreatment with AEO, suggesting that they could protect the three antioxidant enzymes in CCl4-damaged livers. The protective effect of AEO was potent in terms of the SOD and GSH-Px activities, which is most likely related to the elimination of toxic metabolites (Fig. 1D and F). SOD is a manganese-containing enzyme in the mitochondria and converts the dismutation of superoxide anions into hydrogen peroxide (H2O2) (Reiter et al., 2000). GSH-Px is an important enzyme that catalyzes the reduction of H2O2 and hydroperoxides and terminates the chain reaction of lipid peroxidation by removing lipid hydroperoxides from the cell membrane (Ai et al., 2013). Similarly, GSH is an important water-phase antioxidant and an essential cofactor for antioxidant enzymes; it protects the mitochondria against endogenous oxygen radicals (Han et al., 2006). The endogenous antioxidant GSH peroxidase inhibits oxidative stress by quickly removing superoxide radicals (Chaudiere et al., 1999) and plays an important role in clearing intracellular hydrogen peroxide and lipid peroxides. Therefore, the level of GSH in the body is considered to be an important indicator of antioxidative capacity (Campo et al., 2001; Iwamoto et al., 2002). Fig. 1E showed that CCl4 treatment induced a significant decrease in the level of GSH in liver homogenates compared to control livers. The treatment of mice with AEO at 50 and 100 mg/kg significantly increased the hepatic GSH content compared with the CCl4-treated group. This finding indicated that the free radicals released in the liver were effectively scavenged during AEO treatment. All of these data suggest that the hepatoprotective effects of AEO on CCl4-induced liver damage in mice are associated with its capacity for free radical scavenging and antioxidation.

Histopathological examination of mouse livers

Furthermore, the histopathological observations substantiated the biochemical analysis, as presented in Fig. 2. The histology of the liver sections from the normal control group showed normal hepatic cells with a well-preserved cytoplasm and legible nucleus, hepatocytes that were radially arranged around the central vein, and a well-defined sinusoidal line (Fig. 2A). CCl4 caused damage to the hepatic architecture and produced histological changes, such as severe hepatocellular degeneration and necrosis around the central vein, sinusoidal dilatation, loss of cellular boundaries, inflammatory cell infiltration, and cytoplasmic vacuolation, all of which confirmed the successful establishment of liver injury (Fig. 2B). In contrast, CCl4-intoxicated mice pretreated with BT showed near normalization of liver tissues with no significant changes in hepatocytes (Fig. 2C). The liver damage was noteworthy and dose-dependently reduced by pretreatment with AEO at different doses, as indicated by the significant reduction in the number of ballooning-degenerated hepatocytes and significantly decreased necrotic area. In the group pretreated with a low dose of AEO (Fig. 2E), the liver sections showed moderate hypertrophy of hepatocytes with a relatively intact central vein, a shrunken sinusoidal area and reduced inflammatory cells. The administration of a high dose of AEO (Fig. 2D) induced a near-normal appearance, suggesting that AEO at a dose of 100 mg/kg was more effective compared to the dose of 50 mg/kg and that AEO could protect the liver from acute CCl4-induced hepatic damage. This was in good agreement with the results from the serum biochemical markers.

Fig. 2 Effects of AEO on the histological changes of the liver after CCl4 treatment in mice (original magnification 400×): (A) control group; (B) CCl4-intoxicated group (model group); (C) bifendatatum (10 mg/kg) + CCl4; (D) AEO (100 mg/kg) + CCl4; (E) AEO (50 mg/kg) + CCl4

In summary, the results from this study clearly demonstrate that AEO is effective for the prevention of CCl4-induced hepatic injury in mice. This study indicates the potential for the development of AEO as a potential hepatoprotective agent in cases of CCl4 induced acute liver injury.

Acknowledgements

Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (Project No. 2013BAI05B01).

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Received: July 13, 2015; Accepted: January 11, 2016

* Corresponding authors. E-mails:xueyanfu2661@163.com (X. Fu), tungbuo@163.com (W. Sun).

Conflicts of interest

The authors declare no conflicts of interest.

1

These authors equally contributed to this manuscript; they should be considered first author.

Author contributions

QG, LY and XZ contributed to performing the laboratory work. YZ and XZ contributed to the estimation of the chemical composition. BW contributed to the analysis of the data. LY wrote the manuscript. QG and XW contributed to the critical reading of the manuscript. XF and WS designed the study, supervised the laboratory work and contributed to the critical reading of the manuscript. All of the authors have read the final manuscript and approved its submission.

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