The hepatoprotective activities of Kalimeris indica ethanol extract against liver injury in vivo

Abstract Kalimeris indica (L.) Sch. Bip. is a traditional Chinese medicine (TCM) and a portion of food used for cooking in China. It has been demonstrated that an ethanol extract of K. indica has an anti‐inflammatory effect by inhibition of nitric oxide (NO) production on murine macrophage RAW264.7 cells after lipopolysaccharide (LPS) induction. In this study, the hepatoprotective effects of the total phenolics of K. indica (TPK), the total triterpenes of K. indica (TTK), and the total flavones of K. indica (TFK) from ethanol extracts of K. indica were evaluated in Bacille Calmette–Guerin (BCG)/LPS‐induced liver injury in vivo. The treatments of TPK, TTK, and TFK improved liver injury in mice. Additionally, all treatments significantly not only reduced the hepatic malondialdehyde (MDA) content and hepatic total nitric oxide synthase (tNOS) but also induced the hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) activity. The treatments of TPK and TTK significantly reduced the hepatic inducible nitric oxide synthase (iNOS). The treatments of TPK, TTK, and TFK reduced the serum total bilirubin (T‐Bil), and only TFK treatment reduced the serum alanine aminotransferase (ALT). Our results suggest that TPK, TTK, and TFK from ethanol extracts of K. indica might play an essential protective role against BCG/LPS‐induced liver injury in vivo.

Kalimeris indica (L.) Sch. Bip., an agricultural product, belongs to the family Asteraceae (Compositae) and is found in the eastern Asian countries of China, Taiwan, Korea, and Japan, and in America (Wang et al., 2015(Wang et al., , 2017. Kalimeris indica is also named Ma Lan, Ji Er Chang, and Tian Bian Ju (Wang et al., 2017). The plant of K. indica has been used in traditional Chinese medicine (TCM) and as food for cooking (Wang et al., 2015(Wang et al., , 2010. The major clinical applications of K. indica include the treatments of the acute gastric abscess, acute orchitis, blood vomiting, conjunctivitis, cold, diarrhea, and gastric ulcer (Wang et al., 2015(Wang et al., , 2010(Wang et al., , 2017. Chemical investigations of K. indica have been found to exert the sixty compounds or phytochemicals, including flavonoids, triterpenes, phenolics, and polysaccharides. (Wang et al., 2017).
Our previous study has shown the hepatoprotective effects of K. indica on carbon tetrachloride (CCl 4 )-induced acute liver injury in primary cultured hepatocytes (Wang et al., 2010). However, the hepatoprotective effects of the ethanol extracts from K. indica on BCG/LPS-induced liver injury of mice are still unclear. In this study, the underlying hepatoprotective effects were investigated to find the total phenolics of K. indica (TPK), the total triterpenes of K. indica (TTK), and the total flavones of K. indica (TFK) from ethanol extracts of K. indica in BCG/LPS-induced liver injury mice model in vivo.

| Preparation of crude extracts of Kalimeris indica
The air-dried herbs of K. indica (500 g) were treated with 40% ethanol concentration (16.5:1) three times for 2 hr each time. The extracts were evaporated under reduced pressure. The flow rate of purification concentration by macroporous resin (HDP-300) was 0.1 g/ml, and the sampling mount was 2 ml/min. The concentration and volume of elution ethanol were 90% and 2 bed volume (BV). The total flavones of K. indica (TFK) yielded 21.69 g. With rutin as the reference substance, the content of TFK was 56.29%. The air-dried herbs of K. indica (500 g) were crushed and extracted four times with 40% ethanol (1:18) for 1 hr each time. The extracts were evaporated under reduced pressure. The flow rate of purification concentration by macroporous resin (HDP-500) was 0.02 g/ml, and the sampling mount was 2 ml/min. The concentration and volume of elution ethanol were 70% and 2 BV. The total triterpenes of K. indica (TTK) yielded 64.86 g.
With oleanolic acid as the reference substance, the content of TTK is 17.72%. The air-dried herbs of K. indica (500 g) were crushed and extracted six times with 53% ethanol l (1:12) two times for 2 hr each time. The extracts were evaporated under reduced pressure. The flow rate of purification concentration by macroporous resin (HDP-300) was 0.004 g/ml, and sampling mount was 4 ml/min, respectively.
The concentration and volume of elution ethanol were 90% and 3 BV. The total phenolics of K. indica (TPK) were to yield 55.90 g. With chlorogenic acid as the reference substance, the content of TPK was 24.80%. The crude extracts were dried by freeze-drying, as previously described (Wang et al., 2015(Wang et al., , 2017. The procedures of preparation of each K. indica extract can be seen in Figure 2.

| Animal experiments and biochemical assays
A total of 120 male Kunming mice (25 ± 5 g) were obtained from the Experimental Animal Center at Anhui Medical University. The animals were treated in accordance with the "Guide for the Care and Use of Laboratory Animals, 8th edition" and approved by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine. The mice were kept in an environmentally controlled breeding room (temperature 20 ± 2°C, humidity: 60 ± 5%, 12-hr light/dark cycle) and used for our study when they reached 5 weeks of age. All mice had free access to tap water.
They were randomly divided into 12 groups (ten mice per group).
The mice were fed a basal diet for 5 days before being used in this study. All of the mice, apart from those in the mock group (normal mice), After administration, the mice were fasted for 16 hr, and then, blood from the eyeballs and liver was taken. After the blood was left to stand for 45 min, the serum was harvested after centrifugation at 252

| Histological examination
The liver tissue was collected and immediately fixed in 10% formaldehyde. The samples were then dehydrated in graded ethanol (50%-100%) series and cleared in xylene prior to embedding in paraffin. The sections (4-5 μm) were prepared and stained with hematoxylin and eosin (HE) dyes. The areas of necrotic lesions were microscopically evaluated to check the liver injury using a microcomputer image device (MCID) Image analyzer (Imaging Research Inc., St.).

| Statistical analysis
The differences between the mean values of the treatment were determined via one-way analyses of variance (ANOVA) followed by Student's t test using SPSS ver. 21.0. software (SPSS, Inc.), and the values were expressed as the mean ± standard deviation (SD). The level of significance was uniformly set at p < .05.

| Effects of TPK, TTK, and TFK on liver histopathology in mice
The results of HE staining showed that the liver lobule structure of the normal group was clear. The central cells were the hepatocyte plate, and the cytoplasm was deeply stained. There was no inflammatory cell infiltration in the portal area. The HE staining section of the model group found the central vein around the liver ( Figure 4). Liver injury is shown to exert cell swelling and degeneration, partial hepatocyte rupture, massive interstitial hyperemia, large amounts of inflammatory cell infiltration in the portal area, and the surrounding area and large area of flaky necrosis (Kleiner, 2017). The high (600 mg/kg), medium (400 mg/kg), and low dose (200 mg/kg) groups of TPK, TTK and TFK significantly reduced F I G U R E 3 Experimental design of hepatoprotective activities of Kalimeris indica in vivo. The mice were infected by intravenously (I.V.) injecting BCG and then orally (P.O.) treated with K. indica ethanol extracts. After administration, all mice were sacrificed, and the liver tissue and serum were harvested to further investigate enzyme activity and biochemical assays hepatocyte injury and the extent of necrosis (Figure 4). Also, the degree of inflammatory cell infiltration is lighter than the control group (Table 1).

| Effects of TPK, TTK, and TFK on MDA, SOD, GSH-Px, tNOS, and iNOS levels of liver injury in mice
The MDA, tNOS, and iNOS levels were significantly increased, and the SOD and GSH-Px levels were significantly decreased in the liver injury un-treatment group (control) compared with the mock group ( Figure 5).
All of the treatment groups saw the MDA level in the liver homogenates effectively reduced (Figure 5a). The TPK at 600 mg/kg and TTK at 400 and 600 mg/kg groups all saw an effective increase in the SOD level ( Figure 5b). All the groups effectively increased the GSH-Px level

| Effects of TFK, TTK, and TPK on ALT, ALP, NO, and T-Bil levels in the serum of mice
Compared with the mock group, the ALT, NO, and T-Bil levels of the serum in the liver injury un-treatment group (control) were significantly increased. Compared with the control group, the TPK and TTK groups were ineffective in the ALT level, and only the TFK (200, 400, and 600 mg/kg) groups significantly reduced the ALT level (Figure 6a). However, there were no significant differences in ALP levels among the all groups treated with or without TFK, TTK, and TPK (Figure 6b). In comparison with control group, the TPK at 600 mg/kg and TTK at 600 mg/kg groups significantly reduced the NO level (Figure 6c). The TPK at 400 and 600 mg/kg groups, the TTK at 600 mg/kg, and the TFK at 400 and 600 mg/ kg groups were able to significantly attenuate the high T-Bil levels ( Figure 6d). F I G U R E 4 Histological image of liver tissues from mice after each Kalimeris indica ethanol extract exposure. All liver sections were subjected to histological study via staining with hematoxylin and eosin (HE). The liver of normal mice showed no histological abnormality. The negative control indicated severe degeneration of hepatocytes with marked mononuclear infiltration (liver injury). The bifendate (150 mg/kg) group was used as a positive control, and the liver injury mice were orally administrated (P.O.) with TPK (200, 400, and 600 mg/kg), TFK (200, 400, and 600 mg/kg), and TTK (200, 400, and 600 mg/kg)

| Effects of TPK, TTK, and TFK on TNF-α, INF-γ, and IL-4 levels in mice
The levels of TNF-α, IFN-γ, and IL-4 in the serum were determined via ELISA. The TNF-α, IFN-γ, and IL-4 levels in the serum in the control group (liver injury un-treated mice) were slightly increased.
The TPK at 600 mg/kg, the TTK at 600 mg/kg, and all of the TFK TA B L E 1 Effects of TPK, TTK, and TFK on the pathological grading changes in BCG/LPS-induced liver injury in mice F I G U R E 5 Effects of Kalimeris indica ethanol extracts on liver enzyme activity in BCG/LPS-injured mice. The levels of (a) MDA, (b) SOD, (c) GSH-Px, (d) tNOS, and (e) iNOS were detected, as described in the MATERIAL AND METHODS section. Data were presented as mean ± SD (n = 10). *p < .05 compared with mock (normal) group, and # p < .05 compared with control group

| D ISCUSS I ON
Kalimeris indica is a traditional Chinese medicine (TCM) and an agricultural product used for food, especially in China and Taiwan.
Our results indicated that the TTK significantly reduced the hepatic MDA at 400 and 600 mg/kg dosages (Figure 5a), hepatic tNOS, hepatic iNOS at 400 and 600 mg/kg dosages (Figure 5c and  shown that the triterpenes compounds, including α-amyrin (Singh, Arya, Sharma, Dobhal, & Gupta, 2015), have hepatoprotective effects on CCl 4 -induced liver injury. Our result suggests that α-amyrin maybe is the major compound of TTK on hepatoprotective active fractions.
The hepatic MDA is the major product of lipid peroxidation in liver, and the MDA can reflect the change that is liver damage (Smathers, Galligan, Stewart, & Petersen, 2011). The levels of SOD and GSH-Px are two important key enzymes in the anti-oxidative system, and those can reflect the change in the anti-oxidation effect on the liver (Dunning et al., 2013). Our results demonstrated that the TPK, TTK, and TFK significantly reduced the hepatic MDA ( Figure 5a). TPK, TTK, and TFK also significantly induced the hepatic SOD ( Figure 5b) and GSH-Px activities. (Figure 5c). It has been reported that flavonoids, phenolics, and triterpenes have an anti-oxidation activity by regulating SOD and GSH-Px activities in liver injuries (Ai, Huang, Liu, Han, & Chen, 2016;Ali et al., 2018;Zhang et al., 2018;Zhao et al., 2012).
In a BCG/LPS-induced liver injury in vivo system, the hepatic tNOS and hepatic iNOS are vital enzymes to regulate the pro-inflammatory cytokine production or NO signal transduction (Soskic et al., 2011).
The level of iNOS was to generate NO by oxidation of L-arginine to L-citrulline. Inhibition of tNOS or iNOS enzyme activities protected the liver against BCG/LPS-induced injury (Abdel-Salam et al., 2017).
Our results showed that TPK, TTK, and TFK significantly reduced the hepatic tNOS or iNOS activities (Figure 5c and d). Flavonoids, phenolics, and triterpenes have hepatoprotective actions by suppressing iNOS expression in liver injury (Ai et al., 2016;Farombi, Shrotriya, & Surh, 2009;Vicente-Sanchez et al., 2008;Xin et al., 2016). However, the molecular mechanisms of iNOS gene regulation in the BCG/LPS liver injury in vitro model indeed need to further study.
In conclusions, our results are in agreement with the measurements of biochemical indicators in the serum and anti-oxidative indexes in liver homogenate, which confirms that ethanol extracts of K. indica possess hepatoprotective action in BCG/LPS-induced liver injury mice, as illustrated in Figure 8.

ACK N OWLED G M ENTS
The work was supported by the National Natural Sciences Foundation Botanical Garden, Chinese Academy of Sciences (PCU201804).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

This study was approved by the Experimental Animal Ethics
Committee of Anhui University of Chinese Medicine.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.