Baccharis crispa attenuates toxic hepatitis induced by acetaminophen and carbon tetrachloride in mice

Aerial parts of Baccharis crispa are used as digestive and diuretic and are considered useful for hepatic, renal, and bladder diseases among others in Paraguayan folk medicine. The purpose of this study was to evaluate the effect of crude extract of B. crispa (CEBc) on acute hepatitis induced by acetaminophen and carbon tetrachloride in mice treated orally as a part of the validation procedure of the popular use. The hepato-protective effect of B. crispa against acute hepatitis induced by acetaminophen and carbon tetrachloride in mice were performed and liver function marker, aspartate transaminase (GOT), alanine transaminase (GPT) and alkaline phosphatase(ALP) were estimated. Increased serum levels of aspartate transaminase (AST = GOT), alanine transaminase (ALT = GPT), and ALP induced by both acetaminophen (350 mg/kg, i.p.) and CCl4 (1% in sunflower oil v/v, i.p.) given to mice were ameliorated by oral pretreatment with CEBc (50.0, 100.0, 200.0, and 300.0 mg/kg). Therefore, based on results, CEBc provoked a protecting effect on chemically induced acute liver injury, considering the transaminases and ALP serum level in two models of liver injury. The active compound(s) and mechanism of protection of B. crispa remain unclear and encourage us to pursue complementary chemical and pharmacological studies.


INTRODUCTION
There is little doubt about the health value of traditional medicine around the world. According to the World Health Organization (World Health Organization, 2013), herbal medicine, traditional treatments, and traditional practitioners are the main source of healthcare and sometimes the only source of care. In Paraguay, herbal medicine is culturally accepted, accessible, in proximity or cultivated into home garden, affordable, and trusted by the population. However, unfocalized efforts from either health policymakers, academicians, or researchers contribute poorly to the availability of traditional medicine of proven quality, safety, and efficacy. In Paraguay, where medicine is lacking, medicinal plants are collected from wild sources and there is no plan for good agronomical practice, thus compromising conservation and sustainability of future medicine.
Many herbs have been used in clinical practice for centuries as natural remedies, either for preventing or for the treatments of liver diseases in both western and eastern populations. Among the many hepatoprotective herbs/compounds, Silybum marianum (milk thistle), glycyrrhizin, Bupleurum chinense, Schisandra chinensis, and Phyllanthus amarus have been most widely studied and documented (Wang et al., 2007).
The genus Baccharis is claimed to possess a pain reliever activity in the southern area of Brazil (Stolz et al., 2014). Baccharis crispa, Baccharis trimera, and Baccharis microcephala are commercialized in Paraguay under the Guarani name of jaguareteka'a. Preparation from aerial parts is used as digestives and diuretics (Ibarrola et al., 2011) and is considered useful for hepatic, renal, and bladder disease among others in Paraguayan folk medicine (Basualdo et al., 2004;Caceres and Machaín, 2000). In Brazil, it is claimed popularly that B. crispa is useful in diabetes mellitus (Salgueiro et al., 2018).The hepatoprotective effect of B. trimera against acetaminophen (APAP, N-acetyl-p-aminophenol)induced toxic hepatitis has been proved (Pádua et al., 2014). Also, a significant free radical scavenging activity in aqueous extracts obtained from aerial parts of Baccharis cordifolia and B. crispa (Asteraceae) correlating to a significant antioxidant activity was found (Desmarchelier et al., 1997). Besides, an antimicrobial effect of B. trimera against gram-positive and gram-negative bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) and fungus (Candida albicans) has been verified (da Silva et al., 2018). Gastroprotective and curative effects of B. trimera in rats submitted to experimental ulcers induced by ethanol and acetic acid were denoted (Aparecida et al., 2016). Phytochemical analysis of B. crispa revealed the presence of saponins, flavonoids, sugar, and glycosides. Recently, we were able to show a gastrointestinal prokinetic activity of crude extract of B. crispa (CEBc) in mice (Velázquez et al., 2019).
Oxidative damage and lipid peroxidation are common chemical injuries induced to liver cells. Drugs commonly responsible for chemical injuries to liver cells are antibiotics, nonsteroidal anti-inflammatories, and psychotropic drugs (Mayoral and Lewis, 2000). Acetaminophen and carbon tetrachloride are common chemicals used for the induction of acute hepatitis for an experimental purpose (Lancaster et al., 2015;McGill and Jaeschke, 2018). Rats are very resistant to acetaminophen-induced hepatic damage in comparison to mice (McGill et al., 2012). The aim of this study was to evaluate the influence of B. crispa on acute hepatitis induced by acetaminophen and carbon tetrachloride in mice treated orally as a part of the validation procedure of its popular use.

Plant material and extract preparation
Aerial parts of cultivated B. crispa Spreng were collected in Villeta, Paraguay. A voucher specimen was authenticated at the herbarium of the Faculty of Chemical Sciences, National University of Asunción, and was deposited under code CC & AV 1,322. Fresh samples were dried in an oven at 38°C and ground to fine powder. This powder was extracted with ethanol by a conventional reflux method for 1 hour, then filtered, and evaporated under reduced pressure. The ethanolic extract of B. crispa (Bc) was stored at room temperature in a desiccator until use. This semisolid extract was used in all biological tests.

Reagents and drugs
Chemicals of analytical grade were used for the pharmacological assay. Sodium chloride (WAKO Pure Chemical Industries Ltd., Japan), carbon tetrachloride (Biopack), paracetamol and silymarin (Sigma Chemical Company, St. Louis, MO), sodium pentobarbital (Nembutal Abbott Laboratory, Japan), and ethanol (submitted to glassware distillation before use) and propylene glycol were acquired locally.

Animals
Swiss albino male mice weighing 25 and 35 g were obtained from the Bioterium of the Faculty of Chemical Sciences (National University of Asunción). The animals were kept in a room with controlled light/dark cycles (12 hours), temperature (23°C ± 2°C), and relative humidity (50%-60%) to avoid environmentally originated factors affecting animal behavior. The animals received commercial foods and were fasted overnight before the experiments; they had free access to drinking water during the trials. All experimental practices performed in the present work were directed in agreement with the international standards of animal welfare and the protocol was previously approved by the institutional Ethical Committee in Scientific Research of the Faculty of Chemical Sciences on March 2, 2015 (CEI-188-15).

Acetaminophen-induced hepatotoxicity and treatment
The animals were randomly allocated into seven different experimental groups of six mice each. A pilot study was performed to set up the effective dose of acetaminophen (APAP, N-acetyl-p-aminophenol or paracetamol), which increased serum transaminases and alkaline phosphatase levels in mice. Druginduced acute hepatotoxicity was provoked by an intraperitoneal injection of 350.0 mg/kg of APAP as described in Wu et al. (2008), with small modifications. Briefly, two groups received vehicle (2.5% of ethanol in distilled water) and five other groups received, respectively, silymarin (100.0 mg/kg p.o.) and CEBc (50.0, 100.0, 200.0, and 300.0 mg/kg, p.o.) as pretreatments. Thirty minutes later, all groups except the first one (vehicle) were given intraperitoneally 350.0 mg/kg of acetaminophen (APAP). After 4 hours, all animals were submitted to total blood collection according to individual timing. Previously, a deep pentobarbital (50.0 mg/kg, i.p.) anesthesia was induced and an intracardiac puncture was done, followed by cervical dislocation. Serum was separated and aspartate transaminase (AST=GOT), alanine transaminase (ALT=GPT), alkaline phosphatase (ALP) were measured.

Carbon tetrachloride-induced hepatotoxicity and treatment
The animals were assigned randomly into seven different groups of six mice each. Acute hepatotoxicity was provoked by oral administration of 1% carbon tetrachloride (v/v) dissolved in sunflower oil as described in Sun et al. (2018), with some modifications. Briefly, two groups received vehicle (2.5% of tween 80 in distilled water) and distilled water (0.1 ml/10 g bw), respectively. The other five groups received silymarin (100.0 mg/ kg, p.o.) and CEBc (50.0, 100.0, 200.0, and 300.0 mg/kg, p.o.), respectively, as pretreatments once a day for 5 days. One hour after the last administration, the control group was treated orally with sunflower oil (0.1 ml/10 g bw) and all other groups were administered orally with 1% carbon tetrachloride (0.1 ml/10g bw). After 4 hours, all animals were submitted to total blood collection according to individual timing. Previously, a deep pentobarbital (50.0 mg/kg, i.p.) anesthesia was induced and an intracardiac puncture was done, followed by cervical dislocation. Serum was separated and aspartate transaminase (AST=GOT), alanine transaminase (ALT=GPT), ALP levels were measured

Statistical analysis
The results were expressed as mean ± standard deviation (SD) and one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparisons test, which was performed using GraphPad Prism version 7.0 for Windows, GraphPad Software, La Jolla, CA. The values of p < 0.05 were considered as statistically significant.

Effect of CEBc on acetaminophen-induced hepatotoxicity in mice
Considering the serum level of GPT, GOT, and ALP of mice treated with different doses of CEBc, a clear liver protective effect was denoted. Certainly, CEBc given orally to mice (50.0, 79 ± 10.8; 100.0, 75.2 ± 16.8; 200.0, 52.8 ± 13; and 300.0, 58.33 ± 5.6, mg/kg and U/L, respectively; p < 0.0001) significantly reduced the APAP-induced elevation of the serum level of GPT (U/L) as far as 59%. Similarly, 100.0 mg/kg of silymarin (64 ± 6.2 U/L, positive control) induced a significant protecting effect on liver damage visualized by a reduced serum GPT level in comparison to pathological control (129.2 ± 31.3U/L) by 50%. Besides, the group of animals treated with APAP (350.0 mg/kg) denoted an increase in GPT serum level by 420%, significantly different from vehicletreated animals (30.83 ± 5.6 UI/L; p < 0.0001), both validating the methods used ( Fig. 1). Interestingly, 200.0 and 300.0 mg/kg of CEBc provoked a similar hepatoprotective effect as silymarin.

Effect of CEBc on carbon tetrachloride-induced hepatotoxicity in mice
A clear liver-protective effect was denoted since increased serum levels of GPT, GOT, and ALP induced by CCl 4 in mice were attenuated with different oral doses of CEBc. In concordance with the protective effect observed with CEBc in the liver injury induced by APAP, undoubtedly oral administration of CEBc (50.0, 234.3 ± 55.37; 100.0, 594.3 ± 280.9; 200.0, 560 ± 280.3; and 300.0, 679.6 ± 179.1, mg/kg andU/L, respectively; p < 0.0001) for 5 days to mice significantly reduced the CCl 4 -induced elevation of the serum level of GOT by 73%. Similarly, 100.0 mg/kg of silymarin (287 ± 81.77 U/L; positive control) induced a significant protecting effect on liver damage visualized by a reduced serum GPT level in comparison to pathological control (859.5 ± 287.3 U/L) by 67%. Besides, the group of animals treated with CCl 4 (1%) denoted an increase in GOT serum level by 687%, significantly different from vehicle-treated animal (109.2 ± 19.87 U/L; ****p < 0.0001), both validating the method used (Fig. 4). The dose of 50.0 mg/kg of CEBc provoked a more intense (73%) protection than silymarin (67%).

Figure 2.
Influence of B. crispa (CEBc) given orally on serum GOT activity (U/L) of mice after acute hepatitis induced by acetaminophen (APAP 350 mg/ kg). Each bar represents the mean ± SD (n= 6). Statistical analysis was performed using ANOVA, followed by Dunnett's multiple comparison test. ***p < 0.0002; *p < 0.0332, significantly different from vehicle (Veh.). mg/kg, p.o.) provoked protection at a similar intensity compared with silymarin. In contrast, 5 days of treatment with higher doses of CEBc (100.0, 927 ± 62; 200.0, 634 ± 253; and 300.0, 579 ± 109, mg/kg; U/L, respectively) did not modify the GPT level which increased after liver injury induced by CCl 4 . We have no knowledge about this response and speculate that this maybe is due at least to the severity of injury induced by CCl 4 in comparison to damage induced by APAP.

DISCUSSION
The administration of hepatoprotective drugs may induce the hepatocytes to resist the toxic effect of toxic agents, such as APAP and CCl 4 among others (Somasundaram et al., 2010). Measuring the serum level of GPT, GOT, and ALP usually is the initial step involving the evaluation of hepatic conditions after viral or chemically induced injuries (Sun et al., 2018). A significant elevation of such enzymes induced by APAP and CCl 4 was observed in this study, showing both hepatocellular and biliary injuries (Essien et al., 2013). Indeed, CEBc showed significant hepatoprotective activity, which was comparable with the standard liver-protecting drug silymarin. Therefore, a significant reduction of the APAP and CCl 4 -induced elevation of the serum level of these parameters was observed in mice treated with CEBc, revealing a significant protective effect in comparison to APAP-and CCl 4 -treated groups.
In the literature, comparable findings to our results have been reported for Artemisia asiatica (Ryu et al., 1998), Cassia fistula (Bhakta et al., 2001), Cassia occidentalis (Yadav et al., 2010), Litchi chinensis (Bhoopat et al., 2011), and Sorbus pohuashanensis (Yin et al., 2019), among others. The serum rises in transaminases (GPT and GOT) and ALP, enzymes of cytoplasmic location, have been attributed to the damaged structural integrity of the liver (Ryu et al., 1998), allowing this enzyme to release into the blood stream after cell injury or death. Reversing the effects induced by treatment using phytopharmaceuticals or plants are common practices in traditional medicine (He et al., 2004). Phytochemicals such as citrus flavonoids, red wine polyphenols, Garcinia, Gymnema, Ginkgo, Ephedra sinica, Camellia sinensis, Silybum, Guarana, Eleuthero, Allium sativum, and Ocimum basilicum extracts were studied against the massive liver injuries induced by APAP with an auspicious counteracting effect on transaminases, oxidative stress, and DNA fragmentation in rats (Ray et al., 2006). It was observed that curcumin and its derivatives (octahydrocurcumin and tetrahydrocurcumin) possess a satisfactory hepatoprotective effect on mice through restoring antioxidant status, inhibiting CYP2E1, and activating Keap1-Nrf2 pathway, which might represent effective antioxidants for clinical use (Luo et al., 2019). Mice with a chronic liver injury and fibrosis induced by CCl 4 were markedly ameliorated by polydatin (a glucoside of resveratrol) through inhibition of oxidative stress and inflammation. The levels of serum transaminases (GPT and GOT) and inflammatory mediators responsible for fibrosis were clearly modified by polydatin, reducing chronic oxidative stress and inflammation induced by CCl 4 treatment (Zhao et al., 2017). A protective mechanism of aqueous leaves extract of Eucalyptus obliqua, probably due to its ability to reinforce the antioxidant defense system of Wistar rats submitted to CCl 4 -mediated liver damage, was established by Sabiu et al. (2017). Total flavonoids from Cyclocarya paliurus exert a potent protective effect against CCl 4 -induced acute liver injury in mice. The effect appears to be closely associated with the activity of flavonoids from C. paliurus that attenuated the transaminase increase induced by CCl 4 and improved the defense antioxidant status of liver (Xie et al., 2018).
The mechanism of attenuating effect of B. crispa in acute chemically induced liver injury in mice is unknown. Our findings showed a protective effect of B. crispa using two different liver injury models. However, the presence of flavonoids, saponins, steroids, and triterpenes (Velázquez et al., 2019) could induce a minimizing oxidative stress in the liver in a similar way as mentioned by several studies present in the literature. The protective effect of B. crispa against acetaminophen-induced liver damage appears to be in a dosedependent manner according to the serum transaminases and ALP level. However, the attenuating effect of B. crispa on the elevation of transaminase and ALP induced by CCl 4 was not dosedependent. At least, these divergences probably are mainly due to the hepatotoxicity models used. Severity of liver damage induced by CCl 4 was greater than APAP, considering the observed serum level of transaminases and ALP. CCl 4 , at a suitable dose, induced widespread necrosis in the centrilobular region of the liver around the central veins (Bhoopat et al., 2011). Indeed, the potency of doses of hepatotoxins used in this study is not comparable, and also, the sensitivity of animals submitted to injuries was not evaluated. The mechanism of injuries is also complex with shared (oxidative stress and generation of reactive oxygen species) and uncommon (tissue factor activation) mechanisms of liver damage (Abdel-Bakky et al., 2015;Woolbright and Jaeschke, 2018). In the same genus, a hepatoprotective effect of B. trimera against acetaminophen-induced toxic hepatitis was found (Pádua et al., 2014). Also, a significant free radical scavenging activity in aqueous extracts obtained from aerial parts of B. cordifolia and B. crispa (Asteraceae) correlating to a significant antioxidant activity was found (Desmarchelier et al., 1997). Understanding of pathophysiology of drug-induced damage and the principles of liver protective activity of phytochemicals could guide future drug development and help prevention of clinical trial failure. Nevertheless, it is known that silymarin has poor solubility resulting in low bioavailability. This property has conditioned the great advances made on the development of novel delivery systems and pharmaceutical formulation with improved bioavailability for this bioactive natural compound and its derivatives (Bilia et al., 2017;Theodosiou et al., 2014;Yang et al., 2015). The bioavailability of CEBc is unknown and probably reduced activity of higher dose could be explained by a potential poor absorption from intestinal tract. Therefore, a good candidate for hepatoprotective drug and bioavailability of phytomedicine should be well defined for a safe and effective clinical use. Furthermore, the use of new delivery systems or pharmaceutical formulations that enhances bioavailability of poorly water-soluble compounds may improve effectiveness already obtained (Domitrović and Potočnjak, 2016). Finally, complementary molecular and histological studies are in a way needed to clarify the mechanism of attenuating effect of B. crispa on acute liver injury using mice.

CONCLUSION
Based on the results, it was evident that crude extract obtained from the aerial part of B. crispa has a protecting effect on chemically induced acute liver injury, considering the transaminase and ALP serum level in two models of liver injury. The active compound(s) and mechanism of protection remain unclear and are in a way to be solved soon in new chemical and pharmacological studies.