Essential oils of green cumin and chamomile partially protect against acute acetaminophen hepatotoxicity in rats

Essential oils of green cumin (Cuminum cyminum) and chamomile (Chamomilla recutita) have antioxidant and anti-inflammatory effects. Acetaminophen, N-acetyl-p-amino-phenol, is an over-the-counter analgesic and antipyretic. Despite being safe at therapeutic doses, acetaminophen overdose is a leading cause of acute liver failure. This study aimed to compare the possible protective effects of cumin and chamomile essential oils against acute acetaminophen hepatotoxicity in male rats. Cumin oil (400 mg/kg) and chamomile oil (250 mg/kg) were orally administered for 2 weeks prior to a single acetaminophen dose (1g/kg). Serum liver function enzymes, oxidative stress markers in the liver and histopathological features were evaluated. Acetaminophen caused marked damage to hepatocytes evidenced by a significant rise in the levels of liver function enzymes, including alanine aminotransferase and aspartate aminotransferase, and disruption to the liver antioxidant protective system. Whereas the cumin oil normalized acetaminophen-induced liver enzymes elevation, the chamomile oil slightly attenuated the increase in alanine aminotransferase levels in acetaminophen-intoxicated rats. The Chamomile oil moderately ameliorated glutathione depletion and the decrease in superoxide dismutase activity in the liver of acetaminophen-administered rats. The Cumin oil preserved the liver structure to a greater extent than chamomile oil in acetaminophen-intoxicated rats. Essential oils of cumin and chamomile partially counteracted acute acetaminophen hepatotoxicity.


INTRODUCTION
Acetaminophen (APAP) has been widely used as an over-the-counter analgesic and antipyretic (Leung 2012).Clinically, APAP's daily dosage should not exceed 4 g in divided doses according to British National Formulary 70 (Royal Pharmaceutical Society 2015).Despite being generally safe at its therapeutic levels, lower APAP doses for geriatric patients with insufficient liver function and accurate APAP doses for pediatric patients are highly recommended to avoid APAP-induced liver injury (Royal Pharmaceutical Society 2015).APAP hepatotoxicity has been a leading cause of acute liver failure and could necessitate liver transplantation (Lee 2013, Woolbright andJaeschke 2016).APAP at toxic plasma levels has been found to induce a MOHAMED E. EBADA state of oxidative stress featuring marked depletion of reduced glutathione (GSH), propagation of lipid peroxidation and substantial distortion to hepatic antioxidant enzymes (El Morsy andKamel 2015, Ghanem et al. 2016).Upon exposure to APAP overdose, decreased liver GSH content renders hepatocytes highly exposed to APAP's reactive metabolite N-acetyl-p-benzoquinone-imine, generated by cytochrome P450 metabolizing enzymes, i.e. mainly CYP2E1 (Gonzalez 2007), resulting in liver injury with a subsequent increase in the serum levels of liver rich enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (El Morsy andKamel 2015, Ghanem et al. 2016).In experimental animals, APAP toxicity is associated with overproduction of reactive oxygen species such as superoxide anions (Woolbright and Jaeschke 2016), removed by superoxide dismutase (SOD), which aggressively attack hepatocytes resulting in marked congestion in the central vein, inflammation, hepatocellular degeneration and necrosis (Xie et al. 2016).On the other hand, SH-donating compounds such as N-acetylcysteine are effective antidotes against APAP hepatotoxicity (Kisaoglu et al. 2014).
APAP overdose has been repeatedly implemented as a model of drug-induced liver injury to investigate antioxidant/hepatoprotective effects of compounds of natural origin (El Morsy and Kamel 2015, Hasanein and Sharifi 2017, Kumari and Kakkar 2012, Xie et al. 2016).Given their popular use and relative safety profiles, the use of herbal remedies has been encouraged as a prophylactic approach to safeguard against oxidative-distress implicated pathologies such as drug-induced liver injury (Madrigal-Santillan et al. 2014, Singh et al. 2016) and malignancies (Miyanishi et al. 2015).Green cumin (Cuminum cyminum L., Umbelliferae) (Mnif and Aifa 2015) and chamomile (Chamomilla recutita L., Compositae) (Srivastava et al. 2010) are preferably consumed for their spicy taste, mainly cumin, and their multiple therapeutic benefits.The chamomile essential oil (CHEO) possesses anti-inflammatory, antioxidant and anticancer effects (Agatonovic-Kustrin et al. 2015, McKay andBlumberg 2006).Major constituents in CHEO including α-bisabolol and chamazulene have been found to mediate such medical effects (Cavalieri et al. 2011, Flemming et al. 2015, Rocha et al. 2011).In addition, CHEO's sesquiterpenoid trans-β-farnesene content was reported to possess hepatoprotective effects (Vinholes et al. 2014).On the other hand, the cumin essential oil (CUEO) contains around 30 components and it has antioxidant, antineoplastic, antibacterial, anti-inflammatory and hypoglycemic effects (Allahghadri et al. 2010, Patil et al. 2013, Wei et al. 2015).Major ingredients in CUEO such as cuminaldehyde, α-pinene and ɣ-terpinene contribute to its anti-inflammatory and antioxidant properties (Chen et al. 2014, Rufino et al. 2014, Tomy et al. 2014).Two previous studies, evaluating biochemical alterations and/or selected markers of oxidative stress, reported complete protective effects mediated by the whole Cuminum cyminum seeds against hepatotoxicity induced by chronic APAP administration (Elhabib et al. 2007), and by the Chamomilla recutita extract against acute acetaminophen hepatotoxicity (Gupta and Misra 2006).However, those studies showed certain limitations such as unequal dosing between animals, being APAP or cumin seeds mixed with diet/drinking water in one study (Elhabib et al. 2007), and the administration of a very small and single APAP dose (200 mg/kg) to rats in another study (Gupta and Misra 2006).It has been well documented that 1 g/kg APAP is the highest tolerable dose in rats (Jin et al. 2012) and, therefore, the acute oral APAP dose required to produce considerable liver injury in rats has been found to be 1g/kg or more in most studies (El Morsy andKamel 2015, Kiran et al. 2012).Other limitations were the lack of the liver histopathological examination in the chamomile's study (Gupta and Misra 2006) and the absence of evaluating the impact on the liver antioxidant protective system in the cumin's study (Elhabib et al. 2007).It has been suggested that marked elevation of liver enzymes is not always associated with severe liver injury or impaired liver antioxidant system (Contreras-Zentella and Hernández-Muñoz 2016).Generally, biochemical alterations, histological features and markers of oxidative stress are essentially evaluated to judge potential hepatoprotective effects.The present work aimed to compare the possible hepatoprotective impact of CUEO and CHEO administered for 2 weeks prior to a single acetaminophen toxic dose (1g/kg body weight) in male rats.

ANIMALS
Twenty four adult male Wistar rats weighing 180-200 g were obtained from the Holding Company for Biological Products and Vaccines (VACSERA), Egypt.The animals were kept under controlled laboratory conditions of ventilation/humidity and 12:12 light/dark cycle throughout the period of the study.Rats were housed in groups of 3 and were provided with free access to standard diet and water.All experiments were carried out in accordance with the UK Animal Scientific Procedures Act 1986 and approved guidelines set for animal care and use at National Organization for Drug Control and Research (NODCAR), Giza, Egypt.

EXPERIMENTAL DESIGN
After one week of acclimatization, rats were assigned to 4 groups (n = 6).For 14 days (Treatment I), groups I (control) and II (toxic) received the vehicle of essential oils (1% Tween 80 in distilled water), and groups III and IV received CUEO (400 mg/kg/day) and CHEO (250 mg/kg/ day), respectively (Al-Musa and AL-Hashem 2014, Jagtap and Patil 2010).Tween 80 at 1% was used as a solubilizing agent to aid diluting concentrated essential oils, and it was reported that repeated oral administration of more concentrated Tween 80 solution (up to 10%) for 2 weeks was not associated with liver injury (El Morsy and Kamel 2015).The CHEO dose was selected based on previous studies demonstrating that treating rats for 4 weeks with increasing doses (≤ 500 mg/kg/day) of the Chamomile recutita extract did not cause significant toxicity (Al-Musa and AL-Hashem 2014).On the other hand, the CUEO dose used in the present study was found to improve pancreatic antioxidant enzymes in diabetic rats (Jagtap and Patil 2010).On Day 13, rats had no access to food for 18 hours (El Morsy and Kamel 2015), as fasting may potentiate APAP hepatotoxicity (Whitcomb and Block 1994).On Day 14, APAP MOHAMED E. EBADA 1g/kg (Treatment II), suspended in 1% sodium carboxymethylcellulose, was administered to rats in groups II-IV (Gunduz et al. 2015, Jin et al. 2012), one hour after Treatment I's fourteenth dose.APAP and essential oils were freshly prepared and their concentrations were adjusted so that each 100 g animal body weight received 0.5 ml solution.All treatments were orally administered.

BLOOD AND LIVER SAMPLING
Twenty four hours after APAP administration, blood samples were collected into non-heparinized tubes, permitting blood coagulation and serum separation, by puncturing the retro-orbital venous plexus (Sorg and Buckner 1964), and were left for 1 hour at the room temperature before being centrifuged at 3000 rpm for 20 min for serum collection.Rats were euthanized, and livers were rapidly dissected and rinsed with ice cooled saline (0.9% NaCl).Liver specimens were fixed in 10% neutral buffered formalin for histological examination.The liver tissue remainder and sera were kept at -80 °C for further analysis.

DETERMINATION OF MARKERS OF OXIDATIVE STRESS IN THE RAT LIVERS
The frozen liver tissues were left at room temperature for thawing, weighed and homogenized into ice-cooled 1.17% KCl solution (pH adjusted to be 7.4 by adding K 2 HPO 4 and KH 2 PO 4 ) to prepare 10% (w/v) homogenates.The liver homogenate was divided into three aliquots.The first part was used for the estimation of the liver GSH content (Ahmed et al. 1991).Briefly, equal volumes of the homogenate and the ice-cold precipitating solution (12% trichloroacetic acid + 6 mM EDTA) were vortex-mixed and centrifuged at 3000 rpm for 5 minutes.Then, the supernatant was treated with Ellman's reagent dissolved in 1% sodium citrate, and the absorbance was measured spectrophotometrically at 412 nm.The second aliquot was centrifuged at 3000 rpm for 15 minutes and the supernatant was used for the determination of the total protein (Weichselbaum 1946) and the lipid peroxidation degradation product malondialdehyde (MDA) levels (Uchiyama and Mihara 1978).The supernatant was mixed with 1% orthophosphoric acid (pH ~2) and 0.6% thiobarbituric acid, and the mixture was firstly heated in a boiling water bath for 45 minutes, and was later left to cool at the room temperature before being mixed well with n-butanol.The absorbance of the separated n-butanol layer, containing a pink colored substance formed as a result of the reaction between MDA and thiobarbituric acid, was measured at 520 nm and 535 nm, and the difference between the two readings corresponded to MDA levels.The third aliquot was centrifuged at 85,000 rpm for 15 minutes at 4 °C and the supernatant was used for the determination of cytosolic SOD activity (Marklund and Marklund 1974).One unit of SOD was defined as its amount which inhibits 50% of pyrogallol autoxidation.In the cuvette, 0.2 mM pyrogallol and Tris HCl buffer (pH = 8.2), containing 50 mM Tris(hydroxymethyl)aminomethane, 1 mM diethylenetriaminepentaacetic acid and 200 mM HCl, were added to 10 µl sample.The absorbance of the mixture was measured at 420 nm every minute for 2 minutes, and the difference between the two readings accounted for SOD activity.Following the same procedures, 1,1,3,3-tetramethoxypropane, standard GSH and SOD were used to generate standard curves for the determination of MDA and GSH levels and SOD activity, respectively.Levels of GSH (mg) and MDA (nmol) were expressed relative to mg protein and SOD activity was expressed as U/min/mg protein.

HISTOPATHOLOGICAL EXAMINATION
Twenty four hours after necropsy, formalin preserved liver specimens were left under running water overnight to wash off the fixative.Fixed tissues were dehydrated with a graded series of alcohols before being cleared in methyl benzoate and embedded in paraffin.Later, 3 µM thick sections were cut by a microtome, and were stained with hematoxylin and counter stained with eosin (Gabe 1968).Dry sections were examined under the optical microscope and representative images were captured using a digital camera attached to the microscope.Five histopathological features (necrosis, inflammatory cells infiltration, degeneration, central vein congestion and hemorrhage) were evaluated and scored in each group (Lobenhofer et al. 2006).

STATISTICAL ANALYSIS
Data are expressed as mean ± SEM and were analyzed using one way analysis of variance (ANOVA) followed by post-hoc Tukey's test for multiple comparisons.Also, the percent change of each group relative to the control group was calculated for all variables.IBM SPSS package version 19 was used to perform statistical analyses, and significance was considered when p was <0.05.

EFFECTS OF 14-DAY TREATMENT WITH CUEO OR CHEO ON ACUTE APAP-INDUCED ELEVATION OF LIVER FUNCTION ENZYMES
One way ANOVAs revealed significant differences between groups in serum levels of ALT [F(3.20)=17.60;p<0.001] and AST [F(3.20)=11.64;p<0.001].Tukey's post-hoc tests detected that rats treated only with APAP in the toxic group exhibited a significant rise in the serum levels of ALT (p<0.001) and AST (p=0.006),compared to the control group (Figure 1).In contrast, CUEO markedly attenuated APAP-induced rise in ALT (p<0.001) and AST (p=0.002).To lesser extent, CHEO decreased ALT levels (p=0.024) in APAP-treated rats (Figure 1).Clearly, there were no significant effects of CHEO on AST levels (p=0.98) in APAP-administered rats.CUEO reduced serum levels of ALT (p=0.049) and AST (p=0.001) to a greater extent than CHEO in APAP-intoxicated rats.The percent change in serum ALT and AST levels, relative to control, for each group is indicated above the relevant bars.

EFFECTS OF REPEATED ADMINISTRATION OF CUEO OR CHEO ON SELECTED MARKERS OF OXIDATIVE STRESS INDUCED BY A SINGLE APAP DOSE IN THE RAT LIVER
Oxidative stress in the liver was statistically detected among all APAP-administered groups, evidenced by significant changes in the liver content of GSH [F(3.20)=5.52;p=0.006] and MDA [F(3.20)=8.24;p=0.001] as well as the liver SOD activity [F(3.20)=4.17;p=0.019].Whereas a significant drop in the liver GSH levels of rats treated only with APAP (p = 0.013) or with APAP and CUEO (p = 0.011) was reported, CHEO treatment moderately ameliorated the liver GSH depletion in APAPintoxicated rats (p = 0.37), compared to the control group.The percent change in the liver GSH levels of each group relative to control is mentioned in Table II.On the other hand, all APAP-treated groups showed a significant rise in the liver MDA levels compared to the control group (Toxic, p = 0.049, CUEO+APAP and CHEO+APAP, p = 0.002).Consequently, the percent changes in the liver MDA levels of APAP-treated groups relative to control were as follows: Toxic (92.64%),CUEO+APAP (141.86%) and CHEO+APAP (142.45%).Similar to its effect on the liver GSH content, acute APAP administration caused a significant decrease in the liver SOD activity in the toxic group (p = 0.03) and CUEO+APAP treated rats (p = 0.044), but not in CHEO+APAP treated rats (p = 0.62), compared to the control group.Whereas % change in the liver SOD activity was substantially high for rats treated solely with APAP (106.72%) or with CUEO+APAP (100.16%),CHEO+APAP treated rats exhibited only 42.99% reduction in the liver SOD activity relative to the control.

IMPACT OF 2-WEEK TREATMENT WITH CUEO OR CHEO ON ACUTE APAP-INDUCED LIVER HISTOPATHOLOGY
Microscopic examination revealed normal pattern of hepatocytes in the control rats.In contrast, acute APAP treatment caused massive degeneration and necrosis to hepatocytes, central vein congestion, hemorrhage and infiltration of nonspecific inflammatory cells between hepatocytes.On the other hand, CUEO moderately preserved the liver structure by limiting the necrosis of hepatocytes and by mitigating the migration of inflammatory cells between hepatocytes to a greater extent than CHEO in APAP-intoxicated rats; Figure 2a-d illustrates representative microscopic images of the rat liver for all groups (I-IV).The five observed criteria of the histopathological evaluation were scored to describe the magnitude of the liver injury as appropriate (Table III).

DISCUSSION
The present study demonstrated that repeated treatment with CUEO or CHEO partially counteracted acute APAP-induced liver injury in rats.Whereas CUEO markedly reduced the elevated serum levels of ALT and AST, CHEO moderately ameliorated GSH depletion and reduced SOD activity in APAP-intoxicated rats.Essential oils of green cumin and chamomile failed to suppress MDA generation, although the histopathological features associated with acute APAP hepatotoxicity were alleviated to a greater extent by prior CUEO treatment.
As stated earlier, acute APAP toxicity caused massive destruction to hepatocytes, resulting in elevation of serum ALT and AST levels, and induced a state of oxidative stress manifested by the accumulation of MDA, depletion of GSH and reduction of SOD activity in the liver.These findings were in agreement with a wide range of studies investigating potential hepatoprotective effects of herbal products against APAP hepatotoxicity (El Morsy and Kamel 2015, Hamza and Al-Harbi 2015, Xie et al. 2016).Previous research revealed that APAP hepatotoxicity was associated with diminished activity of liver antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione peroxidase (El Morsy and Kamel 2015, Hamza and Al-Harbi 2015, Xie et al. 2016).There are three SOD isoforms, including copper/zinc SOD (cytosolic and major form) and manganese SOD (mitochondrial form).The third form is located in extracellular and nuclear compartments (Weydert and Cullen 2010).The activity of the liver manganese SOD in APAPintoxicated rats was reported to be higher than control rats 6 hours after APAP administration, but this initial rise in the manganese SOD activity dropped near the control level 24 hours after APAP exposure (Mladenovic et al. 2009).This suggests that acute APAP toxicity could rather temporarily activate the liver antioxidant defense system.As mentioned earlier, previous studies undertaken by Gupta and Misra (2006) showed that the Chamomilla recutita extract (400 mg/kg, p.o., for 7 days) completely counteracted the increase in serum liver enzymes (ALT and AST) as well as the elevated MDA levels and the depleted glutathione content in the liver of rats sacrificed 48 hours after being treated with a single APAP dose (Gupta and Misra 2006).In fact, it was difficult to draw a definite conclusion about the hepatoprotective potential of the chamomile extract against APAP toxicity in the previous study owing to the small and single APAP dose administered to rats (200 mg/kg, p.o.), and the absence of histopathological studies and components analysis of the prepared chamomile extract (Gupta and Misra 2006).In another study, the chamomile flower extract, whose constituents were identified, received for 10 days, showed dose-dependent marked protection against liver injury induced by a single ethanol dose (6g/kg) in rats, through maintaining the hepatic antioxidant system, preserving the liver structure and counteracting the increase in serum ALT and AST levels as well as liver MDA accumulation (Sebai et al. 2015).It was also documented that CHEO could inhibit the activity of certain cytochrome P450 enzymes such as CYP3A4 (Ganzera et al. 2006) whose inhibition could safeguard against APAP hepatotoxicity (Wolf et al. 2007).With regard to the potential hepatoprotective effects of  III.
green cumin, it was demonstrated that rats which were chronically fed a diet mixed with cumin seeds (6% of the diet weight) and APAP (500 mg/kg diet) exhibited an improvement in APAP-induced liver necrosis and increased serum ALT and AST levels (Elhabib et al. 2007).In the previous study, the possible role of oxidative stress in mediating chronic APAP hepatotoxicity was not investigated.The current findings revealed that CUEO did not affect evaluated markers of oxidative stress altered by acute APAP hepatotoxicity, although it abolished the increase in the serum levels of ALT and AST.Also, the results of histopathological examination indicated that prior CUEO treatment provided some protection against necrosis of hepatocytes and infiltration of inflammatory cells induced by acute APAP hepatotoxicity.In agreement with effects of CUEO on the liver function enzymes in response to drug-induced liver injury, chronic treatment (45 days) with the cumin powder suspension ameliorated ethanol-induced rise in the AST, ALP and gamma glutamyl transferase in rats (Aruna et al. 2005).CUEO was found to possess powerful anti-inflammatory activity via interfering with the expression of pro-inflammatory cytokines such as interleukin 1, interleukin 6 and nuclear factor kappa B (Wei et al. 2015), a transcription factor which regulates various aspects of inflammatory cascades, such as the expression of pro-inflammatory cytokines (Baldwin 1996, Hanada andYoshimura 2002).It was also reported that CUEO suppressed lipopolysaccharide-induced activation of extracellular signal regulated kinase and c-Jun N-terminal kinase (Wei et al. 2015), which could play a role in APAP-induced cell death (Ghanem et al. 2016).
One unclear point that needs further investigation was the lack of any effects of CUEO and CHEO on the elevated MDA levels in the liver of APAP-treated rats.Instead, both CUEO and CHEO insignificantly aggravated the formation of MDA in the liver of APAP-intoxicated rats.Obviously, improving the liver protective antioxidant system has been widely reported to be accompanied with reduced MDA levels following acute APAP toxicity (El Morsy andKamel 2015, Xie et al. 2016).In particular, CHEO had moderate improving effects on the reduced SOD activity and depleted GSH content in the livers of APAP-intoxicated rats, the case was opposite for acute APAP-induced liver MDA accumulation.This is in disagreement with the aforementioned study in which the Chamomilla recutita extract rescued APAP-lowered liver Na + Ka + ATPase activity, a marker of membrane integrity, in rats (Gupta and Misra 2006).Importantly, the APAP dose in the previous study was one fifth of the standard APAP toxic dose used in the present study, and, therefore, this could not support the marked hepatoprotective effects of the chamomile extract, reported in Gupta and Misra (2006), against acute APAP hepatotoxicity particularly in the absence of histopathological studies.It is worth noting that constituents and their strength in the cumin or chamomile extract could be different from those isolated in their essential oils, and this could be one reason why CUEO and CHEO showed limited hepatoprotective effects in the present study.

CONCLUSIONS
The findings of this study suggest that CUEO and CHEO could offer some protection against acute APAP hepatotoxicity at 1 g/kg body weight.CHEO seems to have partially counteracted acute APAP-induced liver injury through its antioxidant effects.In contrast, effects of CUEO were greater in preserving the liver histology, hindering migration of inflammatory cells between hepatocytes, and abolishing the rise in the liver function enzymes in APAP-intoxicated rats.Further studies are encouraged to determine whether previously reported anti-inflammatory properties of CUEO contribute to its hepatoprotective effects against APAP toxicity.