In vivo protective effect of cinnamon aqueous extract in carbon tetrachloride-treated male albino rats

The liver as an organ is important for the metabolism of drugs and toxins. However, it is not immune from environmental insults. Exposure of liver cells to carbon tetrachloride (CCl4) results in the generation of tricholoromethyl radicals, which induce liver toxicity. This study aims at investigating the ameliorative effect of the cinnamon aqueous extract (CAE) against CCl4-induced hepatotoxicity in male albino rats. Hepatotoxicity was induced in rats through the intraperitoneal administration of 0.5 mL kg body weight of CCl4. The analyses of the results obtained showed significant reduction in the levels of serum biochemical markers for 400 and 600 mg kg bw of CAE protected rats as compared with CCl4 group. In addition, CAE administration reversed liver tissue damaged via increased antioxidants markers. Histopathological examination of CAE treatment on rats showed improved changes to the liver damage caused by CCl4 with no evidence of steatosis and inflammation. This result hence suggests that CAE has marked hepatoprotective and healing activities against CCl4-induced liver damage and could serve as a suitable candidate in drug discovery for the treatment of liver toxicity.


Introduction
The liver, an important organ in the body that help sustain human life. It plays an important role to dominate numerous physiological processes which include different vital activities such as metabolism of drugs and toxins, secretion and storage (Shanmugasundaram & Venkataraman, 2006). Different stressors such as infectious agents, environmental pollutants, and hepatotoxins (carbon tetrachloride) are known to cause liver injuries (Kim et al., 2014). Carbon tetrachloride (CCl 4 ) is classified as one of the most harmful substances which is usually employed for the induction of liver injuries in experimental rats (Kim et al., 2014). The exposure to CCl 4 results in the generation of tricholoromethyl radicals which induce toxicity in rat liver (El-Sayed, Fouda, Mansour, & Elazab, 2015), raises the levels of hepatic lipid peroxidation and ultimately leading liver damage (Weber, Boll, & Stampfl, 2003). Reactive oxygen species in addition to the decrease in the body's inhibitory and scavenging mechanism are responsible for its increased oxidative stress. For this, the liver cells have developed a defense mechanism to combat oxidative stress such as catalase, superoxide dismutase and glutathione peroxidase as well as antioxidants substances such as vitamin E, ascorbic and GSH (Kaplowitz & Tsukamoto, 1996).
When the orally ingested chemicals and drugs inter into the body system, they firstly go to the liver organ which represent the target part for the toxicity of these substances (xenobiotics and drugs), in which they are metabolized into a substances with intermediate toxicity. Therefor a huge numbers of xenobiotics and drugs are probably described as a hepatotoxic substances (Ajith, Hema, & Aswathy, 2007).
When the liver is exposed to different kinds of stress, it becomes damaged and it is thus incapable of performing its routine functions. Due to that, an urgent medical care is essentially needed when the liver is damaged. This damage occurs gradually and accumulates over many years which leads to liver disease (Garcia-Tsao & Lim, 2009). Traditional plants are common medications employed by most of the population (Garcia-Tsao & Lim, 2009;Ghashghaii, Hashemnia, Nikousefat, Zangeneh, & Zangeneh, 2017;Goorani et al., 2018). The impression that traditional plants and their role in prevention and treatment cannot be dispensed (Najafi et al., 2017;Zangeneh, Zangeneh, Tahvilian, & Moradi, 2018).

Plant materials
The barks of Cinnamon (Cinnamomum zeylanicum) were obtained from a local traditional market in Jeddah, Saudi Arabia and were identified by a Taxonomist. The barks were air dried at room temperature, washed with distilled water, dried again and grounded to fine powder. For the preparation of 10% decoction, 10 g of the Cinnamon bark powder was dissolved in 100 mL of distilled water and allowed to boil for about 30 minutes. The decoction was then cooled at room temperature, filtered and dispensed into a clean sterilized bottle; the bottles were then put into a refrigerator for storage until use.

Chromatographic analysis of Cinnamon extract (CE) by GC-MS
For the identification of bioactive compounds, GC-MS Chromatographic analysis of CE was performed using Agilent Technologies 7890B GC Systems equipped with 5977A Mass Selective Detector. Capillary column (HP-5MS Capillary; measuring 30.0m × 0.25 mm ID × 0.25 μm film) with helium as a carrier gas following a flow rate of 1.7 mL min. -1 and 1 μl injection. Analysis of the sample was carried out with the column held for a period of 4 min. at 40˚C post injection, followed by elevation in temperature to 300°C (20°C min. -1 heating ramp) along with a 3.0 min. hold. A split-less mode injection was applied at 300°C. MS scan range was (m z -1 ): 50 -550 atomic mass units (AMU) under electron impact (EI) ionization (70 eV). The bioactive compounds were identified by comparing the retention indexes of these compounds together with their spectra mass with the NIST library in the GC-MS system.

Animals and treatment
Male Albino rats weighing 203.30 ± 0.93 g were bought from the animal house of Department of Biology, King Abdulaziz University, Jeddah and housed to acclimatized in the laboratory for one week. They were kept under normal laboratory temperature (23 ± 2ºC) with daily 12 hours of light on/off cycle, fed with standard feeds and body weight gained was recorded weekly. All the experimental studies relating to the animals were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH 1985); and in accordance with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals.
The rats were separated into 8 groups with n = 6 has outlined below: Group 1 (control): normal diet and water only. Group 2 (CCl 4 ): subcutaneous injection of 0.5 mL Kg -1 bw (20% CCl 4 in paraffin oil) as previously described (Hesami et al., 2014) and commences from the start of the experiment.
Group 6 (CCl 4 + PCAE400): This post-treatment group was subcutaneously injected with 0.5 mL Kg -1 bw of CCl 4 (20% CCl4 in paraffin oil) on the 14th day and then received oral dose of 400 mg kg bw CAE till experiment ends.
Group 7 (CCl 4 + PCAE600): This post-treatment group was subcutaneously injected with 0.5 mL Kg -1 bw of CCl 4 (20% CCl 4 in paraffin oil) on the 14th day and then received oral dose of 600 mg . kg bw CAE till experiment ends Group 8 (Silm): Oral dose of 100 mg kg -1 bw silymarin daily for 4 weeks (positive control).

Oxidative stress markers
The liver homogenate was done by homogenizing the liver tissue in 10 volumes of ice-cold 100 mM phosphate buffer containing 1 mM EDTA pH 7.4 using a homogenizer and centrifuged for 15 min at 10,000 rpm and 4°C. The resulting supernatant was utilized to determine the activities of liver enzymes markers of oxidative stress (SOD, GST, CAT) and Malondialdehyde (MDA) levels of lipid peroxidation. These assays were performed using commercial kits (Bioassay Technology Laboratory, Shanghai Korian Biotech CO).

Histopathological analysis
Liver samples were fixed in 10% neutral buffered formalin solution once sacrificed, routinely processed, paraffin embedded, sectioned at a thickness of 4 μm using a microtome, and hematoxylin and eosin (H&E) stained. All morphological alterations were examined using light microcopy.

Statistical analysis
Statistical analysis of all data was done with SPSS version 20 software. Test of significance was achieved by employing Student t-test. Significance level p was set at p < 0.05.

GC-MS investigation of cinnamon extract (CE)
Analysis of CE using GC-MS showed the phytochemical components as shown in Figure 1 and Table 1. The GC-MS record described that the occurrences of many bioactive compounds which are arranged alongside with retention time, relative area (%), molecular weight and chemical formula (Table 1). By matching the spectra mass of the components against the NIST library, 14 major compounds peaks were identified from the 32 phytochemicals in CE. The major bioactive compound was Cinnamaldehyde (17.22%) followed by alpha-Muurolene (15.81%), 9-Octadecenoic acid (Z)-, methyl ester (15.61%), Pentadecanoic acid (9.31%), 14methyl-, methyl ester and gamma.-Muurolene (5.54%).

Body weight
As illustrated in Table 2 below, a significant increase in body weight was observed in all experimental groups except in rats treated with CCl 4 , wherein a significant reduction in body weight (4.03 ± 1.39) was observed.

Effects of CAE on serum biochemical parameters
The serum level of rats in the CCl 4 treated group, showed a significant rise in the levels of ALP, ALT, AST, total bilirubin, cholesterol, GGT, glucose, LDL triglyceride, uric acid and urea whereas there were significant reduction in the HDL, TP and ALB levels in comparison with the control group (p < 0.001) (Table 3). However, rats pretreated with 400 and 600 mg kg -1 of CAE for four weeks as well as posttreatment with 400 and 600 mg kg -1 of CAE two weeks after the injection of CCl 4 , showed a significant reduction in the serum levels of ALT, AST, ALP, TC, TG, LDL, and glucose and a rise in the levels albumin and HDL (Table 3). Acta Scientiarum. Health Sciences, v. 43, e52826, 2021 104.85 ± 8.51** Data were stated as mean ± SEM and test of significance was achieved using Student t-test with significant differences at p < 0.01, p < 0.05, p < 0.001 as shown for (*),(**) and (***) with respect to normal control and by (#),(##),(###) with respect to CCl4 treated group respectively.

Effect of CAE on CCl 4 -induced oxidative stress
Animals treated with CCl 4 revealed a significant reduction (p < 0.001) in the hepatic homogenate levels of SOD, CAT and GSH accompanied by significant increase in lipid peroxidation (p < 0.001) has quantified in the MDA levels in the hepatic tissue homogenate in comparison with the animals in the normal control group (Figure 2). Moreover, pretreatment with 400 and 600 gm kg -1 bw CAE resulted in a significant rise (p < 0.001) in hepatic tissue homogenate levels of SOD, CAT and GSH in comparison with CCl 4 treated group. In addition, treatment with 400 and 600 gm kg -1 bw CAE significantly decreased (p < 0.001) the elevated hepatic tissue homogenate levels of MDA in comparison with CCl 4 group (Figure 2).

Histopathological analysis
The micrographs of the histopathological analyses using haematoxilin-eosin staining is shown in (Figure 3 A-H).
The micrograph of rats in the normal control group as well as the CAE treated groups showed a normal aspect, healthy, cellular construction and clear nucleus within its cytoplasm ( Figure 3A and 3C). However, exposure of liver tissue to CCl 4 intoxication revealed deterioration in the feathery of the hepatic cells, steatosis, inflammation of the portal, sustained venous congestion and fibrosis ( Figure 3B). Histopathological examination of liver tissue exposed to CCl 4 as well as daily oral dose of 400 mg/kgbw of CAE showed evident sustained venous congestion with feathery deterioration, absence of steatosis or small vesicular steatosis, minimal parenchymal and portal inflammation ( Figure 3D). In addition, findings on liver sample from animals in CCl 4 group as well as daily oral dose of 600 mg kg -1 bw of CAE revealed an enhancement of the structural changes to the liver; with absence of sustained venous congestion, absence of steatosis, minimal parenchymal inflammation ( Figure 3E).

Discussion
The current study revealed the hepatoprotective, antioxidant and restorative effects of aqueous extract of cinnamon on CCl 4 -induced liver damage in rats. The liver as an important organ is responsible for drugs and xenobiotics metabolism. Various studies illustrated that CCl 4 is commonly utilized in the induction of liver injury since cytochrome P450 is responsible for its metabolism in hepatic cells, producing a strongly reactive trichloromethyl radical, that initiates a sequential events of lipid peroxidation, cellular impairment and thus leading to liver fibrosis (Shenoy, Somayaji, & Bairy, 2001;Weber et al., 2003;Fang, Lai, & Lin, 2008;Halliwell & Gutteridge, 2015). Apart of lipid peroxidation, CCl 4 is also a culprit in the depletion of tissue's CAT, SOD and GSH actions, and this could be due to oxidative alteration of those proteins (Augustyniak, Waszkiewicz, & Skrzydlewska, 2005). This damage was related to loss of the liver function stated as a substantial rise in the total bilirubin content in the serum and reduction in albumin, which are proposed as markers of hepatic function (Renugadevi & Prabu, 2010).
The results of this study revealed that oral administration of aqueous extract of cinnamon efficiently protected against the harmful loss of those antioxidant actions after CCl 4 injection, besides it is recognized to serve various biological functions, comprising cell protection from oxidative stress mediated by reactive oxygen species and free radicals (Nakamura, Torikai, & Ohigashi, 2001;Gäbele et al., 2009;). Bioactive compounds present in cinnamon were found to stimulate production of detoxifying agents and antioxidant enzymes in the transcriptional stage, through response elements of antioxidant (Masella, Di Benedetto, Varì, Filesi, & Giovannini, 2005) and to increase synthesis of -glutamylcysteine (Kim et al., 2007). Elevated serum levels of transaminases and ALP in CCl 4 -treated rats reveal liver damage because those enzymes outflow from hepatocytes into the blood circulation at the event of tissue injury, which is permanently accompanied with hepatic necrosis (Naik & Panda, 2008). As a result of administration of cinnamon extract, the serum profile of the liver enzymes were almost restored to normal or a little above normal, demonstrating the protective effect against hepatic damage. The effectiveness of the hepatic cells in linked to the action of alkaline phosphatase. Hence, inhibition in the elevated serum ALP proposes the continuous biliary dysfunction in the liver of rat throughout prolonged hepatic intoxication with CCl 4 . Depletion of albumin and total protein induced by CCl 4 injection is an additional indication of hepatic injury (Bies & Bonate, 2006). Cinnamon aqueous extract has ameliorated serum total protein level to near normal, which proves hepatoprotective activity of cinnamon. Enhancement of protein synthesis was progressive as a contributing hepatoprotective mechanism, enhancing the repairing phase and hepatocytes proliferation (Tadeusz, Teresa, & Krzysztof, 2001). Moreover, histopathological alterations were also observed demonstrating hepatic damage as a result of CCl 4 treatment. It has been reported earlier that CCl 4 injection leads to hepatic necrosis (Sun et al., 2003), fibrosis (Natsume et al., 1999) infiltration seen in mononuclear cell (Natsume et al., 1999), steatosis and disintegration of hepatocytes, increased mitotic activity (Theocharis et al., 2001) and liver cirrhosis (Zalatnai, Sarosi, Rot, & Lapsis, 1991). In addition, liver apoptosis has been linked to CCl 4 (Sun et al., 2001). Consequently, histological hepatic alterations owing to CCl 4 injection are consistent with former studies. Preand post-treatment involving cinnamon aqueous extract significantly ameliorated hepatocyte structure. The present findings thus suggest that treatment with cinnamon extract obviously reversed hepatic intoxication induced by CCl 4 injection. GC-MS screening of cinnamon revealed the occurrence of the most major compounds: cinnamaldehyde (17.22%), alpha-Muurolene (15.81%) and 9-Octadecenoic acid (Z), methyl ester (15.61%). Cinnamaldehyde is a major component in various food prepartions and the presence of this active compound could explain the effective role of cinnamon as an heaptoprotective, lipo-protective and reducing powers (Sharma et al., 2017;Sharma, Sharma, & Pandey, 2016). Further, different studies revealed that Cinnamaldehyde act as anti-inflammatory key in vivo and in vitro studies including neuro cells inflammation (Hwa et al., 2012) and cardiotoxicity (Pyo et al., 2013). The other bioactive compounds like octadecenoic acid (El-Raey, 2016), alpha-muurolene (Lubsandorzhieva, Boldanova, & Dashinamzhilov, 2013), flavonoids (Baek, Kim, Kyung, & Park, 1996) and alkaloids (Vijayan et al., 2003) were recognized to have hepatoprotective activity. In the current study, the existence of Octadecenoic acid in cinnamon extract could be the reason for its antioxidative efficacy and thus hepatic protectio n activity. The antioxidant capability of flavones is owing to the strong hydroxyl radical reactive potentials, and its ROS scavenging ability is a function of the number of the functional groups on its B -ring (Pannala, Chan, O'Brien, & Rice-Evans, 2001;Heim, Tagliaferro, & Bobilya, 2002).

Conclusion
The current study demonstrated that CAE is an important compound in the inhibition of the harmful effects arising from CCl 4 . CAE treatment could considerably improve the level of lipid peroxidation largely while restoring the anti-oxidative enzymes and shifts the functioning of the liver to optimum level. Furthermore, CAE decreased hepatic lipid, vacuolar disintegration, reduced hepatic architectural damage. This finding thus demonstrated the hepatoprotective activity of CAE. Therefore, the active phytochemicals of this plant could possibly be used as a future candidate drug for the treatment of liver toxicity.