Beneficial Effects of Ocimum gratissimum Aqueous Extract on Rats with CCl4-Induced Acute Liver Injury

Ocimum gratissimum (OG) is known as a food spice and traditional herb, which has been recommended for the treatment of various diseases. To investigate the hepatoprotective effect of OG aqueous extract (OGAE), male Wistar rats challenged by carbon tetrachloride (CCl4) were used as the animal model of chronic hepatic injury. Significantly increased serum catalase and DPPH levels were detected in CCl4-administrated rats that were treated with OGAE or silymarin as compared to those rats that were treated with saline or CCl4. In contrast, significantly decreased stress proteins including HSP70 and iNOS were observed in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. Moreover, significant decreases of MMP-9/MMP-2 ratio, uPA, phosphorylated ERK (p-ERK) and NF-κB (p-P65) were detected in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. These findings imply that OGAE can efficiently inhibit CCl4-induced liver injuries in rats and may therefore be a potential food or herb for preventing liver injuries.


Introduction
The liver is the largest organ in human body and necessary for metabolism of drugs and exogenous toxins. Liver damage is a prevalent pathology that involves a variety of disorders including oxidative stress, steatosis, hepatitis, fibrosis, cirrhosis, apoptosis, and hepatocellular carcinoma [1]. However, liver damage due to natural, industrial toxins or drugs is common but rarely recognized [2]. Various xenobiotics are known to cause hepatotoxicity such as carbon tetrachloride (CCl 4 ) [3], which alters the antioxidant profile of the liver including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST) [4]. Moreover, marked increases of serum matrix metalloproteinase (MMP9) [5], aminotransferases, tumor necrosis factor-alpha (TNF-α) [6] hepatic HSP70 [7], and inducible nitric oxide synthase (iNOS) protein [6] were detected in animal models after CCl 4 challenging.
Although a wide range of drugs is currently employed in the management of hepatic disorders, alternative approaches from traditional medicinal systems are increasingly popular 2 Evidence-Based Complementary and Alternative Medicine in recent days [8]. In traditional systems of medicine, many herbs including Ocimum species have been recommended for the treatment of various diseases [9]. The Ocimum speciesare widely found in tropical and subtropical regions and commonly used as food spice and traditional herb. To avoid the side effects by administration of western medicines, growing studies of Ocimum species were performed to investigate their therapeutic potentials on hepatic disorders [10][11][12][13][14].
Essential oils obtained from Ocimum species showed various medicinal potentials in chemopreventive, anticarcinogenic, free radical scavenging, and radioprotective uses [10][11][12]. Additionally, ethanolic extract of Ocimum gratissimum (OG) leaf also revealed significant chemopreventive effects on chemical-induced papilloma genesis by modulating metabolizing enzymes such as cytochrome P450, glutathione-s-transferase, and aryl hydrocarbon hydroxylase [15,16]. Moreover, a recent study indicated that administered orally aqueous extract of OG leaf could reduce oxidative and toxicant activity and enhance specific activities of hepatic antioxidant enzymes in rats [13]. Notably, our recent study also indicated that OG leaf aqueous extract (OGAE) may be important in protecting H9c2 cells from H 2 O 2induced cell death by inhibiting the mitochondrial dependent apoptosis pathway [14]. Although these studies strongly implicated the medicinal effects of OG, there are only few studies for the beneficial effects of OGAE on chemicalinduced hepatic injury. Herein, we investigated the effects of OGAE on reducing hepatic injuries in rats after CCl 4 challenging.

Preparation of Ocimum gratissimum Aqueous Extract and Composition
Analysis. Extract of OG was prepared as described elsewhere [14,17]. Briefly, leaves of OG Linn were harvested, cleaned with distilled water, and homogenized with distilled water by using polytron. The homogenate was incubated at 95 • C for 1 hour (h) and then filtered through two layers of gauze. The filtrate was centrifuged at 20000 g for 15 min at 4 • C to remove insoluble pellets, and the supernatant was collected, lyophilized, and stored at −70 • C until use. The contents of polyphenol in OGAE were analyzed as indicated in our previous paper [14,17] and shown in Table 1, revealing the final extract composition of 11.1% polyphenolic acid and 4.5% flavonoids.

Animal Model and Treatments.
Thirty-two male Wistar rats (4 weeks old) were obtained from the National Animal Breeding and Research Center, Taipei, Taiwan and acclimatize for 1 week under controlled conditions. The animals were kept under a 12-h light-dark cycle, and ambient temperature was maintained at 25 • C. Animals were free access to water and standard laboratory chow (Lab Diet 5001; PMI Nutrition International Inc., Brentwood, MO, USA). Animal welfare and experimental procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional   Figure 1: Detection of (a) catalase activity and (b) eliminated DPPH in serum of rats with different treatment.

OGAE Increases the Serum Catalase and DPPH Levels in
Serum of Rats Treated with CCl 4 . To examine the effects of OGAE on antioxidant activities, the levels of catalase and DPPH in serum of rats with different treatment were examined. Significant decreases of serum catalase and DPPH were detected in rats treated with CCl 4 (E group) as compared to those rats from control group (Figures 1(a) and 1(b)).
In contrast, significant increases of catalase and DPPH levels were observed in serum of rats from OGAE and S groups as compared to those rats from E group (Figures 1(a) and 1(b)).

OGAE Decreases the Expressions of HSP70 and iNOS Proteins in Livers of Rats Treated with CCl 4 .
To examine the effects of OGAE on stress proteins after CCl 4 challenging, Western blots were performed to detect the expressions of HSP70 and iNOS proteins in livers of rats with different treatments. Significant increases of HSP70 were detected in livers of rats from E group as compared to those rats from N group (Figure 2(a)). However, significant decreases of HSP70 proteins were observed in livers of rats from both OGAE and S groups as compared to those rats from E group  ( Figure 2(a)). Quantified results were shown in Figure 2(b). In addition, similar results were observed in iNOS expression. Significant increases of iNOS proteins were detected in livers of rats from E group as compared to those rats from control group (Figure 3(a)). In contrast, significant decreases of iNOS proteins were detected in livers of rats from both OGAE and S groups as compared to those rats from E group (Figure 3(a)). Quantified results were shown in Figure 3(b).

OGAE Reduces MMP-9 Activity and uPA Protein Expression through Inhibiting ERK and NF-κB Signaling in Rats
Treated with CCl 4 . MMP-9 is known as an indicator playing important roles in hepatic disorders. To investigate the effects of OGAE on MMP-9, gel zymography was performed to detect the MMP-9 activity. Significant increase of MMP-9/ MMP-2 ratio was detected in liver of rats from E group as compared to those rats from N group (Figure 4(a)). Notably, significant decreases of MMP-9/MMP-2 ratio were observed in livers of rats from both OGAE and S groups (Figure 4(a)).
Quantified results were shown in Figure 4(b). In addition, the expression of uPA protein, an upstream activator of MMP-9, was also examined by Western blot. As shown in Figure 5(a), significant increase of uPA protein was observed in liver of rats from E group as compared to those rats from N group ( Figure 5(a)). In contrast, significant decreases of MMP-9/MMP-2 ratio were observed in livers of rats from both OGAE and S groups as compared to those rats from E group ( Figure 5(a)). Quantified results were shown in the lower panel of Figure 5(b). To further investigate the influence of OGAE on MMP-9 signaling, presence of ERK protein and its phosphorylated form were examined. Significantly increased ratio of p-ERK/ERK was detected in liver of rats from E group as compared to those rats from N group (Figure 6(a)). In contrast, significant decreases of p-ERK/ERK ratio were observed in livers of rats from both OGAE and S groups as Evidence-Based Complementary and Alternative Medicine 7 compared to those rats from E group (Figure 6(a)). Quantified results were shown in Figure 6(b). Moreover, similar results were observed in phosphorylation of NF-κB (p-P65).
Significantly increased ratio of p-65/β-actin ratio was detected in liver of rats from E group as compared to those rats from N group (Figure 7(a)) whereas significant decreases of p-65/β-actin ratio were observed in livers of rats from both OGAE and S groups as compared to those rats from E group (Figure 7(a)). Quantified results were shown in Figure 7(b).

Discussion
Although growing evidences have indicated the therapeutic potentials of Ocimum species on hepatic disorders, only few studies for the therapeutic effects of OGAE on chemicalinduced hepatic injury were performed. In current study, we further reported the beneficial effects of OGAE on increasing serum catalase and DPPH levels and reducing hepatic HSP70 and iNOS protein in livers of CCl 4 -administrated rats. In the meantime, we found that OGAE also reduces the ratio of MMP-9/MMP-2, uPA protein level via ERK, and NF-κB phosphorylation signaling. Liver injury induced by CCl 4 is a well-known experimental model [21][22][23]. The hepatic toxicity of CCl 4 is mainly through the generation of trichloromethyl free radical in liver microsomes and consequently induces lipid peroxidation [24]. Marked reduction of antioxidant levels was observed in animal models after CCl 4 challenging, including SOD, CAT, GPx, GR, and GST [4]. Meanwhile, various hepatic damaged markers such as HSP70 and iNOS were also elevated [6,7]. In current study, our experimental results indicated that OGAE and silymarin exhibited a significant hepatoprotective effect as evident from the increase of serum CAT and DPPH as compared with control group. In addition, significantly reduced hepatic damaged markers, HSP70 and iNOS, were detected in both OGAE or silymarin-treated rats as compared to control group. These findings implied the potentials of OGAE on increasing antioxidant activity and reducing inflammatory associated proteins in livers of rats after CCl 4 challenging as well as the silymarin does.
MMP9 is a member of the MMP protein family and plays a crucial role in various hepatic disorders [25], including inflammatory processes [26], fibrogenesis [27], and cancers [28,29]. A variety of studies have indicated that CCl 4 could induce hepatic injury via elevating MMP9 protein level and activity [30][31][32][33]. In addition, urokinase-type plasminogen activator (uPA) has been demonstrated to upregulate MMP-9 expression in both gene transcription and protein synthesis [34]. Decreased expression of MMPs by inhibiting the uPA system could provide the microvascular protection in animal model of cerebral ischemic rats [35]. Moreover, ERK1/2 and NF-κB signaling is known to play crucial roles in upregulation of MMP9 [29,36]. These studies suggested the importance of MMP9 signaling in liver injuries. Notably, our experimental results exhibited a significant hepatoprotective effect of OGAE by decreasing MMP9, uPA, p-ERK/ERK ratio, and phosphorylated P65 in livers of CCl 4 -challenging rats as compared to controls. Polyphenols from plant extracts have been indicated as being major therapeutic components for oxidative stress. Although the cellular mechanisms underlying the actions of polyphenols and their metabolites have not been completely interpreted, it is believed that their properties including antioxidant activity, free radical scavenging, and anti-in-flammation should be involved [37]. Silymarin is known as a purified extract from Silybum marianum (L.) Gaertn and composed of silibinin, isosilibinin, silydianin, and silychristin. This extract has been wildly used as a remedy for nearly 2000 years and remains being used as a medicine for many types of acute and chronic liver diseases [38,39]. However, various side effects of siymarin such as nausea, mild headache, diarrhea, vomiting, and joint pain were reported [38,40]. Recently, similar components of polyphenols and effects on hepatic protection were reported in studies of O. gratissimum [11][12][13][14]. As illustrated in Figure 8, our results revealed that both silymarin and OGAE have very similar effects on hepatic protection by increasing antioxidant activities, reducing stress-related proteins and MMP9 activity through ERK and NF-κB signaling in CCl 4 -challenging rats. Additionally, the beneficial effects of OGAE were observed along with the administration of CCl 4 in this study. Therefore, we consider that the potential benefit of OGAE should be preventive or neutralizing on CCl 4 -induced acute liver injury rather than therapeutic. However, further study is merited to investigate whether OGAE has therapeutic effects on CCL 4 -induced liver injury. Altogether, the current study shows that OGAE supplement to CCl 4 -administrated rats leads to several beneficial alternations at multiple levels in livers and suggest the potential of OGAE in protective application. Although silymarin and OGAE share the similar effect and effectiveness in terms of anti-injury of liver caused by CCl 4 ; herein we indeed provide another possible health food or alternative medicine for alleviating acute liver injuries.