Background
Hepatotoxicity is a growing public health concern in modern society due to the increasing incidence of alcoholism, cigarette smoking, drug abuse and other unhealthy lifestyle options such as consuming high fructose-containing beverages and high fat containing foods. Liver damage is characterized by a progressive development of steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [
1,
2]. Oxidative stress plays a central role in the development of liver diseases. Carbon tetrachloride (CCl
4) is a widely used solvent in chemical industries and well known for its hepatic and renal toxic actions. It is also used to establish experimental animal model of hepatic dysfunction in the laboratory [
3,
4]. The metabolism of CCl
4 occurs mainly in the liver by CYP450 enzyme system into trichloromethyl (
•CCl
3) and peroxy trichloromethyl (
•OOCCl
3) free radicals which have been reported to cause hepatotoxic effects, like fibrosis, steatosis, necrosis, and hepatocarcinoma [
3,
5]. Chronic insult to the liver due to hepatotoxins, alcohol consumption and high-calorie diet triggers inflammation and fibrosis [
4]. Liver fibrosis is a dynamic process where oxidative stresses are responsible for the activation of hepatic stellate cells (HSCs) [
6]. HSCs are the major cell types responsible for the deposition of a large amount of extracellular matrix (ECM) and collagen in liver [
7]. CCl
4-mediated liver fibrosis is characterized by activation of Kupffer cells and induction of an inflammatory response by secreting cytokines, chemokines and other pro-inflammatory factors [
8]. CCl
4 treatment also attracts more inflammatory cells in liver apart from Kuffer cells and further contributes to liver necrosis [
8]. High level of lipid peroxidation products and decreased antioxidant levels were also noted in CCl
4 mediated hepatic dysfunction in animal [
9]. As oxidative stress plays a central role in liver pathologies and their progression, the use of antioxidants would have been an alternative therapeutic approach to counteract the liver damage. Citrus fruits are a rich source of natural antioxidants and showed beneficial role in various degenerative diseases [
10]. Caffeic acid was found in high amount compared to other phenolic compounds in
Citrus macroptera peel which is a strong antioxidant compound [
11]
. Caffeic acid also showed hepatic protection and prevented fibrosis in various experimental animal models [
12‐
14]. This hepatoprotective activity of caffeic acid is linked to improved antioxidant defense and inflammatory state in liver [
11,
15].
Citrus macroptera is known as ‘Satkara’ in Bengali and ‘Wild orange’ in English. It is a semi-wild species of citrus fruits which is native to the regions of Southeast Asia and found in large amount in Sylhet Division of Bangladesh. The fruit is used as a cooking ingredient in different kinds of meats and as an aromatic vegetable in Bangladesh. Meats cooked with satkara are now served in many Bangladeshi/Indian restaurants in the United Kingdom.
Citrus macroptera (Satkara) fruit is also used as an appetite stimulant and in the treatment of fever as reported in the traditional system of medicine in Bangladesh. The previous report suggested that
Citrus macroptera contains mainly lupeol and stigmasterol [
16]. Other studies conducted on the bark and leaves of the
Citrus macroptera reported the antioxidant and antimicrobial activities [
16,
17]. A recent investigation also suggests that
Citrus macroptera extract showed in-vitro α-amylase inhibitory activity and hypoglycemic activity in normal rats [
18]. We recently reported the hepatoprotective activity of
Citrus maxima peel powder in CCl
4 treated rats [
19]. However, limited data and few scientific literatures are available on any therapeutic effect of
Citrus macroptera in liver diseases. Therefore, this current research was designed to understand the possible anti-inflammatory and anti-fibrotic activity of
Citrus macroptera peel powder in hepatic dysfunction in CCl
4 treated rats.
Methods
Chemicals
Arbutin (AR), gallic acid (GA), hydroquinone (HQ), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), Syringic acid (SA), (−)-epicatechin (EC), vanillin (VL), p-coumaric acid (PCA), trans-ferulic acid (FA), rutin hydrate (RH), ellagic acid (EA), benzoic acid (BA), rosmarinic acid (RA), myricetin (MC), quercetin (QU), trans-cinnamic acid (TCA), and kaempferol (KF) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC), methanol (HPLC), acetic acid (HPLC), and ethanol were obtained from Merck (Darmstadt, Germany).
Plant material
Citrus macroptera fruits were collected from Sylhet, Bangladesh and authenticated by Sarker Nasir Uddin, Senior Scientific Officer, National Herbarium, Mirpur, Dhaka. A voucher specimen (Acc. No. 40847) was deposited in the herbarium for future reference. The peels were removed and dried for further processing. The dried peels were grinded to fine powder using an electric grinder machine and mixed with the food directly as supplementation. A portion of the dried peel powder was used to prepare crude ethanol extract and used for polyphenol detection and quantification.
Detection and quantification of selected phenolic compounds in the ethanol extract were determined by HPLC-DAD analysis as described by Ismet et al. [
20] with some modifications. It was carried out on a Dionex UltiMate 3000 system equipped with quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). Separation was performed using Acclaim® C
18 (5 μm) Dionex column (4.6 × 250 mm) at 30 °C with a flow rate of 1 ml/min and an injection volume of 20 μl. For the preparation of calibration curve, a standard stock solution was prepared in methanol containing arbutin, (−)-epicatechin (5 μg/ml each), gallic acid, hydroquinone, vanillic acid, rosmarinic acid, myricetin (4 μg/ml each), caffeic acid, Syringic acid, vanillin,
trans-ferulic acid (3 μg/ml each),
p-coumaric acid, quercetin, kaempferol (2 μg/ml each), (+)-catechin hydrate, ellagic acid (10 μg/ml each),
trans-cinnamic acid (1 μg/ml), rutin hydrate (6 μg/ml) and benzoic acid (8 μg/ml). The UV detector was set to 280 nm for 22.0 min, changed to 320 nm for 28.0 min, again change to 280 nm for 35 min and finally to 380 nm for 36 min and held for the rest of the analysis period while the diode array detector was set at an acquisition range from 200 nm to 700 nm.
Animals and treatment
Ten to 12 weeks old, 24 Long Evans female rats (150–170 g) were obtained from Animal breeding unit of Animal House at the Department of Pharmaceutical Sciences, North South University and were kept in individual cages at room temperature of 25 ± 3 °C with a 12 h dark/light cycles. They had free access to standard laboratory feed and water, according to the study protocol approved by Ethical Committee of Department of Pharmaceutical Sciences, North South University for animal care and experimentation. To evaluate the hepatoprotective effect of
Citrus macroptera, rats were equally divided into four groups (six rats in each group). The groups are as follows-
-
Group I- Animals were treated with 1 ml/kg of saline (0.85%) and olive oil (1 ml/kg) intragastrically twice a week for 2 weeks
-
Group II- Animals were treated with 1 ml/kg of saline (0.85%) and olive oil (1 ml/kg) intragastrically twice a week for 2 weeks. Animals of group II also received Citrus macroptera fruit peel powder supplementation mixed with food (0.5% of the diet, w/w).
-
Group III – Animals were treated with CCl4 (1:3 in olive oil) at a dose of 1 ml/kg intragastrically twice a week for 2 weeks.
-
Group IV- Animals were treated with CCl4 (1:3 in olive oil) at a dose of 1 ml/kg intragastrically twice a week for 2 weeks. Animals of group IV also received Citrus macroptera fruit peel powder mixed in food (0.5% of the diet, w/w) respectively, in addition to CCl4 treatment, twice a week for 2 weeks.
Animals of group I were served as control animals. Animals were checked for the body weight, food and water intake on a daily basis. After 14 days, all animals were weighted and sacrificed, collected the blood in citrate buffer containing tubes and all the organs such as heart, kidney, spleen, and liver were havested. Immediately after collection of the organs, they are weighted and one part was stored in neutral buffered formalin (pH 7.4) for histological analysis and another part was kept in the refrigerator at − 20 °C for further studies. The collected blood was centrifuged at 8000 rpm and separated the plasma and stored in a refrigerator at − 20 °C for further analysis.
Assessment of hepatotoxicity
Liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were estimated in plasma by using Diatec diagnostic kits (Hungary) according to the manufacturer’s protocol.
Preparation of tissue sample for the assessment of oxidative stress markers
For determination of oxidative stress markers, 0.2 g of liver tissue was homogenized in 1.8 mL Phosphate buffer (pH 7.4) and centrifuged at 10000 rpm for 30 min at 4 °C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below.
Estimation of lipid peroxidation
Lipid peroxidation in liver was estimated colorimetrically measuring thiobarbituric acid reactive substances (TBARS) followed by previously described method [
21].
Assay of nitric oxide (NO)
Nitric oxide (NO) was determined according to the method described by Tracey et al. as nitrate [
22]. In this study, Griess-Illosvoy reagent was modified by using naphthyl ethylenediamine dihydrochloride (0.1%
w/
v) instead of 1-naphthylamine (5%).
Advanced protein oxidation products (APOP) assay:
APOP levels were measured by a modification of the method of Witko-Sarsat et al. [
23] and Tiwari et al. [
24]. APOP concentrations were expressed as nmol·mL
− 1 chloramine-T equivalents.
Catalase assay (CAT)
CAT activities were determined using previously described the method by Chance and Maehly [
25]. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. [
26]. 1.0 ml sample of 10% homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4 °C for 1 h and then centrifuged at 4000 rpm for 20 min at 4 °C. The total volume of 3.0 ml assay mixture composed of 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM). The mixture which was developed as yellow color, UV absorbance was taken immediately at 412 nm on a Smart SpecTM plus Spectrophotometer and the content of GSH was expressed as ng/mg protein.
Histopathological determination
For microscopic evaluation, liver tissues were fixed in neutral buffered formalin and embedded in paraffin, sectioned at 5 μm and subsequently stained with hematoxylin/eosin to see the architecture of hepatic tissue and inflammatory cell infiltration. Liver injury score in the Hematoxylin/eosin stained sections were evaluated as follows: 0, minimal or no evidence of injury; 1, mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; 2, moderate to severe injury with extensive nuclear pyknosis and loss of intercellular borders and 3, severe necrosis with disintegration of hepatic cords, hemorrhage and neutrophil infiltration. All evaluations were made in 3 fields per section and 3 sections per liver.
Sirius red staining for fibrosis and Prussian blue staining for iron deposition were also done in liver sections. Sections were then studied and photographed under a light microscope (Zeiss Axioscope) at 40 magnifications. Collagen deposition was semiquantitatively measured using NIH Image J free software (Version 1.48v).
Statistical analysis
All values are expressed as a mean ± standard error of the mean (SEM). The results were evaluated by using the One-way ANOVA followed by Bonferroni test using Graph Pad Prism Software, version 6. Statistical significance was considered p < 0.05 in all cases.
Discussion
Currently, public awareness program against liver diseases such as fibrosis and cirrhosis are being aggressively focused [
27]. The liver is the main organ where almost all kinds of toxins, foods as well as drugs are detoxified [
28]. CCl
4 is metabolized in phase-I reaction and activates the ROS producing system [
29]. In the current study, CCl
4 administration in rats developed hepatic damage by increasing lipid peroxidation and oxidative stress. CCl
4 administration in rats also increased inflammatory cells infiltration and fibrosis in liver tissues. Our investigation also suggests that
Citrus macroptera peel powder supplementation ameliorated most of the deleterious effects developed in CCl
4 administered rats.
The active metabolite of CCl
4 lead to hepatocyte damage and may initiate the leakage of a lot of hepatic enzymes (AST, ALT, and ALP) in blood circulation which was associated with immune cells infiltration, massive centrilobular apoptosis, ballooning degeneration and finally cell death [
30]. Elevated liver marker enzymes activities (AST, ALP, and ALT) indicate the dysfunction and damage in the liver during disease condition [
31]. This study also revealed the increased liver marker enzymes (AST, ALP, and ALT) activities in CCl
4 administered rats which were decreased or normalized by
Citrus macroptera supplementation. The previous study reported that reduction of liver enzyme activity would be beneficial in case of hepatic damage protection [
32]. Earlier investigations also showed that derivative of caffeic acid also prevented the liver damage and decreased the liver marker enzymes activities [
33,
34].
One of the major markers of hepatic damage is oxidative stress-mediated lipid peroxidation which plays irreversible damage to hepatocellular components. MDA is the outcome of lipid peroxidation in cell membranes and damages the integrity of cells. MDA development also causes an imbalance to lysosomes and helps to oxidize the protein [
35]. Our study showed that
Citrus macroptera supplementation decreased lipid peroxidation level in plasma and liver tissue compared to CCl
4 administered rats. The strong antioxidant can scavenge the free radical and superoxide anions from a biological system. Caffeic acid is a strong antioxidant showed protection against free radical-mediated lipid peroxidation in experimental animals reported previously [
34].
Previous studies also reported that anti-oxidant system can reduce inflammatory, pro-inflammatory signaling by inhibiting inducible nitric oxide synthase (iNOS) which is a key factor for immunity responder [
36,
37]. iNOS is considered as the major source of NO in stress condition and generates nitrosative stress. NO is a vasodilator which maintains regular vascular tone in blood vessels. It also works as a signaling molecule to produce immunological substances when the body needed [
38]. However, increased production of NO in oxidative stress may turn into peroxynitrite (
−ONOO•) radical formation which further initiate nitrosative stress in tissues. In this study, we found that CCl
4 increased the level of NO in plasma but not in tissue, which was normalized by the supplementation of
Citrus macroptera peel powder. These findings are also supported by a recent investigation, suggested that derivative of caffeic acid prevents NO production in liver [
39].
Furthermore, free radicals-mediated oxidative stress also reduces the amount of natural anti-oxidant like SOD, catalase, and GSH which fights against any free radicals-mediated damage in tissues. It is well reported that superoxide radicals (O
2•-) are removed by SOD which produces hydrogen peroxide (H
2O
2) [
40]. H
2O
2 then further converted into water by another antioxidant enzyme known as catalase [
40]. Moreover, CCl
4 administration also depleted intracellular-reduced GSH levels, suggesting that GSH loss might result from detoxification of CCl
4 by GSH conjugation [
40]. GSH also breaks H
2O
2, ROOH and helps against protein oxidation [
41]. Our study revealed that administration of oral CCl
4 reduced the amount of anti-oxidant capacity in both plasma and liver tissue which was restored by
Citrus macroptera supplementation. Our previous study also demonstrated that iron overload could lead to cause hepatic damage. Free iron in tissues may start Fenton like reaction resulted in a hydroxyl free radicals generation [
42]. Supplementation with
Citrus macroptera peel powder supplementation reduced iron deposition in the liver of CCl
4 treated rats.
The most noteworthy pathological characteristics of CCl
4 intoxicated hepatotoxicity are inflammation and fibrosis. Hepatic fibrosis is usually initiated by hepatocyte damage, leading to the recruitment of inflammatory cells and platelets with the subsequent release of cytokines, chemokines, and growth factors [
7]. Inflammatory cells infiltration further lead to activation of HSCs and their transformation into myofibroblast-like cells [
43]. Chronically activated HSCs produce large amounts of ECM proteins and enhance fibrosis followed by TGF-β1 mediated signaling pathways [
1]. Free radicals may also activate HSC which further help to secret collagenase-1 from local fibroblast [
1,
44]. Our investigation showed that CCl
4 administration significantly enhanced inflammatory cell infiltration and fibrosis in the liver of rats. These findings are in agreement with previously published literature which showed that CCl
4 mediated oxidative stress is responsible for the inflammation and fibrosis in liver [
45,
46]. Moreover,
Citrus macroptera peel powder supplementation reduced inflammatory cells infiltration and fibrosis in the liver of CCl
4 treated rats. This antifibrotic activity of
Citrus macroptera peel powder could be attributed to the presence of the antioxidant and anti-inflammatory compound, caffeic acid. Recent findings also suggested that caffeic acid administration in experimental animals may prevent hepatic fibrosis [
14,
39].
Researchers are still working on natural plants to discover new compounds of high biological activity. It is reported by World Health Organization (WHO) that more than 80% people in developing countries basically depends on herbal medicines [
47]. Researchers are more focusing on plant-derived polyphenols, seeing that the synthetic anti-oxidants have several adverse effects [
48]. Our investigation showed that caffeic acid; syringic acid and vanillic acid are present in
Citrus macroptera peel powder. The previous report also suggests that syringic acid and vanillic acid which are important polyphenols showed anti-oxidants, anti-inflammatory, anti-fibrogenic and hepatoprotective activities in CCl
4 induced rats [
49].
Citrus macroptera or wild orange is occasionally used for increasing appetite, reducing fever and making meat preparation [
50]. A recent investigation also suggests that
Citrus macroptera is extremely safe, even methanolic extract at a dose of 1000 mg/kg did not produce toxic effects in rats considering biochemical as well as histological assessment [
51]. Our investigation also supports this notions that
Citrus macroptera peel powder supplementation in normal rats did not alter any of the biochemical as well as histological parameters compared to control rats.