Methods
Chemicals, antibodies, and kit
CC14, Tris-HCl, thiobarbituric acid (TBA), oxidized and reduced glutathione, reduced β-nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate, 1-chloro-2,4-dinitrobenzene (CDNB), glutathione reductase, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), potassium persulfate, sulfosalicylic acid (SSA), bovine serum albumin (BSA), hydrogen peroxide (H2O2), flavin adenine dinucleotide (FAD), 2,6-dichloroindophenol, trichloroacetic acid (TCA), Tween 20, and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit polyclonal antibody specific for 8-hydroxy-2′-deoxyguanosine (8-8-OHdG), rabbit polyclonal antibody specific for 4-hydroxy-2-nonenal (HNE), rabbit polyclonal antibody specific to tumor necrosis factor alpha (TNF-α), rabbit polyclonal antibody specific to interleukin 6 (IL-6), rabbit polyclonal antibody specific to prostaglandin E2 (PGE2), EnVision™ + System/horseradish peroxidase (HRP), Rb (DAB+), target retrieval solution, and antibody diluent were purchased from Dako (Agilent Technologies Company, Denmark).
Plant collection and extraction
The plant was collected from the lowlands of Papar, Sabah, Malaysia. Plant identification was confirmed by Mr. Kulip and Mr. Johnny Gisil from the Institute of Tropical Biology and Conservation, Universiti Malaysia Sabah. A voucher specimen (MDS003) was deposited at the Tropical Biology and Conservation Herbarium, Universiti Malaysia Sabah. Sixty grams of dry powder was extracted with 300 ml of methanol by the Soxhlet method (50–60 °C and 72 h). Methanol residues were removed from the extract using a vacuum rotary evaporator. The samples were kept at − 80 °C for 24 h and then lyophilized using a freeze dryer. The freeze-dried samples were then stored in the freezer for further analysis [
7].
Total phenolic content
The total phenolic content in the
C. nudiflora methanol extract was determined by the Folin-Ciocalteu method [
8] with slight modifications. A stock solution of 1 mg/ml was prepared from the extract. A Folin-Ciocalteu reagent was prepared by tenfold dilution (ratio 1:9). The reagent reacts with phenolic and non-phenolic reducing substances to form chromogens that can be measured spectrophotometrically [
8]. Briefly, 1.5 ml of Folin-Ciocalteu reagent was mixed with 0.2 ml of assay samples and mixed vigorously. After 5 min, 1.5 ml of sodium carbonate (60 g/l) was added to the mixture. Finally, the mixture was allowed to stand for 90 min in the dark at room temperature. The absorbance was measured at 725 nm against a blank. Gallic acid was used as a standard for the quantification of phenolic compounds. Concentrations of 0.01, 0.02, 0.04, 0.08, and 0.1 mg/ml of gallic acid were used to plot the standard calibration curve. The concentration of the total phenolic content was estimated as milligrams of gallic acid equivalent by using an equation obtained from the gallic acid calibration curve.
2,2-Diphenyl-2-picrylhydrazyl assay
The antioxidant activity of
C. nudiflora methanol extracts was determined via 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay [
9]. A plant extract was prepared at a concentration of 1 mg/ml. Various concentrations (0.012, 0.025, 0.050, 0.1, and 0.5 mg/ml) of plant extract were used. During the process, 0.3 ml of plant extract was mixed with 2.7 ml of DPPH (6 × 10
−5 M) in methanol and left in the dark for 60 min. Absolute methanol was used as a blank. The absorbance was measured at 512 nm using a spectrophotometer. Ascorbic acid was used as a standard. The radical scavenging activity was calculated according to the formula summarized below
$$ \%\mathrm{RSA}=\left[\left({A}_{\mathrm{B}\ \mathrm{control}}-{A}_{\mathrm{A}\ \mathrm{sample}}\right)/{A}_{\mathrm{B}\ \mathrm{control}}\right]\times 100 $$
where % RSA is the percentage of radical scavenging activities,
A
A is the absorbance values of the extract sample, and
A
B is the absorbance values of the control sample.
Gas chromatography mass spectrometry analysis of C. nudiflora
A small quantity of C. nudiflora methanol extract was injected into a gas chromatography mass spectrometry (GCMS) system, which consisted of an Agilent 7890A gas chromatograph system coupled with an Agilent 5975C mass spectrometry detector. A capillary column, HP-5MS (30 m × 0.25 mm) of 0.25 μm film thickness of coated material, was used. The injector temperature was set at 250 °C whereas the temperature settings were as follows: start at 40 °C and hold for 3 min; from 40 to 200 °C with 3 °C/min and then hold for 3 min. A post-run of 5 min at 200 °C was sufficient for the next injection. A gas chromatography was performed in splitless mode. Helium gas was used as a carrier gas and maintained at a 1.0 ml/min constant flow rate. Identification of various compounds was carried out by referring to the NIST library, and the chemical makeup was computed with reference to the abundance of the compounds in a chromatogram. Each analysis was carried out in triplicate, together with a blank solvent.
Experimental protocol
Male Sprague Dawley rats weighing 150–250 g were utilized throughout the experiment. The animals were acquired from the Animal Breeding House, Biotechnology Research Institute, Universiti Malaysia Sabah. The animals were maintained at a room temperature (25 °C) in a temperature-controlled room and allowed access to food (normal laboratory chow) and tap water ad libitum. All the animals were treated humanely and well maintained under standard ethical principles as per university regulations (UMS/IP7.5/M3/4-2012). They were acclimatized to laboratory conditions for 7 days before experiments started. CCl4 was prepared at a dose of 1.0 ml/kg body weight with corn oil (1:1). A suspension of plant extract was prepared in distilled water, and different doses of C. nudiflora extract (150, 300, and 450 mg/kg body weight) were administered to the animals by gastric gavage needles. Thirty-six adult male rats were taken and distributed randomly into six groups of six animals each. Group 1 served as a normal control; group 2 was treated with CCl4 (1.0 ml/kg body weight); groups 3, 4, and 5 were treated with C. nudiflora (150, 300, and 450 mg/kg body weight, respectively) + CCl4 (1.0 ml/kg body weight); and group 6 was treated only with high doses of C. nudiflora (450 mg/kg body weight).
The rats were pretreated with selected doses of C. nudiflora methanol extracts continuously for 14 days, followed by an administration of CCl4 on the 13th and 14th days. The doses of the plant extract (150, 300, and 450 mg/kg body weight) and CCl4 (1.0 ml/kg body weight) were administered. All of these rats were sacrificed 24 h after the last dose of CCl4 within a period of 1 h. Blood was collected by cardiac puncture using sterile disposable syringes, while serum was obtained by centrifugation at 2000×g for 15 min. The livers of these animals were removed immediately and cleaned with chilled saline (0.85% w/v, sodium chloride) to remove an extrinsic material. The liver tissues were stored at − 80 °C for biochemical studies while a small portion of the tissues was kept in a 10% neutral buffered formalin solution for histopathological and immunohistochemical analyses.
Preparation of post-mitochondrial and cytosolic supernatant
The hepatic homogenate was prepared by the method proposed by Mohandas et al. [
10], as described by Iqbal et al. [
11]. The rat livers were homogenized in an ice-cold phosphate buffer (0.1 M, pH 7.4) containing KCl (1.17%
w/
v) using a homogenizer (Polytron PT 1200E, Switzerland). The nuclear debris was removed from the liver homogenate by centrifugation at 3000×
g for 10 min at 4 °C. The post-mitochondrial supernatant (PMS) was obtained by centrifugation at 12,000×
g for 30 min at 4 °C, which was utilized for the measurement of malondialdehyde (MDA) and reduced glutathione (GSH) content, as well as a source of antioxidant enzymes. A part of PMS was further ultracentrifuged at 105,000×
g for 1 h to obtain cytosolic fractions to determine quinone oxidoreductase assay activity.
Biochemical assays
Assay of reduced glutathione
Reduced glutathione in liver PMS was estimated according to the method described by Jollow et al. [
12]. Briefly, 1.0 ml of hepatic PMS (10%
w/
v) was reacted with 1.0 ml of sulfosalicylic acid (4%
w/
v). The samples were kept at 4 °C for 60 min and then centrifuged at 3000×
g for 30 min at 4 °C. The assay mixture contained 0.2 ml filtered supernatant, 2.6 ml phosphate buffer (0.1 M, pH 7.4), and 0.2 ml DTNB (4 mg/ml of 0.1 M phosphate buffer, pH 7.4) in a total volume of 3.0 ml. The development of a yellow color was read immediately at 412 nm on a spectrophotometer (model 4001/4). Results were expressed as micromoles of reduced glutathione per gram of liver tissue.
Assay of lipid peroxidation
Hepatic lipid peroxidation in PMS was performed by the method described by Buege and Aust [
13]. Briefly, 1.0 ml of PMS was mixed with 0.5 ml of trichloroacetic acid (10%
w/
v) and centrifuged at 800×
g for 30 min, and 1.0 ml of the supernatant was reacted with 1.0 ml of thiobarbituric acid (0.67%
w/
v). All the tubes were placed in a boiling water bath for a time period of 20 min. The tubes were then transferred to an ice bath and allowed to cool for 5 min. The amount of MDA formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm using a spectrophotometer (model 4001/4). The results were expressed as nanomoles of MDA formed/gram tissue using a molar extinction coefficient of 1.56 × 10
5 M × 1 cm × 1.
Assay of glutathione peroxidase activity
Glutathione peroxidase activity in liver PMS was carried out according to the method by Mohandas et al. [
10] as described by Iqbal et al. [
14]. Briefly, 0.025 ml of 10%
w/
v hepatic PMS was added to a reaction mixture that consisted of 1.51 ml of 0.1 M phosphate buffer (pH 7.4), 0.1 ml EDTA (0.5 mM), 0.1 ml sodium azide (1.0 mM), 0.05 ml glutathione reductase (1.0 EU/ml), 0.1 ml GSH (1.0 mM), 0.1 ml NADPH (0.1 mM), and 0.01 ml hydrogen peroxide (30%), forming a total volume of 2.0 ml. Enzyme activity was calculated as nanomoles of NADPH oxidized/minute/milligram protein using a molar extinction coefficient of 6.22 × 10
3 M × 1 cm × 1.
Assay of glucose-6-phosphate dehydrogenase activity
Glucose-6-phosphate dehydrogenase activity was measured by the method of Zaheer et al. [
15] as described by Iqbal et al. [
14]. Briefly, a reaction mixture of 3.0 ml, consisting of 0.5 ml of 0.05 M Tris-HCl buffer (pH 7.6), 0.05 ml NADP (0.1 mM), 0.05 ml glucose-6-phosphate (0.8 mM), 0.25 ml MgCl
2 (8 mM), 0.1 ml of 10%
w/
v hepatic PMS, and 2.0 ml of distilled water, was prepared. The changes in absorbance were noted at 340 nm, and the enzyme activity was calculated as nanomoles of NADP reduced/minute/milligram protein using a molar extinction coefficient of 6.22 × 10
3 M × 1 cm × 1.
Assay of glutathione reductase activity
Glutathione reductase activity was determined by the Carlberg and Mannervik method, [
16] as described by Iqbal et al. [
14]. Briefly, 0.05 ml of 10%
w/
v hepatic PMS was mixed with 1.7 ml of phosphate buffer (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1 mM), and 0.1 ml NADPH (0.1 mM) and the reaction mixture was read at 340 nm. Enzyme activity was calculated as nanomoles of NADPH oxidized/minute/milligram protein using a molar extinction coefficient of 6.22 × 10
3 M × 1 cm × 1.
Assay of catalase activity
Catalase activity was determined by the Claiborne [
17] method, as described by Iqbal et al. [
14]. Briefly, a reaction mixture of 2 ml, consisting of 0.99 ml of 0.05 M phosphate buffer (pH 7.0), 1.0 ml of 0.019 M hydrogen peroxide, and 0.01 ml of hepatic PMS (10%
w/
v), was prepared. The changes in absorbance of the reaction solution were noted at 240 nm, and the enzyme activity was calculated as nanomoles of H
2O
2 consumed/minute/milligram protein using a molar extinction coefficient of 6.4 × 10
3 M × 1 cm × 1.
Assay of glutathione S-transferase activity
Glutathione
S-transferase activity was measured by the Habig et al. [
18] method, as modified by Athar and Iqbal [
19]. The assay system was obtained by the addition of 2.75 ml phosphate buffer (0.1 M, pH 6.5), 0.1 ml reduced glutathione (1.0 mM), 0.1 ml CDNB (1.0 mM), and 0.25 ml of hepatic PMS (10%
w/
v). The absorbance was determined at 340 nm, and the enzyme activity was calculated as nanomoles of CDNB conjugate formed/minute/milligram protein using a molar extinction coefficient of 9.6 × 10
3 M × 1 cm × 1.
Assay of NAD(P)H: quinoneoxido reductase activity
Quinone reductase activity was determined by the Benson et al. [
20] method, as modified by Iqbal et al. [
21]. The assay mixture consisted of 2.0 ml of Tris-HCl buffer (0.025 M, pH 7.4), 0.7 ml BSA (1 mg/ml), 0.1 ml FAD (150 μM), 0.02 ml NADPH (0.1 mM), 0.02 ml Tween 20 (1%
w/
v), 0.05 ml of cytosolic fraction (10%
w/
v), and 0.05 ml of 2,6-dichlorophenolindophenol (2.4 mM) in a final volume of 3.0 ml. The enzyme activity was determined at 600 nm and calculated as nanomoles of 2,6-dichlorophenolindophenol reduced/minute/milligram protein using a molar extinction coefficient of 2.1 × 10
4 M × 1 cm × 1.
Assay of serum alanine transaminase and aspartate transaminase
Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels were determined by the method described by Reitman and Frankel [
22]. Briefly, 0.5 ml of α-ketoglutarate (2 mM) and α-
l-alanine (200 mM) for ALT, and α-ketoglutarate (2 mM) and
l-aspartate (200 mM) for AST, was incubated in a water bath for 10 min at 37 °C; 0.1 ml of serum was added, and the volume was made up to 1.0 ml with a sodium phosphate buffer. The reaction mixture was incubated for exactly 30 and 60 min for ALT and AST, respectively. After that, 0.5 ml of DNPH (1 mM) was added to the reaction mixture and kept for another 20 min at room temperature. Finally, the change in the color was noted by the addition of 5.0 ml of NaOH (0.4 N) and the final product was read at 510 nm after 30 min.
DPPH assay
The free radical scavenging activity of rat tissue and serum samples was determined by the DPPH method, described by Brand-Williams et al. [
23] with slight modifications. Briefly, 100 μl of each sample was added to 2.7 ml of DPPH in an ethanol solution (6 × 10
−5 M) in a test tube. The tubes were kept in the dark for 1 h. After that, the samples were treated with 1 ml of chloroform and centrifuged at 1107×
g for 5 min. The absorbance of clear solution was recorded at 517 nm using a spectrophotometer. An ethanol solution of DPPH (6 × 10
−5 M) without the sample was used as a control, and the percentage of DPPH radical scavenging activity was calculated according to the following equation:
$$ \%\mathrm{DPPH}=\left[\left({A}_{\mathrm{B}\ \mathrm{control}}-{A}_{\mathrm{A}\ \mathrm{sample}}\right)/{A}_{\mathrm{B}\ \mathrm{control}}\right]\times 100 $$
where
A
A is the absorbance values of the extract sample and
A
B is the absorbance values of the control sample.
ABTS assay
The free radical scavenging activity of our samples was performed via ABTS assay, as proposed by Re et al. [
24]. Briefly, a stock solution of ABTS (7 mM) was prepared in water. An ABTS radical cation (ABTS
S+) was obtained by treating ABTS stock solution with 2.45 mM potassium persulfate (final concentration). The mixture was kept in the dark overnight (12–16 h). The ABTS
S+ solution was then diluted with phosphate buffer saline with a pH value of 7.4 (PBS) to an absorbance of 0.70 at 734 nm. After that, 20 μl of tissue or serum was added to 2 ml of diluted ABTS
S+ solution. The mixture was incubated for 6 min at 30 °C, and the absorbance was recorded at 734 nm. ABTS solution without the sample was used as a control. The percentage inhibition of ABTS
S+ by the sample was calculated according to the given equation:
$$ \%\mathrm{ABTS}=\left[\left({A}_{\mathrm{B}\ \mathrm{control}}-{A}_{\mathrm{A}\ \mathrm{sample}}\right)/{A}_{\mathrm{B}\ \mathrm{control}}\right]\times 100 $$
where
A
A is the absorbance values of the extract sample and
A
B is the absorbance values of the control sample.
Histopathological studies in liver tissue
Light microscopy analysis
For histopathological studies, a fixed portion of rat liver in 10% neutral buffered formalin solution was dehydrated in alcohol and embedded in paraffin. Cut thin sections (5–6 μm) were placed on glass slides and stained with hematoxylin and eosin (H&E) stains. The slides were examined under a light microscope by an expert pathologist who was not aware of the sample assignments to experimental groups for the pathological symptoms of hepatotoxicity, such as necrosis, hepatocyte derangement, fatty degeneration, and blood vessel congestion.
Transmission electron microscopy analysis
Small sections of hepatic tissues were fixed in 4% glutaraldehyde in 0.1 M of phosphate buffer for electron microscopy evaluations. The samples were washed and post-fixed in 1% osmium tetroxide (0.1 M phosphate buffer). Afterwards, the tissues were dehydrated in increasing concentrations of alcohol. Finally, the tissues were washed with 0.1 M of phosphate buffer and embedded in epoxy-resin embedding media. Ultrathin sections were obtained on copper grids, stained with uranyl acetate and lead citrate, and viewed under a transmission electron microscope.
Immunohistochemistry
Immunohistochemical studies were conducted using a Dako Kit. During the process, five antigens, which were HNE-modified protein adducts, 8-OHdG, TNF-α, IL-6, and PGE2, were screened. The hepatic tissues were fixed in 10% phosphate-buffered formalin for 24 h and embedded in paraffin. The paraffin sections were then de-paraffinized and rehydrated through a xylene and graded ethanol series. Antigen retrieval was performed using a water bath at 97 °C for 20 min. The samples were further treated with 3.0% hydrogen peroxide for 5 min and rinsed with Tris-buffered saline (TBS). The tissue sections on the slide were highlighted by wax pens in order to define the spot for the application of various staining reagents. The samples were then incubated for 20 min at room temperature with diluted antibodies, rinsed with TBS three times for 10 min, and applied with HRP for 20 min. The slides were rinsed again with TBS three times for 10 min, and freshly prepared DAB working solution was applied for 5–10 min, followed with a rinse by deionized water three times. The slides were counterstained by Harris hematoxylin for one dip and dehydrated through a graded ethanol and xylene series. The staining intensities of these markers were evaluated semi-quantitatively (weak/strong) and compared among the six groups.
Assay of protein
Protein concentration in all samples was determined by a bicinchoninic acid (BCA) protein assay kit using BSA as a standard.
Statistical analysis
The data was analyzed using SPSS 17.0 Windows statistical package software (SPSS, Inc., Chicago, IL, USA). Significant differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All the results were presented as means ± standard error (S.E.) of the mean. p values less than 0.05 were considered as significant.
Discussion
In this study, a GCMS analysis of the bioactive compounds in
C. nudiflora extracts revealed the presence of bioactive compounds with antioxidant, anti-inflammatory, anticancer, antimicrobial, and hepatoprotective properties. It is widely recognized that dietary antioxidants have positive influences in reducing the risk of development of nutrition-related chronic diseases [
25], and this fact has stimulated the search for new antioxidant sources. In the present study, the hepatoprotective effects of
C. nudiflora were first investigated by suppressing CCl
4-induced oxidative stress and inflammation in the livers of rats and attenuating the morphological changes caused by CCl
4. Our findings contribute to the understanding that
C. nudiflora treatment reduces the occurrence of liver oxidative injuries, and this nutritional strategy presents an alternative to current pharmaceutical approaches aimed at reducing hepatotoxicity.
Herein, nine bioactive compounds have been identified from methanol extracts of
C. nudiflora. Among the identified compounds is phenol, a simple phenolic compound. It has been reported in the ethanol bark extract of
Ficus religiosa Linn with antioxidant, antiseptic, and antibacterial properties [
26]. Benzyl alcohol, an aromatic compound, has been also noticed in extracts of basil leaves (
Ocimum basilicum L.) and reported with antioxidant and antimicrobial activities [
27‐
29]. Eugenol, a phenolic compound, has been also detected in extracts of basil leaves (
O. basilicum L.) and
Eugenia caryophyllata. The antioxidant and anti-inflammatory activities of the compound have been reported [
27‐
31]. Phenol,2,4-bis(1,1-dimethylethyl), an alkylated phenol, has been detected in extracts of
Plumbago zeylanica,
Hybanthus enneaspermus, and
Tephrosia tinctoria and reported with antioxidant, anticancer, and antimicrobial properties [
32,
33]. The abovementioned bioactive compounds have been reported with antioxidant and anti-inflammatory properties. Thus, we believe that these compounds have played a vital role in the protection of liver by neutralizing the free radicals produced by the administration of CCl
4.
Dodecanoic acid, a lauric and saturated fatty acid, has been found in extracts of
Vitex altissima L. The compound has been reported with antimicrobial, antioxidant, antiviral, and hypocholesterolemic properties [
34‐
36]. Hexadecanoic acid (ethyl ester), a palmitic acid ester, has been detected in
Vitex negundo and reported with antioxidant and hypocholesterolemic properties [
37].
N-Hexadecanoic acid, a palmitic acid (saturated fatty acid), has been noticed in
Centaurea aladagensis and reported with antioxidant, hypocholesterolemic, and hemolytic activities [
37,
38]. Phytol, a diterpene alcohol, has been also found in the
H. enneaspermus and reported with antimicrobial, anticancer, anti-inflammatory, hepatoprotective, and diuretic activities [
37‐
39]. 9,12-Octadecadienoic acid, commonly known as linoleic acid (unsaturated fatty acid), has been also detected in
Scotia brachypetala and reported with anti-inflammatory, anticancer, hypocholesterolemic, and hepatoprotective properties [
37,
40,
41].
The study also demonstrates the hepatoprotective and antioxidative effects of
C. nudiflora against CCl
4-induced oxidative hepatic damage in rats. CCl
4 is a known hepatotoxin, mostly utilized for the induction of hepatic injuries in experimental animals [
42]. In our study, it is indicated that CCl
4 results in a moderate decrease in body weight as compared to the control group. During the experiment, a constant increase in the body weight in the control group was noticed for 15 days with no mortality in all groups. However, sudden changes were noticed in the food and water intake of the CCl
4-administered group alone after CCl
4 treatment on the 13th and 14th days, which resulted in a decrease in body weight. The changes were less obvious in rats which were pretreated with
C. nudiflora as compared to the CCl
4-treated group. However, no changes were noticed in the food and water intake of the plant-treated control group (Table
2). Our results are in agreement with previous studies [
43]. Thus, it is indicated that the methanol extract of
C. nudiflora is effective in reducing the toxicity induced by CCl
4.
CCl
4 elevated levels of ALT and AST in the serum of the CCl
4-treated rats, showing hepatic injury as these enzymes leak out from the liver into the blood due to hepatic tissue damage, which is always associated with hepatonecrosis [
44,
45]. With the administration of
C. nudiflora at various doses, the levels of these marker enzymes were restored in a dose-dependent fashion. The recovery of these damages may be due to the stabilization of the plasma membrane and the repair of hepatocytes. The results indicate that treatment with methanol extracts of
C. nudiflora could protect the liver against damage caused by CCl
4.
Lipid peroxidation is the main mechanism of hepatic damage. CCl
4 is biotransformed by the catalytic activity of the liver cytochrome P450 in the endoplasmic reticulum to generate free radicals, mainly trichloromethyl (CCl
3). Free radicals react with oxygen to produce trichloromethyl peroxyl (CCl
3O
2·) radicals. The resulted toxic metabolites have the potential to bind to various proteins or lipids and initiate the peroxidation of lipids [
46,
47]. In the current study, both trichloromethyl and peroxyl radicals seem to initiate the degradation of membrane lipids. This causes the generation of lipid peroxides, which, in turn, give MDA products that result in a loss of cell membrane integrity and liver injury [
46,
47]. Pretreatment of
C. nudiflora methanol extracts markedly reduced the generation of the lipid peroxidation end product (MDA) in a dose-dependent manner. This shows that the administration of
C. nudiflora methanol extracts effectively minimized lipid peroxidation induced by CCl
4.
GSH, a well-known antioxidant, plays an important role against CCl
4-induced injury by covalently binding to CCl
3· radicals and enhancing the activities of glutathione peroxidase and glutathione reductase [
46,
48]. Pretreatment with
C. nudiflora methanol extracts resulted in elevating the liver GSH levels by 23, 34, and 38% as compared with the CCl
4-treated group. The GSH levels in animals pretreated with
C. nudiflora show no significant differences when compared with the saline-treated control group.
CCl
4 intoxication also affects the activities of hepatic antioxidant enzymes. All oxygen-utilizing organisms are equipped with well-organized antioxidant systems to prevent damage caused by free radicals. These enzymes include catalase, glutathione peroxidase, glutathione reductase, glutathione-6-phosphate dehydrogenase, glutathione
S-transferase, and quinone reductase. These enzymes act as the first line of defense to counteract free radical-induced oxidative stress [
49]. Glutathione peroxidase is an enzyme which minimizes the production of peroxide radicals (hydrogen peroxide and alkyl hydroperoxides) in association with GSH [
50]. Catalase neutralizes harmful H
2O
2 to oxygen and water. Glutathione
S-transferase and quinone reductase (phase II detoxification enzymes) increase cellular GSH levels and protect cells against the toxicities of electrophiles [
51]. Glutathione-6-phosphate dehydrogenase is involved in the generation of NADPH through pentose phosphate pathways. NADPH is required for the production of GSH, which is necessary for cell protection against free radical damage [
52]. The pretreatment of rats with various doses of
C. nudiflora methanol extracts significantly increased the activities of these antioxidant enzymes, as compared with the CCl
4 alone-treated group. Using DPPH and ABTS assays, we found that methanol extracts of
C. nudiflora increased DPPH and ABTS levels in the liver and serum of CCl
4-treated rats. Our findings are in agreement with previously published data [
53].
Light microscopy analysis demonstrated that animals treated with CCl4 alone showed marked destruction of liver architecture with extensive fatty degeneration, blood vessel congestion, derangement of the hepatic cells, and necrosis, while the saline- and plant-treated control groups showed normal hepatic cells with intact cytoplasm, prominent nuclei, and visible central veins. However, the pretreatment of animals with various doses of C. nudiflora reduced the histopathological changes and resulted in less-pronounced destruction of liver architecture, which indicates that pretreatment with methanol extracts of C. nudiflora reduced liver injuries.
Ultrastructural findings that were pointed out in the CCl
4-treated group include dilations and irregular organization of membranes, large lipid droplets, and glycogen loss. The changes in organelle structure and edematous cytoplasmic matrix were probably due to alterations in membrane structure caused by lipid peroxidation. Our results are in agreement with previous reports [
54]. MDA levels of the CCl
4-treated group were more elevated when compared to the normal control group, which further supports these histological results. Administration of
C. nudiflora markedly reduced the cellular damage in CCl
4-treated rats.
The administration of CCl
4 also markedly increased the formation of HNE-modified protein adducts and 8-OHdG. HNE is a major aldehyde product of lipid peroxidation and displays a variety of cytopathological properties, including the inhibition of enzymes and proteins, as well as DNA and RNA synthesis [
55]. The aldehyde is highly toxic to hepatocytes. In addition to this, it also has genotoxic and mutagenic effects. It is believed that the toxicity of HNE is due to its reactive aldehyde group. On the other hand, 8-OHdG is a major product of oxidative DNA damage. Toyokuni et al. [
56] and Uchida et al. [
57] have reported that the process of lipid peroxidation has been involved in promoting the development of 8-OHdG by the production of HNE-modified protein adducts. Thus, we were interested to investigate whether various doses of
C. nudiflora were able to block the increase in formation of HNE-modified protein adducts and 8-OHdG in the livers of CCl
4-treated rats. In the current research, the productions of HNE-modified protein adducts and 8-OHdG are indicated by yellow and brown coloration due to staining. Normal and plant-treated control groups reveal no yellow coloration which signifies that protein adducts and 8-OHdG are not produced, while the CCl
4-intoxicated group displays intense yellow coloration as compared to normal and plant-treated control groups, which proves that CCl
4 intoxication triggers the production of four HNE-modified protein adducts and 8-OHdG. Meanwhile, the administration of
C. nudiflora methanolic extracts reduced the yellow coloration in CCl
4-intoxicated groups in a dose-dependent fashion as compared to the CCl
4 only-treated group (Figs.
8 and
9). Our results indicated that prophylactic treatments of
C. nudiflora to rats can efficiently attenuate this increase. This shows that phytochemical compounds may be responsible for the biological effects of
C. nudiflora.
In the current study, we also investigated the inhibitory effects of
C. nudiflora on the expression of proinflammatory markers such as TNF-α, IL-6 (cytokines), and PGE2. Cytokines act as central regulators, controlling genes, and are accountable for causing either apoptosis or protective action on cells by stimulating the proliferation of hepatocytes. They also play an important role in the constitution of a complex network involved in the regulation of inflammatory responses. Important hepatotoxic mediators in various experimental models of hepatic damage are TNF-α, IL-6, and PGE2. These markers are formed and released from several cells under physiological and pathological stress, and the liver is highly vulnerable to the action of these markers [
58]. These markers have been selected due to an important role in inflammation, vascular permeability, as well as proliferation [
59‐
61]. In our findings, the markers were expressed in inflammatory cells. The overexpression of proinflammatory markers is indicated by intense yellow and brown coloration due to staining (Figs.
10,
11, and
12). The hepatic sections of the normal and plant control groups showed a complete absence of immunostaining of TNF-α, IL-6, and PGE2 which implies that no proinflammatory markers were expressed. On the other hand, a large amount of immunopositive cells was noticed in the hepatic tissue sections of the CCl
4-administered rats as compared to the control rats. This demonstrates that more proinflammatory markers were expressed in the CCl
4-treated group. The overexpression of TNF-α, IL-6, and PGE2 in the CCl
4-administered rats was markedly reduced by
C. nudiflora in a dose-dependent manner. Thus, this demonstrates that administration of
C. nudiflora had anti-inflammatory effects via suppression of proinflammatory mediator expressions.