Introduction
The liver is a site of metabolism and detoxification of chemicals, drugs, and harmful environmental toxicants. It protects the whole-body organs from injury by those harmful toxins. The exposure to liver toxins in food, water, air and medicines for a long time causes acute liver injury, cirrhosis, fibrosis, fatty liver and cancer. These diseases are worldwide spread nowadays. In some critical cases, urgent liver transplantation is required to save the life of patients [
1].
Halogenated alkanes such as carbon tetrachloride (CCl
4), chloroform and iodoform are examples of chemical toxins that have been restricted due to their high risk of hepatotoxicity [
2,
3]. Liver enzymes activate the halogenated alkanes (CCl
4) into highly reactive free radicals such as trichloromethyl and trichloromethyl peroxyl radicals (
·CCl
3 and
·CCl
3O
2). These radicals interact with hepatic cell proteins, fats and nucleic acids which harm the normal cellular processes such as lipid metabolism, hepatic enzyme reactions, initiation of cancer, liver fibrosis, and finally cell death [
2]. Therefore, antioxidants such as plant polyphenols (flavonoids and tannins) have an important role in scavenging the free radicals (
·CCl
3 and
·CCl
3O
2) [
4] and protect the hepatic cells from their liver toxicity [
5,
6].
Kupffer cells are the main players in liver inflammation. They are localized macrophages in the liver and are responsible for the expression and release of cytokines upon their activation during liver injury. They exhibit markers such as M1-like macrophages or M2-like macrophages that regulate liver inflammation [
7]. Several inflammatory cytokines such as chemokines, IL-6, IL-12, IL-18, and TNFα are released from Kupffer cells in response to liver injury, alcoholic liver disease, and other hepatic toxins [
7]. Other bone-marrow derived macrophages, such as M1 and M2 macrophages, play a role in liver inflammation by releasing inflammatory cytokines (TNF-α, IL-1, and IL-6) [
7]. After liver inflammation, hepatic macrophages such as Kupffer cells and macrophage-TGF-β1 activate myofibroblasts (hepatic stellate cells) to produce fibrosis [
7].
CCl
4, a liver toxin, induces Kupffer cells to release several inflammatory mediators such as TNFα, TGF-ß, and IL-1, nitric oxide, IL-10 and IL-6 which are responsible for necrosis and fibrosis of hepatic cells [
8]. Polyphenols (flavonoids and tannins) are promising anti-inflammatory compounds that inhibit the release of the inflammatory mediators by several mechanisms and hence, protect the hepatic cells from necrosis and fibrosis [
9].
Several plant extracts exhibited potent in vivo hepatoprotective activity against CCl
4 induced hepatotoxicity. For example, the methanol and chloroform extracts as well as some isolated phenolics from
Aframomum melegueta seeds exhibited hepatoprotective activity on CCl
4 induced hepatotoxic rats. Suppression of inflammatory response of cytokines, apoptosis and enhancement of the antioxidant defense activity were the mode of action [
10].
Lannea stuhlmannii and
Lannea humilis tannins rich extracts displayed a potent hepatoprotective effect through the enhancement of the anti-apoptotic protein Bcl-2 [
11]. Strawberry (
Fragaria ananassa) juice showed enhancement of the anti-apoptotic Bcl-2 protein and controlled the pro-apoptotic Bax and caspase-3 proteins with a clear reducing collagen areas in hepatic tissue [
12]. Phenolic extract of barley (
Hordeum vulgare) exhibited a hepatoprotective effect on hepatotoxic mice induced by CCl
4 [
13]. The elevation of liver antioxidant enzymes and reducing the hepatocyte apoptosis were the mechanism of action of the phenolic extract of barley [
13].
Calamus rotang L. (Rattan palm) a monocot climbing shrub, belongs to family Ericaceae. It is native to South-West Asia. In India, the shrub is cultivated for its’ edible fruits and medicinal importance especially in Ayurveda. The shoots are used as anthelmintic and the leaves are used for eye problems, skin diseases, pruritus and cough [
14‐
16].
Few biological studies on
C. rotang (CR) were previously reported. For example, the aqueous extract of leaves showed immunoadjuvant activity against hepatitis B surface antigen [
17]. The in vitro antioxidant study on fruits and leaves methanol extracts revealed that the leaves have more polyphenolics than the fruits [
18]. The methanol extract of seeds displayed in vivo antidiabetic and hypoglycemic activities [
19]. The phytochemical study on
C. rotang rhizome led to isolation of ( +) –afzelechin,
β-sitosterol and
β-sitosterol glucoside [
20]. No chemical study was previously reported on the leaves, fruits and seeds. The current study aimed to determine the total phenolics, total flavonoids, antioxidant property and in vivo hepatoprotective activity of the CR extract on CCl
4 induced hepatotoxic rats. HPLC chemical characterization of the polyphenols (flavonoids and phenolics) in CR extract was performed. The binding ability of the identified compounds to the pro-apoptotic Bcl-2: Bim (BH3) protein by molecular docking was studied.
Material and methods
Solvents and chemicals
The solvents used in this study for extraction (methanol) and chromatography (methanol and acetonitrile) were HPLC solvents (Thermo Fisher Scientific, Pittsburgh, USA). The chemicals, such as Folin-Ciocalteu’s phenol reagent, gallic acid, quercetin, AlCl3, DPPH, and ascorbic acid, used in the determination of phenolic and flavonoid contents and antioxidant activity were purchased from Sigma (Sigma-Aldrich, Burlington, MA, USA). Hematoxylin and eosin kits and toluidine blue dye used in the histopathological study were bought from Abcam (Cambridge, UK). The following antibodies: anti-TNFα antibody (sc-52746), anti-PPARα antibody (sc-398394), anti-arginase (sc-271430), and anti-Bcl-2 antibody (sc-7382) used in the immunohistochemistry study were purchased from Santa Cruz Biotechnology (Texas, USA).
Plant materials
The leaves of C. rotang L. were used in the current study. They were collected in October 2018 from Aswan Botanical Garden, Aswan, Egypt. The plant was kindly identified by Dr. Amr M. M. Mahmoud, Director of Aswan Botanical Garden, Hort. Res. Institute, Agriculture centre, Egypt. Voucher specimen of the plant leaves was placed in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Egypt (Voucher no. A20220906).
Extraction and fractionation
A hundred grams of dried powdered leaves were macerated in methanol (500 mL) at room temperature (25 °C) with shaking for 24 h. The extraction process was repeated twice until exhaustion. The methanol extract was combined and concentrated under reduced pressure using rotavapor. The fractionation of the dried methanol extract (10 g) was performed by suspending it in 250 mL of 10% methanol in water using a 1L separating funnel. Liquid–liquid extraction to fractionate the aqueous extract using dichloromethane and ethyl acetate solvents (250 mL × 3 for each solvent) was performed. The dichloromethane and ethyl acetate fractions were collected and concentrated at reduced pressure to yield dichloromethane extract (3 g), ethyl acetate extract (2 g), and aqueous extract (5 g). The preliminary screening test for flavonoids demonstrated that the ethyl acetate fraction is the richest flavonoid fraction.
Total phenolic contents
The total phenolic content of CR extract was determined according to the method previously reported [
21]. Briefly, gallic acid was used as a standard phenolic compound. Serial dilution of 100 ppm gallic acid solution was prepared (100–10 ppm) in distilled water. Folin-Ciocalteu’s phenol reagent (0.2 mL) was mixed with all gallic acid solutions and tested CR ethyl acetate extract (1:1). One mL of sodium carbonate and 1.6 mL distilled water were added to the mixtures after 5 min. All tested and standard mixtures were kept on dark for 30 min. After that, the samples were centrifuged, and their colorimetric absorbance was measured in triplicates at 750 nm using UV2000 spectrophotometer (Ray Wild Limited company, L569 Gottingen, Germany). Standard curve of gallic acid was obtained (Fig. S
3) and the total phenolic content of CR extract GAE mg/g dry weight was calculated as a mean of triplicate measurements ± SD.
Total flavonoid contents
The total flavonoid content of CR extract was determined according to the method previously reported [
22]. Serial dilution of 100 ppm quercetin solution was prepared (100–10 ppm) in methanol. AlCl
3 2% reagent (0.6 mL) was mixed with all quercetin solutions and tested CR ethyl acetate extract (1:1). All tested and standard mixtures were kept for 60 min at room temperature. After that, the samples were measured for their colorimetric absorbance in triplicates at 415 nm using UV2000 spectrophotometer (Ray Wild Limited company, L569 Gottingen, Germany) Standard curve of quercetin was obtained (Fig. S
4) and the total flavonoid content of CR extract QE mg/g dry weight was calculated as a mean of triplicate measurements ± SD.
Antioxidant DPPH assay
The capacity of CR extract to scavenge free radicals was calculated by DPPH previously reported method [
23]. DPPH stock solution of 200 µM, tested extract samples (60—1000 µg/mL ethanol) and ascorbic acid reference samples (60—1000 µg/mL ethanol) were prepared. The antioxidant reaction was performed by adding 300 µL DPPH solution to 300 µL of each tested or control sample on dark for 30 min at room temperature. The colorimetric absorbance was measured at 517 nm. The percentage antioxidant activity was calculated by the following equation SA% = (A
0—A
s/A
0) × 100 (A
0 = Absorbance of DPPH solution in ethanol, A
s = Absorbance of CR extract and DPPH).
HPLC analysis of CR ethyl acetate extract
Detection of phenolic compounds
Twenty-five microliter of CR was injected in HPLC-Agilent 1100 with UV/Vis detector (Agilent technology, USA). The phenolic compounds were separated on C18 column (125 mm × 4.60 mm, 5 µm) using gradient solvent elution. Two solvents were used as mobile phase for separation of phenolics, solvent A: acetic acid in water (1:25) and solvent B: 100% methanol. The gradient solvent method was started with 100% of solvent A for 3 min, followed by 50% solvent B for 5 min, 80% solvent B for next 2 min and finally 50% solvent B for 5 min. Phenolic compounds were detected at 280 nm. They were detected using Agilent ChemStation software (Agilent technology, USA) and were identified by using authentic samples.
Detection of flavonoids compounds
Twenty-five microliter of CR was injected in HPLC-Agilent 1100 with UV/Vis detector (Agilent technology, USA). The phenolic compounds were separated on C18 column (250 mm × 4.60 mm, 5 µm) using isocratic solvent elution in 15 min. Two solvents were used as mobile phase for separation of flavonoids, solvent A: formic acid in water (1%) and solvent B: 100% acetonitrile. Seventy % of solvent B was used as isocratic eluted mobile solvent of the experiment. The detector wavelength was 320 nm. The identification of flavonoids was performed by using authentic samples.
Animals and experimental design
Eighteen Wistar albino rats weighting 180 to 250 g (aged 6 weeks) were obtained from Animal Housing Center, Faculty of Medicine, Assiut University. They were housed, provided feed and water ad libitum under standard conditions for 7 days before experiments begun. This experiment was carried out in accordance with relevant guidelines and regulations. It was approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmacy, Assiut University (Approved No. S27-22). The rats were divided into three groups; control, CCl
4 intoxicated and CR extract treated groups. CCl
4 was chosen to intoxicate the hepatic cells in the positive and treated groups based on a previously published protocol of CCl
4 induced hepatotoxicity in rats [
24‐
27]. The rats of treated (CR extract) group were supplied orally by 0.3 mL of 350 mg/kg CR extract once a daily for 21 days [
28]. The dose of ethyl acetate extract was determined in the range of the previous reported doses of hepatoprotective medicinal plants rich in flavonoids and phenolics [
29,
30]. The rats of other groups were supplied orally by 0.3 mL of saline once a daily for 21 days. The rats of all groups except the control were supplied orally with 0.25 mL of 1% CCl
4 in olive oil per day from day 10. The rats were fasted for 4 h and the animals killed at the end of the corresponding experimental periods. The animals were anesthetized by pentobarbital (50 mg/kg, i.p.). After the rat loss the reflexes, the rat’s thoracic cages had been incised and transcardially perfused with normal saline followed by 4% paraformaldehyde fixative. Liver specimens were obtained after whole-body perfusion of experimental rats with 4% paraformaldehyde. All methods are reported in accordance with ARRIVE guidelines (
https://arriveguidelines.org).
Histopathological examination
The dissected samples were processed and stained with hematoxylin & eosin and Crossmon’s trichrome (Fig. S
5) according to the standard protocols [
31]. Histopathological studies were performed using an Olympus CX 41RF light microscope (Olympus Corporation, Tokyo, Japan). Also, specimens from liver were used for semithin sections and stained with toluidine blue [
32].
Immunohistochemical examination
Immunohistochemistry staining was performed according to the previous reported strudy [
33]. The sections were incubated overnight at 4 °C with the following antibodies: anti-TNFα antibody (sc-52746), anti-PPARα antibody (sc-398394), anti-arginase (sc-271430) and anti-Bcl-2 antibody (sc-7382). Immunohistochemical staining was evaluated by Olympus CX 41RF light microscope (Olympus Corporation, Tokyo, Japan).
Quantitative analysis of TNFα, Arginase I, BCL2, PPARα immunostaining
The morphometric studies carried out on the immunohistochemical images of the liver of all groups using Image-J software. The measurements were done according to the previous study [
34].
Statistical analysis
The data of immunohistochemical analysis were summarized in figures using “GraphPad Software” (Version 6.05, International Scientific Community) to compare between different variables in CCl4, CR extract treated animals in relation to control group. Differences were considered significant if P < 0.05 (*). All data were statistically analyzed using Tukey’s test.
Molecular docking
Molecular docking was conducted by using Autodock [
35] vina 1.5.6 software to assess the binding affinities of the selected polyphenols with antiapoptotic protein Bcl-2: (BH3). The crystal structure of BH3 with its inhibitor with PDB ID: 4B4S was retrieved from Protein Data Bank [
36]. The interaction residues of the binding site of BH3 that was used for the docking study were identified based on the previous literature [
11,
36]. The ligand and all the water molecules were removed from the crystal structure. The polar hydrogen atoms were added and pdbqt file was generated by using AutoDock tools-1.5.6 [
37]. The site of the grid box was set at -8.425, 9.62, and -2.654 Å (for × , y and z) by means of a grid of 40, 40, and 40 points (for × , y and z). The binding affinities of polyphenols with Bcl-2 were predicted based on the average of the lowest energy of docking. Chimera [
38] 1.12 software was used to analyze and visualize the best-scored conformation.
Discussion
The CR leaf extract displayed potent DPPH scavenging activity, with IC
50 = 0.12 mg/mL. The DPPH scavenging of the CR increased with concentration. The higher the concentration of the CR extract, the greater the scavenging of DPPH radicals (Fig. S
1).
The most natural plant components that are responsible for antioxidants are flavonoids and phenolics [
42,
43]. The first quantitative determination of flavonoids and phenolics in the CR leaf extract was reported in the current study. The total phenolic content of the CR extract was 804 ± 0.18 mg GAE/100 g, and the total flavonoid content was 1760 ± 0.69 mg QE/100 g (Fig. S
1). Therefore, CR is a rich source of flavonoids and phenolics in comparison with the previously reported data on antioxidant plants [
44‐
46]. The HPLC characterization of phenolics in CR extract led to the identification of 6 simple phenolic acids; gallic acid, ellagic acid, syringic acid,
p-coumaric acid, caffeic acid and ferulic acid in addition to one phenolic compound (pyrogallol) as shown in Fig.
1. Additionally, seven flavonoid compounds of different classes; flavonols (Rutin, quercetin, and kaempferol), flavones (Apigenin, 7-OH flavone and myricetin) and a flavanone (Naringin) were identified (Fig.
1). The detection of compounds was achieved by using Agilent ChemStation software (Agilent technology, USA). Authentic compounds were used to identify and quantify flavonoids and phenolics in CR extract. This study is the first to report the polyphenol composition of CR leaf extract.
The histopathological and immunohistochemical studies proved that CR extract greatly protects the liver tissue from the steato-cirrhotic effect of CCl
4. The exposure of the rats to CCl
4 causes hepatocytes vacuolation, and induces hepatic inflammation and fibrosis as previously reported [
47]. The CR extract significantly decreased the collagen deposition, and suppressed the inflammatory cellular infiltrations. Tumor necrosis factor α (TNF α) is a one of cytokines produced mainly by macrophages, that stimulates cellular response such as production of the inflammatory mediators and cell death [
48]. In current study, the CCl
4 treated group showed a significant increase in TNF α in comparison to CR treated group. This was attributed to the role of CR extract as an anti-inflammatory agent. Arginine is a substrate for the synthesis of urea and nitric oxide, arginase has a catalytic activity which converts arginine to urea [
40]. In the present work, the arginase showed a significant decrease in CR treated group in comparison to CCl
4, and its elevation in the CCl
4, intoxicated group occurred as a result of the up regulation of TNF α [
49].
The accumulation of reactive oxygen species (ROS) within the cells along with a decrease in the cellular antioxidants provokes mitochondrial damage [
50]. This led to the release of the apoptogenic factors (Cytochrome c) through the mitochondrial membrane. Bcl-2 is an anti-apoptotic member which inhibits or decreases apoptosis [
51]. The CR extract exhibited a significantly increase in Bcl-2 positive hepatocytes which indicates its role in the protection of the hepatocytes from the programed cell death. Additionally, our results showed a significant increase in PPAR α in the CCl
4 intoxicated group due to the elevation of intercellular lipid accumulation. PPAR α mainly controls the lipid metabolism and regulates the inflammatory response in liver [
41]. In this regard, the increase in the number of PPAR α positive cells is due to the excess of the inflammatory cell’s infiltration after the exposure to CCl
4 [
52]. PPARα levels decreased in the CR extract group [
53]. Thus, CR ethyl acetate extract has a prospective anti-steatosis activity.
Molecular docking is a powerful approach that has been employed in drug discovery studies to screen the binding affinities of the drugs with its possible target proteins [
54]. Therefore, we used this approach to study the inhibitory role of the identified polyphenols on Bcl2: Bim (BH3) as a crucial pro-apoptotic protein. Interestingly, naringin, rutin, hydroxy flavone, and ellagic acid showed a promising binding affinity with Bcl2: Bim (BH3). These high binding affinities are attributed to the unique structure of polyphenols that combine the aromatic rings and the substituted polar groups that allow them to form various non-covalent interactions with the target proteins. These findings suggested the antiapoptotic role of polyphenols through the inhibition of BH3 protein.
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