Protective role of polyphenols from Bauhinia hookeri against carbon tetrachloride-induced hepato- and nephrotoxicity in mice

Abstract

The hepatoprotective and nephroprotective activity of a polyphenol-rich fraction (BHPF) obtained from Bauhinia hookeri was investigated against CCl₄-induced acute hepatorenal toxicity in mice. BHPF was administered (100, 200 and 400 mg/kg/day) for 5 days, then CCl₄ was administered. BHPF pretreatment significantly (p < 0.001) inhibited the CCl₄-induced increase in ALT, AST, ALP, LDH, total bilirubin, cholesterol, creatinine, uric acid, urea and malondialdehyde in a dose-dependent manner. In contrast, BHPF pretreatment markedly increased the contents of glutathione and superoxide dismutase in the liver and kidney tissues, indicating the strong in vivo antioxidant activity of BHPF. Pretreatment with BHPF preserved the hepatic architecture and conferred marked protection against necrosis and ballooning degeneration. Pretreatment with BHPF reduced the inflammatory cell aggregation and degenerative changes in the lining epithelium of the kidney tubules. It can be concluded that BHPF has a remarkable hepato- and nephroprotective activity by enhancing the antioxidant defense status, reducing lipid peroxidation and protecting against the histopathological changes induced by CCl₄ in the liver and kidney tissues.

• Antioxidant
• epicatechin gallate
• HPLC-PDA-ESI/MS/MS
• hepatoprotective
• kidney
• liver
• nephroprotective
• proanthocyanidins

Introduction

The kidney is the main organ involved in the excretion of xenobiotics.1^,2 The liver is the primary organ of metabolism; therefore, the toxic effects of chemicals usually appear primarily in the liver and kidney tissues.3^,4 Oxidative stress has a crucial role in many diseases including renal failure, liver damage, atherosclerosis, inflammation and carcinogenesis.5^,6 Oxidative stress is caused by cellular excess of reactive oxygen species (ROS). Lipid peroxidation of cell membranes induces cell injury.7–9 Under normal homeostasis, the cells maintain the ROS levels with endogenous non-enzymatic and enzymatic-antioxidants.10–12 The risk of cell injury may also be prevented by natural antioxidants including dietary polyphenols. Dietary polyphenolic compounds have received a great deal of attention because of their beneficial effects on health, including protection against oxidative stress and degenerative diseases.13 Dietary polyphenolic compounds are known to restore the balance between the natural antioxidants and free radicals by direct scavenging of ROS and by enhancing the activity of natural antioxidant enzymes.13 Proanthocyanidins are naturally occurring antioxidants that are abundant in many plant foods such as fruits, vegetables and legumes.14 Proanthocyanidins exert a wide spectrum of pharmacological and therapeutic activities against oxidative stress. Grape seed proanthocyanidin extract (GSPE) is marked as a dietary supplement due to its health benefits, including hepatoprotective, cardioprotective, anti-fibrogenic and chemopreventive activities.15

The genus Bauhinia (family Fabaceae) comprises 300 species, and is commonly known as “cow's paw” tree, because of the shape of their leaves.16 Different species are used in folk medicine worldwide.16 Diverse range of pharmacological activities has been reported for many Bauhinia species, including hepatoprotective, antioxidant, anti-inflammatory and anti-hyperlipoidemic effects.17^,18 Bauhinia hookeri F. Mull, is a small ornamental tree native to Australia.19 The current study was designed to investigate the possible mechanisms of the hepato- and nephroprotective action of a proanthocyanidin-rich fraction obtained from B. hookeri (BHPF) against the acute toxicity of CCl₄ in mice. Hepatoprotection was determined by assaying the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, cholesterol and protein in the serum of control and treated mice in an acute model of CCl₄ intoxication. Nephroprotection was determined by estimating the serum level of uric acid, urea and creatinine. The lipid peroxidation and antioxidant parameters [glutathione (GSH) and superoxide dismutase (SOD)] were estimated in the liver and kidney homogenates to determine the possible mechanisms of the hepato- and nephroprotective activity. A histopathological examination of liver and kidney sections was conducted to confirm the hepato- and nephroprotective effects. The identification of B. hookeri polyphenols was achieved utilizing the high-performance liquid chromatography coupled with diode array detection-electrospray ionization tandem mass spectrometry (HPLC-PDA-ESI/MS/MS) technique.

Material and methods

Chemicals

Carbon tetrachloride (CCl₄) was obtained from El Nasr Pharmaceutical & Chemical Company (Cairo, Egypt). Silymarin was purchased from Sedico Pharmaceutical Company (6th October City, Egypt). All the assay kits were purchased from Biodiagnostics Company (Cairo, Egypt). The assay kit of LDH was purchased from Randox Laboratories Ltd (Crumlin, UK). All other chemicals were of analytical grade. LC–MS grade solvents were used in HPLC-PDA-ESI/MS/MS analysis. Sephadex LH-20 was obtained from Amersham Biosciences (Uppsala, Sweden), RP-C18 from Sigma-Aldrich GmbH (Darmstadt, Germany) and pre-coated silica gel TLC GF254 was obtained from Riedel-De Häen-AG (Seelze, Germany).

Plant material

The leaves of B. hookeri were collected in July 2011 from the botanical garden of the Faculty of Agriculture, Cairo University, Cairo, Egypt. The plant was botanically identified by Eng. Therese Labib, the taxonomy specialist at the herbarium of El-Orman Botanical Garden, Giza, Egypt. A voucher specimen of B. hookeri was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU BHF2011).

Extract preparation and fractionation

The air-dried powdered leaves of B. hookeri (1700 g) were extracted three times with 80% aqueous ethanol (EtOH). The total extract was concentrated and freeze-dried to obtain a dry powder, which was dissolved in absolute EtOH. The EtOH-soluble portion was concentrated and freeze-dried to obtain a dry powder (140 g). Column fractionation of part of the extract (100 g) was performed using Sephadex LH-20 (5 × 100 cm), eluted with H₂O followed by H₂O–MeOH mixtures of decreasing polarities. Fractions are combined based on their analytical HPLC profiles to afford eight major fractions. The fraction eluted with 60% aqueous methanol (MeOH) was concentrated and freeze-dried to obtain a dry powder of BHPF (3 g).

HPLC-PDA-ESI/MS/MS method

Part of the fraction was dissolved in 20% MeOH (20 mg/mL) and the solution was filtered through 0.2 μm PTFE membranes. LC–HRESIMS was performed on a Bruker micrOTOF-Q quadrupole time-of-flight mass spectrometer (Bremen, Germany), coupled to a 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with an auto sampler, a binary pump and a diode-array detector. Chromatographic separation was performed on an XBridge C18 (2.1 mm × 100 mm; 3.5 μm) column (Waters, Dublin, Ireland). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The elution profile was 0–3 min, 100% B (isocratic); 3–30 min, 0–30% A in B; 30–45 min, 30–70% A in B; 45–55, 70% A in B (isocratic) with a flow rate of 0.2 mL/min. The HPLC system was controlled by Hystar software (version 3.2; Bruker BioSpin, Rheinstetten, Germany). The mass spectrometer was controlled by the Compass 1.3 for micrOTOF software package (Bruker Daltonics). The ionization technique was an electrospray. The mass spectrometer was operated in negative mode. Mass detection was performed in full scan mode in the mass range m/z 50–2000. The following settings were applied to the instrument: capillary voltage, 4000 V; end plate offset, −500 V. Heated drying gas (N₂) flow rate was 12 L/min; the drying gas temperature was 200 °C. The gas flow to the nebulizer was set at a pressure of 1.6 bar. For collision-induced dissociation MS/MS measurements, the voltage over the collision cell varied by collision sweeping mode from 20 to 70 eV. Argon was used as a collision gas. Sodium formate was used for calibration at the end of each LC/MS run. The data were analyzed using Compass Data Analysis Software (version 4.0 SP5; Bruker Daltonics).

Animals

Male Swiss Albino mice weighing 27.5 ± 2.5 g were purchased from the Animal House Facility, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. The mice were housed in cages in a ventilated room under the controlled laboratory conditions of temperature (25 ± 2 °C) and under 12 h light/dark cycles. The mice were fed a standard rodent pellet chow and water ad libitum. The animals were acclimatized for at least l week before use. All the animal experiments were approved by the ethical committee of the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (201405).

Acute toxicity study

Male Swiss Albino mice weighing 27.5 ± 2.5 g were used to determine the acute oral toxicity of BHPF according to the reported method.20 BHPF was diluted in saline, and the mice (n =  8 per group) were treated with high doses (500, 1000 and 2000 mg/kg body weight p.o.). The animals were observed for 24 h to record toxicity symptoms and mortality rates. The animals were also observed for possible toxicological or behavioral changes for further 14 days after BHPF treatment.

Experimental design

Forty-eight Swiss Albino mice were divided into six groups (n =  8 per group). Group (I) served as the normal control and received saline only. Group II was treated intraperitoneally with a sublethal dose of CCl₄ at a dose of 0.5 mL/kg body weight (20% v/v in corn oil) at the end of the experiment and served as the positive control. The mice in groups (III, IV and V) were treated orally with 100, 200 and 400 mg/kg body weight of BHPF, respectively. Group VI was treated with standard silymarin at a daily dose of 200 mg/kg.21 All tested material and silymarin were administered orally, once daily, for 5 consecutive days. On the sixth day, liver and kidney injury was induced in animals (all groups except the normal group) by a single i.p. injection of 20% CCl₄ v/v in corn oil (0.5 mL/kg bw).21 Blood collection was done by direct cardiac puncture under diethyl ether anesthesia 24 h after the last treatment dose. The collected blood was left to clot at room temperature and the sera were separated by centrifugation at 3000 rpm for 15 min, then the sera were stored at −20 °C for biochemical analysis. The animals were then sacrificed by decapitation under diethyl ether anesthesia and the liver and kidney were excised rapidly. One part of each tissue was perfused with a 50 mM ice-cold phosphate buffered saline (pH 7.4), containing 0.1 mM ethylenediaminetetraacetic acid to remove any red blood cells and clots. These tissues were then homogenized ice-cold phosphate buffered saline in (5–10 mL/g tissue), centrifuged at 5000 rpm for 30 min and stored at −80 °C for the estimation of lipid peroxidation, GSH content and SOD activity. Another hepatic and renal tissue part was preserved in 10% formalin for the histopathology.

Serum biochemical analysis

The already separated sera were used for the estimation of serum liver biomarkers according to the manufacturer's protocol and previously reported methods for ALT, AST,22 ALP,23 LDH,24 total bilirubin,25 cholesterol26^,27 and total proteins.28 The biochemical markers of kidney damage were estimated according to reported methods for creatinine,29 urea30 and uric acid.31

Evaluation of tissue lipid peroxidation and antioxidant parameters

Lipid peroxidation was evaluated by measuring the MDA content in hepatic and renal tissues.32 The antioxidant parameters were assayed according to previous methods for the SOD activity33 and GSH.34

Histopathological examination

Liver and kidney specimens from all experimental groups were fixed in 10% formol saline for 24 h. Washing was done by the tap water followed by serial dilutions of alcohol (methyl, ethyl and absolute ethyl). The specimens were cleared in xylene and embedded in paraffin at 56 °C in a hot-air oven for 24 h. Sections were prepared (4 μm thick) by a sledge microtome and then stained with hematoxylin and eosin. The liver and kidney sections were examined for pathological changes by a light microscope.

Statistical analysis

All the data were expressed as the means ± SEM. The statistical analysis of the data was performed using the one-way ANOVA test followed by Tukey's post hoc test to determine the difference between the mean values of the different groups. All statistical analyses were performed using the GraphPad InStat software (Version 3.06, La Jolla, CA). p-Values < 0.05 were considered statistically significant.

Results

Identification of the constituents of BHPF by HPLC-PDA-ESI/MS/MS

The individual constituents of BHPF were identified using the HPLC instrument coupled to a PDA detector and a mass spectrometer. The combination of the PDA and mass spectrometry (MS/MS) data provided a sensitive method for the characterization of the constituents of BHPF (Table 1 and Figure 1). Compounds 1–3 were tentatively identified based on their molecular ions [M−H]⁻ at m/z 729.16 and 881.18, respectively, and the MS/MS ions at m/z 407.09, 289.08, 245.08, 169.02, 125.03, of which the first three ones are typical for dimeric procyanidins.35^,40 The MS/MS ions at m/z 169.02 indicated the presence of galloyl groups.41 Compounds 1 and 2 produced the MS2 base peak at m/z 407.09 by the loss of a gallic acid moiety (−170 amu) followed by a retro-Diels-Alder fragment (−152 amu).42 The position of the galloyl residue was confirmed to be attached to the C3′ of the base unit of the procyanidin molecule based on the presence of the fragment ion at m/z 407.08, while the fragment ions at m/z 425, 577, 559 were not detected. These fragments are more predominant if the galloyl residue is attached to the C3 of the top unit. The molecule loses the galloyl residue or gallic acid and produces the MS2 base peak at m/z 577 and a secondary peak at m/z 559, respectively.42 Similarly, compounds 4–9 were identified based on their [M−H]⁻ and fragment ions as well as from the distinctive PDA data.36–38 Compounds 10 and 12 were tentatively identified based on their molecular ions and their distinctive PDA of flavonoids.39

Figure 1. HPLC chromatogram of BHPF and the fragmentation pattern of its constituents.

Table 1. LC-PDA-ESI/MS/MS Identification of the major constituents of BHPF.

No.	tR	PDA	[M−H]^−	Fragments (MS/MS)	Tentative structural assignment	References
1	22.8	205, 280	729.16	407.09, 289.08, 245.08, 169.02, 125.03	(Epi)catechin-3′-O-galloyl (epi)catechin (procyanidin dimer gallate)	Jaiswal et al.12, Reed et al.35
2	24.1	205, 280	729.16	407.09, 289.08, 245.08, 169.02, 125.03	(Epi)catechin-3′-O-galloyl (epi)catechin (procyanidin dimer gallate)	Jaiswal et al.12, Reed et al.35
3	26.1	205, 280	881.18	407.09, 289.08, 245.08, 169.02, 125.03	3-O-galloyl (epi)catechin-3′-O-galloyl (epi)catechin (Procyanidin dimer digallate)	Jaiswal et al.12, Reed et al.35
4	26.6	200. 215, 225 sh, 278	441.09	289.08, 245.09, 169.02, 125.03	Epicatechin-3-O-gallate	Khallouki et al.36
5	27.6	205, 280	605.14	441.09, 289.08, 245.09, 169.02, 125.03	Epicatechin gallate derivative
6	29.6	200, 280	425.10	273.08, 255.07, 229.09, 169.02, 125.03	Afzelechin-3-O-gallate	de Souza et al.37
7	30.0	200, 278	589.15	437.13, 425.35, 273.08, 255.07, 229.09, 169.02, 125.03	Afzelechin gallate derivative
8	31.0	200, 220 sh, 280	1005.27	502.13 [M−2H]^2−, 853.25, 415.10, 271.07, 169.02	Unidentified
9	31.4	200, 278	409.10	289.08, 245.09, 145.48, 137.03	(Epi)catechin hydroxybenzoate	Hartisch and Kolodziej38
10	34.3	200, 250sh, 270 sh, 350	285.05	241.06, 217.05, 151.01, 133.03	Luteolin	Mabry et al.39
11	38.2	200, 220, 265, 370	285.05	233.66, 229.05	Unidentified
12	41.0	210, 250, 270, 340	283.07	255.04, 145.48, 117.04	Methyl apigenin	Mabry et al.39
13	41.4	198, 220, 280, 335	297.08	255.08, 179.04, 135.05, 117.04	Unidentified
14	42.0	210, 250 sh, 280, 340	327.09	312.07, 284.07, 145.48	Unidentified methylated flavonoid

Acute toxicity

No adverse behavioral changes, toxicity symptoms or mortality were observed in mice at doses up to 2000 mg/kg of BHPF. Based on these findings, the oral 50% lethal dose (LD₅₀) value of BHPF is greater than 2000 mg/kg.

Hepato- and nephroprotective effects

A significant increase in the activity of the serum markers of liver damage ALT, AST, ALP and LDH (p < 0.001) was observed in the CCl₄-intoxicated group compared with the negative control group (Figure 2). The pretreatment of intoxicated mice with BHPF produced a marked hepatoprotective effect and reduced the activity of serum hepatic biomarkers in a dose-dependent manner. The percentage decrease in the liver marker enzymes at the treatment doses (100, 200 and 400 mg/kg/day) was 16%, 42% and 53% for ALT, 20%, 45% and 58% for AST, 21%, 49% and 57% for ALP and 22%, 49% and 60% for LDH, respectively, compared with the CCl₄-treated group. Notably, BHPF pretreatment at a dose of 400 mg/kg markedly reduced the levels of ALT and AST, which were comparable and non-significant to those in the silymarin-treated group (Figure 2). The levels of the ALP and LDH in the group treated with 400 mg/kg of BHPF were comparable and not significantly different from the normal control and silymarin groups (Figure 2). Similarly, a remarkable and dose-dependent decrease of the serum bilirubin and cholesterol levels was observed in the BHPF pretreated groups (Table 2). The percentage decrease in the serum bilirubin and cholesterol of the treated groups compared with the CCl₄-treated group is listed in Table 2. In contrast, the total protein level was significantly increased in the groups treated with BHPF in a dose-dependent manner. The total protein level in the groups treated with 200 and 400 mg/kg of BHPF were comparable and not significantly different from the normal control and silymarin groups (Table 2). The biochemical markers of kidney damage, uric acid, urea and creatinine were markedly reduced in the BHPF treated groups compared with the CCl₄-intoxicated group (Table 2). These results indicated that BHPF effectively reduced the CCl₄-induced hepatorenal toxicity.

Figure 2. Effect of BHPF and silymarin on the hepatic function tests of CCl₄-intoxicated mice. Data are expressed as the means ± SEM (n = 8). BHPF 1, 2 and 3: 100, 200 and 400 mg/kg of BHPF, respectively. Values having different superscripts are significantly different at p < 0.05.

Table 2. Effect of BHPF on biochemical parameters of CCl₄-intoxicated mice.

Animal groups	Total bilirubin (mg/dL)	Cholesterol (mg/dL)	Total proteins (mg/dL)	Creatinine (mg/dL)	Uric acid (mg/dL)	Urea (mg/dL)
Control	1.22 ± 0.04^a	76.45 ± 3.13^a	7.46 ± 0.14^a	0.31 ± 0.03^a	24.04 ± 1.01^a	24.22 ± 1.13^a
CCl4	1.66 ± 0.04^b	150.82 ± 2.81^b	5.65 ± 0.15^b	6.97 ± 0.40^b	90.43 ± 4.74^b	85.04 ± 4.51^b
BHPF (100 mg/kg)	1.54 ± 0.02^b (−7%)	119.27 ± 4.95^c (−21%)	6.62 ± 0.10^c (17%)	3.75 ± 0.33^c (−46%)	72.59 ± 2.67^c (−20%)	65.06 ± 2.85^c (−23%)
BHPF (200 mg/kg)	1.35 ± 0.04^a (−19%)	108.95 ± 4.09^c (−28%)	7.03 ± 0.14^a,c (24%)	2.52 ± 0.28^d (−64%)	57.16 ± 3.27^d (−37%)	50.36 ± 3.47^d (−41%)
BHPF (400 mg/kg)	1.27 ± 0.04^a (−23%)	79.31 ± 3.54^a (−47%)	7.33 ± 0.10^a (30%)	0.61 ± 0.07^a (−91%)	35.33 ± 1.86^a (−61%)	34.26 ± 1.64^a (−60%)
Silymarin (500 mg/kg)	1.25 ± 0.03^a (−25%)	73.66 ± 2.27^a (−51%)	7.43 ± 0.14^a (31%)	0.48 ± 0.06^a (−93%)	25.78 ± 1.37^a (−71%)	26.56 ± 1.33^a (−69%)

Notes: Data are expressed as the means ± SEM (n = 8). The numbers in parentheses represent the percentage change from the CCl₄-intoxicated group. Values having different superscripts within the same column are significantly different at p < 0.05.

Antioxidant activity and lipid peroxidation

A significant reduction in hepatic and renal GSH and SOD contents was observed in CCl₄-intoxicated mice. In contrast, a significant increase in the MDA levels was evident as compared to the normal control group (Figure 3). Administration of BHPF at the treatment doses (100, 200 and 400 mg/kg/day) produced a marked increase in hepatic GSH (by 42%, 78% and 106%, respectively), as well as in renal GSH (by 22%, 45% and 74%, respectively). In addition, BHPF pretreatment improved the activity of hepatic SOD (by 18%, 61% and 105%, respectively), and the activity of renal SOD (by 44%, 72% and 91%, respectively) relative to the CCl₄-intoxicated group. In contrast, the elevated hepatic level of MDA was reduced by 19%, 45% and 50% and the renal MDA by 22%, 35% and 51% at the tested doses, respectively, compared with the CCl₄-intoxicated group (Figure 3). Notably, the liver GSH content in the groups treated with 200 and 400 mg/kg of BHPF was comparable and insignificant to those in the normal control and silymarin groups. BHPF pretreatment at a dose of 400 mg/kg markedly reduced the hepatic and renal MDA levels, which were comparable and non-significant to those in the normal and silymarin-treated groups. These results clearly indicated the strong in vivo antioxidant activity provided by BHPF.

Figure 3. Effect of BHPF and silymarin on lipid peroxidation and antioxidant parameters in the liver and kidney of CCl₄-intoxicated mice. Data are expressed as the means ± SEM (n = 8). BHPF 1, 2 and 3: 100, 200 and 400 mg/kg of BHPF, respectively. Values having different superscripts are significantly different at p <  0.05.

Histopathological observations

Liver sections of the normal control group showed normal histological structure of the central vein and intact surrounding hepatocytes in hepatic parenchyma. CCl₄ induced severe loss of hepatic architecture with multiple focal necrosis, ballooning degeneration in the hepatocytes all over the hepatic parenchyma. The pathological changes induced by CCl₄ were markedly ameliorated in the groups treated with BHPF at the three treatment doses. Pretreatment with 200 and 400 mg/kg of BHPF conferred marked protection against liver damage as evidenced by the intact hepatic architecture and absence of histopathological lesions. Diffuse Kupffer cells proliferation in between the hepatocytes was observed in the group treated with 100 mg/kg of BHPF (Figure 4). Histological examination of kidney sections revealed normal histological structure of the glomeruli and tubules at the cortex with absence of histopathological alterations in the normal control group. CCl₄ induced marked inflammatory cell aggregation in between the tubules, associated with marked degeneration in the lining epithelium of all the tubules, along with blood vessel congestion. The aforementioned renal pathological changes induced by CCl₄ were markedly reduced in the groups treated with BHPF at the three treatment doses. Notably, pretreatment with BHPF at a dose of 400 mg/kg markedly reduced all the degenerative changes in the lining epithelium of the tubules. Mild inflammatory cell infiltration was noticed in between the tubules in the groups pretreated with 100 and 200 mg/kg of BHPF (Figure 5).

Figure 4. Hepatoprotective effect of BHPF in CCl₄-intoxicated mice. (A) Group I (normal control): showing normal histological structure of the central vein and intact hepatocytes. (B) Group II (CCl₄-treated group): showing severe loss of hepatic architecture with multiple focal necrosis, ballooning degeneration in the hepatocytes. (C) Group VI (CCl₄ + 200 mg/kg of silymarin): showing absence of histopathological alterations. (D and E) Groups V and IV (CCl₄ + 400 mg/kg and CCl₄ + 200 mg/kg, respectively of BHPF): showing normal histological structure. (F) Group III (CCl₄ + 100 mg/kg of BHPF): showing diffuse Kupffer cell proliferation in between the hepatocytes (H&E, × 20).

Figure 5. Nephroprotective effect of BHPF in CCl₄-intoxicated mice. (A) Group I (normal control): showing normal histological structure of the glomeruli and tubules at the cortex with absence of histopathological alterations. (B) Group II (CCl₄-treated group): showing marked inflammatory cell aggregation in between the tubules, marked degeneration in the lining epithelium of all the tubules, and blood vessel congestion. (C) Group VI (CCl₄ + 200 mg/kg of silymarin): showing absence of histopathological alterations. (D) Group V (CCl₄ + 400 mg/kg of BHPF): showing normal histological structure. (E and F) Groups IV and III (CCl₄ + 200 mg/kg and CCl₄ + 100 mg/kg of BHPF, respectively): showing mild inflammatory cell infiltration in between the tubules (H&E, × 20).

Discussion

CCl₄ is a potent environmental hepatotoxin. CCl₄ also causes disorders in kidneys, lungs and brain. In addition, it may lead to acute and chronic renal injuries.43 CCl₄ is widely used in experimental models to induce liver and kidney damage.44^,45 The highly reactive trichloroethyl radicals (CCl_3. and CCl₃O_2.) are formed from CCl₄ by cytochrome P-450. These radicals initiate lipid peroxidation, necrosis and fatty changes of the liver. The trichloroethyl radicals also change the cellular antioxidant capacity by deactivating GSH and the defense antioxidant enzymes.21^,44 In this study, the acute model of CCl₄-intoxication was used because it resembles the acute intoxication in human.43 The present study indicated that CCl₄ administration significantly increased the serum levels of ALP, AST, ALT, LDH, cholesterol and total bilirubin. Moreover, it reduced the total protein level, which indicated severe loss of liver function. Histopathological examination revealed severe loss of hepatic architecture associated with multiple necrosis, marked ballooning degeneration of hepatocytes along with marked inflammatory cell infiltration and degeneration of the kidney tissues. CCl₄ intoxication also induced severe oxidative stress as evidenced by the marked increase in hepatic and renal MDA levels. The SOD and GSH levels in the liver and kidney tissues were markedly reduced in response to CCl₄ intoxication. The results of the present study indicated that pretreatment with BHPF restored the increased MDA levels to their normal values. The inhibitory effect against lipid peroxidation suggested that BHPF could prevent the hepatorenal damage induced by free radicals, along with the subsequent histopathological alterations in the liver and kidney. In contrast, BHPF pretreatment significantly enhanced the GSH and SOD levels as compared to the CCl₄-intoxicated group. Modulation of these antioxidant defenses clearly contributed to the hepato- and nephroprotective activity of BHPF. Based on the results of this study, the hepato- and nephroprotective effect of BHPF is attributed to its ability to reduce the rate of lipid peroxidation, to enhance the antioxidant defense status and to guard against the pathological changes of the liver and kidney induced by CCl₄ administration. The remarkable hepato- and nephroprotective effect of BHPF may, at least partly, be due to the antioxidant effect of its bioactive constituents. The HPLC-PDA-ESI/MS/MS analysis of BHPF revealed the presence of proanthocyanidins and epicatechin gallate (ECG) as the major components. Experimental evidence suggests that whole plant fractions usually have much better pharmacological activities than their single isolated ingredients due to synergistic interactions between the individual components.46 It is also proved that mixtures of antioxidant compounds are more active than the individual components of these mixtures.47

Proanthocyanidins are complex polymers of polyhydroxy flavan-3-ol constitutive units, and are classified according to the hydroxylation pattern of their constitutive units and the linkages between them.35 The most common units are (+)-catechin, (−)-epicatechin in the case of procyanidin type, or (+) gallocatechin and (−)-epigallocatechin, for the prodelphinidin structure.35 Previous studies demonstrated that proanthocyanidins confer greater protection against free radicals and lipid peroxidation than vitamins C, E and β-carotene.14 Many studies confirmed the protective effect of GSPE against chemical assaults in various organs using different models. GSPE exhibited a strong protective effect on thioacetamide-induced hepatic fibrosis in mice,15 and produced hepatoprotective as well as anti-fibrogenic effects against dimethylnitrosamine-induced liver injury in rats.48 In addition, GSPE significantly protected against acetaminophen-induced liver and kidney injury by reducing oxidative stress, ALT activity and by inhibiting apoptotic and necrotic cell death.14 GSPE also conferred protection against cyclosporine A- and cisplatin-induced nephropathy in rats and recovered the kidney functions. The nephroprotective activity of GSPE was attributed to its potent antioxidant activity, the attenuation of renal tubular damage and the enhancement of the regeneration response.49^,50 The remarkable hepato- and nephroprotective effect of BHPF may be related to the presence of different proanthocyanidins, which may confer a synergistic action. ECG, a major component in GSPE, exhibited a protective role against renal failure, along with the ability to reduce serum uric acid, urea, and creatinine concentrations.51 Diverse pharmacological activities have been attributed to ECG, including potent antioxidant, anti-fibrogenic and hepatoprotective activity.13^,52 Besides, it was proved that ECG has more effective antioxidant activity over vitamin C.53 All these attributes might contribute to the hepato- and nephroprotective effect of BHPF. Based on the results of this study, the potent hepatoprotective effect previously reported for the total extract of B. hookeri44 could be attributed to its proanthocyanidin and ECG contents. Furthermore, the results of this study confirmed that B. hookeri proanthocyanidin has a potent nephroprotective effect.

Experimental evidence indicates that oxidative stress is a major link between liver injury and hepatic fibrosis. Persistent hepatocellular damage disrupts the regeneration process of damaged tissues and overwhelms the liver's defensive mechanisms. The injured hepatocytes are potent sources of ROS and lipid peroxidation products that stimulate the transformation of hepatic stellate cells (HSCs) to fibrogenic myofibroblast-like cells, which produce collagen.15^,48 An excessive extracellular matrix is accumulated in liver tissues, which may progress to hepatic fibrosis or cirrhosis. Inactivation of HSCs represents a promising approach to block the progression of hepatic fibrosis.15 The protective effect of BHPF against infiltration by inflammatory cells and other CCl₄-induced pathological changes in the liver, along with the protective effect against oxidative stress, indicated that a dietary supplement of BHPF has hepatoprotective and antifibrotic therapeutic potential.

Acknowledgments

The authors would like to thank Eng. Therese Labib, the taxonomy specialist at the herbarium of El-Orman Botanical Garden, Giza, Egypt for his kind help regard to identification of B. hookeri leaves used in our study.

Declaration of interest

The authors have declared no conflicts of interest. The current research received no fund from any organization.