Modulatory potential of Bacopa monnieri against aflatoxin B1 induced biochemical, molecular and histological alterations in rats

Abstract

Oxidative injury is concerned with the pathogenesis of several liver injuries, including those from acute liver failure to cirrhosis. This study was designed to explore the antioxidant activity of Bacopa monnieri (BM) on Aflatoxin B1 (AFB1) induced oxidative damage in Wistar albino rats. Aflatoxin B1 treatment (200 μg/kg/day, p.o.) for 28 days induced oxidative injury by a significant alteration in serum liver function test marker enzymes (AST, ALT, ALP, LDH, albumin and bilirubin), inflammatory cytokines (IL-6, IL-10 and TNF-α), thiobarbituric acid reactive substances (TBARS) along with reduction of antioxidant enzymes (GSH, SOD, CAT), GSH cycle enzymes and drug-metabolizing enzymes (AH and AND). Treatment of rats with B. monnieri (20, 30 and 40 mg/kg for 5 days, p.o.) after 28 days of AFB1 intoxication significantly restored these parameters near control in a dose-dependent way. Histopathological examination disclosed extensive hepatic injuries, characterized by cellular necrosis, infiltration, congestion and sinusoidal dilatation in the AFB1-treated group. Treatment with B. monnieri significantly reduced these toxic effects resulting from AFB1. B. monnieriper se group (40 mg/kg) did not show any significant change and proved safe. The cytotoxic activity of B. monnieri was also evaluated on HepG2 cells and showed a good percentage of cytotoxic activity. This finding suggests that B. monnieri protects the liver against oxidative damage caused by AFB1, which aids in the evaluation of the traditional usage of this medicinal plant.

Graphical Abstract

Graphical Abstract.

Introduction

In the eagerness to modernize and cater to the needs of the increasing population humans are exposed to several toxicants, of which mycotoxins are the most commonly explored toxicants in daily life.¹ Mycotoxins are metabolites produced by fungi to protect from enemies and may infect inappropriately kept grains and several other staple products. Ingesting these infected food items by people or cattle results in disturbing health and several other clinical manifestations.² Of these mycotoxins, aflatoxins are most studied due to the ubiquity of their producing fungi, which are Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1) and Aflatoxin G2 (AFG2) are naturally found in numerous composites with the core aflatoxins skeleton.³ AFB1 is the most toxic of these toxins. Consumption of foods or exposure to grains contaminated with AFB1 might result in several health problems including liver, kidney, brain, lung, and gastrointestinal cancers, delayed development and genotoxic effects.⁴

AFB1 is a pro mutagen that requires metabolic bioactivation through CYP450 to form an epoxide- AFB1-O, which reacts with intracellular nucleophiles to produce AFB1 toxicity by causing lesions.⁵ Endogenous detoxification requires the conjugation of AFB1-O to compounds such as GSH.⁶ Our current observation is interested in identifying strategies for AFB1 detoxification using B. monnieri.

B. monnieri (BM), frequently known as “Brahmi” is a highly cherished medicinal herb widely used as a memory enhancer, vitalizer, and nerve tonic and is frequently used in Ayurveda, Siddha, and Unani. BM holds important pharmacological properties like antioxidant activity, anti-cancer, anti-inflammatory, neuroprotective, immunostimulatory, and anti-depressant.⁷ It is also used in the treatment of skin infection, diabetes, epilepsy, inflammation and hyperpyrexia.⁸

Herein, we report in-vitro (HepG2 cells) anti-cancer activity of BM and in-vivo hepatoprotective activity against AFB1-induced hepatotoxicity in albino Wistar rats.

Material and methods

Animals and chemicals

Male rats of Wistar strain weighing between 150–200 grams were purchased from Central Animal House, All India Institute of Medical Sciences, New Delhi, India. The animals were housed in a university animal house facility under standard conditions (25 ± 2 °C temp, 60%–70% relative humidity, 14 h light and 10 h dark). Rats were fed on a standard pellet diet and water ad libitum. Animals were treated and cared for by the guidelines recommended by the Committee for Control and Supervision of Experiments on Animals (CPCSEA No-IAEC/JU/56).

Aflatoxin B1 was procured from Sigma Aldrich and Co., USA. All other chemicals were of analytical grade and purchased from Sigma-Aldrich Company, Ranbaxy, New Delhi and Hi-media Laboratories Ltd Mumbai, India.

Collection and preparation of plant extract

The whole plant of BM was collected from the campus of Jiwaji University. The herbarium was submitted to the School of Studies in Botany with accession number 5312/PP-49-50/03/12/2020. After drying the plant was ground to a coarse powder. 10-gram powder was added in 100 mL of double distilled water in an Erlenmeyer flask and boiled in a water bath at 60 °C for 1–2 h. After cooling, the mixture was filtered using a muslin cloth. The filtrate was further filtered through 0.6 μm sized filters, allowed to dry in an oven at 37 °C then stored in an air-tight tube at 4 °C for further analysis.

Phytochemical studies

The aqueous plant extract was subjected to various phytochemical analyses to quantify total phenolic content. H₂O₂ free radical scavenging activity was also detected by Ruch.⁹

Isolation of primary hepatocytes from rat liver by Kiso¹⁰

The study involved isolating primary hepatocytes from a fasting male albino rat of the Wistar strain. Urethane was injected intra-peritoneally to anesthetize the rat, and ethanol was used to disinfect the animal. The liver was exposed through a midline incision, and a teflon catheter was inserted into the hepatic portal vein. Hank's Balanced Salt Solution (HBSS) was used to perfuse the liver for 15 min. The liver was then placed in a petri plate containing HBSS and cut into small pieces. The cells were then filtered and centrifuged for 5 min at 600 rpm. The supernatant was aspirated off, and the cells were re-suspended in HBSS and washed three times. Cell viability was checked using Sulphorhodamine B.

Cell culture and condition

HepG2 cells were procured from the National Centre for Cell Sciences, Pune, India and preserved in a DMEM medium supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin and 100 U/mL penicillin. Cells were sub-cultured once the 70% confluence was achieved.

Cell viability assay

The cytotoxic action of BM was determined in HepG2 cells using an MTT assay. About 5×10⁴ cells were seeded in 96 well plates incubated at 37 °C in a CO₂ incubator for a day. Next-day treatment of BM was given in different concentrations (2, 4, 8, 16, 32, 64 μg/mL) and cells were kept for 24 h. Afterward, 20 μL of MTT was added to each well and further cells were incubated for 4 h in the dark. After incubation, formazan crystals were dissolved by the addition of 100 μL DMSO. After 5 min, absorbance was recorded at λ 570 nm. The percentage of HepG2 cells demise was calculated using the formula:

Further, the IC₅₀ of BM extract was calculated.

In-vivo experimental design

Animals were randomly divided into six groups with six animals in each group. Group, 1 served as the control. Group 2 served as per se and received a higher dose of therapy which was 40 mg/kg. Group 3 received AFB1 (200 μg/kg/day, orally) for 28 days. Groups 4, 5 and 6 received AFB1 for 28 days which was further followed by different doses of BM (20, 30, 40 mg/kg/day, orally) for 5 consecutive days. All animals were euthanized using urethane (0.75 mg/kg i.p.), after 24 h of the last treatment.

Group I	Control
Group II	Toxicant (200 μg/kg)	According to Vipin et al.11 and Gupte et al.1 selected dose
Group III	Bacopa monnieri per se (40 mg/kg)	Kamkaew et al.12
Group IV-VI	B. monnieri (20/30/40 mg/kg) + Toxicant (Group II)	Kamkaew et al.12

Hematological analysis

Blood was collected by puncturing the retro-orbital plexus of anesthetized rats. 10% ethylene diamine tetraacetic acid (EDTA) was mixed in blood to prevent clotting. Complete blood count was determined using apparatus ABX Micros 60.

Serological study

For the serological studies blood was collected in heparin-free centrifuging tubes and clot formation was allowed at room temperature for half an hour. The blood was centrifugated at 3,000 RPM for 15 min to obtain serum for the determination of serological parameters such as triglycerides (TG), cholesterol, bilirubin, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) with the help of their respective kits following the direction given on kit manual (ERBA). Albumin aspartate aminotransferase (AST) and alanine aminotransferase (ALT) according to Reitman and Frankel.¹³

Buffers

Tris buffer (0.5 M, pH 8.23): Tris buffer (6.05 g) was dissolved in a minimum amount of distilled water and pH (8.23) was adjusted by HCl then the volume was made up to 100 mL by distilled water.

Sodium bicarbonate buffer (0.3 M, pH 10.2): NaHCO₃ and Na₂HCO₃ were dissolved separately and the pH was set to 10.2.

Potassium phosphate buffer (50 mM) (pH 7.4): 0.870 g of K₂HPO₄ was dissolved in 50 ml distilled water. pH was adjusted to 7.4 by mixing 0.700 g KH₂PO₄ solution and the final volume was made up to 100 mL with distilled water.

Oxidative stress assessment

To determine lipid peroxidation (LPO), tissue homogenates were prepared in a 10% KCl solution according to the method described by Sharma and Murti.¹⁴ The LPO in the liver was calculated by quantifying the level of thio barbituric acid reactive substances (TBARS).

Antioxidant status assessment in hepatic tissues

Sucrose solution (1%) was used to make homogenates for the evaluation of reduced glutathione (GSH).¹⁵ NaCl solution (0.9%) was used to formulate tissue homogenates to measure superoxide dismutase (SOD)¹⁶ and catalase (CAT) activity.¹⁷

Enzymes of the GSH cycle in hepatic tissue

Levels of glutathione reductase (GR),¹⁸ glutathione peroxidase (GPx),¹⁹ glucose-6-phosphate dehydrogenase (G6PDH)²⁰ and glutathione-S-transferase (GST)²¹ were determined in tissue homogenates prepared in 1.15% KCl solution.

Microsomal CYP2E1 activity

Microsomes were prepared by CaCl₂ precipitation process.²² The CYP2E1 activity was measured in terms of aniline hydroxylase (AH)²³ and Amidopyrine-N-demethylase (AND).²⁴

Membrane-bound enzymes

Membrane-bound enzymes in tissue: adenosine triphosphatase (ATPase)²⁵ and glucose 6 phosphatase (G6Pase) were determined in hypotonic solution.²⁶

Estimation of anti-inflammatory and pro-inflammatory biomarkers

Concentrations of IL-6, IL-10 and TNF-α were evaluated using RayBio ELISA Kit according to the manufacturer’s manual.

Procedure:

• Prepare all reagents, samples, and standards according to the instructions.

• Fill each well with 100 μL of standard or sample. Incubate at room temperature for 2.5 h.

• Fill each well with 100 μL of produced biotin antibody. Incubate at room temperature for 1 h.

• Pour in 100 μL of the prepared Streptavidin solution. Incubate at room temperature for 45 min.

• Fill each well with 100 μL TMB One-Step Substrate Reagent. Incubate at room temperature for 30 min.

• Fill each well with 50 μL Stop Solution. Read at 450 nm right away.

Histopathological observation

Small pieces of liver tissues were fixed in Bouin’s fixative. Fixed tissues were processed and embedded in paraffin wax for cutting of 5 μm sections and sections were placed on glass slides and stained with hematoxylin and eosin (H&E) stain and photographs were captured.

Statistical analysis

Data were expressed as mean ± standard error in each group. The mean using one-way analysis of variance (ANOVA) was completed considering significance at P ≤ 0.05 followed by student t-test.²⁷

Results

Phytochemical studies

Table 1 represents the amount of total phenolics in BM. Gallic acid was used as a standard for comparison of effectiveness (Fig. 1). H₂O₂ scavenging activity was carried out at different concentrations ranging from 10 to 100 mg in Fig. 2. On increasing concentration, H₂O₂ scavenging activity was found to be increased significantly. Ascorbic acid was used as standard.

Table 1.

Phytochemical estimation of aqueous extract Bacopa monnieri plant extracted using conventional solvent extraction method.

S.N.	Aqueous Extract	Total phenolic content (mg Gallic acid equivalent/g plant)
1	B. monnieri	1.64 ± 0.09

Figure 1.

Calibration curve of Gallic acid for estimation of Total phenolic content. Values are the mean ± SE for three observations.

Figure 2.

Hydrogen peroxide (H₂O₂) scavenging activity.

Determination of the antiproliferative effect of test drugs on HepG2 cells

MTT assay

The MTT test was used to investigate the effect of BM on cell growth. BM drastically reduced the growth of HepG2 cells. After 24 h of incubation, the inhibition was detected. Figure 3 depicts the variations in the percentage of cell viability in HepG2 cells treated with BM (2, 4, 8, 16, 32, 64 μg/mL). In BM-treated HepG2 cells, cytotoxicity was concentration-dependent. The inhibitory concentration (IC₅₀) values of the test agents BM against HepG2 cells were held at 64 μg/mL) after 24 h of incubation. The cytotoxic activity against HepG2 cells was also compared to the common anticancer medication 5-Fluorouracil (5-FU).

Figure 3.

MTT {3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium} assay for the determination of cytotoxicity of test drugs against hepatic cancer cells. Values are mean ± SD; n = 2; P ≤ 0.05 versus untreated control. 5-FU = 5-fluorouracil, BM = Bacopa monnieri.

Determination NOAEL of test drugs on cryopreserved normal hepatocytes

The addition of BM had no harmful effect on cryopreserved plated normal hepatocytes, according to this investigation. When different concentrations of BM-treated cells were added, there was no difference in cell viability when compared to the control. The delivery of AFB1 to cryopreserved hepatocytes at a concentration of 5 mM resulted in considerably lower cell viability (13.51%) when compared to the control. However, as demonstrated in Table 2 and Fig. 4, BM therapy prevented AFB1-induced damage. After toxicant exposure, BM demonstrated 52.89%, 40.13%, 23.15%, 19.64%, 15.98% and 10.36% cell viability at 64 μg/mL, 32 μg/mL, 16 μg/mL, 8 μg/mL, 4 μg/mL, and 2 μg/mL concentrations, respectively. Thus, the current investigation demonstrates that the test drugs had no detrimental effects on normal hepatic cells and could protect hepatocytes from the unfavorable effects of AFB1.

Table 2.

No observed adverse effect level.

Treatment	Concentration	Cell viability (%)
Control	0.0	97.31 ± 5.37
BM Per s (μg/mL)	2 4 8 16 32 645 mM	83.75 ± 0.57 85.67 ± 0.88 89.35 ± 1.08 90.12 ± 1.27 92.56 ± 2.21 95.56 ± 2.8213.51 ± 0.74
AFB1
AFB1 (5 mM) + BM (μg/mL)	2 4 8 16 32 64	10.36 ± 0.57 15.98 ± 0.88 19.64 ± 1.08 23.15 ± 1.27 40.13 ± 2.21 52.89 ± 2.82

Determination of NOAEL of test drugs on cryopreserved hepatocytes.

Figure 4.

No observed adverse effect level. Determination of NOAEL of test drugs on cryopreserved hepatocytes. Values are the mean ± S.E. of three wells in each group of three repeated experiments.

Hematological analysis

The results of hematological analysis are shown in Table 3.

Table 3.

Therapeutic effect of Bacopa monnieri against aflatoxin B1 on blood profile.

Treatment	Blood Biochemistry
	WBCs (10,000/3/uL)	RBCs (10,000/3/uL)	HGB (%)	HCT (%)	Platelet (1010/μL)
Control	10.4 ± 0.57	6.10 ± 0.33	16.2 ± 0.89	33.4 ± 1.84	7.61 ± 0.42
BM per se	11.04 ± 0.61	5.91 ± 0.32	15.9 ± 0.87	34.3 ± 1.89	7.32 ± 0.40
AFB1	21.5 ± 1.18#	5 ± 0.27#	13.9 ± 0.76#	24.6 ± 1.35#	4.13 ± 0.22#
AFB1 + BM (20 mg/kg) (% protection)	15 ± 0.82* (58.55%)	5.14 ± 0.28* (12.72%)	14.20 ± 0.78* (13.04%)	28.4 ± 1.56* (43.18%)	5.69 ± 0.31* (44.82%)
AFB1 + BM (30 mg/kg) (% protection)	14 ± 0.77* (67.56%)	5.23 ± 0.28* (20.90%)	14.90 ± 0.82* (43.47%)	30.4 ± 1.68* (65.90%)	6.84 ± 0.37* (77.87%)
AFB1 + BM (40 mg/kg) (% protection)	12.2 ± 0.67* (83.33%)	5.69 ± 0.31* (62.72%)	15.4 ± 0.85* (65.21%)	31.2 ± 1.72* (75%)	7.09 ± 0.39* (85.05%)
F value (at 5% level)	30.69@	2.61 @	1.52@	5.25@	15.75@

Abbreviations: BM = Bacopa monnieri, AFB1 = Aflatoxin B1; Data are Mean ± SE; N = 6; @ = Significant at 5% for ANOVA ^# AFB1 vs Control; ^* AFB1 + Therapy vs AFB1; at P ≤ 0.05.

White blood cells (WBCs) of rats in group 3 were above the reference range, while red blood corpuscles (RBCs), hemoglobin (HGBs), hematocrit (HCTs) and platelet values were found to be decreased. Animals from groups 4 to 6 showed recovery in the above hematological profiling (P < 0.05).

Serological study

Table 4 demonstrated that the activities of triglycerides (TG), cholesterol, bilirubin, albumin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were significantly increased (P ≤ 0.05) in the serum of AFB1-intoxicated rats compared with control and per se. However, activities of the above liver function test markers were significantly decreased towards control values (P ≤ 0.05) in AFB1-intoxicated rats treated with BM in a dose-dependent manner, a higher dose of therapy shows maximum improvement.

Table 4.

Therapeutic efficacy of Bacopa monnieri on lipid profile serum parameters.

Treatment	LFT
	Triglycerde (mg/dL)	Cholesterol (mg/dL)	Bilirubin(mg/dL)	Albumin (mg/dL)	AST (IU/L)	ALT (IU/L)	LDH (U/L)	ALP (mg Pi/h/100 mL)
Control	79.14 ± 4.37	69.1 ± 3.81	0.35 ± 0.01	2.90 ± 0.16	61 ± 3.37	42.67 ± 2.35	42.3 ± 2.33	194.6 ± 10.7
BM per se	81.05 ± 4.05	70.15 ± 3.87	0.34 ± 0.04	2.62 ± 0.14	62.4 ± 3.45	44.05 ± 2.43	43.6 ± 2.41	195.2 ± 10.7
AFB1	415 ± 22.94#	109.2 ± 6.03#	1.83 ± 0.10#	1.6 ± 0.08#	102.02 ± 5.63#	88.94 ± 4.91#	167 ± 9.23#	533 ± 29.4#
AFB1 + BM (20 mg/kg) (% protection)	336 ± 18.57* (23.52%)	84.2 ± 4.65* (62.34%)	0.27 ± 0.01* (38.46%)	1.87 ± 0.10* (20.76%)	89.02 ± 4.92* (31.69%)	76.32 ± 4.21* (27.01%)	89.16 ± 4.92* (62.42%)	314 ± 17.3* (64.71%)
AFB1 + BM (30 mg/kg) (% protection)	246 ± 13.59* (50.31%)	78.3 ± 4.32* (77.05%)	0.31 ± 0.01* (69.23%)	2.07 ± 0.11* (36.15%)	78.82 ± 4.35* (56.55%)	68.47 ± 3.78* (44.24%)	66.33 ± 3.66* (80.72%)	271 ± 14.9* (77.42%)
AFB1 + BM (40 mg/kg) (% protection)	180 ± 9.39* (69.96%)	74.1 ± 4.09* (87.53%)	0.33 ± 0.01* (84.61%)	2.11 ± 0.11* (39.23%)	70.34 ± 3.88* (77.23%)	57.63 ± 3.18* (67.66%)	57.25 ± 3.16* (88.01%)	219 ± 12.1* (92.78%)
F value (at 5% level)	111.9 @	13.08@	18.18@	232.1@	16.32@	31.06@	110.4@	67.49@

Abbreviations: BM = Bacopa monnieri, AFB1= Aflatoxin B1; Data are Mean ± SE; N = 6; @ = Significant at 5% for ANOVA ^# AFB1 vs Control; ^* AFB1 + Therapy vs AFB1; at P ≤ 0.05.

Oxidative stress assessment in hepatic tissue

TBARS level was significantly (P ≤ 0.05) increased and the GSH level was significantly (P ≤ 0.05) decreased in the liver of AFB1 intoxicated rats compared to control. Treatment of BM caused a significant (P ≤ 0.05) decrease in the TBARS level and a significant (P ≤ 0.05) increase in the GSH of liver tissue. Significant recovery was observed after BM therapy at all three doses. Percent protection showed maximum recovery at a higher most 40 mg/kg dose, as shown by ANOVA at a 5% significance level (Table 5).

Table 5.

Therapeutic effect of Bacopa monnieri against aflatoxin B1 on oxidant and antioxidant potential.

Treatments	Lipid Peroxidation (n mole TBARS/mg protein)	Reduced Glutathione (μ mole/ g)	Superoxide Dismutase (U/ min/ mg protein)	Catalase (μ mole H2O2/ min/ mg protein)
Control	0.34 ± 0.018	7.96 ± 0.42	64.30 ± 3.55	63.6 ± 3.51
BM per se	0.36 ± 019	7.77 ± 0.42	61.2 ± 3.38	62.2 ± 3.43
AFB	1.13 ± 0.062#	3.35 ± 0.18#	24.80 ± 1.37#	30.66 ± 1.69#
AFB1 + BM (20 mg/kg) (% protection)	0.82 ± 0.045* (39.24%)	4.39 ± 0.24* (22.55%)	36.32 ± 2.0* (29.16%)	42.24 ± 2.33* (35.15%)
AFB1 + BM (30 mg/kg) (% protection)	0.53 ± 0.029* (75.94%)	5.09 ± 0.28* (37.74%)	40.27 ± 2.22* (39.16%)	55.57 ± 3.07* (75.62%)
AFB1 + BM (40 mg/kg) (% protection)	0.42 ± 0.023* (89.87%)	6.63 ± 0.36* (71.14%)	56.04 ± 3.09* (79.08%)	60.2 ± 3.32* (89.67%)
F value (at 5% level)	87.38@	37.22@	39.91@	23.58@

Abbreviations: BM = Bacopa monnieri, AFB1 = Aflatoxin B1; Data are Mean ± SE; N = 6; @ = Significant at 5% for ANOVA ^# AFB1 vs Control; ^* AFB1 + Therapy vs AFB1; at P ≤ 0.05.

Antioxidant status assessment in hepatic tissues

The SOD and CAT level drastically declined (P ≤ 0.05) in the liver after AFB1administrated rats when compared to the control group. Treatment of BM caused a significant (P ≤ 0.05) elevated towards normal value of SOD and CAT in liver tissue. Therapy of BM showed restoration of these values at all doses. Maximum restoration was seen in a 40 mg/kg dose (Table 5).

Enzymes of the GSH cycle in hepatic tissue (GR, GPx, G6PDH and GST)

Glutathione GSH is a thiol-containing antioxidant, actively involved in drug detoxification. Its homeostasis is maintained via the GSH cycle. The GSH cycle enzymes (GR, GPx, G6PDH and GST) level significantly (P ≤ 0.05) declined in the liver after AFB1 intoxicated rats compared to control. Treatment of BM caused a significant (P ≤ 0.05) recoupment of these enzyme levels in liver tissue (Fig. 5).

Figure 5.

Toxic effect of AFB1 on enzymes of GSH cycle. A) GR, B) GPx, C) G6PDH, D) GST. Abbreviations: BM = Bacopa monnieri, AFB1 = aflatoxin B1; data are mean ± SE; N = 6; @ = significant at 5% for ANOVA ^# AFB1 vs control; ^* AFB1 + therapy vs AFB1; at P ≤ 0.05. ANOVA  GR    GPx    G6PDH    GST. F Value  20.54^@   22.71^@  22.42^@   5.36^@.

Microsomal CYP2E1 activity

AFB1 exposure resulted in a significant decrease in hepatic CYP enzyme activities (AH and AND). Treatment of BM at all three doses showed improvement in AH and AND activity (P ≤ 0.05). Thus, therapeutic agents exhibited a marked hepatoprotective activity (Fig. 6).

Figure 6.

Toxicology consequence of AFB1 on CYP450 enzymes. A) AH, B) AND. Abbreviations: BM = B. monnieri, AFB1 = aflatoxin B1; mean ± SE; N = 6; @ = significant at 5% for ANOVA ^# AFB1 vs control; ^* AFB1 + therapy vs AFB1; at P ≤ 0.05. ANOVA   AH        AND   F values  29.82^@  52.12^@.

Membrane-bound enzymes

The activity of adenosine triphosphatase (ATPase) and glucose 6 phosphatase (G6Pase) was significantly (P ≤ 0.05) reduced after AFB1 administration. Treatment with BM restored the activity of adenosine triphosphatase and glucose 6 phosphatase at all doses. Maximum restoration was seen in a 40 mg/kg dose (Table 6).

Table 6.

Therapeutic efficacy of Bacopa monnieri on membrane bound enzymes.

Treatments	ATPase (mg pi/100 g/min)	G6Pase (μmole pi/min/g liver)
Control	1849.9 ± 102.26	5.93 ± 0.32
BM per se	1843 ± 101.8	5.74 ± 0.31
AFB1	1,147 ± 63.40#	2.53 ± 0.13#
AFB1 + BM (20 mg/kg) (% protection)	1,478 ± 81.70* (47.15%)	5.23 ± 0.28* (79.41%)
AFB1 + BM (30 mg/kg) (% protection)	1,564 ± 86.45* (59.40%)	5.39 ± 0.29* (84.11%)
AFB1 + BM (40 mg/kg) (% protection)	1,650 ± 91.21* (71.65%)	5.53 ± 0.30* (88.23%)
F value (at 5% level)	10.47@	23.28@

Abbreviations: BM = Bacopa monnieri, AFB1 = Aflatoxin B1; Data are Mean ± SE; N = 6; @ = Significant at 5% for ANOVA ^# AFB1 vs Control; ^* AFB1 + Therapy vs AFB1; at P ≤ 0.05.

Estimation of anti-inflammatory and pro-inflammatory cytokines

Figures 7A–C depict the effect of AFB1 and BM treatment on inflammatory markers. Administration of AFB1 alone significantly increased (P < 0.05) IL-6 and TNF-α levels compared to control while decline in IL-10 level. However, BM treatment restores their level towards control in a dose-dependent manner.

Figure 7.

Effect of AFB1 on serum anti-inflammatory and pro-inflammatory cytokines. A) IL-10, B) IL-6, C) TNF-α. abbreviations: BM = Bacopa monnieri, AFB1 = aflatoxin B; data are mean ± SE; N = 6; @ = significant at 5% for ANOVA ^# AFB1 vs control; ^* AFB1 + therapy vs AFB1; at P ≤ 0.05. ANOVA    IL-6       IL-10   TNF- α   F values   91.78^@  49.59^@  164.4^@.

Histopathological observation

In histological studies of the liver, the control group (Fig. 8A) showed regular morphology with intact structure and the boundary between cells is clear. The structure inside the cells is clean without impurities and droplets. Infiltration of inflammatory cells does not exist in the central venous area. Per se group did not show any changes (Fig. 8B). Administration of AFB1 caused central venous congestion with heavy lymphocytic infiltration, hypertrophied nuclei, dilation in sinusoidal spaces, vacuolation, loss of lobular architecture with damaged cellular outlines, nuclear pyknosis and fatty changes (Fig. 8C). Significant liver protection was observed in the liver sections of BM treated group evident by the presence of normal hepatic cords, and the absence of necrosis with very few fatty lobules in a dose-dependent manner (Fig. 8D, E, and F). The cytoplasm of the hepatocyte was eosinophilic as in the control group. Sinusoids were clear and the nucleus showed regenerative features.

Figure 8.

Histological observation of hepatocytes. Abbreviations: A) The liver of the control rat showed well-formed hepatocytes and a clear central vein with maintained sinusoidal space. The well-formed portal vein, hepatic artery and bile ductule were observed (X100, 400). B) Liver of per se group depicts clear central vein and sinusoidal spaces with radially arranged polygonal hepatocytes (X100, 400). C) The group intoxicated with AFB1 (200 μg/kg) revealed infiltration and congestion in the central vein and loss of sinusoidal spaces. Disturbed chord arrangement due to damaged hepatocytes (X100, 400). D) BM (20 mg/kg) showed congestion in a central vein (X100, 400). E) BM (30 mg/kg) showed well-formed hepatocytes and mild improved central vein (CV) (X100, 400). F) Liver of the treated group with BM (40 mg/kg) shows a fairly normal hepatic structure with well-arranged hepatocytes clear central vein and well-formed sinusoidal spaces with intact portal tract (X100, 400).

Discussion

The liver is a complex organ involved in several metabolic functions and because of its central role in xenobiotics metabolism, it is susceptible to direct injury by them and their metabolites. Exposure to xenobiotics such as acetaminophen, carbon tetrachloride, acrylamide, thioacetamide and AFB1 results in damage to the liver.

Hepatic disorders have become a universal health problem due to abnormal lifestyles due to exposure to several xenobiotics.²⁸ In recent years, natural plants and their derived compounds have been increasing interest due to the pleiotropic effects of these compounds on the liver. A previous study has confirmed that BM repressed HCC cellular growth and colony formation,²⁹ similar to our findings.

In the whole world, hepatocellular cancer is the major liver cancer and is the fifth recorded common cancer. Researchers have investigated and been involved in the search for new and effective approaches for the treatment of hepatocellular carcinoma with natural compounds which are having minimal side effects.³⁰ Recently, researchers reported that BM possesses strong activity in therapeutic cancer treatment.³¹ Our results showed concentration-dependent cytotoxicity on HepG2 cells after 24 h of BM treatment. Several studies have reported that BM demonstrated remarkable anticancer properties.^32,33 Also, BM has been shown to induce cytotoxicity by induction of oxidative stress, observed in our cytotoxicity assay.³⁴

Blood is the most important pathological biomarker of xenobiotic-exposed animals' health. Red blood cells, white blood cells or leucocytes, hemoglobin, and hematocrit levels are the most commonly examined parameters during blood toxicity.³⁵ Hematological abnormalities caused by AFB1 indicate blood toxicity of AFB1 and its metabolites, whereas therapy with BM inhibits this modification, demonstrating the extract's therapeutic efficacy.

Our results showed a significant upsurge in liver function test marker enzymes (ALT, AST, ALP and LDH) in the AFB1 group. The upsurge of these liver enzymes (ALT, AST and LDH) indicates damage to hepatocytes and impairment of liver functions due to hepatotoxicity caused by any xenobiotics.³⁶ The mechanism of ALP high level may result from faulty liver discharges or increased ALP generation of hepatic parenchymal cells.³⁷ Our results showed improvement after treatment with BM. Recovery towards normalization of these liver function test marker enzymes following BM treatment after AFB1 exposure suggested that BM has a role in maintaining the structural integrity of the hepatocellular membrane and preventing these enzymes from leaking into circulation. Our results are with support of the hypothesis that transaminase levels return to normal with healing of hepatic parenchyma and regeneration of hepatocytes.³⁸

The mechanism of liver injury resulting from AFB1 has been reported to involve free radicals, resulting in damage and reactive oxygen species (ROS) production.³⁹ AFB1 is the most effective thiol-binding agent for SH groups of endogenous biomolecules. Hence, it is attached to thiol-containing proteins and small molecular weight thiol components such as GSH and cysteine. In addition, free radicals released from AFB1 produce free radicals such as superoxide and hydrogen peroxide, which cause oxidation of protein, lipid and DNA.⁴⁰ LPO has been recognized as a biomarker in the pathogenesis of AFB1 resulting in liver injury. Increased LPO results from the overproduction of ROS and decreased activities of endogen antioxidant enzymes that ultimately lead to tissue injury. Other studies^40,41 In support of our study, it has been documented that AFB1 increases ROS levels and induces oxidative stress. Previous studies have specified that BM has therapeutic effects against liver injury resulting from other toxic agents such as nitrobenzene.⁴² Our data clarifies that BM attenuates free radical damage by preventing oxidative stress induced by AFB1 resulting in lipid peroxidation, clarifying that BM provides safety against AFB1 resulted increased LPO.

Antioxidant molecules like glutathione, SOD and catalase neutralize oxidative stress by satisfying free radicals. SOD eliminates superoxide anions and produces H₂O₂, which was then further decomposed by catalase. BM protects depletion of these antioxidant enzymes by acting as an antioxidant by providing free electrons to free radicals making them stable. GPx harvests electrons from glutathione and neutralizes free radicals by giving their free electron, whereas, GR harvestes electrons by NADPH and reduces oxidized glutathione to make it available further for GPx. Then, G6PDH obtained electrons from glucose 6 phosphate, converting NADP⁺ into NADPH by donating that electron.⁴³ Appropriate regulation of this cycle helped to maintain antioxidant homeostasis levels, but as cellular ROS increases as a result of AFB1 toxicity, components of this cycle depleted in maintaining homeostasis. Glutathione-S-transferase permits conjugation of glutathione with toxic metabolites to eliminate them successfully out from the system. Depletion of glutathione-S-transferase and glutathione resulted as exposure to AFB1 produces several toxic metabolites during its metabolism. These enzymes of the GSH cycle were well maintained by BM as this plant might have the ability to form conjugates with toxic intermediates and scavenge free radicals from the system.⁴⁴

The microsomal drug metabolizing enzymes which are AH and AND are the enzymes of cytochrome P450 system. The damage inflicted by AFB1 on hepatocytes as well as on hepatic microsomal drug-metabolizing enzymes results in loss of the drug-metabolizing capacity of the liver. The liver microsomes originate from the endoplasmic reticulum and are the most common site of oxidative stress.⁴⁵ BM therapy reversed the AFB1-induced damage in hepatocytes evident by AH and AND. Flavonoids and phenolic compounds of BM might encounter the AFB1-induced microsomal inhibition of AH and AND activities; thus, showed protective effects by recovery of the subcellular architecture and functional integrity of hepatic cells, also supported by another author.⁴⁶

The earlier inflammatory response plays a key role in the cascading inflammatory response through the macrophages’ activation as well as the production of these pro-inflammatory mediators like TNF-α, and IL-6 increasing their level while decreasing anti-inflammatory cytokine like IL-10 levels. The gathering of macrophages results in the release of ROS and inflammatory cytokines, responsible for several important biological functions in the development of hepatic injury.⁴⁷ The pro-inflammatory cytokines TNF-α and IL-6 directly indicate the degree of inflammation. This trend was recorded to be inverted with the treatment of BM, which demonstrated that BM effectively ameliorated AFB1 resulting from injury via weakening the inflammation and oxidative stress. The level of IL-10 was found to be normal with BM treatment.

Histology of liver tissues has been considered as the golden standard for finding abnormalities, as it is a direct way of visualizing the inflammatory and architectural position of tissues, confirming that our biochemical parameters correlate with the tissue structure.⁴⁸ Induction of AFB1 demonstrated significant impairment in hepatic cellular structure as observed in histological findings of liver sections with distinct hepatocytes, sinusoidal spaces and central veins while groups that receive BM show restoration in these structures confirming hepatoprotective activity of BM.

Conclusion

The present study suggests that BM has the property to protect the liver against AFB1-induced damage. BM showed significant hepato-protection from AFB1-induced hepatotoxicity in rats by controlling levels of different liver function test markers, inflammatory markers and also histologically. Polyphenolic compounds with good antioxidant stuff might play the answerable role in this hepatoprotective activity. Anticancer activity was also noted. However, further pre-clinical studies are required to understand the proposed hepatoprotective activity.

Author contributions

Arti Rathour (Conceptualization, Formal analysis, Software, Validation, Visualization, Writing—original draft, Writing—review & editing), Shamli S. Gupte (Conceptualization, Data curation, Project administration, Validation, Writing—review & editing), Shubham Singh (Data curation, Investigation, Visualization, Writing—review & editing), Divya Gupta (Data curation, Formal analysis, Writing—review & editing), Sadhana Shrivastava (Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing—original draft, Writing—review & editing), Deepa Yadav (Data curation, Formal analysis, Visualization), and Sangeeta Shukla (Conceptualization, Funding acquisition, Supervision, Writing—original draft, Writing—review & editing).

All authors approve the final manuscript.

Funding

The author is thankful to DRDO (LSRB scheme), New Delhi (No.0/0 DGTM/81/48222/LSRB-340/BTB/2019) for funding the research work and Jiwaji University Gwalior for providing a lab facility.

Conflict of interest statement. All authors declare no conflict of interest related to this paper.

Kits catalogue number

LDH (No. 11760200011730), ALP (No. 11767700011730), Albumin (No. 118275.0001), Bilirubin (No. 103333.0001), Triglycerides (No. 11761000011730), Cholesterol (No.11767900011730), TNF-α (No. 0812160709), IL-6 (No. 0826160724) and IL-10 (No. 0812160704).