Pericopsis laxiflora (Baker) stem bark extract is protective against carbon tetrachloride-induced acute liver damage by modulation of oxidative stress and inflammation

Abstract

The present study aimed to investigate the hepatoprotective activity of the hydroethanolic (HSE) and methanolic extracts (MSE) of Pericopsis laxiflora stem bark on carbon tetrachloride-induced hepatotoxicity in rats. The stem bark of P. laxiflora was extracted with 50% ethanol and methanol. Hepatoprotective activities were assessed using the CCl4 model and tested against extracts at 100, 250, and 500 mg/kg bwt. Biochemical, antioxidant enzymes and proinflammatory cytokines in liver homogenate were assayed. Treatment with HSE and MSE resulted in a significant increase in SOD, CAT, and GSH levels and a significant decrease in MDA and MPO levels in livers of CCl4 treated rats. There was also restoration of liver biomarkers and pro-inflammatory cytokines to near-normal levels. The present investigation suggests P. laxiflora extracts possess good hepatoprotective properties. This could be attributed in part to the inhibitory effect of the extracts on oxidative stress and the suppression of proinflammatory cytokines.

KEYWORDS:

• Biochemical parameters
• carbon tetrachloride
• hepatoprotective
• Pericopsis laxiflora

1. Introduction

The liver is an organ that is commonly known for the production of reactive oxygen species that results in tissue damage through lipid peroxidation or inflammation. These generated free radicals can also be considered as important mediators of pathogens leading to many diseases such as diabetes, cancer, heart infection, and gout [1]. The presence of antioxidant enzymes provides a defence mechanism by scavenging the reactive oxygen species into a less toxic substance [1]. Diseases of the hepatocytes are regarded as a tremendous global health concern, especially in Africa, however, in allopathic medicine, few effective drugs for liver protection against serious disorders have been discovered [2]. Various herbal plants have been recorded to perform a crucial function in the healing and management of different types of liver diseases.

Pericopsis laxiflora is a plant that is regularly used by diabetes patients. In Cameroon, Bum [3] reported P. laxiflora as a medicinal plant that possess anti-diabetic effect when compared to Combretum molle. P. laxiflora continue to remain as one of the most popular and well-known plants with a long history of use among the Ghanaian population for the treatment of jaundice and body weakness [4].

In Ghana, numerous herbal plants have been investigated for their hepatoprotective effects and actions [5,6], however, no research has been done to determine the hepatoprotective effects of Pericopsis laxiflora [7]. Therefore, the purpose of this investigation was to examine the hepatoprotective potential of Pericopsis laxiflora stem bark extract, to help improve the outcomes of patients with liver disorders. Pericopsis laxiflora is a savannah, perennial, deciduous shrub, or tree that belongs to the Leguminosae family. It is mostly planted in the communities and villages due to its fond shade capabilities. It is commonly called Satinwood [7]. Carbon tetrachloride (CCl₄) and certain analgesics, such as acetaminophen, are prominent liver damage models used to test for hepatoprotective drugs. In this investigation, CCl₄ was chosen as the toxicant because of its high reactive metabolite produced. The ability of P. laxiflora stem bark extract to reverse damage caused by CCl₄ to near normal will therefore prove its hepatoprotective role.

Silymarin a flavonolignan molecule has been considered a standard drug for treating liver diseases in many experiments due to its hepatoprotective properties [8]. Silymarin comprises structural components such as silibinin, silydianine, and silychristine and was extracted from the Silybum marianum plant [9]. According to the current study, hepatoprotective effects of silymarin in acute hepatitis poisoning by Amanita phalloides, ethanol, carbon tetrachloride, and thioacetamide were reported [10], however, the hepatoprotective properties of the hydroethanolic (HS) and methanolic (MSE) stem extract of P. laxiflora have not yet been reported. Thus, the present study was carried out to investigate the hepatoprotective properties and mechanism of action 50% hydroethanolic and methanolic extracts of Pericopsis laxiflora on carbon tetrachloride-induced liver damage in rats.

2. Material and methods

2.1 Chemicals and reagents

Ethanol 99.9% was procured from Changshu Hongsheng Fine Chemicals Co. Ltd., China. Ethylenediaminetetraacetic acid (EDTA) was procured from Thermo Fisher Scientific India Pvt. Ltd., (Mumbai, India). All other chemicals and reagents used in the study were of analytical grade and obtained from standard suppliers.

2.2 Collection and authentication of plant materials

P. laxiflora fresh stem bark was obtained in February 2019 from healthy, fully developed plants in the Ejura Savannah forest reserve in Ghana's Ashanti region. (Latitude location – N 070°, 21°, 03.4; Longitude location – W 001°, 22°, 28.9). Mr. Jonathan Dabo, a botanist from the Centre for Scientific and Industrial Research, Kumasi, authenticated the plant samples. A voucher identification number (*******) was assigned, and the voucher specimen was deposited at the herbarium of the Herbal Medicine Department, ********. The phytochemical components is published elsewhere.

2.3 Preparation of extracts

The plant, Pericopsis laxiflora, was separated into the leaves, flowers, and stem barks components. The stem bark was washed, dried in the shade, and milled into a powder. Fifty percent hydroethanolic extract of the stem bark (HSE) was prepared using cold maceration procedure by suspending 100 grams of the powder in 500 ml of 50 percent ethanol (50:50, ethanol: water, v / v). Similarly, methanolic extract of the stem bark (MSE) was prepared by suspending 100 grams of the powder stem bark material in 500 ml of methanol. The extraction was done at room temperature on a shaker for 24 h. Subsequently, the extracts were filtered through cotton wool and concentrated under pressure using a rotary evaporator. The extracts were frozen at – 20^oC freeze-dried and stored until use.

2.4 Animals

HSE and MSE were evaluated for their hepatoprotective effects in the adult male of Sprague–Dawley rats (body weight, 150–200 g) in good health. Animals were purchased from the University of Ghana Medical School in Legon, Accra, and were kept in polypropylene cages coated with wood shavings. They were acclimatized for one week before testing in the Department of Biochemistry and Biotechnology's animal holding facility at ******, where they were kept under normal circumstances (temperature 252°C, relative humidity 65 percent, light/dark cycle 12/12 h). The rats were fed with a normal pellet diet (Agricare, Kumasi), and provided with drinking water ad libitum. For easy identification, they were only painted on their tails using permanent markers. All animal research was carried out by the Committee for the Monitoring and Control of Animal Experimentation's guidelines. Protocol for all animal experiments were verified and approved by a veterinarian on the research team.

2.5 Experimental design

Seventy-five (75) male rats weighing 150–200 g were divided into 15 groups and treated for 7 days (n = 5). Table 1 displays the groups and treatments. All animals were starved for 12 h before the first oral dose was administered, and they had unlimited access to food and freshly distilled water throughout the experiment. After an overnight fast under light ether anesthesia, all rats were slaughtered on the eighth day. Blood samples were collected from the animals’ necks in gel-activated tubes for biochemical analysis and EDTA tubes for haematological examination. In liver homogenates, antioxidant enzymes, oxidative stress markers, and pro-inflammatory cytokines were all assessed.

Table 1. Animal groups and treatments.

S/N.	Group	Treatment
1	Normal (control)	Potable water p.o (1 ml/kg b.wt)
Extracts Only
2	100 mg HSE only	100 mg/kg HSE only (per day, p.o.) for 7 consecutive days
3	250 mg HSE only	250 mg/kg HSE only (per day, p.o.) for 7 consecutive days
4	500 mg HSE only	500 mg/kg HSE only (per day, p.o.) only for 7 consecutive days
5	100 mg MSE only	100 mg/kg MSE only (per day, p.o.) for 7 consecutive days
6	250 mg MSE only	250 mg/kg MSE only (per day, p.o.) for 7 consecutive days
7	500 mg MSE only	500 mg/kg MSE only (per day, p.o.) only for 7 consecutive days
CCl4
8	CCl4	CCl4 i.p (1 ml/kg b.wt, 1:1 v/v olive oil) received on 2nd and 3^rd day.
9	CCl4 + Sily	Silymarin p.o (100 mg/kg per day) received for seven days plus a single dose of CCl4 in olive oil (1:1 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day
10	100 mg HSE + CCl4	100 mg/kg HSE (per day, p.o.) for seven days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day.
11	250 mg HSE + CCl4	250 mg/kg HSE (per day, p.o.) for seven days for seven consecutive days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day.
12	500 mg HSE + CCl4	500 mg/kg HSE (per day, p.o.) for seven days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day.
13	100 mg MSE + CCl4	100 mg/kg MSE (per day, p.o.) for seven days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day.
14	250 mg MSE + CCl4	250 mg/kg MSE (per day, p.o.) for seven days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3^rd day.
15	500 mg MSE + CCl4	500 mg/kg MSE for seven days plus a single dose of CCl4 in olive oil (1:0 v/v, 1.0 ml/kg, i.p.) on the 2nd and 3rd day.

2.6 Effect of extracts on body weight of rats

On the 1st (D1) and 7th days (D7) of treatment, the body weights of all animals were measured using the formula below [11]. $\mathrm{\%}\mathrm{Change}\mathrm{in}\mathrm{Body}\mathrm{Weight}=\frac{\mathrm{Weigh}{t}_n-\mathrm{Weigh}{t}_o}{\mathrm{Weigh}{t}_o}\times 100$where Weight n is the weight on day 7 and Weight o is the weight on day 1.

2.7 Effect of extracts on organ weights of rats

The rats’ livers and kidneys were collected, cleaned in a buffered saline solution, dried on tissue paper, visually evaluated, and weighed to estimate the absolute organ weight (AOW).

The Relative Organ Weight (ROW) of each organ was calculated using the formula below [11]. $\mathrm{ROW}=\frac{\mathrm{AOW}}{\mathrm{Body}\mathrm{Weight}\mathrm{at}\mathrm{Sacrifice}}\times 100$

2.8 Effect of the treatments on haematological parameters of rats

An automated haematological analyser was used to examine the haematological profile of the animals (Sysmex XS-1000i). The following parameters were measured: haemoglobin, white blood cell count (WBC), red blood cell count (RBC), haematocrit (HCT), platelets (PLT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and differential leucocyte count (neutrophils, lymphocytes, eosinophils, monocytes).

2.9 Effect of treatments on serum biochemistry of rats

To extract serum, blood samples in activated gel tubes were allowed to coagulate before being centrifuged at 1500 × g for 15 min. The following biochemical parameters were measured: glucose, creatinine, urea, sodium, potassium, chloride, total protein, albumin, globulin, total bilirubin (TBil), direct bilirubin (DBil), alanine transaminase (ALT), aspartate transaminase (AST), triglyceride (TG), and cholesterol (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol) using an automated biochemistry analyzer (ADVIA 2400, Siemens Healthcare) and analyzer specific reagents Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bilirubin were used to compute percentage protection (TBIL). The following formula was used to compute percentage protection for liver functional indices [12]: $\begin{array}{r}\mathrm{Percent}\mathrm{Protection}=\frac{\begin{array}{c}(\mathrm{Values}\mathrm{of}\mathrm{Toxin}\mathrm{Control}\\ -\mathrm{Values}\mathrm{of}\mathrm{Test}\mathrm{Group})\times 100\end{array}}{\begin{array}{c}(\mathrm{Values}\mathrm{of}\mathrm{Toxin}\mathrm{Control}\\ -\mathrm{Values}\mathrm{of}\mathrm{Normal}\mathrm{Control})\end{array}}\end{array}$

2.10 Antioxidant and oxidative stress biomarkers

After homogenizing the liver (1.0 g) in 10 mL phosphate buffer (0.1 M, pH 7.4) and centrifuging at 10,000 rpm for 20 min at 4°C, the post-mitochondrial fraction was extracted (PMF). The supernatant was collected and utilized for antioxidant assays such as reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) using methods described by Beutler et al. [13], Jollow et al. [14] and Oyagbemi et al. [15], with modifications as described by Genfi et al. [16].

2.11 Inflammatory cytokine assay

Using homogenized liver samples, measurement of anti-inflammatory proteins of specific cytokines, namely Tumour Necrosis Factor-alpha (TNF-α), Transforming Growth Factor-Beta1 (TGF-β1), Nuclear Factor Kappa B (NFkβ), Cyclo-oxygenase 2 (COX-2), Interleukin – 17 (IL-17) and Interleukin – 23 (IL-23) were tested using appropriate ELISA kits. TNF-α, NFkβ, and COX-2 ELISA kits were obtained from RayBio – tech, Inc. (Norcross, GA, USA) and TGF-β1, IL-17 and IL-23 kits were from Aviscera Bioscience, Inc. (Santa Clara, CA, USA).

2.12 Histopathological studies

Each slice of liver tissue was immersed in a 10% buffered formaldehyde solution to guarantee adequate 24 h fixing. Following that, the samples were rinsed with double distilled water and dehydrated using methanol and ethanol dilutions. Specimens were purified in xylene before being embedded in paraffin for 24 h at 56^oC in an oven. Using a sled microtome, paraffin beeswax tissue blocks with a thickness of 4 m were produced for sectioning. For analysis, the tissue slices were mounted on glass slides, deparaffinized, and counterstained with haematoxylin and eosin. The stained tissues were viewed under a microscope at X40 (BX-51) magnification and shot using a charge-couple device (CCD) camera.

2.13 Statistical analyses

All statistical data were analysed using GraphPad Prism for Windows version 9.0 (GraphPad Software, San Diego, CA, USA) with a one-way analysis of variance test, and presented as means ± SEM. The Tukey Multiple Comparison Test was employed and, significant differences between groups were considered as p < 0.05.

3. Results

3.1 Effect of the treatments on rat body weight

Table 2 shows the impact of HSE and MSE on the body weight of normal and CCl₄ – induced rats. On the 7th day of the experiment, there was a considerable loss in body weight in the CCl₄ group when compared to the control group. Co-treated of HSE or MSE with CCl₄ resulted in weight increase when compared with CCl₄ only treatment group.

Table 2. Effect of treatment on body weight of rats.

Treatment	% Change in Body Weight, D7
Normal	3.12 ± 0.52
Extracts only
100 mg HSE only	4.25 ± 0.88
250 mg HSE only	3.97 ± 2.14
500 mg HSE only	4.19 ± 1.19
100 mg MSE only	5.00 ± 1.26
250 mg MSE only	4.85 ± 2.75
500 mg MSE only	5.79 ± 1.89
CCl4
CCl4 only	−2.83 ± 1.00
CCl4 + Sily	2.96 ± 2.57
100 mg HSE + CCl4	3.70 ± 1.89
250 mg HSE + CCl4	3.05 ± 0.31
500 mg HSE + CCl4	2.23 ± 1.16
100 mg MSE + CCl4	1.00 ± 0.00
250 mg MSE + CCl4	2.41 ± 0.64
500 mg MSE + CCl4	0.44 ± 1.46

3.2 Effect of the treatments on relative organ weight

Figure 1 shows the effect of HSE and MSE on the relative weight of liver and kidney samples of CCl₄ treated rats. Administration of CCl₄ resulted in a significant increase in the relative liver (p < 0.05–0.001) and kidney (p < 0.5–0.001) weight of the CCl₄ treated group when compared to the normal (untreated or control) group. CCl₄ and HSE or MSE co-treatment groups did not show any significant changes compared to the normal group, at all doses for both liver and kidney samples, indicating the hepatoprotective effects of HSE and MSE against the CCl4 insult.

Figure 1. Effect of Treatment on Relative Organ Weight. Values are expressed as mean ± SEM (n = 5). Statistical significance “a” at p < 0.05–0.001 compared to Normal; “b” at p < 0.05–0.001 when compared to CCl₄-treated group only.

3.3 Effect of treatment on haematological parameters

The effect of HSE and MSE treatment on hematological indices of CCl₄ – intoxicated rats is shown in Table 3. The hematological indices were measured to assess the effect of the CCl₄, HSE, and MSE on the rats. Generally, the treatments did not cause significant changes to the haematological indices of the rats.

Table 3. Effect of treatment on haematological parameters.

Parameter	Normal	HSE only (100 mg)	HSE only (250 mg)	HSE only (500 mg)	MSE only (100 mg)	MSE only (250 mg)	MSE only (500 mg)	CCl4 only	CCl4+ Sily	CCl4+ HSE (100 mg)	CCl4+ HSE (250 mg)	CCl4+ HSE (500 mg)	CCl4+ MSE (100 mg)	CCl4+ MSE (250 mg)	CCl4+ MSE (500 mg)
WBCx10^3/µL	7.33 ± 0.74	7.83 ± 0.75	7.43 ± 1.16	7.40 ± 0.05	9.40 ± 0.55	8.90 ± 2.30	9.76 ± 1.01	7.2 ± 0.52	7.65 ± 1.46	7.77 ± 2.69	7.20 ± 1.45	7.10 ± 1.15	7.36 ± 0.54	8.07 ± 1.29	8.53 ± 0.50
RBCx10^6/µL	7.78 ± 0.73	7.41 ± 0.15	7.11 ± .49	7.88 ± 0.37	8.02 ± 0.51	8.67 ± 0.34	8.93 ± 0.49	7.76 ± 0.49	7.53 ± 0.49	7.72 ± 0.51	7.99 ± 0.09	7.39 ± 0.65	8.54 ± 0.24	8.26 ± 0.27	8.23 ± 0.50
HGB g/dL	13.57 ± 0.84	13.47 ± 0.35	12.80 ± 0.85	13.77 ± 0.18	14.47 ± 0.68	15.97 ± 0.29	15.80 ± 0.65	11.12 ± 0.73	11.53 ± 2.17	11.90 ± 0.60	13.9 ± 0.26	11.2 ± 0.66	13.33 ± 1.04	13.40 ± 0.55	14.60 ± 0.66
HCT%	54.60 ± 2.62	58.43 ± 0.81	62.07 ± 2.59	59.20 ± 1.25	55.27 ± 2.00	58.53 ± 1.55	59.40 ± 1.75	55.77 ± 3.33	54.86 ± 15.81	59.03 ± 2.24	61.10 ± 1.30	57.56 ± 3.80	60.03 ± 1.20	61.56 ± 1.46	60.87 ± 2.57
MCV fL	70.80 ± 3.58	69.47 ± 0.49	68.27 ± 1.09	66.80 ± 2.05	69.16 ± 2.13	67.53 ± 1.32	66.70 ± 1.91	69.27 ± 0.38	71.17 ± 17.98	67.83 ± 1.53	67.97 ± 0.90	68.73 ± 1.28	70.33 ± 0.92	68.50 ± 0.32	67.03 ± 0.85
MCH pg	17.53 ± 0.62	18.37 ± 0.13	18.47 ± 0.26	17.77 ± 0.57	18.10 ± 0.45	18.43 ± 0.38	17.73 ± 0.23	17.70 ± 0.38	17.87 ± 0.62	18.27 ± 0.52	17.67 ± 0.12	18.20 ± 0.61	19.13 ± 0.85	17.73 ± 0.12	18.03 ± 0.26
PLT uL	925.33 ± 52.45	767.67 ± 54.17	842.67 ± 77.55	900.33 ± 87.12	752.33 ± 68.69	1094.67 ± 99.74	916.00 ± 92.15	824.90 ± 84.56	836.33 ± 59.26	760.67 ± 80.32	1004.67 ± 98.33	1066.33 ± 63.25	942.00 ± 77.08	988.67 ± 107.82	811.33 ± 104.65
LYM%	65.67 ± 8.89	59.23 ± 2.61	59.87 ± 4.79	60.70 ± 3.45	60.60 ± 5.06	50.13 ± 6.08	55.13 ± 3.43	54.07 ± 1.21	60.07 ± 3.22	60.9 ± 2.65	54.37 ± 8.97	56.60 ± 4.86	61.26 ± 2.48	59.73 ± 4.16	59.73 ± 4.16
NEUT%	34.34 ± 8.89	40.77 ± 2.61	40.13 ± 4.79	39.30 ± 3.45	39.40 ± 5.06	49.87 ± 6.09	44.87 ± 3.43	39.93 ± 20.07	37.93 ± 3.22	40.10 ± 2.64	45.62 ± 8.97	43.40 ± 4.87	38.73 ± 2.48	40.27 ± 4.16	40.27 ± 4.16
RDW-SD fL	40.17 ± 1.67	39.40 ± 1.49	39.40 ± 1.46	37.56 ± 1.56	39.17 ± 2.18	40.40 ± 2.80	37.83 ± 2.14	40.23 ± 2.11	40.83 ± 0.52	39.27 ± 2.78	41.67 ± 1.36	39.83 ± 1.68	39.86 ± 1.00	38.67 ± 2.09	38.13 ± 0.03
RDW-CV %	17.93 ± 0.68	14.47 ± 1.17	15.90 ± 0.87	14.50 ± 0.36	14.10 ± 0.55	15.86 ± 1.76	14.40 ± 0.45	16.27 ± 1.98	15.67 ± 0.15	16.93 ± 0.81	17.20 ± 0.62	16.27 ± 0.81	16.50 ± 0.31	15.90 ± 1.62	15.13 ± 0.52
PDW fL	8.47 ± 0.23	8.13 ± 0.22	8.43 ± 0.19	8.53 ± 0.24	8.27 ± 0.09	8.30 ± 0.06	8.17 ± 0.17	9.27 ± 0.07	7.97 ± 0.24	8.90 ± 0.30	8.37 ± 0.33	7.90 ± 0.36	8.43 ± 0.18	8.40 ± 0.15	8.47 ± 0.06
MPV fL	7.30 ± 0.06	7.20 ± 0.15	7.10 ± 0.10	7.13 ± 0.14	7.13 ± 0.03	7.07 ± 0.07	7.13 ± 0.12	7.80 ± 0.6	6.90 ± 0.10	7.33 ± 0.76	7.20 ± 0.12	6.93 ± 0.18	7.17 ± 0.9	7.07 ± 0.07	7.23 ± 0.09
P-LCR %	7.90 ± 0.15	7.50 ± 0.67	6.77 ± 0.65	7.30 ± 0.90	7.50 ± 0.56	6.83 ± 0.39	7.17 ± 0.59	8.50 ± 0.06	6.80 ± 0.61	8.63 ± 0.76	7.96 ± 0.26	7.77 ± 0.26	6.93 ± 0.83	7.37 ± 0.50	8.43 ± 0.91
PCT %	0.67 ± 0.04	0.55 ± 0.05	0.59 ± 0.05	0.64 ± 0.08	0.53 ± 0.05	0.77 ± 0.07	0.65 ± 0.07	0.62 ± 0.09	0.68 ± 0.10	0.55 ± 0.07	0.72 ± 0.08	0.74 ± 0.06	0.67 ± 0.06	0.69 ± 0.07	0.58 ± 0.07

Values are expressed as mean ± SEM (n = 5).

3.4 Effect of treatment on biochemical profile

Table 4 shows the effect of treatment on the biochemical profile of rats after CCl₄ treatment. Administration of CCl₄ resulted in a significant increase in most of the biochemical parameters tested when compared to the normal group. Treatment with 250 mg HSE and MSE separately, resulted in significant decreases in most of the parameters.

Table 4. Effect of treatment on biochemical parameters.

Treatment	AST (U/L)	ALP (U/L)	ALT (U/L)	GGT (U/L)	LDH (U/L)	CK (U/L)	TPro (g/L)	Album (g/L)	TBil. (mmol/L)	DBil. (mmol/L)	Trig. (mmol/L)	TCho (mmol/L)	Creat. (µmol/L)	Urea (mmol/L)
Normal	111.61 ± 6.81	42.43 ± 1.62	51.54 ± 5.74	80.02 ± 8.71	207.62 ± 21.50	418.34 ± 32.23	83.34 ± 7.30	65.35 ± 5.02	6.11 ± 0.63	4.32 ± 0.44	0.74 ± 0.14	3.12 ± 0.40	37.05 ± 1.72	5.65 ± 0.33
Extract only
HSE only (100 mg)	136.29 ± 8.45	46.34 ± 3.34	51.12 ± 5.51	84.74 ± 5.72	229.04 ± 42.12	413.34 ± 31.14	88.04 ± 9.01	65.30 ± 4.54	6.44 ± 0.94	4.34 ± 0.83	0.84 ± 0.23	3.53 ± 0.42	42.66 ± 6.41	5.31 ± 0.42
HSE only (250 mg)	114.84 ± 8.31	41.72 ± 2.91	51.73 ± 2.92	80.04 ± 5.73	288.33 ± 45.91	380.00 ± 20.62	83.36 ± 5.40	60.30 ± 3.20	6.32 ± 0.34	3.78 ± 0.84	0.91 ± 0.14	3.84 ± 0.44	35.74 ± 2.63	5.24 ± 0.61
HSE only (500 mg)	126.70 ± 8.92	46.10 ± 2.43	56.10 ± 2.40	81.00 ± 5.9	225.38 ± 69.70	396.32 ± 14.72	81.64 ± 3.85	62.72 ± 4.63	6.25 ± 0.92	4.16 ± 1.04	0.92 ± 0.14	3.71 ± 0.77	41.04 ± 2.11	5.61 ± 0.52
MSE only (100 mg)	126.32 ± 4.50	42.24 ± 3.50	50.77 ± 5.21	82.63 ± 6.62	266.34 ± 55.90	474.33 ± 35.14	87.37 ± 4.74	64.74 ± 6.95	7.10 ± 1.12	4.52 ± 1.04	0.62 ± 0.11	3.80 ± 0.41	34.33 ± 5.95	5.43 ± 0.24
MSE only (250 mg)	123.57 ± 7.25	48.94 ± 2.26	54.30 ± 6.25	76.04 ± 5.64	220.03 ± 40.91	421.04 ± 27.82	86.06 ± 5.02	63.04 ± 5.34	6.30 ± 0.92	3.84 ± 1.10	0.74 ± 0.14	3.56 ± 0.31	30.32 ± 1.55	5.44 ± 0.45
MSE only (500 mg)	112.60 ± 6.21	55.20 ± 4.33	56.04 ± 7.20	75.35 ± 9.91	231.30 ± 40.41	406.02 ± 28.54	81.74 ± 3.81	65.74 ± 6.44	6.03 ± 1.24	4.65 ± 0.44	0.84 ± 0.34	3.30 ± 0.42	38.74 ± 2.36	5.12 ± 0.82
CCl4
CCl4 only	203.56 ± 11.82 ^a	72.54 ± 6.10^a	107.76 ± 5.73 ^a	106.04 ± 7.15 ^a	448.04 ± 51.72 ^a	667.77 ± 35.50 ^a	32.62 ± 8.23 ^a	41.74 ± 6.12 ^a	14.54 ± 1.05 ^a	9.52 ± 0.45 ^a	1.85 ± 0.14 ^a	14.90 ± 0.75 ^a	62.30 ± 3.55^a	10.15 ± 1.03^a
CCl4 + Sily	158.01 ± 8.73 ^ab	47.00 ± 2.35^b	71.70 ± 4.92 ^ab	83.05 ± 4.60^b	211.74 ± 47.53^b	590.34 ± 33.54 ^a	71.37 ± 7.85^b	66.00 ± 3.15^b	6.04 ± 0.61^b	4.10 ± 0.64^b	1.34 ± 0.12^a	5.02 ± 0.45^b	40.61 ± 2.93^b	5.62 ± 0.74 ^b
CCl4 + HSE (100 mg)	158.03 ± 6.70 ^ab	54.74 ± 4.10^b	71.50 ± 4.54 ^ab	69.02 ± 3.21 ^ab	392.75 ± 13.72 ^a	393.04 ± 14.32^bc	56.04 ± 2.56^ab	72.72 ± 4.62^b	11.44 ± 0.43 ^ac	5.72 ± 0.63^b	0.74 ± 0.13 ^b	10.43 ± 1.41 ^ac	47.24 ± 3.35^b	8.13 ± 0.52 ^ac
CCl4 + HSE (250 mg)	119.32 ± 6.94^bc	38.50 ± 4.32^b	69.74 ± 4.32^b	60.75 ± 5.20 ^abc	367.04 ± 32.04 ^a	287.72 ± 21.24 ^abc	52.32 ± 3.85 ^ab	73.05 ± 4.34^b	9.32 ± 0.55^ab	4.34 ± 0.33^b	0.65 ± 0.12 ^b	10.00 ± 0.64 ^ac	43.61 ± 2.11^b	5.12 ± 0.64 ^b
CCl4 + HSE (500 mg)	143.34 ± 12.01 ^ab	44.20 ± 3.43^b	62.90 ± 6.26 ^ab	75.34 ± 4.34^b	425.30 ± 44.13 ^a	413.05 ± 60.93^b	58.03 ± 3.25 ^ab	65.74 ± 3.42^b	10.74 ± 0.65 ^abc	4.65 ± 0.74^b	0.87 ± 0.24 ^b	12.03 ± 1.22 ^ac	46.72 ± 2.05^b	6.11 ± 0.52 ^b
CCl4 + MSE (100 mg)	132.05 ± 11.74^bc	45.43 ± 4.11^b	72.23 ± 4.35 ^ab	72.36 ± 12.63^b	444.04 ± 32.66 ^a	361.72 ± 23.40^b	48.30 ± 2.56 ^ac	77.75 ± 5.01^b	10.02 ± 1.10 ^abc	5.30 ± 0.89 ^b	1.23 ± 0.14^a	9.31 ± 0.75^ab	36.05 ± 3.26^b	6.35 ± 0.35 ^b
CCl4 + MSE (250 mg)	117.20 ± 5.82^bc	39.92 ± 5.72^b	75.94 ± 3.81 ^b	72.05 ± 6.12^b	262.74 ± 26.08^b	331.75 ± 21.24^bc	61.34 ± 5.57 ^ab	73.36 ± 3.51^b	8.13 ± 0.65^b	5.23 ± 0.61 ^b	0.91 ± 0.12 ^b	8.70 ± 1.05^ab	39.71 ± 4.23^b	5.62 ± 0.42 ^b
CCl4 + MSE (500 mg)	131.76 ± 6.04^bc	41.56 ± 2.94^b	90.44 ± 10.01 ^ac	95.03 ± 9.51 ^a	314.32 ± 26.81 ^a	389.36 ± 30.18^bc	54.62 ± 3.52 ^ab	63.05 ± 5.10^b	8.24 ± 0.65^b	5.67 ± 0.35 ^b	1.24 ± 0.24 ^a	9.83 ± 0.84^ab	44.04 ± 2.65^b	5.15 ± 0.63 ^b

Values are expressed as mean ± SEM (n = 5). Superscript “a” represent significance at p < 0.05-0.0001 when compared to the Normal group. Superscript “b” represents significance at p < 0.05-0.001, when compared with CCl₄, treated group only, “c” at p < 0.05-0.001 compared to Sily + CCl₄ treated group.

3.5 Percentage hepatoprotection

Figure 2 shows the percent protection of silymarin, HSE, and MSE against CCl₄. HSE treatment at 250 mg/kg b.wt recorded the highest liver protection against the CCl₄ – induced hepatotoxicity. Principal indicators used in the calculation included ALT, AST, GGT, and TBIL.

Figure 2. Percent liver protection of HSE, MSE, and Silymarin against CCl₄-induced hepatotoxicity. Each bar is a mean of percentage protection of each indicator of liver protection.

3.6 Effect of treatment on electrolytes

The effects of HSE and MSE treatment on electrolytes are shown in Table 5. CCl₄ administration resulted in a non-significant difference in all electrolytes indices.

Table 5. Effect of HSE on electrolytes.

Treatment	K (mmol/L)	Na (mmol/L)	Cl (mmol/L)
Normal	19.95 ± 0.84	194.48 ± 3.87	145.20 ± 1.18
Extracts only
100 mg HSE only	18.27 ± 1.34	182.00 ± 13.28	146.67 ± 3.53
250 mg HSE only	20.17 ± 0.53	196.33 ± 10.71	138.67 ± 4.63
500 mg HSE only	21.37 ± 1.43	211. 67 ± 5.68	147.00 ± 3.05
100 mg MSE only	21.66 ± 3.38	191.67 ± 8.41	141.33 ± 2.40
250 mg MSE only	21.00 ± 1.53	184.00 ± 5.51	14162 ± 0.88
500 mg MSE only	23.33 ± 0.89	181.67 ± 3.84	144.00 ± 3.61
CCl4
CCl4 only	21.23 ± 0.58	188.00 ± 5.29	143.33 ± 2.91
CCl4 + Sily	21.20 ± 0.42	182.33 ± 3.18	141.67 ± 4.06
100 mg HSE + CCl4	20.67 ± 0.89	178.00 ± 4.35	139.37 ± 9.45
250 mg HSE + CCl4	21.67 ± 1.85	178.00 ± 4.62	138.67 ± 2.03
500 mg HSE + CCl4	21.67 ± 2.18	174.00 ± 3.46	134.67 ± 3.71
100 mg MSE + CCl4	20.63 ± 0.68	180.23 ± 2.72	132.00 ± 2.31
250 mg MSE + CCl4	22.13 ± 1.12	182.32 ± 2.03	135.00 ± 3.21
500 mg MSE + CCl4	22.00 ± 1.52	183.62 ± 1.76	141.33 ± 2.40

3.7 Effect of treatments on inflammation

The effect of treatment on inflammatory cytokine is shown in Figure 3. In the CCl₄ treatment group, TNF-α, TGF-β1, NF kβ1, COX-2, IL-17, and IL-23 levels were significantly higher (p < 0.05–0.001) than the Normal control. Groups treated with extracts only showed no significant difference in TNF-α, TGF-β1, NF kβ, COX-2, IL-17, and IL-23 levels when compared with the normal group.

Figure 3. Effect of HSE on Inflammation. Values are expressed as mean ± SEM (n = 5). “a” represent significant different from Normal at P < 0.05–0.001; “b” represent significant different from CCl₄ at P < 0.05–0.001.

3.8 Effect of treatments on hepatic antioxidants and oxidative stress markers

Table 6 shows the effects of HSE and MSE on antioxidants and hepatic oxidative stress. Administration of CCl₄ resulted in a significant increase in hepatic oxidative stress biomarkers of the CCl₄ treated group when compared to the Normal group. However, groups co-treated with CCl₄ and HSE or MSE at all doses recorded significant changes (p < 0.05) in antioxidants and hepatic oxidative stress biomarkers when compared to the CCl₄ group.

Table 6. Effect of treatments on antioxidants and hepatic oxidative stress.

Treatment	SOD (U/mg Protein)	CAT (U/mg Protein)	GSH (µmol/mg Protein)	MPO (U/mg Protein)	MDA (mmol/mg Protein)
Normal	6.37 ± 0.88	89.67 ± 11.55	549.67 ± 32.43^b	121.67 ± 8.45^b	105.00 ± 4.93^b
Extract only
HSE only (100 mg)	6.13 ± 0.78	102.33 ± 11.35	551.00 ± 75.37	92.33 ± 8.42	123.33 ± 4.67
HSE only (250 mg)	5.60 ± 0.98	117.00 ± 11.72	560.00 ± 26.50	86.67 ± 13.13	105.33 ± 4.81
HSE only (500 mg)	5.37 ± 0.59	128.00 ± 8.08^a	499.67 ± 56.09	104.33 ± 14.07	105.00 ± 13.65
MSE only (100 mg)	8.30 ± 0.91	78.67 ± 5.21	554.67 ± 23.25	108.67 ± 10.41	111.00 ± 7.00
MSE only (250 mg)	8.67 ± 1.45	89.67 ± 12.99	602.33 ± 10.59	105.33 ± 19.24	82.33 ± 9.13
MSE only (500 mg)	12.67 ± 2.03^a	78.00 ± 12.77	612.67 ± 15.89	101.00 ± 12.42	78.67 ± 4.67
Carbon Tetrachloride (CCl4)
CCl4 only	1.23 ± 0.38^a	26.00 ± 4.93^a	239.00 ± 16.19^a	699.67 ± 30.36^a	299.67 ± 10.41^a
CCl4 + Sily	4.27 ± 0.46^a,b	52.00 ± 7.57^a,b	525.67 ± 50.00^b	516.67 ± 9.33^a	153.33 ± 4.81^a,b
CCl4 + HSE (100 mg)	6.90 ± 0.92^b	96.00 ± 5.77^b	412.67 ± 20.08^b	589.67 ± 39.31^a	152.33 ± 5.36^a,b
CCl4 + HSE (250 mg)	6.63 ± 1.34^b	101.67 ± 12.17^b	489.00 ± 51.64^b	533.67 ± 73.63^a	130.67 ± 8.29^a,b
CCl4 + HSE (500 mg)	7.17 ± 1.27^b	113.67 ± 4.48^b	404.67 ± 24.37^b	505.33 ± 30.82^a	139.33 ± 2.96^a,b
CCl4 + MSE (100 mg)	7.17 ± 1.0^b	60.67 ± 5.04^a,b	474.67 ± 24.13^b	491.67 ± 44.12^a	124.00 ± 4.16^b
CCl4 + MSE (250 mg)	8.33 ± 2.03^b	68.67 ± 4.67^a,b	507.33 ± 4.06^b	502.00 ± 58.79^a	108.00 ± 6.56^b
CCl4 + MSE (500 mg)	10.67 ± 2.60^a,b	65.33 ± 3.28^a,b	551.67 ± 5.69^b	532.67 ± 60.95^a	116.33 ± 6.12^b

Values are expressed as mean ± SEM (n = 5). Superscript “a” represent significance at p < 0.05-0.001 when compared to Normal. Superscript “b” represents significance at p < 0.05-0.001 when compared to CCl₄.

3.9 Effect of treatment on liver histology

Figure 4 depicts liver micrographs from both normal and treated rats. While there were no unfavourable morphological changes in the Normal group, the CCl₄ group had significant hepatocellular abnormalities. In animals treated with 250 mg/kg bwt of either HSE or MSE and Silymarin, these changes were reverted to near to normal architecture.

Figure 4. Photomicrographs of liver from male rats administered orally for 7 days Photomicrograph (A) – (G) shows normal hepatocytes with no observable lesion (F0); (H) showed severe destruction of the hepatic architecture with portal fibrous strands. (black arrows, F3); (I) shows no observable hepatic degeneration (F0); (J) shows mild degeneration with sinusoidal dilation (black arrows, F1); (K) recorded no observable lesion (F0); (L) shows mild degeneration with sinusoidal dilation (F1); (M) shows mild necrosis with centrilobular atrophy of hepatocytes (black arrows, F2); (N) shows normal hepatocytes with no observable lesion (F0); (O) shows degeneration of hepatocytes with moderate necrosis (black arrows, F2). (H&E x 400). From Metavir system; level of fibrosis from F0 to F3.

4 Discussion

Liver diseases are a global health concern that causes a high rate of mortality and morbidity in more than two million people yearly [17]. Regardless of the recent improvement in orthodox medicine, effective medicine with hepatoprotective properties and little effects is not easily available. Exposing toxins or chemicals to the liver cells is one good approach hepatoprotective drugs can be developed [18]. In this work, the hepatoprotective property of HSE and MSE were investigated against liver toxicants carbon tetrachloride. Silymarin, a common chemical known to possess a hepatoprotective role [19] was used as a standard drug in the study.

Extensive research has not yet been carried out on P. laxiflora. Hence information on the plant is scanty, The hepatoprotective ability of hydroethanolic (HSE) and methanolic (MSE) extracts of P. laxiflora at different doses (100, 250, and 500 mg/kg doses) on CCl₄ treated rats, was comparable to silymarin. The difference in organ weight is mostly considered to be a benchmark to investigate the toxic effect of toxins substances was used in the experiment [20]. HSE and MSE at all doses resulted in increased body weight gain following acute liver toxicity, however, CCl₄ treated group recorded a significant reduction in body weight on termination of the experiment as expected. Loss of appetite has a direct effect on body weight loss because it causes a disturbance in the carbohydrate, protein, and fat metabolism of an individual [17]. The significant decrease in body weight was attributed to the toxic effect of CCl₄ while the reported increases following administration of HSE and MSE were probably due to the averting effect of CCl₄ by the extracts. Increases in weight of rat livers and kidneys were possibly a result of enlargement occurring from the toxicity of CCl₄, which resulted in the infiltration of chemicals such as fatty acids and glycerol into the hepatic and nephron cells [21]. Earlier reports have indicated that treatment with CCl₄ caused an increase in the weight of organs such as the liver and kidney [4–6,18].

The concentration of biomarkers such as AST, ALT, ALP, and GGT in blood serum is usually used to determine the normal functioning and cellular integrity of the liver [4–6].

In earlier studies, a group of animals treated with CCl₄ only showed a significant increase in AST, ALT, ALP, and GGT (at P < 0.05–0.0001) when compared to the normal or control group. Thus, the presence of CCl₄ was suggested to destroy the liver cells resulting in the leaking of liver enzymes into the blood serum [4]. Co-treatment with standard drug, silymarin and HSE or MSE was able to significantly decrease the levels of AST, ALT, GGT, and ALP, thus bringing them to near normal range. In this work, the treatment with CCl₄ resulted in increased activity of these enzymes. This may lead to the destruction of the hepatocytes and adverse effects on membrane permeability. These findings lend support to a previously reported study [5]. Co-treatment with the extracts and silymarin resulted in a reduction of these enzymes concentration to near-normal levels, implying that the extracts have a beneficial role in ameliorating liver damage. The current study also revealed a significant increase in lactate dehydrogenase (LDH) (P < 0.01) and creatine kinase (P < 0.001) in the CCl₄ treated group when compared to the normal group. Previous studies by Kotoh and colleagues [20] indicated that free radicals generated by CCl₄ caused oxidative damage to the liver cell membrane resulting in increased serum concentration of LDH and CK. CCl₄ toxicity resulted in a similar outcome in the brain of treated rats. Pre-treatment with HSE or MSE and silymarin were however able to significantly reduce the level of LDH and CK to near normal at 250 mg/kg b.wt. dose of HSE, showing the greatest significant reduction when compared to the normal levels. This suggests the protective effects of this extract. CCl₄ treatment also resulted in a significant increase in blood creatinine and urea levels, thus suggesting possible kidney damage. The combined reduction in albumin, creatinine, and urea level occurring in rats treated with the extracts suggested the protective role of HSE and MSE. Cholesterol plays a key role in the cell membrane and hormone formation. The level of total cholesterol mostly depends on the balance between the body’s rate of production and the rate of absorption from the diet. Hence liver damage may arise when there is a high level of cholesterol accumulation. In this study, significant increases in total cholesterol and triglyceride levels by the CCl₄ insults were reversed to normal by pre-treatment with either HSE or MSE. These results suggest that the extracts are promising hepatoprotective agents against CCl₄ toxicity, with 250 mg/kg b.wt. of HSE treatment dose having the highest activity.

The CCl₄ treatment could also cause the generation of free radicals which ultimately results in rapid haemolysis of red blood cells. Excessive breakdown of RBC can also lead to hyper-bilirubin or a high accumulation of bilirubin in the liver beyond the capacity of the liver cells.

Breakdown of haem results in the formation of a yellowish pigment called bilirubin. Hyper-bilirubin mostly occurs when there is a deficiency in erythropoiesis [22].

Hyper-bilirubin is a build-up of bilirubin due to the endangered bile duct or damaged liver cells, thus resulting in the liver’s inability to excrete a normal amount of bilirubin through the bile duct. A significant increase (P < 0.5–0.0001) in both total bilirubin and direct bilirubin was observed after CCl₄ treatment. On the other hand, a significant reduction was reported after co-treatment with silymarin and HSE or MSE at all doses.

At all doses, the ability of HSE and MSE to prevent an increase in organ weight, body weight, and liver enzymes in blood plasma caused by CCl₄ was an important marker of the hepatoprotective potential of these extracts. Further investigation on the protection by 250 mg/kg b.wt. HSE and MSE are warranted. Free radicals generated from the treatment with CCl₄ result in lipid peroxidation which eventually damages the liver cells [4]. Therefore, hepatoprotective drugs must possess antioxidant properties to facilitate the scavenging of free radicals produced by hepatotoxicants. The HSE and MSE contain known antioxidants such as flavonoids, alkaloids, and Phenols [7].

In the current study, significant changes in levels of CAT, GSH, SOD, MPO, and MDA after CCl₄ treatment, thus suggesting a potential liver injury caused by oxidative stress. Superoxide dismutase plays a key role in the antioxidant defence system. A significant increase in SOD activity in the hepatocytes was caused by P. aculeate, thus preventing oxidative damage to the liver which is usually caused by the high production of free radicals [23]. In the present study, the activity of SOD was significantly reduced in the liver of the CCl₄ treated group compared with the control group. Co-treatment of CCl₄ with HSE or MSE at all doses normalized the activity of the SOD enzyme, thus resulting in a significant difference when compared to the CCl₄ group and a non-significant difference when compared to the normal group. Treatment with CCl₄ also led to a significant reduction in CAT protein concentration than the normal group. Subsequent, treatment of the rats with HSE or MSE at all doses resulted in a significant increase (P < 0.05–0.001) in CAT level compared to the CCl₄ treated group. Elevated levels of MPO and MDA (which are considered indices of hepatic infiltration) were observed in the CCl₄ treatment group, which was also associated with lipid peroxidation enhancement. Pre-treatment with HSE and MSE showed a significant reduction in a hepatic infiltration index (MPO and MDA) when compared with the CCl₄ treatment group. The reduction of MPO and MDA by HSE and MSE could partially be due to the antioxidant property of the extracts.

Histological examination of liver microstructure corroborated the protective effect of HSE and MSE extracts at 250 mg/kg bwt. Histology of CCl₄-treated liver showed massive necrosis, also the micrographs of the HSE and MSE – treated groups at 100 and 500 mg/kg b.wt. corroborated the low percentage of protection, however HSE and MSE co-treated groups at 250 mg/kg b.wt. respectively showed that they prevented cellular organelle degeneration by reversing the damage. In accordance according to the biochemistry results, MSE treatment at 500 mg/kg b.wt also provided the least protection for hepatocytes and hepatic enzymes as shown in the liver histology.

There is also proof that free radical-mediated oxidative stress is accountable for CCl₄ toxicity [7,24]. We proposed that the pathway of defence by HSE and MSE may be due to the inhibition of reactive oxygen species (ROS) through the suppression of cytochrome P450 from activating CCl₄ to its reactive form (trichloromethyl free radical) and highly reactive metabolites such trichloromethyl peroxyl radical.

The capacity of HSE and MSE to mitigate CCl₄-induced hepatic damage may be partly attributed to its ability to decreased phosphorylation of Erk1/2 expression while subjected to oxidative stress [25]. The toxicity of CCl₄ can also relate to pro-inflammatory cytokine generation in an experimental model using rats [26]. This was concomitant with the high increased expression of TNF-α, NF kβ, COX-2, TGF-β, IL-17, and IL-23 after CCl₄ administration. These pro-inflammatory cytokines play important roles in the formation of liver injury, especially in the activation of hepatic stellate cells (HSCs), which is an essential initiator for liver cell damage [27]. This means that NF kβ signalling is involved in CCl₄-induced hepatotoxicity. Oxidative stress is one of the activators of NF kβ which is released from cytosolic inhibitors, transported to the nucleus, and eventually to NF kβ target genes for expression [28]. Cyclooxygenase-2 (COX-2) is an isoenzyme that is regulated by growth factors and many inflammatory cytokines such as IL-6 and TNF; hence, it can be upregulated during inflammation [29].

The cytokines, IL-17 and IL-23, which are involved in inflammation, immune response, muscle injury, and haematopoiesis control, were highly expressed in response to CCl₄ [30]. Treatment with HSE or MSE and silymarin blocked the NF kβ inflammatory cascade (slightly due to the brevity of the experiment), resulting in suppression of the pro-inflammatory cytokines COX-2, IL-17, and IL-23.

Thus, in this study, co-treatment of CCl₄ with HSE or MSE at all doses resulted in a significant reduction in the levels of pro-inflammatory cytokines. The hepatoprotective effect of P. laxiflora was partly due to the presence of phytochemicals such as alkaloids, flavonoids, cyanogenic glycosides, triterpenes, saponins, polyphenols, tannins, and phytosterol, as reported in our previous studies.

5. Conclusion

This study found that the plant extracts HSE and MSE have the potential to stabilize free radicals produced by CCl₄, reduce oxidative stress and inhibit the NF kβ inflammatory pathway through COX-2, IL-17, and IL-23 downregulation. The hepatoprotective effects of the extracts were similar to that of the known standard silymarin. This study found that the free radical – scavenging activity, the enhancement of the antioxidant enzyme system, and the mild inhibition of the NF kB inflammatory pathway are all potential pathways of hepatoprotection by P. laxiflora with HSE at 250 mg/kg b.wt giving the highest protection potential. One key limitation was the inadequate of funds which prevent us from investigating into other molecular markers such as vascular endothelia growth factor and alpha smooth muscle actin. Future work may probable look into exploring other molecular markers.

Disclosure statement

No potential conflict of interest was reported by the author(s).