Hepatoprotective Effects of Tetrapleura tetraptera (Schum. & Thonn.) Taub. Stem Bark Extracts in Drug-induced Toxicities in Rats

Kusi Stephen; Christopher Larbie; Frederick Sarfo-Antwi

Hepatoprotective Effects of Tetrapleura tetraptera (Schum. & Thonn.) Taub. Stem Bark Extracts in Drug-induced Toxicities in Rats

by Kusi Stephen^1,2, Christopher Larbie^1*, Frederick Sarfo-Antwi³

¹Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

²Department of Science, Prempeh College, Kumasi, Ghana

³Department of Biochemistry and Molecular Medicine, School of Medicine, University for Development Studies, Tamale, Ghana

*Corresponding Authors: Christopher Larbie, Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Received Date: 25 August 2023

Accepted Date: 15 September 2023

Published Date: 2 October 2023

Citation: Kusi, S., Larbie C, Sarfo-Antwi F (2023) Hepatoprotective Effects of Tetrapleura tetraptera (Schum. & Thonn.) Taub. Stem Bark Extracts in Drug-induced Toxicities in Rats. Curr Res Cmpl Alt Med 7: 201. https://doi.org/10.29011/2577-2201.100201

Abstract

Hepatotoxicity is an issue of global concern because of numerous liver-related deaths over the years. In addition, the conventional hepatoprotective and hepatocurative drugs used are more expensive, not easily accessible, and sometimes have serious adverse effects. These reasons account for why many have resorted to traditional plant medicines. This study evaluated the hepatoprotective effects of aqueous and hydroethanolic stem bark extracts of Tetrapleura tetraptera, a commonly used traditional medicinal plant, in paracetamol and carbon tetrachloride-induced hepatotoxicities in rats. Pulverized stem bark samples of 500 g each were soaked in 1000 mL boiled water and 1000 mL 50% hydroethanol respectively for 24 hours and filtered. Filtrates were concentrated over a water bath and designated as aqueous stem bark extract (ASE) and hydroethanolic stem bark extract (HSE). Extracts were evaluated for LD₅₀value, phytochemical constituents, free radical scavenging effects and heavy metals. Fifty-seven male Wistar albino rats weighing 120-180 g were divided into nineteen groups (n=3/group). The LD₅₀ for both extracts were found to be above 5000 mg/kg b.wt. This study showed HSE had higher amounts of phytochemicals than ASE. Again, this study also showed that HSE and ASE had greater levels of liver percentage protection than Silymarin. Furthermore, the liver antioxidant profile study proved that HSE and ASE could protect the liver. These results suggest that ASE and HSE, through their antioxidant activities, have hepatoprotective and hepatocurative effects against paracetamol and carbon tetrachloride-induced hepatotoxicities in rats and could be developed as potential liver protective agents.

Keywords: Hepatotoxicity, Hepatoprotective, Hepatocurative, Antioxidant, Tetrapleura tetraptera

Introduction

The liver is the largest visceral organ that regulates different bodily functions, such as metabolism, secretion, storage and detoxification [1]. It is continuously and variedly exposed to elevated amounts of xenobiotics like drugs and environmental pollutants which enter the body and are delivered to it via the hepatic portal vein after intestinal absorption.

Xenobiotics undergo biotransformation which is mostly a detoxification process, the many oxidative reactions in this process produce reactive metabolites that are more toxic than the parent compound and can induce lesions within the liver [2]. These metabolites bind covalently to cellular macromolecules resulting in increased lipid peroxidation, reduced cellular ATP production and oxidative damage to cause liver diseases.

Naturally, the liver protects itself from harmful reactive metabolites, and ultimately liver diseases through its antioxidant system. However, it is overpowered by the excessive production of reactive metabolites resulting in liver diseases. Several reports indicate a continuous increase in the incidence rate of hepatic diseases [3,4] despite the advances in modern medicine. Arhoghro et al.,[5] reported that conventional drugs used in disease treatment are inadequate and have serious adverse effects. These reasons may partly be responsible for the high incidence rate of liver diseases. Globally, many people, especially those from developing countries, have resorted to medicinal plants since they consider these plants to be natural, have less toxicity, simple accessibility and simple assimilation in the body and are economical [6-8].

Tetrapleura tetraptera (Schum. & Thonn.) Taub. (Family: Leguminosae) is a well-known and indispensable medicinal plant with proven efficacy in the management of numerous health conditions [9,10]. It is known in English as Aidan or Aridan and among the Akans of Ghana as ‘prεkesε’. Ethnomedically, all parts of T. tetraptera have been reported to be used for medicinal purposes [11]. The stem bark is used to treat cardiovascular disorders, gastrointestinal disorders, malaria and other fevers, low body immunity, reproductive disorders, cancer of the breast and uterus, general body pains and weakness, dental disorders Kemigisha et al., [11] and wound Tsala et al., [12]. The leaves are used to treat convulsions and epilepsy, malaria and other fevers, reproductive disorders, general body pains and weakness. The fruits have various applications and are extensively used for the management of an array of human ailments including cardiovascular disorders, hypertension, arthritis, diabetes mellitus, epilepsy, convulsion, leprosy, inflammation and rheumatoid pains, gastrointestinal disorders, malaria and other fevers, asthma and chest pain, low body immunity, flu and colds, reproductive disorders, cancer of the breast and uterus, skin disorders of newly born babies, wounds and burns, back and general body pains and weakness, dental disorders, spherocytosis, and iron deficiency anaemia [13-15,11].

The ability of T. tetraptera to heal diseases and provide for significant normal body functioning has been attributed to its high number of essential phytochemicals and nutrients including flavonoids, triterpenoid, glycoside, tannins, alkaloids, saponins, steroids and phenolic compounds, potassium, iron, calcium, zinc and phosphorus [16,17]. These phytochemicals and minerals have been identified in the stem bark, leaves and fruits of T. tetraptera in varying amounts and have been proven pharmacologically to be anti-inflammatory Onda et al., [18] and antioxidant activities [19,10]. Though literature revealed that anti-inflammatory and antioxidant properties are vital for hepatoprotective activity, no information was found regarding the hepatoprotective activity of stem bark extracts of T. tetraptera. This present study, therefore, sought to evaluate the hepatoprotective effects of aqueous and hydroethanolic stem bark extracts of Tetrapleura tetraptera in drug-induced toxicities in rats.

Materials and Methods

Materials and reagents

Legalon Flordis, Australia, supplied all solvents used in the extraction process. All chemicals and reagents used in the study were of analytical grade.

Plant collection and identification

The fresh stem bark sample of the T. tetraptera plant was obtained in September 2019, from healthy, fully grown plants in Ghana’s Ashanti Region (latitude 6^o 58^’12.0” N and longitude 1^o38”23.9” W). The sample was authenticated at the Department of Herbal Medicine, KNUST, with a voucher specimen (KNUST/ HM1/2019/SB013) deposited at the Herbarium for reference purposes.

Preparation of Extracts from the Stem Bark

The stem bark was washed, dried in the shade, and milled into powder. The aqueous and hydroethanolic extractions were prepared using 500 g of pulverized stem bark each in 1000 mL boiled water and 50% hydro-ethanol respectively, at room temperature with continued shaking for 24 hours. The extracts were then filtered through cotton wool and concentrated under 60^∘C pressure using a rotary evaporator (Buchi R205, Switzerland) and designated as aqueous stem bark extract (ASE) and hydroethanolic stem bark extract (HSE).

Qualitative Phytochemical Screening

Phytochemical constituents of Tetrapleura tetraptera stem bark extracts were determined using standard methods as described by Evans [20]. Alkaloids, flavonoids, terpenoids, saponins, coumarins, reducing sugars, sterols, and tannins were the phytochemicals examined.

In vitro Antioxidant Activity

Total Phenol Content (TPC) Determination

The total phenolic content was determined by the FolinCiocalteu method Singleton et al., [21] with some modifications. About 50 μL of each sample (extracts and gallic acid [0.00, 0.25, 0.50, 0.75 and 1 mM]) was mixed with 3 mL of distilled water and 250 μL of the Folin-Ciocalteu reagent. The mixture was made to stand for a minimum of 5 minutes and 750 μL of 20% Na₂CO₃ was added. The mixture was then vigorously vortexed for 2 minutes followed by 30 minutes of incubation at room temperature. The absorbance of the solutions was measured at 760 nm using a UVVIS spectrophotometer (Shimadzu Corporation, 1201, Kyoto, Japan). Each sample was tested in triplicate and a calibration curve with six data points for gallic acid was obtained. The results were compared with a gallic acid calibration curve. The total phenolic content was expressed as Gallic Acid Equivalent (GAE) (mg GAE/g extract).

Total Tannins Content (TTC)

The quantity of tannins in extracts was determined by the Folin–Ciocalteu method with slight modifications [22]. One hundred microlitres of extract was added to 5 mL of distilled water, 500 μL of Folin–Ciocalteu reagent and 1 mL of 35% Na₂CO₃ solution. The mixture was shaken well and kept at room temperature for 30 min and absorbance was measured against the blank at 725 nm. The total tannin content was determined from the calibration curve of gallic acid. Results were expressed in terms of mg GAE/100 g. All determinations were performed in triplicates.

Estimation of Total Flavonoids Content (TFC)

The total flavonoid content in the samples was estimated by the aluminium chloride colourimetric assay method Zhishen et al., [23] using quercetin as the standard. An aliquot of 500 μL of each extract was mixed with 1.5 mL of 99.9% ethanol, 100 μL of 1 M potassium acetate, 100 μL of 10% aluminium chloride and 3 mL of distilled water. The mixture was vigorously shaken and left to stand in the dark at room temperature. The resulting mixture samples were incubated for 30 min at room temperature and the corresponding absorbance was measured at 415 nm. The total flavonoid content of each extract was determined from the standard curve and the results were expressed as mg QE/100 g.

DPPH Scavenging Activity

The DPPH free radical scavenging ability of the extracts was evaluated as described by Oliveira et al. [24] with some modifications as previously described by Donkor et al. [25]. Two hundred microlitres of each extract were added to 3.8 mL of 0.004% DPPH methanolic solution and incubated at room temperature in the dark for 60 minutes. The absorbance was measured at 517 nm against a methanol blank.

Heavy Metals

Each extract and a raw sample of mass 1 g were weighed into a correspondingly labelled Pyrex digestion tube. Each sample was wet digested using a mixture of 1 mL water, 2 mL HCl, 5 mL 1:1 HNO₃:HClO₄, and 2 mL of H₂SO₄ for 20 minutes on a heating block at 150°C. The digest was allowed to cool, transferred to a volumetric flask, and diluted with distilled water to 50 mL. Heavy metals including lead, copper, nickel, zinc, and iron were analysed using an atomic absorption spectrometer (Varian AA 240FS) with a long path air acetylene burner and cathode lamp for respective metals.

Toxicity assessment of Tetrapleura tetraptera stem bark extract

Fifty-seven maleWistar albino rats weighing between 120 and 180 grams were obtained from the Animal House of the School of Medical Sciences, University of Ghana, Legon, Accra. They were then kept in propylene cages with wood shavings bedding and allowed to acclimatize for 14 days. Throughout the study, the animals were maintained under standard environmental conditions (room temperature and 12-hour light), fed with standard rat feed (Agricare, Kumasi, Ghana) and given distilled water ad libitum except for the overnight fast before the start of the study and termination. Rats were weighed and identified on their tails using a permanent marker to allow for efficient dosing.

Acute oral toxicity

The OECD guidelines 425 [26] was adopted in the acute oral toxicity study of HSE and ASE using six male Wistar albino rats in two groups (120–180g; n=3/group). ASE and HSE at a maximum dose of 5000 mg/kg body weight (b.wt.) were dissolved in normal saline and administered by gavage (p.o.) Following the administration of extracts, the animals were observed for signs of toxicity and mortality for the first critical 4 hours and thereafter daily for 7 days [26,27]. This was used to guide the selection of two doses (100, and 250 mg/kg b.wt) for the hepatoprotective studies.

Evaluation of Hepatoprotective Activity

Hepatotoxicity was induced in animals using carbon tetrachloride (CCl₄; Sigma Aldrich) and acetaminophen (Para, Trade Winds Pharmaceuticals, Kumasi, Ghana). Carbon tetrachloride only treated group received oral administration of 1.0 mL/kg b.wt CCl₄ (1/1 v/v olive oil, intraperitoneal [i.p.]) for two successive days, day 2 and day 3. For the Para (Paracetamol IV Pfizer Solution) only treated group, rats received 500 mg/kg b.wt p.o. daily for 7 days [28].

Table 1: Experimental grouping and treatment of Animals.

S/N.	Group	Treatment
1	Normal (control)	Rats were orally treated with 1.0 mL/kg distilled water each day for 7 days.
		Extract only
2	100 mg/kg HSE only	Rats were orally treated daily with only 100mg/kg HSE for 7 days.
3	250 mg/kg HSE only	Rats were orally treated daily with only 250 mg/kg HSE for 7 days.
4	100 mg/kg ASE only	Rats were orally treated daily with only 100 mg/kg ASE for 7 days.
5	250 mg/kg ASE only	Rats were orally treated daily with only 250 mg/kg ASE for 7 days.
		Treatment with CCl₄
6	CCl₄	CCl₄ i.p (1 ml/kg b.wt, 1:1 v/v olive oil) received on 2^nd and 3^rd day.
7	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 2^nd and 3^rd day
8	100 mg/kg 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 2^nd and 3^rd day.
9	250 mg/kg HSE + CCl₄	250 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 2^nd and 3^rd day.
10	100 mg/kg ASE + CCl₄	100 mg/kg ASE (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 2^nd and 3^rd day.
11	250 mg/kg ASE + CCl₄	250 mg/kg ASE (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 2^nd and 3^rd day.
		Treatment with Para
12	Para only	500 mg/kg Para (i.p) for 7 consecutive days
13	Para+Sily	Silymarin p.o (100 mg/kg per day) received for seven days plus 500 mg/kg Para (i.p) for 7 consecutive days
14	100 mg/kg HSE + Para	100 mg/kg HSE (per day, p.o.) for seven days plus 500 mg/kg Para (i.p) for 7 consecutive days
15	250 mg/kg HSE + Para	250 mg/kg HSE (per day, p.o.) for seven days plus 500 mg/kg Para (i.p) for 7 consecutive days
16	100 mg/kg ASE + Para	100 mg/kg ASE (per day, p.o.) for seven days plus 500 mg/kg Para (i.p) for 7 consecutive days
17	250 mg/kg ASE + Para	250 mg/kg ASE (per day, p.o.) for seven days plus 500 mg/kg Para (i.p) for 7 consecutive days

Experimental design

On termination, the animals were fasted overnight, and sacrificed under cervical decapitation. The animals were quickly slit at the neck, and blood samples were collected into gel-activated tubes for biochemical analyses and EDTA tubes for haematological analyses. The animals were dissected, and their livers excised, freed of fat, washed with normal buffered saline and blotted with clean tissue paper.

Relative liver weights

The relative organ weights of the liver (ROW) of the rats for each group were determined as:

ROW = AOW; Absolute Organ Weight of excised liver

Effect of treatments on haematological parameters of rats

The evaluation of haematological parameters was done using Sysmex XS-1000i Auto Haematology Analyser (Japan). Parameters measured include white blood cell (WBC) count, red blood cell (RBC) count, haemoglobin (HGB) concentration, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet (PLT), lymphocyte (LYM), neutrophils (NEUT), red blood cell distribution width standard deviation (RDW-SD), red blood cell distribution width coefficient of variation (RDW-CV), platelet distribution width (PDW), mean platelet volume (MPV), platelet-large cell rate (P-LCR) and platelet crit (PCT).

Effect of treatments on serum biochemistry of rats

Blood samples in activated gel tubes were allowed to clot and centrifuged at 1500 g for 15 min to obtain blood serum. Biochemical analysis was carried out using Selectra E (Vital Scientific) and reagents from ELITECH. Parameters determined were aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (Y-GT), Total protein (TP), albumin (ALB), globulin (Glo), total bilirubin (T-bil), direct bilirubin (D-bil), and indirect bilirubin (I-Bil).

Percentage Protection

Percentage protection was calculated based on the principal indicators of liver protection; AST, ALT, GGT and TBil using the formula below:

Percentage Protection = ×100%

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 minutes 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. [29], Jollow et al. [30] and Oyagbemi et al. [31], with modifications as described by Genfi et al. [28].

Statistical Analysis

The results were assessed by Two Way ANOVA followed by Tukey’s multiple comparisons test and analysed using GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA). Data were presented as mean ± SEM at a 5% significance level.

Results

Preliminary Phytochemical screening

As shown in Table 2, preliminary phytochemical screening of Raw powder, HSE, and ASE revealed the presence of major phytochemical groups.

Table 2: Phytochemical constituents of T. tetraptera stem bark extracts.

Phytochemical	HSE	ASE	RAW
Saponins	+	+	+
Coumarins	+	+	+
Reducing Sugars	+	+	+
Tannins	+	+	+
Alkaloids	+	-	+
Terpenoids	+	-	+
Sterols Flavonoids	- +	- +	+ +
Key: present (+);		Absent (-)

The total phenolic, tannin and flavonoid contents of the ASE and HSE are shown in Figure 1. HSE had the highest amounts of total phenols, flavonoids, and tannins. In addition, the radical scavenging activity levels obtained for HSE, ASE and gallic acid standard were 87.98%, 19.23% and 94.15% respectively.

Figure 1: The total phenolic, tannin and flavonoid contents of the stem bark extracts of T. tetraptera.

Heavy Metal Content

Table 3 shows the heavy metals content including copper (Cu), zinc (Zn), nickel (Ni), lead (Pb) and iron (Fe) in raw stem samples and extracts.

Table 3: Heavy Metal Content of Stem Bark and Extracts of T. Tetraptera.

Samples			Concentration (mg/L)
Samples	Cu	Fe	Ni	Pb	Zn
RAW	0.25 ± 0.02	1.87 ± 0.02	B D L	1.33 ± 0.02	0.58 ± 0.02
ASE	0.25 ± 0.03	0.78 ± 0.01	B D L	2.63 ± 0.04	0.40 ± 0.02
HSE	0.55 ± 0.05	8.98 ± 0.12	B D L	2.68 ± 0.04	0.49 ± 0.02
Note: BDL means below the detection limit of 0.00001 mg/L

Toxicity Profile Studies

No death was reported in the acute toxicity investigations within the first 4 hours of continuous monitoring and after the first 24 hours for both extracts. No fatal side effects were found. Hair, skin, eyes, and nose morphological features appeared normal. Drooling, diarrhoea, lethargy, or any other odd behaviour were absent. According to an estimate of the LD₅₀ of 5000 mg/kg, it is safe.

Effects of Treatments on the Relative Liver Weights

There were significant increases in the relative organ weights among rats treated with CCl₄ in both HSE and ASE models when compared with the normal group. However, treatment with Para only recorded no significant changes as shown in Figure 2.

Figure 2: Effects of treatments on Relative Liver Weights. Values are presented as mean ± SEM, n = 3; a = significant difference from normal, b = significant difference between CCl₄only group, c = significant difference from Para only group; p<0.05 – 0.0001.

Effect of treatment on haematological parameters

Tables 4 and 5 show treatment effects of CCl₄, Para, HSE and ASE on haematological parameters.

Table 4: Effect of Stem Bark Hydroethanolic Extract on Haematological Profile.

Parameters	Normal	100 mg only	250 mg only	CCl₄only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	100 mg + CCl₄	250 mg+ CCl₄
WBC (x10³/ uL)	10.67±0.94	13.13±1.64	11.33±2.42	6.93±0.58	10.50±0.35	8.90±1.96	9.03±0.07	9.47±1.63	11.23±0.94	11.33±0.30	111.57±1.71
RBC(x10⁶/ uL)	6.63±0.75	8.49±0.12	7.59±0.30	9.35±0.37	8.81±0.38	8.85±0.89	7.52±0.20	7.46±0.09	7.58±0.06	7.35±0.18	7.34±0.58
HGB (g/dL)	12.97±0.70	13.73±0.26	12.57±0.38	16.60±0.51	15.20±0.56	16.23±1.21	14.37±0.33	13.77±0.18	14.03±0.27	13.47±0.18	13.73±0.82
HCT (%)	43.10±5.80	48.23±1.13	47.87±2.25	57.80±1.40	53.03±1.87	56.00±4.83	47.67±0.46	47.43±0.67	48.00±1.01	48.80±1.18	48.00±2.69
MCV (fL)	64.73±2.09	56.83±1.05	63.10±1.91	61.93±1.13	60.23±0.49	63.47±1.77	63.47±1.39	63.77±1.43	63.33±1.16	66.73±0.03	64.63±1.52
MCH (pg)	19.87±1.37	16.20±0.15	16.60±0.55	17.77±0.37	17.27±0.13	18.40±0.46	19.10±0.17	18.47±0.24	18.53±0.29	18.47±0.20	18.33±0.35
MCHC (g/ dL)	30.83±2.70	28.47±0.32	26.30±0.50	28.67±0.24	28.63±0.09	29.03±0.71	30.13±0.50	28.97±0.33	29.23±0.07	27.70±0.32	28.57±0.09
PLT (x10⁶/ uL)	961.33± 137.42	1001.67± 55.32	679.00± 184.91^a	1846.33± 97.36^a	834.00± 70.12	681.00± 130.02^ab	795.00± 34.95^a	1368.33± 40.04^c	1197.33± 193.04^ac	1280.00± 69.86^ab	1462.67± 201.24^ab
LYM (%)	69.40±7.54	81.80±6.51	79.97±6.17	52.73±2.30	78.83±3.02	68.87±3.04	79.10±3.04	73.87±1.85	65.10±3.71	67.07±1.82	68.80±5.44
NEUT (%)	30.60±7.54	18.20±6.51	20.03±6.17	47.27±2.30	21.17±3.02	31.13±3.04	20.90±3.04	26.13±1.85	34.90±3.71	32.67±1.79	30.67±5.43
LYM (x10³/ uL)	7.57±1.39	10.83±1.95	9.00±2.01	3.67±0.33	8.30±0.52	6.23±1.64	7.13±0.28	6.93±0.99	7.27±0.55	7.67±0.03	7.67±0.55
NEUT(x10³/ uL)	3.10±0.47	2.30±0.75	2.33±0.79	3.27±0.32	2.20±0.25	2.67±0.32	1.90±0.26	2.53±0.64	3.97±0.64	3.73±0.29	5.27±0.29
RDW-SD (fL)	51.80±3.27	37.90±0.87	45.03±2.53	38.27±3.05	24.57 ±10.60	34.80 ±2.23	32.87±1.07	38.33±1.43	36.13±0.80	38.50±1.01	35.57±0.80
RDW-CV (%)	25.77±4.39	20.03±0.20	20.43±1.62	18.30±0.85	16.10±0.26	14.53±1.47	15.20±2.54	16.10±0.44	15.47±0.65	15.03±0.38	14.53±0.03
PDW (fL)	11.77±0.55	9.30±0.38	10.30±0.68	9.53±0.34	8.40±0.15	9.33±0.34	8.53±0.12	8.10±0.21	7.93±0.19	9.20±0.52	9.20±0.06
MPV (fL)	8.07±0.32	7.70±0.21	8.10±0.47	7.90±0.21	7.17±0.12	7.77±0.09	7.23±0.09	6.90±0.06	6.83±0.15	7.60±0.35	7.70±0.06
P-LCR (%)	15.07±1.41	11.37±1.27	13.40±3.19	10.97±1.17	6.33±0.41	10.07±0.79	6.90±0.50	5.90±0.21	5.57±0.72	8.90±1.97	10.00±0.29
PCT (%)	0.77±0.11	1.42±0.04	0.94±0.20	0.54±0.14	0.60±0.06	0.53±0.11	0.57±0.03	0.95±0.04	0.82±0.12	0.96±0.01	1.08±0.16
a = significant difference between normal and others, b = significant difference between CCl₄ only, c = significant difference between para only, p<0.05 - 0.0001.

Table 5: Effect of Stem Bark Aqueous Extract on Haematological Profile.

Parameters	Normal	100 mg only	250 mg only	CCl₄only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	250 mg + CCl₄
WBC (x10³/uL)	10.67±0.94	10.33±2.33	8.53±1.80	6.93±0.58	10.50±0.35	8.90±1.96	9.03±0.07	9.70±0.06	11.87±0.70	10.33±0.81
RBC(x10⁶/uL)	6.63±0.75	7.91±0.37	8.11±0.30	9.35±0.37	8.81±0.38	8.85±0.89	7.52±0.20	7.09±0.27	7.36±0.12	7.49±0.05
HGB (g/dL)	12.97±0.70	14.03±0.38	14.60±0.20	16.60±0.51	15.20±0.56	16.23±1.21	14.37±0.33	13.30±0.31	13.57±0.35	13.47±0.18
HCT (%)	43.10±5.80	51.57±2.17	51.27±0.95	57.80±1.40	53.03±1.87	56.00±4.83	47.67±0.46	47.03±0.20	46.57±1.26	48.43±0.67
MCV (fL)	64.73±2.09	65.23±0.94	63.33±1.92	61.93±1.13	60.23±0.49	63.47±1.77	63.47±1.39	66.50±2.32	63.27±1.35	65.80±0.06
MCH (pg)	19.87±1.37	17.77±0.38	18.07±0.61	17.77±0.37	17.27±0.13	18.40±0.46	19.10±0.17	19.10±0.29	18.43±0.38	18.10±0.06
MCHC (g/dL)	30.83±2.70	27.27±0.43	28.47±0.15	28.67±0.24	28.63±0.09	29.03±0.71	30.13±0.50	28.37±0.50	29.13±0.03	27.53±0.18
PLT (x10⁶/uL)	961.33± 137.42	1052.33± 139.85	1079.00± 68.51^a	1846.33± 97.36^a	834.00± 70.12^a	681.00± 130.02^ab	795.00± 34.95^a	1241.67± 83.91^ac	1231.00± 63.26^ac	1423.33± 95.05^ab
LYM (%)	69.40±7.54	73.03±7.70	64.63±6.82	52.73±2.30	78.83±3.02	68.87±3.04	79.10±3.04	65.33±3.18	68.03±1.66	75.93±1.74
NEUT (%)	30.60±7.54	26.97±7.70	35.37±6.82	47.27±2.30	21.17±3.02	31.13±3.04	20.90±3.04	34.33±3.18	31.97±1.66	24.43±1.75
LYM (x10³/uL)	7.57±1.39	7.20±0.75	5.30±0.49	3.67±0.33	8.30±0.52	6.23±1.64	7.13±0.28	6.30±0.31	8.07±0.64	7.87±0.43
NEUT(x10³/uL)	3.10±0.47	3.13±1.58	3.23±1.33	3.27±0.32	2.20±0.25	2.67±0.32	1.90±0.26	3.27±0.33	3.80±0.10	2.67±0.39
RDW-SD (fL)	51.80±3.27	36.30±2.40	35.33±1.35	38.27±3.05	24.57±10.60	34.80±2.23	32.87±1.07	38.03±1.03	34.73±0.35	38.70±0.21
RDW-CV (%)	25.77±4.39	14.70±1.46	14.17±0.61	18.30±0.85	16.10±0.26	14.53±1.47	15.20±2.54	15.17±0.81	14.80±1.01	16.13±0.29
PDW (fL)	11.77±0.55	8.93±0.32	8.33±0.42	9.53±0.34	8.40±0.15	9.33±0.34	8.53±0.12	7.70±0.26	8.03±0.09	8.60±0.35
MPV (fL)	8.07±0.32	7.47±0.19	7.07±0.19	7.90±0.21	7.17±0.12	7.77±0.09	7.23±0.09	6.77±0.09	6.93±0.12	7.23±0.26
P-LCR (%)	15.07±1.41	8.40±1.15	6.67±0.89	10.97±1.17	6.33±0.41	10.07±0.79	6.90±0.50	5.03±0.41	5.97±0.66	7.47±1.22
PCT (%)	0.77±0.11	0.79±0.11	0.76±0.04	0.54±0.14	0.60±0.06	0.53±0.11	0.57±0.03	0.84±0.07	0.85±0.03	1.03±0.03
a = significant difference between normal and others, b = significant difference between CCl₄ only, c = significant difference between para only, p<0.05 - 0.0001.

Effect of treatment on biochemical parameters

Tables 6 and 7 show the treatment effects of CCl₄, Para, HSE and ASE on biochemical parameters. Table 6 reveals significant increases in ALT, AST, GGT and Total Bilirubin levels for the CCl₄-only and Para-only treated groups when compared with the normal. However, HSE treatment resulted in some significant decreases compared with the CCl₄-only treatment. Table 7 also shows that significant increases in ALT, AST, GGT and Total Bilirubin following CCl₄-only and Para-only treatments were restored to near-normal levels with ASE treatment

Table 6: Effect of Stem Bark Hydroethanolic Extract on Biochemical Parameters.

Parameter	Normal	100 mg only	250 mg only	CCl4 Only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	100 mg + CCl₄	250 mg + CCl₄
ALT (U/L)	54.70±0.42	56.77±0.75	52.57±3.39	636.60± 47.73^a	109.43± 11.15^a	326.90± 46.89^abc	111.53± 13.62	42.83 ±4.42	54.83 ±5.94	75.90 ±0.90^b	68.65 ±1.05^b
AST (U/L)	302.47± 8.26	295.87± 1.32	299.50± 6.07	976.57± 73.86^a	383.67± 2.43^a	723.53± 43.40^ab	289.50± 30.77^c	298.40± 8.16^c	305.90± 25.85^c	291.30± 0.80^b	381.90± 60.50^b
AST/ALT	5.53±0.12	5.21±0.06	5.76±0.48	1.57±0.13	3.57±0.35	2.33±0.39	2.63±0.23	7.12±0.75	5.78±1.02	3.85±0.05	5.55±0.75
ALP (U/L)	232.20± 5.20	196.30± 49.41	329.60± 37.80^a	268.20± 15.08	286.93± 80.36	306.80± 31.48^a	261.27± 10.11	222.13± 14.05^c	197.77± 26.60^c	259.70± 1.60	203.20± 50.00^c
γ-GT (U/L)	1.23±0.07	1.20±0.35	1.13±0.12	10.20±1.48^a	10.07±0.94^a	2.73±0.07	2.50±0.51^c	3.33±0.48^c	5.10±0.12^c	8.55±1.25^a	3.80±0.10^b
T-bil (µmol/L)	0.59±0.13	0.92±0.10	0.95±0.12	6.41±0.14^a	2.40±0.31^a	2.80±0.11^a	2.35±0.15^a	11.62 ±1.67^a	5.87±1.28^a	8.85±0.74^a	8.41±0.59
D-bil (µmol/L)	0.18±0.05	0.45±0.07	0.54±0.09	1.60±0.37^a	1.08±0.17	1.22±0.20	0.89±0.03	1.60±0.87	0.79±0.28	1.04±0.31	1.32±0.16
I-Bil (µmol/L)	0.41±0.15	0.47±0.07	0.41±0.04	4.81±0.49^a	1.31±0.40	1.57±0.10	1.45±0.13	10.03±1.87	5.07±1.01	7.80±0.40	7.05±0.75
TP (g/L)	66.33±2.76	67.07±3.01	57.27±2.98	94.13±2.38^a	94.47±2.05^a	96.30±6.21^a	104.77± 2.80^a	67.10±2.00^c	70.23±2.44^c	66.90±1.00^b	67.80±0.00^b
ALB(g/L)	33.37±1.71	31.37±0.29	28.53±2.17	40.07±0.87	39.67±1.63	35.70±3.18	45.43±1.18	34.13±0.88	34.43±1.02	33.30±1.40	35.30±0.50
Glo(g/L)	32.97±1.27	35.70±3.27	28.73±4.49	54.07±1.54	54.80±3.40	60.60±3.76	59.33±2.02	32.97±2.73	35.80±1.53	33.60±0.40	32.50±0.50
A/G	1.00±0.06	0.90±0.10	1.07±0.23	0.73±0.03	0.73±0.07	0.60±0.06	0.77±0.03	1.06±0.12	0.96±0.02	0.99±0.05	1.09±0.03
		a = significant difference between normal and others, b = significant difference between CCl4 only, c = significant difference between para only, p<0.05 -0.0001

Table 7: Effect of Stem Bark Aqueous Extract on Biochemical Parameters.

Parameters	Normal	100 mg only	250 mg only	CCl4 only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	250 mg + CCl₄
ALT (U/L)	54.70±0.42	64.90±5.51	63.90±6.84	636.60± 47.73^a	109.43± 11.15^a	326.90± 46.89^abc	111.53± 13.62^b	78.70±4.10^c	61.87±3.90^c	70.00±5.20^b
AST (U/L)	302.47± 8.26	284.97± 23.54	312.20± 33.28	976.57± 73.86^a	383.67± 2.43^a	723.53± 43.40^ab	289.50± 30.77^c	299.70± 37.70^c	294.57± 32.37^c	266.80± 0.90^b
AST/ALT	5.53±0.12	4.40±0.46	4.89±0.14	1.57±0.13	3.57±0.35	2.33±0.39	2.63±0.23	3.80±0.30	4.73±0.25	3.83±0.27
ALP (U/L)	232.20± 5.20	289.20± 120.57	233.20± 11125.65	268.20± 15.08	286.93± 80.36	306.80± 31.48^a	261.27± 10.11	270.60± 60.50	183.20± 11.89^c	297.80± 20.30
γGT (U/L)	1.23±0.07	4.47±1.07	3.57±0.44	10.20±1.48^a	10.07±0.94^a	2.73±0.07^b	2.50±0.51^c	4.75±0.05^c	4.70±0.35^c	4.25±0.35^b
T-bil (µmol/L)	0.59±0.13	1.53±0.38	1.64±0.90	6.41±0.14^a	2.40±0.31^a	2.80±0.11^a	2.35±0.15^a	2.29±0.40^a	2.91±1.23^a	2.05±0.78^a
D-bil (µmol/L)	0.18±0.05	1.37±0.06^a	1.01±0.16^a	1.60±0.37^a	1.08±0.17^a	1.22±0.20^a	0.89±0.03^a	0.47±0.01^a	0.44±0.05^a	0.66±0.00^a
I-Bil	0.41±0.15	0.16±0.06	0.63±0.08	4.81±0.49^a	1.31±0.40^a	1.57±0.10^a	1.45±0.13^a	1.80±0.30^a	2.47±0.20^a	2.47±0.80^a
TP (g/L)	66.33±2.76	71.93±4.68	74.30±1.50	94.13±2.38^a	94.47±2.05^a	96.30±6.21^a	104.77±2.80^a	75.15±0.35	70.43±1.51	79.10±7.30
ALB(g/L)	33.37±1.71	32.33±1.23	36.03±1.19	40.07±0.87	39.67±1.63	35.70±3.18	45.43±1.18	35.00±0.30	35.37±1.42	35.65±2.55
Glo(g/L)	32.97±1.27	39.60±5.90	38.27±0.38	54.07±1.54	54.80±3.40	60.60±3.76	59.33±2.02	40.15±0.05	35.07±2.70	43.45±4.75
A/G	1.00±0.06	0.86±0.14	0.94±0.02	0.73±0.03	0.73±0.07	0.60±0.06	0.77±0.03	0.87±0.01	1.03±0.12	0.82±0.03
	a	= significant difference between normal and others, b = significant difference between CCl4 only, c = significant difference between para only, p<0.05 -0.000							1.

Percentage Protection of the Stem Bark Extracts

Treatments with HSE and ASE showed protection against Para and CCl₄, comparable to that of standard silymarin (Figure 3). The HSE showed the highest protection level (80.70 %) against CCl₄group at 250 mg; the same dose was protective against Para at 44.59%. This was followed by the Sily + Para group (49.49%). For the ASE, the 250 mg treatment showed high protection against CCl4 (63.7%) and Para (58.84%).

Figure 3: Percentage Protection of HSE and ASE. Values are presented as mean ± SEM, n = 3; a = significant difference from normal, b = significant difference between CCl₄ only group, c = significant difference from Para only group; p<0.05 – 0.0001.

Effect of treatments on Antioxidant and Oxidative stress biomarkers

Tables 8 and 9 show the effect of treatments on antioxidant and oxidative stress biomarkers.

The CCl₄-only treatment group caused significant decreases in GPX and GSH but significant increases in GST and MDA when these were compared with the normal (Tables 8 and 9). However, co-treatments of CCl₄with HSE and ASE caused significant increases in GPX and GSH to near-normal levels while causing decreases in MDA, also to near-normal levels. Para-only treatment also resulted in significant decreases in GPX and GSH levels above Normal but was significantly increased to near-normalcy by co-treatments with HSE and ASE.

Table 8: Effect of Stem Bark Hydroethanolic Extract on Oxidative stress parameters.

	Normal	100 mg only	250 mg only	CCl₄only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	100 mg + CCl₄	250 mg + CCl₄
GPX	58.36 ±4.16	57.87 ±1.94	59.96 ±2.79	41.30 ±1.49^a	41.80 ±2.61^a	40.60 ±0.90^a	41.94 ±2.38^a	55.77 ±2.97^c	50.13 ±1.72^c	52.40 ±1.91^b	55.76 ±2.22^b
GSH	172.94 ±12.68	120.64 ±7.07^a	186.33 ±17.49	133.31 ±15.84^a	141.15 ±7.93^a	154.12 ±11.93^a	184.83 ±18.01	127.12 ±17.99^a	195.32 ±9.91^c	197.58 ±3.72^c	152.86 ±20.42^b
GST	0.07±0.02	0.07±0.01	0.13±0.03	0.26±0.03^a	0.09±0.03	0.18±0.03	0.18±0.04	0.13±0.04	0.06±0.02	0.19±0.06	0.00±0.00
SOD	7.37±0.50	5.54±0.39	7.73±0.65	6.16±0.60	6.29±0.49	7.08±0.56	8.79±0.86	7.57±0.28	9.55±0.57^ac	10.42±0.49^b	8.31±0.88
H2O2	29.98 ±1.43	60.43 ±10.26^a	57.83 ±7.11^a	40.40 ±4.53	36.27 ±4.96	34.36 ±1.37	47.38 ±2.74c	25.68 ±2.63	24.95 ±1.70	23.58±0.70	27.12 ±2.18
MDA	1.45±0.36	0.93±0.06	1.31±0.19	2.57±1.14	1.91±0.72	0.68±0.06	1.30±0.13	1.88±0.74	1.06±0.21	0.52±0.03	0.61±0.03
a = significant difference between normal and others, b = significant difference between CCl₄ only, c = significant difference between Para only, p<0.05 -0.0001.

Table 9: Effect of Stem Bark Aqueous Extract on Oxidative stress parameters.

	Normal	100 mg only	250 mg only	CCl₄only	Para only	Sily + CCl₄	Sily + Para	100 mg + Para	250 mg + Para	250 mg + CCl₄
GPX	58.36 ±4.16	59.46 ±2.40	60.45±1.77	41.30 ±1.49^a	41.80 ±2.61^a	40.60 ±0.90^a	41.94 ±2.38^a	52.24 ±1.51^c	60.49 ±2.58^c	63.47 ±5.66^b
GSH	172.94 ±12.68	127.81± 15.41^a	128.14 ±11.96^a	133.31 ±15.84^a	141.15 ±7.93^a	154.12 ±11.93	184.83 ±18.01^c	162.84 ±10.83	130.64 ±16.14^a	137.29 ±16.88^a
GST	0.07±0.02	0.07±0.02	0.11±0.03	0.26±0.03^a	0.09±0.03	0.18±0.03	0.18±0.04	0.10±0.01	0.09±0.01	0.11±0.01
SOD	7.37±0.50	7.11±0.62	6.96 ±0.44	6.16±0.60	6.29±0.49	7.08±0.56	8.79±0.86	8.80±0.70	6.38±0.82	7.63±0.41
H2O2	29.98± 1.43	34.77 ±4.90	32.93 ±1.74	40.40 ±4.53	36.27 ±4.96	34.36 ±1.37	47.38± 2.74^a	28.31 ±3.10	34.39 ±1.73	32.95 ±3.28
MDA	1.45±0.36	0.60±0.07	0.56±0.04	1.57±0.30	1.91±0.72	0.68±0.06	1.30±0.13	0.57±0.04	0.63±0.06	0.59±0.05
		a = significant difference between normal and others, b = significant difference between CCl₄ only, c = significant difference between para only, p<0.05 -0.000.

Discussion

Drugs like paracetamol and carbon tetrachloride induce varied degrees of injury to the liver, which is the primary organ for xenobiotic processing. To protect the liver against these injuries, medicinal plants, and other materials with hepatoprotective properties are utilized. The hepatoprotective effect of aqueous (ASE) and hydroethanolic (HSE) stem bark extracts of Tetrapleura tetraptera, a medicinal plant, was tested in rats against liver injury induced by paracetamol and carbon tetrachloride.

Preliminary phytochemical analysis showed the presence of saponins, coumarins, reducing sugars, tannins, and flavonoids in both ASE and HSE. In addition, the HSE had terpenoids and alkaloids. These findings are consistent with the results of Larbie et al., [10], concerning their aqueous and hydroethanolic stem bark extracts of T. tetraptera. Saponins, coumarins, tannins, terpenoids and flavonoids are strong antioxidants and account for the observed levels of DPPH radical scavenging activity, percentage protection and antioxidant activity of HSE and ASE in this present study. These are confirmed by Reiter [32], who reported that antioxidants scavenge free radicals and help to protect cells and tissues from damage and diseases. These phytochemical antioxidants found in HSE and ASE may also contribute to their levels of hepatoprotection against Para and CCl₄. This is by the suggestion by Gupta et al., [33] that extracts with antioxidants may have hepatoprotection through the elimination of deleterious effects of toxic metabolites from paracetamol or CCl₄.

Furthermore, quantitative analyses revealed the HSE to have higher phenol, tannins, and flavonoids than the ASE. These findings indicate that 50% hydroethanol, being an organic solvent, is better at extracting these phytochemicals which are also organic. This may account for HSE having a greater hepatoprotection in albino rats in this study than ASE. It is therefore not surprising the HSE had a higher DPPH scavenging activity than the ASE. This is confirmed by Koma et al. [34] who stated that phenols donate hydrogen ions to react with reactive oxygen and nitrogen species. Phenols, tannins and flavonoids are powerful antioxidants and according to Parr and Bolwell [35] and Cos et al., [36], they can chelate metal ions involved in the production of free radicals and also inhibit some enzymes involved in the generation of radicals. The HSE’s DPPH radical inhibition level (87.98%) is comparable to the level (28.74% to 85.26%) obtained by Famobuwa et al. [37] in a similar DPPH scavenging activity by the ethanolic stem bark extract of T. tetraptera, where they concluded that the extract’s efficacy in some of their bioactivities could be attributed to its favourable antioxidant potential.

The quantities of Zn, Cu and Fe were all found to be below the tolerable upper intake (TUIL) limits (mg/L) of 40, 10 and 45 respectively for powdered samples and both extracts [38]. This suggests that both extracts and powdered samples are safe for consumption. Copper is a vital trace element that prevents damage to cells due to its antioxidant action and as a component of many enzymes; it helps in the production of energy from carbohydrates, protein, and fat [39]. It is essential for iron utilization, and melanin production Ullah et al., [40] as well as for the formation of bone, connective tissues, and red blood cells [41]. Zinc, a very useful medicinal trace element for the human body, offers a natural protective mechanism against viruses that cause respiratory tract infections [42]. It is extensively used in warfare against HIV and it is believed to delay the integration of HIV in the blood [43]. These may confirm the belief that the plant cures colds, HIV, and other viral infections. Zinc is also described to play another vital role in the development and functioning of the pituitary gland, the gonads and the reproductive organs [44]. This may confirm the use of stem bark extracts in wound healing and as an aphrodisiac. Zinc is involved in the function of enzymes, improves immune function, helps blood clotting, maintains a sense of taste and smell, keeps skin healthy, and facilitates bone formation, wound healing, brain development and normal growth and development processes [41, 45]. Iron is another important trace element needed as a component of haemoglobin and as such said to be involved in the regeneration of lost blood. Iron in haemoglobin binds to oxygen and then transports it from the lungs to all parts of the body. It forms part of many enzymes and is essential for the growth, healing, immune function and synthesis of DNA [41]. Lead, a non-essential metal, was found in both stem bark extracts at levels higher than the RDA of 0.10 mg/L [38]. High amounts of it have been related to liver and kidney damage, reduced gestation in humans, mental retardation, and decreased hearing [46]. Severe anaemia, lasting brain damage, neurological disorders, reproductive issues, reduced intelligence, and a variety of other ailments have all been linked to high levels [47]. The high levels of lead in these extracts therefore suggest a likelihood of exposure to poisoning. Further determination of different plant material sources as well as on the fruits will be required to rule out the species as a source of lead poisoning.

The toxicity profile of a drug or extract is established through acute toxicity studies. The purpose of acute toxicity is to determine the nature and extent of the adverse reactions to a single dose or an overdose of a drug [48]. In this study, a single dose of 5000 mg/kg b.w.t of HSE and ASE of T. tetraptera, based on OCED guidelines [26], proved safe based on the absence of mortality, or signs of toxicity after seven days. This, according to Lorke, [49] is indicative of low toxicity. This suggests that both extracts of T. tetraptera have a very high margin of safety and hence tolerable.

Kharasch [50] reported that organ toxicity occurs when changes in cellular structure or function persist beyond the administration and elimination of a substance. Such cellular structural changes affect organ morphology as well as its function. This is confirmed by Piao et al. [51] who reported that organ weight is one of the most sensitive drug toxicity indicators and its changes often precede morphological changes. This suggests that the increases in liver weight by Para and CCl₄-only treatments are strongly indicative of hepatotoxicity. The death of all rats in the 100 mg + CCl₄stem bark aqueous group further confirmed the strength of CCl₄-induced hepatotoxicity.However, there were significant decreases in liver weight with extract co-treatment in both CCl₄and Para-treated animals, a clear indication of hepatoprotection by hydroethanolic and aqueous stem bark extracts of T. tetraptera based on liver weight.

Observations in haematological assessment of normal and treated animals showed only significant differences in PLT levels for some treatment groups. Platelets control blood loss through blood clotting following vessel damage; they are also crucial to wound healing. A high number of platelets in the blood is known as thrombocytosis and may result in thrombosis while a low level is thrombocytopenia. Thus, the significant increases in PLT levels for CCl₄only, and other treated groups when compared with normal suggests a possible thrombocytosis. Thrombocytosis is caused by chronic inflammatory diseases, haemorrhage, iron deficiency anaemia, etc. [52]. It can be inferred from the above that the hepatotoxic drug (i.e., CCl₄treatment group) induced inflammation responsible for the significantly high platelet levels. The observed decreases in PLT levels following silymarin, HSE and ASE cotreatments suggest these extracts had antioxidant activities against CCl₄. Thrombocytopaenia represents significantly reduced platelet count which may be caused by issues in platelet making or faster destruction of platelets than normal. According to Cheesbrough [52], these may be caused by infections, immune destruction of platelets and drugs like aspirin or deficiency of folate or vitamin B₁₂. In this study, Para caused insignificantly low PLT levels for HSE and ASE when compared to the normal. Thus, Para might have caused some level of liver injury and hence thrombocytopenia. This is confirmed by the report that Para causes thrombocytopenia which correlates with the severity of liver dysfunction and fibrosis in many studies [53-55]. The liver is a major organ that secretes thrombopoietin to stimulate platelet production so damage to the liver means decreased thrombopoietin secretion and hence a low platelet count.

WBC differential parameters namely neutrophils and lymphocytes function in phagocytosis and antibody or toxin production respectively to protect an animal from diseases. Their increases and decreases in the blood are usually an indication of a bacterial infection or tissue or organ damage Cheesbrough [52] caused by drugs among others. In the current study, all extracttreated groups non-significant changes in total WBC and its differential parameters including neutrophils and lymphocytes when compared with the normal group, implying that the treatment groups including the extract were safe.

The degree of liver damage induced by Para and CCl₄intoxication shows parallelism with the serum level of liver biomarkers. In this study, both CCl₄ only and Para only caused significant increases in ALT, AST, GGT and Total Bil levels when compared with the normal group. ALT is a more specific and sensitive liver biomarker than AST [56]. The current study revealed that CCl₄ and Para-only treated groups experienced liver necrosis with significantly high AST levels indicative of excessive levels of liver toxicity though other organs like the heart, muscles and kidneys also produce AST and hence it is not specific to the liver alone [56]. Thus, AST alone cannot confirm liver damage. Though GGT has a low concentration in the liver as compared to other organs Ozer et al., [57], it is described to be a more specific biomarker of cholestasis and other biliary defects [58]. Thus, the significantly high level of GGT in CCl₄ and Para-only treated groups suggests the incidence of cholestasis and other biliary defects caused by the CCl₄ and Para. Increased levels of bilirubin could be due to decreased hepatic clearance and ultimately, cholestasis. This goes on to show that the Para and CCl₄ caused cholestasis and demonstrate the extent of liver damage being associated with Para and CCl₄treatment.

In this current study, significant decreases in ALT levels were observed following HSE and ASE treatments when compared with CCl₄only proved an indication of hepatoprotection. These nearnormal levels in ALT given by HSE and ASE treatment groups mean these extracts were restoring liver function to normal levels. The percentage protection studies confirm the above deductions for the hepatoprotective levels of HSE and ASE.

HSE and ASE had greater levels of protection than Silymarin. HSE had the highest protection level of 80.70% at 250 mg/kg b.wt as against the CCl₄groupcompared with the ASE protection level of 63.7% against the CCl₄groupat 250 mg/kg b.wt. For Para, the HSE level of protection was 44.59% at 250 mg/kg b.wt. while ASE had 58.84% at 250 mg/kg b.wt. Sily only had a protection level of 49.49%.

The mechanism of defence of the liver against free radicals requires GPX, GSH, GST, and SOD. GPX is a selenium cytosolic enzyme that catalyzes the reduction of hydrogen peroxide to water and oxygen and peroxide radicals to alcohol and oxygen [59,60]. GSH is a substrate for glutathione-related enzymes like GST and GPX where it acts to remove free radicals. The hepatotoxic drugs, Para and CCl₄, produced harmful intermediate metabolites to consume GSH. The significant decreases in GPX and GSH levels for CCl₄ and Para-only groups suggest liver damage caused by these two hepatotoxic drugs. However, co-treatment with HSE and ASE treatment gave near to normal but insignificant values of GPX and GSH indicating their ability to reduce oxidative stress caused by the toxicants.

Acetaminophen (paracetamol) has been reported to be safe and non-hepatotoxic in low doses since its metabolism produces a minor amount of the harmful metabolite N-acetyl-pbenzoquinoneimine (NAPQI) which is electrophilic and binds to the sulfhydryl group of reduced glutathione (GSH) to form APAP-GSH, and ultimately excreted in the urine as cysteine and mercapturic acid conjugates (APAP-cys) [61]. However, the continuous administration of a dose of 500 mg/kg b.wt Para for 7 days might have resulted in the production of excess NAPQI. This inflammation caused by excess NAPQI may be responsible for the high levels of PLT in some Para-treated groups. The inflammation of the liver coupled with its damage caused an increase in ALT, AST, GGT, ALP, Tbil and TP in blood circulation. Thus, significantly high levels of ALT, AST, GGT, T-bil and TP are due to inflammation by NAPQI. According to Liu et al. [62], the development of therapeutic agents to shorten the injury phase for inflammatory targets by HMGB1 is a potential clinical strategy to improve liver injury caused by Para. Considering this, it may be said that the phytochemicals such as flavonoids, tannins, phenols, and saponins and heavy metals like Cu and Zn in ASE and HSE increased the antioxidants in rats to prevent oxidative stress and inflammation caused by NAPQI. It has been reported that antioxidant and anti-inflammatory properties are responsible for hepatoprotection [63,64]. Thus, ASE and HSE of T. tetraptera have a hepatoprotective activity where their phytochemical antioxidants act through antiinflammation by mopping up NAPQI.

Carbon tetrachloride toxicity has been described to be dependent on dosage and duration of exposure. According to Knockaert et al. [65], covalent binding and lipid peroxidation result in hepatocyte swelling and may account for the significant increase in the relative liver weight of rats administered CCl₄ only in this study. It is also reported that some products of lipid peroxidation such as 4-HNE may induce inflammations by activating NF-κB pathways which may lead to cell apoptosis [66]. These inflammations are responsible for the significantly high PLT level for CCl₄ only and confirmed by Cheesbrough [52], who stated that inflammations cause high PLT levels. Furthermore, high levels of biochemical parameters are associated with cell damage. Thus, for CCl_4, the cell damage is caused by covalent binding and lipid peroxidation which resulted in the significantly increased levels of ALT, AST, GGT, TP and T-Bil in this present study. ASE and HSE acted against CCl₄-induced liver damage by mediation through its antioxidant phytochemicals. The radicals and the products of lipid peroxidation consume antioxidants to result in oxidative stress. In this study, the significantly low levels of GSH, and GPX for the CCl₄-only group are due to the excessive building of oxidants in liver cells. Oxidative stress resulted in the depletion of GSH required for the proper functioning of GPX and GST. As realized from the above discussion, the covalent binding of carbon tetrachloride radical plays the most significant role in liver damage by lipid peroxidation. This suggests that the prevention of carbon tetrachloride radical production or its covalent binding will prevent liver damage. It is therefore suggested that extracts of T. tetraptera moped up the radicals generated, modulated the synthesis of GSH for effective function of GPX, inhibited related inflammation and further improved liver function.

In conclusion, this study showed that the aqueous and hydroethanolic stem bark extracts of Tetrapleura tetraptera have hepatoprotective effects against paracetamol and carbon tetrachloride (CCl₄) induced hepatotoxicities in rats through their antioxidant phytochemicals and metals mopping up or inhibiting free radicals’ production by Para and CCl₄respectively, modulating the synthesis of reduced glutathione and function of glutathione peroxidase and reducing inflammation.