Antioxidant and drug detoxification potential of aqueous extract of Annona senegalensis leaves in carbon tetrachloride-induced hepatocellular damage

Abstract

Context: Despite the myriad uses of Annona senegalensis Pers. (Annonaceae) leaves in folklore medicine of Nigeria, the basis is yet to be substantiated by scientific investigations.

Objectives:  To investigate the antioxidant (in vitro and in vivo) and drug detoxification potential of aqueous extract of A. senegalensis leaves in CCl₄-induced hepatocellular damage.

Materials and methods: In vitro antioxidant activity of the aqueous extract of A. senegalensis leaves was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), H₂O₂, superoxide ion, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and ferric ion models while in vivo antioxidant and drug detoxification activities of the extract at 100, 200, and 400 mg/kg body weight were done by assaying the levels of enzymic and non-enzymic indices in CCl₄-induced hepatocellular damage.

Results:  The extract at 1 mg/mL scavenged DPPH, H₂O₂, superoxide ion, and ABTS radicals, whereas ferric ion was significantly (P <0.05) reduced. The levels of alkaline and acid phosphatases, alanine and aspartate aminotransferases, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, reduced glutathione, vitamins C and E, glutathione S-transferase, nicotinamide adenine dinucleotide (reduced):Quinone oxidoreductase, uridyl diphosphoglucuronyl transferase, malondialdehyde, and lipid hydroperoxide that decreased in CCl₄ treated animals were significantly attenuated by the extract in a manner similar to the animals treated with the reference drug.

Discussion and conclusion: The ability of the aqueous extract of A. senegalensis leaves to scavenge free radicals in vitro and reversal of CCl₄-induced hepatocellular damage in rats suggest antioxidant and drug detoxification activities. Overall, this study has justified the rationale behind some of the medicinal uses of the plant in folklore medicine of Nigeria.

Keywords::

• Annona senegalensis
• Annonaceae
• antioxidant
• carbon tetrachloride
• drug detoxification
• hepatocellular damage
• radical scavenger

Introduction

Carbon tetrachloride (CCl₄) is a hepatotoxic solvent that produces several biochemical effects on animal organs. Hepatocellular toxicity of CCl₄ requires activation by the liver microsomal mixed function oxidase (CYP 2E1) to produce trichloromethyl radical (CCl₃^•) (Albano et al., 1982). In the presence of oxygen, however, CCl₃. forms highly reactive trichloromethyl peroxyl radicals (CCl₃O^•) which oxidizes membrane polyunsaturated fatty acids via lipid peroxidation (Slater, 1982). Antioxidative and free radical scavenging mechanism plays an important role in the protection against CCl₄-induced hepatic damage (Xiong et al., 1998).

Several agents such as N,N'-diphenyl-p-phenylenediamine, α-tocopherol (vitamin E) and superoxide dismutase have been found to decrease or prevent CCl₄ toxicity in animals. Again, reduced glutathione has also been implicated to play a role in detoxifying the toxic metabolites in animals produced during the metabolism of CCl₄. Patchoo et al. (2008a) evaluated and compared the in vitro antioxidant potentials of the leaf extracts of aqueous methanol and ethyl acetate of A. senegalensis growing in Togo and Burkina Faso. They concluded that the aqueous methanol extract from Togo had significantly higher total polyphenol and total flavonoids while the species from Burkina Faso showed an improved antioxidant activity than the Togo specimen and this was attributed to the presence of flavonoids. Since this study did not take into consideration the species growing in Nigeria, and coupled with the lack of information in the scientific literature on the antioxidant activity of the species growing in Nigeria, there is the need to continue the investigation of the mechanism by which botanicals protect or prevent tissue from damage by chemical compounds such as CCl₄. One botanical that has been widely used in the management of vast diseased conditions in the folklore medicine of Nigeria is Annona senegalensis Pers. (Annonaceae).

Annona senegalensis, referred to as sour sop (English), abo (Yoruba, Western Nigeria), uburu-ocha (Igbo, Eastern Nigeria) and gwandar (Hausa, Northern Nigeria), is widely distributed across Nigeria (Neuwinger, 1996). The stem bark is silvery grey or grey-brown in color. The leaves are simple, alternate, oblong and are without hairs on top but often with brownish hairs underside. Phytochemical constituents of A. senegalensis leaf included epicatechin, catechin, rutin, isoquercetrin, anthocyanosides, saponosides, tannins, carotenoids, sterols, triterpenes, alkaloids, and cardiac glycoside (Potchoo et al., 2008b). The leaves are used in the treatment of diarrhea, disease of the joints, respiratory diseases, conjunctivitis, wounds, snakebites, trypanosomiasis, jaundice, hemorrhoids, feminine barrenness, convulsions, ovarian cancer, fever, and asthenia (Neuwinger, 1996), some of which may be the consequence of free radical damage.

Despite the myriad of uses of this plant, there is no adequate information in the scientific literature that addresses the antioxidant and drug detoxification potentials of A. senegalensis growing in Nigeria. The present study thus investigates the antioxidant (in vitro and in vivo) and drug detoxification potential of aqueous extract of A. senegalensis growing in Nigeria using albino rats as a model.

Materials and methods

Plant material

A. senegalensis leaves, collected at Idu Industrial Layout, Idu-Abuja, Nigeria, in February, 2008 were identified by Grace Ugbabe of the Herbarium Unit, Medicinal Plant Research and Traditional Medicine Department, National Institute for Pharmaceutical Research and Development, Abuja, Nigeria. A voucher specimen was deposited at the Herbarium of the Department.

Animals

Apparently healthy, male albino rats (Rattus norvegicus) of Wistar strain, weighing 195.28 ± 4.2 g were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Nigeria. The animals were kept in clean aluminum cages placed in well-ventilated house conditions (temperature: 28–31°C; photoperiod: 12 h natural light and 12 h dark; humidity: 50–55%) with free access to rat pellets (Bendel Feeds and Flour Mills, Ewu, Nigeria) and tap water. The animals were handled according to the guidelines of the National Institutes of Health on the care and use of laboratory animals (NIH, 1985), and the experiment was carried out following approval from the Ethical Committee on the care and use of experimental animals of the Department of Biochemistry, University of Ilorin, Nigeria.

Chemicals

The assay kits for alkaline and acid phosphatase were products of Human Gesellschaft fur Biochemica und Diagnostica, Wiesbaden, Germany. Glutathione peroxidase assay kit was a product of Randox Laboratories, Crumlin, County Antrim, Northern Ireland. All other reagents used were products of Sigma-Aldrich, St. Louis, MO, USA.

Preparation of extract

The leaves were air dried at room temperature for 2 weeks to constant weight. The dried pieces were then pulverized using a Beltone Luinohun Blender/Miller III (model MS-223, Taipei, Taiwan). The powder (100 g) was extracted in 1 L of distilled water for 48 h at room temperature. This was later filtered with Whatman No. 1 filter paper (Maidstone, Kent, UK) and the resulting filtrate concentrated on a steam bath to give a yield of 12.04 g.

Determination of some antioxidant phytoconstitutents of the extract

Total phenolic compound

The concentration of phenolic compounds in the extract of A. senegalensis leaves was determined using the method described by Spanos and Wrolstad (1991). Briefly, 2.5 mL of 10% Folin-Ciocalteu reagent and 2 mL of Na₂CO₃ (2% w/v) were added to 0.5 mL each of the solution of the extract (1 mg/mL). The resulting mixture was incubated at 45°C with constant shaking for 15 min. The absorbance of the samples was read at 765 nm. This was done in triplicate. The total phenolic content in the extract was expressed as mg of epicatechin (0-0.5 mg/mL) dissolved in distilled water.

Total flavonoids

Aluminum chloride based colorimetric method was used for the determination of flavonoids. Briefly, the extract (1 mL) was mixed with 3 mL of methanol, 0.2 mL of 10% aluminum chloride, 0.2 mL of 1 M potassium acetate and 5.6 mL of distilled water. The mixture was allowed to stand at room temperature for 30 min. The absorbance of the reaction mixture was read at 420 nm. The concentration of flavonoids in mg/mL was obtained from the calibration curve of epicatechin solution (0-0.8 mg/mL) in distilled water.

Total proanthocyanidins

Total proanthocyanidins in the extract was determined using the procedure described by Sun et al. (1998). The extract, 0.5 mL (1 mg/mL) was added to a mixture of 3 mL of vanillin-methanol (4% v/v) and 1.5 mL of hydrochloric acid. This was thereafter vortexed and the resulting mixture allowed to stand for 15 min at room temperature. The absorbance was read at 500 nm and total proanthocyanidin content was expressed as epicatechin equivalent (mg/mL) from the calibration curve.

In vitro antioxidant assay

DPPH radical scavenging activity

The antioxidant activities of A. senegalensis leaf extract was based on the capacity of the extract to bleach a purple-coloured ethanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as described by Turkoglu et al. (2007). Briefly, 2 mL of various concentrations of the extract (0.2–1 mg/mL) in ethanol was added to 2 ml of 0.2 mmol/L DPPH in ethanol. After a 30 min incubation period at room temperature, the absorbance was read at 517 nm. Percentage inhibition rate [I (%)] on DPPH radical was obtained using the expression:

where A_o was the absorbance of the control reaction (containing all reagents except the extract) and A_s was the absorbance of the extract.

Hydrogen peroxide scavenging activity

The ability of the extract to scavenge hydrogen peroxide was determined according to the procedure described by Ruch et al. (1989). Briefly, varying concentrations of the extract (0.2–1 mg/mL) in 4 mL of distilled water was added to 0.6 mL of hydrogen peroxide solution (4 mM of H₂O₂ in phosphate buffer saline, pH 7.4). The solution was allowed to stand for 10 min at room temperature after which the absorbance was read at 230 nm.

Superoxide radical scavenging activity

Superoxide anion radical scavenging activity of A. senegalensis leaf extract was determined using the method described by Hseu et al. (2008). In this experiment, superoxide radicals were generated in 3 mL of Tris-HCl buffer (16 mM, pH 8.0) containing 0.5 mL of 300 µM nitroblue tetrazolium solution, 0.5 mL of 936 µM NADH solution, after which 0.5 mL of A. senegalensis leaf extract (0.2–1 mg/mL) was added. The reaction was started by the addition of 0.5 mL of 120 µM phenazine methosulfate solution. The reaction mixture was later incubated at 25°C for 5 min, and the absorbance was read at 560 nm.

TEAC Assay

The 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) free radical-scavenging activity of the extract was determined according to the method described by Pavel and Vlastimil (2006). ABTS⁺ was generated by persulfate oxidation of ABTS. A mixture (1:1 v/v) of ABTS (7 mM) and potassium persulfate (4.95 mM) was allowed to stand overnight at room temperature in the dark to form radical cation ABTS⁺. A working solution was diluted with phosphate buffer solution to absorbance values of l and 1.5 units at 734 nm (constant initial absorbance values must be used for standard and samples). An aliquot (0.1 mL) of the extract containing 0.2–1 mg/mL was mixed with 3.9 mL of the working solution. The absorbance was read at 734 nm after 10 min at 37°C in the dark. Aqueous phosphate buffer solution (3.9 mL, without ABTS⁺ solution) was used as the control. The ABTS⁺ scavenging activity of the extract was obtained from the expression:

where Ac and As were the absorbance of the control and the extract, respectively.

Reducing power

The reducing power of A. senegalensis leaf extract was evaluated using the method described by Oyaizu (1986). Varying amounts of the extract was suspended in 1 mL of distilled water and mixed with 2.5 µL of 0.2 M phosphate buffer, (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min after which 2.5 µL of trichloroacetic acid was added. Following centrifugation at 3000 rpm for 10 min, 2.5 µL of the supernatant was mixed with an equal amount of distilled water and 0.5 mL of 0.1% FeCl₃ after which the absorbance was read at 700 nm. Increase in absorbance of the reaction mixture indicated enhanced reducing power.

In vivo study

Animal grouping and administration of compounds

Thirty, male albino rats were completely randomized into six groups (A–F) of five rats each and were treated for 14 days as follows: Group A (the control) received orally 0.5 mL of distilled water (vehicle) once daily. Rats in Group B were intraperitoneally treated with 0.5 mL of CCl₄ solution only (prepared by dissolving 0.5 mL of CCl₄ in 1.5 mL of physiological saline). Groups C, D and E were simultaneously treated with 0.5 mL containing 100, 200 and 400 mg/kg bodyweight of the extract and CCl₄, while rats in group F received 300 mg/kg bodyweight of butylated hydroanisole (BHA) and CCl₄ simultaneously.

Preparation of serum and tissue homogenate

The rats were sacrificed 24 h after their last daily doses using the anesthetic method described by Yakubu et al. (2005). Under diethyl ether anesthesia, rats were made to bleed through their cut jugular veins (slightly displaced to prevent blood from being contaminated with interstitial fluid) into centrifuge tubes. The blood samples were allowed to clot for 15 min and centrifuged at 33.5 g × 15 min to obtain the sera. The sera were stored frozen and used within 12 h of preparation for the biochemical assay. Liver excised from the animals were blotted in tissue paper, cut thinly with sterile scalpel blade and then homogenized in ice-cold 0.25 M sucrose solution (1:5 w/v). The homogenates were centrifuged at 800 g for 10 min at 4°C to obtain the supernatant that was kept frozen at -20°C before being used for the various biochemical assay.

Determination of biochemical parameters

Biochemical parameters were assayed as described for acid phosphatase (ACP) (Hillmann, 1971), alkaline phosphatase (ALP) (Wright et al., 1972), alanine aminotransferase (ALT) (Bergmeyer et al., 1986a), aspartate aminotransferase (AST) (Bergmeyer et al., 1986b), superoxide dismutase (SOD) (Misra & Fridovich, 1972), catalase (CAT) (Beers & Sizer, 1952), glutathione peroxidase (GPx) (Rotruck et al., 1973) and glutathione reductase (GR) (Mavis & Stellwagen, 1968). Other parameters included reduced glutathione (GSH) (Ellman, 1959), vitamin C (Omaye et al., 1979), vitamin E (Desai, 1984), uridyl diphosphoglucuronosyl transferase (UDPGT) (Fishman & Bernfeld, 1955), glutathione S-transferase (Habig et al., 1974), NADH:quinone oxidoreductase (Brower & Woodbridge, 1970), protein (Gornall et al., 1949), lipid peroxidation (Buege & Aust, 1978).

Statistical analysis

Data were expressed as the mean ± SD of five determinations. Mean was analyzed between the groups using a one-way ANOVA and Duncan multiple range test with SPSS 15.0 version. Differences were considered statistically significant at P <0.05.

Results

In vitro study

Preliminary phytochemical screening of some antioxidant compounds in the extract of A. senegalensis leaves revealed the presence of total phenol (4.64 ± 0.3 mg/mL), flavonoids (2.74 ± 0.5 mg/mL) and proanthocyanidins (1.92 ± 0.2 mg/mL). The extract produced dose related effects on DPPH and H₂O₂ radical scavenging activities similar to the reference drugs (BHA and α-tocopherol) (Figures 1 and 2). Specifically, the extract produced activities higher than the reference drugs with the highest doses of the extract producing 96.9% of DPPH radical and 77.54% of H₂O₂ scavenging activities, respectively. In addition, A. senegalensis leaf extract scavenged superoxide ion generated by PMS-NADH producing 97.4% at 1000 μg/mL. In this model, the extract produced the highest scavenging effect when compared with the reference drugs (Figure 3). Similarly, A. senegalensis leaf extract produced the most pronounced scavenging effect on the ABTS radical than the reference drugs. For instance, the 1000 μg/mL of the extract produced 96.7% scavenging activity on the ABTS radical (Figure 4). The reducing effect of A. senegalensis leaf extract on ferric ion was concentration-related and was similar to the reference drugs. Whereas the highest reducing effect on ferric ion was produced by the extract, BHA gave the least (Figure 5).

Figure 1.  Scavenging effect of Annona senegalensis on DPPH radical.

Figure 2.  Scavenging effect of Annona senegalensis on H₂O₂.

Figure 3.  Scavenging effect of Annona senegalensis on superoxide ion.

Figure 4.  Scavenging effect of Annona senegalensis on ABTS radical.

Figure 5.  Reducing effect of Annona senegalensis on ferric ion.

In vivo study

Administration of CCl₄ alone resulted in significant reduction (P <0.05) in ALP, ACP, ALT and AST activities of the liver. The reductions in these enzymes were accompanied by corresponding increases in the serum enzymes (Table 1). This trend was reversed when the extract at various doses was simultaneously administered with CCl₄ as the activity of the liver and serum enzymes compared favourably (P >0.05) with that of the distilled water treated group. Similarly, the activity of the liver and serum enzymes of the animals simultaneously administered with BHA and CCl₄ compared well with the distilled water treated animals (Table 1).

Table 1.  Effect of aqueous extract of A. senegalensis leaves on the activities of some phosphatases and aminotransferase activities of rat liver.

Treatment groups	Alkaline phosphatase		Acid phosphatase		Alanine aminotransferase		Aspartate aminotransferase
	Liver	Serum	Liver	Serum	Liver	Serum	Liver	Serum
Distilled water	1.89 ± 0.06^a	0.31 ± 0.03^a	3.82 ± 0.02^a	0.23 ± 0.02^a	45.36 ± 3.4^a	9.42 ± 1.28^a	63.51 ± 2.32^a	13.18 ± 0.5^a
CCl4 only	0.52 ± 0.03^b	2.63 ± 0.15^b	1.41 ± 0.03^b	2.14 ± 0.32^b	10.4 ± 0.92^b	35.29 ± 1.80^b	15.67 ± 2.01^b	49.65 ± 0.35^b
100 mg/kg body weight of extract + CCl4	1.33 ± 0.02^c	0.61 ± 0.01^c	3.25 ± 0.05^c	0.83 ± 0.05^c	37.33 ± 0.46^c	13.08 ± 0.25^c	54.22 ± 1.35^c	14.99 ± 0.4^a
200 mg/kg body weight of extract + CCl4	1.55 ± 0.1^d	0.32 ± 0.09^a	3.78 ± 0.02^a	0.22 ± 0.02^a	44.02 ± 1.39^a	10.29 ± 0.23^a	59.46 ± 4.12^a	15.22 ± 0.5^a
400 mg/kg body weight of extract + CCl4	1.74 ± 0.09^a	0.35 ± 0.01^a	3.79 ± 0.06^a	0.24 ± 0.07^a	43.11 ± 1.25^a	11.12 ± 0.12^a	61.89 ± 1.25^a	14.3 ± 1.4^a
300 mg/kg body weight of BHA + CCl4	1.82 ± 0.02^a	0.34 ± 0.03^a	3.73 ± 0.02^a	0.28 ± 0.05^a	41.82 ± 2.2^a	11.91 ± 1.05^a	65.96 ± 0.5^a	14.89 ± 0.5^a

Data are means ± SD of five replicates.

Enzyme activities are expressed as nmol min⁻¹mg⁻¹protein.

^a-cValues for the test groups carrying superscripts different from the distilled water control group for each enzyme are significantly different (P < 0.05).

Administration of CCl₄ alone decreased the activities of SOD, CAT, GPx, GR and G6PDH in the liver of the animals by 26.97, 40.32, 52.77, 38.42 and 54.42%, respectively (Table 2). This trend of reduction in the hepatic enzymes was reversed following the simultaneous administration of the extract at various doses and CCl₄ as well as the BHA and CCl₄ (Table 2).

Table 2.  Effect of aqueous extract of A. senegalensis leaves on some antioxidant enzymes of rat liver.

Treatment groups	Superoxide dismutase	Catalase	GSH-Reductase	GSH-Peroxidase	G6P Dehydrogenase
Distilled water	60.8 ± 0.2^a	37.2 ± 2.1^a	1.8 ± 0.06^a	68.2 ± 1.6^a	22.53 ± 1.02^a
CCl4 only	44.4 ± 2.5^b	22.2 ± 1.8^b	0.85 ± 0.02^b	42.0 ± 0.8^b	10.27 ± 2.25^b
100 mg/kg body weight of extract + CCl4	58.6 ± 1.8^a	35.6 ± 3.5^a	1.78 ± 0.02^a	68.5 ± 1.8^a	20.01 ± 3.12^a
200 mg/kg body weight of extract + CCl4	58.8 ± 3.1^a	37.9 ± 4.4^a	1.82 ± 0.08^a	68.6 ± 2.5^a	21.14 ± 1.43^a
400 mg/kg body weight of extract + CCl4	62.8 ± 1.0^a	39.6 ± 2.7^a	1.84 ± 0.03^a	68.3 ± 1.2^a	22.01 ± 1.2^a
300 mg/kg body weight of BHA + CCl4	59.6 ± 1.2^a	36.6 ± 2.0^a	1.81 ± 0.09^a	66.2 ± 2.5^a	18.9 ± 4.1^a

Data are means ± SD of five replicates. Enzyme activities are expressed as nmol min⁻¹mg⁻¹protein.

^a-bValues for the test groups carrying superscripts different from the distilled water control group for each enzyme are significantly different (p < 0.05).

CCl₄ administration decreased the levels of GSH, vitamins C and E of the liver of the animals by 63.81, 45.79, and 60.74%, respectively. These decreases were reversed by the simultaneous administration of the extract and CCl₄ in a manner similar to the BHA and CCl₄ treated animals (Table 3).

Table 3.  Effect of aqueous extract of A. senegalensis leaves on some nonenzymic antioxidants of rat liver.

Treatment groups	Glutathione reduced (nmol mg^−1 protein)	Vitamin C (mgdL^−1)	Vitamin E (mgdL^−1)
Distilled water	28.32 ± 0.2^a	57.0 ± 3.5^a	24.2 ± 1.8^a
CCl4 only	10.25 ± 1.2^b	30.9 ± 1.5^b	9.5 ± 0.6^b
100 mg/kg body weight of extract + CCl4	26.41 ± 2.2^a	56.2 ± 4.1^a	20.1 ± 4.2^a
200 mg/kg body weight of extract + CCl4	27.62 ± 0.9^a	58.5 ± 1.5^a	22.2 ± 0.8^a
400 mg/kg body weight of extract + CCl4	28.89 ± 1.5^a	58.2 ± 2.4^a	23.4 ± 0.8^a
300 mg/kg body weight of BHA + CCl4	26.2 ± 2.4^a	59.1 ± 0.5^a	22.7 ± 2.2^a

Data are means ± SD of five replicates. Enzyme activities are expressed as nmol min⁻¹mg⁻¹protein.

^a-bValues for the test groups carrying superscripts different from the distilled water control group for each non-enzymic antioxidants are significantly different (p <0.05).

Administration of CCl₄ alone decreased glutathione S-transferase, NADH: quinone oxidoreductase, and UDPGT activities (Table 4). This trend was reversed towards the distilled water treated control group by the simultaneous administration of the extract and CCl₄ as well as BHA and CCl₄. In particular, the simultaneous administration of 400 mg/kg body weight of the extract and CCl₄ increased the activities of glutathione S-transferase, NAD(H):quinone oxidoreductase, and UDPGT by 80.92, 38.79, and 128. 11%, respectively (Table 4).

Table 4.  Effect of aqueous extract of A. senegalensis leaves on some drug detoxification enzymes of rats.

Treatment groups	Glutathione S-transferase	NADH:quinone oxidoreductase	Uridyl diphosphoglucuronyl transferase
Distilled water	32.5 ± 1.4^a	67.8 ± 1.9^a	28.1 ± 1.2^a
CCl4 only	23.5 ± 1.2^b	39.5 ± 0.3^b	15.8 ± 0.3^b
100 mg/kg body weight of extract + CCl4	39.2 ± 1.0^c	66.7 ± 0.8^a	42.1 ± 0.6^c
200 mg/kg body weight of extract + CCl4	46.3 ± 0.8^d	70.9 ± 3.2^c	47.1 ± 2.1^d
400 mg/kg body weight of extract + CCl4	51.1 ± 2.5^c	83.3 ± 2.0^d	49.8 ± 0.2^d
300 mg/kg body weight of BHA + CCl4	33.4 ± 2.4^a	61.8 ± 2.4^a	28.9 ± 1.0^a

Data are means ± SD of five replicates. Enzyme activities are expressed as nmol min⁻¹mg⁻¹protein.

^a-dValues for the test groups carrying superscripts different from the distilled water control group for each enzyme are significantly different (p < 0.05).

The levels of malondialdehyde (MDA) and lipid hydroperoxides (LHP) in the liver of the animals were significantly (P <0.05) elevated following the administration of CCl₄ alone (Table 5). In contrast, the simultaneous administration of 100 mg/kg body of the extract and CCl₄ reduced the MDA and LHP by 56.69 and 54.52%, respectively. Again, the 200 mg/kg body weight of the extract and CCl₄ reduced the levels of MDA and LHP by 60.13 and 57.85%, respectively. The simultaneous administration of BHA and CCl₄ produced similar reduction in the levels of MDA and LHP by 60.37 and 58.79%, respectively. In addition, the levels of the lipid peroxidized products obtained with the simultaneous administration of 400 mg/kg body weight of the extract and CCl₄ compared well (P >0.05) with those of the distilled water control group.

Table 5.  Effect of aqueous extract of A. senegalensis leaves on lipid peroxidized products of rat liver.

Treatment groups	Malondialdehyde (nmol mg^−1 protein)	Lipid hydroperoxide (nmol mg^−1 protein)
Distilled water	442.3 ± 2.1^a	423.1 ± 0.1^a
CCl4 only	1234.8 ± 0.8^b	1146.4 ± 2.5^b
100 mg/kg body weight of extract + CCl4	534.8 ± 2.5^c	521.4 ± 2.5^c
200 mg/kg body weight of extract + CCl4	492.3 ± 2.2^d	483.2 ± 1.2^d
400 mg/kg body weight of extract + CCl4	440.2 ± 3.2^a	434.2 ± 1.5^e
300 mg/kg body weight of BHA + CCl4	489.3 ± 2.1^e	472.4 ± 3.2^f

Data are means ± SD of five replicates. Enzyme activities are expressed as nmol min⁻¹mg⁻¹protein.

^a-fValues for the test groups carrying superscripts different from the distilled water control group for each lipid peroxidized products are significantly different (p < 0.05).

Discussion

The plant kingdom offers a wide range of natural antioxidants. Dietary plants with proven antioxidant properties may function as a direct anti-radical chain breaking of free radical propagation, interaction with transition metals, inhibition of reactive oxygen species (ROS) generating enzymes as well as inducing specific antioxidant enzymes. These are made possible by the presence of phenolic compounds and flavonoids. Although, the relevance of in vitro assay of antioxidant compounds can be different with respect to the physiological conditions, the presence of phenolic compounds, flavonoids, and proanthocyanidins may confer antioxidant activity on the plant extract. Similarly, studies by Potchoo et al. (2008a, 2008b) attributed the antioxidant activity of aqueous methanol and ethyl acetate extract of the leaves of A. senegalensis obtained from Togo and Burkina Faso to the presence of these phytochemicals. Therefore, these phytochemicals may also be responsible for the observed antioxidant effect in this study.

DPPH, superoxide ion and ABTS are very stable free radicals widely used to evaluate in vitro antioxidant activities within a relatively short time. Similarly, metal ion can initiate lipid peroxidation and start a chain reaction that leads to deterioration of food. For example, the catalysis of ferrous ion, the most effective pro-oxidant, has been correlated with the incidence of free radical related diseases such as diabetes, arthritis, cancer, cardiovascular and liver diseases (Halliwell et al., 1995). Therefore, the scavenging activity on DPPH, H₂O₂, superoxide ion, and ABTS as well as the reducing effect on the ferric ion by the aqueous leaf extract of A. senegalensis may be attributed to interference with lipid peroxidation, possibly terminating free radical chain reactions (Bran-Williams et al., 1995). Furthermore, the most profound scavenging activity displayed by the highest dose of the extract suggests that it was more effective than the known reference antioxidants (BHA and α-tocopherol) used in this study. In addition, the ability of the extract to scavenge for in vitro ROS such as superoxide ion and H₂O₂ implied that the extract could be used in counteracting the deleterious effects of ROS produced in vivo during chemical assault. The extract when consumed may complement the in vivo free radical scavenging activity of SOD, CAT and GPx, when the internal enzymatic mechanism fails or is inadequately efficient. This effect is thus likely to prevent redox imbalance arising from the increased production of ROS in vivo.

ALP, a “marker” enzyme for the plasma membrane, is often used to assess the integrity of the organelle (Akanji et al., 1993). The loss of ALP from the liver and the corresponding increase of the enzyme in the serum of the animals following the administration of CCl₄ alone suggest that ALP has leaked from the plasma membrane of the hepatocytes into the serum in this study. Enzymes from damaged or diseased tissues find their way into the serum by leakage (Reichling & Kaplan, 1988). Therefore, the leakage of ALP from the liver to the serum of the animals may be attributed to disruption of the ordered lipid bilayer of the plasma membrane. This may be the consequence of labilization of the plasma membrane by trichloromethyl radical (CCl₃^•) and trichloromethyl peroxyl radical (CCl₃O^•) generated during the metabolism of CCl₄, which eventually resulted in peroxidation of the plasma membrane. Similarly, the loss of ACP, a “marker” enzyme of the lysosomal membrane from the hepatocyte to the serum of animals treated with CCl₄ only may be attributed to peroxidation of unsaturated fatty acids present on the membrane by the free radicals generated during the metabolism of the organic solvent. Again, the loss of AST and ALT (cytosolic enzymes) from the liver to the serum of animals treated with CCl₄ is quite understandable since it is in close proximity to the plasma membrane. Damage to plasma membrane will consequentially lead to leakage of cytosolic content of the cell to the external milieu. The ability of the extract to reverse this trend in a manner similar to BHA suggests antioxidant potential of the extract. This amelioration may be adduced to the ability of the extract to scavenge ROS (O₂⁻, OH⁻ and H₂O₂), or act as chain breaker in free radical reactions. This may contribute, in part, to the mechanism by which the plant finds application in the management of diseased conditions in the folklore medicine of Nigeria.

The antioxidant enzyme system (SOD, CAT, GR, GPx, and G6PDH) plays a coordinated role in preventing oxidative damage by O₂⁻ and H₂O₂. These enzymes prevent lipid peroxidation, base modification, DNA-crosslinks and covalent binding to protein when ROS are scavenged. The reduction in the levels of the hepatic antioxidant enzymes following the administration of CCl₄ alone suggests that the natural defense mechanisms of the animals have been overwhelmed by free radicals generated by CCl₄. The reversal of the levels of these antioxidant enzymes by the extract of A. senegalensis suggests that the extract prevented the free radicals from overwhelming the natural defense system of the animals. These are indications of antioxidant potentials of aqueous extract of A. senegalensis leaves.

The non-enzymic antioxidants complement the activity of the enzymic antioxidants in preventing animals from excessive oxidative stress by free radicals. Vitamins C and E prevent oxidative stress in cells by acting as free radical scavengers (Pesh-Imam & Recknagel, 1977). Similarly, GSH scavenges singlet oxygen, O₂⁻ and hydroxyl radicals, and thus protect lipids, proteins, and nucleic acids from the attack of electrophilic compounds by reacting with the electrophilic center through the thiol group (-SH) (Singh & Ahluwalia, 2003). The levels of these molecules decreased significantly following the treatment of rats with CCl₄. Depletion of hepatic GSH has been shown to be associated with an enhanced toxicity to chemicals including CCl₄ (Ko et al., 1995). Depletion in GPx and GR observed in this study has a direct relationship to GSH, as these enzymes maintain high intracellular GSH:GSSG ratio and thus protect cellular macromolecules from oxidative attack (Kozer et al., 2003). Therefore, the preservation of non-enzymic antioxidant system by A. senegalensis leaf extract suggests antioxidant role for the extract.

Drug metabolizing enzymes are a diverse group of proteins that are responsible for metabolizing a vast array of xenobiotics such as drugs, environmental pollutants, and endogenous compounds. Phase II drug detoxification mechanisms involves the conjugation of toxic agents with compounds such as glutathione and glucuronic acid. Administration of CCl₄ alone depleted the levels of these enzymes, which were not only restored by the A. senegalensis leaf extract, but also induced. James et al. (2003) implicated UDPGT in the elimination of CCl₄. Therefore, the significant induction in the levels of these enzymes by the extract might enhance the elimination/clearance of CCl₄ from the body before it is metabolized to potential toxic agent(s). Similarly, the induction in the level of glutathione S-transferase by the extract which was absent in the group of animals administered with the BHA might also enhance detoxification through conjugation.

MDA and LHP are compounds derived from lipid peroxidation and exist in biological matrices both in the free and bound forms (Esterbauer et al., 1991). The measurement of the levels of MDA-thiobarbituric acid adducts and LHP are widely used to monitor oxidative stress including lipid peroxidation (Gomez et al., 1998). Therefore, the 3.27- and 2.71-folds in MDA and LHP of the CCl₄ treated animals further indicated that peroxidation of polyunsaturated fatty acids present on the plasma membrane of the hepatocytes have occurred. This also buttressed the disruption of plasma membrane earlier observed in this study which led to the loss of the membrane bound and cytosolic enzymes from the hepatocytes to the serum as a consequence of peroxidation of unsaturated fatty acids. The significant reduction in the levels of MDA and LHP of the animals following the simultaneous administration of the extract of A. senegalensis and CCl₄ suggested protective effect by the extract against the oxidant. Therefore, the components of the extract such as flavonoids, total phenolics and proanthocyanidins might be responsible for the antioxidant activity of the extract.

Conclusion

Overall, it is evident from this study that aqueous leaf extract of A. senegalensis attenuated the CCl₄-induced oxidative damage in the hepatocytes of the animals by acting as free radical scavenger/chain breaker. The ability to scavenge in vitro and in vivo free radicals was made possible by the antioxidant phytoconstituents. The antioxidant potential of aqueous extract of Annona senegalensis leaves as well as the ability to induce de novo synthesis of some drug detoxifying enzymes may explain the rationale behind the use of the plant in the management of several ailments.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.