Acute toxicity and anti-inflammatory activity of hydro-methanol leaves extract of Allophylus africanus Beauv in rats

Introduction Traditional medicine includes diverse health practices, approaches, knowledge and beliefs (1). The use of traditional medicines and medicinal plants in most developing countries as therapeutic agents for the maintenance of good health has been widely observed (2), with herbal medicine being an integral element, meet about 75% of the population’s health care needs in Nigeria and up to 80% in Africa (3). The only true medicines ever used initially are plants (4) and some chemically useful plant drugs have been identified from the lead provided by their ethno-medical uses (5). Inflammation is a protective response that involves immune cells, blood vessels and molecular mediators and its disorders bring about enormous array of human diseases (6). The major classes of drugs to suppress inflammation are non-steroidal anti-inflammatory drugs (NSAIDS) and corticosteroids but their toxic adverse effects such as epigastric distress, peptic ulceration, osteoporosis, and iatrogenic Cushing’s syndrome have limited their uses (7,8). There is hence, a need to intensify researches for plant based antiinflammatory agents that are efficacious, safe, affordable, and accessible for patients. Allophylus africanus is a specie of the genus Allophylus of the family Sapindaceae. It is a shrub that grows in the reverine thickest, open wood land and forest edges at http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.20


Introduction
Traditional medicine includes diverse health practices, approaches, knowledge and beliefs (1).The use of traditional medicines and medicinal plants in most developing countries as therapeutic agents for the maintenance of good health has been widely observed (2), with herbal medicine being an integral element, meet about 75% of the population's health care needs in Nigeria and up to 80% in Africa (3).The only true medicines ever used initially are plants (4) and some chemically useful plant drugs have been identified from the lead provided by their ethno-medical uses (5).Inflammation is a protective response that involves immune cells, blood vessels and molecular mediators and its disorders bring about enormous array of human diseases (6).The major classes of drugs to suppress inflammation are non-steroidal anti-inflammatory drugs (NSAIDS) and corticosteroids but their toxic adverse effects such as epigastric distress, peptic ulceration, osteoporosis, and iatrogenic Cushing's syndrome have limited their uses (7,8).There is hence, a need to intensify researches for plant based antiinflammatory agents that are efficacious, safe, affordable, and accessible for patients.Allophylus africanus is a specie of the genus Allophylus of the family Sapindaceae.It is a shrub that grows in the reverine thickest, open wood land and forest edges at altitudes of 960-1540 m.It is widely distributed throughout tropical Africa (9).In Nigeria, it is locally known as Akanro, Akaraesu (in Yoruba), Akaito (in Igbo), Karki (in Hausa) and Ebe/ukpe (in Esan tribe in Edo state).In ethno-medicine, the boiled leaves are used in aches, fever and rheumatic pains (10), in gout, hemorrhoids (9) while root and twigs are used for diarrhea, dental and oral health care (10).Previous pharmacological studies on the leaves extract of this plant showed that it possessed antimalarial, antimicrobial and anti-oxidant activities (11).The present study was carried out to evaluate the acute toxicity and anti-inflammatory effects of the extract in animal model, so as to validate the ethno-medicinal claim of the use of the plant in treatment of inflammation.

Materials and Methods
Collection, identification and preparation of the plant material Fresh leaves of A. africanus were collected from "Samaru" village, Zaria, Kaduna State, Nigeria in March, 2014.The plant leaves were identified by Mallam Musa of the herbarium section of the Department of Biological Sciences, Ahmadu Bello University Zaria and a voucher specimen was deposited there with number 1540.The plant leaves were cleaned, air dried under shade and powdered using mortar and pestle to a suitable size and stored in appropriate container for further use.
Extraction procedure Analytical grade solvents and freshly prepared solutions were used throughout the study.One thousand gram (1000 g) of the air dried powdered plant material was extracted by cold maceration in hydro-methanol mixture (methanol-water 80:20 v/v) for 120 hours using 5 L. The filtrate obtained was concentrated to dryness in a rotor evaporator under vacuum and later in oven (45°C).The dried powder was used for toxicological tests and anti-inflammatory activity (12).Qualitative phytochemical screening Preliminary phytochemical screening of the aqueous methanol leaf extract was carried out for detection of carbohydrates, tannins, steroids/triterpenes, glycosides, flavonoids, alkaloids using standard test methods (13)(14)(15).

Experimental animals
Adult rats of both sexes (95-105 g) were obtained from National Institute for Trypanosomiasis Research (NITR), Kaduna State, Nigeria and kept in the animal house of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria.The animals were maintained under standard conditions (12 hours light/12 hours dark cycle, temperature of 37 ± 2°C, 35-60% humidity).The rats were fed with standard (grower) mash (Vital feed, Jos, Nigeria) and water -ad libitum.Ethical rules guiding the use of animals for the experimentation were strictly adhered to (16) and with approval of Animal Ethics and Care Committee, Ahmadu Bello University, Nigeria (ABUCAUC/2016/018).

Acute toxicity study
The acute toxicity study of the aqueous methanol leaves extract of A. africanus was determined using previously described method (17).The study was carried out in two phases.The first phase consisted of 9 rats divided into 3 groups of three rats each and were treated with the aqueous methanol extract at doses of 10, 100 and 1000 mg/kg body weight per oral.They were observed for 24 hours for signs and symptom of toxicity and death.In the second phase, three rats divided into 3 groups of one rat each were treated with the aqueous methanol extract at doses of 1600, 2900 and 5000 mg/kg body weight per oral and observed for 24 hours for signs and symptom of toxicity and death.The oral median lethal dose (LD 50 ) was calculated as the geometric mean of the minimum toxic dose and maximum tolerated dose.Anti-inflammatory study Thirty adult rats of both sexes of weight ranges (85-105 g) were divided into five groups of six animals each.Stock solution of the extract, aspirin and carrageenan were prepared.To group 1 of the animals, 1 mL/kg of normal saline (negative control) was administered to each animal.To group II, aspirin (positive control) at dose of 300mg/ kg was administered.To groups III, IV and V, the extracts at doses 250, 500 and 1000 mg/kg were administered, respectively.After an hour, 0.1ml of sterile saline suspension of 1% carrageenan was injected into the sub planter surface of the left hind paw of all animals in each group.Paw size was measured using venier caliper at time 0, 1, 2, 3, 4 and 5 hours after carrageenan administration.The percentage inhibition (PI) at each time interval was calculated (18).100 Control group treated group PI Control group

Statistical analysis
The results were expressed as mean ± standard error of the mean (SEM) for all values.The data were statistically analyzed using one-way ANOVA (SPSS version 20.0) followed by Tukey's post hoc multiple comparison tests.The results were considered to be significant at P<0.05.

Extraction yield
The total solid of A. africanus crude extract recovered from maceration was 71 g.The extract was dark green in color with pleasant smell and hard.

Qualitative phytochemical screening
The results of the phytochemical screening of aqueous methanol leaves extract of A. africanus revealed the presence of secondary metabolites namely carbohydrates, tannins, steroids/triterpenes, flavonoids, alkaloids, cardiac glycosides and absent of saponins as shown in Table 1.

Acute toxicity
The acute toxicity of the extract was done per oral in rats.
The LD 50 was found to be 3807.89mg/kg (the following formula) and there was death of an animal at dose 5000 mg/kg in phase II (Table 2).

Anti-inflammatory activity of aqueous methanol leaves extract of Allophylus africanus
In the normal saline treated rats, sub-planter injection of 1% carrageenan suspension produced a local edema reaching its maximum effect at 3 hours.The aqueous methanol extract at doses 250 mg and 1000 mg/kg was able to significantly produce anti-inflammatory effect (at the 3rd, 4th and 5th hours).At the 4th and 5th hours, the effect was dose dependent.The extract at dose 1000 mg/ kg showed higher inhibition of induced edema compared to standard drug (aspirin) at the 5th hour as shown in Table 3.

Discussion
Preliminary phytochemical screening provides a brief idea about the qualitative nature of active phytochemical constituents present in plant extract.The result of the preliminary phytochemical screening of the aqueous methanol extract indicated the presence of carbohydrates, steroids, triterpenes, glycosides, tannins, flavonoids and alkaloids (Table 1).These results other than conforming the previous findings (11) which reported the presence of tannins, saponins, flavonoids and carbohydrates in A. africanus plant, were necessary for standardization of the plant.These secondary plant metabolites are known to have various pharmacological effects and may be responsible for various activities of A. africanus.
The acute toxicity of the extract was done per oral in rats.
The median lethal dose LD 50 of aqueous methanol leaves extract of A. africanus was found to be 3.807.89mg/kg.This suggests that the extract is slightly toxic, based on the recommended classification (19) which states that substances with LD 50 values of 2000 to 5000 mg/kg body weight is slightly toxic.The hydro-methanolic leaf extract of A. africanus was able to significantly (P < 0.05) produce anti-inflammatory effect at the peak of carrageenan induced edema but with less activity than that of the standard anti-inflammatory agent (aspirin).The extract at doses 250 mg/kg and 1000 mg/kg was able to significantly produce anti-inflammatory effect at the 3rd, 4th and 5th hours with the effect being dose dependent at the 4th and 5th hour.The extract however showed higher inhibition significantly (P < 0.01) of induced edema compared to the standard drug (Aspirin) at the 4th and 5th hour with percentage inhibition of 62.57% and 82.88% respectively (Table 3).Inflammation induced by carrageenan is thought to be biphasic (20).The early phase is mediated by histamine, serotonin, and prostaglandin increased synthesis in the damaged surrounding tissues.The late phase is sustained by prostaglandins produced by tissue macrophages and prostaglandins released and mediated by polymorhphonuclear cells, leukotrines and bradykinins.Mona et al (6) reported that secondary metabolites like flavonoids, tannins, saponins, alkaloids found in plants have anti-inflammatory effects.The mechanism underlying the anti-inflammatory effects of tannins includes the scavenging of radicals and inhibition of the expression of inflammatory mediators such as some cytokines, inducible nitric-oxide synthase and COX-2 (21).Some flavonoids have anti-inflammatory property and produce activity by inhibiting molecular targets of pro-inflammatory mediators in inflammatory responses (21).Wang et al (22) has reported that the antiinflammatory effects of steroids could be attributed to a counter-irritation effect.

Conclusion
The aqueous methanol extract of A. africanus leaves had

Acute toxicity
The acute toxicity of the extract was done per oral in rats.The LD50 was found to 3807.89mg/kg (the following formula) and there was death of an animal at dose 5000 m in phase II (Table 2).

Anti-inflammatory activity of aqueous methanol leaves extract of A. africanus
anti-inflammatory activity which may explain the basis of its use in traditional medicine in the management of inflammation and related inflammatory disorders.The significant anti-inflammatory activity of the extract observed might be associated to the secondary metabolites present in the plant and this may proffer scientific basis for its use.But, systemic important dose should be avoided due to the toxicity risk.Further bioassay-led fractionation and isolation of active principles are to be undertaken.

Table 1 .
Qualitative phytochemical constituent of aqueous methanol extract of Allophylus africanus leaves

Table 2 .
Median lethal dose (LD 50 ) of aqueous methanol leaves extract of Allophylus africanus administered orally

Table 2 :
Median Lethal dose (LD50) of aqueous methanol leaves extract of A. africa administered orally

Table 3 .
Effect of the aqueous methanol extract of Allophylus africanus on carrageenan induced paw edema in rats P < 0.05, compared with normal saline group, **P < 0.01 compared with normal saline group (One-way ANOVA followed by Tukey's post hoc multiple comparison test).Data expressed as Mean ± SEM. values given in parentheses represent PI -Percentage inhibition. *