Antioxidant and antiulcer potential of aqueous leaf extract of Kigelia africana against ethanol-induced ulcer in rats.

Ethnobotanical claims regarding Kigelia africana reported antiulcer properties as part of its medicinal application. In this work, aqueous leaf extract from K. africana was investigated for its phytochemical constituents and antiulcer potential against ethanol-induced ulcer in rats. The participation of oxidative stress on ethanol-induced ulcer and the potential protective antioxidant activity of K. africana extracts were investigated by determining vitamin C and thiobarbituric acid reactive species (TBARS) contents in the gastric mucosa of rats. The HPLC analysis showed the presence of gallic acid, chlorogenic acid, caffeic acid and also the flavonoids rutin, quercetin and kaempferol in the aqueous plant extract. Oral treatment with K. africana extract (1.75; 3.5; 7 and 14 mg/kg) one hour after ulcer induction with ethanol decreased in a dose dependent manner the ulcer index. Ethanol increased significantly stomachal TBARS levels and decreased vitamin C content when compared to the control animals. K. africana blunted the ethanol-induced oxidative stress and restored vitamin C content to the control levels. The present results indicate that the aqueous leaf extract from K. africana possesses antiulcer potential. The presence of flavonoids in plant extract suggests that its antiulcerogenic potential is associated with antioxidant activity. Of particular therapeutic potential, K. africana was effective against ethanol even after the induction of ulcer, indicating that it can have protective and curative effects against gastric lesion.

Introduction

The use of traditional medicine is expanding globally. It continues to be used not only for primary health care in developing countries, but also in countries where conventional medicine is predominant in the national health care system (WHO, 2000[32]). In South Africa, 60-80 % of the population relies solely or partially on traditional herbal medicines to treat a variety of animal and human diseases (Dausdardt, 1990[2]). In the late 1980s, an estimated 65 % of the world's population depended on or at least used medicines derived from plants in health care (Farnsworth, 1988[3]).

Kigelia africana (Sausage tree) belongs to the family Bignoniacae and in the South-western part of Nigeria hot-infusion preparations of its leaves are popularly used to treat stomach ulcer. This plant is widely spread across Africa and is abundantly found in wet savannah where it occurs in abundance. Traditional remedies prepared from crushed dried fruits are used for emollient, antieczema, antipsoriasis, as dressing for ulcers and wounds, treatment of skin cancer, as anaphrodiasic and also as an active ingredient in skin lightening and breast firming formulations (Maisiri and Gundidza, 1999[17]). The polar extract of K. africana fruit contains verminoside as the major constituent and a series of other phenols (Verbascoside, Caffeic Acid, p-Coumaric acid and Caffeic acid methyl ester) (Picerno et al., 2005[26]). Remedies from root bark are also used for the treatment of venereal diseases, hemorrhoids, and rheumatism (Oliver-Bever, 1986[25]). However, little is known about the pharmacological properties of its leaves.

Previously, preliminary data from our laboratories have indicated that the ethanolic leaf extract of K. africana has antiulcerogenic potential against aspirin-induced ulcer (Olaleye, 2005[22]). However, the molecular mechanism involved in the protection, namely, the role of oxidative stress was not investigated. Furthermore, there is dearth of information as regards the antiulcerogenic potential of the leaf extract of this plant against ethanol-induced ulcer. Pharmacological and scientific validation of the ethnobotanical claims regarding the plant is essential to move towards the use of the plant as a drug. Since the literature reports suggest the ethnobotanical use of this plant in stomach disorders and skin ulceration, it becomes important to study the antiulcer potential of the extract against ethanol-induced gastric ulcer.

Ethanol-induced gastric ulcers have been widely used for the evaluation of gastroprotective activity of different compounds (Al-Shabanah, 1997[1]; Hwang et al., 2008[12]; Ineu et al., 2008[13]). Furthermore, oxygen radicals and lipid peroxidation are thought to be involved in the ethanol-induced gastric damage and antioxidant compounds can have gastroprotective properties (Ineu et al., 2008[13]). Of particular pharmacological importance, K. africana has both in vitro and in vivo antioxidant properties (Olaleye and Rocha, 2007[24], 2008[23]) and there is no work about participation of the antioxidant effect of this plant on its antiulcer properties. This work is therefore designed to evaluate the potential protective effect of aqueous leaf extract from K. africana against ethanol-induced ulcer and the possible protection role of the extracts against oxidative stress in ethanol-induced ulcer in rats.

Materials and Methods

Sample collection and preparation

The plant sample Kigelia africana (Sausage tree) was collected from Ado in Ekiti State, Nigeria. Authentication was done at the Biology Department of the Federal University of Technology, Akure, Nigeria. The leaves of the plants were cleaned, air dried at room temperature for two weeks, ground to a powder and boiled in hot water for 3 hours, filtered and concentrated to dryness. The % yield for the extract was about 20 %.

Bioassay

Adult Wistar albino rats (160-250 g) were collected from our own breeding colony. They were all clinically healthy and maintained in standard environmental conditions of temperature (24 + 2 °C), 12 h dark/light cycle. They were fed a standard diet and water ad libitum. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria RS, Brazil. The gastric lesions were induced with ethanol according to the method described by Robert (1979[28]). After a 36 h fasting, the animals received a single dose of 0.5 ml of ethanol or water (control group) orally by gavage. Then, after one hour animals received 1 ml of water (control and ethanol group) or 1 ml of one of the doses of aqueous leaf extract of K. africana by gavage, resulting in six groups of five animals each as follows:

Group I: Control

Group II: Ethanol group [treated with 0.5 ml ethanol (70 %) only]

Group III: Induced with 0.5 ml ethanol (70 %) and treated with Kigelia africana at 1.75 mg/Kg)

Group IV: Induced with 0.5 ml ethanol (70 %) and treated with Kigelia africana at 3.5 mg/kg

Group V: Induced with 0.5 ml ethanol (70 %) and treated with Kigelia africana 7 mg/Kg

Group VI: Induced with 0.5 ml ethanol (70 %) and treated with Kigelia africana at 14 mg/kg

One hour after the administration of extracts, the animals were anaesthetized and euthanized. Stomach was removed and cut open along the greater curvature. The stomachs were rinsed under a slow stream of water and pinned flat on a cork board. The stomachs were examined macroscopically to assess the degree of ulceration. The ulcerative lesion index (UI) of each animal was calculated according to the method of Gamberini et al. (1991[4]). The stomach linings were scraped weighed and homogenized in 0.1 M phosphate buffer in 1:10 wt/ vol and centrifuged for 15 minutes at 2000 ×g. The homogenates were used to assay for TBARS and Vitamin C.

Lipid peroxidation

The extent of gastric lipid peroxidation was determined using the method described by Ohkawa et al. (1979[21]) as slightly modified by Puntel et al. (2007[27]).

Ascorbic acid determination

Ascorbic acid determination was performed as previously described by Jacques-Silva et al. (2001[14]). Stomach protein content was precipitated in 10 volumes of cold 4 % trichloroacetic acid solution. An aliquot of the sample in a final volume of 1 mL of the solution was incubated for 3 h at 38 °C, and subsequently 1 mL H2SO4 65 % (v/v) was added to the medium. The reaction product was determined using a color reagent containing 4.5 mg/mL dinitrophenyl hydrazine and CuSO4 (0.075 mg/mL) at 520 nm (Spectrophotometer U-2001 Hitachi, Japan).

HPLC analysis of Kigelia africana crude extract

All chemical were of analytical grade. Methanol, acetic acid, gallic acid, chlorogenic acid and caffeic acid purchased from Merck (Darmstadt, Germany). Quercetin, rutin and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High performance liquid chromatography (HPLC-DAD) was performed with the HPLC system (Shimadzu, Kyoto, Japan), Prominence Auto Sampler (SIL-20A), equipped with Shimadzu LC-20AT reciprocating pumps connected to the degasser DGU 20A5 with integrator CBM 20A, UV-VIS detector DAD (diode) SPD-M20A and Software LC solution 1.22 SP1.

Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm x 250 mm) packed with 5 µm diameter particles; the mobile phase was water containing 2 % acetic acid (A) and methanol (B), and the composition gradient was: 5 % of B until 2 min and changed to obtain 25 %, 40 %, 50 %, 60 %, 70 % and 100 % B at 10, 20, 30, 40, 50 and 80 min, respectively, following the method described by Laghari et al. (2011[15]) with slight modifications. The aqueous extract of the plant was dissolved in water and analyzed at a concentration of 4 mg/mL. The presence of six phenolic compounds was investigated, namely, gallic, chlorogenic and caffeic acids and the flavonoids quercetin, rutin and kaempferol. Identification of these compounds was performed by comparing their retention time and UV absorption spectrum with those of the commercial standards. The flow rate was 0.6 ml/min, injection volume 40 µl and the wavelength were 254 nm for gallic acid, 325 nm for caffeic and chlorogenic acids, and 365 nm for quercetin, rutin and kaempferol. All the samples and mobile phase were filtered through 0.45 µm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.031-0.250 mg/ml for kaempferol, quercetin and rutin; and 0.006-0.250 mg/ml for gallic, caffeic and chlorogenic acids. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 500 nm). Calibration curve

for gallic acid:

Y = 11707x + 1459.8 (r = 0.9996);

chlorogenic acid:

Y = 15882x + 1326.3 (r = 0.9993);

rutin:

Y = 12473x – 1075.7 (r = 0.9999);

quercetin:

Y = 16135x – 1092.6 (r =0.9997) and

kaempferol: Y = 16923x – 1353.9 (r = 0.9998).

All chromatography operations were carried out at ambient temperature and in triplicate.

Statistical analysis

Data were analyzed statistically by one-way ANOVA, followed by Duncan’s multiple range tests when appropriate. The results were considered statistically significant at p < 0.05.

Results

Ulcer index

Ethanol caused a statistically significant increase in the ulcer index when compared to the control group (p < 0.05) and K. africana treatment caused a dose dependent decrease in this index (Table 1(Tab. 1)). In fact, K. africana extract was effective at the lowest dose tested (1.75 mg/kg) and it had a maximal effect at the dose of 7 mg/kg (Table 1(Tab. 1)).

Table 1

Indexes of ethanol-induced stomachal ulcer in albino rats pos-treated with leaf extract of Kigelia africana as compared to the control

Vitamin C

Ethanol caused a significant decrease of about 20 % in stomach vitamin C content (p < 0.05) when compared to the control group. K. africana leaf extract (1.75 to 7 mg/kg) restored the vitamin C content to control levels. The dose of 14 mg/kg of K. africana extract increased vitamin C levels but this effect was not statistically significant (Figure 1(Fig. 1)).

Figure 1

Vitamin C content of control, ethanol and K. africana treated rats. Values are expressed as means ± SE of 5 replicates. The letter “a” indicates significant difference from control group (p < 0.05). Columns marked with the same letter are not statistically different.

TBARS

Ethanol caused statistically significant increase in the TBARS formation as compared to the control and treatment with K. africana abolished the pro-oxidant effect of ethanol (Figure 2(Fig. 2)).

Figure 2

TBARS levels of control, ethanol and K. africana treated rats. Values are expressed as means ± SE of 5 replicates. The letter “a” indicates significant difference from control group (p < 0.05).

HPLC analysis

HPLC fingerprinting of Kigelia africana aqueous extracts revealed the presence of the

gallic acid (tR = 17.07 min; peak 1),

chlorogenic acid (tR = 30.11 min peak 2),

caffeic acid (tR = 34.27 min peak 3),

rutin (tR = 47.94 min; peak 4),

quercetin (tR = 55.83 min; peak 5) and

kaempferol (tR = 64.56 min; peak 6) (Figure 3(Fig. 3) and Table 2(Tab. 2)).

Figure 3

Representative high performance liquid chromatography profile of K. africana. Detection UV was at 325 nm.

Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), rutin (peak 4), quercetin (peak 5) and kaempferol (peak 6). Chromatographic conditions are described in the Methods section.

Table 2

Phenolics and flavonoids composition of aqueous extract of leaves from K. africana

Discussion

Plant extracts are among the most attractive sources for developing new drugs and have been shown to produce promising results in the treatment of gastric ulcer (Hiruma-Lima et al., 2000[10][11]; 2001[9]). Accordingly, in traditional medicine several plants with antioxidant properties have been used to treat gastrointestinal disorders (Olaleye and Rocha, 2007[24], 2008[23]; Sabir and Rocha, 2008[30]; Sabir et al., 2012[29]; Leite et al., 2009[16]) including gastric ulcers (Toma et al., 2002[31]). Plants continue to play a major role as therapeutic remedies in primary health care in developing countries and the use of aqueous extracts are very common for the population around the world (McDonald et al., 2001[19]). Here, we have demonstrated that aqueous extract of Kigelia africana protected the gastric mucosa decreasing in a dose dependent manner the ulcer index caused by ethanol.

Of particular importance, literature data have indicated the antiulcer properties of flavonoids (Gonzalez and Di Stasi, 2002[6]; Gracioso et al., 2002[7]). The analyses of the plant extract by HPLC revealed the presence of several poliphenolic compounds such as Gallic acid, Chlorogenic acid. Caffeic acid, rutin, quercetin and kaempferol. The flavonoids rutin, quercetin and kaempferol, which were found in relatively high concentrations in aqueous extract of K. africana, are secondary metabolites present in plants and have attracted the attention of many researchers because the wide range of their biological activities (Harborne, 1996[8]; Middleton et al., 2000[20]; Manthey et al., 2001[18]; Zaveri, 2006[33]).

Reactive oxygen species (ROS) can cause deleterious effects at the cellular level which can culminate in cell death. In line with this, the increase in gastric TBARS contents and the decrease in vitamin C levels after ethanol administration suggest that oxidative stress play an important role in ethanol-induced ulcer, which is in accordance with literature data (Ineu et al., 2008[13]; Glavin and Szabo, 1992[5]). The ability of K. africana extract to ameliorate the gastric damage and to normalize vitamin C and TBARS contents indicate that its antiulcer effect was mediated, at least in part, by its antioxidant activity (Olaleye and Rocha, 2007[24], 2008[23]).

Taken together, the results obtained here indicated the antiulcer potential of K. africana, which validates its popular use in the treatment of gastrointestinal ailments. Importantly, the protective effect of K. africana crude extract was observed one hour after the injection of ethanol, indicating that this plant can have protective and curative effects against ethanol-induced ulcer. Furthermore, part of the antiulcer effect of K. africana can be attributed to the presence of antioxidant flavonoids and more studies are need to determine which other components of the aqueous plant extract could be also involved in K. africana gastroprotective effects.

Acknowledgements

We gratefully acknowledge the financial support and the offer of Post doctoral fellowship granted Mary T. Olaleye by Conselho Nacional de Pesquisa (CNPQ) and the Academic of Science for Developing Countries (TWAS). Work supported by CNPq, CAPES-PROCAD/SAUX/PROAP/NOVOSTALENTOS, INCT for excitotoxicity and neuroprotection, FINEP-IBNet, FINEP and CNPQ/TWAS to M. Olaleye-J.B.T. Rocha, CNPq-PRO-AFRICA.