Phytochemical screening, antioxidant, antiulcer and toxicity studies on Desmodium adscendens (Sw) DC Fabaceae leaf and stem

Purpose: To assess the phytochemical profile, toxicity, as well as the antioxidant, and antiulcer activities of the methanol extracts of Desmodium adscendens stem and leaf. Methods: Maceration procedure was employed in the preparation of the methanol extracts. Phytochemical characterization of the extracts was carried out according to standard methods. In vitro antioxidant activity was evaluated using 2, 2-diphenyl-1-picryhyldrazy (DPPH l) and ferric reducing antioxidant power assay (FRAP). Antiulcer activity was investigated using ethanol-induced ulcer model, while toxicity was assessed by observing the mice for mortality. Results: Phytochemical analysis indicate the presence of glycosides, alkaloids, tannins, flavonoids, and saponins in the stem and leaf. Methanol extracts of the plant exhibited antioxidant activity, with DPPH assay results showing median inhibitory concentration (IC50) of 87.59 (leaf), 108.87 (stem), 28.52 (alpha-tocopherol), and 5.05 μg/mL (ascorbic acid). The FRAP assay results for the stem and leaf extracts were 1483 and 1953 μM Fe/g dry plant, respectively, while for ascorbic acid it was 3463 μMFe/g. The extracts showed significant antiulcer activity, with 14.27 and 15.18 % ulceration inhibition for the leaf extract, and 12.31 and 13.36 % for the stem extract at administered doses of 100 and 200 mg/kg, respectively. Cimetidine and omeprazole (standards) showed ulceration inhibition of 5.53, and 8.26 % at 5.7 and 0.57 mg/kg doses, respectively. Conclusion: The methanol extracts of Desmodium adscendens stem and leaf offer significant protective activity against ethanol-induced gastric ulceration in rats, and the activity may be related to their antioxidant effect.


INTRODUCTION
Desmodium adscendens (Family Fabaceae) is a climbing woody stem plant which grows in an uncultivated land on the West Coastal region of Africa. The species of Desmodium adscendens (D. adscendens) is frequently found in Nigeria, Cameroon, Zimbabwe, and tropical America. It is a sole runway hedge, preferring damp locations and it is found in forest areas and on the border of savannas [1].

Animals
Male Albino rats (180 -190 g), and Swiss albino mice (18 -20 g) were used in the study. The animals were acquired from the Animal care house at the Physiology department of the College of Medicine, Lagos University Teaching Hospital, Idi araba campus, Lagos, Nigeria. They were harboured in a dirt free cage with the rats separated from the mice and preserved in a well ventilated room. They fed on standard pellet diet (Livestock Nigeria brand) and tap water was made available ad libitum. The animals were maintained under standard condition of (23 ± 1 o C), on a light/day cycle for 12 h and the cage cleaned frequently. The study procedure was in compliance with the nationally accepted guidelines for laboratory animal use and care and consent was given by Health Research Ethics Committee (HREC), of the College of Medicine, University of Lagos, Nigeria, with approval number CMUL/HREC/07/17/212. The Guide for the Care and use of Laboratory Animal was followed for the research [7].

Collection of plant material
D. adscendens leaves and stems were gathered from Ibadan, Nigeria. The materials were authenticated by Mr.
Adeleke at the Pharmacognosy department, University of Lagos, and a sample was kept at the herbarium of Pharmacognosy department, University of Lagos, Nigeria with the voucher number PCGH164. The samples were dried at ambient temperature and milled into a uniformly dried powder.

Preparation of extracts
Dried powdered leaf sample (200 g) was immersed in 1000 mL of 96 % (w/v) methanol for 3 days. The extract was sieved through Whatman filter paper no. 42 (125 mm) and the filtrates collected were dried using a rotary evaporator at 40 o C. It was suitably labelled and kept in a cool place until use. The same process was replicated for the stem sample.

Phytochemical screening
The qualitative phytochemical examination of the extracts of D. adscendens stem and leaf was conducted by using standard methods [8,9]. The extracts were examined for the following phytochemicals; flavonoids, glycosides, alkaloids, tannins, and saponins using appropriate reagents.

DPPH radical scavenging activity
The scavenging activity of D. adscendens extracts against 2, 2-diphenyl-1-picrylhydrazyl radical was ascertained by reading the UV absorbance at 517 nm, as stated by the modified procedures of Amaeze et al, and Brand-Williams et al [10,11]. Different concentrations of the extracts (0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL) were prepared with methanol, using alphatocopherol, and ascorbic acid as standards. For each prepared solutions, 2 ml was transferred into test tubes and added to it was 0.5 ml of 1 mM 2, 2-diphenyl-1-picrylhydrazyl solution in methanol. The tests were done in triplicates. The prepared solution was agitated and allowed to rest for 15 min at ambient temperature, and the absorbance was measured spectrophotometrically at 517 nm [12]. DPPH inhibitory activity (D) was computed as in Eq 1. Reduction in absorbance measurement indicates the extent of antioxidant activity of the solutions.
where A 0 is blank absorbance, A 1 is test sample absorbance and D is the DPPH inhibitory activity.

Total antioxidant assay (FRAP assay)
An adapted method of Benzie and Strain [13] was assumed for the ferric reducing antioxidant power (FRAP) assay. The principle involved relies on the capability of the test sample to decrease the ferric tripyridyltriazine (Fe [III]-TPTZ) complex to ferrous tripyridyltriazine (Fe [II]-TPTZ) at low a pH. Fe [II]-TPTZ has an intense blue colour which was taken at 593 nm. Newly prepared FRAP solution (1.5 mL), containing 25 mL of 300 mM acetate buffer at pH 3.6, 2.5 mL of 10 mm 2,3,6-TPTZ in 40 mM HCl, and 2.5 ml of 20 mM Iron(lll)chloride hexahydrate (FeCl 3 .6H 2 O) solution was transferred to 1 mL of the extracts and absorbance taken at 593 nm [14]. Calibration plot was linear between 50 -300 μM and it was prepared with FeSO 4 .7H 2 O solution. Results are indicated in μM Fe (II)/g dry plant material and were juxtaposed with that of ascorbic acid.

Total phenolic content
The phenolic content of the extract of D. adscendens was determined on the authority of Folin and Ciocalteau method [15] with slight modifications. The calibration curve was derived by adding gallic acid in ethanol (1 mL, 0.025 mg/mL) to 5 ml Folin-Ciocalteau reagent (on ten folds dilution) and Na 2 CO 3 (4 mL, 0.7 M). Absorbance was determined at 765 nm. One ml of the extracts in ethanol (5 mg/ml) was added to the reagent, mixed and after 2 h the absorbance was read to confirm the phenolic content. Each test was done in triplicate. The result was exhibited as gallic acid equivalent (GAE) mg/g, using the equation derived from the calibration curve: y = 11.523x + 0.678, R 2 = 0.999.

Total flavonoid content
The flavonoid content was studied with the procedure of Miliauskas et al [16]. To 2 ml of the extracts in methanol (concentrations of 0.1 and 1 mg/mL), was added 2 ml of 2% AlCl 3 in ethanol and the absorbance was read at 420 nm after 1h of incubation at room temperature. The concentrations of rutin prepared in methanol (0.01, 0.02, 0.04, 0.08 and 0.10 mg/mL) were used to establish the calibration plot. Total flavonoid content was calculated as rutin equivalent (RE) in mg/g using the equation derived from the calibration curve: y = 0.5451 x + 0.0203, R 2 = 0.9205.

Toxicity studies
Acute toxicity study was conducted by using gradient doses of each extracts in albino mice. They were splitted into four groups, each of seven mice each, the stem extract were administered orally in graded doses (5,000, 10,000, 15,000 and 20,000 mg/kg body weight). They were observed at various time intervals (30 min, 1, 2, 3 and 4 h) for toxic symptoms and mortality.
Sub-acute toxicity was conducted by a single administration of the dose to the animals with daily observation and weighing of the mice for one week [17].

Anti-ulcer activity study
Ulcer was established by administering ethanol, and the route of administration of prepared solutions was by oral. The adult male albino rats were made to abstain from food for 36 h prior to the administration of ethanol. The animals were split into seven groups, each made up of four rats except for the control group which contains three rats: Group I: animals were administered distilled water only and this represents the control group After about 30 min, all animals were orally given 1 ml of ethanol and thereafter kept in a specially built cage to prevent coprophagia. They were afterwards decapitated after 1 h of administration; the stomach was incised along the greater curvature and cleansed in physiological saline solution to remove debris. It was laid flat and gastric mucosa lesions immediately traced on acetate paper using a magnifying lens. Ulcer protection (U) was calculated as in Eq 2.

Statistical analysis
The data are presented as mean ± standard error of mean (SEM). Comparisons were done between the treated groups by two-way analysis of variance (ANOVA) using SPSS. The level of statistical significance was set at p < 0.05.

RESULTS
Phytochemical evaluation of the D. adscendens stem and leaf unveiled the confirmation of flavonoids, glycosides, saponins, tannins, and alkaloids.
DPPH assay data showed inhibitory activity ranging from 3.17 -91.37 % for leaf extract, 2.87 -61.65 % for stem extract, 43.93 -92.58 % for alpha tocopherol, and 95.97 -97.97 % of ascorbic acid (Figure 1)    Phytochemical quantification showed the phenolic content of D. adscendens leaf and stem as 10 mg GAE/g and 24 mg GAE/g of plant material, respectively, while the flavonoid content was 2.76mg/g and 2.54mg/g for leaf and stem extracts respectively. This is similar to the results from the work of Konan et al [19] who found total phenolics (3.76 mg GAE/g) and (2.15 mg GAE/g) for the leaf and stem; also 0.077 and 0.02 % of flavonoids were found in the leaf and stem.
The scavenging property of D. adscendens stem and leaf extract may be ascribable to the phenolic compounds that can supply the necessary component as a radical scavenger. The IC 50 value for the leaf (87.59 µg/mL) was lower than the stems extract (108.87 µg/mL), which reflects a good demonstration of its antioxidant activity. While the research of Konan et al demonstrates that the ethyl acetate extracts of the leaf of D. adscendens has IC 50 260 µg/mL for radical scavenging property based on DPPH assay [19]. Free radicals are often generated as a result of biological reactions or from exogenous factors, its implication in the progression of some disease conditions is well established and has been published [20]. The antioxidant property of D. adscendens stem and leaf could be related to the high amount of flavonoid and phenolic compounds in this plant extract [21].
Ethanol-induced intestinal ulcer is a suitable model for exploring the gastroprotective effects of herbal drugs. The causes of peptic ulcer are still unknown in most cases; nevertheless, it has been recommended that it results from a variation between some destructive elements and the preservation of mucosal cohesion through the internal defense mechanisms. To restore this balance, different therapeutic agents like H2-blockers, proton pump inhibitors, and plant based substances have been used [22]. D. adscendens stem and leaf extracts used in this study to evaluate the anti-ulcerogenic in ethanol induced ulcers in rats showed a significant dose dependent ulceration inhibition activity at 100 and 200 mg/kg respectively. This could arise from a cytoprotective effect of the extracts through anti-oxidant effects. The anti-ulcer effect of D. adscendens stem and leaf could arise from two factors: reductions in gastric acid release and gastric cytoprotection. The remarkable increase in the anti-ulcer effect of D. adscendens leaf and stem extracts might be accredited to the confirmation of saponins, glycosides, tannins, and flavonoids compounds. Flavonoids are in the group of cytoprotective substance where its antiulcer efficacy has been studied [23]. Hence, the anti-ulcer activity of D. adscendens may be assigned to its flavonoids content.
It is well known that ethanol causes disorder in gastric secretion, destruction of the mucosa alters the permeability of the gastric membrane, gastric mucus depletion, and free radical production. This has been accredited to the release of free hydroperoxy and superoxide anion radicals produced during metabolism of ethanol. Free radicals derived from oxygen have been indicated in the mechanism of action of acute and chronic ulceration in the mucous membrane of the gastrointestinal tract [24]. Ethanol induction of gastric ulcer which causes the development of necrotic features of tissue lesion and haemorrhage, may arise from inactivity in gastric blood flow. The alcohol rapidly infiltrates the intestinal mucous membrane and causes destruction to the plasma and cell membrane, which leads to increased intracellular membrane permeability to sodium and water.
Furthermore, an enormous intracellular increase of calcium correlates with a vital pace in the development disease state of gastric mucosal lesion [22]. It is indicated that active phytochemical compounds would be able to stimulate mucus, bicarbonate and the prostaglandin secretion and, counteracts the distorting activity of reactive oxidants in the stomach and intestinal lumen [25]. Hence, this research recommends that the methanol extract of D. adscendens stem and leaf may be used to forestall gastric lesions. Thither is a demand to identify specific molecular species present in the extracts which are responsible for its gastroprotective effect, nevertheless, this would warrant further investigations.

CONCLUSION
The findings of this study indicate that methanol extracts of Desmodium adscendens stem and leaf are rich in phenolics and flavonoids, with high antioxidant activities, and also may offer a new gastroprotective treatment against ethanolinduced ulcers.