Plant materials

Stem bark and leaves of P. nitida (Voucher number 45138/HNC) were collected in May 2009 in the Bakassa village (West Region, Cameroon) and the S. oleraceus (Voucher number 37069/HNC) whole plant harvested in the University of Yaoundé I Campus (Centre Region, Cameroon). The identification of both plants species were confirmed by the Cameroon National Herbarium in Yaoundé where Voucher specimens were deposited.

Extract preparation

The different plant parts collected were air-dried in the dark and ground to powder. For the methanol extraction, 200 g each of S. oleraceus powder whole plant and P. nitida stem bark and leaves, were separately macerated in 500 mL of methanol for three days with thorough homogenization twice per day. Regarding the hydroethanol extraction, 500 mL of distilled water - 95% alcohol (1:1, v/v) was used to macerate 200 g each of powder from S. oleraceus and similarly as for methanol extraction. At the end, each macerate was filtered with Whatman No 1 filter paper, and the filtrate evaporated to dryness using a rotatory evaporator (Bűchi 011, USA).

Phytochemical analysis

The extracts were screened for detection of different chemical families according to the methods previous described by Odebeji and Safowara [10]. In brief, phenolic compounds were detected using the ferrocyanide reaction; triterpenes and sterols were revealed by their reactivity with anhydrous acetate and sulphuric acid. Alkaloids were detected using sulphuric acid, whereas the presence of saponins revealed based on their foaming property. Tanins and flavonoids were revealed using ferric chloride and hydrochloric acid, respectively. Anthraquinones were detected in extracts by the chloroform/petroleum system, while the presence of lipids was assessed on filter paper.

DPPH radical-scavenging activity

The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radical-scavenging activity of the extracts as described by Hatano et al. [11] with slight modification [12]. Different concentrations (0.25, 0.5, 1 and 2 mg/mL) of each extract were prepared in distilled water, 30 μL of each solution mixed with 1 mL of ethanol solution of DPPH (0.1 mM) and incubated for 30 min in the dark. At the end of this period, the absorbance was recorded at 517 nm using a spectrophotometer, and the antiradical activity of each concentration calculated as percentage reduction of DPPH concentration, with reference to the optical density at the start, as followed:

% scavenged [Free radicals] = [(A o − A1)/A o ] × 100 where A o was the absorbance of the control and A 1 was the absorbance in the presence of the sample of extract or standard.

The IC 50 values were then generated by extrapolation from the curve of activity versus concentration.

Reduction potential of the extracts

In order to investigate the reduction potential of the different extracts, their polyphenol contents were determined using the method described of Folin-Ciocalteu as described earlier by Singleton and Rossi [13], with some modifications [14]. In brief, 30 μL of extract of known concentration were thoroughly mixed with 10 mL of Folin-Ciocalteu (Sigma) 0.2 N and the absorbance measured at 750 nm, after 30 min incubation at room temperature in the dark. Catechine solutions in methanol at 10, 20, 30, 40 and 60 μM were used as standards.

Diabetes induction in rats

The animals were kindly provided by the Animal House of the Department of Animal Biology and Physiology, Faculty of Science at the University of Yaoundé I (Cameroon). The antidiabetic profiles of the extracts with significant antiradical and antioxidant activities was assessed using two complementary approaches: the glucose intolerance test (hypoglycaemic activity), and the evaluation of activity in rats with induced diabetes.

Hypoglycaemic activity

A total of 45 three month-old Wistar rats (20 males, 25 females) with body weight of 285-310 g, were divided into 9 groups with comparable average body weight. The different treatments were administered orally as indicated in Table 1. During the period of treatment, water was available to the mice ad libitum. Fasting blood sugar (FBS) was first measured for all the animals before administration of the different treatments. The different extracts and Glibenclamide (positive control) were then administered orally 30 min after administration of glucose. FBS was then taken 0.5, 1.5, 2.5, 3.5 and 4.5 hours post hyperglycaemia induction. Each time, FBS was measured from a blood drop collected from the rat’s tail, using a glucometer (Accucheck, USA). The hypoglycaemic potential of the extracts was evaluated as their ability to correct the hyperglycaemia 4.5 hours after induction.

Table 1 Treatment of different groups of mice for the hypoglycaemic activity assay Full size table

Antidiabetic activity

The acute and sub-acute activities of the different extracts were determined in rats with streptozotocin-induced diabetes, following the method of Al-Shamaony et al. [15]. Male rats were subjected to overnight fasting prior to the diabetes induction. Each of them subsequently received an intravenous injection of 50 mg/Kg streptozotocin (Sigma) dissolved in 0.1 citrate buffer at pH 4.5. After 3 days, the animals with at least 250 mg/dL FBS were considered “diabetic” and used for the assays.

Acute activity

Seven groups of 5 rats each were based on their body weight, and treated as shown by Table 2. FBS was measured immediately before drug administration (0 h), and subsequently 1, 3 and 5 hours after drug administration, with all the animals maintained fasting and receiving water ad libitum. The acute antidiabetic potential of the extracts was determined as the ability of the single dose treatment to drop FBS in 5 hours.

Table 2 Treatment of different groups of mice for the antidiabetic activity assay Full size table

Sub-acute activity

A total of 35 rats were divided into 7 groups of 5 each and treated similarly as for the acute activity study. But in this case, the different drugs were administered daily, for 14 consecutive days, and FBS measured on days 0 (before drug administration), 4, 8 and 14 (end of the assay). At the end of the assay, the rats were sacrificed, blood collected in heparin to prepare plasma. The liver and kidneys were equally extracted and ground to form homogenates. The plasma and organ homogenates prepared were stored at -20°C until required for measurement of the markers of oxidative stress (Malondialdehyde, Catalase activity and hydrogen peroxide).

Titration of markers of oxidative stress in treated rats

Malondialdehyde

Serum MDA levels were estimated by the method of Yagi [16] using thiobarbituric acid (TBA). According to this method, the acid reacts with MDA to form a stable pink colour with maximum absorption at 535 nm. The reagent was prepared by mixing 375 mg of thiobarbituric acid, 20 g of Trichloroacetic acid, 10 mg of butyl-hydroxytoluene, 20 mL of HCl 1 N, in 50 mL of distilled water. The mixture was heated at 40°C until total dissolution of crystals, and the volume filled up to 100 mL with distilled water. 400 μL of this reagent was mixed with 300 μL of homogenate (10% w/v) and tightly capped in a glass tube protected from light with aluminium foil. The mixture was then boiled in a water bath at 100°C for 15 min, and the tubes allowed getting cold for about 30 min on ice. The mixture was subsequently centrifuged at 3000 rpm for 5 min and the absorbance of the supernatant recorded at 532 nm. The MDA content of the sample was calculated as follow:

MDA mol / L = A o – A 1 / ϵ x l x V

Where A 1 was the absorbance of the test sample; A o, absorbance of the negative control; ℇ = 1.53.105 M-1/Cm; l the diameter of the cuvette (l = 1 cm); and V was the total volume of the supernatant collected.

Hydrogen peroxide

The hydrogen peroxide content was determined by a methods previously described by Jiang et al. [17]. Briefly, the (FOX) ferrous oxidation in xylenol orange) reagent was prepared by mixing 88 mg of butylated hydroxytoluene, 7.6 mg of xylenol orange, 9.8 mg of ammonium sulphate, 90 mL of methanol and 10 mL of sulphuric acid, 250 mM. The mixture was then thoroughly homogenized before used. In a test tube, 100 μL of sample or distilled water (blank) was added to 900 μL of FOX reagent, homogenized and incubated at 37°C for 30 min. At the end, the absorbance was recorded at 560 nm and the hydrogen peroxide concentration was determined as followed:

H 2 O 2 μmol / L = A o – A 1 / ϵ x l x V

Where A 1 was the absorbance of the test sample; A o, the absorbance of the blank; ℇ = 1.53.105 M-1/Cm; l the diameter of the cuvette (l = 1 cm), and V was the total volume of the mixture.

Catalase

The catalase activity in blood and organ homogenate was measured by the method of Sinha [18]. For each of the tubes in a series of 3 per sample, 100 μL of the sample or distilled water (blank) was added to 250 μL of 1 time phosphate buffer and 200 μL of H 2 O 2 , and the reaction stopped after 0, 30 and 60 seconds for tube 1, 2, and 3 respectively. The reaction was stopped by adding 1 mL of dichromate-acetic acid mixture (100 mL of potassium dichromate dissolved in 300 mL of acetic acid). The tubes were then boiled at 100°C for 10 min, allowed to get cold and the absorbance recorded at 620 nm, for each incubation time. The OD values obtained were plotted against time in Microsoft Excel 2010 and the catalase activity calculated.

Statistical analysis

Each test was conducted at least in triplicate and all the replicate values pooled together into Mean ± Standard variation. The different test groups were compared to each other using ANOVA, whereas variations within the same group were evaluated using pair-t test. Correlations between the different activities were assessed. All the statistical analysis was conducted in SPSS at 95% and 99% confidence intervals.