Plant material
The leaves of C. myxa were collected from May to November 2015 from El-Orman botanical garden, Cairo, Egypt and identified by Prof. Dr. Nasser Barakat, Professor of Botany, Faculty of Science, Minia University. A voucher sample (Mn-Ph-Cog-023) was kept in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Minia University, Minia, Egypt.
Preparation of the extract and fractions
The air dried powdered leaves (4.24 Kg) of C. myxa. Were extracted by maceration with 95% ethanol at room temperature with occasional agitation for 7 days till exhaustion, and then concentrated under reduced pressure till dryness. The concentrated ethanol extract (275 g) was suspended in the least amount of distilled water, transferred to a separating funnel and partitioned successively by liquid/liquid extraction with petroleum ether, dichloromethane, ethyl acetate and finally with n-butanol. The fractions were concentrated under reduced pressure at 40 °C to afford petroleum ether (107 g), dichloromethane (13.2 g), ethyl acetate (11.74 g) and n-butanol (49.92 g) fractions.
Animals
The animals used in this study include female and male albino rats weighing 200 ± 50 g and mice weighing 30 ± 5 g, obtained from animal house of Faculty of Medicine, Assiut University. They were housed under standardized environmental conditions, and fed with standard diet and water. The study was conducted following approval by the Institutional Animal Ethical Committee of Faculty of Pharmacy, Minia University, Minia, Egypt.
Acute toxicity
The acute toxicity of the total ethanolic extract of Cordia myxa leaves was evaluated by measuring the lethal dose for 50% of the laboratory animals (LD50) [5]. Different dose levels (1, 2, 2.5, 3 up to 3.5 g/ kg, p.o) of the total ethanolic extract (suspended in 0.5% CMC) were orally administrated to different groups of mice (30 ± 5 g, each containing six mice). The control group received an equivalent dose of the vehicle (0.5% CMC). Both the test and control groups were noticed for 24 h under normal environmental conditions, with free access to food and water.
Evaluation of antioxidant study
Each sample was dissolved in 95% methanol to make a concentration of 40 mg/ml from total ethanolic extract and different fractions except the ethyl acetate fraction which was prepared as 10 mg/ml and then diluted to prepare the series concentrations for antioxidant assays.
Estimation of total phenolic content
The content of total phenolic compounds for total ethanol extract and different fractions of C. myxa leaves were determined by Folin–Ciocalteu method [6]. Analysis was performed by adding 3.5 ml of deionized water, 50 μl of sample extract, 50 μl Folin-Ciocalteu reagent (2 N) and 300 μl of sodium carbonate (10%). The reaction was left for 30 min. and then the absorbance was measured in triplicate at 730 nm. The blank consisted of all reagents excluding the sample extract. A standard curve was made using gallic acid so that total phenolic concentration was expressed as mg of gallic acid equivalents per gram dried fraction.
Estimation of total flavonoid content
Total flavonoid content was determined following a method in literature [6], where 0.3 ml of extracts, 3.4 ml of 30% methanol, 0.15 ml of NaNO2 (0.5 M) and 0.15 ml of AlCl3.6H2O (0.3 M) were mixed. After 5 min, 1 ml of NaOH (1 M) was added. The solution was mixed well and the absorbance was measured in triplicate against the reagent blank at 506 nm. The standard curve for total flavonoids was made using rutin standard solution at different concentration under the same procedure. The total flavonoid content was expressed as milligrams of rutin equivalents per gram of dried fraction.
Phosphomolybdate assay (total antioxidant capacity)
The total antioxidant capacity of the fractions was determined by phosphomolybdate method using ascorbic acid as a standard [6]. An aliquot of 0.3 ml of sample solution was mixed with 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a water bath at 95 °C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. A typical blank contained 3 ml of the reagent solution and the appropriate volume of the solvent and incubated under the same conditions. A standard curve was made using ascorbic acid; hence, the antioxidant activity was expressed relative to that of ascorbic acid. All determinations were done in triplicate.
DPPH radical scavenging activity assay
The free radical scavenging activity of the fractions was measured using 1,1- diphenyl-2-picryl-hydrazyl (DPPH) [6]. Briefly, 200 μl of each of the extract or fractions at various concentrations was added to 2 ml of DPPH solution (0.1 mM), The reaction mixture was shaken well and incubated in the dark for 15 min at room temperature. Methanol was used instead of the extract and fractions as a control. Then the absorbance was measured in triplicate at 517 nm. The capability to scavenge the DPPH radical was calculated using the following equation:
DPPH scavenging effect (%) = [(A0 -A1/A0) × 100].
Where A0 was the absorbance of the control reaction and A1 equal the absorbance in the presence of the extract. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of DPPH scavenging effect against extract concentration.
Anti-inflammatory activity
The total ethanol extract and different fractions of C. myxa leaves were evaluated for their anti-inflammatory activity using the carrageenan-induced paw edema method [7]. Female albino rats (200 ± 50 g) were randomly divided into seven groups (six animals per group). The specified dose of extract, fractions, and standard drug were suspended in 0.5% CMC solution. The –ve control group administered the vehicle (0.5% CMC solution), while the standard drug indomethacin (+ve control) was given orally at a dose level of 8 mg/kg. The total ethanol extract and different fractions were administered orally at a dose of 350 mg/kg through 2 h after carrageenan injection 0.1 ml, 1% w/v in normal saline, s.c.) into the sub-plantar tissue of the right hind paw. The paw thickness (mm) was measured using a vernier calliper at 0, 0.5, 1, 2, 3, 4 and 5 h after administration of the tested extract, fractions and standard drug. The percentage inhibition of the rat paw edema was calculated as follows [8]:
$$ \%\mathrm{Inhibition}=\frac{\left(\mathrm{Control}\ \mathrm{mean}-\mathrm{treated}\ \mathrm{mean}\right)}{\mathrm{Control}\ \mathrm{mean}}\mathrm{X}\ 100 $$
Analgesic activity
The analgesic activity of the total ethanol extract and different fractions of C. myxa L. leaves was evaluated using hot plate method [9]. Mice (30 ± 5 g) were grouped into seven groups (six animals each). The –ve control group administered the vehicle (0.5% CMC solution) and the standard drug paracetamol 100 mg/kg, p.o. (+ve control). The tested extract and different fractions were suspended in 0.5% CMC solution and were administered orally at a dose level of 350 mg/kg. The animals were placed on a hot plate and the temperature of the metal surface was maintained at 54 °C. The time (sec) of the response produced by the animal as tail withdrawn, licking paws or jumping due to radient heat is noted and recorded at 0, 0.5, 1, 2, 3, 4 and 5 h after the administration of the tested extract, fractions and the standard drug.
The percentage of protection against thermal stimulus was calculated as follows [10]:
$$ \%\mathrm{protection}\ \mathrm{against}\ \mathrm{thermal}\ \mathrm{stimulus}=\frac{\mathrm{Test}\ \mathrm{mean}\left(\mathrm{Ta}\right)\hbox{-} \mathrm{Control}\ \mathrm{mean}\left(\mathrm{Tb}\right)}{\mathrm{Control}\ \mathrm{mean}\left(\mathrm{Tb}\right)}\mathrm{X}\ 100 $$
Antipyretic activity
The total ethanol extract and different fractions of C. myxa L. leaves were evaluated for their antipyretic activity using yeast-induced pyrexia method [11, 12]. The test was performed on female albino rats (200 ± 50 g) by subcutaneous injection (in the back, below the nape of the neck) of 20% aqueous suspension of yeast in a dose of 10 ml/kg to induce pyrexia. The pyretic animals were grouped into seven groups (six animals each). The –ve control group orally administered the vehicle (0.5% CMC solution), while the +ve control was given the reference drug acetylsalicylic acid at a dose level of 330 mg/kg, p.o). The tested extract and different fractions were suspended in 0.5% CMC solution and were administered orally at a dose level of 350 mg/kg through 2 h after yeast injection. The rectal temperature of each animal was recorded by inserting a thermometer 2 cm into the rectum at 0, 0.5, 1, 2, 3, 4 and 5 h following the administration of the tested extract, fractions and the reference drug.
Anti-diabetic activity
The total ethanol extract and different fractions of C. myxa L. leaves were evaluated for their anti-diabetic effects using streptozotocin-induced hyperglycemia method [12]. The test was performed on adult male albino rats (200 ± 50 g) by intraperitoneal injection of streptozotocin (80 mg/kg). Blood glucose level was measured after 3 days up to 1 week for assessment of hyperglycemia. Rats with blood glucose level above (200 mg/dl) were considered to be diabetic and were used in this study. The diabetic rats were divided into seven groups (six rats each). The control group was administered the vehicle (0.5% CMC solution) and the standard drug vildagliptin 50 mg/kg p.o. (+ve control). The tested extract and different fractions were suspended in 0.5% CMC solution and were orally administered at a dose level of 350 mg/kg. Blood glucose levels were measured at intervals of 0 (fasting), 0.5, 1, 2, 3, 4 and 5 h by collecting blood samples from the tail vein (caudal vein). The percentage of change in blood glucose level was calculated by the following formula [12, 13]:
$$ \%\mathrm{lowering}\ \mathrm{of}\ \mathrm{blood}\ \mathrm{glucose}\ \mathrm{level}=\frac{\mathrm{Wc}\left(\mathrm{fasting}\right)-\mathrm{Wt}\left(\mathrm{test}\right)}{\mathrm{Wc}\left(\mathrm{fasting}\right)}\mathrm{X}\ 100 $$
Statistical analysis
Results of all biological studies were expressed as means ± S.E.M. One-way analysis of variance (ANOVA) followedby Dunnett’s test was used to determine significance when compared to the control group. p values less than 0.05, 0.01, and 0.001 were considered significant (*p < 0.05, **p < 0.01,*** p < 0.001). Graph Pad Prism 5 was used for statistical calculations (Graph pad Software, San Diego California, USA).
