Solvent, reagents, and chemicals
The solvents, chemicals, and reagents used were of analytical grade. Sigma-Aldrich (St Louis, MO, USA) provided the Bovine serum albumin, epinephrine, 20% tricloroacetic acid, 1% thiobarbituric acid and Dithionitrobenzene. Hematoxylin and eosin were obtained from ScyTek Laboratories Inc. (West Logan, UT, USA). Analytical grade methanol, ethanol, n-hexane, ethylacetate, and butanol were obtained from BDH Chemical Ltd (Poole, England).
Drug
Omeprazole BP 20 mg (OMEFAST-20) Greenlife Pharmaceutical Limited, Lagos, Nigeria.
Experimental animals
Male Wistar rats weighing 120–180 g were obtained from the animal house of the Department of Veterinary Medicine (University of Nigeria Nsukka, Nigeria). They were raised under ideal conditions of temperature (25 °C), humidity (50–60%), and a 12-h light–dark cycle. They were allowed unrestricted access to standard feed and water. They were allowed 14 days to familiarize with the lab conditions prior to the commencement of the experiment. The Faculty of Pharmaceutical Sciences, University of Nigeria’s ethics committee (FPSRE/UNN/19/00015), gave approval of the experimental procedure in accordance with the National Code of Conduct for Animal Research Ethics (NCARE).
Plant collection and preparation
In February 2020, young leaves of C. paniculatum were collected along the bank of a stream during its flowering stage at Obukpa, Nsukka Local Government Area, Enugu State, Nigeria. It was identified, validated, and issued voucher number PCG/UNN/0321 to the plant. Fresh leaves were cleaned twice with water and placed on dry water soaking papers for drying under open, airy, and shady place for five days. The shade-dried leaves were then coarsely milled using a laboratory blender.
Plant extraction and fractionation
Cold maceration in methanol for 48 h with occasional agitation was used to extract crushed leaves (2.6 kg). A filter cloth and No. Whatman filter paper (1) was used to filter the mixture. The marc was then rinsed several times with the new solvent until the filtrate became transparent. To obtain the methanol extract of C. paniculatum (MECP), the filtrate was placed in a stainless tray and left to air-dry. The resultant dry extract was collected, weighed, and stored in an amber-colored container in a refrigerator.
MECP was further fractionated to obtain the n-hexane fraction (HFCP), ethyl acetate fraction (EAFCP), and butanol fraction (BFCP). The MECP (50 g) was weighed and crushed using a mortar and pestle, and a paste was formed using a small quantity of water. A greater quantity of water was then added until the desired consistency was achieved. The resultant solution was transferred into a separating funnel, an equal quantity of n-hexane was added to the separating funnel, and the mixture was shaken properly. The mixture was allowed to stand undisturbed using a retort stand until a clear separation was achieved. Using a beaker, the aqueous phase was separated from the n-hexane phase. This process was repeated until a clear n-hexane phase was obtained. This procedure was repeated for ethyl acetate and butanol.
$${\text{Yield}}\left( \% \right)\;{\text{of}}\;{\text{extract}} = \frac{Weight \;of\;extract\; \left( g \right)}{{Weight \;of\;powdered\;plant\; material\; used\; \left( g \right)}} \times 100$$
(1)
$${\text{Yield}}\left( \% \right)\;{\text{of}}\;{\text{fraction }} = \frac{Weight \;of\;fraction\; \left( g \right)}{{Weight\; of\;extract\;used\; \left( g \right)}} \times 100$$
(2)
Qualitative phytochemical screening of extract and fractions
Qualitative phytochemical analysis of powdered plant material samples was performed according to standard methods [17,18,19].
Quantitative phytochemical analysis of extract and fractions
Standard procedures for quantitative phytochemical analyses have been employed [18,19,20,21,22].
Acute toxicity test
The acute toxicity studies on MECP were performed using Lorke’s method, which involves two stages. The toxic range was determined in the first stage by administering 10, 100, and 1000 mg/kg MECP to three groups of mice (n = 3). Treated mice were occasionally monitored for symptoms of toxicity and death for 24 h. The dosages used in the second stage were determined based on the mortality trend of the previous stage. As no death occurred and no signs of toxicity were observed in the first phase, the second phase was initiated. A new group of four mice was orally administered 1000, 1600, 2900, and 5000 mg/kg MECP. The treated mice were monitored intermittently for 24 h to assess the symptoms of toxicity and mortality. The LD50, geometric mean of the highest non-lethal dose, and least toxic dose were obtained [23].
Absolute ethanol-induced ulcer
The absolute ethanol-induced ulcer study was performed according to previously described methods [3]. The rats were fasted for 24 h but allowed unrestricted access to water prior to the study and were allotted into five groups of six rats each. Group 1 received 3% Tween 80 (5 ml/kg) as a negative control, Group 2 received the standard drug omeprazole (20 ml/kg) with strength 5 mg/ml whereas Groups 3–5 received 100, 200, and 400 mg/kg of methanol extract. After 1 h, the animals received absolute ethanol (1 ml each) and were sacrificed 1 h later using chloroform. The number and length of gastric lesions were determined by dissecting and opening their stomachs along the greater curvature, rinsing them under running water, and pinning them on a corkboard. The ulcer score was further measured using a ruler and divider. An absolute ethanol-induced ulcer scoring method was used.
The lesions were viewed macroscopically using magnifying lens (× 10) and the size of the lesions were measured with the aid of a vernier caliper.
The scoring method of An et al., 2007, was adopted with slight modification [24].
The total score for each stomach divided by a factor of 10 was calculated for each animal and expressed as ulcer index (UI) and the average taken as mean ulcer index for each group. The degree of ulcer protection for each treated group was calculated as a percentage of the mean ulcer index of the negative control group.
A similar study was also performed with solvent fractions at three dose levels (100, 200, and 400 mg/kg).
Oxidative stress
Preparation of tissue homogenate
Following sacrifice, the stomach tissue was immediately excised, weighed, and completely homogenized in phosphate buffer (10 mM KH2PO4-K2HPO4 buffer, pH 7.4; 0.1 mM EDTA). The homogenates were centrifuged at 5000 rpm for 20 min. The supernatant was used to test for oxidative stress markers. Using bovine serum albumin (BSA) as a reference, the protein content of the supernatant was measured using the Lowry et al. technique [25].
Tissue catalase test
Tissue catalase activity was determined according to a method described previously [26]. When dichromate in acetic acid is heated in the presence of hydrogen peroxide (H2O2), it is reduced to chromic acetate, creating chromic acid as an unstable intermediate. The amount of hydrogen peroxide produced in the reaction was proportional to the amount of chromic acetate produced. The generated chromic acetate was quantified spectrophotometrically at 570 nm. Briefly, 0.04 ml of tissue homogenate was mixed with 2.96 ml of H2O2 (0.2 M)-phosphate buffer (0.01 M, pH 7). Dichromate/acetic acid reagent (2 ml) was added to this mixture to terminate the reaction after a 1 min interval. To eliminate precipitated proteins, the tubes were heated to 100 °C for 10 min, cooled, and centrifuged at 2500×g for 5 min. A spectrophotometer (Jenway 6305; Jenway, Essex, UK) was used to record variations in absorbance at 570 nm against a reagent blank. Tissue catalase activity (µmol H2O2/min/mg protein) was determined from a standard curve prepared similarly with 0–200 mMol H2O2.
Tissue reduced glutathione (GSH)
The presence of reduced glutathione in the tissue was evaluated using a previously described technique [27]. When dithionitrobenzene (DTNB) combines with acid-soluble sulfhydryl groups (non-protein thiols), a yellow-colored complex forms, with reduced glutathione accounting for > 93% of the total. The absorbance of the colored complex was determined at 412 nm. The tissue homogenate (0.5 ml) was combined with 0.1 ml of 25% TCA and placed on ice for a few minutes. The supernatant (0.3 ml) was then combined with 0.7 ml of 0.2 M sodium phosphate buffer (pH 8) and 2 ml of 0.6 mM DTNB after centrifugation at 3000 g for 10 min. A spectrophotometer (Jenway 6305; Jenway, Essex, UK) was used to determine the yellow color acquired after 10 min against a reagent blank at 412 nm. GSH concentrations (0–100 nMoles) were used to generate a standard graph. Using this standard graph, GSH content was computed and represented as nmol/mg protein.
Tissue malondialdehyde (MDA)
Malondialdehyde levels, a lipid peroxidation end-product, were determined using a spectrophotometric method. The reaction of thiobarbituric acid with MDA produces a persistent chromogen, which can be measured using spectrophotometry. The color intensity of the chromogen was measured at 532 nm, which is directly proportional to MDA concentration. The production of thiobarbituric acid reactive substances (TBARS) was used to measure lipid peroxidation in tissues using the Stocks and Dormandy's approach [28]. At room temperature, an equal volume of tissue homogenate was mixed with 20% trichloroacetic acid (1:1) and incubated. The samples were then centrifuged at 2500×g for 10 min. The samples were then placed in a boiling water bath (100 °C) for 15 min with 1.0 ml of 1% thiobarbituric acid added to the supernatant. The contents were cooled on ice before centrifugation at 2500×g for 15 min. A spectrophotometer (Jenway 6305; Jenway, Essex, UK) was used to measure absorbance (A) of the supernatant against a reagent blank at 532 nm. A standard graph was created using MDA concentrations ranging from 0 to 20 nM (Sigma-Aldrich). Louis, MO, USA). The degree of lipid peroxidation (TBARS) was measured in nanomoles per mg of protein.
Tissue superoxide dismutase (SOD)
The activity of superoxide dismutase (SOD) in tissues was measured as previously described [29]. SOD's capacity to prevent the autoxidation of epinephrine to adrenochrome at pH 10.2 is the basis for the test. The homogenate (0.5 ml) was diluted with an equal volume of distilled water, then 0.25 ml ice-cold ethanol and 0.15 ml ice-cold chloroform were added. This was combined well in a cyclo-mixer before being centrifuged for 10 min at 2500 rpm. 1.5 ml carbonate buffer (0.05 M, pH 10.2) and 0.5 ml 0.5 mM EDTA solution were added to the supernatant. The reaction was started by adding 0.4 ml of 3 mM epinephrine (Sigma, St. Louis, MO, USA) and measuring the change in absorbance per minute at 480 nm against a reagent blank. The enzyme unit was defined as the change in absorbance per minute when superoxide dismutase inhibited the conversion of epinephrine to adrenochrome by 50%. Using 0–195 units of SOD (Sigma, St. Louis, Mo, USA). An enzyme calibration curve was generated. SOD activity was measured in units per mg of protein.
Histopathological studies
Tissue preparation
Histopathological analysis of the stomach sections was performed. Samples were fixed in 10% phosphate-buffered formalin for 48 h. The tissues were subsequently trimmed, dehydrated in four grades of alcohol (70, 80, 90, and absolute alcohol), cleared in three grades of xylene, and embedded in molten wax.
On solidification, the blocks were sectioned, 5 µm thick with a rotary microtome, floated in a water bathe, and incubated at 60 °C for 30 min. The 5-µm-thick sectioned tissues were subsequently cleared in three grades of xylene and rehydrated in three grades of alcohol (90, 80, and 70%). The sections were then stained with hematoxylin for 15 min. Blue was performed using ammonium chloride. Differentiation was performed using 1% acid alcohol before counterstaining with eosin. Permanent mounts were made on degreased glass slides using DPX mountant.
Slide examination
The prepared slides were viewed using ×4, ×10, and ×40 objective lenses on a Motic™ compound light microscope. Photomicrographs were obtained at ×160 magnification with a Motic™ 5.0 megapixels microscope camera.
Statistical analysis
GraphPad Prism version 7.0 was used to analyze the data. One-way ANOVA was used, followed by Dunnett's multiple comparisons post hoc test. Data are presented as mean ± standard error of the mean (SEM). P < 0.05, 0.01, and 0.001 were considered statistically significant.
