Piper guineense fresh leaves, were harvested from a farm land in Naze, Owerri, Imo State, Nigeria, during rainy season in the month of August, 2018. The plant material was identified, authenticated and deposited as voucher specimen (Herbarium Number: UPH/P/251) for further reference in the Department of Plant Science and Biotechnology, University of Port Harcourt, Nigreia.

Healthy adult Wistar rats of 12-15 weeks old that weighed between 170 and 190g and healthy adult albino mice of 12-15 weeks old that weighed between 20 and 22g were employed in the study. The animals were reared in Animal Facility Unit of Madonna University, Nigeria, under well-ventilated room temperature. They were housed in secured cages, floored with adequate wood shavings/saw-dusts, fed freely with commercial poultry growers feed (Top feeds^R, Nigeria) and supplied with clean drinking tap water. Animal ethics approval (Reference number: MAU/SREC/A/17) was granted by University Senate Research and Ethics Committee of Madonna University, Nigeria

Animal handling compliances as published earlier 21 were strictly followed.

All studies that involved the use of animals were conducted at the Animal Facility Unit, Madonna University, Nigeria, Elele Campus.

Drugs and reagents employed in this work included the following: Histamine (Fine Chemicals, Mumbai, India), Phenolphthalein (Fine Chemicals, Mumbai, India) Cimetidine (Cadila Pharmaceuticals Pvt Ltd, India), Chloroform (Super Tek Chemicals, India), Sodium Hydroxide (Rankem Mumbai, India), Tween 80 (3%v/v) (Super Tek Chemicals, Germany), Ethylacetate (Rankem, Mumbai, India), Dragendorff’s reagent (Super Tek Chemicals, Germany), n-Hexane (Sigma Aldrich Chemie, Germany) Tween 80 (3%v/v) (Super Tek Chemicals, Germany), 96% Ethanol (Gungsdong Guandgua Chemical Factory, China), Topfer’s reagent (Fine Chemicals, Mumbai, India)

As indicated in our earlier study, 22 about three kilograms of matured fresh leaves of Piper guineense were properly washed in water to remove foreign matters and air-dried under shade for two weeks to obtain a dry mass for powdering. Using a roller grinder, the dried leaves were ground into coarse powder, and about 500g was soaked in two liters of ethanol (80%) at ambient temperature. The soaked material was occasionally stirred four times a day (every six hours) for three days. The resultant solution was filtered through filter papers (Whatmann, No 1) to separate the filtrate from the marc. The marc was re-soaked and re-filtered. After filtration, all the obtained filtrates were gathered together in a previously weighed clean empty beaker. The beaker and its content were placed in an oven at 40^oC until ethanol was evaporated and dried solid residue obtained.

Thin-layer chromatography (TLC) 23 and column chromatography 24 as reported earlier 22 were employed in the preparation of fraction extracts from ethanol-extracted residue. Preliminary thin-layer chromatography (TLC) was performed on eight solvent systems constituted in different ratios, to obtain the one that would give the best resolution. 22 The solvent system (Chloroform /Ethylacetate/Ethanol; 7:2:1) that gave the best resolution was subsequently used in column chromatography to obtain the fraction extracts and to determine their TLC mobility (R_f values). In column chromatographic procedure, 10g of ethanol-extracted residue was dissolved in 10ml ethanol, placed on the column and then continuously eluted with Chloroform /Ethylacetate/Ethanol (7:2:1) solvent system. Seventeen-10ml fractions were collected (labeled FE-1 to FE-17) and their TLC mobility (R_f values) was calculated as follows 22

Rf=Distance (cm) travelled by the spot from starting point in TLCDistance (cm) travelled by the solvent front in TLC

Fractions with similar color reaction and R_f value were appropriately gathered together in a previously weighed clean beaker, then labeled as: pooled fraction-1 (PF-1); pooled fraction-2 (PF-2); pooled fraction-3 (PF-3); pooled fraction-4 (PF-4) and pooled fraction-5 (PF-5). Each beaker and its content were placed in an oven at 40^oC to expel the eluting solvent system to obtain a dried solid mass. After drying, the labeled containers were stored in the refrigerator until when needed.

Various beakers and their dried content were re-weighed, compared with the initial weight of empty beakers, and difference in weight was noted. Using the formula proposed by, 25 percent yield was calculated as the ratio of weight (g) of the extracted residue to weight (500g) of the soaked powdered material multiplied by hundred

Both the ethanol extract and fraction extracts were tested for acute toxicity, to ascertain doses that could be safe in subsequent whole animal experiment. Healthy adult albino mice (20-22g) of 12-15 weeks old that fasted over night were used. A method involving two phases as demonstrated by 26 that used a minimal number of thirteen (13) animals was employed. In this method, defined doses of 10mg/kg, 100mg/kg and 1000mg/kg of the extracts were respectively administered orally to three groups of animals (n=3) in the phase one of the experiment, after which, the animals were observed for 24 hours for any sign of toxicity and death. Based on the result obtained in phase one, the phase two of the experiment was conducted, in which defined doses of 1000mg/kg, 1600mg/kg, 2900mg/kg and 5000mg/kg of the extracts were respectively administered to four groups of animals (n=1) and then observed for sings of toxicity or death for another 24 hours

Fifty six healthy adult Wistar rats of 12-15 weeks old that weighed between 170 and 190g were randomly placed into eight experimental groups comprising of six test groups (labeled A to F) and two control (positive and negative) groups (labeled G and H respectively). Each test and control groups consisted of seven animals per group (n=7). Each group was fasted for 24 hours prior to the study but was allowed liberty to drink water until two hours to the experiment. Drug and extracts were orally administered through intra-gastric tube and the dose of extracts administered was safe, as ascertained in acute toxicity study. Various groups of animals were treated in following way:

Each rat in Group A received at once, 400mg/kg PF-1, orally

Each rat in Group B received at once, 400mg/kg PF-2, orally

Each rat in Group C received at once, 400mg/kg PF-3, orally

Each rat in Group D received at once, 400mg/kg PF-4, orally

Each rat in Group E received at once, 400mg/kg PF-5, orally

Each rat in Group F received at once, 400mg/kg EE, orally

Each rat in Group G (positive control) received at once, 100mg/kg cimetidine, orally

Each rat in Group H (negative control) received at once, 5ml/kg 3% v/v Tween 80, orally

Following the respective treatment above (i.e. 30 minutes later), ulcer was induced in each animal via intragastric administration of a dose of 40mg/kg histamine. 27

Six hours after the induction of ulcer, the animals were humanely sacrificed under anesthesia and their stomachs dissected. Gastric content of each animal was drained into a separate graduated tube while the stomach was washed under tap water.

Using hand lens (magnification: x10), the washed stomachs were pinned flat on a board and examined for ulcer/lesion formation. The number of ulcers was counted and scoring made as proposed by 28 as follows:

Normal colored stomach = 0;

Red coloration = 0.5;

Spot ulcer = 1;

Hemorrhagic streak = 1.5;

Deep ulcer (ulcer > 3 but ≤ 5mm) = 2;

Perforation(> 5mm) = 3

Equations proposed in, 29 were used to calculate the ulcer index and percent ulcer inhibition as follows:

Ulcer Index=Total ulcer scoreNumber of animals ulcerated

% Ulcer Inhibition=Ulcer index control negative-Ulcer index test groupUlcer index control negative group x 100

Gastric pH was determined according to procedure specified by 27. In this procedure, an 1ml aliquot of gastric juice was diluted with 1ml of demineralized water, and pH of the resulting solution was measured using pH meter (ADWA AD 8000).

The gastric content of each animal was centrifuged at 3000 rpm for ten minutes, after which, the supernatant portion was decanted and measured using measuring cylinder to obtain the gastric volume.

Determination of level of free acidity and total acidity of the gastric content were done according to Topfer’s method of gastric analysis as described by 30. In this method, 1ml aliquot was pipette out into 100ml conical flask, two drops of Topfer’s reagent (Dimethyl-amino-azo benzene) were added and titrated with 0.01N Sodium hydroxide until all traces of pink color disappeared and the color of the resulting solution turned into yellowish orange. The quantity of Sodium hydroxide added was recorded and this was taken to correspond to free acidity. Titration was continued until pink color of the obtained solution reappeared. Again the total quantity of Sodium hydroxide added was recorded and this volume was taken to correspond to total acidity. Using the equation proposed by³¹, acidity was calculated as follow:

Where V = volume of Sodium hydroxide

N = Normality of Sodium hydroxide

The calculated values of free and total acidity are expressed as mEq/L 31

Column chromatographic separation produced a total of seventeen-10ml fractions. Based on the R_f values, FE-1, FE-2 and FE-3 were pooled together as pooled fraction-1(PF-1); FE-4 and FE-5 were pooled together as pooled fraction-2 (PF-2); FE-6, FE-7, and FE-8 were pooled together as pooled fraction-3(PF-3); FE-9, FE-10, FE-11, FE-12, and FE-13 were pooled together as pooled fraction-4 (PF-4); FE-14, FE-15, FE-16 and FE-17 were pooled fraction-(PF-5).

As shown in Table 1, the quantitative yield of ethanol extract was 21.08g. Among the pooled fractions, pooled fraction-4 (PF-4) gave the highest yield of 3.81g.

Table 1

Extract	Yield (g)	% Yield
Ethanol	21.08	4.2
Pf-1	1.23	12.3
Pf-2	0.92	9.2
Pf-3	1.84	18.4
Pf-4	3.81	38.1
Pf-5	2.11	21.1

The acute toxicity study did not produce any toxicity symptoms at 5000mg/kg in mice. No mortality was observed within 48 hours in the treated groups, hence, the LD₅₀ of the extracts of Piper guineense leaf could be greater than 5000mg/kg.

The effects of the extracts and standard drug (cimetidine) on histamine ulcer model in Wistar rats were studied by comparing the percent inhibition produced by the extract-treated groups (test groups) with that produced by the 3%v/v Tween 80-treated group (negative control). From the result obtained, cimetidine at 100mg/kg and the extracts (ethanol and fractions) at 400mg/kg, significantly (P < 0.05) inhibited ulcer induced with histamine (40mg/kg). PF-3 produced the highest ulcer inhibition of 20.85% as shown in Table 2

Table 2

Treatment group	Dose (oral)	Ulcer index	% Inhibition
A (PF-1)	400mg/kg	5.78 ± 0.18	15.74*
B (PF-2)	400mg/kg	5.64 ± 0.21	17.78*
C (PF-3)	400mg/kg	5.43 ± 0.17	20.85*
D (PF-4)	400mg/kg	5.50 ± 0.31	19.83*
E (PF-5)	400mg/kg	5.79 ± 0.26	15.60*
F (EE)	400mg/kg	4.93 ± 0.30	28.13*
G (positive control)	100mg/kg	2.07 ± 0.93	69.83*
H (negative control)	5ml/kg	6.86 ± 0.28	—

Values represent mean ±SEM of seven animals in each group

Table 3 reveals that both the ethanol and fraction extracts significantly (p< 0.05) increased gastric acid pH. Pooled fraction-2 (PF-2) produced the highest increase (41.94%) in gastric pH.

Table 3

Treatment group	Dose (oral)	Gastric pH	% increase pH
A (PF-1)	400mg/kg	3.7 ±0.05 *	19.35*
B (PF-2)	400mg/kg	4.4 ±0.1 *	41.94*
C (PF-3)	400mg/kg	3.6 ±0.04 *	16.13*
D (PF-4)	400mg/kg	4.2 ±0.07*	35.48*
E (PF-5)	400mg/kg	3.4 ±0.03*	9.68*
F (EE)	400mg/kg	3.5 ±0.04*	12.90*
G ( Positive control)	100mg/kg	4.8 ±0.05 *	54.84*
H ( Negative control)	5ml/kg	3.1 ±0.08	-

Value represent mean ±SEM of seven animals in each group

Table 4 shows that the ethanol extract (EE), pooled fraction-3 (PF-3), pooled fraction-4 (PF-4) and pooled fraction-5 (PF-5) significantly reduced gastric volume (46.01%, 44.15%, 48.94% and 43.09% respectively); free acidity (54.90%, 47.84%, 33.82% and 35.05% respectively) and total acidity (34.54%, 34.60%, 46.18% and 29.35% respectively) compare to negative control (3% v/v Tween 80). Although gastric content volume and total acidity were significantly (P<0.05) reduced by pooled fraction-1 (PF-1), it did not reduce free acidity.

Table 4

Treatment group	Dose (oral)	Gastric volume(ml)	Free Acidity(mEq/L)	Total Acidity (mEq/L)
A (PF-1)	400mg/kg	2.99 ± 0.18(20.48%)*	40.68 ± 0.62 (2.35%)	62.57 ± 0.74(24.16%)*
B (PF-2)	400mg/kg	3.17 ± (15.69%)*	25.24 ± 1.17 (39.41%)*	45.17 ± (45.16%)*
C (PF-3)	400mg/kg	2.10 ± 0.15 (44.15%)*	21.73 ±0.65 (47.84%)*	53.86 ±0.28 (34.60%)*
D (PF-4)	400mg/kg	1.92 ± 0.17 (48.94%)*	27.57 ± 0.41(33.82%)*	44.33 ±1.80 (46.18%)*
E (PF-5)	400mg/kg	2.14 ± 0.17 (43.09%)*	27.06 ± 0.80 (35.05%)*	58.19 ±0.52 (29.35%)*
F (EE)	400mg/kg	2.03 ± 0.13 (46.01%)*	18.79 ±0.58 (54.90%)*	53.91 ±0.26 (34.54%)*
G (positive control)	100mg/kg	1.29 ± 0.20 (65.69%)*	5.64 ± 0.51(86.46%)*	11.83 ±0.53 (85.64%)*
H (negative control)	5ml/kg	3.76 ± 0.09 (0.00%)	41.66 ± 0.63 (0.00)	82.36 ±0.41 (0.00%)

Values represent mean ±SEM of seven animals in each group

This study reveals that the quantitative yield of the ethanol-extracted residue is low (21.08g) when judged with the amount (500g) of soaked powdered material. Pooled fraction-4 (PF-4) although comprised of five fractions, gave a low yield (3.81g) in relation to the proportional amount of soaked material. This finding conforms to the previous report 32 that active biological principles usually occur in plants in low concentration.

Acute toxicity test was conducted to assess the adverse effects that may occur within a short time after administrating a single dose of a test substance 33. Although earlier reports 34, 35 suggest that medicinal plants have advantages of toxicity considerations because of their long term use by humans, we conducted acute toxicity test of the extracts with the principal aim of ascertaining safe dose that could be used in further pharmacological screening. The result obtained indicated that up to LD₅₀ of 5000mg/kg of the leaf extracts, no symptom of toxicity was noticed, and therefore, it was judged to be safe for use in further pharmacological screening. This finding is substantiated by the report that compounds that do not show adverse effects when given in doses of 3000mg to 5000mg per kilogram body weight are essentially non-toxic 36.

Earlier report 37 showed that histamine plays an essential role in the pathogenesis of ulcer since it is a potent stimulator of H₂-receptors of parietal cells. Further report 29 indicated that histamine causes gastric ulcers by its potent stimulation of parietal cells that secrete gastric acid, and by its vasodilating capability, which leads to increased vascular permeability. These bio-pharmacological actions enable its use as an ulcer model for evaluating agents with anti-secretory and/or H₂-receptor-blocking potential 38. In this sutydy, the ability of Piper guineense ethanol and fraction extracts to block histamine-induced ulcer may suggest H₂-receptor blockade and/or anti-secretory as the mechanism of action. This finding is similar to earlier report 39 that Cudrania tricuspidata ethanol extract prevents H₂-receptor related diseases like hyperacidity and reflux esophagitis.

Earlier report 40 suggested that elevated concentration of the hydrogen ion is a destructive factor that promotes gastric damage via decreasing pH of the gastric juice. The result of this study shows that pre-treatment with Piper guineense leaf extract significantly (P<0.05) elevated gastric pH levels with simultaneous decrease in gastric secretion (volume); an effects that support anti-secretory and/or H₂ receptor-blocking activity of the leaves. These findings correlate with the earlier report 41 on Cuphea ignea, which significantly increased gastric pH, and reduced gastric volume.