INTRODUCTION:

Peptic ulcer disease is a problem of the gastrointestinal tract characterized by mucosal damage secondary to pepsin and gastric acid secretion. It usually occurs in the stomach and proximal duodenum; less commonly, it occurs in the lower esophagus, the distal duodenum, or the jejunum, as in unopposed hyper-secretory states such as Zollinger-Ellison syndrome, in hiatal hernias (Cameron ulcers), or in ectopic gastric mucosa (e.g., in Meckel's diverticulum)¹.

Peptic ulcer disease had a tremendous effect on morbidity and mortality until the last decades of the 20^th century, when epidemiological trends started to point to an impressive fall in its incidence.

Two important developments are associated with the decrease in rates of peptic ulcer disease: the discovery of effective and potent acid suppressants, and of Helicobacter pylori. With the discovery of H pylori infection, the causes, pathogenesis, and treatment of peptic ulcer disease have been rewritten. We focus on this revolution of understanding and management of peptic ulcer disease over the past 25 years. Despite substantial advances, this disease remains an important clinical problem, largely because of increasingly widespread use of non-steroidal anti-inflammatory drugs (NSAIDs) and low-dose aspirin. We discuss the role of these agents in the causes of ulcer disease and therapeutic and preventive strategies for drug-induced ulcers. The rare but increasingly problematic H pylori-negative NSAID-negative ulcer is also examined.² The maximal capacity for acid production by the stomach reflects total parietal cell mass. Both parietal cell mass and maximal acid secretion are increased up to twofold in patients with duodenal ulcers. However, there is a large overlap with normal values and only one third of these patients secrete excess acid.

Accelerated gastric emptying, a condition that might lead to excessive acidification of the duodenum, has been noted in patients with duodenal ulcers. However, as with other factors, there is substantial overlap with normal rates. Normally, acidification of the duodenal bulb inhibits further gastric emptying.

MATERIALS AND METHODS:^3-15

Collection of plant:

The herb Calycopteris floribundas Lam were collected from Palakkad, Kerala. The plant material was taxonomically identified and authenticated by the botanist Dr. Maya. C. Nayar, M.Sc, PhD, Associate professor, Department of botany, Govt. Victoria College, Palakkad -578001 Authentication number: VS001

Extraction of plant material:

The powdered plant materials were successfully extracted with petroleum ether (40-60ºC) by hot continuous percolation method in Soxhlet apparatus (Harborne, 1984). for 24 hrs. Then the marc was dried and then subjected to ethyl acetate extraction (76-78ºC) for 24 hrs, then marc was dried and then it was subjected to methanol extraction (80ºC) for 24hrs. The solvent from the extracts was recovered under reduced pressure using rotary evaporator and subjected to freeze drying in a lyophilizer until dry powder was obtained.

Preliminary phytochemical screening:

Natural products are the source of synthetic and traditional herbal medicine.The medicinal importance of plant due to the presence of some special substances like alkaloids, glycosides, tannins, flavonoids, saponins etc.,were determined by performing chemical tests.

Pharmacological screening:

Male wistar rat (average body weight 200 –300g) were used for antiulcer study. The rats were procured from, department of pharmacology, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore. The study protocol was approved by the IAEC No: RVSCOPS/IAEC-2020- 2021 and all procedures were performed in accordance with the recommendations for the proper care and use of laboratory animal.

Acute toxicity studies:¹⁸

All rats were monitored continuously for 24hour after dosing for signs of toxicity. For the remainder of the Calycopteris floribunda Lam (5g/kg) 14 days study period, animals were monitored and any additional behavioural or clinical signs of toxicity. Animal’s body weight was measured prior to dosing and on days 7 and on all animals was killed and at the end of the study LD₅₀ value was established. Clinical observations and gross pathological examination was carried out.

Stress ulcer by cold water immersion:

Cooling of rats in water during the restraint period accelerates the occurrence of gastric ulcers and shortens the time of necessary immobilization. The lengths of the longest diameters of the lesions are measured and summated to give a total lesion score (in mm) for each animal, the mean count for each group being calculated.

Ethanol induced gastric ulcer:

Intra gastric application of absolute ethanol is a reproducible method to produce gastric lesions in experimental animals (Robert et al., 1979; Szabo et al., 1981). These lesions can be at least partially inhibited by various drugs, such as some prostaglandins. The protective effect against various irritants has been called cytoprotective activity.

Stress ulcer through immobilization stress:

Psychogenic factors, such as stress, play a major role in the pathogenesis of gastric ulcers in man. The experimental model resembles the psychogenic factors in the pathogenesis of gastric ulcers in patients. Therefore, it is not surprising that not only antacids, anticholinergic, H₂-– antagonists, proton pump inhibitors, but also psychotropic drugs like neuroleptics have been found to be effective in this test.

Estimation of Physical and biochemical parameter:^19,20

Physical Parameters:

Ulcer index is measured by using following formula:

U_I = U_N + U_S + U_P × 10^-1

Where,

U_I = Ulcer index

U_N= Average of number of ulcer per animal

U_S= Average of severity score

U_P = Percentage of animal with ulcers

Total acidity, pH, and free acidity, acid volumes were determined.

Biochemical parameters:

In this study, all the groups were treated with C. floribunda and standard drugs. After the treatment period, the animals were anaesthetized and the blood was collected from Cardiac Puncture by using capillary into a centrifugation tube for serum biochemical parameters. Serum was separated by centrifugation at 3000rpm for 10 min and analyzed for SGOT (or AST – Aspartate Aminotransferase) and SGPT (or ALT- Alanin aminotransferase), total protein and super oxide dismutase were determined.

Histopathological analysis:

After the study period, the rats were anaesthetized. Stomach was dissected out perfused with chilled saline to remove blood and blood clots and fixed in 10% w/v formalin saline. Paraffin blocks were prepared and tissue was stained with hematoxyline and eosin, and subjected to routine histopathology. The photographs were taken under 100× magnification.

Statistical analysis:

Data were expressed as mean±standard error mean (SEM) of six replicated determination and then analysed by Graph pad prism – Version 8.0.2 computer software program. Significance was calculated by one-way ANOVA followed by Dunnett’s multiple comparison tests. The significance of difference wasaccepted at P<0.01.

RESULT AND DISCUSSION

Screening of anti ulcer activity

Table No. 1: Stress ulcer by cold water immersion (Physical parameters)

Treatment	No of animals	Dose (mg/kg)	Total acidity (m/eq)	Ulcer index (mm²/rat)	Free acidity (m/eq) 100 g	pH	% Inhibition
Control	6	-	73.13±1.70	14.20±0.21	37.33±1.80	3.20±0.44	-
Pantoprazole	6	20	28.46±0.76	4.69±0.11	14.40±0.36	5.37±0.98	59.76%
(100mg/kg p.o) (C.floribunda)	6	100	69.06±0.72	8.30±0.16	24.66±0.66	4.16±0.87	45.23%
(200mg/kg p.o) (C.floribunda)	6	200	39.83±1.31*	5.62±0.11*	22.60±0.93*	5.00±0.45	61.42%

Table No. 2: Stress ulcer by cold water immersion (Biochemical parameters)

Group and Treatment	No of animals	Dose (mg/kg)	SOD (mmol/min/mg)	Total protein (g/dl)	AST OR SGOT (U/L)
Control	6	-	140.67±2.60	6.377±0.415	168.33±6.01
Pantoprazole	6	20	216.00±5.30**	5.390±2.60**	129.67±4.26**
(100mg/kg p.o) (C.floribunda)	6	100	170.67±4.91**	5.823±0.104***	151.66±4.41**
(200mg/kg p.o) (C.floribunda)	6	200	212.67±2.73***	3.840±0.096*	131.66±4.41**

Table No. 3: Ethanol induced gastric ulcer method (Physical parameters)

Treatment	No of animals	Dose (mg/kg)	Total acidity (m/eq) 100 g	Gastric volume (ml)	pH	Ulcer index (mm²/rat)	%Inhibition (%)
Control	6	-	112.1±1.13	7.33±0.21	2.2±0.10	11.85±0.30	-
Pantoprazole	6	20	58.5±0.84**	4.73±0.33*	4.9±0.16**	4.80±0.42**	72.52%
(100mg/kg p.o) (C.floribunda)	6	100	87.5±0.22*	6.05±0.11*	3.32±3.05	6.55.00±0.28	46.02%
(200mg/kg p.o) (C.floribunda)	6	200	63.2±0.516*	5.05±0.09*	4.05±4.77**	4.91±0.004*	65.67%

Table No. 4: Ethanol induced gastric ulcer method (Biochemical parameters)

Group and Treatment	No of animals	Dose (mg/kg)	Glutathione	AST OR SGOT(U/L)	ALT OR SGPT(U/L)
Control	6	-	4.08±0.09	152.67±5.04	97.00.±2.65
Pantoprazole	6	20	6.7±0.15**	116.00±0.06**	63.00±5.04**
(100mg/kg p.o) (C.floribunda))	6	100	5.95±0.25**	133.67±3.38**	75.33±2.60**
(200mg/kg p.o) (C.floribunda))	6	200	5.01±0.25**	120.33±2.60*	60.33±3.16**

Table No. 5: Stress ulcer induced by immobilization (Physical parameters)

Treatment	No of animals	Dose (mg/kg)	Total acidity (m/eq) 100g	Gastric volume (ml)	pH	Ulcer index (mm²/rat)	Inhibition (%)
Control	6	-	117.05±2.5	9.4±0.3	2.44±0.14	15.6±1.2	-
Pantoprazole	6	20	37.32±1.3**	2.1±0.14**	4.8±0.12*	2.4±0.04**	86
(100mg/kg p.o) (C.floribunda)	6	100	67.45±3.2**	3.1±0.06**	3.6±0.12**	3.9±0.81**	76
(200mg/kg p.o) (C.floribunda)	6	200	61.23±2.4*	2.65±0.04***	4.2±0.11***	2.64±0.81***	82

Table No. 6: Stress ulcer induced by immobilization (Biochemical parameters)

Group and Treatment	No of animals	Dose (mg/kg)	AST OR SGOT(U/L)	ALT OR SGPT(U/L)
Control	6	-	172.67±8.04	99.00.±2.65
Pantoprazole	6	20	136.00±1.06**	73.09±5.94*
(100mg/kg p.o) (C.floribunda)	6	100	153.67±0.38*	85.39±2.80**
200mg/kg p.o) (C.floribunda)	6	200	140.33±9.60*	70.30±3.66**

Histopathological studies:

Methods	Group-I Control	Group –II (pantaprazole)	Group III (C.floribunda) *(100mg/kg p.o)*	Group-IV (C.floribunda) *(200mg/kg p.o)*
Cold water immersion induced stress ulcer
Ethanol induced gastric ulcer
Immobilization induced stress ulcer in model

Figure 1-12: Histopathology of stomach

DISCUSSION:

The plant Calycopteris floribunda Lam is widely distributed in Bangladesh and India. The antiulcer effect of Calycopteris floribunda Lam leaf have been never studied. Hence the objective of the study is determining this effect from the Calycopteris floribunda Lam leaf extract.

The preliminary phytochemical screening of leaf extracts indicate in presence of flavonoid, alkaloid, tannins, and glycosides may accounts anti-ulcer potential.

The antiulcer effect is screened in leaf extract of Calycopteris floribunda Lam on cold water immersion, ethanol and immobilization induced dose dependent ulcer study. The results get from these study have been shown that leaf extract of Calycopteris floribunda Lam produce antiulcer effect in cold water immersion, ethanol and immobilization induced ulcer models. In cold water immersion, ethanol and immobilization induced model, there is reduction in ulcer index, total acidity, total volume of gastric contents, total protein concentration and higher concentration of glutathione content and pH of gastric secretion they compared with control treated group.

Pantoprazole used as a standard comparison agents. Pantoprazole is a proton pump inhibitors blocker, is significantly reduce about 90% of basal, food induced and hormonal mediated gastric acid, which again induced by gastrin, and parasympathomimetic drugs. In cold water immersion method and immobilization, stress ulcer is induced. Continous exposure to the stress cause depletion of protective neurotransmitters such as noradrenaline (NE), dopamine (DA), and γ-aminobutyric acid (GABA), which exert a protective effect on the gastric mucosa through an inhibitory control on acid secretion and motility. Due to the depletion of protective neuropeptides, the activation of the parasympathetic system characterized by ulcerogenic release factors such as acetylcholine and thyrotropic releasing hormone (TRH) and, as consequence, we assist to an increase in gastric motility and acid secretion. The treatment of leaf extract showed reduces the total acidity of the gastric contents.

On ethanol administration, the mucosal mast cells mediate to secretion of vasoactive mediators containing histamine. Histamine is mediated to stimulate the synthesis of cyclic AMP through activation of the enzyme adenyl cyclase which mediate the activation of gastric proton pump and secrete of hydrogen ions. The treatment of leaf extract showed reduce the total acidity of the gastric contents.

Serum protein including albumin and globulin. In the peptic ulcer the total protein concentration of serum or gastric secretion are increased. This may be due to leakage of plasma protein in to the gastric secretion or serum with lower mucosal resistance/barrier of the gastric mucosal layer After treatment with C. floribunda extract there was a significant reduction in protein concentration of gastric juice which enhancing leakage of plasma proteins.

Acid volume is amount (in ml) of acid release in the gastric content release contain HCl, pepsinogen enzyme, mucus secretion, bicarbonates concentration, intrinsic factor and proteins. Amount of acid release is an important factor responsible for the production of ulcer mediated by exposure of the unprotected lumen of stomach by concentrated acids. C. floribunda extract treatment showed decrease in the acid volume of the gastric secretion.

Increased pH shows a lower concentration of the hydrogen ion. The hydrogen ion is a major triggering factor responsible for the etiologic factor for ulcer and gastric damage. C. floribunda extract treatment indicates higher concentration of pH of the gastric juices. This values directly shown the C. floribunda extract reduce possibility of ulcer and has a protective effect of surface of the gastric mucosa.

SGOT (AST) and SGPT (ALT) are liver enzyme, this levels are increased which indicates injury caused to stomach tissue, cell membrane gets damaged, these enzymes leak into blood stream causing damage to the gastric mucosa in the ulcer induced model whereas it showed in SGOT (AST) and SGPT (ALT) levels are decreased in C. floribunda extract treated groups.

In gastric ulcer tissues, Glutathione (g-glutamyl cysteinyl glycine, GSH) levels were found to be decreased. Ethanol-induced genesis of free radical concentration reduces the cysteine concentration which mediated for GSH released. Values from this study responsible for depletion of gastric GSH are related with induction of gastric lesion in the rats. GSH is a tripeptide and having a superoxide radical scavenger and it protect thiol protein contents essential for release the integrity of tissue against oxidation reaction. In my present study, Calycopteris floribunda Lam treatment showed increase in the glutathione content. Pre-treatment with floribunda extract treatment reduce the ulcer index value, total acidity concentration, total volume of acid release and total protein and increase value pH and glutathione content when compared with control groups.

CONCLUSION:

In conclusion, the present study provided preliminary data for the Calycopteris floribunda Lam. possess significant antiulcer activity in animal models. It has a gastric antisecretory and acid neutralizing effect that are comparable to standard drug Pantaprazole. The antiulcer activity is probably due to the presence of bioactive compounds like Flavonoids, Tannins. These compounds protect and strengthen the mucosal barrier which may be responsible for the antiulcer activity.

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Received on 20.03.2024 Revised on 31.05.2024

Accepted on 02.07.2024 Published on 07.12.2024

Available online on December 30, 2024

Res.J. Pharmacology and Pharmacodynamics.2024;16(4):277-282.

DOI: 10.52711/2321-5836.2024.00048

Evaluation of Anti Ulcer activity of Calycopteris floribunda Lams leaf extract in wistar rats