INTRODUCTION:

Peptic ulcers (PU) are caused by imbalances in the human gastrointestinal tract, affecting the protective stomach mucosal barrier and causing symptoms like pain, nausea, vomiting, bloating, and appetite disturbances. Pathophysiological factors include Helicobacter pylori, NSAIDs, smoking, stress, caffeine, alcoholic beverages, and inheritance¹. Antiulcer drugs neutralize stomach acid, reduce gastric acid output, and prevent ulcers, but they are not preventative measures².

Alcohol damage to stomach mucosa is attributed to ethanol-produced free radicals, which cause oxidative damage and ulceration. Ethanol, a necrotizing substance, also reduces gastrointestinal mucus release, alters cell permeability, and stops stomach mucus function, making gastric mucosal cells more vulnerable to free radicals. Studies have shown functional and morphological alterations in experimental animals with ethanol treatment^3,4. A standard medication, pantoprazole, is used to treat peptic ulcers, but it has drawbacks like asymptomatic hypomagnesemia, hypocalcemia, and hypernatremia. Scientists are working to find a natural substance or therapeutic plant to replace artificially produced PPIs⁵. Ocimum americanum L. is an indigenous plant used in Chinese and Ayurvedic medicine for managing digestive system issues. Its flavonoids, phenols, and tannins have been shown to be useful in treating gastrointestinal problems and PU. Tulsi leaves contain flavonoids and phenolic acids, including rosmarinic acid and quercetin, which have anti-ulcer properties. Research suggests that Ocimum americanum leaves may have potential anti-ulcer activity^6,7. This study aims to determine if OALE has an anti-ulcer effect against stomach mucosal damage in mice generated by ethanol.

MATERIALS AND METHODS:

Chemicals and Reagents:

All reagents and compounds were purchased at a drugstore. Pantoprazole was dissolved in water and administered orally by gavage to animals in laboratories at a dose of 40mg/kg.

Animals:

A set of 30–40g Swiss albino mice was bought. Before beginning the experiment, the mice were housed in appropriate cages and given a week to get used to the lab environment. A 12hour light/dark cycle with a 25°C temperature was maintained. The animals had unrestricted access to water and standard feeding pellets. Our institutional ethics committee, which adhered to CCSEA (Committee for Control and supervision of Experimental on animals) criteria, approved the testing protocol (HRFT/IAEC/2023-2024/11).

Plant material:

Ocimum americanum L. leaves was collected from the surrounding villages in Ahmednagar, Maharashtra, India, and dried in the shade. The plant was authenticated by Mr. Mahesh Atale, MSc. Botany, Alarsin Pioneers in Ayurvedic Research, Andheri (E), Mumbai – 400093.

Preparation of extract:

Using a Soxhlet apparatus, the air-dried powdered Ocimum americanum L. leaves samples (50g/250mL) was extracted successively with ethanol at 55–85°C for 8–10hours. After being air dried and concentrated with a rotary vacuum evaporator, the extract was stored for later use at 4°C. After every extraction, the yield percentage was computed by weighing the amount of recovered extract from it⁸.

Phytochemical investigation:

Tests were conducted to determine whether the OALE included the following constituents: flavonoids, saponins, glycosides, alkaloids, carbohydrates, tannins, and phenols⁹.

Acute Toxicity study (LD50 determination):

The literature review suggests that Ocimum americanum L. LD50 was previously reported to be 4000mg/kg^10,11.

Dose selection:

According to OECD guidelines as the LD50 of Ocimum americanum L. was found to be 4000mg/kg, then dose was selected for further evaluation as 1/10th i.e. 400 mg/kg, 200mg/kg, 100mg/kg.

In-Vitro Anti-Oxidant Studies:

a) DPPH Assay:

The antioxidant potential of the sample compounds was assessed by measuring their capacity to scavenge free radicals using DPPH (1, 1-Diphenyl-2, Picryl-Hydrazyl) oxidative damage. 1ml of the experimental chemical water has been transferred to the test tube. The samples was treated with 1ml of 0.1% ethanolic DPPH and kept in the dark for half an hour. Following that, specimens then was scrutinized for discoloration; colors ranging from purple to yellow and light pink was classified as strong and weak positives, respectively, and the mixture's absorbance was measured at 517nm¹².

b) Alkaline DMSO Method:

Sodium hydroxide was added to air-saturated DMSO to produce superoxide radical. At room temperature, the produced superoxide transforms nitroblue tetrazolium (NBT) into formazan dye, which may be detected at 560 nm. The superoxide stays stable in solution. Following the addition of 300µL of drug samples at 1000µg/ml and 1000µg/ml of standard ascorbic acid to the reaction mixture that included 1000µL of alkaline DMSO at various concentrations of substances, 100µL of NBT (0.1mg) was added. The absorbance was measured at 560nm¹³.

Assessment of anti-ulcer activity:

a) Induction of gastric ulcer:

Mice were housed in metabolic cages having raised flooring constructed of wide mesh to inhibit coprophagy, which impacts the development of stomach ulcers. The animals were fasted for twenty-four hours so order to increase the amount of gastric acid in their stomachs and clear their stomachs of food, which made it simpler for the ethanol to damage the stomachs. Prior to the studies, water was also restricted for one hour. Single oral dose of ethanol (1ml/kg) caused gastric mucosal damage.

b) Ethanol induced ulcer in mice model:

For this study, 30–40g Swiss albino mice were used. There were six groups of all the species, with six animals in each group. Normal: Vehicle (Water) 10 ml/kg p.o; Ulcer Control: Vehicle 10ml/kg + Ethanol (1 ml/kg) p.o; Standard: Pantoprazole 40mg/kg + Ethanol (1ml/kg) p.o; Test I: OALE 100mg/kg (For 07 days) + Ethanol (1ml/kg) p.o; Test II: OALE 200mg/kg (For 07 days) + Ethanol (1ml/kg) p.o; Test III: OALE 400 mg/kg (For 07 days) + Ethanol (1ml/kg) p.o.

The test sample was administered for seven days. On day seven after the last test extract dose, the mice were fasted for a 24hours. On eight day animals were given ethanol at a dosage of 1ml/kg, and after an hour, they were euthanized using CO2 asphyxiation. Subsequently, the stomach had been removed and its contents were transferred inside sterile tubes and centrifuged. Supernatant was collected for measuring biological parameters. All animals' stomachs were then further cut with a greater curvature, and the inside surface was examined for ulcers by assigning a score, and the severity was evaluated under a microscope¹⁴.

c) Determination of pH:

After adding 1ml of distilled water and 1ml of stomach fluid to an aliquot, the mixture's pH was measured using a pH meter.

d) Macroscopic analysis of the stomach:

The stomachs were opened along their greater curvature, cleaned with saline to get clear of any blood clots and gastric contents, and then examined under a 5x magnifying lens to check for ulcer development. The number of ulcers was measured. Ulcer scoring was carried out in accordance with. The scores were: 0= no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation. Ulcer index was measured by using following formula.

UI =UN+US +UP ×10⁻¹; UI = Ulcer Index; UN= Average number of ulcers per animal; US =Average number of severity score; UP =percentage of animals with ulcers

Percentage inhibition of ulceration was calculated as below:

% Inhibition of Ulceration = (Ulcer index Control-Ulcer index Test) × 100/Ulcer index Control¹⁵.

Biochemical study:

a) Determination of malonaldehyde (MDA) content:

Once the stomach ulcer homogenate from the tissue was ready, combine 0.4ml of the 10% stomach ulcer homogenate with 1.5ml of 8.1% sodium dodecyl sulphate. 1.5ml of 20% buffered acetate (pH 3.5) and 1.5 ml of 0.8% TBA reagent was added to the combination as previously stated. The mixture was heated to 95°C for 60 minutes, and then it was cooled to room temperature add 5 ml of nbutanol-pyridine (15:1). After complete vortexing the mixture, let it remain until the aqueous and organic layers did not separate. After that, measured the organic layer's absorbance at 532nm using a UV-visible spectrophotometer¹⁶.

b) Determination of total nitrite/nitrate (NO) content:

To prepare the stomach ulcer tissue homogenate, 0.4 ml of 10% stomach ulcer homogenate was combined with 50μl of 30% ZnSO4 for protein precipitation. After that, the precipitated protein was separated using a 15-minute centrifugation process. A 300μl solution of the resulting supernatant was made with water, and 300μl of vanadium trichloride (0.8g% in 1 M HCl) was added immediately. Next, 150μl of sulphanilamide (2% in 5% HCl) and 150μl of N-1-(naphthyl) ethylenediamine dihydrochloride (0.1%) was added. When incubating for 30 minutes at 37°C, the mixture was cooled. After that Recorded absorbance at 540nm on a UV-visible spectrophotometer¹⁷.

Histopathological Examination:

For histological analysis, stomach tissues were embedded in paraffin and stored in a 10% buffered formalin solution. Then evaluate mucosal abnormalities and lesions in the stomach tissue lining.

Statistical Analysis:

Graph Pad Prism (version 10.2.2) was used to examine the data collected through animal experiments. The Mean±SEM was used to express it. The data were subjected to Dunnett's multiple comparison after analysis of variance (ANOVA) for statistical analysis. At (P≤0.05), the results were deemed statistically significant.

RESULTS AND DISCUSSION:

Preliminary phytochemical screening:

Table 1 displays the preliminary phytochemical data for OALE. The phytochemicals discovered in this study's OALE included flavanoids, saponins, alkaloids, glycosides, and tannins.

Table 1: Qualitative phytochemical analysis of Ocimum americanum leaves extract

Phytochemicals	Test	Inference
Alkaloids	Mayer’s Test	Present
Alkaloids	Wagner’s Test	Present
Flavonoids	Shinoda Test	Present
Flavonoids	Ferric Chloride Test	Present
Glycosides	Keller-Kilani Test	Absent
Tannins	Ferric chloride Test	Present
Tannins	Lead acetate	Present
Phenol	FeCl3 Test	Present
Saponin	Foam Test	Present
Carbohydrates	Molisch’s Test	Absent
Carbohydrates	Benedict’s Test	Absent

In vitro Antioxidant Activity:

Anti-Oxidant activity by DPPH assay:

The antioxidant capacity of OALE was carried out by DPPH assay using ascorbic acid as a reference standard antioxidant. In table 2 show that extracts are active in DPPH radical scavenging. OALE (IC50 = 224.56μg/ml) demonstrated stronger antioxidant capacity.

Table 2: Observation of % scavenging activity by DPPH assay

Sr. No.	Concentration	% SCV of Ascorbic acid	% SCV of OALE
1.	12.5	11.29	25.97
2.	25	16.20	31.91
3.	50	25.18	26.31
4.	100	44.90	31.40
5.	200	66.29	47.53
6.	400	91.25	71.30

Figure 1: Standard curve of Ascorbic acid

Figure 2: Standard curve of OALE

Anti-Oxidant activity by alkaline DMSO assay:

The antioxidant capacity of OALE was carried out by the alkaline DMSO assay using ascorbic acid as a reference standard antioxidant. In table 3 the OALE sample strongly inhibited the superoxide radical generation.

Table 3: Observation of % scavenging activity by alkaline DMSO assay

Sr. No.	Concentration (µg /ml)	% Scavenging Activity
Sr. No.	Concentration (µg /ml)	OALE	Ascorbic acid
1.	50	12.5	27.6
2.	100	21.9	75.27
3.	150	74.4	80.17
4.	200	79.8	96.79
5.	250	89.7	97.97

Figure 3: % scavenging activity OALE and ascorbic acid by DMSO assay

In vivo Antiulcer Activity:

It was found that 400mg/kg of OALE dose shows significant inhibition of ulcers when compared with the ulcer control group. Table 4 shows that the % of the inhibition of ulcers in the stomach is significantly increased by the standard drug pantoprazole and 100 mg/kg, 200mg/kg, and 400mg/kg. [F (5, 29) = 9.01; p ≤ 0.05, Figure 4].

Low pH is responsible for more damage in the gastric portion. In this study, the pH is increased by a significant level in both the standard and test (100 mg/kg, 200mg/kg, 400mg/kg of OALE) group as compared to the ulcer control group [F (4, 25) = 8.996; p ≤ 0.05, Figure 5].

Figure 4: Effect of OALE on UI in ethanol-induce ulcer in mice

Figure 5: Effect of OALE on pH in ethanol-induce ulcer in mice

Table 4: Effect of OALE on gastric parameters in ethanol-induce ulcer in mice

Treatment	Dose (mg/kg)	Ulcer Index	% inhibition of Ulcer	pH of Gastric Content
Vehicle	10 ml/kg	-	-	5.6 ± 0.07
Ulcer Control	1ml/kg	46.63 ± 5.712	-	1.41 ± 1.02
Pantoprazole	40 mg/kg	19.63 ± 3.687**	57.90	5.21 ± 0.11 ****
OALE	100 mg/kg	27.25 ± 5.484	41.56	3.20 ± 0.09
OALE	200 mg/kg	26.89 ± 4.924	42.33	3.90 ± 0.12
OALE	400 mg/kg	24.19 ± 6.322 *	48.12	4.0 ± 0.08**

Each group consists of six animals; Data is presented in Mean±SEM (p ≤ 0.05) in comparison to the ulcer control group.

Morphology and Histopathology of Stomach:

Ethanol-induced gastric damage showed gross mucosal lesions, including long haemorrhage bands and lesions. Blood flow stasis and microvascular disruption are caused by ethanol because it increases vascular permeability and exposes the stomach mucosa to the proteolytic and hydrolytic actions of pepsin and HCL. The pantoprazole (Std.) group had fewer haemorrhages, damaged epithelial structure and celluler infiltration showing nearly normal gastric mucosa. OALE (100 mg/kg) and OALE (200mg/kg) showed moderate haemorrhages, and structural damage whereas, OALE (400mg/kg) showed mild haemorrhages and cellular infiltrations as well as less structural damage.

Figure 6: Histopathological samples illustrating stomach tissue from various experimental groups: (A)Normal control; (B)Standard (Pantoprazole); (C)Ulcer control; (D)OALE 100 mg/kg; (E)OALE 200mg/kg and (F)OALE 400mg/kg. Note: Structural loss of tissue (arrows), Hyperemia (circles).

Biochemical Studies:

Administration of OALE at doses of 100, 200, and 400 mg/kg, similar to the pantoprazole group exhibited a dose-dependent decrease in MDA level. As shown in table 5, ulcer control group increased the gastric MDA level compared to the vehicle group [F (5, 30) = 19.85; p ≤ 0.05, Figure 7]. Administration of OALE at doses of 100, 200, and 400mg/kg, similar to the pantoprazole group exhibited a dose-dependent increase in nitric oxide level. As shown in table 5, ulcer control group decreases the gastric nitric oxide level compared to the vehicle group [F (5, 30) = 11.8; p ≤ 0.05, Figure 8].

Table 5: Effect of OALE on biological parameters in ethanol-induce ulcer in mice

Sr. No.	Treatment	MDA	NO
1	Vehicle	0.010 ± 0.001	20.10 ± 0.243
2	Ulcer Control	0.036 ± 0.0003	7.11 ± 0.021
3	Pantoprazole (40 mg/kg)	0.0153 ± 0.0007 ****	14.95 ± 0.128 ***
4	OALE (100 mg/kg)	0.0293 ± 0.0007	8.10 ± 0.0140
5	OALE (200 mg/kg)	0.0275 ± 0.0006	9.03 ± 0.087
6	OALE (400 mg/kg)	0.0209 ± 0.0006 **	13.04 ± 0.203 **

Each group consists of six animals; Data is presented in Mean ± SEM (p ≤ 0.05) in comparison to the ulcer control group.

Figure 7: Effect of OALE extract (100,200 and 400 mg/kg) on the level of malondialdehyde (MDA) gastric tissue in ethanol induce ulcer in mice.

Figure 8: Effect of OALE extract (100,200 and 400 mg/kg) on the level of nitric oxide (NO) gastric tissue in ethanol induce ulcer in mice.

SUMMARY AND CONCLUSION:

In this work, we investigated the antiulcer effects of an extract from Ocimum americanum leaves on mice that had ulcers caused by ethanol. An initially phytochemical examination of an extract from Ocimum americanum leaves revealed the presence of tannins, alkaloids, phenols, flavonoids, and saponins. The extract contains various flavonoid responsible for anti-oxidant and free radical scavenging activity. This was analyzed by performing antioxidant assays like DPPH and DMSO on the extract before the animal study. OALE and the pantoprazole were administrated to mice and receving ethanol for inducing ulcer. Our current study's findings indicate that pretreatment with pantoprazole and OALE may significantly reduce stomach lesions and histological indications of damage. The gastric level of MDA was found to decrease, and nitrite levels increased, which may be shows the anti-inflammatory and muco-protective nature of Ocimum americanum leaves extract. OALE has a great potential usage on ethanol-induced gastric lesions as the higher dose (400 mg/kg) demonstrated a gastroprotective effect equivalent to that seen with pantoprazole. Further studies are required to confirm the extract mechanism underlying for the ulcer healing and protecting property of extract.

ACKNOWLEDGMENT:

We are thankful for their guidance and help of our Principal Dr. Mohan Kale, my guide Dr. Baban Thawkar, as well as to the Department of Pharmacology, Konkan Gyanpeeth Rahul Dharkar College of Pharmacy and Research Institute, Karjat, Maharashtra 410201 and Milind Bagul Head, Bioanalytical at Raptim Research Pvt Ltd.

CONFLICTS OF INTREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 06.09.2024 Revised on 11.12.2024

Accepted on 05.02.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2555-2560.

DOI: 10.52711/0974-360X.2025.00365

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