INTRODUCTION:

The term peptic ulcer is allocated to local distraction of mucosa or inner wall of the stomach (known as gastric ulcer) or upper position of small intestine (known as duodenal ulcer). Peptic ulcer commonly arises when there is an imbalance between destructive gastric factors (refluxed bile salts, pepsin, acid and Helicobacter pylori) and self-protective mucosal factors (bicarbonate secretion, gastric mucosal barrier, high blood stream and rapid cell turnover)¹. The agents used to treat peptic ulcer act by decreasing aggressive factors, which are responsible for the damage of mucosa lining of stomach and duodenum. It is stated that sulfhydryl (SH) non-protein endogenous complexes contribute in gastric mucosa cryoprotection².

HCl, pepsin, lipid peroxidation and reactive oxygen species (ROS) are the endogenous components and while excess NSAIDS, alcohol, smoking are the exogenous elements responsible for destruction of stomach. It also stated that stress and infection by Helicobacter pylori bacterial is the leading cause for peptic ulcers. The self-protective components are antioxidant enzymes, cell growth factors, prostaglandins (PGs), mucin secretion, mucus-bicarbonate barrier, nitric oxide (NO), surface phospholipids, and mucosal blood flow^3,4.

The presence of oxidative stress, during the progression of gastric ulceration, leads to increases the generation ROS, that can abolish epithelial cell integrity. An over generation of ROS metabolites, may leads to the suppression of endogenous elements⁵. Moreover, reactive oxygen species gathers neutrophils in mucosa tissue, during the process of gastric ulceration. Various investigations have established that proinflammatory cytokines initiate the neutrophils activation and are solid supporters for the ulcer formation^6.7. For constructing a SAR (Structure activity relationship), target molecule and mechanism of action of numerous natural compounds are needed to study. Molecular docking and interaction analysis studies can be carried out to study the interaction of these compounds with numerous target molecules of antiulcerogenic activity.

Salvia fruticosa (Family Lamiaceae) also named as “Greek sage” or “Lebanese sage”. It is perennial herb, also known as “East Mediterranean sage” as it is native to eastern Mediterranean⁸. Various species of the Salvia have been utilizing since antiquated occasions as folk medicine and economically important, which have been exposed to broad pharmacogenetic exploration to recognize naturally dynamic constituents. The plant parts are boiled and served as a tea for the relief of diverse pains, viral infections, colds, and many other ailments.

The leaves are utilized as a flavor or as an adulterant of sage (Salvia officinalis). Slightly substandard in quality to sage but it is simpler to grow indoors⁹. The leaves constitute about 50 - 95% of economically dried sage leaves. Fascomiglia ''fragrant tea” is made by infusing of sage leaves¹⁰. The leaves are used as antiseptic, antihydrotic, antispasmodic, carminative, astringent, cholagogue, expectorant, febrifuge, tonic, stimulant and vasodilator. Sega parts are utilized internally in the treatment of respiratory and digestive and disorders, menstrual issues, infertility, depression and anxiety¹¹. This remedy ought not to be endorsed to pregnant ladies. The leaves of Salvia officinalis can be collected as required and utilized fresh, or they can be collected before the blossoms open and dried or refined for their basic oil. This Lebanese plant have been additionally utilized for enlightening memory and as antidiabetic agent with reported antioxidant activity¹². The anti-inflammatory effect of flavonoids and phenolics has additionally been demonstrated as a result of their antioxidant potentials¹³. The objective of the present study is to carry out phytochemical studies, computational interaction analysis and anti-ulcerogenic activity of Salvia fruticosa hydroalcoholic extract against ethanol induced gastric ulcers.

MATERIALS AND METHODS:

Protein preparation:

Diffracted X-Ray crystal structure of the stomach proton pump complexes with vonoprazan, deposited by Abe and co-workers¹⁴, was retrieved from the PDB (5Ylu) for these studies, with a resolution of 2.80. The structure was first verified for missing amino acids and alternative confirmation, and then enhanced using the BIOVIA DS clean protein process. CHARMm force field was applied and the energy of protein was minimized to make the proteins Tab form using the smart minimizer algorithm (1000 steps of Steepest Descent, followed by Conjugate Gradient minimization) with an RMS gradient tolerance of 0.1, the energy of the proteins was minimised to produce the proteins Tab shape. Following minimization, the protein's binding site was determined using two algorithms: eraser and flood-filling method to capture contiguous space living in vacant, connected grid points.

Phytoconstituents preparation:

Sixty-eight identified phytoconstituents were collected^15,16,17from the herb Salvia officinalis which are hypothesized as effective against Ulcer. The phytoconstituent’sformat files were retrieved from the PubChem public domain, and the CHARMm force field was applied with CVFF partial charges using the CHARMm force field. The smart minimizer algorithm with 5000 steps was used to transform the energy of these forcefield typed molecules to local minima. The docking approach was applied to energy-minimized molecules.

CDOCKER protocol and interaction analysis setup:

The BIOVIA discovery studio's CDOCKER docking module was used to execute the lock-key interaction studies¹⁸. The algorithm was ran with the following protocol parameters (Table 1) in the CDOCKER frame to predict the binding affinities between the proton pump and natural phytoconstituents. –CDocker interaction energy, –CDocker energy, Angle Energy, Bond Energy, Solvation Free Energy, Urey-Bradley Energy, Van der Waals Energy, Dihedral Energy, Electrostatic Energy, and Hydrogen Bond Energy were all included to this protocol run. After the docking process was completed, the results of the docked pose were analysed using the view interaction tool.

Table 1: CDOCKER protocol parameter set up

Parameters	Values
Input Receptor	5Ylu.pdb
Input Ligands	67 Phytoconstituents+1 std Drug
Input Site Sphere (X, Y, Z and r)	-34.1899, 26.0506, 4.77289, 9.9
Top Hits	1
Pose cluster Radius	0.1
Random Conformations	10
Dynamics Steps	1000
Dynamics Target Temperature	1000
Include Electrostatic Interactions	True
Orientations to Refine	10
Maximum Bad Orientations	800
Orientation vdW Energy Threshold	300
Simulated Annealing	True
Heating Steps	2000
Heating Target Temperature	700
Cooling Steps	5000
Cooling Target Temperature	300

Eventually, the results and amino acid interactions were analysed and discussed to identify the effective phytoconstituents. Interaction pattern was distinguished by different colours as following Table 2.

Table 2: Common colour pattern for non bonded interactions

Type	Color	Type	Color
Conventional Hydrogen Bond		Pi-Pi T-Shaped
Carbon Hydrogen Bond		Alkyl
Pi-Cation		Unfavourable
Pi-Pi Stacked

Experimental Animals:

30 Wister strain rat’s weight about 160±10g were obtained from Nizam institute of pharmacy animal house. During experimental period rats were kept at 20 - 28˚C temperature in well ventilated and disinfected cages and exposed to 12 hours day and 12 hours dark environment. The animals are freely permitted to access feed and water ad libitum. The experimental research proposal was approved by CPCSEA and also by IAEC meeting held at Nizam Institute of Pharmacy and Research Centre, Hyderabad with registration number (1330/AC/10/CPCSEA).

Drugs and Chemicals:

Ranitidine sample was procured from Aurobindo Pharmaceuticals. Ethanol, chloroform, normal saline, dimethyl sulfoxide was obtained from chemical store house of Nizam Institute of Pharmacy and Research Centre, Deshmukhi.

Acute Oral Toxicity Studies:

In compliance with OECD 423 guideline the oral acute toxicity study was studied. Three of the test animals (rats) were abstained from feed for overnight and weighed individually. Test doses of Salvia fruticosa hydroalcoholic leaves extract were determined comparable to the body weight of each fasted rats and extract were administered by means of oral gavage at a dose of 2000mg/kg. Rats were normally and independently observed for behavioural fluctuations and signs of general toxicity after dosing, for the initial 24 h, with unique consideration being given throughout the primary 4 h. Then, observation was continuous daily for a period of total 14 days¹⁹.

Plant material and extract preparation:

The plant material Salvia fruticosa was gathered from Tirupati, authentified by Botanist, Dr.Madhav Chetty from S.V. University, Tirupati. The plant material Salvia fruticosa were shade dried and extracted bycold maceration method. Plant material were dried out under shade and extraction were completed by cold maceration technique. The Salvia fruticosa leaves powderwas soaked in ethanol and distilled water in ratio of 1:1(1g of leaves powder per 5ml of solvent) for a period of 4-5 days in 250ml round bottom flask at normal temperature with constant shaking. The extract was centrifuged for 20 min and then filtered through Whatman filter paper (No.1).Under reduced pressure the filtrate was concentrated in rotary vacuum evaporator to obtain semi-solid material, which was then dried at 45ºC to get a solid powder.

Phytochemical investigation:

The principal qualitative phytochemical studies were done for testing the diverse phytoconstituents such as phenols, flavonoids, glycosides, diterpenoids, carbohydrates, tannins, coumarins, volatile oils, alkaloids and saponins²⁰.

Experimental Design:

After acclimatization period, animals were randomly divided into five groups of six animals each²¹.

Group I: Control group; Group II: Ethanol (1ml/kg.p.o); Group III: Ethanol (1ml/kg.p.o) + Salvia fruticosa (250mg/kg.p.o); Group IV: Ethanol (1ml/kg.p.o) + Salvia fruticosa (500mg/kg.p.o); Group V: Ethanol (1 ml/kgp.o) + Standard drug (Ranitidine 200mg/kg,p.o)

Group I received saline, Group III and IV received Salvia fruticosa (250mg/kg) and (500mg/kg), Group V received Ranitidine 200mg/kg by gastric gavage for 4 days. On the day five, after 24hrs of starvation, 1 hour after the administration of test samples the animals of Group II-V received absolute ethanol (1ml/kg, p.o.). The animals were sacrificed, one hour after the administration of ethanol. The stomach was isolated and cut along the greater curvature, then washed cautiously with 5.0ml of normal saline (0.9% NaCl) and ulcer scoring were done by a person who is unaware of the experiment protocol in the glandular portion of the stomach.

Macroscopic estimation of ulcers in the stomach:

After sacrificing the animal, along the greater curvature the stomach was opened, washed cautiously with normal saline solution to evacuate blood clots and gastric contents and observed by 5x magnifying lens to measure the formation of ulcers. The total number of ulcers in the animal were counted. Ulcer scoring was completed according to the procedure described standard method²². By using following formula, Ulcer index was measured.

UI = UN + US + UP × 10-1

Were,

UI = represent “ulcer index”; UN= represent “Average number of ulcers per animal” ;US = represent “Average number of severity score”; UP= represent “Percentage of animals”;

Ulcer scoring

0 for “Normal colored stomach” ;0.5 for “Red coloration”, 1.0 for “Spot ulcers”, 1.5 for “Hemorrhagic streak”, 2.0 for “Ulcers”, and 3.0 for “Perforation”.

Statistical analysis:

The statistical significance was measured using ANOVA (one-way analysis of variance) followed by Dunnett’s comparison test. The values were stated as mean±SEM, and p<0.05 were considered as significant.

RESULTS:

Results of the phytochemical studies showed the presence of phenols, flavonoids, glycosides, diterpenoids, carbohydrates, tannins, coumarins, volatile oils, while alkaloids and saponins are reported to be absent (Table 3).

Table 3: Phytochemical screening of Salvia fruticosa

Phytochemical compounds	Present/Absent
Phenols	+
Flavonoids	+
Glycosides	+
Carbohydrates	+
Diterpenoids	+
Tannins	+
Coumarins	+
Volatile oils	+
Alkaloids	-
Saponins	-

+ = Presence; - = Absence

Proton pump (Lock) – inhibition (Key) studies:

Inhibiting the Gastric Proton pump(GPPIs) blocks gastric acid secretion and it aid to heal peptic ulcers and gastroesophageal reflux disease (GERD) etc. The preparation results of protein and Phytoconstituent showed that the energy of 5Ylu protein brought to local minima of -76128.6 from the 442130.5 kcal/mol. Similarly, phytoconstituent energy was minimized to the range of -67.2147 kcal/mol to 95.7487 kcal/mol (Table 4). The binding site sphere of proton pump continent XYZ 48.846752, -15.529343, -3.557968 and radius of 7.066863 Ǻ (Figure 1) selected from the PDB input by flood-filling algorithm.

Figure 1. Crystal structure of the gastric proton pump with defined binding site using vonoprazan PDB ligand.

Interaction interpretation of proton pump and phytoconstituents:

The result of CHARMmdocker protocol run revealed that among 69 compounds, 68 formed proper poses within the binding site. The molecule Sagerinic acid unable to form the refined pose inside the active site of proton pump. Rosmarinic acid was found that the highest dock energy of -54.1556kcal/mol, Oleanolic acid showed less dock score of -120.304kcal/mol and slandered drug ranitidine interact with the score of - 40.4806kcal/mol. Based on the theoretical and docking analysis point out the 4 phytoconstituent of herb Salvia fruticosa will be the potent lead molecules to treat ulcer patient as presented in Table 4.

Table 4: Energy profile of phytoconstituents after minimization and the dock score.

Name	Int.pot. Energy	CHARMm	Int.RMS	RMS	electrostatic	VdW	Dock score
Rosmarinic acid	24.2392	-67.2147	39.039	0.00961	-80.7804	-0.4249	-54.1556
Stearic_acid	-3.20445	-15.8016	13.4883	0.00581	-8.41937	-9.1492	-45.8874
luteolin	74.1407	-3.77795	42.8549	0.00802	-13.0619	2.11008	-38.4799
Nepetin	63.3176	9.01196	41.753	0.0091	-4.38187	4.34354	-35.0734
apigenin	70.5436	24.3032	43.7713	0.00837	13.6628	4.56444	-34.1459
Genkwanin	70.9089	20.6431	42.008	0.00879	8.78885	4.71405	-31.0405
Gallic_acid	-5.86117	-27.2416	39.8829	0.00727	-31.5768	2.04043	-30.7802
Hispidulin	70.4918	16.4747	42.8042	0.00961	3.1844	4.43437	-30.6037
Chlorogenic_acid	44.0817	-23.9264	31.5096	0.0096	-63.02	1.85715	-30.3838
Cirsimaritin	74.0554	17.8734	39.7468	0.00868	1.52517	5.03996	-28.5713
Luteolin-O-glucuronide	108.644	35.9713	35.0026	0.00934	-1.08532	-0.8954	-27.8558
Cinaroside	164.876	17.2247	53.4854	0.00952	-26.3545	-0.0901	-27.2726
Apigenin-O-glucuronide	148.277	20.3369	51.3443	0.00742	-19.454	-2.2937	-26.7899
cis-calamenene	24.6591	1.52275	24.9418	0.00872	-4.17335	-3.1274	-26.7743
Caffeic_Acid	7.67883	-15.5185	37.0292	0.00603	-20.4613	2.40869	-26.6917
Ferulic_acids	16.9581	-8.57443	33.8148	0.00835	-15.5741	2.61681	-24.5116
Thymol	22.1807	0.30549	29.4666	0.00918	-1.97936	-0.8842	-23.7823
Cosmosiin	162.753	30.3991	53.3657	0.00981	-11.1867	-0.4311	-23.2812
p-cymene	16.1133	-1.90812	30.159	0.00883	-5.23677	1.17512	-22.6274
o-cymene	28.6981	-0.08681	33.5962	0.00892	-1.74007	-0.0162	-22.0026
Carnosic_acid	67.7886	-3.80958	31.6058	0.00808	-27.8009	-5.5397	-20.4663
12-O-Methylcarnosicacid	76.5397	9.37116	26.1704	0.00801	-16.9241	-6.7467	-18.2109
a-thujone	26.3995	8.94723	19.27	0.00972	-3.98125	-4.5969	-12.9811
Homoplontagenin	316.638	31.2583	148.84	0.00961	-13.7716	-1.4985	-12.1905
Trans-Thujone	71.252	8.92144	38.1704	0.00956	-4.0349	-4.6971	-11.7318
Rosmanol	69.5143	10.5568	25.6	0.00719	-20.1703	-6.5326	-11.0414
Anethole	37.6581	12.8337	32.9083	0.00522	-5.12037	2.3281	-9.87707
Carnosol	43.4823	-0.23112	23.897	0.00946	-32.3762	-2.8017	-8.92056
Rosmadial	295.375	19.6254	152.089	0.00953	-15.2127	-1.5662	-8.82167
Glucopyranoside	38.352	16.9187	15.4999	0.00991	-12.4515	0.59572	-5.39275
a-copaene	42.2906	23.3859	14.333	0.00911	-4.56325	-2.2364	-0.82158
Pinene	31.9988	16.227	13.5584	0.00908	-4.59397	0.616	0.987402
rutin	225.305	29.2418	34.5695	0.00976	-46.6146	-2.2022	2.37882
Alloaromadendrene_oxide	60.9061	32.0849	16.6371	0.0096	-4.62811	-7.2261	2.57394
Cineole	134.915	16.076	23.0018	0.00748	-12.3673	-2.0032	3.71371
Terpinen-4-ol	48.1831	14.4906	10.6323	0.00987	-17.1223	-2.9186	5.11778
Sclareol	109.839	27.7424	23.9835	0.00913	-27.1896	-7.6913	9.92647
Manool	74.253	24.6409	22.3376	0.00918	-23.6442	-7.5067	11.8669
Carene	102.56	30.1168	21.2856	0.00984	-3.98483	-2.3849	11.9428
a-maaliene	65.4459	39.6201	17.3636	0.00932	-5.68977	-3.4449	13.3958
Spathulenol	90.6669	31.832	17.4326	0.00988	-14.5766	-5.9952	13.5797
Caryophyllene	790.53	37.8206	586.145	0.00911	-7.35243	-4.3001	13.6342
Epizonarene	75.1544	39.0928	12.2022	0.00986	-3.31519	-1.9475	14.2601
Muurolene	86.5847	40.3199	19.6396	0.00878	-5.60121	-5.3968	14.4281
Cadinene	133.216	41.5858	51.4715	0.0099	-5.71182	-4.3073	15.5008
Valencene	79.8948	37.4214	20.4511	0.00863	-8.76612	-4.6568	16.0966
Calarene	138.279	41.2489	43.5263	0.00874	-5.58776	-4.1037	17.5827
Limonene	48.0234	34.1314	13.8865	0.00716	-4.71849	-1.8159	17.755
Germacrene_d	317.927	76.4868	227.967	0.00955	-5.98291	-3.2825	18.0425
Camphor	207.333	45.7768	50.1883	0.00965	1.06237	-2.5830	22.8762
Viridiflorene	109.688	51.4767	23.9956	0.00991	-4.57064	-5.2939	23.1023
Thujene	64.8822	42.7546	17.5054	0.00986	-3.67948	-3.9395	23.6858
Gurjunene	103.387	55.5772	21.3879	0.00941	-6.87642	-4.6062	28.376
Bicyclogermacrene	113.028	62.3459	23.9691	0.00828	-6.4558	-3.8453	28.9932
Humulene_1-2_epoxide	99.162	50.6711	18.678	0.00985	-6.94782	-3.4065	29.3232
Allo-aromadendrene	215.502	57.429	114.03	0.00831	-5.20651	-5.7144	29.4927
d-cadinene	111.397	58.7753	12.7254	0.00947	-4.97678	-2.8865	32.1542
a-guaiene	117.489	58.3143	14.7522	0.00962	-5.98861	-5.6577	35.9436
Himachalene	100.552	66.4327	14.0208	0.00939	-7.12669	-3.8135	36.4253
Humulene	449.912	64.9832	399.913	0.0088	-7.54333	-2.4507	38.1457
longifolene_aldehyde	133.054	75.3642	16.6244	0.00957	-0.45299	-4.6025	42.9181
Camphene	78.4035	57.5591	13.8961	0.00829	-7.38276	-2.738	44.7589
Germacrene_A	152.419	66.7377	33.2414	0.00916	-7.00426	-2.2008	45.7457
longifolene	213.266	73.3899	24.1943	0.00882	-8.52012	-4.9651	52.5428
Diosmin	303.716	40.1283	72.3927	0.04003	-33.3072	-4.1699	91.3546
Ursolic_acid	387.328	95.7487	116.129	0.00943	-25.1396	-6.8440	113.415
Olenolic	217.099	69.8865	39.0989	0.00981	-27.8681	-8.4903	120.304
Ranitidine	49.8633	18.6776	21.3582	0.09581	15.1362	-4.4795	-40.4806
5YLU	44,130.50	-76,128.6	6,562.68	1.08649	-78,121	-8,450.0	-56.6578

Analysis of Standard Drug-Ranitidine:

Interaction view analysis showed that the ranitidine formed totally 8 bonds with active site amino acids of proton pump. Especially it formed 2 conventional hydrogen bond, 5 Carbon hydrogen bond and 1 π-alkyl interaction. These three orientational interactions leads the potent against the proton pump (Figure 2).

Figure 2 (a) Ranitidine binding inside the proton pump pocket, (b) interaction of amino acids with the active site amino acids, (c) 2D interaction view of interaction, and (d). Orientational binding view of ranitidine.

Analysis of high affinity and Low affinity phytoconstituent:

Rosmarinic acid formed higher number of 7 hydrogen bonds, 1 carbon hydrogen bond and two kinds of π interactions such as π-π stacked and π-alkyl. Hydroxyl groups of this molecule increase the hydrogen bond to make higher interaction with 7 active site amino acids. The week binding compound of oleanolic acid formed the multiple week electrostatic alkyl-alkyl and π-alkyl interactions only, which made this molecule bind less strong inside the binding pocket (Figure 3).

Figure 3: a and b; Interactions of high affinity rosmarinic acid phytoconstituent. C & d; Interactions of less affinity olenolic acid phytoconstituent.

Effect of Salvia fruticosa extracts in ethanol-induced gastric ulcers:

The acute oral toxicity study of Salvia fruticosahydroalcoholic leaves extract showed no mortality up to 2000mg/kg and the extract found to be safe for animal dosing. The hydroalcoholic leaves extracts of Salvia fruticosahas exhibited a significant decrease in ulcer score and ulcer index, when compared to control group (p< 0.05) and results were expressed in Table 5 and Figure4.

Table 5: Effect of Salvia fruticosaextracts in ethanol-induced gastric ulcers

Treatment	Ulcer index	Ulcer score
Control	0.0 ± 0.0	0.0 ± 0.0
Ethanol	5.66±0.51	11.03±044^**
Ethanol +Salvia fruticosaExtract(250mg/kg)	3.24±0.044^**	3.5±0.2226^**
Ethanol + Salvia fruticosaExtract (500mg/kg)	1.25 ± 0.045^**	1.3± 0.2108^**
Ethanol + Ranitidine (200mg/kg)	1.10± 0.025^**	0.9± 0.1212^**

Values were expressed in mean±SEM, **p<0.05

Figure 4: Effect of Salvia fruticosa extracts on (a) Ulcer index, and (b) Ulcer score

Histopathology of the rat’s stomach:

Histopathology of the rat’s stomach in Group 1: showed no lesions, Group II: showed gastric lesions, Group III-V: showed the maximum inhibition of both the number and length of gastric lesions (Figure 5).

Figure 5: a)Group I: Control group; b) Group II: Ethanol (1 ml/kg,p.o); c) Group III: Ethanol (1ml/kg,p.o) + Salvia fruticosa (250mg/kg); d) Group IV: Ethanol (1ml/kg,p.o) + Salvia fruticosa (500mg/kg); e) Group V: Ethanol (1ml/kg, p.o) + Standard drug (Rantidine 200mg/kg).

From the histopathology, it is observed that ulcer protective activity was seen for Salvia fruticosa extract (at 250 and 500mg/kg doses) and standard ranitidine as the reference drug

DISCUSSION:

The Salvia fruticosa was found to be a rich source of antioxidants compounds. It is reported that ethyl acetate root extract of Salvia fruticosa contains high concentration of phenols and also possess the highest radical scavenging property. In addition to antioxidant property the aerial parts of Salvia fruticosa are reported with highest anti-inflammatory effect. Both the roots and aerial parts extract had exhibited significant protection against carrageenan-induced mouse paw edema. Previous literature shows that different plant extract of Salvia fruticosa had reported anti-Inflammatory and antioxidant activities²³.

Structural analysis of high affinity molecules and the standard drug revealed that the –OH groups of rosmarinic acid contributed to the better interaction against the proton pump. These group release the electron inside the benzene ring of the molecule, which lead to increase the electron density²⁴of the benzene ring as shown in Figure 6. In addition, the oxygen and hydrogen atom free electrons share the electrons with the neighbour amino acids which interact the orbitals to form various types of bonds²⁵. Most of hydrogen bonds of formed with the -C=O groups of the amino acids and hydrogen atoms of the hydroxyl fragment. And then carboxylic group of rosmerinic acid form the two interaction with an asparagine amino acid like amide bond. These bonds made the modification of the natural folding of the proton pump leads the denature and inactivation. Hydroxyl groups (-OH), ketogenic groups (C=O) substituted in the ring system sharing their electron to form the various conventional and carbon hydrogen bonds

Figure 6: Core skeletons of high affinity molecules and their interactions with the active site amino acids of proton pump.

For investigating of gastroprotective drugs, ethanol induce gastric ulcer is a suitable and usual animal model. Ethanol induces progressive destruction of mucosal membrane microcirculation and ischemia leads to release of endothelium and free radicals²⁶. Formation of ulcer is the sign of the imbalance between protective and aggressive factors²⁷. In the existing study, the oral administration of Salvia fruticosa exhibited protective effects against ethanol induced gastric lesions. However, studies carried out on other species of Salvia (S. verticillate) methanol extract displayed the highest free radical scavenging activity. This scavenging effect was due to the presence of phenolic compounds in different proportions (gallic acid and rosmarinic acid) which occurred in higher amount and was identified by TLC screening process²⁸. This finding may prove the popular use of Salvia fruticosa worldwide for treating several illnesses. Definitely, the antioxidant effect is considered one of the important biological activities of Salvia plants, mainly to the presence of terpenes and polyphenols²⁹.

Gastric lesions induced by ethanol are assumed to arise as a result of direct damage of gastric mucosal cells³⁰. Both psychological and physiological factors induce stress ulcer, which leads to increase in accumulation of pepsin and acid leading to gastric mucosa auto digestion³¹. The anti-ulcer activity of hydroalcoholic leaves extract of Salvia fruticosawas evaluated in absolute ethanol induced lesions in rats. This model evaluates the drug capacity to protect the gastric mucosa, distinguish the severity of gastric lesions. Ethanol induced gastric mucosal injury might be due to stasis in gastric blood flow, which contributes to the development of the necrotic and haemorrhagic aspects of tissue injury. In addition, ethanol also induces solubilization of constituents of mucus, decreases the difference of potential in mucosa thus cumulative the flow of K⁺andNa⁺ to the lumen and pepsin secretion, and also increases histamine and H⁺ ions³². The results show that the tested hydroalcoholic extracts have an important protective effect for gastric mucosa, at a dose of 250 and 500mg/kg extracts, they were effective in reducing ulcer lesions.

The preliminary phytochemical screening of Salvia fruticosaleaves revealed the presence of phenols, flavonoids, glycosides, diterpenoids, carbohydrates, tannins, coumarins, volatile oils³³. Previous studies had proved that volatile oils and tannins possess significant anti-ulcer activity in experimental animal models³⁴. The literature shows that Salvia fruticosa contains phenolic and flavonoids compounds, in addition the flavonoids(luteolin, rutin and apigenin) and phenolic acids includes; gallic, rosmarinic acid and ferulic acids were also obtained also from aerial parts. It is also previously mentioned that these compounds were responsible for the antioxidant activity of Salvia¹⁵.

The antioxidant properties of natural compounds have been found to play a role in gastric mucosa protection through radical scavenging mechanisms³⁵. Phenolic compounds are key compounds that possess this scavenging property due to their hydrogen-donating ability³⁶. The presence of phenolic compounds in Salvia fruticosamay also be responsible for its anti-ulcer activity. It is mostly acknowledged that gastric ulcers result from an imbalance between aggressive factors and the maintenance of the mucosal integrity through the endogenous defence mechanism. The role of free radicals in the indication of ulcers is also reported. Hydroalcoholic leaves extract of Salvia fruticosaat the dose of 250mg/kg and 500mg/kg, reduces ulcer occurrence significantly (P<0.05) when compared to the control as evident by decrease in ulcer score. Protection against ulcerations in ethanol induced gastric ulcer model indicates the gastro protective action of Salvia fruticosaand it might be due the presence of phenolic compounds.

CONCLUSION:

The present study concluded that hydroalcoholic extract of Salvia fruticosahas potent antiulcer effect, which is not superior to the effect shown by Ranitidine. Salvia fruticosashowed its ulcer protective activity. The antiulcerogenic effect of the extract were found to be dose dependent with no adversative effects. Computational studies proved the presence of phenolic compounds in Salvia fruticosaextract might be responsible for its anti-ulcer activity. Further research is expected to decide the specific mechanism of activity of the different constituents in the extract concentrates.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

I would like to thank to all staffs of Nizam Institute of Pharmacy and Research Centre, Hyderabad, India, and College of Applied Medical Sciences in Jubail, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia for helping for carrying this research successfully.

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Received on 05.11.2021 Modified on 08.06.2022

Research J. Pharm. and Tech 2023; 16(1):314-322.

DOI: 10.52711/0974-360X.2023.00056