Antiulcer Potential of Psidium guajava Seed Extract Supported by Metabolic Profiling and Molecular Docking

One of the most severe human health problems is gastric ulceration. The main aim of our study is to explore the gastroprotective effect of the Psidium guajava seeds extract (PGE). Metabolic profiling based on LC-HRMS for the extract led to the dereplication of 23 compounds (1–23). We carried out a gastric ulcer model induced by indomethacin in male albino rats in vivo and the extract of PGE was investigated at a dose of 300 mg/kg in comparison to cimetidine (100 mg/kg). Furthermore, the assessment of gastric mucosal lesions and histopathology investigation of gastric tissue was done. It has been proved that Psidium guajava seeds significantly decreased the ulcer index and protected the mucosa from lesions. The antiulcer effect of Psidium guajava seed extract, which has the power of reducing the ensuing inflammatory reactions, can counteract the inflammation induced by indomethacin by the downregulation of relative genes expression (IL-1β, IL-6, and TNF-α). Moreover, PGE significantly downregulated the increased COX-2, TGF-β, and IGF-1 relative genes expression, confirming its beneficial effect in ulcer healing. Moreover, the possible PGE antioxidant potential was determined by in vitro assays using hydrogen peroxide and superoxide radical scavenging and revealed high antioxidant potential. Additionally, on the putatively annotated metabolites, an in silico study was conducted, which emphasized the extract’s antiulcer properties might be attributed to several sterols such as stigmasterol and campesterol. The present study provided evidence of Psidium guajava seeds considered as a potential natural gastroprotective agent.


Introduction
Psidium guajava (Family: Myrtaceae) is a tropical edible fruiting plant. They are evergreen trees indigenous to subtropical and tropical climatic regions, it originated from Latin

Plants Material
The seeds of Psidium guajava were collected in September 2021 from Minia area, Egypt. Mr. Abdallah Salem authenticated the plant (at Minia University, Minia, Egypt). A voucher specimen (PS 2-2021) was conserved at Deraya University.

Extraction of Psidium guajava Seeds
One kilogram of dried Psidium guajava seeds were extracted three times by maceration in methanol at an ambient temperature. The alcoholic extract was vacuum-dried to produce a dense residue (100 g).

Metabolic Profiling
LCMS was performed by using a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer (Waters, Milford, CT, USA). One mg of the sample was injected into the BEH C18 column, at 40 • C, and connected to the guard column. A gradient elution was used, starting from no solvent A and 0.1% formic acid in water to 100% acetonitrile as solvent B. MZmine 2.12 San Diego, USA was used to investigate MS data using mzML format converted with ProteoWizard Palo Alto, CA, USA [27].

Docking Study
Investigated compounds 3D structures were downloaded from PubChem and prepared as reported earlier [28]. Compounds were docked against a gastric proton pump (PDB 5YLU), M3 muscarinic receptor (PDB 5ZHP) obtained from http://www.rcsb.org/ (accessed on 23 March 2022) and against a H2 receptor as obtained from GPCR data base. Docking was done in the active sites of target protein as recognized by co-crystalized ligands. Docking was done using Autodock Vina [29] using cubic grid box with 25 Å side and exhaustiveness of 16. Similarly docking was also done using the same procedure against other targets involved in inflammation including COX-1 (PDB 1EQH), COX-2 (PDB 3LN1), TNF-α (PDB 2AZ5), TGF-β (PDB 5E8S), EGFR (PDB 1M17) and IGFR (PDB 5XFS).

Animal Model
The experiment was conducted on 150-200 g Albino rats (Wistar) kept under normal conditions (room temperature 24-27 • C, moisture 60-65%) with a 12-h light-dark cycle. Rats were fasted for 24 h before the test to guarantee an empty stomach. The animals were housed in standard cages with free access to water and food. The animals were acclimatized for seven days prior to the experiment start day, and all circumstances were designed to eliminate animal suffering. To eliminate differences owing to diurnal rhythms as possible regulators of stomach functions, all rats were used in the experiment at the same time of day [30,31]. The rats were separated into four groups: A, B, C and D, each of which had 6 rats. The normal control group, Group A received only the vehicle (0.5 percent CMC solution). Group B (positive control) was given the vehicle (0.5 percent CMC solution). Group C (market comparable reference group) was administered oral cimetidine (100 mg/kg p.o) (oral administration), whereas Group D was given guava seed extracts of 300 mg/kg p.o. (dissolved in a 0.5% CMC solution). After 1 h, groups B, C, and D received a single acute dosage of indomethacin (IND) (40 mg/kg) orally to induce gastric ulcers. All animals of the four groups were killed four hours after starting the experiment [32,33]. The stomach was cut and the larger curvature of the stomach was sliced apart. The stomachs were placed flat on a cork board after being washed under a steady stream of water. The ulcer index was obtained by counting the number of ulcer spots in the glandular region of the stomach in both control and drug-treated animals [30,34].

Gastric Mucosal Lesions Evaluation
The ulcer index (UI) was determined using a 0-3 scoring system and an inspection of the stomachs using an eyepiece. The severity factor was determined as follows: level 0 = no lesions, level 1 = lesions, level 2 = lesions, level 3 = lesions. Lesions with a length of less than 1 mm were classified as level 1, lesions with a length of 2-4 mm were classified as level 2, and lesions with a length of more than 4 mm were classified as level 3. Each rat's lesion score was determined by multiplying the number of lesions by their respective factor. The drug's preventative index (PI) was determined by the use the following formula: PI = (UI of indomethacin group − treated group) × 100\(UI of indomethacin group) [35].

Histopathological Examination
The stomach tissue was preserved in a 10% ethanol formalin solution and treated with graded ethanol, paraffin wax, and xylene before being cut into slices using a microtome. The slices were viewed under a microscope after being stained with Eosin stain and Hematoxylin. The various histological indicators were examined [36].

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Quantitative Real-Time Polymerase Chain Reaction is discussed in detail in the Supplementary Materials, as mentioned in Table S1.

In Vitro Antioxidant Activity
The H 2 O 2 and SOD scavenging activities of Psidium Guajava seed crude extract are thoroughly discussed in the Supplementary Materials.

Statistical Analysis
All of the data were set as a mean ± SD. A one-way analysis of variance (ANOVA) was used to evaluate the data of all groups, followed by the Dunnett's test with statistical significance at p < 0.05.

Histopathological Studies
Moreover, the pharmacological and biochemical findings are compatible with histological investigations of stomach tissue. The normal stomach mucosa is shown in Figure 3A. The negative control group ( Figure 3B) showed ulceration and bleeding on the mucosa. The positive control group ( Figure 3C) had cimetidine. Guava seed extract pretreatment of rats resulted in a lack of mucosal ulcers and bleeding, as shown in Figure  3D.

Histopathological Studies
Moreover, the pharmacological and biochemical findings are compatible with histological investigations of stomach tissue. The normal stomach mucosa is shown in Figure 3A. The negative control group ( Figure 3B) showed ulceration and bleeding on the mucosa. The positive control group ( Figure 3C) had cimetidine. Guava seed extract pretreatment of rats resulted in a lack of mucosal ulcers and bleeding, as shown in Figure 3D.

Docking Study
In this study, we used a molecular docking technique to predict potential mechanisms of action of the identified compounds acting as an antiulcer. Twenty-three compounds were docked against three antiulcer targets including the gastric proton pump, muscarinic receptor and H2 receptor. Two of these targets are available on the protein date bank, which are the proton pump and muscarinic receptor under 5YLU and 5ZHP, respectively. The structure of the H2 receptor was obtained in homology model form in the GPCR data base. Validation of docking procedure was done by redocking of the co-crystalized ligand and calculation of RMSD between docked and crystalized poses. An RMSD of less than 2 Å is considered acceptable ( Figure 4A shows and example of the validation of the gastric proton pump). Docking results are shown in Table 2, and show that several sterol compounds have docking scores better than the co-crystalized ligand vonoprazan in cases with the gastric proton pump. These compounds include β-Sitosterol, campesterol, stigmastanol and stigmasterol. The scores of these compounds with gastric proton pump is better than their scores with the other two targets. In addition to these sterols, Quercetin-4′-glucuronide showed a docking score with the same target, the gastric proton pump, that is very close to the co-crystalized ligand. The docked sterols were able to maintain the same hydrogen bond that is formed by vonoprazone with A339 of the gastric proton pump ( Figure 4B). Among those sterols, campesterol showed the best docking score (−9.9 kcal/mol) with proton pump protein. Its docking pose overlapped with the docking position of vonoperazone, forming the same hydrogen bonds as well as hydrophobic interactions, as shown in Figure 4C. Another example is the next sterol, stigmasterol (−9.7 kcal/mol), that showed a similar docking pose to campesterol, as shown in Figure 4D. These results suggest that these sterols might be responsible for the antiulcer effect through the inhibition of the gastric proton pump, which might require further

Docking Study
In this study, we used a molecular docking technique to predict potential mechanisms of action of the identified compounds acting as an antiulcer. Twenty-three compounds were docked against three antiulcer targets including the gastric proton pump, muscarinic receptor and H2 receptor. Two of these targets are available on the protein date bank, which are the proton pump and muscarinic receptor under 5YLU and 5ZHP, respectively. The structure of the H2 receptor was obtained in homology model form in the GPCR data base. Validation of docking procedure was done by redocking of the co-crystalized ligand and calculation of RMSD between docked and crystalized poses. An RMSD of less than 2 Å is considered acceptable ( Figure 4A shows and example of the validation of the gastric proton pump). Docking results are shown in Table 2, and show that several sterol compounds have docking scores better than the co-crystalized ligand vonoprazan in cases with the gastric proton pump. These compounds include β-Sitosterol, campesterol, stigmastanol and stigmasterol. The scores of these compounds with gastric proton pump is better than their scores with the other two targets. In addition to these sterols, Quercetin-4 -glucuronide showed a docking score with the same target, the gastric proton pump, that is very close to the co-crystalized ligand. The docked sterols were able to maintain the same hydrogen bond that is formed by vonoprazone with A339 of the gastric proton pump ( Figure 4B). Among those sterols, campesterol showed the best docking score (−9.9 kcal/mol) with proton pump protein. Its docking pose overlapped with the docking position of vonoperazone, forming the same hydrogen bonds as well as hydrophobic interactions, as shown in Figure 4C. Another example is the next sterol, stigmasterol (−9.7 kcal/mol), that showed a similar docking pose to campesterol, as shown in Figure 4D. These results suggest that these sterols might be responsible for the antiulcer effect through the inhibition of the gastric proton pump, which might require further follow-up studies to investigate this effect. In addition, the same twenty-three identified compounds were also docked in the active sites of several inflammatory targets and mediators which include COX-1, COX-2, TNF-α, TGF-β, EGFR, and IGFR. This was conducted to investigate whether the antiulcer effect of these compounds could be mediated through the modification of any of these inflammatory mediators. The results of this docking study are also shown in Table 2. Several sterols were able to hit some of these inflammatory targets; for example, stigmasterol had good docking scores with TGF-β (−9.6 kcal/mol) and EGFR (−9.4 kcal/mol). Another example of a nonsteroidal compound that showed good results with one of these inflammatory mediators is quercetin-4 -glucuronide, which showed its best docking score with TGF-β (−9.5 kcal/mol). These results suggest that other mechanisms could also be involved in the antiulcer effect of the study extracts and open the door for further investigation in this important area.

Effect of Psidium guajava Seed Extract on Expression of TNF-α, IL-1β, IL-6, TGF-β, COX-2, and IGF-1
After mucosal damage, the healing of a stomach ulcer is a complex physiological process including tissue auto-repair, reperfusion, and regeneration. A stomach ulcer is a lesion comprised of connective and granular tissues with fibroblasts and macrophages, as well as the generation of proliferating microvessels at the ulcer base and ulcer edge via angiogenesis. This lesion is surrounded by non-necrotic mucosa, which creates the ulcer edge that heals too quickly, resulting in the formation of an ulcer scar. There are four stages to the ulcer healing process: Indomethacin-induced inflammation, ulcer formation as evidenced by inflammatory cell infiltration, tissue necrosis, and lastly, the creation of granulation tissue and ulcer margin [43].
Upon activation of immune cells, a variety of proinflammatory cytokines such as interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are secreted from macrophages. A proinflammatory cytokine, IL1β, regulates a variety of genes involved in the inflammatory reaction and related tissue damage, such as compromising the function of entero-chromaffin-like cells. In addition, IL-1β inhibits the proliferation of gastric epithelial cells [44], an effect that involves tyrosine kinase, protein kinase C, and multiple mitogen-activated protein kinases. The inflammation triggered by IL-1β, Il-6 and TNF-α can be antagonized by the antiulcer action of Psidium guajava seed extract, which attains the capability of suppressing the resultant inflammatory responses, as shown in Figure 5.

Effect of Psidium guajava Seed Extract on Expression of TNF-α, IL-1β, IL-6, TGF-β, COX-2, and IGF-1
After mucosal damage, the healing of a stomach ulcer is a complex physiological process including tissue auto-repair, reperfusion, and regeneration. A stomach ulcer is a lesion comprised of connective and granular tissues with fibroblasts and macrophages, as well as the generation of proliferating microvessels at the ulcer base and ulcer edge via angiogenesis. This lesion is surrounded by non-necrotic mucosa, which creates the ulcer edge that heals too quickly, resulting in the formation of an ulcer scar. There are four stages to the ulcer healing process: Indomethacin-induced inflammation, ulcer formation as evidenced by inflammatory cell infiltration, tissue necrosis, and lastly, the creation of granulation tissue and ulcer margin [43].
Upon activation of immune cells, a variety of proinflammatory cytokines such as interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are secreted from macrophages. A proinflammatory cytokine, IL1β, regulates a variety of genes involved in the inflammatory reaction and related tissue damage, such as compromising the function of entero-chromaffin-like cells. In addition, IL-1β inhibits the proliferation of gastric epithelial cells [44], an effect that involves tyrosine kinase, protein kinase C, and multiple mitogen-activated protein kinases. The inflammation triggered by IL-1β, Il-6 and TNF-α can be antagonized by the antiulcer action of Psidium guajava seed extract, which attains the capability of suppressing the resultant inflammatory responses, as shown in Figure 5.
The induction of COX-2 in fibroblasts, epithelial, parietal, and mononuclear cells of the mucosa, and in response to IL-1β elevation, is related with indomethacin-induced gastritis. The results showed a marked reduction of the elevated COX-2 relative gene expression upon treatment with Psidium guajava seed extract, which further confirms its protective role in ulcer healing, as shown in Figure 5. The induction of COX-2 in fibroblasts, epithelial, parietal, and mononuclear cells of the mucosa, and in response to IL-1β elevation, is related with indomethacin-induced gastritis. The results showed a marked reduction of the elevated COX-2 relative gene expression upon treatment with Psidium guajava seed extract, which further confirms its protective role in ulcer healing, as shown in Figure 5.
IL-1β contributes to the initial upregulation of TGF-β, and IGF-1 in the base of indomethacin-evoked ulcers in a rat stomach [45]. Although TGF-β inhibits cell proliferation in a variety of cells, it induces angiogenesis, cell migration, and increases extracellular matrix (ECM) production, all of which are required for gastrointestinal ulcer healing at the intermediate and late stages of the healing process. It also acts as an antiinflammatory cytokine [46]. Macrophages are activated following indomethacin-induced , and IGF-1 (F) relative gene expression in gastric mucosa tissue of different groups of rats was assessed using quantitative RT-PCR. After normalization to GAPDH, the data reflect a fold difference in expression compared to the normal control group. The bars show the mean ± standard deviation. To see if there is a significant difference between groups, a one-way ANOVA test is performed, with # p < 0.05 compared to the normal control group and * p < 0.05 compared to the indomethacin induced group.
IL-1β contributes to the initial upregulation of TGF-β, and IGF-1 in the base of indomethacin-evoked ulcers in a rat stomach [45]. Although TGF-β inhibits cell proliferation in a variety of cells, it induces angiogenesis, cell migration, and increases extracellular matrix (ECM) production, all of which are required for gastrointestinal ulcer healing at the intermediate and late stages of the healing process. It also acts as an anti-inflammatory cytokine [46]. Macrophages are activated following indomethacin-induced inflammation, which increases the release of active TGF-β with increasing plasmin activation [47].
Furthermore, IGF-1 expression can contribute to the acceleration of ulcer healing in both the early and late phases of the healing process [46]. IGF-1, a major mediator of soft tissue regeneration, is released from platelet granules shortly after damage and plays a crucial role in angiogenesis and epithelialization in gastric ulceration [48]. Accordingly, we found that the IGF-1 mRNA level was significantly increased in the stomach mucosa following ulceration in normal rats. Finally, after treatment with Psidium guajava seed extract, the results showed marked downregulation of the elevated TGF-β, and IGF-1 relative gene expression, as shown in Figure 5. This study looked at the antioxidant activity of Psidium Guajava seed extract as a scavenger potential against (H 2 O 2 ). According to the findings, the maximum H 2 O 2 radical scavenging activity of PGE was 55.55% at a 1000 µg/mL concentration. When compared to conventional ascorbic acid (IC 50 = 182.7 µg/mL), Psidium guajava seed extract suppressed the formation of H 2 O 2 in a dose-dependent manner ( Figure 6). crucial role in angiogenesis and epithelialization in gastric ulceration [48]. Accordingly, we found that the IGF-1 mRNA level was significantly increased in the stomach mucosa following ulceration in normal rats. Finally, after treatment with Psidium guajava seed extract, the results showed marked downregulation of the elevated TGF-β, and IGF-1 relative gene expression, as shown in Figure 5.

Hydrogen Peroxide Scavenging Activity
This study looked at the antioxidant activity of Psidium Guajava seed extract as a scavenger potential against (H2O2). According to the findings, the maximum H2O2 radical scavenging activity of PGE was 55.55% at a 1000 µ g/mL concentration. When compared to conventional ascorbic acid (IC50 = 182.7 µ g/mL), Psidium guajava seed extract suppressed the formation of H2O2 in a dose-dependent manner ( Figure 6). Figure 6. H2O2 radical scavenging activity of PGE at different concentration (1000 µ g/mL, 500 µ g/mL, 250 µ g/mL, and 125 µ g/mL). Bars represent mean ± standard deviation (SD). Significant difference between groups is analyzed by a two-way ANOVA test.

Superoxide Radical Scavenging Activity
The superoxide scavenging (SOD) activity of Psidium guajava seed extract was determined. The scavenging potential of PGE and the standard increase are concentration dependent (Figure 7), with Psidium guajava seed extract showing the maximum superoxide radical scavenging activity. Psidium guajava seed extract had 62.5% superoxide scavenging efficacy at a 1000 μg/mL concentration. The concentration of Psidium guajava seed extract needed for 50% inhibition (IC50) was found to be 147.1 µ g/mL compared to (IC50) 158.8 μg/mL for ascorbic acid. radical scavenging activity of PGE at different concentration (1000 µg/mL, 500 µg/mL, 250 µg/mL, and 125 µg/mL). Bars represent mean ± standard deviation (SD). Significant difference between groups is analyzed by a two-way ANOVA test.

Superoxide Radical Scavenging Activity
The superoxide scavenging (SOD) activity of Psidium guajava seed extract was determined. The scavenging potential of PGE and the standard increase are concentration dependent (Figure 7), with Psidium guajava seed extract showing the maximum superoxide radical scavenging activity. Psidium guajava seed extract had 62.5% superoxide scavenging efficacy at a 1000 µg/mL concentration. The concentration of Psidium guajava seed extract needed for 50% inhibition (IC 50 ) was found to be 147.1 µg/mL compared to (IC 50 ) 158.8 µg/mL for ascorbic acid. Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 14 Figure 7. Superoxide radical scavenging activity of Psidium guajava seed extract at different concentration (1000 µ g/mL, 500 µ g/mL, 250 µ g/mL, and 125 µ g/mL). Bars represent mean ± SD (standard deviation). Significant difference between groups is analyzed by a two-way ANOVA test.

Conclusions
The present research examined the possible gastroprotective effect of Psidium Guajava seed extract against NSAID-induced gastric ulcers, as well as its antioxidant activity (hydrogen peroxide scavenging activity and superoxide radical scavenging activity). Metabolomic profiling via LC-HRMS-based chemical profiling of Psidium guajava revealed the abundance of many putative active compounds. Psidium guajava seed extracts significantly decreased the ulcer index and protected the mucosa from lesions. The antiulcer effect of Psidium guajava seed extract downregulated the cytokines expression (IL-1β, IL-6, and TNF-α). Moreover, PGE significantly downregulated the increased COX-2, TGF-β, and IGF-1 relative gene expressions, confirming its beneficial effect in ulcer healing. Moreover, the identified compounds were also docked in the active sites of several inflammatory targets and mediators, which include COX-1, COX-2, TNF-α, TGFβ, EGFR, and IGFR. The gastroprotective effect could be potentially attributed to the effect of several sterols such as stigmasterol and campesterol, as suggested by docking studies. Our experiment proved the possible use of Psidium guajava seeds as a protective agent against ulceration, and a possible alternative to antiulcer agents. These results might need further investigation to support this potential effect, but they highlight the mechanism of the gastro protective and antioxidant effect of Psidium guajava seeds.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Primer sequence of the genes included in the study [35,[49][50][51][52].  Institutional Review Board Statement: Extreme care was taken and all procedures on rabbits were adopted according to Deraya laboratory guidelines. The approval code is 9/2021.

Informed Consent Statement: Not applicable
Data Availability Statement: Data is contained within the article and supplementary material. . Superoxide radical scavenging activity of Psidium guajava seed extract at different concentration (1000 µg/mL, 500 µg/mL, 250 µg/mL, and 125 µg/mL). Bars represent mean ± SD (standard deviation). Significant difference between groups is analyzed by a two-way ANOVA test.

Conclusions
The present research examined the possible gastroprotective effect of Psidium Guajava seed extract against NSAID-induced gastric ulcers, as well as its antioxidant activity (hydrogen peroxide scavenging activity and superoxide radical scavenging activity). Metabolomic profiling via LC-HRMS-based chemical profiling of Psidium guajava revealed the abundance of many putative active compounds. Psidium guajava seed extracts significantly decreased the ulcer index and protected the mucosa from lesions. The antiulcer effect of Psidium guajava seed extract downregulated the cytokines expression (IL-1β, IL-6, and TNF-α). Moreover, PGE significantly downregulated the increased COX-2, TGF-β, and IGF-1 relative gene expressions, confirming its beneficial effect in ulcer healing. Moreover, the identified compounds were also docked in the active sites of several inflammatory targets and mediators, which include COX-1, COX-2, TNF-α, TGF-β, EGFR, and IGFR. The gastroprotective effect could be potentially attributed to the effect of several sterols such as stigmasterol and campesterol, as suggested by docking studies. Our experiment proved the possible use of Psidium guajava seeds as a protective agent against ulceration, and a possible alternative to antiulcer agents. These results might need further investigation to support this potential effect, but they highlight the mechanism of the gastro protective and antioxidant effect of Psidium guajava seeds.