In Vivo, Proteomic, and In Silico Investigation of Sapodilla for Therapeutic Potential in Gastrointestinal Disorders

This study aims to delineate the effects of Manilkara zapota Linn. (Sapodilla) fruit chloroform (Mz.CHCl3) and aqueous (Mz.Aq) extracts tested through different techniques. Antidiarrheal activity and intestinal fluid accumulation were examined by using castor oil-induced diarrhea and castor oil fluid accumulation models. Isolated rabbit jejunum tissues were employed for in vitro experiments. Antimotility and antiulcer were performed through charcoal meal transient time and ethanol-induced ulcer assay, molecular studies were conducted through proteomic analysis, and virtual screening was performed by using a discovery studio visualizer (DSV). Mz.CHCl3 and Mz.Aq extracts attributed dose-dependent (50–300 mg/kg) protection (20–100%) against castor oil-induced diarrhea and dose-dependently (50–300 mg/kg) inhibited intestinal fluid secretions in mice. Mz.CHCl3 and Mz.Aq extracts produce relaxation of spontaneous and K+ (80 Mm) induced contractions in isolated tissue preparations and decreased the distance moved by charcoal in the gastrointestinal transit model in rats. It showed gastroprotective effect in ulcerative stomach of rats and decreased levels of IL-18 quantified by proteomic analysis. Histopathological results showed ethanol-induced significant gastric injury, leading to cloudy swelling, hydropic degeneration, apoptosis, and focal necrosis in all gastric zones using hematoxylin and eosin (H&E) staining. Moreover, ethanol increased the activation and the expression of tumor necrotic factor (TNF-α), cyclooxygenase (COX-2), and nuclear factor kappa-light-chain-enhancer of activated B cells (p-NFκB). In silico results were comparative to in vitro results evaluated through virtual screening. Moreover, ethanol increased the activation and expression of tumor necrotic factor, cyclooxygenase, and nuclear factor kappa-light-chain-enhancer of activated B cells. This study exhibits the gastroprotective effect of Manilkara zapota extracts in the peritoneal cavity using a proteomic and in silico approach which reveals different energy values against target proteins, which mediate the gastrointestinal functions.


Introduction
Gastrointestinal disorders are highly prevalent among the people of Asia. Health physicians claimed that it is an origin for the occurrence of several other comorbidities. Till now, there is no significant drug that has been discovered for gastrointestinal disorders, which completely eradicates the disease [1]. Moreover, herbal therapies have shown excellent economical and long-lasting potential to treat gastric system disorders [2].
In silico and in vivo evaluation of fruit extracts helps to screen out novel bioactive molecules, and their further processing leads to the development of innovative therapies. Most significant and desirable therapeutic effects can be achieved with the purified form of isolated bioactive constituents that can be formulated into suitable dosage form along with the dosage regimen. Many chronic disorders have been treated by herbal remedies, particularly by consuming fruits as functional foods as well as their active constituents. Researchers have investigated the role of crude fruit extracts, which have the potential to combat gastrointestinal disorders.
is research aimed to delineate Manilkara zapota fruit extracts for antidiarrheal effects, antisecretory effects, isolated tissue preparation, gastrointestinal transient time, and antiulcer effects. On the basis of ethnomedicinal uses, we assumed that extracts modulate the expression of proteins in the stomach and thus potentially promote the molecular and organ/tissue damage associated with diseases [5]. Ulcer is an inflammatory disorder, so we hypothesize here whether Manilkara zapota extract could cure gastric ulcer by inhibiting NFκB-dependent proinflammatory cytokines. e present study aims to evaluate whether Manilkara zapota effects on inflammation and oxidative stress could eventually account for gastric cell protection. Moreover, in silico approach helps us to understand the biochemical mechanisms and unknot the complex signaling network, which controls cellular function including apoptosis and inflammation [8]. e chemical structure of Manilkara zapota constituent is shown in Figure 1.

Materials and Methods
2.1. Materials. Manilkara zapota fruits (4 kg) were purchased from the local market in March 2018 and verified by a taxonomist Dr. Mushtaq Ahmad, at the Department of Plant Sciences, Quaid-a-Azam University, Islamabad. Voucher specimen no. ISL-B-23 was collected after submitting the specimen sample of species to the herbarium. Plants were dried, crushed, and extracted with chloroform (4 × 500 mL). e whole extracts were filtered through a standard procedure using Whatman No. 1 filter paper and then evaporated through a rotary shaker (Tokyo Rikakikai Type A 1000S) under reduced pressure at temperature not exceeding 55°C to yield 30 g dry extract.
e leftover residue was suspended in 4 L of methanol-water (20 : 80) and successively extracted with the aforementioned technique. e solvent was evaporated under reduced pressure to yield 110 g of extract [5].

Experimental Animals and Housing Conditions.
Experiments were performed in compilation with the rules of Research and Ethics Committee of RIPS (Ref. no. REC/ RIPS/2018/021) along with the guidelines of "Principles of Laboratory Animal care." Balb/C mice (20-25 g), rabbits (1.0-2.0 kg), and Sprague-Dawley rats (190-240 g) of either gender were used in the experimentation procedure and were kept in animal house of the Riphah Institute of Pharmaceutical Sciences (RIPS), Islamabad, provided with controlled environment (20-25°C).

Phytochemical Analysis.
Preliminary phytochemical analysis was carried out for the detection of major secondary metabolites such as saponins, glycosides, anthraquinones, alkaloids, steroids, proteins, flavonoids, and tannins according to the standard procedure with slight modifications [9].

Castor Oil-Induced Diarrhea.
e protocol was previously described by Sisay et al. [10]. Balb/C mice were randomly allocated to five groups for Mz.CHCl 3 and Mz.Aq, respectively. Animals were fasted for 24 hours (08:00-08:00) before experimentation. Each floor of cage was lined with blotting sheets in which animals were placed. Negative control group received normal saline (10 mL/kg) while positive control group received loperamide hydrochloride (2 mg/kg), and the remaining groups were given 50, 100, and 300 mg/kg of the extracts orally, respectively. After 1 hour of treatment, all mice received castor oil (10 mL/kg p.o.). Animals were individually observed for a period of 4 hours to analyze the onset of diarrhea and presence and absence of diarrheal dropping. e percentage of diarrheal protection was evaluated according to Rehman et al. [11] by using the chi-square (Χ 2 ) test.

Assessment of Intestinal Fluid Accumulation.
Intestinal fluid accumulation was determined using the method described by Sisay et al. [10]. To study the intestinal fluid accumulation, enteropooling assay was used. Mice fasted overnight for 24 hours (08:00-08:00) were taken and placed in five assigned cages (n = 5). Groups I and II were given normal saline (10 mL/kg) and castor oil (10 mL/kg, p.o.), respectively. Mz.CHCl 3 doses of 50, 100, and 300 mg/kg intraperitoneally were administered to Groups III, IV, and V, respectively. Standard drug atropine at dose 10 mg/kg was given to II group, 1 hour prior induction with castor oil (10 mL/kg, p.o.). e same procedure was repeated for the Mz.Aq extract. Mice were sacrificed after 30 min, and the intestine was removed and weighed.
e results were articulated as (P i /P m ) × 1000, where P i is the weight (g) of the intestine and P m is the weight of the animal, by using GraphPad Prism and analysis of variance one-way ANOVA followed by Tukey's post hoc test.

Isolated Tissue Preparation.
Before experimentation, the rabbit was fasted for 24 hours (08:00-08:00) but had a free access to water. After cervical dislocation, jejunal portion of 2 cm was isolated and washed with Tyrode's solution and suspended in tissue bath containing Tyrode's solution to equilibrate for 30 minutes with environment along with proper supply of oxygen (95% O 2 ) and 5% CO 2 (carbogen) under control temperature conditions before drug administration. Each preparation was stabilized with concentration of ACh (0.3 μM), until constant responses were recorded via a force displacement transducer (model FT-03) coupled with a bridge amplifier and a power Lab 4/25 data acquisition system connected to computer running Lab-Chart 6 software (AD Instrument, Sydney, Australia). Graded doses of Mz.CHCl 3 and Mz.Aq extracts were tested for % change in contractions of jejunum [12]. e graphs were analyzed by using the nonlinear regression curve followed by sigmoidal dose-response (variable slope) by using GraphPad Prism.

Charcoal Meal Transit Time.
e inhibitory activity of the propulsion of a charcoal meal was estimated as described by Shang et al. [13]. Rats were fasted for 24 hours but had a free access to water. Test groups received the doses 50, 100, and 300 mg/kg of Mz.CHCl 3 and Mz.Aq according to body weight, where they as positive control group received atropine sulphate (0.1 mg/kg, i.p.), while the negative control group received normal saline (10 mL/kg, p.o.). After one hour, animals receive charcoal meal (1 mL/ kg). 30 minutes after all treatments, all the animals were sacrificed. e small intestine was excised, and the distance travelled by charcoal meal (5% activated charcoal suspension in distilled water) through the organ was expressed as a percentage of the length of the small intestine according to the following expression. e data were analyzed by using one-way ANOVA followed by Tukey's post hoc test: peristaltic index (PI) % � distance moved by charcoal meal total length of the intestine × 100. (1) For further evaluation of % inhibition, peristaltic index is used: where PIC is the peristaltic index of control and PIT is the peristaltic index of the test group.
2.9. Ethanol-Induced Ulcer Assay. Rats were randomly assigned into groups weighing 190-240 g of either sex and kept for fasting period of 24 hours (09:00-09:00). Group 1 served as a negative control received normal saline 10 mL/ kg body weight, and group 2 received 20 mg/kg (p.o.) omeprazole as standard drug. Groups 3, 4, and 5 received 50, 100, and 300 mg/kg (p.o.) of Mz.CHCl 3 and Mz.Aq extracts, respectively. All the animals were treated with 1 mL/100 g of ethanol (p.o.) after 1 hour of treatment. Animals were sacrificed through cervical dislocation and stomachs were removed and washed with normal saline, and lesion index was estimated by measuring each lesion in mm along its greater curvature. Surface area of each lesion was measured and scoring was done. For each stomach lesion, ulcer index was taken as mean ulcer score (US) (such as 0: no ulcer, 1: US ≤ 0.5 mm 2 , 2: 0.5 < US ≤ 2.5 mm 2 , 3: 2.5 mm 2 < US ≤ 5 mm 2 , 4: 5 mm 2 < US ≤ 10 mm 2 , 5: 10 mm 2 < US ≤ 15 mm 2 , 6: 15 mm 2 < US ≤ 20 mm 2 , 7: 20 mm 2 < US ≤ 25 mm 2 , 8: 25 mm 2 < US ≤ 30 mm 2 , 9: 30 mm 2 < US ≤ 35 mm 2 ; and 10: US > 35 mm 2 ) [14]. For each stomach injury, the sum of the lengths (mm) of all scores was utilized as the ulcer index (UI). e gastroprotective assessment was displayed as an inhibition percentage (I%) calculated by the following formula: where U Sc is the ulcer surface area of control and U St is the ulcer surface area of the test drug group. e data were analyzed by using one-way ANOVA followed by Tukey's post hoc test. e gastric tissues were stored in a biofreezer (− 80°C) for further proteomic analysis.

Enzyme-Linked Immunosorbent Assay (ELISA).
IL-18 expression was measured using Rat IL-18 ELISA kit (E-EL-R0567) according to the manufacturer's instructions (Elabscience). e tissues were homogenized at 15000 using Silent Crusher M (Heidolph), and the supernatant was collected after centrifugation (at 1350 ×g for 1 h). e concentration of IL-18 was determined by using an ELISA microplate reader [15]. Values are expressed as pictograms of cytokines per milliliter (pg/ mL) and evaluated by using two-way ANOVA followed by Tukey's post hoc test.

Hematoxylin and Eosin (H&E) Staining and Immunohistochemical Analysis.
Tissue sections on coated slides were deparaffinized with absolute xylene (100%) and rehydrated with ethyl alcohol (from 100% (absolute) to 70%). e slides were rinsed with distilled water and immersed in hematoxylin for 10 min. e slides were then kept under running water in glass jar for 10 minutes and treated with 1% HCl and 1% ammonia water as previously reported by Gim et al. [16]. e slides were added to eosin solution for 5-10 min. After due time, the slides were rinsed in water and air-dried for some time. e dried slides were dehydrated in graded ethyl alcohol (70%, 95%, and 100%). e slides were cleared with xylene and were mounted with glass cover slip. e slides were pictured with light microscope (Olympus, Japan) and analyzed by ImageJ, a computer-based program. e number of images per slide was five per group, while focusing specifically on gastric cell size and shape, inflammatory infiltrated cells, and vacuolation described by Shah et al. [8]. e TIF images were optimized to the same threshold intensity for all groups and examined in GraphPad Prism.
Immunohistochemical staining was performed as described previously with little modifications done by Gim et al. [16]. After deparaffinization, slides were processed for antigen retrieval step (enzymatic method) and then washed with PBS. e endogenous peroxidase was quenched by applying 3% hydrogen peroxide (H 2 O 2 ) in methanol for 10 min. e slides were incubated with 5% normal goat serum containing 0.1% Triton X-100. After blocking, the slides were incubated overnight with mouse anti-TNF-α, p-NFκB, and mouse anti-COX-2 antibodies (dilution 1 : 100, Santa Cruz Biotechnology). e following morning, after washing with 0.1 M PBS, slides were incubated in biotinylated secondary antibody (dilution 1 : 50) according to the origin of the primary antibody and serum used. Following secondary antibody treatment, slides were incubated with ABC Elite kit (Santa Cruz Biotechnology) for 1 hour in a humidified chamber. e slides were washed with 0.1 M PBS, stained in DAB solution, washed with distilled water, dehydrated in a graded ethanol series, fixed in xylene, and cover-slipped in mounting medium. Immunohistochemical TIF images (five images per slide) were captured with a light microscope. ImageJ software was used to quantitatively determine hyperactivated p-NFκB, COX-2, and TNF-α, by optimizing the background of images according to the threshold intensity and analyzing p-NFκB, COX-2, and TNF-α positive cells at the same threshold intensity for all groups. e intensity is expressed as the relative integrated density of the samples relative to the saline by using one-way ANOVA followed by Tukey's post hoc test.

Acute Toxicity.
is study delineates lethal versus nonlethal dose of extracts in the animal model by using acute toxicity model. Mice were divided into three groups of five mice each. e test was performed using increasing doses (3 and 5 g) of the Mz extract given in 10 mL/kg volume. Saline (10 mL/kg, p.o., negative control) was administered to one group. Animals were observed for change in behavior for about 4 hours, and mortality rate was studied for 24 hours after drug administration described by Ogwal-Okeng et al. [17].

Computational Studies.
rough DSV, 3-dimensional structures of reference drugs were prepared (2016) by adding polar hydrogen atoms (H-atoms) and converted into PDB file and downloaded from PubChem database. Standard drugs for docking included phenylephrine, pirenzepine, atropine, domperidone, calmidazolium, verapamil, omeprazole, ranitidine, loperamide, and papaverine for receptors. Targets involved in gut physiology were selected and obtained from the website of RCSB protein data bank. Selected targets included adrenergic α 1 receptor (PDB ID: P35348), calmodulin (PDB ID: 1CTR), dopaminergic D 2 (PDB ID: 6CM4), histaminergic H 2 (PDB ID: P25021), H + / K + ATPase (PDB ID: 4UX2), muscarinic M 1 (PDB ID: 5CXV), muscarinic M 3 (PDB ID: 2CSA), mu-opioid (PDB ID: 6DDE), phosphodiesterase enzyme (PDB ID: 6DDE), and voltage gated L-type calcium channel (PDB ID: 1T3S). Structures were purified by removing water molecules and ligands in DSV and were saved in PDB format till further procedure. PyRx is used to perform molecular docking, and the results were analyzed through the best pose by using DSV and energy values (kcal/mol) achieved through PyRx in sdf and csv formats. Only one pose having the lowest value of energy (kcal/mol) was selected for postdock analysis through DSV after the evaluation of top ten conformations. 2D image was assessed to check the ligands and amino-acid residue interactions with the receptor including alanine (ALA), asparagine (ASN), aspartic acid (ASP), arginine (ARG), lysine (LYS), threonine (THR), glycine (GLY), glutamine (GLN), cysteine (CYS), methionine (MET), glutamic acid (GLU), histidine (HIS), phenylalanine (PHE), isoleucine (ILE), proline (PRO), tyrosine (TYR), serine (SER), threonine (THR), tryptophan (TRP), and valine (VAL). Docking signifies the binding of the receptor with the ligand in the physiological mechanism and hence provides a valuable marker for drug binding inside the body.
2.14. Statistical Analysis. Data were expressed as mean-± SEM (n � 5) and median effective concentrations (EC 50 ) having 95% confidence intervals. Statistical analysis of the results was analyzed using one-way ANOVA followed by Tukey's post hoc test. Chi-square test was used in the case of the antidiarrheal data, where p < 0.05 was regarded as significant. Nonlinear regression using GraphPad program (GraphPAD, San Diego, CA, USA) was used to analyze the concentration-response curves.

Effect of Mz.CHCl 3 and Mz.Aq on IL-18 Expression in the
Stomach of Ethanol-Induced Ulcer Models. Figure 5 illustrates the effect of Manilkara zapota chloroform (Mz.CHCl 3 ) and aqueous extract (Mz.Aq) on IL-18 in gastric tissues. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test revealed that Manilkara zapota extracts significantly reduced the IL-18 expression in treated groups as compared to the negative control group ( * p < 0.05 and * * p < 0.01 versus ethanol group) ( # p < 0.001 versus saline). e levels are significantly decreased in treated versus disease group. In Mz.CHCl 3 at doses of 100 and 300 mg/kg, the level of inflammatory mediator release from the tissue was relatively less at higher doses, in contrast to the response shown by Mz.Aq at doses of 100 and 300 mg/ kg. In comparison, Mz.Aq has more significant results with respect to omeprazole.

Histopathology and Immunohistochemistry (IHC)
Analysis. Figure 6 shows IHC and H&E staining in order to distinguish necrotic cells from intact ones. Ethanol produces robust cellular changes in the highly prone areas of gastric cells, while treatment with Mz.Aq extracts attenuates this damage. A substantial difference was observed in the control group relative to the ethanol-administered group. e damaged area exhibits abnormal morphological features including alteration in mucosal cell size and shape, alteration in color staining (cytoplasmic eosinophilia/pyknosis and basophilic nature of nucleus), and vacuolation. No noticeable alterations were noticed in the histological preparations of control animals. Significantly more intact cells were there in the Mz.Aq extract treated groups compared to the ethanol-operated group ( * p < 0.05, * * p < 0.01, and * * * p < 0.001). Severity scores of stomach injury in different groups (n � 5) are calculated via relative density of value (AU) and relative integrated density. Data are mean ± SEM. * * * p < 0.001 versus ethanol group; * p < 0.05 versus ethanol group.

Acute
Toxicity. e extracts did not cause any significant behavioral and pathological changes and did not show any mortality up to the dose of 5 g/kg.

Docking Evaluation.
Energy values and H-bonds are the two main contributing factors for docking evaluation. However, formation of π-π bonds, π-alkyl bonds, π-sigma bonds, and Vander Waal forces plays a leading role between ligand-receptor complexes. In this study, methyl chlorogenate showed variable binding affinities against different protein receptors. Table 4 summarizes the atomic energy values (kcal/mol) and residues involved in H-bonding, π-π bonding, and other hydrophobic interactions of best dock poses of methyl chlorogenate and reference drugs in comparison with adrenergic α 1 receptor, muscarinic M 1 , muscarinic M 3 , dopaminergic D 2 , calmodulin, mu-opioid, voltage gated L-type calcium channel, histaminergic H 2 , H + / K + ATPase pump, and phosphodiesterase enzyme are plotted. Figures 7-16 illustrate the 2D view of interactions of methyl chlorogenate along with standard drugs. Against α 1 adrenergic receptor, methyl chlorogenate showed E-value of − 7.5 kcal/mol and formed 2 H-bonds and 4 hydrophobic interactions whereas phenylephrine showed E-value of − 6.6 kcal/mol and formed 5 H-bonds, 1 π-π bonds, and 4 other hydrophobic interactions. e 2D interactions are shown in Figure 7. Against M 1 muscarinic receptor, methyl chlorogenate showed E-value of − 7.5 kcal/mol and formed 6 H-bonds, 2 π-π bonds, and 1 hydrophobic interaction, whereas pirenzepine showed E-value of − 8.8 kcal/mol and formed 2 H-bonds, 1 π-π bond, and 1 other hydrophobic interaction. Its 2D interactions are shown in Figure 8. Against M 3 muscarinic receptor, methyl chlorogenate showed E-value of − 6.1 kcal/mol and formed 2 H-bonds and 5 hydrophobic interactions, whereas atropine showed Evalue of − 5.6 kcal/mol and formed 2 H-bonds, 1 π-π bond, and 19 other hydrophobic interactions as shown in 2D interactions in Figure 9. Against D 2 dopaminergic receptor, methyl chlorogenate showed E-value of − 7.8 kcal/mol and formed 9 H-bonds, 1 π-π bond, and 1 hydrophobic interactions, whereas domperidone showed E-value of − 9.3 kcal/mol and formed 3 H-bonds, 5 π-π bonds, and 8 other hydrophobic interactions, as shown in Table 4, and 2D interactions as shown in Figure 10. Against calmodulin receptor, methyl chlorogenate showed E-value of − 6.2 kcal/ mol and formed 3 hydrophobic interactions, whereas calmidazolium showed E-value of − 8.2 kcal/mol and formed 20 other hydrophobic interactions given in Table 4 and 2D interactions shown in Figure 11. Against calcium channel receptor, methyl chlorogenate showed E-value of − 7.3 kcal/ mol and formed 2 H-bonds, 1 π-π bond, and 6 hydrophobic interactions, whereas verapamil showed E-value of − 6.7 kcal/mol and formed 1 H-bond, 2 π-π bonds, and 7 other hydrophobic interactions given in Table 4 and 2D interactions as shown in Figure 12. Against H + /K + ATPase receptor, methyl chlorogenate showed E-value of − 7.9 kcal/ mol and formed 6 H-bonds and 7 hydrophobic interactions, whereas omeprazole showed E-value of − 8.1 kcal/mol and formed 2 π-π bonds and 12 other hydrophobic interactions given in Table 4 and 2D interactions as shown in Figure 13.
Against H 2 histaminergic receptor, methyl chlorogenate showed E-value of − 7.4 kcal/mol and formed 3 H-bonds and 4 hydrophobic interactions, whereas ranitidine showed Evalue of − 5.7 kcal/mol and formed 4 H-bonds, 1 π-π bond, and 7 other hydrophobic interactions given in Table 4 and 2D interactions as shown in Figure 14. Against mu-opioid receptor, methyl chlorogenate showed E-value of − 9.2 kcal/ mol and formed 6 H-bonds and 3 hydrophobic interactions, whereas loperamide showed E-value of − 9.5 kcal/mol and formed 1 H-bond, and 4 other hydrophobic interactions given in Table 4 and 2D interactions as shown in Figure 15. Against phosphodiesterase enzyme receptor, methyl chlorogenate showed E-value of − 8.9 kcal/ mol and formed 4 H-bonds, 1 π-π bond, and 4 hydrophobic interactions, whereas papaverine showed E-value of − 8.1 kcal/mol and 2 H-bonds, 2 π-π bonds, and 5 other hydrophobic interactions given in Table 4 and 2D interactions as shown in Figure 16.

Discussion
On the basis of ethnopharmacological utilization of Manilkara zapota in gastric disorders, such as gastritis, constipation, and diarrhea, Manilkara zapota extract was evaluated for its antidiarrheal, antisecretory, antispasmodic, charcoal meal gastrointestinal motility, and antiulcer effects. In vitro, in silico, and proteomic approach were used for the explication of possible underlying mechanisms to rationalize the aforementioned ethnomedicinal uses of the plant. Manilkara zapota fruit extracts exhibited protective effects against castor oil-induced diarrhea, similar to the effect produced by loperamide, a standard drug, while its possible underlying mechanism was estimated through isolated tissue preparations also associated with the reduction in gastric motility. Castor oil is responsible for increasing intestinal fluid as well as diarrheal effect through its active metabolite, i.e., ricinoleic acid [10]. It changes the electrolyte and water transport and generates enormous contractions in the transverse and distal colon. Gastrointestinal motility is maintained through the release of various mediators which are histamine, prostaglandins, acetylcholine, substance P, 5-hydroxytrptamine, nitric oxide, and cholecystokinin which provides stimulatory effects in gut and ultimately increases cytosolic Ca 2+ along with the release of mediators that block the above-described pathways accompanying with nonspecific inhibitory action that would relief gut disorders [11]. Manilkara zapota extracts demonstrated protective effect against castor oil-induced intestinal fluid secretion in mice. Manilkara zapota extracts contain a gut relaxant constituent which mediates antidiarrheal and antisecretory effects.
Papaverine (a standard phosphodiesterase enzyme (PDE) inhibitor), chloroform, and aqueous extracts of plant caused a dose-dependent inhibition of spontaneous and high K + -induced contractions in isolated rabbit preparations by producing a similar pattern of nonspecific inhibitory response whereas verapamil was found more potent against K + (80 mM) induced contractions. ese data proposed that plant extracts produce their relaxant effect through the dual mechanisms with CCB and producing relaxation effect, like PDE enzyme inhibition [18]. Antidiarrheal effect of the Mz fruit extract, mediated possibly through the dual blockade of phosphodiesterase enzyme receptors and Ca 2+ channels, provides evidence for its effectiveness in diarrhea, though additional mechanisms can be ruled out. e phytochemical screening of Manilkara zapota fruit extract revealed the presence of different classes of chemicals, such as flavonoids, polyphenols, alkaloids, carbohydrates, saponins, and anthraquinones. Moreover, polyphenols target gut microbiota activity through its metabolites [19]; hence, the presence of these phytochemicals in Manilkara zapota may be accountable for its efficacy in diarrhea, though further research is required to explore the precise nature of chemical constituents mediating alleged biological activities. For charcoal meal transient time activity, Manilkara zapota extract in the small intestine produces suppression of the propulsion of charcoal marker in test doses just like standard drug atropine sulphate that has prominent anticholinergic effect on intestinal transit. A diminished GIT motility tone causes increase in the stay of substances in intestine, which permits better water absorption. ese findings proposed that Manilkara zapota extract has an effect on the peristaltic movement of intestine which indicates its antimotility effects. In gastrointestinal tract, various aggressive and protective factors play an important role in the production and release of acids. Disturbance in these factors results in rupturing of mucosal protection which exposes gastric lining  Figure 6: Effects of Mz.Aq extract on ethanol-mediated stomach histopathologic changes. Stomach tissues (n � 5) from each experimental group were processed for histological evaluation at 1 hour after ethanol challenge. (a) Representative histological changes of apoptotic markers (TNF-α, P-NFκB, and COX-2) in stomach, scale bar � 20 μm, magnification 40x: control group, ethanol group, Mz.Aq extract group, and omeprazole group. (b, c) Severity scores of stomach injury in different groups (n � 5) calculated via relative density of value (AU) and relative integrated density. Data were analyzed by two-way ANOVA followed by Tukey's post hoc test using GraphPad Prism software. Mean ± SEM. * * * p < 0.001 versus ethanol group; * p < 0.01 versus ethanol group. (d) Histopathological examination of saline group, ethanol group, test group, and omeprazole group.   to different enzyme and acid productions leading to the sores called ulcers [20]. Ethanol-induced gastric model was used to explore the beneficial effect of Manilkara zapota extract through a variety of mechanisms that stimulate ulcers including free radicals OH − , NO production, mucus exhaustion, mucosal damage, release of superoxide anion, which ultimately prolonged the tissue oxidative stress, and release of inflammatory mediators like TNF-α, p-NFκB, and COX-2, which are measured through the proteomic analysis. e potential of Manilkara zapota extract to produce antiulcer effect might be due to its CCB effect, as Ca 2+ antagonist is well known to demonstrate such effects [2]. In pathophysiology of gastric ulcers, oxidative stress plays a vital role. Antioxidant and nitric oxide free radical scavenging activity has been reported by Manilkara zapota, which may be responsible for its effectiveness as antiulcer agent [3]. Furthermore, ELISA was done to quantify the protein expression of IL-18, and Mz.Aq significantly attenuates necrosis in ulcerative tissue as compared to Mz.CHCl 3 achieved through proteomic analysis [15]. Mz.Aq extract administration exerts more protective effect as compared to Mz.CHCl 3 in ulcerative model, so, for IHC and H&E staining, Mz.Aq was selected for further analysis. Mz.Aq protects the gastric cells through the inhibition of inflammatory markers like TNF-α, p-NFκB, and COX-2. e results were quite significant as compared to ethanol. TNF-α, COX-2, and p-NFκB are widely considered as prototypic proinflammatory cytokines due to their principal role in initiating the cascade of the activation of other cytokines and growth factors in the inflammatory response [20]. After injury or during inflammation, TNF-α and NFκB signaling pathway is activated which plays a vital role in inflammation. Ethanol induces inflammation in gastric mucosa in the control group, whereas Manilkara zapota extracts decreased the levels of TNF-α, p-NFκB, and COX-2 as compared to omeprazole when given alone to rats. Manilkara zapota extracts have broader therapeutic index as no mortality was observed at a dose of 5 g/kg. e ligand methyl chlorogenate (phytochemical constituent) was docked in the catalytic active pocket of α 1adrenergic receptor, muscarinic M 1 and M 3 , domperidone D 2 , calmodulin, voltage gated L-type calcium channel, gastric hydrogen potassium ATPase, histamine H 2 , muopioid, and phosphodiesterase enzyme receptor targets that may be possibly associated with the pathophysiology of gastric disorders [21]. Docking is used as a preliminary tool used to evaluate the affinity of ligands to their particular protein targets. Molecular docking has a significant role in drug discovery and development, which includes structurebased evaluation and finding target specificity along with binding affinity. For virtual screening, DSV was used through PyRx [22]. It has gradient optimization method and it predicts the binding mode of ligand-receptor complex more precisely. Affinity of ligands with receptor could be assessed through E-value and hydrogen bonding that relates their influential effect in gastrointestinal diseases [23]. Based on E-value against different protein targets, the order of ligand affinity was found as mu-opioid > phosphodiesterase enzyme > H + /K + ATPase > D 2 > α 1 > M 1 > H 2 > voltage gated L-type calcium channel > Calmodulin receptor > M 3 .

Conclusions
Manilkara zapota Linn. holds a vital place in the traditional system of medicines. e current explored potential of the plant signifies its importance to the pharmaceutical industry due to exhibited antidiarrheal, antisecretory, antispasmodic, antimotility, and antiulcer effects. Most histopathological changes in gastric tissues and associated derangement in related polyclonal antibodies were largely prevented by the Manilkara zapota extract, thus paving the way to a new therapeutic approach towards the management of inflammation-induced gastric mucositis. e plant constituent methyl chlorogenate showed moderate binding affinities (Evalue ≥ − 6.1 kcal/mol) against muscarinic M 1 , muscarinic M 3 , histaminergic H 2 receptors, H + /K + ATPase pump, calmodulin, adrenergic α 1 , and dopaminergic D 2 receptors and voltage gated L-type calcium channels, while it showed high affinities (E-value ≥ 8 kcal/mol) against mu-opioid and phosphodiesterase enzyme.

Data Availability
e data used to support the findings of this study are available from the authors upon request.

Disclosure
is work was performed in partial fulfillment of the requirements for the degree of M.Phil. in Pharmacology by "Sameen Fatima Ansari" at the Faculty of Pharmaceutical Sciences, Riphah International University of Pharmaceutical Sciences, Islamabad, Pakistan.