MATERIALS AND METHODS

This work was done in Lab. of Therapeutical Chemistry Department, National Research Center, El Buhouth St., Dokki, Cairo, Egypt as a PhD study. The total time duration was 2 years starting from February, 2015 till February, 2017.

Drugs and chemicals: Ranitidine hydrochloride was kindly provided by medical union pharmaceuticals (MUP, Ismailia, Egypt). The ELISA kits used for measuring prostaglandin E₂ (PGE₂)_, heat shock protein 70 (HSP₇₀), vascular endothelial growth factor (VEGF) and IL-1β were purchased from Cloud Clone Corp (USA). TNF-α ELISA kit was purchased from Thermo Scientific (USA).

Plant collection and extraction: The BCF and ZSCF were collected from Orman botanical garden, Giza, Egypt. After complete extraction of fresh flowers and fruits (1 kg) with 70% ethanol, the aqueous ethanol was evaporated under vacuum. For the experiments the semi solid residues of extracts were freshly prepared as a suspension in distilled water and administered orally to rats.

Phytochemical screening: The ethanolic extracts for both BCF and ZSCF were tested for the presence of carbohydrates and/or glycosides, tannins, alkaloids, flavonoids, sterols, saponins and coumarins¹⁷.

Total phenolic assay: The total phenolic content (TP) was determined using Folin-Ciocalteu colorimetric method previously described by Karawya and Aboutabl¹⁸. The TP content was expressed as milligrams of gallic acid equivalents (g of the dry plant material.

Total flavonoid assay: The total flavonoid content (TF) was measured using an aluminum chloride colorimetric assay¹⁹. The TFC was expressed as milligrams of rutin equivalents/g of the dry plant material.

HPLC analysis of flavonoids and phenolic compounds: Flavonoids and phenolic compounds of BCF and ZSCF were detected and determined according to the method described by Goupy et al.²⁰ and Mattila et al.²¹ using HPLC instrument. The HPLC analysis was carried out using an Agilent 1260 series. The separation was carried out using C18 column (4.6×250 mm i.d., 5 μm).

Acute toxicity test: The acute toxicity study was used to determine the safe dose for both extracts. About 50 fasted rats (25 males and 25 females) were assigned equally into 5 groups and orally received; vehicle (distilled water), 2 and 5 g kg^–1 of each extract. Food was withheld for a further 3-4 h after dosing. The animals were observed for 48 h after the administration of the extracts for the onset of clinical or toxicological symptoms. Mortality, if any, was reported over a period of 2 weeks.

Experimental design: About 72 adult male albino Wistar rats weighing 150-200 g were fasted for 24 h before the experiment. They were allowed free access to water during the fasting period until 2 h before the start of the experiment²². They were divided into 9 groups each consisting of 8 animals (Fig. 1):

All rats were sacrificed after 2 h, from ethanol administration, in set A or after 1 week in set B. All experiments were carried out after approval of the protocol by the ethics committee of Faculty of Pharmacy, Cairo University and of NRC (Ethical approval No. is BC 1010 and 13163, respectively) and conformed to the ethical guidelines of the 1975 Helsinki Declaration.

Measurement of gastric secretion: Rats were anesthetized, the abdomen was dissected and the stomach contents were collected, measured, centrifuged and subjected to analysis for titratable acidity against 0.01 N NaOH^27,28.

Quantification of ulceration: The glandular portion comprising of the fundic and corpus region of each stomach was opened longitudinally along the greater curvature and examined macroscopically using a 10×magnifying lens to assess the formation of ulcerations. The number and ulcer score (US) in the glandular mucosa were scored from 0-10 according to the method of Tan et al.²⁹.

Fig. 1:	Experimental design

Preparation of tissue homogenate: Longitudinal sections weighing 0.5 g from each stomach were homogenized in 5 mL phosphate buffered saline (pH 7.4). The homogenate was centrifuged at 4000 rpm for 10 min at 4°C and the supernatant was stored at -20°C for further studies.

Biochemical determinations in stomach homogenate: Glutathione (GSH) in the tissue homogenate was measured spectrophotometrically using the method of Moron et al.³⁰. Lipid peroxide, malondialdehyde (MDA), content in gastric mucosal tissues was determined by thiobarbituric acid reaction as described by Ruiz-Larrea et al.³¹. Nitric oxide (NO_x): NO_x metabolites were assayed colorimetrically, after griess reaction, as described by Miranda et al.³². Protein concentration was determined following the method described by Bradford³³ using bovine serum albumin as standard.

Succinate dehydrogenase (SDH), Glucose-6-phosphatase (G-6-Pase) and acid phosphatase (AP) activities were estimated in stomach homogenate by the method of Rice and Shelton³⁴, Swanson³⁵ and Wattiaux and De Duve³⁶, respectively.

PGE₂ was measured by solid phase competitive ELISA kit while VEGF, HSP₇₀, TNF-α and IL-1β levels were measured by solid phase sandwich ELISA kits according to manufacturer instructions.

Histopathological evaluation: Stomach was immediately immersed in 10% formalin-saline, embedded in paraffin and 5 μm sections were prepared, stained with hematoxylin and eosin (H and E) and examined microscopically³⁷.

Statistical analysis: Data were expressed as means±standard error (SEM) of 8 readings. The results were analyzed statistically by one-way analysis of variance (ANOVA) using SPSS (Statistical Package for the Social Sciences, version 16.0.1, Chicago, IL) software. Comparison of numerical variables between the studied groups was done using Scheffe’s test as a post hoc pair wise comparisons. Differences were considered significant at p<0.05.

RESULTS

Phytochemical constituents: Phytochemical screening of BCF and ZSCF ethanolic extract revealed the presence of carbohydrates, tannins, flavonoids, sterols and coumarins. Saponins and alkaloids were present in ZSCF and absent in BCF (Table 1).

Total phenolics and flavonoids content: Table 2 showed that the total phenolic content of BCF and ZSCF were 0.68 and 0.51 mg of gallic acid equivalents g of the dry plant material, respectively. The total flavonoids were 0.55 and 0.21 mg rutin equivalents/g of the dry plant material, respectively.

HPLC analysis of phenolic and flavonoids compounds: The experiment revealed the identification of 12 phenolic compounds in BCF, the most abundant one was naringenin (11.77 mg g^–1) while ZSCF revealed the presence of 8 phenolic compounds. The most abundant one in ZSCF was gallic acid (0.09 mg g^–1). These results were illustrated in Table 3.

Acute toxicity study: During the period of 2 weeks, neither toxic symptoms nor mortality was observed. Likewise, no abnormal behavioral changes or body weight (b.wt.) alteration were observed at both mentioned doses. Both extracts proved to be safe up to 5 g kg^–1 b.wt. These results were illustrated in Table 4.

Table 1:	Phytochemical screening of BCF and ZSCF
+: Presence of the constituent, -: Absence of the constituent, BCF: Bombax ceiba flower, ZSCF: Ziziphus spina christi Fruit

Table 2:	Total phenolic and flavonoid content of BCF and ZSCF
BCF: Bombax ceiba flower, ZSCF: Ziziphus spina christi Fruit

Table 3:	HPLC analysis of phenolic and flavonoid compounds of ethanolic extract of BCF and ZSCF
Rt : Retention time

Table 4:	Symptoms and death of animals treated with BCF and ZSCF
10 animals for control and each dose

Biochemical analysis
Effect of BCF and ZSCF extracts on gastric ulcer indices: Data in Table 5 showed that the greatest decrease in measured gastric ulcer indices was due to BCF extract administration which recorded better results than ZSCF and ranitidine. These results were accentuated by BCF therapeutic effect where it showed the best decrease in the measured gastric ulcer indices.

Effect of BCF and ZSCF extracts on cell organelle marker enzymes in stomach: Stomach ulcer protected by BCF (PB) and ZSCF (PZ) ameliorated the significant increase in all cell organelle marker enzymes as compared with PE group. Here again BCF declared the best significant decrease in all measured enzymes. Therapeutic effect of BCF (TB) and ZSCF (TZ) after ulcer induction confirmed the above results where BCF achieved the best improvement in SDH, G-6-Pase and AP (Table 6).

Effect of BCF and ZSCF extracts on oxidative stress markers in stomach: Gastric ulcer protected for 1 week with either BCF or ZSCF extract improved the significant increase in MDA due to ulcer formation. Conjointly, the 2 extracts ameliorated the significant decrease in both GSH and NO_x levels due to ulceration (Table 7). Treatment of gastric ulcer with BCF (TB) or ZSCF (TZ) for one week affirmed the protected results where BCF succeeded in reaching the best improvement. It corrected the significant increase in MDA and the significant decrease in both GSH and NO_x levels.

Table 5:	Effect of BCF and ZSCF extracts on gastric ulcer indices in stomach
Data are Means±SE of eight rats in each group, ap<0.05 compared to NC group, bp<0.05 compared to PE group, cp<0.05 compared to TE group, dp<0.05 compared to PR group, ep<0.05 compared to TR group

Table 6:	Effect of BCF and ZSCF extracts on cell organelle marker enzymes in stomach
Data are Means±SE of 8 rats in each group, ap<0.05 compared to NC group, bp<0.05 compared to PE group, cp<0.05 compared to TE group, dp<0.05 compared to PR group, ep<0.05 compared to TR group, SDH: Succinate dehydrogenase, G-6-Pase: Glucose-6-phosphatase, AP: Acid phosphatase

Table 7:	Effect of BCF and ZSCF extracts on oxidative stress markers in stomach
Data are Means±SE of 8 rats in each group, ap<0.05 compared to NC group, bp<0.05 compared to PE group, cp<0.05 compared to TE group, dp<0.05 compared to PR group, ep<0.05 compared to TR group, MDA: Malondialdehyde, GSH: Glutathione, NOX: Nitric oxide

Effect of BCF and ZSCF extracts on some inflammatory cytokines in stomach: Data in Fig. 2 showed that BCF and ZSCF significantly decreased the elevated TNF-α, IL-1β and VEGF. This outcome was ascertained by the therapeutic groups.

Effect of BCF and ZSCF extracts on some gastroprotective factors in stomach: Administration of BCF recorded the best increase in the reduced levels of PGE₂ and HSP₇₀, due to ulceration. Similar results were found therapeutically where BCF achieved the best amelioration in PGE₂ and HSP₇₀ (Fig. 3). By comparing prophylactic and treated groups in all measured parameters. The treated groups, either for BCF or ZSCF showed better results than the prophylactic groups. These results were ascertained by the histopathological data.

Histopathological results: Histopathological results presented in Fig. 4 showed that the stomach of normal rats revealed no alterations. Normal histological structure of the gastric mucosa was recorded (a). By contrast, stomach sections from rats of PE group showed disruption of surface epithelium with ulcer formation, marked edema and leucocytes infiltration of the submucosal layer (b).

Prophylactic treatment of PR, PB and PZ improved the alterations in gastric morphology evidenced by the appearance of almost normal surface epithelium, no leucocytes infiltration of the submucosal layer and normal muscle layer for the 3 treatments. Mild edema was showed in PR and PB groups (c and d, respectively), while marked edema was observed in PZ group (e).

Rats treated with ethanol, ranitidine, BCF and ZSCF for 1 week showed almost normal surface epithelium, no leucocytes infiltration of the submucosal layer and normal muscle layer. The TR, TB and TZ showed no edema (g, h and i, respectively) while TE showed mild edema (f).

Fig. 2(a-c):	Effect of BCF and ZSCF extracts on some inflammatory cytokines in stomach, (a) TNF-α: Tumor necrosis factor alpha, (b) IL-1β: Interleukin 1β and (c) VEGF: Vascular endothelial growth factor
	Data are Means±SE of 8 rats in each group, ap<0.05 compared to NC group, bp<0.05 compared to PE group, cp<0.05 compared to TE group, dp<0.05 compared to PR group, ep<0.05 compared to TR group

Fig. 3(a-b):	Effect of BCF and ZSCF extracts on gastroprotective factors in stomach, (a) PGE2: Prostaglandin E2 and (b) HSP70: sHeat shock protein 70
	Data are Means±SE of 8 rats in each group, ap<0.05 compared to NC group, bp<0.05 compared to PE group, cp<0.05 compared to TE group, dp<0.05 compared to PR group, ep<0.05 compared to TR group

Fig. 4(a-i):

Photomicrographs of gastric sections (H and E stain, x100), (a) NC: Normal histological structure of the gastric mucosa, (b) PE: Disruption of surface epithelium with ulcer formation (black arrow), marked edema and leucocytes infiltration of the submucosal layer (red arrow), (c) PR: Almost normal surface epithelium appearance with mild edema (black arrow), (d) PB: Showed almost normal surface epithelium with mild edema (black arrow), (e) PZ: Showed almost normal surface epithelium with moderate to marked edema (black arrow), (f) TE: There was almost normal surface epithelium with mild edema (black arrow) and (g-i) For groups TR, TB and TZ, respectively, there was almost normal histological structure

DISCUSSION

Ethanol administration augments oxidative stress and inflammation while weakens the mucosal protective factors³⁸. The oxidative stress and inflammation were evidenced, in the present study, by depletion of gastric GSH and elevation of MDA, IL-1β and TNF-α-content^39,40. The enzyme leakage was proved by increasing the activity of SDH, G-6-Pase and AP in ulcerated rats.

IL-1β induces acute phase response and release of TNF-α^41,42. The TNF-α enhances ROS generation, expression of nuclear factor kappa (NF-κB) and the production of other cytokines. It suppresses the gastric micro-circulation around ulcerated mucosa thus delaying its healing^43,44.

The observed reduction in NO level may be explained by down regulation of gastric mucosal constitutive nitric oxide synthetase or the consumption of NO in the overproduction of peroxynitrites during ethanol metabolism^45,46. Previously, it was demonstrated that ethanol administration reduces PGE₂ synthesis in gastric mucosa^4,47. The ROS converts prostaglandin into 8-iso-prostaglandin F_2alpha that causes vasoconstriction and platelet aggregation^48,49. The GSH augments prostaglandin action and stabilizes mucus composition by controlling the thiol/disulfide ratio⁵⁰. The observed decrease in HSP₇₀ level is mainly attributed to its damage by the ROS generation^49,51. The elevation of VEGF level may be explained by the increase of; IL-1, TNF-α or increase in hypoxia inducible factor-1α mRNA⁵². These factors reinforce the binding of NF-κB to the VEGF gene promoter resulting in the increase in VEGF expression⁵³. The BCF and ZSCF extracts succeeded in protecting and treating gastric mucosa after ethanol injury more than ranitidine the reference drug⁵⁴.

Due to their antioxidant effect, both extracts protected the cell organelles integrity and combated oxidative stress by augmenting GSH⁵⁵ and quenched the superoxide anion which MDA consumes NO⁵⁶.

Due to their anti-inflammatory and protecting effect, the 2 plants decreased IL-1β, TNF-α and VEGF level while increased PGE₂ and HSP₇₀ levels. Consequently, elevated prostaglandin attenuated the release of IL-1β and TNF-α⁴⁰. HSP₇₀ suppressed mitogenic activated protein kinase (MAPK) which transduces signals to initiate inflammatory cellular response⁵⁷. Finally, the decrease in IL-1β, TNF-α and MAPK lead to decrease in VEGF level.

Both extracts contain rutin, quercetin and gallic acid previously reported to prevent gastric mucosal ulceration in several animal models^58,59. Rutin inhibits Platelet activating factor (PAF) and the gastric proton pump⁶⁰. Quercetin exhibits an antiradical property toward hydroxyl, peroxyl radicals and superoxides anions⁶¹. Gallic acid has the ability to form a protective pellicle by promoting precipitation of protein on the ulcer in order to prevent ulcer development. This pellicle helps in preventing toxic substance absorption and combat the attack of proteolytic enzymes⁶².

The present study revealed that ZSCF was ranked after BCF in almost all measured parameters. This may be due to higher concentration of total phenolics and flavonoids in BCF, as well as it contains additional phytoconstituents. The BCF contains caffeic, ferulic, p-coumaric, syringeic and cinnamic acids which are previously reported of having potent anti ulcer, antioxidant and antilipid peroxidation activities^63-65.

Naringenin was the most abundant flavonoid identified in BCF. Cavia-Saiz et al.⁶⁶ found that naringenin is a strong scavenger of free radicals and prevent lipid peroxidation. Nishimura et al.⁶⁷ found that naringenin exerts its effects by controlling the effector mechanisms of ROS production through ROS scavenging activity^68,69.

Said et al.⁸ isolated and identified mangiferin from BCF. Mangiferin (MF) possesses a gastroprotective effect via its antisecretory and antioxidant activities^70,71. It maintains the cellular oxidation-antioxidant balance as it generates MF phenoxy radicals that binds to metal ions (Fe^2+/3+) and form a stable MF-iron complex. This complex does not allow the generation of hydroxyl (OH^∙) radicals and/or oxo-ferryl groups and scavenges lipid peroxy/alkoxy radicals.

CONCLUSION

It can be concluded that BCF and ZSCF have antiulcer effect, which is evidenced by the decreased levels of ulcer indicies, SDH, G-6-Pase, AP, MDA, TNF-α, IL-1β, VEGF and elevated levels of GSH, PGE₂ and HSP₇₀ in the tissue homogenate and in this respect, it was found that BCF more efficient than ZSCF.

SIGNIFICANCE STATEMENT

This study highlight the use of BCF and ZSCF extracts in the alleviation of ethanol gastric injury. This can be beneficial for introducing new alternatives using natural sources. As both plants showed almost better effect on ulcerated rats than that exerted by ranitidine, they can be formulated as new antiulcer drugs after further studies.

ACKNOWLEDGMENTS

This work was supported by National Research Center, Cairo, Egypt as a PhD study with grant number 2/2/8. The authors would like to thank Professor Dr. Olfat Emam, Department of Pathology, Theodor Bilharz Rersearch Institute for evaluation of histopathological slides.