Evidence of the gastroprotective and anti- Helicobacter pylori activities of β-mangostin isolated from Cratoxylum arborescens (vahl) blume

Figures & data

Figure 1 Chemical structure and purity of BM.

Notes: (A) Represents the chemical structure of BM. (B) Shows the separation of BM by HPLC following the solid phase extraction method (chromatogram of standard BM [10 µg/mL]).
Abbreviations: BM, β-mangostin; HPLC, high-performance liquid chromatography; min, minutes; PDA, photodiode array; mAU, milli absorption unit.

Table 1 Effects of BM administered intraduodenally on the biochemical parameters of gastric juice obtained from pylorus ligature in rats

Treatment	Volume (mL)	pH	Total acidity (mEq/L)
Ulcer control (5% Tween 80 v/v)	6.233±0.30	2.617±0.22	203.3±6.1
Omeprazole (20 mg/kg)	1.867±0.15*	4.617±0.32*	70.00±4.65*
BM (5 mg/kg)	1.883±0.12*	3.527±0.16*	93.33±7.92*
BM (10 mg/kg)	2.000±0.12*	3.795±0.11*	92.50±8.24*
BM (20 mg/kg)	2.367±0.12*	4.528±0.17*	128.3±9.09*

Notes: Results are mean ± SEM (n=6). Statistical comparison was performed using ANOVA followed by Dunnett’s test.

* P<0.05 when compared to control group.

Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; SEM, standard error of the mean.

Table 2 Observed ulcer area and inhibition percentage of BM against ethanol-induced gastric ulcer in rats

Pretreatment	Dose (mg/kg)	Ulcer area (mm^2)	Inhibition (%)
Ethanol ulcer control	–	557.3±9.588	–
Normal control (vehicle)	–	–	100
Omeprazole	20	115.2±11.00*	79.07
BM	5	236.2±49.93*	57.62
BM	10	89.28±50.15*	83.98
BM	20	148.3±31.47*	73.39

Notes: Data are expressed as mean ± SEM (n=6),

* P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons.

Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; SEM, standard error of the mean.

Figure 2 Macroscopic evaluation.

Notes: The macroscopic appearance of the gastric mucosa of the rats of the normal control (A) exhibit the intact mucosal appearance with no lesions as seen. Whereas, the rats of the ulcer control pretreated with only Tween 80 (B) showed that the ethanol-induced severe visible lesions to the gastric mucosa appeared as elongated bands of hemorrhage (black arrows). However, the pretreatment with omeprazole at 20 mg/kg (C) showed mild lesions as seen in the gastric mucosa (black arrow). The pretreatment with BM at 5 mg/kg (D) showed moderate lesions as seen in the gastric mucosa (black arrows). The pretreatment with BM at 10 mg/kg (E) showed very mild lesions as seen in the gastric mucosa (black arrow). The pretreatment with BM at 20 mg/kg (F) showed mild lesions as seen in the gastric mucosa (black arrow) (magnification: ×1.8).
Abbreviation: BM, β-mangostin.

Table 3 Serum biochemical analysis of animal pretreated by BM against ethanol-induced gastric ulcer

Parameter	Normal control	Ulcer control	Dose administered (mg/kg bw)
			OMP 20	BM 5	BM 10	BM 20
AP (IU/L)	60.4±1.44	95.7±1.02	69.67±2*	80.80±9.3*	76.80±8.5*	90.4±8.3*
ALT (IU/L)	30.4±0.51	66.0±1.14	38.2±2.5*	51.8±1.62*	51.0±17.3*	48.0±5.62*
AST (IU/L)	152.0±3.0	192.0±2.0	100.8±4*	106.8±5.3*	108.4±3.5*	183.8±9.6*
Sodium (mmol/L)	139.1±0.1	137.3±1.79	137.8±1.9	143.8±0.1*	139.0±1.1*	138.8±0.4*
Potassium (mmol/L)	4.80±0.15	3.42±0.12	4.16±0.0*	4.44±0.04*	4.56±0.12*	4.58±0.08*
Chloride (mmol/L)	102.8±0.2	99.80±1.4	101.0±1.7	103.4±0.3*	100.8±1.2*	100.8±0.1*
Carbon dioxide (mmol/L)	22.88±0.2	20.98±1.2	22.6±0.7*	21.96±0.5*	21.86±0.6*	21.24±0.4*
Anion gap (mmol/L)	18.0±0.45	21.5±0.97	19.0±0.4*	20.0±0.96*	20.8±0.6*	20.6±0.41*
Urea (mmol/L)	6.0±0.08	7.7±0.56	6.06±0.3*	6.58±0.59*	6.82±1.0*	6.62±0.61*
Creatinine (mmol/L)	34.16±0.2	49.67±0.2	34.3±1.5*	37.2±4.3*	36.20±2.9*	36.40±2.5*
Total protein (g/L)	66.3±0.2	61.75±1.5	64.0±0.7*	62.8±1.19*	63.8±1.83*	64.2±1.74*
Albumin (g/L)	39.60±0.4	12.0±0.54	36.33±1*	29.6±0.41*	32.0±0.36*	34.4±0.41*
Globulin (g/L)	25.20±0.2	50.75±1.1	35.6±0.4*	44.2±1.07*	35.8±1.51*	37.8±1.75*
Triglyceride (mmol/L)	0.36±0.01	0.45±0.08	0.36±0.0*	0.40±0.08*	0.40±0.02*	0.46±0.04
Total cholesterol (mmol/L)	1.84±0.08	1.40±0.10	1.46±0.1*	1.42±0.12*	1.47±0.07*	1.42±0.14*
HDL cholesterol (mmol/L)	1.29±0.07	1.08±0.07	1.17±0.1*	1.18±0.1*	1.09±0.17	1.18±29.6*

Notes: Data are expressed as mean ± SEM (n=6),

* P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparison.

Abbreviations: ALT, alanine aminotransferase; ANOVA, analysis of variance; AP, alkaline phosphatase; AST, aspartate aminotransferase; BM, β-mangostin; HDL, high-density lipoprotein; OMP, omeprazole; SEM, standard error of the mean; bw, body weight.

Table 4 Lesion score of rat gastric tissue pretreated with BM against ethanol-induced gastric damage

Pretreatment	Hemorrhagic damage (score 0–4)	Submucosal edema (score 0–4)	Epithelial cell loss (score 0–3)	Inflammatory cells (score 0–3)	Total scoring (scores 14)
Normal control (vehicle)	0	0	0	0	0
Ethanol ulcer control	4 (3–4)	3 (2–4)	3 (2–3)	1 (0–3)	11 (4–14)
Omeprazole (20 mg/kg)	0 (0–2)*	2 (1–2)*	1 (0–2)*	0 (0–1)*	4 (2–5)*
BM (5 mg/kg)	3 (2–4)	3 (1–4)	2 (1–3)	1 (0–1)*	9 (2–10)
BM (10 mg/kg)	0 (0–2)*	1 (0–2)*	0 (0–1)*	0 (0–1)*	2 (0–4)*
BM (20 mg/kg)	1 (1–3)*	2 (1–3)*	1 (1–2)*	1 (0–1)*	5 (2–8)*

Notes: Data are expressed as mean ± SEM (n=6),

* P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons.

Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; SEM, standard error of the mean.

Figure 3 Histological evaluation.

Notes: The microscopic appearance of gastric mucosa of the rats of the normal control (A) showed normal histological arrangement of the gastric mucosa and submucosa with no tissue damage. The gastric mucosa of the rats of the ulcer control pretreated with only Tween 80 (B) showed that the ethanol-induced severe disruption to the surface epithelium and necrotic lesions penetrate deeply into mucosa (white arrow) and extensive edema of submucosal layer and leukocyte infiltration are present (black arrow). However, the pretreatment with omeprazole at 20 mg/kg (C) showed mild disruption of the surface epithelium mucosa and slight mucosal damage (white arrow) but no submucosal edema and leukocytes infiltration are seen. The pretreatment with BM at 5 mg/kg (D) showed moderate disruption of the surface epithelium mucosa and mucosal damage (white arrows) with submucosal edema and leukocytes infiltration as seen (black arrow). The pretreatment with BM at 10 mg/kg (E) showed very mild disruption of the surface epithelium mucosa and slight mucosal damage (white arrow) but no submucosal edema and leukocytes infiltration are seen. The pretreatment with BM at 20 mg/kg (F) showed mild disruption of the surface epithelium mucosa and slight mucosal damage (white arrow) but no submucosal edema and leukocytes infiltration are seen (H&E stain: ×20).
Abbreviations: BM, β-mangostin; H&E, hematoxylin and eosin.

Figure 4 PAS staining.

Notes: The microscopic appearance of gastric mucosa of the rats of the normal control (A) showed the positive PAS stain noted as a bright-magenta color to the mucus cells lining the gastric pits (black arrow). The gastric mucosa of the rats of the ulcer control pretreated with only Tween 80 (B) showed that the ethanol-induced complete depletion to the mucous layer as seen by the absence of the PAS stain. However, the pretreatment with omeprazole at 20 mg/kg (C) showed intense PAS stain noted as a bright-magenta to the mucus cells lining the gastric pits due to the carbohydrate-rich, viscous mucus they secrete (black arrow). The pretreatment with BM at 5 mg/kg (D) showed moderate expansion of a substantial continuous PAS-positive mucous gel layer that lining the entire gastric mucosal surface observed histologically as a bright-magenta-stained area lining the mucosa (black arrow). The pretreatment with BM at 10 mg/kg (E) showed intense PAS stain and increased expansion of a substantial continuous PAS-positive mucous gel layer that lining the entire gastric mucosal surface observed histologically as a bright-magenta-stained area lining the mucosa (black arrow). The pretreatment with BM at 20 mg/kg (F) showed increased expansion of a substantial continuous PAS-positive mucous gel layer that lining the entire gastric mucosal surface observed histologically as a bright-magenta-stained area lining the mucosa (black arrow) (PAS stain: ×20).
Abbreviations: BM, β-mangostin; PAS, periodic acid–Schiff.

Figure 5 Effects of BM (BM; 5, 10, and 20 mg/kg bw) and omeprazole (OMP; 20 mg/kg bw) on (A) gastric mucosal PGE₂ levels and (B) alcian blue binding to free gastric mucus against ethanol-induced gastric ulcer in rat.

Notes: The rats of the ulcer control showed marked (P<0.05) lower gastric mucosal levels of PGE₂ and alcian blue binding to free gastric mucus. The gastric mucosal levels of PGE₂ and alcian blue binding to free gastric mucus were significantly (P<0.05) higher in the pretreated rats with BM than that of ulcer group. Each column represents data as mean ± SEM (n=6), *P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons.
Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; OMP, omeprazole; PGE₂, prostaglandin E2; SEM, standard error of the mean; bw, body weight.

Figure 6 Effects of BM (5, 10, and 20 mg/kg bw) and OMP (20 mg/kg bw) on (A) GSH levels, (B) MDA level, (C) SOD activity, and (D) CAT activity against ethanol-induced gastric ulcer in rat.

Notes: The rats of the ulcer control showed marked (P<0.05) lower gastric mucosal levels of GSH, CAT, and SOD, and significantly (P<0.05) higher MDA. The gastric mucosal levels of GSH, CAT, and SOD were significantly (P<0.05) higher in the pretreated rats with BM than that of ulcer group. The pretreated rats with BM were also expressed marked (P<0.05) lower gastric mucosal levels of MDA than that of ulcer group. Each column represents data as mean ± SEM (n=6), *P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons.
Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; CAT, catalase; GSH, glutathione; MDA, malondialdehyde; OMP, omeprazole; SEM, standard error of the mean; SOD, superoxide dismutase; bw, body weight.

Figure 7 Effects of BM (5, 10, and 20 mg/kg bw) and OMP (20 mg/kg bw) on gastric mucosal NP-SH content against ethanol-induced gastric ulcer in rat.

Notes: The rats of the ulcer control showed marked (P<0.05) lower gastric mucosal levels of NP-SH. The gastric mucosal levels of NP-SH was significantly (P<0.05) higher in the pretreated rats with BM than that of ulcer group. Each column represents data as mean ± SEM (n=6), *P<0.05; significant vs ulcer control. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons.
Abbreviations: ANOVA, analysis of variance; BM, β-mangostin; NP-SH, nonprotein sulfhydryls; OMP, omeprazole; SEM, standard error of the mean; bw, body weight.

Figure 8 HSP70 IHC.

Notes: The microscopic appearance of gastric mucosa of the rats of the normal control (A) showed insignificant HSP70 expression in the normal gastric tissue. The gastric mucosa of the rats of the ulcer control pretreated with only Tween 80 (B) showed very mild HSP70 expression in the gastric tissue (yellow arrow). However, the pretreatment with omeprazole at 20 mg/kg (C) showed increased upregulation of HSP70 expression appeared histologically as an intense brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with BM at 5 mg/kg (D) showed moderate upregulation of HSP70 expression appeared histologically as an intense brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with BM at 10 mg/kg (E) showed highly increased upregulation of HSP70 expression appeared histologically as an intense brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with BM at 20 mg/kg (F) showed increased upregulation of HSP70 expression appeared histologically as an intense brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow) (IHC: ×20).
Abbreviations: BM, β-mangostin; HSP70, heat shock protein 70; IHC, immunohistochemistry.

Figure 9 Bax IHC.

Notes: The microscopic appearance of gastric mucosa of the rats of the normal control (A) showed insignificant Bax expression in the normal gastric tissue. The gastric mucosa of the rats of the ulcer control pretreated with only Tween 80 (B) showed highly increased Bax expression appeared histologically as an intense brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with omeprazole at 20 mg/kg (C) showed very mild upregulation of Bax expression (yellow arrow). The pretreatment with BM at 5 mg/kg (D) showed moderate upregulation of Bax expression noted as brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with BM at 10 mg/kg (E) showed very mild upregulation of Bax expression noted as brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow). The pretreatment with BM at 20 mg/kg (F) showed mild upregulation of Bax expression noted as brown color to the positive-stained-antigen site in the gastric tissue (yellow arrow) (IHC: ×20).
Abbreviations: BM, β-mangostin; IHC, immunohistochemistry.