Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement

Figures & data

Table 1 The experimental design and specifications

Group	Description	Pretreatment	Treatment
A	Normal control	5% Tween^® 20 (5 mL/kg)	5% Tween 20
B	Ulcer control	5% Tween 20 (5 mL/kg)	Absolute ethanol
C	Low dose	EEAM (200 mg/kg)	Absolute ethanol
D	High dose	EEAM (400 mg/kg)	Absolute ethanol
E	Omeprazole control	Omeprazole (20 mg/kg)	Absolute ethanol

Abbreviation: EEAM, ethyl acetate extract of Annona muricata leaves.

Figure 1 A chromatogram analysis of EEAM, characterized with the GC-MS-TOF.

Notes: GC-MS-TOF analysis demonstrated the presence of two major compounds, namely (1) caryophyllene and (2) α-copaene.
Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; GC-MS-TOF, gas chromatography–time-of-flight mass spectrometry; AIC, analytical ion chromatogram.

Table 2 Effects of EEAM on renal and liver function tests of rats after 14 days of acute toxicity study did not show any significant differences between vehicle control and EEAM treated groups

Renal function test
Group	Sodium (mM/L)	Potassium (mM/L)	Chloride (mM/L)		CO2 (mM/L)	Anion (mM/L)	Urea (mM/L)	Creatinine (μM/L)
Vehicle	141.5±0.6	3.91±0.05	105.7±0.9		23.1±0.2	17.3±0.4	6.3±0.2	30.6±1.5
1 g/kg	140.6±0.3	4.14±0.07	104.3±1.1		24.4±0.3	16.6±0.3	5.8±0.1	30.7±1.2
2 g/kg	140.7±0.4	4.22±0.03	104.4±1.0		25.1±0.2	16.9±0.6	5.6±0.2	31.8±1.1
Liver function test
Group	Total protein (g/L)	Albumin (g/L)	Globulin (g/L)	TB (μM/L)	AP (U/L)	ALT (U/L)	AST (U/L)	GGT (U/L)
Vehicle	70.5±0.7	35.4±0.8	27.3±0.8	3.1±0.1	160.4±2.4	27.4±0.9	25.1±2.2	1.1±0.1
1 g/kg	71.4±0.3	36.6±0.4	26.5±0.4	3.2±0.2	159.3±1.9	28.2±1.1	27.5±1.5	1.0±0.1
2 g/kg	71.7±0.8	37.1±0.2	26.1±0.2	3.2±0.1	158.5±2.5	28.9±1.5	26.3±1.4	1.2±0.1

Notes: Values expressed as mean ± SEM. A value of P<0.05 was considered significant.

Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; TB, total bilirubin; AP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; SEM, standard error of the mean.

Figure 2 Histopathology analysis (H&E staining, 20×) of kidney and liver from control, EEAM at 1 g/kg, and EEAM at 2 g/kg demonstrated no significant differences in the structures of kidney and liver between vehicle control and EEAM-treated groups.

Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; H&E, hematoxylin and eosin.

Figure 3 Gross appearances of stomach from five groups of rats, namely: (A) normal control, (B) lesion control, (C) low dose of EEAM, (D) high dose of EEAM, and (E) omeprazole control. Quantitative analysis of ulcer index showed significant reduction in the ulcer index after pretreatment with EEAM (200 mg/kg and 400 mg/kg), and omeprazole.

Notes: Data are reported as means ± SEM of six animals per group. Statistical comparison was performed using the Kruskal–Wallis test, followed by the Dunn test. A value of *P<0.05 was considered significant.
Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; SEM, standard error of the mean.

Figure 4 Measurement of (A) GWM (μg of alcian blue per gram of tissue) and (B) pH in five groups of rats, namely: (A) normal control, (B) lesion control, (C) low dose of EEAM, (D) high dose of EEAM, and (E) omeprazole control.

Notes: Data for GWM are reported as means ± SEM of six animals per group. Statistical comparison for the median pH values was performed using the Kruskal–Wallis test, followed by the Dunn test. A value of *P<0.05 was considered significant.
Abbreviations: GWM, gastric wall mucus; EEAM, ethyl acetate extract of Annona muricata leaves; SEM, standard error of the mean.

Table 3 Effect of EEAM at (C) 200 mg/kg and (D) 400 mg/kg doses, and (E) omeprazole on enzymatic activities of stomach tissue homogenate. (A) and (B) are presenting the normal control and lesion control groups, respectively

Animal group	CAT nM/min/mL	GSH (μM/mg protein)	NO (μM/g protein)	SOD (U/mg protein)	MDA (μM/g protein)	PGE-2 (ng/mg protein)
A	116.11±4.17	16.49±0.44	11±0.9	19.66±0.25	72.23±1.1	4.07±0.09
B	70.14±3.21	9.19±0.38	5.7±0.09	10.19±0.55	168.23±4.98	2.12±0.03
C	81.53*±2.11	12.51*±0.18	7.7*±0.7	12.89*±0.56	123.88*±6.71	2.8*±0.1
D	103.23*±4.21	13.49*±0.12	9.1*±1.1	14.79*±0.61	95.22*±0.22	3.4*±0.7
E	113.23*±5.1	15.02*±0.33	10.1*±1	17.11*±0.7	89.23*±0.41	3.8*±0.06

Notes: Data are reported as means ± SEM of six animals per group. A value of *P<0.05 was considered significant.

Abbreviation: EEAM, ethyl acetate extract of Annona muricata leaves; CAT, catalase; GSH, glutathione; NO, nitric oxide; SOD, superoxide dismutase; MDA, malondialdehyde; PGE-2, prostaglandin E2; SEM, standard error of the mean.

Figure 5 Histopathological staining of gastric tissues after treatment with EEAM at (C) 200 mg/kg and (D) 400 mg/kg doses, and (E) omeprazole, using H&E staining (20×). (A) and (B) are presenting the normal control and lesion control groups, respectively. In the lesion control group, there are clear signs of severe damage to the surface epithelium (white arrow) and leukocyte infiltration (yellow arrow).

Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; H&E, hematoxylin and eosin.

Figure 6 Histopathological staining of gastric tissues after treatment with EEAM at (C) 200 mg/kg and (D) 400 mg/kg doses, and (E) omeprazole, using PAS staining (20×). (A) and (B) are presenting the normal control and lesion control groups, respectively. PAS staining illustrated the glycogen accumulation (red arrow) in rats pretreated with EEAM and omeprazole.

Abbreviations: EEAM, ethyl acetate extract of Annona muricata leaves; PAS, Periodic acid–Schiff.

Figure 7 Immunohistochemical analysis of Hsp70 expression from five groups of rats, namely: (A) normal control, (B) lesion control, (C) low dose of EEAM, (D) high dose of EEAM, and (E) omeprazole control. Immunohistochemistry staining showed upregulation of Hsp70 in groups C–E. (A) and (B) are presenting the normal control and lesion control groups, respectively. The yellow arrow shows Hsp70 accumulation in gastric tissue (20×).

Abbreviation: EEAM, ethyl acetate extract of Annona muricata leaves.

Figure 8 Immunohistochemical analysis of Bax protein expression from five groups of rats, namely: (A) normal control, (B) lesion control, (C) low dose of EEAM, (D) high dose of EEAM, and (E) omeprazole control. Immunohistochemistry staining showed downregulation of Bax in groups C–E. (A) and (B) are presenting the normal control and lesion control groups, respectively. The red arrow shows Bax protein accumulation in gastric tissue (20×).

Abbreviation: EEAM, ethyl acetate extract of Annona muricata leaves.