Unveiling antioxidant and anti-cancer potentials of characterized Annona reticulata leaf extract in 1,2-dimethylhydrazine-induced colorectal cancer in Wistar rats

Abstract

Background: Colorectal cancer (CC) is the third most common cancer in the world. Annona reticulata (AR) also known as bullock's heart, is a traditional herb. AR leaf extract was initially investigated for its anti-bacterial, anti-inflammatory, anti-malarial, anti-helminthic, anti-stress, and wound healing properties. Only a few in vitro cancer studies have been conducted on AR. Although few studies have linked AR leaf extract to many cancers, comprehensive studies addressing regulation, biological functions, and molecular mechanisms leading to CC pathogenesis are clearly lacking.

Objectives: The present study aimed to explore the antioxidant and anti-cancer potentials of AR leaf extract in CC.

Materials and methods: The MTT assay was used to test the anti-proliferative activity of AR leaf extract in vitro on the HCT116 cell line. Qualitative and quantitative phytochemical characterization was carried out using gas chromatography: mass spectrometry (GC-MS). 1,2-dimethylhydrazine (DMH) was used to establish CC model in female Wistar rats. The acute toxicity of AR leaf extract was tested in accordance with OECD guidelines. Aberrant Crypt Foci (ACF) count, organ index, and hematological estimations were used to screen for in vivo anti-cancer potential. The antioxidant activity of colon homogenate was determined.

Results: The alcoholic leaf extract (IC50, 0.55 μg/ml) was found to be more potent than the aqueous extract. Using GC-MS, a total of 108 compounds were quantified in the alcoholic leaf extract. The LD 50 value was found to be safe at a dose of 98.11 mg/kg of body weight. AR alcoholic leaf extract significantly (p < 0.05) decreased ACF count and normalized colon length/weight ratio. AR leaf extract increased RBC, hemoglobin and platelets levels. The AR alcoholic leaf extract reduced the DMH-induced tumors and significantly (p < 0.05) increased the activity of endogenous antioxidant enzymes such as catalase, reduced glutathione, superoxide dismutase, and decreased the lipid peroxidase activity. AR leaf extract reduced the inflammation caused by DMH and helped to repair the colon's damaged muscle layers.

Conclusion: Based on the findings from the present study, it can be concluded that the alcoholic leaf extract of AR has antioxidant and anti-proliferative properties and can aid in the prevention of CC development and dysplasia caused by DMH.

Keywords: 1,2-Dimethylhydrazine; Annona reticulata; Anticancer; Antioxidant; Colorectal cancer.

Fig. 1

GC–MS chromatogram for alcoholic leaf extract of AR. Legend: GC–below in MS chromatogram, contents from the alcoholic leaf extract of AR. A total of 108 compounds were recorded and represented in peaks. The X-axis represents R. time and Y-axis the percentage of area.

Fig. 2

Showing the development of tumors in various groups. Legend: DMH induced tumors in the colon region, AR leaf extract inhibited tumor formation, and the healthy group had no tumors.

Fig. 3

A) Graph represents the effect of AR alcoholic leaf extract on the ACF count index in different groups. B) ACF pathology figures. Legend: all the values are mean ± SEM of six samples, where ^a∗∗∗p < 0.001 compared to Healthy (no ACF formation observed in healthy animals), ^b∗∗∗p < 0.001 compared to DMH control.

Fig. 4

Effect of AR leaf extract on colon length/weight ratio. Legend: values are expressed as mean ± SEM of six samples, where ^a∗∗p < 0.01 compared to healthy control, ^b∗∗p < 0.01 compared to DMH control.

Fig. 5

Impact of AR leaf extract on CAT activity. Legend: values are expressed as mean ± SEM of six samples, where ^a∗∗∗p < 0.001 compared to Healthy control, ^b∗p < 0.05 compared to DMH control, ^b∗∗p < 0.01 compared to DMH control.

Fig. 6

Impact of AR leaf extract on GSH activity. Legend: values are expressed as mean ± SEM of six samples, where ^a∗∗p < 0.05 compared to Healthy control, ^b∗∗∗p < 0.001 compared to DMH control, ^b∗∗p < 0.01 compared to DMH control.

Fig. 7

Impact of AR leaf extract on SOD activity. Legend: values are expressed as Mean ± SEM of six samples, where ^a∗∗p < 0.01 compared to Healthy control, ^b∗∗p < 0.01 compared to DMH control, ^b∗∗∗p < 0.001 compared to DMH control.

Fig. 8

Impact of AR leaf extract on LPO activity. Legend: values are expressed as mean ± SEM of six samples, where ^a∗∗p < 0.01 compared to healthy control, ^b∗p < 0.05 compared to DMH control, ^b∗∗p < 0.01 compared to DMH control.

Fig. 9

Histopathological examination of colon. Legend: (a) & (b) are healthy control group, (c) & (d) are DMH control group, (e) & (f) are standard- 5flurouracil, (g) & (h) are treatment with 5 mg/kg and (i) & (j) are treatment with 10 mg/kg. Objective used 4×.