Abstract
We report the radioprotective attitude of Annona muricata (AM) leaf extract as antioxidant material to prevent cellular damage in the ileum tissue. The protective effects of an ethyl acetate extract of AM leaves are comprehensively investigated against radiation-induced ileal damage in numerous rats. Thirty-two adult female rats were separated into 4 groups (3 intervention groups and 1 control) as follows: controls received 0.01 mL/kg distilled water, the AM group received 300 mg/kg AM leaf extract, the ionizing radiation (IR) group received a single dose of whole body radiation (8.3 Gy) after 0.01 mL/kg saline treatment, and the AM + IR group received 300 mg/kg AM leaf extract treatment and were subjected to whole body radiation (8.3 Gy) 1 h after the last gavage. All treatments are administered by oral gavage once a day for 9 days. At the end of the experiment, biochemical total oxidant status (TOS, interleukin-6, and caspase) and histological examinations are performed on blood samples as well as ileum tissue. TOS levels are found to be significantly high in rats, which received irradiation, and those in the AM group when compared to controls. These findings suggest that AM has radioprotective effects on ileum tissue, likely because of its antioxidative properties. The findings of this research may contribute to the minimizing of major side effects induced by excessive radiation exposure in patients undergoing radiotherapy and may serve as a significant impetus for further assessments. However, future studies are highly recommended to confirm safety and to determine extraction technique and dosage before human use can be considered.
1 Introduction
The concept of ionizing radiation (IR) has found a broad variety of applications and has enabled significant advancements in numerous concepts from energy to health over the years. Diagnostic and therapeutic uses of medical radiation have a clear and indispensable position in healthcare. Moreover, the utilization of high-energy X-rays is an essential and inseparable component of cancer treatment, referred to as radiotherapy or radiation therapy. Approximately 50% of cancer patients benefit from radiation for the treatment of localized disease, while those with the incurable disease may get symptom relief or control of disease spread [1,2]. However, the use of high-dose radiation to treat cancer causes reactive oxygen species (ROS) production, which leads to permanent cell damage and oxidative stress [3].
On the other hand, ROS can interact with biological macromolecules, such as DNA, proteins, and lipids, and can lead to various pathological manifestations due to alterations in the structure and function of these macromolecules. These inflammation-related changes can result in the development or worsening of cardiovascular disease, atherosclerosis, hypertension, cardiomyopathy, and stroke [4,5].
The gastrointestinal tract (GIT) is particularly radiation sensitive, and injury of the tract may negatively affect overall health conditions. Intestine irradiation may manifest as gastrointestinal mucosa loss, mucositis, hemorrhages, and ulcers, resulting in damage to the integrity of the intestinal barrier [6]. Fortunately, numerous radioprotective materials have been investigated in recent years, and their highly promising contributions have been well documented, such that these situations have a limited impact on the patient’s overall health state [7,8,9,10,11,12,13,14,15].
Meanwhile, Annona muricata (A. muricata; AM), commonly known as “graviola” or “soursop,” a member of the Annonaceae family, is a tropical tree [16] widely distributed in Central and South America. Its leaf, root, and fruit have found use as traditional medicines for various conditions, such as neuralgia, arthritis, diarrhea, abscess, and parasitic infections [17]. A number of pharmacological activities have been reported, including anti-cancer [18], antidiabetic [19], anti-inflammatory, and antioxidant activities [10,11]. Antioxidant and anti-inflammatory activities assessed by radical-scavenging activity and other methods have been demonstrated in animal models [20]. Previous studies revealed that AM leaf extract also has protective and healing effects on the GIT. These effects include gastroprotective activity against ulcers and mucosal inflammation [21] and antiparasitic and antibacterial activities against GIT infections [22].
Taking into consideration the oxidative stress caused by tissue irradiation, the sensitivity of the ileum, and the proven antioxidant abilities of AM leaf extract, we hypothesized that the use of AM leaf extracts prior to radiotherapy would have protective effects against radiation-induced damage to the ileum. As such, we carried out this study to investigate the possible protection conferred by AM leaf extract in a rat model of radiation-induced ileum injury. Even though numerous studies have evaluated the effects of AM in different pathophysiological states, we could not find any study investigating the effect of AM leaf extract in radiation-induced ileum injury. The findings of this research may contribute to the minimizing of major side effects induced by excessive radiation exposure in patients undergoing radiotherapy and may serve as a significant impetus for further assessments.
2 Materials and methods
2.1 Animals
Animal studies were carried out in compliance with institutional norms and a protocol authorized by Bulent Ecevit University’s Ethics Committee. Our study was conducted in Bulent Ecevit University Faculty of Medicine Experimental Animal Research Unit with 32 adult female Wistar Albino rats weighing 250 ± 20 g with similar physiological characteristics. Figure 1 depicts the positioning of the rat on the treatment couch. Animals were initially randomized into four groups, one control, and three experimental groups. Throughout the experimental period, animals had constant access to tap water and standardized pellet feeds containing 21% crude protein. Environmental conditions were the routinely utilized optimum conditions for rats (22 ± 1°C temperature, 45–65% humidity, and 12 h light/dark cycle).
Positioning of rat on the treatment couch.
2.2 Radiotherapy procedure
All procedures of whole body irradiation were conducted in the Radiation Oncology Department of Bulent Ecevit University Faculty of Medicine. Subsequently, the required dose of irradiation to create measurable radiation damage was calculated. Following the treatment planning, total dose of 8.3 Gy X-rays was applied to the whole body in a single fraction through a linear accelerator device Varian Clinac IX (see Figure 2). Meanwhile, Figure 3 demonstrates the details of the abovementioned planning procedure along with the given doses and treatment details.
Treatment room and Varian Clinac IX – Radiation Oncology Department of Bulent Ecevit University Faculty of Medicine.
Planning procedure along with the given doses and treatment details.
2.3 Preparation of AM extract
AM (graviola) has many medicinal properties and is used widely in traditional medicine for treatment of various disorders in Africa. The present study was conducted to evaluate the protective effects of graviola leaves against the harms of chemotherapy to organs, which are sold in the Mali (Bomaco) market. These leaves were collected fresh from the graviola trees around Bomaco/Mali and dried hygienically at room temperature. The graviola tree whose leaves were collected is a fruitless tree. Figure 4 depicts the appearance of AM extract along with the leaves. The AM leaves were cleaned with distilled water and chopped into little pieces after being air-dried. Samples of leaves (25 g) were extracted in 50:50 ethanol solution with intermittent shaking throughout 7 days. Concentration was performed using a rotary evaporator to remove ethanol (Heidolph, Germany), followed by overnight lyophilization (Telstar, LyoQuest, Spain) to produce the extract in a dry form. The dry extract was stored at −20°C until application.
Appearance of Annona muricata extract along with the leaves.
2.4 Experiments in a murine model
Thirty-two adult female Wistar albino rats were classified into 4 different groups: Group 1 (n = 8) (control group): 0.01 mL/kg distilled water was given by oral gavage for 9 days (once daily). Group 2 (n = 8) (AM group): 300 mg/kg AM leaf extract was given by oral gavage for 9 days (once daily). Group 3 (n = 8) (IR group): 0.01 mL/kg of physiologic serum (saline) was given by oral gavage for 9 days (once daily), and a single dose of whole body radiation (8.3 Gy) was applied 1 h after the last gavage. Group 4 (n = 8) (AM + IR group): AM leaf extract of 300 mg/kg was given by oral gavage for 9 days (once daily), and whole body radiation (8.3 Gy) was applied 1 h after the last gavage. The animals were sacrificed 96 h after the last treatment, under anesthesia with intraperitoneal injection of 90 mg/kg ketamine and 10 mg/kg xylazine (Ketalar-Eczacibasi and Rompun-Bayer, Turkey, respectively). Blood samples and tissues from all animals were collected and stored at −80°C until the analytical assays were performed.
2.5 Biochemical analysis
Before processing, tissue rinsing with cold saline solution was performed twice, samples were deposited in glass vials, labeled, and kept in a deep freezer (−80°C). Using a glass-Teflon homogenizer, phosphate buffered saline was used to homogenize tissue samples (pH 7.4) (Ultra Turrax IKA T18 Basic). The homogenates were centrifuged at 5,000g for 15 min at 4°C, and the supernatants were analyzed instantly. Interleukin-6 (IL-6) and caspase levels were determined using ELISA immunoenzymatic assays (Cloud-Clone Corp., Wuhan, China). A commercial kit (Rel Assay Diagnostic, Turkey) was used to determine the total antioxidant status (TAS) and total oxidant status (TOS).
2.6 Histopathological evaluation
All ileum tissues (n = 32) that had been stored for histopathological evaluation were processed routinely with automated tissue-processing equipment (Leica ASP300S, Wetzlar, Germany), followed by paraffin embedding. Blocks were cut to 4 μm thickness by using a rotary microtome (Leica RM2255, Germany). Hematoxylin & eosin (HE) staining was performed. Histopathological changes were evaluated by a blinded pathologist using a histological score derived from the standard Chiu system [23]. Each specimen was rated based on the degree of histological damage detected, and each criterion was given a score [24].
2.7 Statistical analysis
The analyses were conducted using SPSS v21 (IBM, Armonk, NY, USA) with significance set as p < 0.05. The Shapiro–Wilk test was employed to assess normal distribution. For continuous variables, data are presented as mean value ± SD (standard deviation) or median (1st–3rd quartiles) in accordance with distribution normality. Variables having a normal distribution were evaluated using a one-way analysis of variance. The Tamhane test was used to conduct pairwise comparisons of these variables. Using the Kruskal–Wallis test, non-normally distributed variables were examined.
3 Results
3.1 Biochemical analyses
The highest levels of TOS were found in the IR group which significantly varied compared to the control and AM + IR groups (p = 0.004, p = 0.028, respectively). The AM group’s TOS level was likewise substantially greater than that of the control group (p = 0.036) (Table 1). Caspase 3, Caspase 8, and Caspase 9 concentrations were not significantly different amongst the groups (Table 1).
Biochemical findings according to treatment groups
| Treatment groups | |||||
|---|---|---|---|---|---|
| Control (n = 5) | AM (n = 6) | IR (n = 4) | AM + IR (n = 8) | p | |
| TOS (µmol H2O2 Eq/L) | 8.40 ± 0.39 | 9.56 ± 0.65a | 9.77 ± 0.32ab | 8.40 ± 0.85 | 0.003 |
| Caspase 3 (ng/mL) | 0.39 (0.26–0.49) | 0.28 (0.18–0.32) | 0.36 (0.24–0.39) | 0.26 (0.21–0.30) | 0.541 |
| Caspase 8 (ng/mL) | 1.22 ± 0.53 | 1.29 ± 0.78 | 2.62 ± 2.80 | 0.93 ± 0.43 | 0.193 |
| Caspase 9 (ng/mL) | 0.10 (0.10–0.15) | 0.16 (0.10–0.18) | 0.14 (0.10–0.20) | 0.14 (0.11–0.17) | 0.890 |
| Chiu score | — | — | — | — | — |
| 0 | 5 (100%) | 1 (16.7%) | 0 (0%) | 0 (0%) | N/A |
| 1 | 0 (0%) | 3 (50%) | 0 (0%) | 0 (0%) | — |
| 2 | 0 (0%) | 2 (33.3%) | 0 (0%) | 0 (0%) | — |
| 3 | 0 (0%) | 0 (0%) | 0 (0%) | 6 (75%) | — |
| 4 | 0 (0%) | 0 (0%) | 2 (50%) | 2 (25%) | — |
| 5 | 0 (0%) | 0 (0%) | 2 (50%) | 0 (0%) | — |
Data are given as mean value ± standard deviation or median (1st quartile – 3rd quartile) for continuous variables according to normality of distribution, and as frequency (percentage) for categorical variables. a and b: significantly different from the control group and AM + IR treated group, respectively.
AM: Annona muricata, IR: irradiation, TOS: total oxidant status.
3.2 Histopathological findings
Before the histopathological examination, the relevant tissues were successfully removed with a careful surgical procedure. Figure 5 represents a picture of a part of the surgical procedure. The mucosa, lamina propria, and villi intestinalis of the control group had normal morphology, according to light microscopic assessment. Therefore, rats in this group received a Chiu score of 0 (Figure 6a). The ileum samples from the AM-treated rats showed a light epithelial lift in apical regions. There was minimal degeneration in epithelial cells and normal scattered goblet cells. With these findings, rats in this group had Chiu scores of 1 or 2 (Figure 6b and c). The ileum samples in the IR group showed dense mucosal injury. The epithelial layer was largely separated from the lamina in most specimens, the tips of the villi were denuded, and villous height was diminished. There was considerable degeneration in epithelial cells. Mucin and goblet cells were decreased. There was a dense capillary congestion in the villus, denudation of the Lieberkühn crypts, intense inflammatory cells, and areas of edema in the lamina propria. Ulcers were observed only in this group. The majority of rats in this group received a Chiu score of 5, the highest score (Figure 6d–f). The ileum samples from the AM + IR group showed thickened and irregular villi and the epithelial layer demonstrated lifting (separation) from the lamina. When compared with Group 1, the rise in goblet cells and mucins were evident in this group. Of note, the dense degenerative findings observed in the AM group were not present. Most specimens in this group received a Chiu score of 3, due to the presence of moderate to severe epithelial loss (Figure 6g–i).
Surgical procedure before the histopathological assessment.
Photomicrographs of ileal sections stained with HE. (a) The ileum samples from the control group showed regular morphology and revealed minimum Chiu score of 0. (b and c) The ileum samples from the AM-treated rats showed slight thickening and irregularity in some villi, light epithelial lift in the apical regions of some villi, minimal degenerative changes in the epithelial cells, generally normal scattered goblet cells, and revealed Chiu score of 1 and 2. (d–f) The ileum samples from the IR group showed thickened and irregular villi, heavy degenerative changes in the epithelial cells, breakage in the epithelial cells, decrease in the goblet cells and mucins, capillary congestion in the villus, denudation of the Lieberkühn crypts, intense inflammatory cells, ulceration, and revealed maximum Chiu score of 5. (g–i) The ileum samples from the AM + IR group showed thickened and irregular villi, lifting of epithelial layer from the lamina propria, increase in the goblet cells and mucins, and revealed Chiu score of 3; (a, c, g, and i: Scale bars: 200 μm, b, d, e, f, and h: Scale bars: 100 μm).
4 Discussion
In this investigation, we examined the effect of IR on the ileum and the potential protective effects of AM against radiotherapy-induced harm. Damage to DNA and other cellular structures caused by cellular exposure to radiation generates a complex cascade of downstream reactions in almost every cellular component, including the modification of the cell cycle, DNA repair, ROS generation/defense, cytokine release, and apoptosis [25]. The GIT is extremely sensitive to radiation and injuries occur frequently. The findings of our research show decreased TOS levels and relatively better histopathological findings in the AM + IR group than in the IR group. However, the AM group had higher TOS levels compared to controls, suggesting a toxic effect of the extract itself, which may be associated with dosage. The analysis of caspases did not reveal any remarkable differences between groups. Upon exposure to radiation, GIT damage arises from both direct and indirect effects, and the damaging effects may increase the likelihood of organ dysfunction or failure in multiple systems [6]. Therefore, studies endorse the concept of utilizing radioprotective drugs to mitigate the indirect effects of radiation by removing the free radicals generated in response to radiation [26]. AM is one of the most investigated phytotherapeutic substances, and its antioxidant and anticancer activities have been shown [18,27]. In the current study, the group of irradiated rats showed a significant increase in the TOS level and administration of AM leaf extract decreased TOS values in the AM + IR group. Based on this result, the use of AM prior to irradiation may be effective in reducing oxidative stress in radiotherapy recipients. This is in line with prior studies proving the antioxidant properties of AM [28,29]. As mentioned before, TOS levels were elevated in the AM group. This brings the suspicion that an AM dosage of 300 mg/kg may have toxic effects on the ileum in healthy rats, or that the extraction technique (ethyl acetate) could be introducing deleterious compounds into the final active form. This is a critical result, particularly for studies evaluating the protective effects of AM in various clinical states, and indicates that the dosage and extraction techniques should be assessed for resultant toxicity. Although the use of all parts of the plant is considered very safe, there are also other studies in the literature that have reported toxicity [30]. In keeping with the literature, we found that Arthur et al. evaluated the acute and sub-chronic safety of AM in animals, and they reported that the extract did not produce any toxic effects on tissues at low and moderate doses. Even high doses of 2,500 mg/kg/day used for a short time (up to 7 days) were reported to be tolerated. However, the use of AM in moderate and high doses for a period of up to 14 days could cause weight loss and kidney damage [31]. The dose we applied (300 mg/kg) is believed to be very safe, but a period of 9 days may be enough to cause an inflammatory or oxidative effect in the body. When taken together, these data show that the toxicity of AM extracts may vary depending on dosage, the solvent employed, the part of the plant used, and duration of application. Effectively eliminating tumor cells using IR is constrained by the need to avoid harm to normal tissues, which limits the dosage that may be delivered. The differential IR sensitivity of the majority of tissues is directly proportional to their proliferation rates. Failure to heal irradiation-induced damage, which is especially visible in sensitive tissues such as the hematological system and small intestine epithelium, is the cause of IR-induced death [32]. It is clear from prior research that multi-organ involvement and mortality/morbidity are associated with irradiation-induced gut injury [6]. Potten and Grant have shown that small intestinal crypts are exceedingly sensitive to radiation [33]. Our study analyzed histopathological changes according to the scale defined by Chiu and colleagues. In the current study, the ileum samples in the IR group showed dense mucosal injury with thickened and irregular villi and ulcers (only present in this group); additionally, the highest Chiu scores were recorded in this group. These findings are consistent with previous studies showing that radiation exposure damages the mucosal epithelial structure and could manifest as obstructive changes in the fine vasculature [6,34]. The administration of AM prior to radiotherapy appears to ameliorate these changes, as determined by mild histopathological findings in the AM + IR group. Caspase-9 is involved in the formation of ROS in relation to mitochondrial changes. This caspase-9 activation leads to caspase-3 stimulation and causes suppression of the generation of ROS, which is necessary for the proper execution of apoptosis [35]. Apoptosis becomes overwhelming in tissues with rapid proliferation of cells, such as hematopoietic cells, hair follicles, intestinal epithelium, and dermis. In normal intestinal epithelium, apoptosis can be demonstrated to be effective in the crypt and at the luminal surface [36]. In this study, caspase levels were similar in all experimental groups. In both human and animal research, many diseases are associated with increased epithelial apoptosis in the gut [37,38,39,40]. In an interesting study, Moghadamtousi et al. had concluded that an ethyl acetate extract of AM leaves was able to induce apoptosis in cancer cells through the mitochondria-mediated pathway, and treatment of rats with AM before irradiation was determined to have inhibited radiation-induced apoptosis [41]. Therefore, in the current study, treatment of rats with AM before irradiation could have also inhibited radiation-induced apoptosis; however, since there was no difference between the groups, it is impossible to conclude the presence of such an effect from our data. The assessment of such an effect would require further investigations, including the measurement of other parameters associated with apoptosis.
5 Conclusion
Utilizing natural plants for radioprotective purposes has shown to be beneficial to humanity [42,43,44,45]. In vitro, in vivo, and human randomized controlled studies have shown that radioprotective drugs minimize DNA damage. The implementation in clinical medicine to minimize DNA damage and lipid peroxidation may reduce carcinogenesis and teratogenesis and improve morbidity and mortality rates among patients. In conclusion, our novel study demonstrates that single-dose of 8.3 Gy irradiation elevated oxidative stress markers and caused ileum injury in rats, as revealed by Chiu scoring. Pretreatment with 300 mg/kg AM leaf extract prior to irradiation decreased TOS levels and appeared to ameliorate damage to the ileum tissue. However, TOS elevation in the AM group (compared to controls) suggests possible toxic effects that may be associated with dosage or extraction technique. Nevertheless, AM appears to have a radioprotective effect in ileum tissue, likely in relation to antioxidative properties. However, further studies are needed to approve the use of plant as a radioprotective modality in humans. Further toxicity testing should be conducted to confirm its safety and evaluate extraction techniques.
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Funding information: The work of Antoaneta ENE and the APC were supported by Dunarea de Jos University of Galati, Romania through the grant no. RF 3621/2021.
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Author contributions: O.E., H.H.K.S., and H.O.T.: – conceptualization and methodology; E.K., B.G., G.A., and R.U.E.: validation; G.A. and H.M.H.Z.: formal analysis; O.E., H.H.K.S., E.K., B.G., and H.O.T.: writing and review; O.E., H.H.K.S., E.K., B.G., and A.E.: investigation; A.E.: funding acquisition through APC by “Dunarea de Jos” University of Galati, Romania through the grant no. RF 3621/2021.
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Conflict of interest: No conflict of interest was declared by the authors.
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Ethical approval: Ethical Approval of this study has been obtained from Bulent Ecevit University, Animal Experiments Local Ethical Committee (2020-17-02/07).
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Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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