Pre-clinical evidence for the therapeutic effect of Pitaya (Hylocereus lemairei) on diabetic intestinal microenvironment

Abstract Intestinal glucose absorption plays a central role in the regulation of glucose plasmatic; however, current clinical management does not target the gut for treating diabetes. This study evaluated the effects of peel and pulp aqueous extract from Hylocereus lemairei on human enterocytes under high glucose concentration. Anti-hyperglycemic and antiobesity activities in vitro were also evaluated. Extracts did not cause cytotoxicity at 1 to 500 μg/mL. Moreover, they were effective in attenuating oxidative stress (DCFH-DA assay) and inflammation (•ON production) caused by high glucose. Intestinal enzymes (α- glucosidase and pancreatic lipase) were inhibited by pulp and peel extracts (>60% and >95%, respectively). Extracts exhibited a redox capacity superior to ascorbic and chlorogenic acids, presenting high phenolic content, mainly anthocyanins. The main compounds for both extracts were chlorogenic acid and naringin, and peel stood both qualitatively and quantitatively. Data suggest red Pitaya has potential as a new medicine for diabetes.


Introduction
Diabetes mellitus (DM) is one of the largest causes of morbid mortality globally.
Conventional medication is unable to prevent its complications, which are associated with oxidative stress, and remain ('metabolic memory'), even in medicated patients (Testa et al. 2017).
It is already known that intestinal glucose absorption plays an important role in the regulation of glycemia (Guo et al. 2020). Intestinal cells suffer the most from excess glucose dietary and recent studies have shown that the redox imbalance in the enteral microenvironment can contribute to the etiology of DM (Patterson et al. 2016). These are previous evidence indicating that positive intestinal manipulation can contribute to DM management. However, to date, no medicine has been designed in this direction. Therefore, it is important to highlight research using natural products. This is supported by the crescent global use of medicinal plants, by nearly 80% of the population, according to the World Health Organization (Pappis et al. 2021).
Red Pitaya, Hylocereus lemairei (Hook) Britton & Rose, is a cactus plant originating in the Americas, popularly known as 'Dragon Fruit'. Its peel, covered with bracts, has a red color, as well as its pulp, which is a source of fiber, vitamins A, C, and E, phosphorus, and calcium, rich in water and low in lipids. This striking coloration is due to the betalain, a nitrogenous pigment, and also by anthocyanins, a phenolic compound. Phenolic compounds are distributed in the peel, pulp, and seeds of red Pitaya. The fruit is a powerful antioxidant, which also has anti-inflammatory, and anti-diabetic properties (Joshi and Prabhakar 2020). Specifically concerning antidiabetic activity, a previous study showed that the ingestion of pulp red Pitaya (400 g/day) was able to control blood glucose levels and maintain a good lipid profile in type 2 diabetic patients (Hadi et al. 2012). However, the deepest mechanisms on this effect need further investigation. Moreover, there are no supplemental studies investigating the antidiabetic potential of H. lemairei pulp. Concerning the peel, its antidiabetic effect was not studied yet. This is particularly important since it is a part usually discarded in consumption, nevertheless has a significative phenolic content (Suh et al. 2014;Hua et al. 2018). The most recent data available points that almost a third of all agricultural production in the world is lost/wasted, representing 1.3 billion tons year (FAO. 2017) and fruits are one of the major drivers of it. Therefore, research that uses parts considered residuals, such as peel, represents both socioeconomic and environmental valuable strategy.
Therefore, this study aimed to investigate the efficacy and safety of aqueous extract from Pitaya pulp and peel in human enterocytes under high glucose exposition. Antihyperglycemic and antiobesity activities in vitro also were investigated.

Results and discussion
Total phenolic content of the aqueous extract of Pitaya pulp was 178.83 GAE ± 4.151 mg/100g. For peel, 392.31 ± 10.876 mg/100g was obtained. Anthocyanin content in pulp was 56.75 ± 2.78 mg/100g, constituting 32% of its total phenolic content. For peel, it was 26.27 ± 0.079 mg/100g, representing about 7%. Extracts were analyzed via HPLC, and the different identified compounds are shown in Table S2. The main molecules comprising pulp and peel chemical matrix were chlorogenic acid and naringin (Figure S1B-C). Peel extract displayed a greater variety of polyphenols and higher levels when compared to the pulp. In addition, it was not detected hesperidin, ferulic acid, and catechin in the pulp extract. Compared to the values described in the Phenol Bank (Phenol Bank 2020) a database that presents the composition of different foods, the content of anthocyanins was higher than reported for plums and blackberries, which are well-known fruits for their preeminent phenolic composition. Concerning peel, total phenolic content was higher than described for red grapes.
Since phenolic compounds exhibit antioxidant activity, it was evaluated through the reducing capacity to neutralize the DPPH radical (Table S3). Pitaya has a redox capacity superior to that shown for ascorbic and chlorogenic acids, two isolated compounds with recognized antioxidant potential. It was also observed that the peel is more antioxidant than the pulp. Total phenolic content and anthocyanins exhibited by pulp and peel were positively correlated with their antioxidant activity (r ¼ 0.999, p ¼ 0.034; r ¼ 0.998, p ¼ 0.039, and r ¼ 0.999, p ¼ 0.035; r ¼ 0.997, p ¼ 0.050, respectively). These data demonstrate that Pitaya is a rich and accessible source of polyphenols, especially anthocyanins, with important antioxidant activity, and highlight the need for the full use of the fruit in nutraceutical and/or pharmaceutical compositions, for example for treating DM.
DM type 2 is characterized by the occurrence of continuous hyperglycemia, hypoinsulinemia, hyperlipidemia, and hypertriglyceridemia, which are central factors in the pathogenesis of diabetic complications (Vlachos et al. 2020). Changes in postprandial hyperglycemia are a result of a complex interaction, including joint action of carbohydrate (a-glucosidase and a-amylase) and lipid (pancreatic lipase) metabolism enzymes. So, achieving good glycemic control is of utmost importance for DM2 patients. Table S4 shows the inhibitory effects of extracts on the important metabolic enzymes. No statistical significance was observed between the extracts; however, it is important to mention that peel concentration was lower, which indicates its best effectiveness.
Extracts had higher inhibitory effects against the pancreatic lipase, followed by a-glycosidase, suggesting an intestinal-targeted action. Regarding a-amylase, a discrete effect was observed. Pulp and peel extracts were more effective than acarbose and orlistat, used as standard. Thus, we found that Pitaya exhibit anti-hyperglycemic and antiobesity activities, therefore indicating a potential to face DM.
DM is a complex pathophysiology that can affect different organs. Until now, diabetes-related functional impairments of the gut have been insufficiently researched. Uptake of intestinal glucose regulates insulin secretion by the pancreas and vice versa. Therefore, this study also evaluated, for the first time, the effects of glucose on human intestinal cells differentiated into enterocytes. Caco-2 cells express morphological and biochemical characteristics present on the human small intestine, such as columnar form, brush border, junction complexes, active transport, and digestive enzymes in the membrane (Sun et al. 2008). These cells are adapted to 5.5 mM glucose normally. Hence, this concentration was used as the normoglycemic-like state. Extracts were tested at concentrations of 1 to 1000 lg/mL ( Figure S2) for 24 h. There was no significant change in cell viability at concentrations of 1, 10, and 100 lg/mL for both extracts. Cellular viability assay was consistent with the microscopy images ( Figure  S3). No damage to cell morphology at doses of 1-100 lg/mL was found. Therefore, the safety doses of 1, 10, and 100 lg/mL were selected for the other experiments. After confirming the safety, it was evaluated whether the extracts would be able to avoid mortality, ROS production, and inflammation induced by high glucose. Glucose exposition induced 18% of reduction in cell viability compared to control. This reduction was avoided when the pulp extract was added, at the three doses, with the best effect observed at 10 lg/mL ( Figure S4A). For peel, the lowest dose (1 lg/mL) had the best response to cell recovery ( Figure S4B). Cells treated only with the extracts did not increase ROS production. Exposure to glucose increased the production of ROS by almost 20%; but 10 lg/mL of pulp reduced by 12% this increment ( Figure S4C). Pitaya peel at concentrations 1 and 10 lg/mL, exhibited a decrease of 17 and 15%, respectively, in ROS levels ( Figure S4D).
The presence of high glucose acts as a modifier of the intestinal barrier, and the state of postprandial hyperglycemia in diabetic patients interferes negatively with the integrity of the epithelial mucosa (Thaiss et al. 2018). Continuously increasing glucose availability can lead to systemic oxidative stress and activation of the inflammatory cascade, by causing, among others, overproduction of nitric oxide ( ON). Pulp extracts (1 and 10 lg/mL) did not significantly alter the ON levels compared to control. Exposure of cells to high glucose resulted in a significant increase (22%) in ON levels. When the extract (1 and 10 lg/mL) was added, there was a significant decrease, with a maximum reduction of 20% at the highest dose ( Figure S4E). Pell extracts (1 and 10 lg/mL) also were able to reduce ON production by 13% and 17%, respectively. In the normoglycemic-like state, these doses did not change the ON levels ( Figure S4F). Although its main production site is the endothelium, intestinal cells also produce ON under inflammatory stimulus (Cavicchi and Whittle 1999;Chen and Kitts 2015). This messenger also plays an important role in peroxynitrite ( ONOO) formation via reaction with superoxide anion radical (O 2 -) generated mainly by the mitochondrial complex I and there is no antioxidant defense to counter ONOO. Thus, the neutralization of these species could block the vicious cycle between inflammation and oxidative stress, and it was observed Pitaya can target it. Effects observed in this study can be attributed, at least in part, to the phenolic compounds. Chlorogenic acid can attenuate insulin resistance and modulate glucose uptake (Chen et al. 2019), as well as protect the intestinal barrier (Xue et al. 2019). Ferulic acid has antiatherogenic activity and regulates hypertension and insulin resistance associated with metabolic syndrome (El-Bassossy et al. 2016). Gallic acid displays antidiabetic activity, mediated by the regulation of pAkt, PPAR-c, and Glut4 (Variya et al. 2020). Among the flavonoids, naringin stands out as anti-diabetic. Its mechanisms of action include inhibition of gluconeogenesis through upregulation of AMPK with metformin-like effects (Nyane et al. 2017). Hesperidin, which is a flavanone glycoside, is effective against diabetic neuropathic pain, and, together with insulin, was effective in controlling hyperglycemia and hyperlipidemia (Hajialyani et al. 2019). Catechin can decrease fasting blood glucose level, lipid parameters, and Hb1c levels (Nazir et al. 2020), while rutin can decrease carbohydrate absorption in the gut (Ghorbani 2017). Such evidence indicates that compounds present in Pitaya peel and pulp have the potential for managing diabetes. Nonetheless, despite its per se efficacy, the polyphenol mixture seems to be more effective (Gertsch 2011;Branco et al. 2019a;2019b), since the synergic effect could be responsible for a multiplicity of cellular targets.

Experimental
All the experimental procedures carried out in this study are described in the supplementary material of this article.

Conclusion
Red Pitaya can control the overproduction of ROS and ON by enterocytes, suggesting the extracts could positively modulate the intestinal inflammation pathway against excess glucose. In addition, extracts were able to target intestinal enzymes that are dysregulated in diabetes. Although promising, these results still need to be validated in future in vivo studies.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Coordenac¸ão de Apoio de Pessoal de N ıvel Superior (CAPES), the Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and the Conselho Nacional de Desenvolvimento Cient ıfico e Tecnol ogico (CNPq). The authors thank UCS Writing Center for the grammatical revision of this paper.