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Hypoglycemic and hypolipidemic effects of two mangrove plants in a streptozotocin-induced animal model of diabetes

Objective: This study aims at evaluating the anti-diabetic, hypolipidemic, and pancreatic histopathological changes of Rhizophora mucronata and Avicennia marina. Materials and Methods: The experimental rats were divided into eight groups (n = 15 each). Streptozotocin was used to induce diabetes. Daily oral administration of an aqueous extract from the leaves of R. mucronata and A. marina at 400 mg/kg BW, and a mixture of the two extracts for 6 weeks was assessed. The measurements of serum glucose, insulin, and lipid profile were carried out. Pancreatic specimens were collected from all groups and processed for pathological studies. Results: The study revealed that the plant extracts restored the levels of diabetic markers and lipid profiles of diabetic rats, with no significant changes in non-diabetic ones. The extract of R. mucronata exhibited more promising anti-diabetic and hypolipidemic effects than A. marina singly or combined. Conclusion: Leaf extracts from R. mucronata, singly or combined, and A. marina, induced a potent anti-diabetic and hypolipidemic potential in diabetic rats.


Introduction
Diabetes mellitus (DM), a metabolic disorder that represents one of the five leading causes of death worldwide, is characterized by hyperglycemia linked with lipid metabolic abnormalities, in addition to disturbances in protein and carbohydrate metabolism [1][2][3]. At the present time, 171 million people are diagnosed with diabetes, and this is proposed to reach 366 million by 2030 [4]. This metabolic disorder, DM, is attributed to decreased insulin secretion or increased cellular resistance to insulin, as well as the induction of oxidative stress and disturbance in metabolism-regulating enzymes of glucose and lipid [1,5]. Recently, efficient foods and their bioactive compounds replaced the available diabetic drugs in use, which have numerous restrictions due to associated undesirable effects, such as hypoglycemia, cell injury (necrosis), and high rates of secondary failure [6].
The use of medicinal plants has been embraced worldwide since it is a critical part of public healthcare.
Rhizophora mucronata and Avicennia marina are mangrove plants that grow in both humid and subtropical climates. These vulnerable plants require protection for their significance in the cure of diabetes and other disease conditions. The two plants have been proven to have antiviral and antibacterial characteristics [7]. Plant extracts of different species have also been tested to have anti-diabetic potential, including Cordia dichotoma fruits [8] and Pedicularis longiflora Rudolph, which additionally showed antioxidant properties [9].
Although the hypoglycemic potential of R. mucronata [10] and A. marina extracts was reported [11], the hypoglycemic effect of R. mucronata extract combined with A. marina extract is not investigated until now. To our knowledge, no studies have investigated the anti-diabetic and hypolipidemic effect of the combination of R. mucronata and A. marina extracts in an animal model of diabetes. Therefore, the current study was carried out to evaluate the efficacy of the aqueous extract of Saudi R. mucronata This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 Licence (http://creativecommons.org/ licenses/by/4.0) and A. marina leaves administrated to rats on improving the impact of diabetes on the glycemic state and lipid metabolism impairment.

Material and Methods
The experiment was carried out on 120 Wistar male albino rats (between 200 and 250 gm average body weight). The rats were individually kept in cages at a constant temperature (24°C ± 1°C), in alternating 12-h light/dark cycle. The animals were fed the standard chow and water. The rats were used according to the guidelines of the Animal Care and Use Committee of King Abdulaziz University [approval number 172060302].

Plant extraction
The aqueous extract of R. mucronata and A. marina leaves was prepared according to previous reports [12,13]. The leaf extracts of R. mucronta and A. marina were administrated orally at a dose of 400 mg/kg BW/day each, while the mixture of both plants included 200 mg/kg BW/day of each extract.

Preparation of streptozotocin (STZ) and induction of DM
Overnight-fasted adult male rats (6 weeks old) were administrated an intraperitoneal injection with a single dose (60 mg/kg) of freshly prepared STZ [14]. After three days, the levels of fasting blood glucose (FBG) were assessed in blood samples taken from the rats' tails by using the One Touch Ultra Glucometer (Lifescan, Johnson and Johnson, Milpitas, CA). The rats that had an FBG level of ≥ 250 mg/dl were assigned as diabetic rats [10].

Experimental design
Rats were randomly divided into eight groups (15 rats each). Group 1 included normal rats and group 2 included untreated diabetic rats. Groups 3-5 included diabetic rats treated with extracts of R. mucronta, A. marina, or a mixture of both plants. Group 6 included normal rats treated with the extract of R. mucronata, group 7 included normal rats treated with the A. marina extract, and group 8 included normal rats treated with a combination of both plant extracts. The daily administration of the extracts started on the fourth day after injecting STZ and lasted for 6 weeks.

Measurement of serum glucose, insulin, and lipid profile
Blood was drawn from the retroorbital venous plexus of the animals during the experiment, and serum was prepared by centrifugation for 20 min. Blood glucose and lipid profile were assessed in the sixth week post-treatment using kits (Roche Cobas Diagnostics, USA). The serum levels of the low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) were estimated as previously described [15]. The serum insulin level was assessed using insulin Enzyme-linked Immunosorbent Assay (ELISA) kits that comprised an enzyme immunoassay for the quantitative determination of insulin in the sera of rats (Cat. no. Ezrmi-13 Kelisa, Billerica, MA) according to the methods described previously [16,17].

Histopathological study
Pancreatic specimens were collected in the sixth week post-treatment from the sacrificed rats of all groups, kept in neutral formalin, and processed in an automated tissue processor. Paraffin sections were stained with hematoxylin and eosin for pathological studies [18].

Statistical analysis
The obtained data in this study are presented as mean ± SEM. One-way analysis of variance (Statistical Package for the Social Sciences 24) was used to determine the differences between the groups. The values were considered to be significantly different when the p-value was < 0.05.

Effects of extracts on glucose, insulin, HOMA-IR, and HOMA-β levels
A significant (p < 0.001) rise in FBG level and a decrease in serum insulin were recorded in diabetic rats compared to that in the normal rats in the sixth week (Table 1, Fig. 1). These parameters were improved in diabetic rats that received herbal extracts (groups 3-5) compared to the STZinduced diabetic rats. Improvement was more remarkable in R. mucronata-treated rats than in those treated with A. marina. The normal rats that received plant extracts alone or in combination showed no significant changes when compared with the control rats.

Effects of plant extract treatments on lipid profile
Rhizophora mucronata and A. marina extracts and the combination of both when administered to the diabetic rats for 6 weeks resulted in a significant reduction (p < 0.05, 0.01, and 0.001) in the serum triglycerides, cholesterol, LDL-C, and VLDL-C levels compared to the STZ-induced diabetic group. In addition, it markedly raised (p < 0.001) the high-density lipoprotein (HDL-C) level compared to the STZ-induced diabetic group (Fig. 1). The marked change (p < 0.001) in the lipid profile was seen in the group 3 rats. Therefore, the diabetic rats receiving R. mucronata extract showed near-normal levels of all lipogram parameters. On the contrary, those treated with A. marina extract singly or combined with R. mucronata (groups 4 and 5) showed enhancements in serum cholesterol, LDL-C, and VLDL-C levels.

Histopathological examinations of pancreatic tissue
Examination of the pancreas from control rats (group 1) revealed intact histo-morphological structures (Fig. 2). The pancreas of diabetic rats (group 2) showed characteristic changes consistent with decreased densities of the islet cells and degenerative changes in the β-cells of the islets, mainly cloudy swelling and hydropic degenerations. In addtition, necrotic changes in moderate numbers of β-cells were a pathognomonic lesion, as the cells entirely or partially lost their nuclei and/or the cytoplasmic components (Fig. 2). The exocrine pancreas showed minor changes, mainly cystic dilatation and fibrosis of the affected ductal walls. Regarding the treatment of diabetic rats, pancreatic sections from groups 3-5 were healthy in most parts; however, a few histopathological lesions were encountered in diabetic groups receiving R. mucronata extract (group 3) (Fig. 2). Diabetic rats receiving A. marina extract (group 4) revealed moderate degenerative and necrotic changes in both α-and β-cells of the pancreas (Fig.  2). Diabetic rats receiviing a mixture of the two extracts (group 5) showed normal pancreatic islet β-cells; however, a few degenerated α-and β-cells were encountered (Fig.  3). Normal rats that were administered the plants extract, as single or in combination (groups 6-8), showed no significant histo-morphologic changes compared with the normal rats (Fig. 3).

Discussion
In the current study, hyperglycemia and hypoinsulinemia were the characteristic findings in diabetic rats. The gradual rise in serum glucose levels appeared to be due to limited insulin, as a result of β-cell damage induced by STZ [19,20]. Therefore, it is known that when β-cell dysfunction is responsible for the occurrence of diabetes, insulin resistance and deficiency are also required for hyperglycemia to occur [21]. The HOMA model is a commonly utilized method to assess insulin resistance and β-cell function [22,23].
The anti-diabetic and hypolipidemic effects of the currently used plant extracts may be attributed to the increased expression of the nuclear receptor, peroxisome proliferator-activated receptor-gamma, commonly recognized to enhance insulin sensitivity, and is thus being utilized in targeting the treatment of type 2 DM [24,25].
The findings of this study are supported by several other previous reports [26,27]. The administration of STZ induced a higher degree of apoptosis in the islets of Langerhans [28].
Treating the diabetic groups with the plant extracts showed potent anti-diabetic activity, especially in R. mucronata-treated rats. The effect of R. mucronata as an anti-diabetic compound could either be due to the improvement of insulin secretion by β-cells of the pancreatic islets [29] or due to the existence of a similar insulin protein in the plant extracts [30]. The extracts of R. mucronata cause strong hypoglycemic and antihyperglycemic reactions; these findings have been further confirmed by other researchers [31,32].
An analysis of the lipid levels in the animals revealed a significant increase in the concentrations of cholesterol, triglycerides, LDL, and VLDL, and a reduction in blood serum HDL in the diabetic group when compared to the control rats. These findings point out significant dyslipidemia in diabetic animals. Such diabetic dyslipidemia and hyperglycemia are thought to be a prognosticator of cardiovascular complications [33,34]. Increased triglycerides and cholesterols in rats with diabetes may be attributed to activating hormone-sensitive lipase, which enhances fatty acids mobilization from triacylglycerols stored in adipocytes [35,36]. Thus, the large number of fatty acids recurring to the liver are reunited to triacylglycerols and excreted as VLDL. Insulin deficiency and/or insulin resistance may be responsible for hyperlipidemia [15].
Controlling hyperlipidemia is a requirement to prevent diabetic microvascular changes, such as retinopathy, neuropathy and nephropathy, and macrovascular changes like cerebral vascular disease, ischemic heart disease, and arteriosclerosis [34]. Interestingly, the current study further demonstrates that lipid and lipoprotein irregularities were opposed by the plant extracts in diabetic rats. The hypolipidemic effects of R. mucronata might be due to the impact of its diverse bioactive components, and other consitituents [37] or the downregulation of LDL epitopes and prevention of 3-hydroxy 3-methylglutaryl coenzyme A reductase [38]. Indeed, low doses of A. marina had been used as anti-cholesterolemic agents due to its contents in bioactive constituents that might have an antioxidants-like effect, scavenger free radical, and enhance lipid profile and organ function [39]. Similar findings regarding the anti-diabetic and hypolipidemic efficiency of an extract of Quercus dilatata fruit had been reported by Shaheen et al. [40].
The flavonoids constituents of R. mucronata could have a vital role in preventing β-cell apoptosis, promotion of β-cell proliferation, increase in secreting insulin, and enhancement of insulin bioactivity [7]. Similar findings were obtained by Abdel-Daim et al. [41]. They reported that Moringa oleifera leaf extracts prevented the histo-architecture changes of the pancreatic tissues of diabetic rats and reduced the percentage of pancreatic apoptotic cells. The extracted form of R. mucronata alone or combined with A. marina induced a powerful anti-diabetic and and a few degenerated α-cells (blue arrow) and β-cells (yellow arrows). Group 6(B) is the group treated with R. mucronta showing normal islets of Langerhans, regarding populations, distributions, and sizes (blue arrows). The α-(red arrow) and β-cells (yellow arrow) are normal, and the capillary network (black arrow) is seen among islet cells. H&E. Scale bars, 50 um. Group 7(C) is the group treated with A. marina showing normal populations, distributions, sizes, and structures of islets of Langerhans (blue arrows) and normal pancreatic acini with active secretory granules (brown arrow). The α-(red arrow) and β-cells (yellow arrow) of the pancreatic islets are normal and in a good active condition with a dispersed capillary network among them. H&E. Scale bars, 50 um. Group 8(D) is the group treated with a mixture of R. mucronta showing normal pancreatic islets, regarding populations, sizes, and distributions (yellow arrows) with normal β-cells (blue arrows), vacuolated cytoplasm of some α-cells (brown arrows) and normal capillary network among islets cells (red arrows). H&E. Scale bars, 50 um. hepatoprotective activities against liver disorders. It addition, they observed to alleviate the histopathological and immunohistochemical changes induced by diabetes on the liver in the animal model of diabetic rats [42]. In this study, the mechanism behind the hypoglycemic and hypolipidemic and the potential of R. mucronata and A. marina was not explored in depth, and this is one of the limitations of this study.

Conclusion
The leaf extracts from R. mucronata, alone or combined, with A. marina, induced an effective anti-diabetic and hypolipidemic activity in the used animal model of diabetic rats.