The Antidiabetic Effects and Modes of Action of the Balanites aegyptiaca Fruit and Seed Aqueous Extracts in NA/STZ-Induced Diabetic Rats

Diabetes mellitus (DM) is a chronic metabolic disorder that threatens human health. Medicinal plants have been a source of wide varieties of pharmacologically active constituents and used extensively as crude extracts or as pure compounds for treating various disease conditions. Thus, the aim of this study is to assess the anti-hyperglycemic and anti-hyperlipidemic effects and the modes of action of the aqueous extracts of the fruits and seeds of Balanites aegyptiaca (B. aegyptiaca) in nicotinamide (NA)/streptozotocin (STZ)-induced diabetic rats. Gas chromatography–mass spectrometry analysis indicated that 3,4,6-tri-O-methyl-d-glucose and 9,12-octadecadienoic acid (Z,Z)- were the major components of the B. aegyptiaca fruit and seed extracts, respectively. A single intraperitoneal injection of STZ (60 mg/kg body weight (b.w.)) 15 min after intraperitoneal NA injection (60 mg/kg b.w.) was administered to induce type 2 DM. After induction was established, the diabetic rats were treated with the B. aegyptiaca fruit and seed aqueous extracts (200 mg/kg b.w./day) via oral gavage for 4 weeks. As a result of the treatments with the B. aegyptiaca fruit and seed extracts, the treated diabetic-treated rats exhibited a significant improvement in the deleterious effects on oral glucose tolerance; serum insulin, and C-peptide levels; liver glycogen content; liver glucose-6-phosphatase and glycogen phosphorylase activities; serum lipid profile; serum free fatty acid level; liver lipid peroxidation; glutathione content and anti-oxidant enzyme (glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase) activities; and the mRNA expression of the adipose tissue expression of the insulin receptor β-subunit. Moreover, the treatment with fruit and seed extracts also produced a remarkable improvement of the pancreatic islet architecture and integrity and increased the islet size and islet cell number. In conclusion, the B. aegyptiaca fruit and seed aqueous extracts exhibit potential anti-hyperglycemic and anti-hyperlipidemic effects, which may be mediated by increasing the serum insulin levels, decreasing insulin resistance, and enhancing the anti-oxidant defense system in diabetic rats.


Introduction
Diabetes mellitus (DM) is a chronic metabolic syndrome with a number of different etiologies. It severely affects the life of patients and heightens the risk of developing other Group I (Normal group): This group was assigned as the normal control group, and rats included in this group was given distilled water daily (5 mL/kg b.w./day) via oral gavage for 4 weeks.
Group II (Diabetic control): This group was assigned as the diabetic control group, and the diabetic rats within this group were given distilled water daily (5 mL/kg b.w./day) via oral gavage for 4 weeks.
Group III (Diabetic rats treated with the B. aegyptiaca fruit extract): This group consisted of diabetic rats that were treated daily with B. aegyptiaca fruit extract at a dose level of 200 mg/kg b.w./day via oral gavage for 4 weeks.
Group IV (diabetic rats treated with the B. aegyptiaca seed extract): This group consisted of diabetic rats that were treated daily with B. aegyptiaca seed extract at a dose level of 200 mg/kg b.w./day via oral gavage for 4 weeks.
At the day before sacrifice, oral glucose tolerance (OGT) test (OGTT) was performed by administering glucose solution (3 g/kg b.w.) to overnight-fasted rats via oral gavage. Successive blood samples were then obtained at 0, 30, 60, 90, and 120 min. Blood samples were left to coagulate and centrifuged. Sera were separated via centrifugation at 3000 rpm for 15 min, and the serum glucose levels were determined. One day after the end of the experiment, blood samples were collected from the jugular vein under diethyl ether inhalation. Moreover, the rats were euthanized and dissected for the excision of various organs.

Blood Sampling and Tissue Sampling
The blood obtained from the jugular vein of each rat was left to coagulate at room temperature. Serum was separated for 15 min via centrifugation at 3000 rpm and stored at −20 • C pending its use for the determination of insulin and C-peptide levels as well as other biochemical parameters. The rats were rapidly dissected. Visceral adipose tissues were excised and kept at −70 • C until use for ribonucleic acid (RNA) extraction and the detection of the messenger RNA (mRNA) of adiponectin and resistin via reverse transcription-polymerase chain reaction (RT-PCR). The liver was excised for the determination of oxidative stress parameters and glycogen content and glycogen metabolizing enzymes. The pancreas was also excised for histological investigation.

Biochemical Analysis
The serum glucose levels were measured using the reagent kits purchased from Spinreact Company (Spain) using the method of Trinder et al. [21]. The serum insulin level was measured using sandwich enzyme-linked immunosorbent assay (ELISA) using kits purchased from Linco Research, USA, in accordance with the manufacturer's instructions. Similarly, the serum C-peptide level was measured using the ELISA kits purchased from Linco Research, USA, in accordance with the manufacturer's instructions. Homeostatic model assessment (HOMA)-insulin resistance (IR), HOMA-insulin sensitivity (IS) [22], and HOMA-β cell function [23] were calculated using the following formulas, respectively: The liver glycogen content was measured using the method of Seifter et al. [24]. The liver glucose-6-phosphatase and glycogen phosphorylase activities were measured using laboratory-prepared chemicals and the methods of Begum et al. [25] and Stallman and Hers, respectively [26]. The serum cholesterol level was assayed using the method of Allain et al. and the reagent kits purchased from Spinreact Company (Spain) [27].
The serum triglyceride level was determined using the reagent kit purchased from Reactivos Spinreact Company (Girona, Spain) and Fossati and Prencipe's method [28]. The serum high density lipoprotein (HDL)-cholesterol level was measured using the method of Allain et al. (1974) and the reagent kit obtained from Spinreact Company, Spain [27]. The serum low density lipoprotein (LDL) cholesterol level was calculated using the formula of Friendewald et al. [29]: LDL cholesterol = total cholesterol − triglycerides/5 − HDL cholesterol Serum very low density lipoprotein (vLDL)-cholesterol was calculated using Norbert's formula [30]: vLDL cholesterol conc. = triglycerides/5 The serum free fatty acid (FFA) level was determined using Duncombe's method [31].

RNA Isolation and RT-PCR
RNA was isolated from visceral adipose tissue using the GeneJet RNA purification kit produced by Thermo Fisher Scientific Inc., Branchburg, NJ 08876, USA, according to the procedures of Chomzynski and Sacchi [32] and Boom et al. [33]. The isolated RNA was quantified and qualified [34,35]. Thermo Scientific Verso 1-Step RT-PCR ReddyMix was used to produce cloned DNA that was amplified in the presence of specific forward and reverse primers using a Techne thermal cycler, Cole-Parmer, IL 60061, USA [35]. The primer pair sequences for the insulin receptor β-subunit were F: 5 CTGGAGAACTGCTCGGTCATT3 and R: 5 GGCCA-TAGACACGGAAAAGAAG3 [36], and those for β-actin re F: 5 TCACCCTGAAGTACCCCATGGAG3 and R: 5 TTGGCCTTGG GGTTCAGGGGG3 [37,38].

Determination of Oxidative Stress and Anti-Oxidant Defense Parameters
The glutathione content (GSH) in the liver was determined [39]. Moreover, glutathione-S-transferase (GST) activity in the liver was measured using Mannervik and Gutenberg's method [40]. The liver glutathione peroxidase (GPx) activity was determined using the method of Matkovics et al. [41]. The superoxide dismutase (SOD) and lipid peroxidation (LPO) were determined using the methods of Marklund and Marklund [42] and Preuss et al. (1998), respectively [43].

Histological Investigation
The pancreas from each rat was rapidly excised after dissection and then fixed in 10% neutral buffered formalin for 24 h. The organs were routinely processed and sectioned at a thickness of 4 to 5 µm. The sections of the pancreas were stained with hematoxylin and eosin [44,45].

Statistical Analysis
The results were analyzed using the PC-STAT Program [46]. One-way analysis of variance (ANOVA) was followed by the least significant difference (LSD) test to compare various groups. Data were described as the mean ± SE. A p value of >0.05 was considered nonsignificantly different, whereas p values of <0.05 and <0.01 were considered significant and highly significant, respectively.

Effects on OGT
The OGT curve of the diabetic rats exhibited a significant (p < 0.01; LSD) elevation at all tested periods (0, 30, 60, 90, and 120 min) after oral glucose intake compared with that of normal animals. The oral administration of B. aegyptiaca fruit and seed extracts to diabetic rats induced a potential amelioration of elevated values at all tested points of the OGT curve. However, the seed extract was more potent at 30 and 60 min after oral glucose intake ( Figure 3). The F-probability of OGTT data indicated that the effect between groups was very highly significant (p < 0.01).

Effects on OGT
The OGT curve of the diabetic rats exhibited a significant (p < 0.01; LSD) elevation at all tested periods (0, 30, 60, 90, and 120 min) after oral glucose intake compared with that of normal animals. The oral administration of B. aegyptiaca fruit and seed extracts to diabetic rats induced a potential amelioration of elevated values at all tested points of the OGT curve. However, the seed extract was more potent at 30 and 60 min after oral glucose intake ( Figure 3). The F-probability of OGTT data indicated that the effect between groups was very highly significant (p < 0.01).

Effects on Serum Insulin and C-Peptide Levels
As indicated in Table 3, the diabetic rats showed a highly significant decrease (p < 0.01; LSD) in the insulin and C-peptide levels in serum. The treatments of the diabetic rats with the fruit and seed extracts caused a highly significant increase (p < 0.01; LSD) in   Table 3, the diabetic rats showed a highly significant decrease (p < 0.01; LSD) in the insulin and C-peptide levels in serum. The treatments of the diabetic rats with the fruit and seed extracts caused a highly significant increase (p < 0.01; LSD) in these lowered levels. The diabetic rats treated with the B. aegyptiaca fruit extract exhibited no significant effects on the insulin and C-peptide levels in comparison with the diabetic rats treated with the B. aegyptiaca seed extract. However, the effects of the fruit extract was more potent in increasing the serum insulin levels. The F-probability indicated that the general effect between groups was very highly significant (p < 0.01).

Effect on HOMA-IR Cell Function, HOMA-IS, and HOMA-β Cell Function
In diabetic rats, the HOMA-IS and HOMA-β cell functions were highly significantly (p < 0.01; LSD) decreased, whereas HOMA-IR was highly significantly (p < 0.01; LSD) increased. The treatment of diabetic rats with B. aegyptiaca fruit and seed extracts induced a highly significant increase in HOMA-β cell function and HOMA-IS. In contrast, HOMA-IR was highly significantly decreased after the treatments with the B. aegyptiaca fruit and seed extracts.
Although the effects of the B. aegyptiaca fruit and seed extracts on HOMA-IR and HOMA-IS were more or less similar, the effects of the fruit extract on HOMA-β cell function was more potent than that of the seed extract (Table 4). One-way ANOVA revealed that the effect between groups on the HOMA-IR, HOMA-IS, and HOMA-β cell function was very highly significant (p < 0.01; F-probability).

Effects on Liver Glycogen Content and Glucose-6-phospatase and Glycogen Phosphorylase Activities
The diabetic rats exhibited a highly significant (p < 0.01; LSD) depletion in liver glycogen content and a highly significant (p < 0.01; LSD) elevation in liver glucose-6phosphatase and glycogen phosphorylase activities. The treatments with the B. aegyptiaca fruit and seed extracts highly significantly improved (p < 0.01; LSD) the reduced liver glycogen content of the diabetic rats and the elevated glucose-6-phosphatase and glycogen  (Table 5). One-way ANOVA revealed that the effect between groups on liver glycogen content and glucose-6-phosphatase and glycogen phosphorylase activities was very highly significant (p < 0.01; F-probability). Table 5. Effects of the B. aegyptiaca fruit and seed extracts on liver glycogen content and glucose-6phosphatase and glycogen phosphorylase activities in NA/STZ-induced diabetic rats.

Effects on Serum Lipid Profile
The total cholesterol, triglycerides, LDL-cholesterol, vLDL-cholesterol, and FFA levels in serum exhibited a highly significant elevation (p < 0.01; LSD) in diabetic rats compared with those in the normal group. The treatment of diabetic rats with the B. aegyptiaca fruit and seed extracts produced a highly significant improvement in the altered lipid profile in the serum. Moreover, the HDL-cholesterol level was affected in an inverse pattern, as it was highly significantly decreased (p < 0.01; LSD) in diabetic rats.
Conversely, the treatment with the fruit extract induced a significant increase (p < 0.01; LSD) compared with the diabetic control, whereas the treatment with the seed extract had no significant effect (p > 0.05; LSD) ( Table 6). The seed extract was more effective in decreasing the elevated total cholesterol and triglyceride levels in diabetic rats than the fruit extract, whereas the fruit extract was more potent in decreasing the elevated LDLcholesterol, vLDL-cholesterol, and FFA levels and increasing the lowered HDL-cholesterol level ( Table 6). The F-probability revealed that the effect on serum lipid profile between groups was very highly significant (p < 0.01).

Effect on Insulin Receptor β-Subunit
The densitometric analysis of the electrophoretogram showed a highly significant (p < 0.01; LSD) decrease in the mRNA expression of the adipose tissue insulin receptor β-subunit in diabetic rats compared with that in the normal group. The treatment of diabetic rats with the B. aegyptiaca fruit extract produced a highly significant (p < 0.01; LSD) amelioration of the insulin receptor β-subunit mRNA expression (Figure 4), whereas the treatment with the B. aegyptiaca seed extract did not show a significant effect (p > 0.05; LSD). One-ANOVA indicated that the effect between groups on the mRNA expression of insulin receptor β-subunit was highly significant (p < 0.01; F-probability).
β-subunit in diabetic rats compared with that in the normal group. The treatment of abetic rats with the B. aegyptiaca fruit extract produced a highly significant (p < 0.01; L amelioration of the insulin receptor β-subunit mRNA expression (Figure 4), whereas treatment with the B. aegyptiaca seed extract did not show a significant effect (p > 0 LSD). One-ANOVA indicated that the effect between groups on the mRNA expression insulin receptor β-subunit was highly significant (p < 0.01; F-probability).

Effect on Oxidative Stress and Anti-Oxidant Defense Parameters
The liver LPO exhibited a highly significant (p < 0.01; LSD) increase in diabetic rats compared with that in normal rats. The treatment with the B. aegyptiaca fruit and seed extracts in diabetic rats resulted in a highly significant (p < 0.01; LSD) amelioration in LPO; the effects of the fruit extract were the most potent.
The GSH content as well as the GPx, GST, and SOD activities showed a highly significant (p < 0.01; LSD) decline in diabetic control rats compared with those in the normal rats. The treatments with the B. aegyptiaca fruit and seed extracts successfully improved the GSH content and GPx, GST (p < 0.01; LSD), and SOD activities (p < 0.05; LSD) ( Table 7). While the effects of the fruit and seed extracts on GSH content and anti-oxidant enzyme activities were more or less similar, the seed extract was more potent in decreasing the elevated LPO. The F-probability indicated that the effect on the liver LPO, GSH content, and GPx, GST, and SOD activities between groups was very highly significant (p < 0.01; F-probability)

Histological Changes in the Pancreas
The histopathological examination of the control pancreas sections showed closely packed lobules of the pancreatic acini. The islet of Langerhans is composed of numerous compactly arranged cells (alpha, beta, and delta cells) occurring as dense cords ( Figure 5; Photomicrographs A and B). The diabetic control revealed the histopathological changes of endocrine portions represented by a marked decrease in the size of the islets of pancreas (pancreatic shrinkage) and decreased number of the cells.  The islets also exhibited cytoplasmic vacuolations (v) and necrosis (nc) (Figure 5; Photomicrographs C and D). The islets of Langerhans exhibited nearly normal and organized architecture, and the necrotic and degenerative changes were markedly improved in rats treated with the B. aegyptiaca fruit ( Figure 5; Photomicrographs E and F) and seed extracts ( Figure 5; Photomicrographs G and H) compared with those in the diabetic control rats; the seed extract was found to be the most potent.

Discussion
DM is a group of metabolic diseases characterized by chronic hyperglycemia due to defects in insulin secretion, insulin action, or both [47]. STZ is known for its selective pancreatic islet β-cell cytotoxicity and has been frequently used to induce DM in animals. NA/STZ-induced DM is selected to be a model of type 2 DM (T2DM) to assess the effects of the B. aegyptiaca fruit and seed aqueous extracts. In this model, when NA is injected prior to the administration of STZ, the severity of DM will be reduced to a certain extent, leading to a T2DM-like condition with deteriorated IS [20,35,[48][49][50].
Several traditional medicines have been discovered for DM. Isolated substances and extracts isolated from various natural resources particularly plants have always been a rich arsenal for the control and treatment of DM problems and complications [9]. Plants have rich sources of antidiabetic as well as anti-hyperlipidemic and anti-oxidant substances, such as flavonoids, amino acids, gallotannins, and many other related polyphenols [51]. B. aegyptiaca is a plant widely used as a hypoglycemic agent in Egyptian folkloric medicine [15]. However, only few studies have investigated the antidiabetic effects and the mechanisms of action of B. aegyptiaca, especially its seeds.
Many of the constituting ingredients of the B. aegyptiaca fruit and seed aqueous extracts exhibit many biological activities. Of these, 9,12-octadecadienoic acid (Z,Z)have anti-inflammatory, hepatoprotective, cancer preventive, and hypocholesterolemic effects [20,52,53]. The derivative 9-octadecenoic acid was reported to have antitumor and anti-inflammatory activities [53]. Isothiocyanates have been demonstrated to have both anti-inflammatory and antioxidant activities [54].
OGTT is a well-known and common assay used to determine the anti-hyperglycemic activity of antidiabetic agents [55]. OGTT is considered the gold standard test for the diagnosis of DM by the World Health Organization [56]. The data in the present study revealed a marked increase in the serum glucose levels of the diabetic groups compared with those of the normal rats. These findings are consistent with those of Akhani et al. [57], Ahmed [58], Schaalan et al. [59], Ahmed et al. [60], Ahmed et al. [61], and Ali et al. [62].
The increase in glucose level may be due to the decreased glucose consumption in the peripheral, muscle, and adipose tissues [63] and increased glycogen breakdown [64], gluconeogenesis, and production of hepatic glucose [65]. Furthermore, Powers confirmed that IR in T2DM induces an increase in blood glucose because of the same causes [66]. In the present study, the treatment of diabetic rats with the B. aegyptiaca fruit or seed extracts caused a potential improvement in OGT. The decrease in the elevated serum glucose levels is in accordance with the results of Zaahkouk et al. [67], Helal et al. [68], and Al-Malki et al. [69] who verified the anti-hyperglycemic effects of the B. aegyptiaca fruit.
The ameliorative effects of the B. aegyptiaca extracts may be associated with insulinomimetic activities [70], stimulation and potentiation of insulin secretion, increased affinity of insulin receptors [71], improved concentration of hepatic glycogen, accelerated glucose metabolism, reduced production of intestinal glucosidase, and decreased gluconeogenesis of the liver [15]. These actions may be attributed to the active constituting ingredients found in the B. aegyptiaca fruit and seed extracts. In this regard, a previous publication found that B. aegyptiaca may contain interketones, organic constituents, rutin, and oils (fatty acids and volatile oils) present in the internal kernel according to the phytochemical investigation [72].
Furthermore, Baragob et al. attributed the hypoglycemic effects of the aqueous extract to its constituents, such as saponins, rutin, and organic constituents [73]. In the present study, the GC-MS analysis indicated the presence of many organic ingredients, which have several biological activities, including antidiabetic potencies.
In the present study, the NA/STZ-induced diabetic rats exhibited a significant decrease in serum insulin and C-peptide levels. It is important to note the relationship between decreased insulin and C-peptide levels in diabetic rats and decreased size and number of the islets of Langerhans that have a decreased number of β-cells, necrosis, and vacuolations. These decreases are also correlated with the calculated HOMA-β cell function.
The administration of the B. aegyptiaca fruit and seed aqueous extracts produced a significant increase in serum insulin and C-peptide levels of diabetic rats, and this finding is consistent with that of Abou Khalil et al. [74], El-Bayomy et al. [75], and Hassan [76]. In this regard, Abdel-Moneim [70] hypothesized that the hypoglycemic action of the B. aegyptiaca aqueous extract stimulated the β-cells of the pancreatic islets to secrete insulin, potentiate glucose-stimulated insulin secretion, and increase the number and sensitivity of insulin receptors and post-receptor effects in peripheral tissues.
Furthermore, the B. aegyptiaca seeds contain diosgenin [77], which may be useful in ameliorating the glucose metabolic disorder related with DM and obesity as reported by Ulbricht et al. [78]. C-peptide is formed during insulin biosynthesis, and the two peptides, insulin and C-peptide, are then released to the circulation in equal amounts [79]. An increase in C-peptide levels in diabetic rats treated with B. aegyptiaca corresponds well with the increase in insulin secretion (endogenous secretion), which is possibly due to the regeneration of the β-cells of the islets of Langerhans.
This association is demonstrated in the present study by the significant increase in HOMA-β cell function and marked improvement in the histological architecture and number of β cells of the pancreatic islets as a result of the treatment with the B. aegyptiaca fruit and seed extracts.
The level of liver glycogen may be regarded as the best marker for evaluating the anti-hyperglycemic activity of any drug [80]. The present study showed that the diabetic rats exhibited a significant depletion of liver glycogen content correlated with a marked increase of glucose-6-phosphatase and glycogen phosphorylase activities. These results are consistent with those of Sundaram et al. [81] and Mahmoud et al. [82]. In the present study, the treatment with the B. aegyptiaca fruit and seed extracts significantly improved the lowered liver glycogen content and the elevated hepatic glucose-6-phosphatase and glycogen phosphorylase activities.
These ameliorations may be secondary to the increase in the insulin levels in the blood and the enhanced IS. These results are consistent with those of Helal et al. [68] who found that the regeneration of β-cells led to an increase in the insulin level and improvement in IS, both of which can lower the glucose levels in the blood. The present results support this finding as the treatment of diabetic rats with the B. aegyptiaca fruit and seed extracts significantly increased the HOMA-β cell function and HOMA-IS but significantly decreased HOMA-IR ( Figure 6).
Hypoinsulinemia and IR clearly shown in untreated diabetic rats are considered the main cause of the recorded dyslipidemia represented by hypertriglyceridemia and hypercholesterolemia associated with the increased production of vLDL cholesterol and LDL cholesterol and decreased HDL cholesterol level. These findings are consistent with those of Abdel-Moneim et al. [83] who reported a marked increase in the levels of serum triglycerides, cholesterol, and LDL cholesterol of diabetic rats. This increase may be due to a decrease in lipoprotein lipase function caused by insulin deficiency [84].
In addition, Goodman and Gilman [85] reached the same results, which were explained by inhibition of lipoprotein lipase transcription inside the capillary endothelium as a result of insulin deficiency. The data of the present study are consistent with those of Harvey and Ferrier [86] who reported that the metabolic abnormalities of T2DM as a result of IR lead to dyslipidemia in the liver where fatty acids are converted into triacylglycerol, which, in turn, are packaged and secreted in vLDL. Both accumulations of lipids, especially triglycerides, and reduced anti-oxidant activity contribute to the development of oxidative stress in diabetic rats [87].
ti-oxidant defense system may have an important role in the improvement of the architecture and tissue IS of the pancreatic islets, which in turn result in the effective management of diabetes. These findings are consistent with those of Hassanin et al. [111] who indicated that B. aegyptiaca exerted hypoglycemic, hypolipidemic, and insulinotropic actions associated with the reduction in oxidative stress, enhancement in the anti-oxidant defense system, and reduced apoptosis in pancreatic β-cells.
In conclusion, the B. aegyptiaca fruit and seed aqueous extracts have potent antidiabetic potencies, which may be mediated via improvements in the insulin secretory response, β-cell function, tissue IS, and anti-oxidant defense system ( Figure 6).  The results of the present study are consistent with those of previous studies [88][89][90], which revealed a reduction in the HDL cholesterol level in the diabetic control group. The present results of the serum lipid profile are consistent with the findings of Stanfield [91] who stated that DM increases the number of LDL particles that transport lipids, including cholesterol to peripheral tissues, and decreases the number of HDL particles, which transport lipids and cholesterol to the liver.
Simultaneously, the elevated serum triglyceride level in the diabetic group of the present study may be related to the decreased clearance and increased production of endogenously synthesized main triglyceride transporters [92]. Moreover, the expansion of the cholesterol pool in DM could be explained by the increased input into the system by accelerating the synthesis of intestinal cholesterol or by increasing the rate of absorption of intestinal cholesterol [93].
Indeed, the improvement in dyslipidemia through B. aegyptiaca treatment may be related to the increased level and sensitivity of insulin. The present results are not in concordance with those of Matter and Helal [94] who reported that the level of serum triglycerides and cholesterol was not significantly different when compared with the control group after treatment with the B. aegyptiaca seed extract.
In accordance with the findings of Abd El-Rahman and Al-ahmari [95], the improvement of lipid profile may be due to the presence of saponins in its extract, indicating antihypercholesterolemic and hypoglycemic activities. Moreover, diosgenin in the B. aegyptiaca seed kernels plays an important role in the regulation of cholesterol metabolism [96]. In the present study, the GC-MS analysis indicated the presence of many organic ingredients, which have several biological activities, including antihypercholesterolemic properties.
The elevated serum FFA level recorded in diabetic rats in the present study is consistent with that estimated in many preceding studies [35,62,97,98]. The elevated release of FFAs from the adipose tissue can be attributed to the lipolysis of visceral adipose depots; this effect can result in IR, excessive endogenous glucose formation, and progression to T2DM [99]. Thus, decreasing the plasma FFA level is recommended as a method for IR prevention and treatment [100].
During the treatment of diabetic animals with B. aegyptiaca aqueous extracts, a reduction in the amount of serum FFA levels that could be associated with the insulin-sensitizing activity of the extract was observed [101]. Furthermore, several studies have found significantly low levels of resistin mRNA in the adipose tissue in various obese mouse models, such as db/db or high-fat diet-induced obesity, as well as in rat models with IR [102].
The present study showed a significant reduction in the mRNA expression of insulin receptor β-subunit in the adipose tissues of the diabetic group in comparison with that of the normal group. This result is consistent with that of Ali et al. [62] and Abdel Aziz et al. [51] who demonstrated that the mRNA expression of insulin receptor β-subunit was significantly decreased in NA/STZ-induced diabetic rats. This effect provides evidence of the presence of IR and impaired IS in such animal models, which in turn is a suitable model of T2DM. In the present study, the treatment with the B. aegyptiaca fruit aqueous extract produced a significant increase in the mRNA expression of insulin receptor β-subunit reflecting the ability of this extract to reduce IR and enhance IS in the adipose tissues ( Figure 6).
Oxidative stress is an important factor in DM etiology and pathogenesis, causing interactions with polyunsaturated fatty acids that contribute to LPO [103]. According to Randle's theory on glucose-fatty acids [104], the excessive release of free fatty acids from the adipose tissue for oxidation induces the production of metabolites that prevent tissue use of glucose. Such metabolites are reactive oxygen species and hydrogen peroxide, which are involved with the glucose-fatty acid process [105].
Belfort et al. [106] showed that the increase in plasma FFA caused a dose-dependent inhibition of insulin-stimulated glucose disposal and insulin signaling in the skeletal muscle of lean healthy individuals. The present findings showed a significant increase in LPO in the liver. Additionally, the GSH level and anti-oxidant enzyme defenses decreased simultaneously in the liver of diabetic rats. These findings are consistent with those of several studies [35,89,89,107,108]. GSH plays a multifaceted role in the defense against anti-oxidants. It actively scavenges free radicals or indirectly detoxifies reactive species via GST and GPx [109].
The treatment of diabetic rats with B. aegyptiaca extracts significantly decreased MDA, which is attributed to the increased levels of anti-oxidants that fight free radicals [110] and markedly increased GSH level and SOD and GPx activities. Thus, it is worth noting that the improvement in the glycemic state, lipid profile, and insulinotropic and insulinsensitizing effects is associated with the suppression of oxidative stress and enhancement of the anti-oxidant defense system. This indicated that the decrease in oxidative stress and enhancement of the antioxidant defense system may have an important role in the improvement of the architecture and tissue IS of the pancreatic islets, which in turn result in the effective management of diabetes. These findings are consistent with those of Hassanin et al. [111] who indicated that B. aegyptiaca exerted hypoglycemic, hypolipidemic, and insulinotropic actions associated with the reduction in oxidative stress, enhancement in the anti-oxidant defense system, and reduced apoptosis in pancreatic β-cells.
In conclusion, the B. aegyptiaca fruit and seed aqueous extracts have potent antidiabetic potencies, which may be mediated via improvements in the insulin secretory response, β-cell function, tissue IS, and anti-oxidant defense system ( Figure 6).