Specimens Aloe vera (L.) plant, commonly known as Aloe vera, was cultivated and purchased in June, 2024 from a garden in Taif, Kingdom of Saudi Arabia. In this investigation, fresh leaves from this cultivated plant were used. The Aloe vera powder was prepared according to the protocol mentioned by Can et al. [9]. In brief, approximately 70 cm length of mature, healthy and fresh green leaves were cut transversely into several parts and then washed thoroughly with clean tap water and air-dried after the thick epidermis removal. The fleshy, colorless, semi-solid mucilaginous gel in the center of the leaves was extracted with the help of a sterilized knife and cut into small parts. The homogenate, which was thick and straw in color, was lyophilized, which was extracted with ethanol (95%) and water, filtrated, collected and freeze-dried by (ALPHA 1-4 LSC, Germany) to dryness. A non-metallic electric grinder (JH-E01, China) was used to crush the dried gel into powder and it was sieved through 300 µm. The gel powder was stored in dry sterilized vacuumed tubes and kept at 4 °C until further use.

Approximately 3 month old male albino 150–200 g Wistar strain rats were employed. Rats were obtained from the Animal House in Jeddah, Kingdom of Saudi Arabia and selected as the animal models. Rats were maintained under the optimal laboratory conditions at 25 °C, 12 hrs light/dark cycle and had unlimited access to rat chow and tap water. The Gulf Countries Association of Sciences in Animal Experimentation granted Ethics Committee Number (8901/3151) approval for the 30 days animal study. Streptozotocin (STZ) a glucose analogue (2-deoxy-2-(3-methyl-3-nitrosouredio)-D-glucopyranose), sitagliptin and glibenclamide was obtained from Sigma, USA. All other chemicals, reagents and kits used were of analytical grade.

Fig. 1 shows the design of the protocol and the treatment schedule. After acclimatization for a week for any observation of untoward symptoms, the rat models were randomly distributed into six main groups of ten rats each in separate cages as follows:

• Vehicle group: Healthy rat models.

• Diabetic group: Diabetic rat models Type 1 (induced with 30 mg/kg of STZ).

• Normal+Aloe vera gel (AG) group: Healthy rat models were given Aloe vera gel extract (300 mg/kg) daily by using gavage needles for 21 days.

• STZ+AG group: Diabetic rat models were given Aloe vera gel extract (300 mg/kg b.wt.) daily by using gavage needles for 21 days.

• STZ+glibenclamide (GLIB) group: Diabetic rat models were given glibenclamide (600 µg/kg) daily by using gavage needles for 21 days.

• STZ+sitagliptin (SIAG) group: Diabetic rat models were given sitagliptin (10 mg/kg) daily by using gavage needles for 21 days.

Fig. 1.

Design of the protocol and the treatment schedule. AG, Aloe vera gel; STZ, streptozotocin; GLIB, glibenclamide; SIAG, sitagliptin.

After the first seven days of the examination of rat models, the weight of rats was recorded after overnight fasting. Consequently, approximately 30 mg/kg of STZ in citrate buffer (0.1 M) with acidity (pH 4.5) was managed by a single intraperitoneal injection. The vehicle group of healthy rat models received an equal intraperitoneal dose of citrate buffer [19]. Blood glucose was assessed following a 16 hrs fast with free access to tap water. Overnight, the rats were given a 5% glucose solution to help them overcome the hypoglycemia caused by the medication. A glucometer (Bionime®, Switzerland) was used to measure fasting blood glucose levels and confirm the hyperglycemia before and 24 hrs following injection for diabetes approval and 7 days following injection for diabetes consolidation from the tails of the rat models. Fasting blood glucose levels were detected at weekly intervals on the 7th and 14th day with the tail prick assay. The body weight of each rat was then recorded every on the (1st, 7th, 14th and 21st) day by an electronic balance. The current study included STZ-treated animals that were diagnosed with diabetes if their fasting plasma levels were greater than 200 mg/dL with glucosuria. In order to prepare the Aloe vera gel extract, 300 mg of powdered Aloe vera gel was combined with 5 mL of water to create a solution. After the standardizing of the STZ dose, the experiments were conducted on animal groups to see the effect of natural Aloe vera gel and other diabetes medications, such as sitagliptin and glibenclamide, on rat models. Rat models in all groups were treated orally by using gavage needles every day in a single dose for 21 days.

After a continuous three-week treatment period, the animals were fasted overnight to reduce experimental variability and normalize any possible changes in feeding behavior. The purpose of this fasting period was to improve the consistency of experimental outcomes by preventing recent food intake from influencing metabolic and physiological parameters. Necropsy was carried out right after euthanasia of thiopental sodium (40 mg/kg) in order to gather the organs and tissues required for further examination [20].

Blood samples were drawn from the tail vein following 24 hrs fasting with unrestricted access to water. A sharp, sterilized blade was used to cut the tail tip in a single stroke. Heparinized tubes were used to collect blood, the plasma was separated from the blood to detect the essential measurements by centrifuging it for 15 min at 2500 rpm. Approximately, 40 µL was taken to measure glucose concentration according to the glucose oxidase assay (Reflotron 1, Boehringer, Germany). While hemoglobin and glycosylated hemoglobin were detected [21]. Homeostatic Model Assessment of Insulin Resistance (HOMA IR and $\beta$ cell) functions were detected [22]. The Dipeptidyl Peptidase-4 (DPP-4) enzyme activity was evaluated against the pNA standard graph after the incubation for 30 min at 37 °C to release the p-Nitroaniline. DPP IV enzyme activity was calculated as (U/min/mL) after absorbance at 405 nm by using a thermo scientific microplate (357) [23].

The fat profile of blood, such as triglycerides (TG) and total cholesterol, was determined by the enzymatic colorimetric assays. The High-Density Lipoprotein (HDL) and Very Low-Density Lipoprotein (VLDL) of cholesterols were detected [24].

The biochemical and antioxidant enzyme evaluations for plasma and liver samples were detected [20]. Kits for blood and liver markers were purchased from Sigma, Darmstadt, Germany. Alkaline phosphatase (ALP) and Alanine Transaminase (ALT) were expressed as (U/L); Glutathione (GSH) and malondialdehyde (MDA) were expressed as (nmol/mg); Thiobarbituric Acid Reactive Substances (TBARS) and hydroperoxides were expressed as (mM/100 g); while antioxidant enzyme activities as glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) were expressed as (U/mg).

ANOVA test was used as the primary statistical analysis to compare differences between groups, followed by post-hoc test (Bonferroni) to identify specific group differences, with significance set at p 0.05. The SPSS program version 26.0 (IBM Corp., Armonk, NY, USA) was applied for the statistical analysis. The results for all rat groups were presented as Mean $\pm$ SD.

Body weights were severely decreased in the diabetic group and STZ+SIAG group compared to the vehicle group on the 21st day (149.48, 161.84 and 179.89 g), respectively. Increases in body weights were improved by STZ+AG group or STZ+GLIB group treatments (189.33 g and 195.22 g) (Fig. 2).

Fig. 2.

Effects of various medications on body weights in rat models. n = 60; each group n = 10.

Fasting blood glucose levels were increased during the 21 days of post-diabetic inductions after the injection of the STZ dose. The level of the STZ+AG group consequently dropped down to reach 128.14 mg/dL on the 7th day, which further decreased to normal levels on the 14th and 21st day. Treatments of the STZ+AG group caused a remarkable reduction level of 100.01 mg/dL compared to the diabetic group 277.13 mg/dL (Fig. 3). On the other hand, the other two diabetes medications sitagliptin and glibenclamide as did not show the ability to decrease the fasting blood glucose levels 201.42 and 115.14 mg/dL after 3 weeks compared with the vehicle group, respectively.

Fig. 3.

Effects of various medications on fasting blood glucose levels in rat models. n = 60; each group n = 10.

Hemoglobin levels rise in a glucose level-dependent manner in response to the severity of hyperglycemia. After 21 days, the administration of natural Aloe vera gel and other diabetes medications such as sitagliptin and glibenclamide (STZ+SIAG group) caused a reduction in hemoglobin level (12.04 g/dL), while STZ+AG group and STZ+GLIB group detected the same value and tended to reach the vehicle group (12.3 g/dL) compared to the diabetic group (11.25 g/dL) (Fig. 4). On the same trend, STZ+AG group and STZ+GLIB group detected the same value of glycosylated hemoglobin and tended to reach the vehicle group (7.05% Hb) compared to the diabetic group (12.41% Hb), while STZ+SIAG group had higher values (8.14% Hb) at the end of the experimental period (Table 1). The DPP IV enzyme activity demonstrated an increase in diabetic rat models from 4.85 U/min/mL in the STZ+SIAG group to 10.11 U/min/mL in the diabetic group compared to the vehicle group recorded 2.56 U/min/mL. While administration of glibenclamide decreased the DPP IV enzyme activity to reach 5.01 U/min/mL followed by the STZ+AG group (6.23 U/min/mL). The increases in (HOMA IR and $\beta$ cell) functions ranged as (74.44–1010.02) and (58.76–955.32) in both vehicle and diabetic groups, respectively. STZ+AG group detected a higher HOMA IR value (49.03) followed by the STZ+SIAG group which detected (48.46). On the same trend, the STZ+AG group detected a higher $\beta$ cell value (600.07) followed by the STZ+SIAG group which detected (488.76) (Table 1). The STZ+GLIB group reported the lowest values for HOMA IR and $\beta$ cell (46.73 and 474.02), respectively. Moreover, a clear reduction in HOMA $\beta$ cells was detected in the normal+AG group to reach (889.08).

Fig. 4.

Effects of various medications on blood analysis in rat models. Different letters mean significant differences among rat models group. Dipeptidyl Peptidase-4 (DDP IV), n = 60; each group n = 10.

The fat profile values among all rat models are presented in Table 2. An increase in TG and VLDL values in the diabetic group (802.00–115.41 mg/dL) compared with that in the vehicle group (568.32–82.34 mg/dL), respectively. The STZ+AG group maintained TG and VLDL values (420.51–59.12 mg/dL) than the STZ+GLIB group (289.40–50.02 mg/dL) and STZ+SIAG group (245.42–56.32 mg/dL), respectively. The STZ+AG group preserved HDL value to reach 27.03 mg/dL compared with the vehicle group’s 28.14 mg/dL. The STZ+SIAG group normal+AG group reported higher values of total cholesterol at 83.30 mg/dL followed by the normal+AG group, which reached 81.04 mg/dL. In the current work, supplementation of Aloe vera decreased the TG and VLDL levels; however, no obvious changes occurred in total cholesterol and HDL levels, indicating that lipid metabolism may be improved by gel. The highest values for TG and VLDL were noticed in the normal+AG group, which fed on Aloe vera only at 510.75 and 78.03 mg/dL, respectively (Table 2).

Some biochemical parameters for the liver functions were determined such as ALP and ALT activities (Fig. 5). The ALP activity was increased in the diabetic group, compared to the vehicle group. The ALT activity was reduced in the diabetic rat models given Aloe vera gel to reach 59.10 U/L. It was noticed that the STZ+AG group got protective effects comparable to STZ+GLIB and STZ+SIAG groups against hepatotoxicity. Fig. 6 presents liver tissue GSH and MDA values in the liver for all rat models. There was a great reduction in GSH values among diabetes rats compared to the vehicle group. The Treatments with Aloe vera and glibenclamide medication raised the GSH values to reach 32.57 and 29.03 nmol/mg, respectively. As shown in Fig. 6, the diabetic group detected an increase in MDA level 13.23 nmol/mg compared to the vehicle group 7.16 nmol/mg. It is known that higher levels of MDA are an indicator of increased oxidative stress. The administration of natural Aloe vera gel, sitagliptin and glibenclamide as diabetes medications to the diabetic rat models resulted in a great reduction of MDA levels as 9.11, 10.12 and 11.44 nmol/mg, respectively. Fig. 7a,b presents TBARS and hydroperoxides values in the liver of rat models. There were great elevations in tissue TBARS and hydroperoxides for diabetic rats (1.82 and 95.47 mM/100 g) compared to the vehicle group (0.90 and 70.05 mM/100 g), respectively. The administration of natural Aloe vera gel, glibenclamide and sitagliptin as diabetes medications to the diabetic rat models resulted in great reductions as 0.95, 1.51, 1.49, 75.21, 82.04 and 87.92 mM/100 g, respectively.

Fig. 5.

Effects of various medications on liver tissue as Alkaline phosphatase (ALP) and Alanine Transaminase (ALT) activities in rat models. Different letters mean significant differences among rat models group, n = 60; each group n = 10.

Fig. 6.

Effects of various medications on liver tissue as Glutathione (GSH) and malondialdehyde (MDA) in rat models. Different letters mean significant differences among rat models group, n = 60; each group n = 10.

Fig. 7.

Effects of various medications on liver tissue in rat models. (a) Thiobarbituric Acid Reactive Substances (TBARS) and (b) Hydroperoxides, n = 60; each group n = 10.

Table 3 presents the enzyme activities of GPx, SOD, CAT and GST in the liver tissues of rat models. The GPx activity recorded a considerable reduction from 1.74 U/mg in the diabetic group to 1.01, 1.17 and 1.20 U/mg for STZ+AG, STZ+GLIB and STZ+SIAG groups, respectively. The effect of Aloe vera gel was more pronounced which reached 6.17, 0.67 and 4.04 U/mg compared with the diabetic group that recorded (3.88, 0.48 and 1.23 U/mg) for SOD, CAT and GST activities, respectively.