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. 2025 Jul 4;15(8):239. doi: 10.1007/s13205-025-04403-5

Effects of a green method olive leaf extract on the metabolic disorders and the pancreatic immunohistopathology in diabetic Wistar rats

Hedya Jemai 1,✉, Salah Ibidhi 1, Oumayma Labidi 2, Zouhaier Bouallagui 3, Sameh Hadj Taieb 4, Imen Hammami 5, Roua Ben Dassi 1, Ouajdi Souilem 2, Moncef Feki 4, Ameur Cherif 1, Rim Driouich Chaouachi 1
PMCID: PMC12227409  PMID: 40621578

Abstract

The present study evaluated the antidiabetic potential of a green, oleuropein-rich extract from olive leaves in alloxan-induced diabetic Wistar rats. Diabetes was induced by a single intraperitoneal injection of alloxan (180 mg/kg b.w.), followed by oral administration of the extract for 30 days. Treatment significantly decreased blood glucose levels from 26 mmol/L in diabetic controls to 5 mmol/L and increased plasma insulin concentrations from 4.65 µU/mL to 13.25 µU/mL. Improvements in body weight, antioxidant defense, and lipid and liver enzyme profiles were also recorded. Histological examination revealed structural restoration of pancreatic tissue, while immunohistochemical analysis indicated enhanced expression of the anti-apoptotic protein Bcl-2, suggesting a protective and regenerative effect on pancreatic β-cells. The solvent-free extraction method preserved the bioactivity of the compound and supports its safe use for therapeutic purposes. Collectively, these findings highlight the efficacy of the oleuropein-rich extract in restoring glycemic balance and pancreatic function, suggesting its promise as a natural and sustainable option for diabetes management.

Keywords: Antioxidant, Diabetes, Green extraction, Oleuropein, Olive leaf

Introduction

Diabetes is a major health issue that affects a large number of people worldwide (Saeedi et al. 2019). Such metabolic alteration is in continuous and alarming rise with a prediction of 700 million affected person by 2045 (WHO 2021). Diabetes is characterized by an elevated level of glucose in the blood and is associated with a range of serious health problems, including heart disease, stroke, kidney failure, and nerve damage (American Diabetes Association 2021). Furthermore, as the number of people affected by diabetes continues to grow, there is an increasing need for safe and effective treatments to help manage the disease and improve the quality of life such as natural compounds (Jemai et al. 2009; Emiru et al. 2020). Besides, diabetes is generally accompanied by an increase in the production of free radicals or altered antioxidant defenses, which causes an oxidative stress state that largely participates in the development and progression of diabetes and its complications (Tiwari et al. 2013; Daher et al. 2019).

One promising natural treatment for diabetes could be oleuropein, which is the major polyphenol found in olive leaves (Ghanbari et al. 2012; Souilem et al. 2017). In fact, oleuropein is a phenylethanoid, a type of phenolic compound found in olive leaves and olive oil. It has been shown to have various health benefits, including antioxidant, anti-inflammatory, cardiovascular protective effects and blood sugar-lowering properties (Jemai et al. 2008, 2009; Ghanbari et al. 2012), making it a potential candidate for the treatment of the metabolic disorders like diabetes. Subsequently, in order to study the effects of oleuropein on diabetic rats, researchers have used extracted oleuropein, which allows the administration of a standardized dose.

Nowadays, the pursuit of sustainable biochemical processes of extraction is driving the development of innovative, environmentally friendly solvents and methods. Recently, a new category known as natural deep eutectic solvents has emerged as a promising green alternative to conventional organic solvents (Ben Dassi 2024). Moreover, some methods have been developed that eliminate the use of solvents by leveraging the chemical specificities and affinities of the desired compounds (Bouallagui et al. 2019).

This type of extraction was adopted as a strategy to facilitate the use of the obtained extract for therapeutic purposes. The method used is considered safe, as it does not involve chemical solvents that could potentially alter the properties of the extracted compound. Moreover, the water-based extraction process allows the preservation of the bioactive compounds, including their antioxidant and anti-inflammatory properties (Ghanbari et al. 2012). Furthermore, this approach also presents a more sustainable option compared to traditional solvent-based extraction methods. Ensuring the use of a standardized extraction method is crucial for obtaining consistent results and enabling comparisons across studies. While numerous studies have explored polyphenols using chemical and enzymatic methods, this study aimed to validate the therapeutic benefits of polyphenols, particularly oleuropein, for diabetes treatment by employing a green extraction method. This approach not only ensures the safety but also facilitates any potential use in preclinical assays.

Material and methods

Plant material

Olive leaves from Chemlali cultivar were collected from southern Tunisia in September 2022. Afterward, leaves were washed then dried in a microwave oven for 6 min at 600 watts and then stored at −80 °C.

Extraction of phenolic compounds from olive leaves

A simple and eco-friendly extraction process was employed to extract phenolic compounds from olive leaves. Initially, the leaves were infused in hot water at 90 °C for 10 min, with periodic stirring, following the method of Bouallagui et al. (2019). The solution was gauze filtered, then centrifuged at 8000 rpm for 10 min, and the supernatant was recovered and stored at −80 °C for later analysis of its chemical composition and biological activities.

High-performance liquid chromatography with diode array detector HPLC–DAD

High-performance liquid chromatography (HPLC) analyses were performed using an HP 1100 Series instrument from Agilent Technologies (Palo Alto, CA, USA). The system is equipped with a UV diode array detector (DAD). The samples were run on a Zorbax C18 column (250 mm X 4.6 mm X 3.5 mm) thermostated at 40 °C. The mobile phase consists of acetonitrile (solvent A) and water with 0.1% acetic acid (solvent B), at a flows of 0.5 mL/min. The gradient was as follows: 10% A at 0–10 min, 40% A at 10–18 min, 80% A at 18–22 min, 100% A at 22–27 min, and 10% A at 27–30 min. The injection volume was 10 μL. Oleuropein was identified and quantified by its retention time and UV spectrum compared to the reference compound (purity ≥ 98.0%, Extrasynthese, France).

Experimental design

Animals

This study was conducted on 24 male Wistar albino rats, aged 7 weeks and weighing between 104 to 139 g and 200 g at the beginning of the experiment. These animals were obtained from the Pasteur Institute of Tunis and were raised in the animal facility at the National School of Veterinary Medicine (ENMV) in Sidi Thabet under standard environmental conditions. The animals were kept in a breeding room with a controlled environment (temperature of 20 ± 2 °C, humidity of 40 (± 5%), 12 h of darkness/12 h of light). Rats were divided into 4 groups of 6 each.

Animal welfare and ethic statement

This study was conducted in compliance with the ethical standards and guidelines for animal welfare. The protocol was approved by the National Ethical Committee on Medical and Animal Research of the National School of Veterinary Medicine in Tunisia (Approval Code: CEEA-ENMV 61/23), and all procedures followed the “Guide for the Care and Use of Laboratory Animals” established by the US National Institutes of Health. Every effort was made to ensure the well-being of the animals and minimize their suffering, as well as to minimize the number of animals used in this study.

Animal treatment

Two groups were injected with a single intraperitoneal dose of alloxan at 180 mg/kg body weight. The experiment was conducted for 4 weeks starting 5 days following the induction of diabetes. The animals were allocated as follows:

  • C: Control rats (n = 6)

  • D: Diabetic rats (n = 6)

  • D + OL: Diabetic rats treated with phenolic extract based on oleuropein (n = 6)

  • C + OL: Control rats supplemented with phenolic extract based on oleuropein (n = 6)

The extract was diluted in drinking water and the dose was referred to its oleuropein content leading to 16 mg/kg b.w. At the end of the experiment, the rats were sacrificed and blood samples were collected to determine plasma lipid profile, while pancreas and liver samples were removed and stored at −80 °C for further analyses.

Weight and blood glucose monitoring

Glycemia and body weight were measured weekly. Blood samples were collected by retro-orbital sinus puncture with a capillary tube and glycemia was estimated using an Accu-Chek Blood 210 Glucose Monitor (Roche, Manheim, Germany), by placing a drop of blood on a test strip. The animals were considered diabetic when their blood glucose level was greater than 200 mg/dL (Prashant et al. 2005).

Biochemicals parameters, hepatic enzymes, and insulin measurements

At the end of the experimentation, the animals were euthanized through decapitation under anesthesia. Fresh blood was immediately collected into heparinized tubes (BD Vacutainer System) and subsequently centrifuged at 2200 × g for 15 min to separate red blood cells from plasma. The pancreas was excised delicately, weighed, rinsed with saline solution, and then fixed in a 10% buffered formalin solution. Both pancreas and serum samples were stored at −80 °C for subsequent analyses.

The enzymatic colorimetric method was used to assess the serum concentrations of total cholesterol (TC), total triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) using the reagent kits on an Architect C8000 analyzer (Abbott Laboratories, Abbott Park, IL, USA) in Rabta Hospital. Additionally, the atherogenic index (AtI) was calculated for each group using the Friedewald equation (Martins et al. 2023). Moreover, plasma insulin was estimated by using a radioimmunoassay kit (Linco Research Inc., USA).

Histology and immunohistology

At the time of sacrifice, pancreas samples were excised, fixed in Bouin solution for 24 h, and subsequently embedded in paraffin. The paraffin-embedded tissues were then sectioned at a thickness of 5 μm and stained with hematoxylin and eosin. The resulting sections were examined under a light microscope to identify any potential histopathological changes. For immunohistochemical staining, we used pancreatic tissue sections obtained from paraffin blocks. These sections were deparaffinized through different baths: three baths of 5 min each in xylene, two times 5 min in 100% ethanol, 3 min in 95% ethanol, and then 3 min in 70% ethanol. The slides were then rinsed twice for 3 min in running water and twice for 3 min in PBS.

Endogenous peroxidases were then inhibited by a 20 min bath in PBS-H2O2 (75ml PBS + 1125µL 30% H2O2). After two washes of 3 min in PBS, pancreatic sections were circled with Dakopen. The slides were then saturated for 15 min in BSA. 300µL of primary antibody diluted in 5% BSA was applied, and the slides were placed in a humid chamber overnight at 4 °C. The primary antibodies included a rabbit polyclonal antibody against Bcl-2. Thereafter, the reaction was detected with a streptavidin peroxidase Histostain SP kit. After washing twice for 3 min in PBS, the slides were incubated with the secondary antibody for 45 min at room temperature. The slides were again washed twice for 3 min in PBS and once for 3 min in PBS-BSA. Then, they were revealed by a revelation solution (250 µl AEC, 1 µL 30% H2O2, 4750 µL 0.1 M acetate buffer; pH = 4.9). Counterstaining with hematoxylin (4 s) was performed after rinsing with running water. The slides were stored at 4 °C before observation under an optical microscope (Olympus Corporation, Tokyo, Japan). As a result, brown-yellow spots indicated a positive staining that was quantified using the ImageJ software.

Statistics analysis

GraphPad Prism 6 for Windows was used to analyze the results (mean ± SD). The significance of differences was determined by one-way ANOVA and Tukey’s multiple comparison post hoc test. Statistical significance was determined at a p-value below 0.05.

Results and discussion

Characterization of the phenolic extract

Through the use of spectroscopic analyses with HPLC, major phenolic compounds present in the extract of olive leaves were identified. Specifically, the analysis revealed the presence of some molecules, mainly, oleuropein which was the major compound detected at 254 nm (Fig. 1). Besides, apigenin-7-O-glucoside, luteolin-7-glucoside, and verbascoside are present in minor concentration in concordance with the results of Bouallagui et al. 2019.

Fig. 1.

Fig. 1

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HPLC–DAD profile of the polyphenol-rich extract of Chemlali olive leaves at 254 nm

In fact, oleuropein contains hydroxyl (-OH) functional groups, making it highly polar (Pérez-Bonilla et al. 2013). This polarity allows it to interact with other polar molecules and can influence their bioactivity and properties in biological systems. Moreover, apigenin-7-O-glucoside, luteolin-7-glucoside, and verbascoside are also polar compounds which explain their presence in the infusion extract of olive leaves (Fiorito et al. 2024).

Body weight evolution and anthropometric parameters

The results of body weight variation in Wistar rats from different groups were assessed in terms of both percentage of weight gain (WG) and percentage of initial weight (W/iW). The initial weight for all animals ranged from 104 to 139 g. This study revealed that rats in the control group, adhering to a normal diet, experienced a typical weight evolution relative to their initial weight, with a modest weight gain of approximately 30%. In contrast, diabetic rats exhibited a decrease in comparison to their initial weight, indicating weight loss. Additionally, diabetic rats treated with oleuropein demonstrated a significant reduction in weight compared to their initial weight, experiencing a weight loss of approximately 25%. Conversely, treatment with oleuropein resulted in a weight gain of around 30% (Fig. 2).

Fig. 2.

Fig. 2

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Anthropological parameters, Body weight, and Food intake evolution, in the Control, Diabetic, Diabetic + OL and Control + OL groups. Effect of the green olive leaves extract supplementation

Furthermore, the weight loss observed in both the diabetic group and the diabetic treated group during the initial seven days of treatment was attributed to catabolism, specifically the breakdown of lipids through adipose tissue lipolysis due to insulin deficiency (Vats et al. 2004; Oliveira et al. 2008). In contrast, the control group treated with oleuropein exhibited a substantial increase in initial weight, experiencing a weight gain (WG) of 70%. The results demonstrated that treatment with the extract from Olea europaea leaves improved body weight in diabetic rats treated with the aqueous extract. This improvement may be explained, in all likelihood, by the extract’s ability to stimulate muscular glycogenesis and/or inhibit muscular glycogenolysis and lipolysis in adipose tissues (Chatterjee and Shinde 2002). Moreover, the administration of oleuropein extract (D + OL) showed positive effects on the weight of animals, leading to an overall improvement in weight.

Food intake

During the experimentation, the dietary behaviors of Wistar rats was analyzed, with a specific focus on comparing the diabetic animals to the control group. Especially, we observed a significant difference in food intake patterns between these groups throughout the experimental period. In fact, diabetic rats displayed an increased food intake, indicative of polyphagia resulting from heightened hunger. However, a significant change occurred in diabetic rats undergoing treatment, where there was an initial surge in food consumption followed by a trend toward stability. This observation implies a positive impact of the phenolic extract treatment on food consumption patterns. The subsequent discussion will delve into the implications of these findings, shedding light on the effects of diabetes and the potential ameliorative influence of the administered treatment, as supported by existing literature (Vats et al. 2004).

Kinetic monitoring of glycemia

The blood glucose levels of the control rats, as depicted in Fig. 3, remained within the normal range during the experimental period. In contrast, the diabetic group showed a significant increase in blood glucose compared to the control group. However, treatment with oleuropein resulted in a notable and significant decrease in blood glucose levels, nearly reaching those of the control group. This aligns with studies by Wainstein et al. (2012), investigating the impact of Olea europaea leaf extract on streptozotocin-induced diabetic rats, revealing a positive impact of olive leaf extract through reduction in blood glucose levels.

Fig. 3.

Fig. 3

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Glycemia evolution during experimentation (A) and its values after sacrifice (B) in the different groups. Effect of the Green Olive leaves extract supplementation. Values are given as mean ± S.D. Value bars with different letters (a, b) are significantly different at p < 0.05

The findings indicate a decrease in blood glucose levels in rats treated with the aqueous extract from olive leaf. This is consistent with Eidi et al.’s (2009) results, highlighting the significant role of Olea europaea extract in reducing hyperglycemia. These observations are also in agreement with Jemai et al.’s (2009) study on the effects of olive leaf extract.

By evaluating the efficacy of oleuropein-rich extract regarding glycemic elevation and its impact on diabetes, it becomes evident that this extract exhibits a noticeable hypoglycemic effect. These results are consistent with investigations by Jemai et al. (2009), elucidating oleuropein’s antidiabetic attributes through the modulation of blood glucose concentrations. El and Karakay (2009) provided additional insights into the hypoglycemic mechanism of oleuropein in diabetes, highlighting its influence on glucose-induced insulin secretion and its facilitation of increased peripheral glucose absorption. Several studies have explored oleuropein’s effects in diabetic rats, demonstrating its potential therapeutic benefits in managing diabetes and its complications confirming that oleuropein significantly lowered blood glucose levels in diabetic rats by increasing insulin sensitivity, which was associated with the downregulation of pro-inflammatory cytokines (Aggul et al. 2020).

Furthermore, emerging evidence suggests that combining certain compounds, particularly those extracted from olive leaves, such as oleuropein, luteolin-7-O-glucoside, apigenin-7-O-glucoside, and verbascoside, may have synergistic effects due to their antioxidant and anti-inflammatory properties. The obtained results are consistent with other studies reporting that the combination of these compounds exhibited greater bioactivity than that for each compound alone. These findings suggest that the interactions between these compounds may enhance their overall effectiveness (Khelouf et al. 2023).

Blood biochemical parameters

Lipid metabolism profile

Total cholesterol

The outcomes of plasma cholesterol assays, illustrated in Fig. 4, revealed the following results: the control groups exhibited normal cholesterol concentrations, while the diabetic group showed a significant increase in total cholesterol. In addition, these findings confirmed the hypocholesterolemic effect of olive leaf extract in diabetic rats. Eidi et al.’s (2009) results demonstrated a significant decrease in serum lipid levels in diabetic rats treated with alcoholic olive extract. Furthermore, other studies suggested also that Olea europaea has hypolipidemic effects in diabetic rats (Somova et al. 2003). The reduction in hypercholesterolemia may be attributed to insulin secretion stimulation, which in turn activates LCAT (Lecithin Cholesterol Acyl Transferase), an enzyme responsible for the esterification of free cholesterol, thereby reducing its plasma concentration (Arii et al. 1997).

Fig. 4.

Fig. 4

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Effect of the Green Olive leaves extract supplementation on the Biochemical parameters, TC, LDL, HDL, TG, and AI in the Control, Diabetic, Diabetic + OL, and Control + OL groups. Values are given as mean ± S.D. Value bars with different letters (a, b) are significantly different at p < 0.05

Triglycerides

Similarly, the results of triglyceride levels (Fig. 4) indicate that control rats exhibited normal values. In contrast, diabetic rats displayed significantly elevated triglyceride levels compared to the control group. However, the treatment with aqueous olive leaf extract resulted in triglyceride levels nearly comparable to the control and treated control groups. These data suggest a significant effect of olive leaf aqueous extract in reducing triglyceride concentrations. El Amin et al.’s (2013) studies demonstrated that treatment with aqueous olive leaf extract effectively reduced triglyceride levels in streptozotocin-induced diabetic rats. The decrease in triglyceridemia in diabetic rats treated with oleuropein could be attributed to improved insulin secretion, as this hormone inhibits the activity of the hormone-sensitive lipase, an enzyme active in adipocytes (Somova et al. 2003).

HDL and LDL

The results depicted in Fig. 4 reveal that HDL levels in diabetic rats exhibited a significant decrease in HDL levels compared to control groups. In contract the LDL level in diabetic rats showed a significant increase compared to control rats, while treatment with olive leaf extract significantly reduced LDL levels in the diabetic group. In summary, these alterations may be linked to disruptions in insulin activity, which activates lipoprotein lipase, hydrolyzing triglycerides. Additionally, insulin inhibits lipolysis, so its absence or reduction could increase lipolysis, ultimately leading to hyperlipidemia (Shirwaikar et al. 2004).

Atherogenic index variation

The atherogenic index, assessed by the classic total cholesterol/HDL ratio, signifies an elevated risk when this index increases. This index involves the assessment of total cholesterol, HDL-C (High-Density Lipoprotein Cholesterol), LDL-C (Low-Density Lipoprotein Cholesterol), and triglycerides (TG). Defined as the ratio of LDL-C to HDL-C, the atherogenic index exhibits a significant increase in diabetic rats compared to the control group. Furthermore, the administration of olive leaf extract has successfully reduced the atherogenic index in diabetic rats (Fig. 4). In conclusion, it can be stated that olive leaf extract exerts an effective impact on reducing the atherogenic effects occurring with diabetes (Korhonen et al. 1996).

Liver enzymes: AST and ALT

The obtained results indicate a significant increase in the serum activity of both AST and ALT in the diabetic group compared to the control (Fig. 5). This increase can be attributed to hepatic cell destruction (hepatocytolysis) induced by toxic substances, such as the toxic effect of alloxan (Barbosa et al. 2006). Additionally, the elevation of transaminases is associated with the accumulation of amino acids like alanine and glutamic acid in the blood, originating from protein breakdown in the body. Our results revealed that the treatment of diabetic rats with the aqueous extract from olive leaf reduced the enzymatic activity of transaminases compared to untreated diabetic rats. Subsequently, this suggests that the aqueous olive leaf extract inhibits hepatic damage caused by the alloxan.

Fig. 5.

Fig. 5

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Effect of the Green Olive leaves extract supplementation on the Hepatic enzymes AST and ALT in the Control, Diabetic, Diabetic + OL and Control + OL groups. Values are given as mean ± S.D. Value bars with different letters (a, b) are significantly different at p < 0.05

The evolution of the relative organ weight

Table 1 shows the relative body weight of the pancreas, the liver, and the adipose tissue in the different groups. The pancreas of rats in the control group exhibits a normal weight. On the other hand, in the diabetic group, there is a decrease in pancreas relative weights compared to the control. This reduction is linked to diabetes induction by alloxan. Indeed, this chemical component has a destructive effect on the β cells of Langerhans (Liu et al. 2008). However, the group treated with the studied extract shows a higher weight compared to the control group. This could signify that the administration of the extract has regenerated the β cells of the Langerhans islets destroyed by Alloxan (Liu et al. 2008).

Table 1.

Effects of the oleuropein-rich extract on the plasma insulin

Groups Plasma insulin (µU/ml)
Control 14.53 ± 0.9
Diabetic 4.65 ± 0.78+
Diabetic + OL 13.25 ± 0.56*
Control + OL 14.76 ± 0.71*
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Values are given as mean ± S.D. Values are statistically significant at p < 0:05. * vs. Diabetic rats and + vs. control rats.

Concerning the liver, the relative weights in the control rats displayed substantially normal weights. However, the relative weights of diabetic rats’s livers have significantly increased compared to that of the control rats, which could be explained by the toxicity of alloxan. For the relative weight of the liver in the diabetic treated group and the control treated group, it is almost equal to that of the controls. This result supports the hepatotoxicity of alloxan (Liu et al. 2008). Furthermore, as a key organ in the physiopathology of the diabetes, the adipose tissue relative weight was investigated. Our results indicate that the weight of adipose tissue in diabetic rats has undergone a significant decrease compared to the control and treated control groups. However, for diabetic rats treated with the extract, a highly significant decrease in weight is observed compared to control rats. This might be explained by the shift in energy metabolism in diabetic rats, which resort to the hydrolysis of fatty acids instead of glucose (Dilworth et al. 2021).

Effects of olive leaf extract on lipid peroxidation in the liver

The levels of thiobarbituric acid reactive substances (TBARS) serving as an indicator of lipid peroxidation resulting from oxidative stress were determined in the liver. The results (Fig. 6) prove that diabetes significantly induced lipid peroxidation in the liver of diabetic rats compared to control rats. However, the administration of olive leaf extract significantly reduced the levels of TBARS in the liver. These findings align with the study by Jemai et al. (2009), which showed that hyperglycemia is associated with the establishment of marked oxidative stress, indicated by increased hepatic lipid peroxidation translated by excessive TBARS formation (Dorsemans 2018).

Fig. 6.

Fig. 6

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Effect of the Green Olive leaves extract supplementation on the Endogenous antioxidant CAT and the Lipid peroxidation evolution in the Control, Diabetic, Diabetic + OL and Control + OL groups. Values are given as mean ± S.D. Value bars with different letters (a, b) are significantly different at p < 0.05

Effects of olive leaf extracts on catalase activity

Catalase, as a crucial antioxidant enzyme, plays a vital role in mitigating oxidative stress by decomposing hydrogen peroxide into water and oxygen. Evaluating catalase activity provides insights into the antioxidant capacity of olive leaf extracts, shedding light on their potential to modulate the oxidative balance in the liver. The results in Fig. 6 indicate that the catalase activity exhibited a decrease of approximately 72% in diabetic rats compared to control rats. Administration of olive leaf extract significantly adjusts this decrease. It is well known that hyperglycemia leads to the overproduction of free radicals and non-enzymatic protein glycation, exerting deleterious effects on various organs involved in glycemic regulation, such as the liver.

In this study, we observed a decrease in the activity of the hepatic antioxidant enzyme catalase (CAT) and an increase in the level of TBARS in diabetic rats. These results align with other findings showing that hyperglycemia is accompanied by an increase in oxidative stress, as evidenced by a significant rise in hepatic lipid peroxidation leading to the formation of TBARS and a significant decrease in hepatic antioxidants, including CAT activity (Jemai et al. 2009; Dorsemans 2018). Subsequently, the obtained results confirm the potential beneficial effect of oleuropein-rich extract in attenuating oxidative stress.

Effects of the oleuropein-rich extract on the plasma insulin

Table 2 shows the level of plasma insulin in normal and experimental groups of rats. This level has significantly decreased in the diabetic rats when compared to the control group. This result is directly related to diabetes induction mechanism using alloxan having as target the beta cells in the pancreatic islets. The values were 4.65 µU/mL in the diabetes rats compared to 14.53 µU/mL in healthy animals. Furthermore, the administration of the oleuropein-rich extract for four weeks has significantly restored the depletion of the plasma insulin in the diabetic rats, and the values increased to reach 13.25 µU/mL which is highly significant compared to the diabetic rats. Moreover, this improvement in plasma insulin concentrations indicates that olive leaf extract stimulates insulin secretion from the remaining β-cells or from the regenerated ones. This finding is very important, and it is consistent with some studies evocating the role of phytotherapy in the management of the diabetes, based on polyphenol compounds (Jemai et al. 2009). Moreover, it is confirmed that natural products may influence diabetes through various mechanisms, including the inhibition of α-glucosidase and α-amylase, enhancing glucose uptake, promoting insulin secretion and the proliferation of pancreatic β-cells and via their antioxidant activities (Governa et al. 2018; Yadav et al. 2018).

Table 2.

Relative body weight (mg/g) of the pancreas, the liver, and the adipose tissue in the different groups

Organ Relative body weight (mg/g)
C D D + OL C + OL
Liver 30.35 ± 0.22 35.29 ± 0.24+ 32.82 ± 0.9* 30.14 ± 0.33*
Pancreas 4.91 ± 0.5 3.77 ± 0.6+ 5.16 ± 0.7* 4.17 ± 0.54*
Adipose tissue 16.8 ± 0.32 14.52 ± 0.33+ 10.93 ± 0.12* 14.95 ± 0.72
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Values are given as mean ± S.D. Values are statistically significant at p < 0:05. * vs. Diabetic rats and + vs. control rats

Histology and immunohistology of the pancreas

The investigation of the histology and the immunohistology of the pancreas is an important part in the comprehension of the effects of the polyphenol extract on the diabetes effects. The results of the histopathological study are depicted in Fig. 7. The histological sections of the pancreas (A (Gx100) and a (Gx400)) of a control rat (C) displayed a normal appearance of pancreatic tissue. Dark regions correspond to serous acini containing secretory portions responsible for the exocrine function of the pancreas, releasing products into the excretory ducts (PD). Clear regions represent the Pancreatic islets (PI), forming a cluster of epithelial cells surrounded by a richly vascularized connective tissue.

Fig. 7.

Fig. 7

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Histology of the Pancreas in the Control, Diabetic, Diabetic + OL and Control + OL groups. Effect of the green olive leaves extract supplementation. The control A, a; Diabetic B, b, C + OL, C, c and D + OL D, d respectively (Gx100) and (Gx400). PA pancreatic acini, PI pancreatic islet, PD pancreatic duct

Nevertheless, the histological sections of the pancreas from a diabetic rat (B (Gx100) and b (Gx400)) revealed alterations in the exocrine region characterized by the presence of glandular cells with dense nuclei and cytoplasm, suggesting necrotic cells. Additionally, there is an alteration in the endocrine region presented by the pancreatic islets, featuring large spaces between endocrine cells, the presence of necrotic cells, deformation of the islets, atrophy of these regions, and significant infiltration of blood cells.

As a reference, supplementation of the phenolic extract to the control (C + OL) rats (Fig. 7C (Gx100) and Fig. 7c (Gx400)) showed histological sections with a normal appearance of pancreatic tissue, indicating intact endocrine and exocrine regions, confirming the safety of the extract and absence of toxicity. Furthermore, in the diabetic group of rats treated with OL (D + OL), (Gx100) and d (Gx400), histological sections displayed a normal appearance of pancreatic tissue with the presence of some necrotic cells in the serous acini (Fig. 7D). The Langerhans cells appeared normal, grouped in clusters and separated by connective tissue. In conclusion, the olive leaf extracts corrected malformations at the level of the Langerhans cells, suggesting that the used extract could provide protection for the β cells and then its direct effects on the insulin releasing.

Additionally, with the aim to further explore the effect of diabetes induction on the pancreas and specifically in the pancreatic islets and the cellular protective effect of oleuropein-rich extract, immunohistochemistry assays, using Bcl-2 antibodies, were performed (Fig. 8).

Fig. 8.

Fig. 8

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Pancreas tissue immunohistochemical staining, with anti Bcl-2, in the control (C), Diabetic (D), D + OL and C + OL groups. Effect of the Green Olive leaves extract supplementation (scale bar: 5 μm; magnification: 400x) PI pancreatic islet

The findings showed an evident effect of diabetes in the pancreatic tissue. In fact, the expression of Bcl-2 was significantly declined in the pancreas of diabetic rats on the contrary to the control, Diabetic + OL and Control + OL groups. Furthermore, Bcl-2 is an anti-apoptotic protein, and its absence in the induced diabetes pancreatic tissue is suggested to be in direct relation with the viability of the beta cells under hyperglycemia conditions. Furthermore, it is well known that apoptosis in beta cells is one of the causes of diabetes type 1(Eizirik and Mandrup-Poulsen 2001). Moreover, these results are confirmed by the plasma insulin values which are significantly low in the diabetic rats compared to the other groups. On the other hand, the Bcl-2 expression was significantly restored by the olive leaves extract rich in oleuropein. This result highlights the relevant effect of such extract on the pancreatic tissue. This activity could be related to its antioxidant power and might extend to confirming its regenerative ability of the pancreatic islets’ cells. These findings are consistent with a previous study confirming the positive effects of oleuropein on Bcl-2 expression in the liver (Jemai et al. 2020). The green extract used in the present study was obtained without chemical solvents, highlighting its potential as a safe and effective therapeutic agent; it improves glycemia by enhancing insulin secretion and increasing insulin sensitivity, likely through its antioxidant and anti-inflammatory properties: Insulin secretion: Oleuropein may stimulate pancreatic beta cells to secrete more insulin, potentially through reducing oxidative stress that impairs beta cell function in diabetes. Insulin sensitivity: It may enhance insulin sensitivity by reducing inflammation, which is known to impair insulin receptor function. By targeting inflammatory pathways, oleuropein could improve cellular response to insulin, facilitating glucose uptake. Finally, antioxidant activity: Oleuropein’s antioxidant effects protect insulin receptors and glucose transporters, promoting better glucose metabolism. (Jemai et al 2008, 2020; Souilem et al. 2017).

Conclusion

The present study investigated the antidiabetic effects of oleuropein-rich extract from olive leaves, using a safe and eco-friendly water-based method, in alloxan-induced diabetic rats. The results showed that oleuropein treatment improved glycemia management and reduced blood glucose levels in the diabetic rats. Additionally, oleuropein treatment increased insulin secretion and improved insulin sensitivity.

These results confirm the antidiabetic effects of the olive leaves extract obtained via a green extraction methods and reveal its regenerative capacity in the pancreatic tissue besides its insulinogenic potential by enhancing the insulin plasma level in the diabetic rats. These findings suggest that oleuropein holds the potential as a natural therapeutic agent for managing diabetes and its complications. Finally, our research, which utilized a solvent-free safe extract, represents an important step toward advanced studies in diabetic patients and encourages further preclinical research in this area.

Acknowledgements

Thanks are due to the support provided by the Tunisian Ministry of Higher Education and Scientific Research and Technology, and to all the collaborator in this research.

Funding

This research received no specific grant or external funding.

Data availability

The data used to support the findings of this study are included within this article.

Declarations

Conflict of interest

The authors declare no conflict of interest.

References

  1. Aggul GA, Gulabglu M, Cetin M, Ozakar E, Ozakar RS, Aydin T (2020) Effects of emulsion formulations of oleuropein isolated from ethanol extract of olive leaf in diabetic rats. Ann Brazil Acad Sci 92(4):e20190810. 10.1590/0001-3765202020190810 [DOI] [PubMed] [Google Scholar]
  2. American Diabetes Association (2021) Standards of medical care in diabetes. Diabetes Care 44(Supplement 1):S4–S1933298411 [Google Scholar]
  3. Arii K, Suehiro T, Yamamoto M, Ito H, Hashimoto K (1997) Suppression of plasma cholesteryl ester transfer protein activity in acute hyperinsulinemia and effect of plasma nonesterified fatty acid. Metabolism 46(10):1166–1173. 10.1016/S0026-0495(97)90211-0 [DOI] [PubMed] [Google Scholar]
  4. Barbosa NBV, Rocha JBT, Wondracek DC, Perottoni J, Zeni G, Nogueira CW (2006) Diphenyl diselenide reduces temporarily hyperglycemia: possible relationship with oxidative stress. Chem Biol Interact 163:230–238. 10.1016/j.cbi.2006.08.004 [DOI] [PubMed] [Google Scholar]
  5. Ben Dassi R, Ibidhi S, Jemai H, Cherif A, Driouich Chaouachi R (2024) Resveratrol: challenges and prospects in extraction and hybridization with nanoparticles, polymers, and bio-ceramics. Phytother Res. 10.1002/ptr.8319 [DOI] [PubMed] [Google Scholar]
  6. Bouallagui Z, Mahmoudi A, Maalej A, Hadrich F, Isoda H, Sayadi S (2019) Contribution of major polyphenols to the antioxidant profile and cytotoxic activity of olive leaves. Anticancer Agents Med Chem 19(13):1651–1657. 10.2174/1871520619666190416101622 [DOI] [PubMed] [Google Scholar]
  7. Chatterjee MN, Shinde R (2002) Textbook of Medical Biochemistry. Jaypee Brothers Medical Publishers. New Delhi, p 317 [Google Scholar]
  8. Daher IM, Joane MM, Abdel Nour AM (2019) Non-nutritive sweeteners and type 2 diabetes. Diabetes Res Clin Pract 155:107786. 10.1016/j.diabres.2019.107786 [DOI] [PubMed] [Google Scholar]
  9. Dilworth L, Facey A, Omoruyi F (2021) Diabetes mellitus and its metabolic complications: the role of adipose tissues. Int J Mol Sci 22(14):7644. 10.3390/ijms22147644 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorsemans AC (2018). Diabète, inflammation et stress oxydatif : impact sur la barrière hématoencéphalique, la neurogenèse et la réparation cérébrale. Santé. Université de la Réunion. Français. ffNNT: LARE0017ff. fftel 01979869.
  11. Eidi A, Eidi M, Darzi R (2009) Antidiabetic effect of Olea europaea L. in normal and diabetic rats. Phytother Res 23(3):347–350. 10.1002/ptr.2629 [DOI] [PubMed] [Google Scholar]
  12. Eizirik DL, Mandrup-Poulsen T (2001) A choice of death: the signal-transduction of immune-mediated β-cell apoptosis. Diabetologia 44:2115–2133 [DOI] [PubMed] [Google Scholar]
  13. El Amin M, Virk P, Elobeid MA, Almarhoon ZM, Hassan ZK, Ali Omer S, Merghani NM, Daghestani MH, Al Olayan EM (2013) Anti-diabetic effect of Murraya koenigii (L) and Olea europaea (L) leaf extracts on streptozotocin induced diabetic rats. Pak J Pharm Sci 26(2):359–365 [PubMed] [Google Scholar]
  14. El SN, Karakay S (2009) Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67(11):632–638. 10.1111/j.1753-4887.2009.00248.x [DOI] [PubMed] [Google Scholar]
  15. Emiru YK, Periasamy G, Karim A, Ur Rehman N, Ansari MN (2020) Evaluation of in vitro α-amylase inhibitory activity and antidiabetic effect of Myrica salicifolia in streptozotocin-induced diabetic mice. Pak J Pharm 33(4):1917–1926. 10.36721/PJPS.2020.33.4.SUP.1917-1926.1 [PubMed] [Google Scholar]
  16. Fiorito S, Collevecchio C, Spogli R, Epifano F, Genovese S (2024) Novel procedures for olive leaves extracts processing: selective isolation of oleuropein and elenolic acid. Food Chem 447:139038. 10.1016/j.foodchem.2024.139038 [DOI] [PubMed] [Google Scholar]
  17. Ghanbari R, Anwar F, Alkharfy KM, Gilani AH, Saari N (2012) Valuable nutrients and functional bioactives in different parts of olive (Olea europaea L.)—a review. Int J Mol Sci 13:3291–3340 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Governa P, Baini G, Borgonetti V, Cettolin G, Giachetti D, Magnano AR, Miraldi E, Biagi M (2018) Phytotherapy in the management of diabetes: a review. Molecules 23(1):105. 10.3390/molecules23010105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jemai H, Bouaziz M, Fki I, El Feki A, Sayadi S (2008) Hypolipidemic and antioxidant activities of oleuropein and its hydrolysis derivative-rich extracts from Chemlali olive leaves. Chem Biol Interact 176(2–3):88–98. 10.1016/j.cbi.2008.08.014 [DOI] [PubMed] [Google Scholar]
  20. Jemai H, El Feki A, Sayadi S (2009) Antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats. J Agric Food Chem 57:8798–8804. 10.1021/jf901280r [DOI] [PubMed] [Google Scholar]
  21. Jemai H, Mahmoudi A, Feryeni A, Fki I, Bouallagui Z, Choura S, Chamkha M, Sayadi S (2020) Hepatoprotective effect of oleuropein-rich extract from olive leaves against cadmium-induced toxicity in mice. Biomed Res Int 2020:4398924. 10.1155/2020/4398924 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Khelouf I, Jabri Karoui I, Lakoud A, Hammami M, Abderrabba M (2023) Comparative chemical composition and antioxidant activity of olive leaves (Olea europaea L.) of Tunisian and Algerian varieties. Heliyon 9(12):e22217. 10.1016/j.heliyon.2023.e22217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Korhonen T, Savolainen MJ, Koistinen MJ, Ikaheimo M, Linnaluoto MK, Kervinen K, Kesaniemi YA (1996) Association of lipoprotein cholesterol and triglycerides with the severity of coronary artery disease in men and women. Atherosclerosis 127(2):213–220. 10.1016/s0021-9150(96)05958-8 [DOI] [PubMed] [Google Scholar]
  24. Liu Z, Li J, Zeng Z, Liu M, Wang M (2008) The antidiabetic effect of cysteinyl metformin, a new lysun the sized agent, in alloxan and streptozotocin induced diabetic rats. Chem Biol Interact 173(1):68–75. 10.1016/j.cbi.2007.11.012 [DOI] [PubMed] [Google Scholar]
  25. Martins J, Steyn N, Rossouw HM, Pillay ST (2023) Best practice for LDL-cholesterol: when and how to calculate. J Clin Pathol 76:145–152. 10.1136/jcp-2022-208480 [DOI] [PubMed] [Google Scholar]
  26. Oliveira HC, Dos-Santos MP, Grigulo R, Lima LL, Martins DTO, Lima JCS, Stoppiglia LF, Lopes CF, Kawashita NH (2008) Antidiabetic activity of Valairea macrocarpa extract in rats. J Ethnopharmacol 115(3):515–519. 10.1016/j.jep.2007.10.025 [DOI] [PubMed] [Google Scholar]
  27. Pérez-Bonilla M, Salido S, Sánchez A, van Beek T, Altarejos J (2013) Effect of extraction conditions on the antioxidant activity of olive wood extracts. Int J Food Sci. 10.1155/2013/719593 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Prashant S, Namakkal HS, Rajasekaran TS, Chandra T (2005) Effects of the antioxidant properties of millet species on oxidative stress and glycemic status in alloxan-induced rats. Nutr Res (n y) 25:1109–1120 [Google Scholar]
  29. Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, Colagiuri S, Guariguata L, Motala AA, Ogurtsova KE, Shaw J, Bright D, Williams R (2019) Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract 157:107843. 10.1016/j.diabres.2019.107843 [DOI] [PubMed] [Google Scholar]
  30. Shirwaikar A, Rajendran K, Dinesh KC, Bodla R (2004) Antidiabetic activity of aqueous leaf extract of Annona squamosa in streptozotocin–nicotinamide type 2 diabetic rats. J Ethnopharmacol 91(1):171–175. 10.1016/j.jep.2003.12.017 [DOI] [PubMed] [Google Scholar]
  31. Somova LI, Shode FO, Ramnanan P, Nadar A (2003) Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea subspecies Africana leaves. J Ethnopharmacol 84:299–305. 10.1016/s0378-8741(02)00332-x [DOI] [PubMed] [Google Scholar]
  32. Souilem S, Fki I, Kobayashi I, Khalid K, Marcos AS, Isoda H, Sayadi S, Nakajima M (2017) Emerging technologies for recovery of value-added components from olive leaves and their applications in food/feed industries. Food Bioprocess Technol 10(2):229–248. 10.1007/s11947-016-1834-7 [Google Scholar]
  33. Tiwari BK, Pandey KB, Abidi B, Rizvi SI (2013) Markers of oxidative stress during diabetes mellitus. J Biomarkers 2013:378790. 10.1155/2013/378790 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vats V, Yadav SP, Grover JK (2004) Ethanolic extract of Ocimum sanctum leaves partially attenuates streptozotocin-induced alterations in glycogen content and carbohydrate metabolism in rats. J Ethnopharmacol 90(1):155–160. 10.1016/j.jep.2003.09.034 [DOI] [PubMed] [Google Scholar]
  35. Wainstein J, Ganz T, Boaz M, Bar Dayan Y, Dolev E, Kerem Z, Madar Z (2012) Olive leaf extract as a hypoglycemic agent in both human diabetic subjects and in rats. J Med Food 15(7):605–610. 10.1089/jmf.2011.0243 [DOI] [PubMed] [Google Scholar]
  36. World Health Organization WHO (2021). Rapport du Sommet mondial sur le diabète.
  37. Yadav RK, Rawat JK, Gautam S et al (2018) Antidiabetic activity of mefloquine via GLP-1 receptor modulation against STZ–NA-induced diabetes in albino Wistar rats. 3 Biotech 8:240. 10.1007/s13205-018-1250-y [DOI] [PMC free article] [PubMed] [Google Scholar]

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Data Availability Statement

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