Yield, quantification of polyphenolics and evaluation of the antioxidant activity
The hydro-ethanolic extract of olive pomace contains phytoconstituents such as tannins, phenolic compounds, and flavonoids, which possess potential antioxidant properties Prior research by [17]. Whereas the chemical composition of olive pomace, as evidenced by various studies including those by [28–29], comprises a range of compounds such as saponins, polyphenols, flavonoids, tannins, quinones, terpenoids, hydroxytyrosol, tyrosol, oleuropein, caffeic acid, benzoic acid, rutin, vanillin, coumaric acid, and quercetin. Aqueous fraction, chosen for its eco-friendliness and effectiveness in extracting antioxidants, yielded concentrations of phenolic compounds, flavonoids, and tannins at 130.812 ± 13.991 mg GAE/g DW, 52.009 ± 3.406 mg QE/g DW, and 10.960 ± 0.966 mg CE/g DW, respectively. Additionally, antioxidant activity measured using the DPPH method showed an EC50 value of 2.460 ± 0.044 mg/mL and a TAC value of 51.191 ± 3.719 mg GAE/mL (Table 1). The slight variations in the yield and the profile of bioactive compounds in the simples can be described to factors such as the selection of solvents, method of extraction, agricultural practices, etc [30–31]. These results indicate the great potential of OP as source of natural antioxidants with potential applications in various industries and pharmaceuticals [28] and [32]. Research suggests OP has health benefits, making it a valuable by product worth further exploration [13–14].
Table 1
Phytochemical determination, yield and antioxidant activity of olive pomace crude extract and fractions
Test | Yield (%) | Total phenolic (mg GAE/g DW) | Flavonoids (mg QE/g DW) | Condensed tannins (mg CE/g DW) | DPPH EC50 (mg/mL) | TAC (mg GAE/g DW) |
HEE | 8.66 | 90.139 ± 15.545*** | 73.97 ± 1.081*** | 7.307 ± 0.966*** | 1.704 ± 0.023*** | 45.41 ± 4.808*** | |
DF | 2.6 | 237.44 ± 43.528*** | 123.43 ± 1.48*** | 17.659 ± 1.838*** | 2.344 ± 0.073*** | 24.668 ± 1.92*** | |
EAF | 0.64 | 107.727 ± 3.109** | 36.984 ± 2.135** | 10.351 ± 1.174*** | 3.815 ± 0.501*** | 23.0574 ± 2.25** | |
AF | 0.064 | 213.257 ± 9.327*** | 113.12 ± 0.102*** | 13.031 ± 1.383*** | 3.340 ± 0.165*** | 21.182 ± 0.696** | |
HEF | 0.046 | 125.316 ± 6.218*** | 34.495 ± 1.735*** | 11.691 ± 1.674*** | 2.0165 ± 0.012** | 17.553 ± 0.434** | ± 0,4335** | | |
AqF | 5.3 | 130.812 ± 13.991*** | 52.009 ± 3.406*** | 10.960 ± 0.966*** | 2.460 ± 0.044*** | 51.191 ± 3.719*** | |
HEE: hydro-ethanolic extract; DF: Dichloromethane Fraction; EAF: Ethyl acetate Fraction; AF: Acetone Fraction; HEF: Hydro-ethanol Fraction; AqF: Aqueous Fraction. GAE: gallic acid equivalent; CE: catechin equivalent; QE: quercetin equivalent; DW: dry weight; TAC: Total antioxidant capacity. |
Data are presented as mean ± SD; *: significant difference at P < 0.05; ***: extremely significant difference at P < 0.001; **: highly significant difference at P < 0.01. |
Acute oral toxicity studies
The hydro-ethanolic extract of olive pomace demonstrates a favorable safety profile in Wistar rats. No acute toxicity was observed after 14 days, even at the highest administered dose (LD50 > 5000 mg/kg) [17]. This safety profile, combined with the extract's potential antioxidant properties, suggests its suitability for long-term administration.
Experimental induction of diabetes
Assessment of blood glucose and body weight
Monitoring changes in blood glucose levels and body weight is a fundamental aspect of diabetes research. These parameters provide vital information about the study substance and its impact on diabetes. Blood glucose was measured using a glucometer following tail vein puncture, and body weight was recorded using a scale graduated in grams (g), all at the end of each week. A significant increase in body weight was recorded after 21 and 28 days of treatment, indicating an upward trend in treated rats, as opposed to untreated diabetic control rats, whose weight exhibited a downward trend. Regarding blood glucose levels, a similar significant decrease was observed in treated rats when compared to the control group.
STZ-induced diabetes is characterized by a severe loss of body weight [33–34]. Indeed, over the 28 days of our experiment, untreated diabetic rats showed a decrease in body weight. This weight reduction in the animals is likely attributable to insulin deficiency, which leads to decreased protein synthesis [35–36]. Furthermore, numerous studies suggest that body weight loss in diabetic rats may be attributed to increased lipid and protein catabolism resulting from cellular carbohydrate deficiency [37–39]. These findings align with those documented in the literature, which indicate that the intraperitoneal injection of 45 mg/kg body weight of streptozotocin causes weight loss in Wistar rats.
In our study, the hydro-ethanolic extract (HEE) and aqueous fraction (AqF) of olive pomace contributed to an increase in body weight in treated diabetic rats. This improvement in body weight was most pronounced during the last two weeks of treatment (Fig. 1). These findings are consistent with those of [16, 40–41]. who observed an increase in body weight for diabetic rats supplemented with hydroxytyrosol and different levels of olive pomace (OP). Other studies have confirmed the beneficial effects of olive pomace on improving body weight in animals fed this by-product [42–43]. These positive effects of this waste product on health are undoubtedly attributed to the high content of phenolic compounds and other antioxidants and nutraceutical molecules [44–45]. In terms of blood glucose levels, a notable decrease was observed in the treated rats, once again in comparison with the control group (Table 2, Fig. 1). This decline in blood glucose levels, beginning in the 2nd week of experimentation in the treated groups and continuing through day 28, underscores the effectiveness of our extracts, with particular emphasis on the aqueous fraction, in ameliorating blood glucose levels. The study conducted by [34] suggests that the dose of streptozotocin administered at 45 mg/kg body weight may be insufficient to sustain the diabetic state. This observation could potentially explain the return to a normal state observed at the end of the experimental period in the streptozotocin-treated animals. [46–47] observed that olive pomace and olive leaf extract reduce blood glucose levels and enhances antioxidant activity in diabetes-induced rats. Additionally, research conducted by Professor Mancini's group [27] demonstrated that a diet rich in olive oil improves blood glucose levels and increases insulin sensitivity. They found also the higher the consumption of olive oil, the lower the fasting blood glucose. Another study realized by [48–49] revealed that olive oil leads to an increase in the secretion of GLP-1 (glucagon-like peptide) in diabetic patients.
Table 2
Variation in blood glucose and body weight in rats pretreated or not with extracts
Rat groups | Body weight (g) | Blood glucose (g/L) |
D0 | D7 | D14 | D21 | D28 | D0 | D7 | D14 | D21 | D28 |
C | 209.6 | 224 | 232 | 241 | 246.5 | 0.72 | 0.8 | 0.78 | 0.75 | 0.79 |
DTrAqF | 237.66 | 204.66 | 199.66 | 207.66 | 224.66 | 0.6 | 3.78*** | 3.44*** | 2.47*** | 1.67 |
DTrHEE | 234.5 | 178 | 183.33** | 189.66* | 203.66 | 0.71 | 3.4*** | 2.82*** | 2.28*** | 1.69 |
DTrMref | 236.5* | 192* | 207.5 | 230.5 | 241 | 0.92 | 3.9*** | 2.79*** | 2.84*** | 2.65*** |
TD | 218 | 202.33 | 189.33 | 161.9*** | 158.66*** | 0.81 | 3.77*** | 3.8*** | 3.64*** | 3.71*** |
DTrAqF: Diabetic treated with aqueous fraction; DTrHEE: Diabetic treated with hydro-ethanolic extract; DTrMref: Diabetic treated with metformin reference molecule; TD: Untreated diabetic; C: Rat treated with physiological water. |
Data are presented as mean ± SD; *: significant difference at P < 0.05; ***: extremely significant difference at P < 0.001; **: highly significant difference at P < 0.01. |
Effect on biochemical parameters
Hydroxytyrosol, a major phenolic phytochemical found in olives, has been reported to have antioxidant, anti-inflammatory, anticancer, and antidiabetic properties [50]. Evidence suggests that hydroxytyrosol may protect against diabetes by affecting glucose and lipid homeostasis [41, 51–52]. In our study, biochemical markers (Table 3) revealed a significant difference between the control group and the groups receiving different doses of the extract. Streptozotocin, a toxic glucose analog, selectively accumulates in pancreatic beta cells. Its diabetogenic effect is attributed to its ability to destroy these pancreatic beta cells [53–54], leading to beta cell necrosis and severe insulin deficiency, causing diabetic hyperglycemia that sets in within 48 hours [55–56]. The increased insulin secretion by beta cells in the groups treated mainly with the aqueous fraction (P < 0.001) would explain the reduction in blood glucose levels [57–59]. In this study, the hypoglycemic effects observed with OP extracts are probably directly related to their antioxidant activities.
Table 3
Effect of OP aqueous fraction and hydro-ethanolic extract on biochemical parameters in diabetic rats
Blood parameters | C | TD | DTrAqF | DTrHEE | DTrMref |
Urea (mg/dl) | 45.85 ± 0.91 | 49.73 ± 5.44 | 46.47 ± 1.06 | 36.84 ± 5.8* | 41.75 ± 2.40* |
Creatinine (mg/l) | 4.25 ± 0.2 | 2.66 ± 0.22 | 4.82 ± 2.19 | 3.6 ± 0.7 | 4.46 ± 0.71 |
AST (UI/l) | 75.42 ± 0.76 | 108.93 ± 2.89*** | 74.37 ± 3.77 | 79.19 ± 1.85 | 80.94 ± 2.30 |
ALT (UI/l) | 34.37 ± 0.84 | 76.12 ± 4.43*** | 44.18 ± 1.94** | 36.31 ± 2.46 | 35.68 ± 1.31 |
Total Prot (g/dl) | 4.52 ± 0.35 | 8.78 ± 0.58*** | 5.08 ± 0.28 | 6.99 ± 0.4** | 4.31 ± 0.26 |
Cholesterol(mg/dl) | 30.86 ± 3.45 | 88.47 ± 3.82*** | 64.54 ± 3.36* | 69.6 ± 5.54** | 64.84 ± 1.6** |
HDL-C(mg/dl) | 63.29 ± 1.67 | 26.97 ± 0.77*** | 52.11 ± 1.35** | 68.51 ± 3.49* | 64.01 ± 0.87 |
TG (mg/dl) | 54.58 ± 4.30 | 81.21 ± 5.22*** | 45.028 ± 3.6 | 66.33 ± 4.98*** | 62.75 ± 2.62* |
Glucose (g/dl) | 1.59 ± 0.41 | 4.91 ± 0.07*** | 1.91 ± 0.22 | 1.82 ± 0.58*** | 2.52 ± 0.13* |
Insulin(µU/mL) | 240 ± 4.06 | 228 ± 0.89*** | 298.01 ± 0.71*** | 229.33 ± 0.62*** | 231.33 ± 2.26** |
DTrAqF: Diabetic treated with aqueous fraction; DTrHEE: Diabetic treated with hydro-ethanolic extract; DTrMref: Diabetic treated with metformin reference molecule; TD: Untreated diabetic; C: Rat treated with physiological water; ALT: alanine aminotransferases, AST: aspartate aminotransferase; TG: triglycerides; HDL-C: High Density Lipoprotein. |
Data are presented as mean ± SD. *: significant difference at P < 0.05; ***: extremely significant difference at P < 0.001; **: highly significant difference at P < 0.01. |
-Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serve as crucial markers of liver function [60–61]. Oleuropein (OLE), and hydroxytyrosol (HT), the major component of olive, ameliorates liver parameters by restoring the expression of enzymes involved in insulin signaling and metabolism [62]. Additionally, HT exhibits hepatoprotective properties by decreasing apoptosis, increasing antioxidant activity, and enhancing hepatocyte viability [63–64]. In our study, in the treated diabetic groups olive pomace extract compared to the untreated group (Table 3), plasma AST activity increased significantly (p < 0.001), whereas a significant decrease (p < 0.05) and/or no significant difference were observed for ALT. These results are comparable to those found by [65] in diabetic rats supplemented with olive pomace.
Diabetes-induced hyperglycemia leads to elevated urea and creatinine levels, which can be considered markers of renal dysfunction. Numerous studies have demonstrated the efficacy of naturel compounds in improving renal function. After 28 days of treatment, urea levels in rats treated with HEE and the AqF of OP did not significantly differ from those in the normal control, whereas they did significantly differ (P < 0.05) from those in the untreated control.
Olive pomace (OP) acts favorably on renal function, probably through its protective effect against disorders caused by hyperglycemia, significantly reducing uremia [16, 41].
No significant difference was observed (Table 3) in creatinine concentration in the blood of treated animals throughout the experiment. This suggests minimal impact on kidney function by the administered extract doses [65]. Further investigation is needed to confirm these findings and explore potential long-term effects, as noted by [5, 16, 41].
It has been reported that hyperlipidemia plays an important role in the pathology of diabetes [65]. Our findings indicate a significant increase (p < 0.001) in plasma cholesterol levels among untreated diabetic rats, accompanied by a significant decrease (p < 0.001) in HDL-C levels. Conversely, no statistically significant difference was observed in the group treated with 50mg/kg of the aqueous fraction. However, there was a significant increase (p < 0.01) in rats treated with 250 mg/kg of the hydro-ethanolic extract, which may indicate positive regulation of cholesterol metabolism. Additionally, a notable increase (p < 0.05) was seen in those treated with 250 mg/kg of metformin, compared to the control group. Diabetic control rats exhibited a significant increase (p < 0.001) in triglyceride concentrations. However, all treatment groups – aqueous fraction, HEE, and metformin – showed significant decreases compared to the control diabetic group (81.21 ± 5.22 mg/dl). These findings align with previous studies demonstrating a hypolipidemic effect in rats treated with olive pomace [29, 66]. From these results, we can deduce that treatment with olive pomace extracts induces hypocholesterolemia and hypotriglyceridemia effects. However, the effect of the aqueous fraction is even more significant. The hypolipidimic and hypoglycemic activity of OP extract may be due to the presence of bioactive molecules such as oleuropein and hydroxytyrosol, which are attributed to the regulation of glucose transporter-4 (GLUT4), potentiation of insulin release, modulation of redox homeostasis, and maintenance of beta cell physiology against oxidative stress. This is in agreement with other studies proving the hepato-protective role of olive oil and olive by-products against pathological changes [41, 16].
Effect on hematological parameters
Hematological parameters of the treated rat’s groups did not vary significantly from those of normoglycemic control rats (Table 4). Consequently, the red blood cell count, mean corpuscular volume, hematocrit, and platelet count of diabetic control rats were significantly (P < 0.01) and/or (P < 0.05) lower than those of normoglycemic control rats. White blood cell, monocyte, and neutrophil counts were significantly (P < 0.05) higher in diabetic control rats than in normoglycemic control rats (Table 4). These results are similar to those produced by Nadir et al. (2016); Elkotb et al. (2017) and Onsiyor et al. (2019). [16, 67, 79].
Table 4
Effect of the aqueous fraction and hydro-ethanolic extract of OP on hematological parameters in diabetic rats
Blood parameters | C | DnTr | DTrAqF | DTrHEE | DTrMref |
HGB(g/dL) | 16.27 ± 0.37 | 12.40 ± 2.14** | 19.9 ± 1.16** | 16.0 ± 0.39 | 16.45 ± 0.49 |
RBC (106/ul) | 8.89 ± 0.16 | 6.99 ± 1.21** | 10.98 ± 0.28** | 9.00 ± 0.51 | 9.27 ± 0.19* |
WBC (103/ul) | 7.29 ± 0.85 | 3.71 ± 0.56* | 6.47 ± 1.59 | 5.8 ± 1.6 | 5.22 ± 0.86 |
HCT % | 57.57 ± 2.54 | 44.02 ± 8.65** | 66.17 ± 3.59 | 51.35 ± 2.85 | 58.9 ± 1.03 |
Eosinophiles | 0.95 ± 0.54 | 1.22 ± 0.41 | 0.97 ± 0.35 | 1.82 ± 0.81 | 1.47 ± 0.27 |
Lymphocytes % | 60.35 ± 3.02 | 37.25 ± 5.24* | 54.97 ± 0.26 | 42.57 ± 7.31 | 47.92 ± 10.57 |
Neutrophils% | 5.6 ± 0.57 | 12.12 ± 1.35** | 4.6 ± 0.25*** | 4.22 ± 3.34 | 8.62 ± 2.37 |
Monocytes% | 20.82 ± 0.57 | 35.6 ± 3.81** | 29.97 ± 0.47 | 24.2 ± 6.71 | 29.4 ± 7.55 |
Basophils% | 1.8 ± 1.77 | 0.77 ± 0.56* | 0.65 ± 0.20* | 4.27 ± 2.13* | 1.57 ± 0.22 |
MCH (pg) | 18.27 ± 0.37 | 16.82 ± 1.26 | 16.45 ± 0.47 | 17.8 ± 0.58 | 17.5 ± 0.85 |
MCHC (g/dl) | 28.67 ± 0.89 | 27.32 ± 1.59 | 29.22 ± 0.43 | 31.17 ± 1.01 | 27.58 ± 0.44 |
VGM | 65.2 ± 2.52 | 42.85 ± 19.58** | 59.57 ± 2.4 | 57.05 ± 0.35 | 62.37 ± 1.64* |
Platelets103/µl) | 695 ± 30.57 | 495.75 ± 169.03 | 743.25 ± 147.52 | 595.75 ± 171.37* | 610.75 ± 67.92 |
CHCM (pg) | 28.12 ± 1.39 | 27.87 ± 0.34 | 26.92 ± 0.67 | 30.62 ± 0.81* | 26.87 ± 0.49 |
DTrAqF: Diabetic treated with aqueous fraction; DTrHEE: Diabetic treated with hydro-ethanolic extract; DTrMref: Diabetic treated with metformin reference molecule; DnTr: Untreated diabetic; C: Rat treated with physiological water; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin; HCT: Hematocrit; VGM: Mean Globular Volume. |
Data are presented as mean ± SD; *: significant difference at P < 0.05; ***: extremely significant difference at P < 0.001; **: highly significant difference at P < 0.01. |
The effects of Olive pomace on hematological analysis, including RBC, HCT, HGB, and PLTs in the plasma of experimental rats treated with AqF and HEE, were as follows: 10.98 ± 0.28 (106/µl), 66.17 ± 3.59 (g/dL), 19.9 ± 1.16%, 743.25 ± 147.52 (103/µL), and, 9.00 ± 0.51 (106/µl), 51.35 ± 2.85 (g/dL), 16.0 ± 0.39%, 595.75 ± 171.37 (103/µL) respectively compared to those of normal control rats, which were 8.89 ± 0.16 (106/µl), 57.57 ± 2.54 (g/dL), 16.27 ± 0.37%, 695 ± 30.57(103/µL). These results align with [67] in rats treated with oleuropein and [16] in rats Supplemented with different levels of olive pomace.
The observed improvement in hematological parameters of rats, especially when compared to the results of olive pomace with leaves, fruits, and oil, underscores the importance of olive as a nutritional and healthy formula. Leukopenia, anemia, and thrombocytopenia were observed in untreated diabetic rats, indicative of disturbed hematopoiesis. Administration of the extract to rats corrected these abnormalities, sometimes in a comparable and even superior manner to the reference drug (metformin), especially for the aqueous fraction.
Effect on oxidant /antioxidant status in pancreas, liver and kidney tissue
Increased blood glucose levels can induce oxidative stress through the formation of reactive oxygen species (ROS) [68]. Several studies have indicated an increase in oxidative stress markers during STZ-induced experimental diabetes [68 ; 69]. The results of our study demonstrate a significant increase in the levels of MDA (P < 0.001), which are indicative markers of oxidative stress (Table 5). Treatment of diabetic rats with olive pomace extract effectively mitigated lipid peroxidation. MDA concentrations in the pancreas of the treated diabetic groups were notably lower compared to the untreated groups (Table 5). These results suggest that this reduction in lipid peroxidation may be attributed to an increased antioxidant status suggested that OP decreases lipid peroxidation either by inhibiting free radical generation or through the high antioxidant activity of olive pomace in diabetic and treated rats [15, 66] noted a reduction in lipid peroxidation, in rats subjected to a cholesterol enriched diet and treated with OP and this may be due to the presence of active compounds [70–71]. Hydroxytyrosol (HT), tyrosol, and other phenolic compounds are found in the highest concentration in olive pomace. Evidence suggests that hydroxytyrosol possesses antioxidant properties, influences glucose and lipid homeostasis, and may provide protection against diabetes [51–52]. In addition to their hypoglycemic and hypolipidemic effects, olive pomace compounds also demonstrate beneficial effects in protecting all organs from radical attacks. They achieve this by increasing the activities of antioxidant enzymes (Cat, SOD, etc.) and stimulating the production of large quantities of GSH, which explains the reduction in lipid peroxidation at the tissue level [51–52, 72].
Table 5
Effect of olive pomace (OP) aqueous fraction and hydro-ethanolic extract on oxidative stress parameters in pancreas, liver, and lysate of diabetic rats
Pancreas | Cat (U/min/g) | GSH (umol/g) | SOD (umol/min/g) | MDA (µmol/g) | CP (umol/g) | O2● (µmol/g) |
C | 26.55 ± 4.11 | 6.29 ± 0.56 | 0.64 ± 0.2 | 0.31 ± 0.01 | 0.149 ± 0.035 | 10.69 ± 0.26 |
DTrAqF | 25.83 ± 0.87 | 3.88 ± 0.34* | 0.59 ± 0.05 | 1.52 ± 0.5* | 0.25 ± 0.027 * | 13.39 ± 0.41*** |
DTrHEE2 | 62.03 ± 1.16*** | 6.79 ± 1.21 | 0.68 ± 0.04 | 1.053 ± 0.31 | 0.263 ± 0.01* | 12.11 ± 0.35*** |
DTrMref | 21.57 ± 1.27 | 4.04 ± 1.17 | 0.58 ± 0.1 | 0.85 ± 0.36 | 0.295 ± 0.031** | 11.37 ± 0.43* |
DnTr | 15.83 ± 0.31* | 2.62 ± 0.04** | 0.254 ± 0.01** | 2.14 ± 0.03** | 0.436 ± 0.09*** | 20.74 ± 0.40*** |
Liver |
C | 15.45 ± 0.11 | 4.83 ± 0.002 | 0.69 ± 0.05 | 0.77 ± 0.02 | 0.28 ± 0.02 | 6.71 ± 0.33 |
DTrAqF | 24.43 ± 2.5** | 3.67 ± 0.51 | 0.24 ± 0.01*** | 2.31 ± 0.83 | 0.28 ± 0.03 | 8.28 ± 0.01*** |
DTrHEE2 | 48.40 ± 2.33*** | 4.76 ± 1.04 | 0.56 ± 0.03*** | 2.03 ± 0.43 | 0.26 ± 0.13 | 9.15 ± 0.5*** |
DTrMref | 20.99 ± 2.3* | 3.45 ± 0.11* | 0.59 ± 0.04** | 0.44 ± 0.15 | 0.26 ± 0.05 | 11.44 ± 0.12*** |
DnTr | 12.09 ± 0.5*** | 2.95 ± 0.37* | 0.129 ± 0.03*** | 4.97 ± 0.6*** | 0.33 ± 0.02*** | 15.76 ± 0.1*** |
Kidneys |
C | 25.51 ± 0.03 | 1.70 ± 0.31 | 0.238 ± 0.05 | 1.08 ± 0.12 | 1.955 ± 0.213 | 14.306 ± 0.09 |
DTrAqF | 15.41 ± 2.13 | 0.21 ± 0.03 | 0.247 ± 0.04** | 0.86 ± 0.16 | 3.44 ± 0.9* | 14.18 ± 0.18*** |
DTrHEE2 | 20.83 ± 2.01 | 0.85 ± 0.13 | 0.25 ± 0.04 | 1.53 ± 0.41* | 3.2 ± 0.35 | 15.20 ± 2.73* |
DTrMref | 25.19 ± 0.56 | 1.12 ± 0.23 | 0.297 ± 0.30 | 0.89 ± 0.06* | 2.52 ± 0.09** | 13.86 ± 0.72 |
DnTr | 13.03 ± 2.38* | 0.26 ± 0.39* | 0.162 ± 0.13*** | 2.53 ± 0.04*** | 5.587 ± 0.30*** | 20.583 ± 0.16*** |
DTrAqF: Diabetic treated with aqueous fraction; DTrHEE: Diabetic treated with hydro-ethanolic extract; DTrMref: Diabetic treated with metformin reference molecule; C: Rat treated with physiological water; DnTr: Untreated diabetic; Cat: Catalase; SOD: superoxide dismutase; GSH: reduced glutathione; CP: carbonyl proteins; O2●: superoxide anion; MDA: malondialdehyde. |
Data are presented as mean ± SD. *: significant difference at P < 0.05; ***: extremely significant difference at P < 0.001 and **: highly significant difference at P < 0.01. |
In our study, the organs (pancreas, liver, and kidneys) of diabetic control rats showed significantly reduced levels of Cat and GSH (P < 0.05), while MDA levels were significantly increased (P < 0.001 and/or P < 0.05), as presented in Table 5. This indicates increased oxidative stress in these organs due to diabetes.
Supplementation with the olive pomace (OP) extract reduced oxidative stress in these organs by stimulating the activity of antioxidant enzymes: SOD and Cat, which neutralize free radicals and increasing the level of glutathione (GSH): A crucial antioxidant molecule. This is likely due to the influence of bioactive compounds and their ability to recycle vitamin E and scavenge free radicals, which may directly contribute to a reduction in GSH utilization [72–73]. Our results showed a significant reduction in MDA level, concomitant with an increase in SOD, GSH and Cat activities, in STZ diabetic rats treated with HEE and the aqueous fraction of OP. These results are comparable with those published by [16, 41].
Our biochemical, hematological, and metabolic results found in our study are in agreement with the histological observations, at the level of all the pancreas, liver and kidneys sections, in untreated and treated diabetic rats.
Histopathology of pancreas revealed β-cell degeneration and islet cellular necrosis with reduced size in the diabetic control rats (Fig. 2d) compared to the treated group. The islet morphology, size, and overall architecture in the treated group (Fig. 2b, 2c and 2e) closely resembled that of the healthy control group (Fig. 2a). In the treated group, the islets displayed an intact cell structure, resulting in a homogeneous appearance and, for the most part, a more regular shape. This suggests an increase in islet cell numbers and restoration of islet contour. These observations are in favor of islet regeneration, under the effect of our extract. These results concur with those of [5, 75].
Other studies showed that treating rat pancreatic tissue with hydroxytyrosol (HT) exhibits β-cell protective properties by stimulating pancreatic insulin secretion and increasing antioxidant activity [72, 75]. For the selected photomicrographs of kidneys, no morphological differences observed in the treated diabetic groups (Fig. 3b, 3e and 3c) which appear similar to the control group (Fig. 3a). Renal glomeruli (G) and convoluted tubes (CT) with cells arranged regularly similar to the control suggesting a healthy kidney without signs of alteration or inflammation. However, in the untreated diabetic group (Fig. 3d), the histological changes in glomeruli observed, are characteristic of diabetic nephropathy. A glomerulus is a structure in the kidney that plays a key role in filtering blood to form urine. The granularity and crescent formation suggest alterations in the normal structure of the glomerulus, likely resulting from the accumulation of abnormal substances. A smaller glomerulus may indicate atrophy or a decrease in the size of this essential structure for renal function. Bowman's space is a part of the glomerulus where filtrate is collected before being transported into the renal tubules. Dilation of this space can result from increased pressure on the glomerular structures.
These histological changes suggest alterations in the glomerulus, characteristic of diabetic nephropathy. These alterations can lead to impairment of renal function, especially in diabetes. Numerous studies using experimental rodent models of diabetes have shown that HT can have beneficial in vivo effects against diabetes showed that HT decreased oxidative damage, enhanced antioxidant capacity and decreased inflammation. These data suggest that HT has the potential to protect organs and tissues from damage caused by diabetes [76].
In the histological analysis of liver tissue for the untreated diabetic rats (Fig. 4d) exhibit alterations highlights lesions in the cytoplasm of hepatic cells, including vascular congestion (CV) associated with the presence of inflammatory cells (CI) between recurrent trabeculae. Additionally, several Centro lobular veins containing red blood cells in their lumen are observed in variable quantities. Foci of cytolysis are also present, covering extensive areas of hepatic tissue. These areas are characterized by cellular destruction at both nuclear and cytoplasmic levels, accompanied by inflammation. [77–78] have shown that diabetes induced in rats triggers morphological and ultrastructural changes in the liver, closely resembling human diseases. For the selected photomicrographs of liver sections from the control group rats (Fig. 4a), histological sections reveal a normal architecture with distinct lobules. A well-defined central vein is observed, bordered by endothelial cells, and hepatocytes with well-defined nuclei are separated by narrow sinusoids. Normal liver architecture is clearly depicted, highlighting the central vein (VC), hepatocytes (H), and sinusoids (S). Furthermore, a normal architecture of a central vein is also represented.
Histological analysis of liver tissue from diabetic rats treated with olive pomace extract and metformin reveals promising results (Fig. 4b, 4e and 4c). These sections show a normal liver architecture, comparable to the healthy control group (Fig. 4a). This indicates the potential efficacy of the extract in mitigating the damaging effects and tissue toxicity induced by streptozotocin (STZ). This observation may not solely reflect the protective nature of the extract against oxidative stress. The STZ dose of 45 mg/kg may not have been sufficient to maintain a robust diabetic state in the animals. Individual variations in the animals' sensitivity to STZ or the potential anti-inflammatory properties of the extract, beyond its antioxidant effects, could also contribute. It is essential to recognize these limitations and continue research to fully understand the mechanisms of action of the extract and confirm its long-term hepatoprotective efficacy.
Although the results are in line with similar observations by [34], further exploration is required to definitively link the extract's bioactive molecules to specific protective mechanisms. In addition, studies by [63–64] demonstrate the hepatoprotective properties of hydroxytyrosol (HT) (an important bioactive molecule in the extract) through mechanisms such as reduced apoptosis, increased antioxidant activity and improved hepatocyte viability. Studying the presence and potential contribution of HT within olive pomace extract could provide a deeper understanding of its observed effects.