The Effect of Vitamin E, Β-Carotene, and Acidophilus Probiotics on The Salivary Gland Flow Rate in Streptozotocin-Induced Diabetes in Rats: An in-vivo Study

Abstract

Purpose

Diabetes is closely linked to reduced salivary flow rate, contributing to oral complications such as infections, dental caries, and periodontal disease. Vitamin E, β-carotene, and probiotics may help counteract these effects by neutralizing reactive oxygen species, reducing cellular damage, and preserving glandular function. This study aimed to evaluate the effects of the effect of vitamin E, β-carotene, and probiotics blend on the salivary glands of rats with Streptozotocin (STZ)-induced diabetes, addressing gaps in current research.

Methods

Eighty rats were induced with diabetes using a single intraperitoneal injection of STZ (60 mg/kg). They were randomly divided into control and experimental groups, which were further subdivided into ten subgroups: 2-week and 4-week saline, oil, vitamin E, β-carotene, and Probiotic Blend. Supplementation was administered daily via oral gavage. Salivary flow rate (SFR) was measured at three time points: before diabetes induction, after induction, and following supplementation, using pre-weighed cotton balls. Data were analyzed using SPSS.

Results

There was a significant reduction in SFR after diabetes induction (pre-diabetes: M=1.1 mL, SD=0.42; post-diabetes: M=0.6 mL, SD=0.24; t=12.925, p=0.000). On the day of sacrifice, Mann–Whitney U-tests showed significant differences in SFR between groups (p=0.000). The Kruskal–Wallis test also indicated significant differences across all groups (p<0.001). After 2 weeks, the highest SFR was observed in the vitamin E group (1.0 mL), followed by β-carotene (0.9 mL) and probiotics (0.8 mL), while the saline group recorded the lowest (0.3 mL). After 4 weeks, vitamin E again showed the highest SFR (1.1 mL), followed by β-carotene (0.9 mL), probiotics (0.7 mL), and oil (0.7 mL), with saline remaining the lowest (0.4 mL).

Conclusion

This study highlights the potential of vitamin E, β-carotene, and probiotics as alternative therapies for managing diabetes-related hyposalivation. Further research is required to validate their role in improving oral health among diabetics.

Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by absolute or relative insulin deficiency, resulting in highly elevated blood glucose levels. It is characterized by complex pathogenesis and heterogeneous clinical manifestations.1,2

According to the International Diabetes Federation, 10.5% of the global population (537 million adults aged 20–79), had diabetes in 2021. This prevalence is projected to increase by 46% by 2045.3

Hyposalivation, defined as oral dryness caused by insufficient or absent salivary secretion, is a prevalent oral manifestation and complication of diabetes.4 It is further classified into true hyposalivation, that arises from salivary gland dysfunction, and pseudo hyposalivation, also known as symptomatic xerostomia, where patients experience a subjective sensation of dry mouth despite normal salivary gland function.5

Current treatments for hyposalivation include salivary stimulants, muscarinic agonists, and artificial salivary substitutes. Systemic options such as pilocarpine and cevimeline, both muscarinic agonists, act on M1 and M3 receptors to alleviate xerostomia but are associated with adverse effects. Despite its broad activity, pilocarpine may cause sweating, gastrointestinal issues, and flushing, and is contraindicated in certain conditions such as uncontrolled asthma and glaucoma.6,7 Cevimeline, with higher M3 receptor affinity, minimizes some systemic effects but affects various physiological systems due to the widespread distribution of muscarinic receptors.8–11 Artificial saliva substitutes temporarily relieve dryness by lubricating the oral mucosa but lack the enzymes and proteins found in natural saliva, necessitating frequent use and incurring higher costs.12–15

Diabetes has been strongly associated with oxidative stress.16–20 Several studies have demonstrated that oxidative stress plays a crucial role in the development and progression of diabetes, along with its complications.21–23 Oxidative stress arises from the detrimental effects of glucotoxicity and lipotoxicity, both closely linked to diabetes and contributing to β-cell dysfunction.24 Furthermore, chronically elevated intracellular reactive oxygen species (ROS) levels in adipocytes, following mitochondrial malfunction, cause insulin resistance by inhibiting insulin signaling.25

Several studies have shown that oxidative stress markers are associated with increased insulin resistance.26,27 Oxidative stress can severely disrupt the function of insulin receptors along with cell damage. This disruption results in diminished insulin sensitivity, and worsening metabolic conditions like type 2 diabetes.

Patients with type II diabetes show altered mitochondrial morphology and impaired respiratory chain function, indicating that mitochondria play a crucial role in diabetes pathogenesis.28 Although the specific effect of mitochondrial dysfunction on salivary glands remains poorly understood, mitochondria are crucial for regulating salivary output via calcium mobilization in submandibular gland cells.29 Consequently, mitochondrial impairment may disrupt salivary gland function, resulting in morphological changes, and adversely affecting oral health.30–32 The evaluation of oxidative stress markers in rats with diabetes mellitus induced by STZ showed oxidative damage to the salivary glands. The results indicated that the major salivary glands underwent increased oxidative stress in STZ-treated rats, regardless of the duration of the disease. Notably, the parotid glands displayed a higher vulnerability, exhibiting a more pronounced extent and diversity of oxidative damage in comparison to the other glands.33

Rats with insulin resistance exhibited elevated markers of oxidative stress alongside a notable reduction in stimulated salivary flow rate. Suggesting that dysfunctions in the salivary glands may be detected early in the progression of this disease, even during the prediabetic phase.34–36 Therefore, addressing oxidative stress through antioxidant mechanisms may offer a practical approach to improving insulin signaling and addressing diabetes and insulin resistance.

Antioxidants are essential for reducing oxidative processes and neutralizing the harmful effects of ROS.37,38 Dietary antioxidants, including vitamins, carotenoids, polyphenols, flavonoids, and bioflavonoids, serve as external barriers against oxidative stress in addition to the body’s internal defensive mechanisms. The synergy between enzymatic and non-enzymatic improve components, along with dietary antioxidants, enhances body’s overall ability to combat oxidation damage. This intricate defense mechanism demonstrates how these elements collaborate to produce a robust and adaptable response to oxidative stress.39

Vitamin C is a potent antioxidant that helps protect salivary gland cells from oxidative stress and damage. By neutralizing free radicals, it supports the overall health and function of these glands, which can enhance saliva production.40 Vitamin C, widely recognized for its role in enhancing immune function, also plays a crucial role in stimulating salivary production through the synthesis of collagen, an essential protein in the structural integrity of salivary glands.41 On the other hand, vitamin E helps protect salivary gland cells from oxidative stress and damage by neutralizing free radicals. Thus, reducing inflammation, enhancing cellular repair and help maintain the balance of electrolytes in saliva This protection supports the overall health and function of the salivary glands, which can enhance saliva production.26,42

Probiotics can positively influence salivary flow rate through several mechanisms including balancing oral microbiome. Certain probiotic strains, such as Levilactobacillus brevis CD2, have been shown to reduce inflammation in the oral cavity. Lower inflammation can lead to better salivary gland function and increased saliva production.43–45 Probiotics are live bacteria that produce various beneficial compounds, including bacteriocins.46 It adds functional value to specific foods. Lactic acid bacteria are commonly included as probiotics because of their health benefits. Several studies have demonstrated the favorable effects of lactic acid bacteria, including their immunoregulatory, antioxidative, and anti-inflammatory provides, making them valuable functional food additives.47–49 Certain strains also contribute to maintaining dental hygiene and salivary gland function.50 Moreover, probiotics such as Lactobacillus have been demonstrated to regulate oral functions, including cleanliness and anti-inflammation.51,52 Probiotic strains have the potential to affect both the rate and composition of salivary flow; however, no biological or physiological mechanisms have been reported to explain these effects. One proposed mechanism of bacteriotherapy involves its ability to inhibit oral pathogens.53 The study aims to evaluate the effect of vitamin E, β-carotene, and Acidophilus probiotics on the salivary gland in rats with Streptozotocin (STZ-induced diabetes), The findings of this study may facilitate the development of innovative alternative strategies for managing diabetes-induced salivary gland dysfunction.

The STZ-induced diabetes model is commonly used to induce diabetes in rodents by selectively destroying pancreatic β-cells, leading to hyperglycemia and metabolic disturbances similar to those seen in humans. It is especially useful for studying diabetic complications, including hyposalivation, under controlled conditions.

However, this model has limitations. As an animal-based, acute-onset model, it does not fully replicate the chronic progression and complexity of human diabetes. Therefore, while the model is valuable for preclinical research, results should be interpreted with caution and further validated in human studies to ensure clinical relevance.

Materials and Methods

Animals and General Housing Conditions

This study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council, Washington, DC, USA). All animal procedures were approved by the local ethics review committee of the Center of Research (KSU-SE-23-63) and were performed in accordance with the local laws and regulations. The experiment was carried out on healthy adult albino Wistar rats weighing (230–380) g aged 2–3 months. They were housed four per cage under standard laboratory conditions at room temperature, with a 12- h light: dark cycle. They were fed standard rat chow and had free access to food and water.

Induction of Diabetes

A single intraperitoneal dose of freshly prepared STZ (Sigma-Aldrich Corp., St. Louis, MO, USA) mixed with freshly prepared citrate buffer solution (pH 4.5) was administered to the overnight-fasted rats. The STZ dosage was 60 mg/kg body weight. 3 days later, fasting blood glucose was measured using a tail blood sample using a blood glucose monitoring system. Diabetes in rats fasted overnight following STZ injection was confirmed by observing blood glucose levels reaching 200 mg/dL or higher. Following confirmation of diabetes, the respective supplements were administered. The dosages of Vitamin E, β-Carotene, and probiotics were selected based on previously established protocols and supporting literature to ensure therapeutic relevance and safety.54–56

Experimental Design

80 diabetic rats were divided randomly into two main control groups and one experimental group, further subdivided into ten groups as follows:

Control Groups

1. 2-week saline control (2wk saline): eight diabetic rats received saline for 2 weeks.

2. 4-week saline control (4wk saline): eight diabetic rats received saline for 4 weeks.

3. 2-week olive oil control (2wk oil): eight diabetic rats received olive oil for 2 weeks, every other day through oral gavage.

4. 4-week olive oil control (4wk oil): eight diabetic rats received olive oil for 4 weeks daily through oral gavage.

Experimental Groups

• 5.2-week vitamin E (2wk vit E): eight diabetic rats received vitamin E for 2 weeks (100 mg/kg BW + olive oil vehicle), daily through oral gavage.
• 6.4-week vitamin E (4wk Vit E): eight diabetic rats received vitamin E for 4 weeks (100 mg/kg BW + olive oil vehicle), daily through oral gavage.
• 7.2-week β-carotene (2wk β-carotene): eight diabetic rats received β-carotene for 2 weeks (10 mg/kg BW + olive oil vehicle), daily through oral gavage.
• 8.4-week β-carotene (4wk β-carotene): eight diabetic rats received β-carotene for 4 weeks (10 mg/kg BW + olive oil vehicle), daily through oral gavage.
• 9.2-week probiotic (2wk probiotic): eight diabetic rats received acidophilus probiotics for 2 weeks (100 mg/kg BW), daily through oral gavage.
• 10.4-week probiotic (4wk probiotic): eight diabetic rats received acidophilus probiotics for 4 weeks (100 mg/kg BW), daily through oral gavage.

(Acidophilus Probiotic Blend: (Lactobacillus acidophilus (La-14), Bifidobacterium lactis (BI-04), Lactobacillus plantarum (Lp-115), Lactobacillus casei (Lc-11), Lactobacillus rhamnosus (Lr-32), Lactobacillus paracasei (Lpc-37), Bifidobacterium breve (Bb-03), Streptococcus thermophilus (St-21), Lactobacillus salivarius (Ls-33), Bifidobacterium longum (BI-05))).

Collection of Saliva

Baseline salivary flow rate (SFR) was measured before the induction of diabetes. Rats were anesthetized using an intraperitoneal injection of ketamine at a dose of 1 mL/kg body weight,57 To stimulate salivation, pilocarpine hydrochloride was administered intraperitoneally at a dose of 2 mg/kg, dissolved in isotonic saline (prepared from 5 mg Salagen tablets). Saliva was collected over a 10-minute period using pre-weighed sterile cotton balls gently inserted into the oral cavity. After collection, the cotton balls were weighed again, and the SFR was calculated by determining the difference between the initial and final weights (Figure 1).

Figure 1.

SFR measurement. (A) Cotton is pre-weighted before SFR collection. (B)Weighed cotton is then placed inside the rat mouth, (C)after saliva collection period, the cotton was weighed again. The Saliva volume produced was determined by subtracting initial weight from the final.

Post-Induction Animal Well-Being

Rats were monitored for the cleanliness of their fur, the consistency of their feces, indications of dehydration, lethargy, significant weight loss, reduced consumption of food or water, skin conditions or infections, and overall signs of distress as assessed by the facility’s veterinarian.58

Statistical Analysis

Data was analyzed using SPSS statistical software (IBM SPSS Statistics for Windows, Version 26.0). Armonk, NY: IBM Corp). Descriptive statistics were generated for each variable. Comparisons between groups were performed using the Mann–Whitney U-test and Kruskal–Wallis test with post-hoc analysis. Statistical significance was set at p<0.05.

Results

The Shapiro–Wilk test was performed to assess the normality of the data. Nonparametric statistical methods were used as the data did not follow a normal distribution. Descriptive statistics were used and the Mann–Whitney U-test and Kruskal–Wallis test with post-hoc analysis were used to compare the mean ranks of SFR and glucose levels among the 10 study groups.

The sample consisted of 80 adult male albino Wistar rats aged 2–3 months, weighing 231–375 g (mean=282.8 g, SD=38.3805). SFR were compared at three distinct time points: prior to diabetes induction (Pre-SFR), after diabetes induction (SFR after DM), and post-intervention on the day of sacrifice (Final SFR). The data are summarized in Table 1. Before diabetes induction, the average SFR was 1.13 mL/10 min (SD=0.42). Following the confirmation of diabetes, the mean saliva production decreased to 0.5 mL/10 min (SD=0.2427) and 0.6 (SD=0.4239) among experimental and control groups respectively, indicating an approximate 50% reduction in saliva production. The mean glucose level on the confirmation day of diabetes was 408.952 mg/dl (SD=52.5959 mg/dL). A Mann–Whitney U-test was performed to compare the average of the final glucose levels between the experimental and control groups, revealing a significant difference (U= 535.5, p=0.019). Significant group differences are represented in Figure 2. Wilcoxon signed-rank test was performed to assess the effect of DM on SFR, indicating a significant difference between the pre-saliva and final saliva (z=−6.052; p=0.003, r= 0.68) and SFR after induction (z=−2.987; p=0.000, r= 0.35).

Table 1.

Mean Salivary Flow Rate of Groups Prior to Induction (Pre-Saliva), After DM Induction (Saliva After DM) and Saliva on Scarification Day (Final Saliva)

Variable	Group		Pre-Saliva	Saliva after DM	Final saliva
Experimentals	Vitamin E 2 Weeks	Mean	1.1000	0.6333	0.9500
		Std. Deviation	0.47509	0.16619	0.41662
	Vitamin E 4 Weeks	Mean	1.0000	0.3400	1.1000
		Std. Deviation	0.22678	0.06761	0.27775
	β -Carotene 2 Weeks	Mean	1.1167	0.6000	0.9400
		Std. Deviation	0.38699	0.18516	0.25967
	β -Carotene 4 Weeks	Mean	1.1000	0.3800	0.8600
		Std. Deviation	0.49570	0.11212	0.49742
	Probiotics 2 Weeks	Mean	1.3667	0.6833	0.8000
		Std. Deviation	0.63845	0.40854	0.49281
	Probiotics Weeks	Mean	0.9833	0.3667	0.6667
		Std. Deviation	0.13540	0.11547	0.20471
	Total	Mean	1.1111	0.5006	0.8861
		Std. Deviation	0.42386	0.24269	0.38105
Controls	Oil 2 Weeks	Mean	1.4800	0.5200	0.4000
		Std. Deviation	0.69364	0.13522	0.16903
	Oil 4 Weeks	Mean	1.0600	0.7000	0.7250
		Std. Deviation	0.23664	0.16903	0.29940
	Saline 2 Weeks	Mean	1.1200	0.8200	0.3000
		Std. Deviation	0.22297	0.20284	0.15119
	Saline 4 Weeks	Mean	1.0000	0.5250	0.4333
		Std. Deviation	0.16903	0.12392	0.03086
	Total	Mean	1.1650	0.6412	0.4646
		Std. Deviation	0.41829	0.19939	0.24060

Figure 2.

Graphical representation of the mean differences of groups’ final glucose level.

The mean SFR of the groups, measured prior to induction, after induction, and on the day of sacrifice is presented in Table 1 and Figure 3.

Figure 3.

Graphical representation of the mean differences of groups’ final SFR level.

Mann–Whitney U-tests indicated a significant difference in SFR between the experimental and control groups on the day of sacrifice (U=251.5, p=0.000).

The Kruskal–Wallis test was performed to determine whether statistically significant differences existed in the final saliva production among the ten study groups. The results indicated statistically significant differences between the groups (p<0.001). This was followed by a post-hoc pairwise comparison to assess the differences among the study groups.

No significant difference was observed in final saliva production between the saline 2wks and saline 4wks group (p=0.293). In contrast, a significant difference was found when comparing the saline 2wks and 2wks probiotics, vitamin E, and β-Carotene (p=0.008, p=0.001 and p=0.000, respectively). However, no significant differences were observed between the intervention groups. The 4wks saline group demonstrated significantly lower final SFR compared to the vitamin E, β-carotene, and oil groups. The results are summarized in Table 2. A borderline significant difference was noted between the 2 and 4wks oil group (p<0.045). Spearman’s rank-order correlation analysis revealed a moderate indirect correlation between final glucose levels and final SFR (r_s=−0.3, p=0.05).

Table 2.

Pairwise Comparisons of Group SFR Mean After Intervention

Weeks	Sample 1-Sample 2	Test Statistic	Std. Error	Std. Test Statistic	Sig.
2 Weeks	Saline-Oil	3.375	5.787	0.583	0.560
	Saline-Probiotics	−15.250	5.787	−2.635	0.008
	Saline-Vit E	19.500	5.787	3.369	0.001
	Saline- β -Carotene	−20.625	5.787	−3.564	0.000
	Oil-Probiotics	−11.875	5.787	−2.052	0.040
	Oil -Vit. E	−16.125	5.787	−2.786	0.005
	Oil - β -Carotene	−17.250	5.787	−2.981	0.003
	Probiotics -Vit. E	4.250	5.787	0.734	0.463
	Probiotics- β -Carotene	5.375	5.787	0.929	0.353
	Vit. E - β -Carotene	−1.125	5.787	−0.194	0.846
4 Weeks	Saline-Probiotics	−10.563	5.824	−1.814	0.070
	Saline -Oil	12.063	5.824	2.071	0.038
	Saline- β -Carotene	−13.813	5.824	−2.372	0.018
	Saline -Vit. E	23.875	5.824	4.099	0.000
	Probiotics-Oil	1.500	5.824	0.258	0.797
	Probiotics - β -Carotene	3.250	5.824	0.558	0.577
	Probiotics-Vit. E	13.313	5.824	2.286	0.007
	Oil - β -Carotene	−1.750	5.824	−0.300	0.764
	Oil -Vite	11.813	5.824	2.028	0.029
	β -Carotene-Vit. E	10.063	5.824	1.728	0.077

Discussion

DM is widely recognized for its negative effects on oral health, particularly saliva production, a relationship substantiated by numerous studies.59–61 In this study, hyposalivation was observed in all rats following diabetes induction, with saliva production decreasing by approximately 50%. This significant reduction in salivary flow aligns with findings from Xiang et al, 2020, who reported similar results in mice Numerous studies have reported salivary gland dysfunction and hyposalivation in patients with diabetes, with varying results.62–64 In two systematic reviews, López-Pintor et al and Marques et al examined the effects on salivary gland function, specifically focusing on flow rate and chemical composition.61,65 While both studies provided evidence of the effect on salivary glands, the overall quality of the research was limited, resulting in weaker evidence. These findings underscore the prevalence of dry mouth and xerostomia in patients with diabetes. It is crucial to acknowledge that many studies did not account for the potential influences of age, glucose control, and medication, because numerous participants with diabetes were undergoing various treatments. Furthermore, most studies relied on subjective assessments rather than objective measurements. Our study revealed that diabetes induces significant changes in salivary production, resulting in varying levels of hyposalivation. Additionally, because the experience of this condition is subjective, objective measurements such as sialometry should be incorporated into the clinical monitoring of diabetes. Adopting multidisciplinary strategies in clinics that provide care for patients with diabetes is essential to improve patient outcomes.

Consistent with the findings of Bernardi et al, our study demonstrated a relationship between elevated glucose concentrations and salivary flow rate, indicating that higher glucose levels correspond to reduced saliva production. Existing literature suggests that individuals with diabetes often exhibit elevated salivary glucose levels, that may have potential diagnostic and monitoring applications for glycemic conditions.66–68 The present findings highlight the potential utility of SFR as an indicator of glucose levels in patients with diabetes. However, there remains a paucity of evidence in the literature regarding this particular association.

The findings of this study demonstrated that Vitamin E supplementation significantly enhanced saliva production, yielding the most favorable outcomes among the tested groups. Although vitamin E demonstrated promising effects in promoting salivation, no statistically significant difference was observed between the 2wks and 4wks assessments. However, an increase in saliva production was observed within the normal range. Additionally, Vitamin E has been associated with positive effects on diabetes and its complications.69,70 Notably effectiveness of Vitamin E supplementation may be modulated by factors such as exercise, dietary intake, and oxidative stress levels. These variables could potentially affect the overall efficacy of supplementation, indicating that increasing the dose or extending the duration of intake may yield further benefits.

Although studies specifically focusing on the effects of vitamin E on diabetes-related hyposalivation are scarce, several investigations have highlighted its beneficial effects on saliva production.71–73 For instance, a randomized controlled trial involving patients with Sjögren’s syndrome revealed that vitamin E interventions significantly improved salivary flow rates.73 Additionally, a double-blind controlled trial assessed the efficacy of a vitamin C/E complex in mitigating radiation-induced xerostomia in patients with head and neck cancer. The findings revealed that participants receiving vitamin supplementation experienced substantial improvements in xerostomia symptoms at 1- and 6-months following radiotherapy compared to those receiving a placebo, underscoring the protective role of vitamin E in combination with vitamin C.71 Notably both the studies utilized a combination of vitamins, making it challenging to attribute the observed effects solely to vitamin E supplementation.

Furthermore, Chitra et al investigated the effects of α-tocopherol on salivary secretion rates and related parameters in oral cancer patients undergoing radiation therapy.72 Their findings indicated an increase in salivary flow rates and preservation of salivary composition with α-tocopherol administration.72 However, the effect on SFR was not definitively conclusive, and the characteristics of the samples were inconsistent.

Research specifically focusing on the effects of β-carotene on saliva production is currently scarce. The present study demonstrates that β-carotene supplementation significantly increased saliva production in diabetic rats. However, after 4 weeks, the outcomes varied compared to that of the other groups, despite the SFR remaining higher than that of the control group. Csepanyi et al reported that low doses of β-carotene are effective in treating diabetes and preventing its complications.74 This suggests that both the dosage and duration are critical factors in determining efficacy. Supporting this, Funegård et al, reported that rats supplemented with α-tocopherol and β-carotene exhibited increased salivary secretion rates and a more stable saliva composition following irradiation, compared to those on a standard diet.75 Overall, these findings indicate that β-carotene, in combination with vitamin E, may serve as a promising therapeutic option for enhancing salivary flow and mitigating diabetes-induced damage to salivary glands. Further research is warranted to explore their combined application for managing xerostomia.

Vitamin E plays a crucial role in protecting polyunsaturated fatty acids, cellular membrane components, and low-density lipoproteins from oxidative damage caused by free radicals. In addition to its well-established antioxidant function, Vitamin E exhibits significant molecular properties, effectively scavenging reactive oxygen and nitrogen species to prevent oxidative damage associated with various diseases.76 By mitigating oxidative stress, Vitamin E may help preserve the structural and functional integrity of salivary gland cells, thereby supporting sustained saliva production and secretion. This antioxidant action may also counteract the ROS-mediated impairment of acinar cells, which are primarily responsible for salivary output.

Similarly, carotenoids are recognized for their potent antioxidant properties. These compounds act as highly efficient scavengers of reactive oxygen species (ROS), particularly by quenching singlet oxygen, one of the most reactive oxygen forms, thereby mitigating oxidative stress.77,78 By reducing ROS levels, carotenoids may indirectly support the physiological function of salivary gland tissues, maintaining or enhancing salivary flow. Furthermore, carotenoids may modulate intracellular signaling pathways related to oxidative stress and inflammation, offering additional protective mechanisms at the cellular level.

Taken together, the antioxidant properties of both Vitamin E and β-carotene provide a plausible explanation for the improvements in salivary flow observed in this study. By neutralizing oxidative stress, these compounds may preserve acinar cell viability, prevent damage to glandular structures, and maintain enzymatic functions critical for saliva secretion. These mechanisms collectively support their potential therapeutic role in managing diabetes-induced hyposalivation.

Probiotic supplementation resulted in a considerable increase in saliva production compared to that in the saline control group after 2 weeks. Although this beneficial effect persisted for 4 weeks, it was not statistically significant, suggesting that probiotics may lose efficacy with prolonged treatment. Studies utilizing Lactobacillus extracts have investigated their effects on dry mouth, demonstrating that Lactobacillus enhances saliva production and holds promise as a valuable probiotic for managing dry mouth.79,80 Probiotics are live microorganisms capable of rapidly responding to environmental changes. Diabetes has been associated with both qualitative and quantitative alterations in the gut microbiota.81 These modifications may prevent the probiotics from colonizing and growing, thereby reducing their therapeutic effects.

Vitamin E may offer more sustained benefits for improving salivary flow. Similarly, supplementation with β-carotene and probiotics has shown potential to improve the SFR and may also influence the regulation of blood sugar levels.82–84 While probiotics can lead to the immediate improvements in saliva production, their long-term effectiveness requires further investigation. Additionally, these findings highlight the potential of olive oil as a natural enhancer of saliva secretion, warranting additional research on its benefits on oral health, particularly for those with dry mouth conditions.

Our investigation indicated an inverse correlation between salivary flow rate and glycemia. Salivary glucose levels is reported to be higher in diabetic patients compared to healthy controls. Unstimulated parotid salivary glucose levels have shown a strong correlation with blood glucose levels, making it a potential indicator for DM. However, the correlation can vary significantly among individuals.67,85 This research is the first report highlighting the inverse relationship between salivation and blood glucose levels.

This study emphasizes the role of antioxidants in providing protective effects. While the precise mechanisms by which antioxidants operate are not completely elucidated, it is hypothesized that they may contribute to the preservation of cellular and vascular integrity. This may occur through the scavenging of free radicals and the neutralization of reactive oxygen species (ROS) generated in chronic diseases.86

STZ induces symptoms in animals that closely mimic those of diabetes in humans. Nonetheless, there are certain challenges and limitations to consider. The STZ model mainly replicate type 1 diabetes by targeting and destroying pancreatic β-cells, whereas the majority of diabetes cases in humans (95%) are type 2 diabetes. In STZ-induced models, diabetes manifests rapidly, unlike the slow onset observed in humans, which affects the study of disease progression. Furthermore, STZ-induced diabetes in rats results in more severe metabolic problems, such as extreme hyperglycemia, and does not represent the insulin resistance characteristic of type 2 diabetes. Although valuable for specific research purposes, the STZ-induced diabetes rat model has significant limitations in accurately representing human diabetes, particularly type 2 diabetes.87–90

Conclusion

The results of this research highlight the potential of vitamin E, β-carotene, and probiotics as antioxidants and possible alternative treatments for managing hyposalivation linked to DM. These natural supplements present a solution with fewer systemic side effects compared to current treatment methods. Antioxidants may help improve salivary flow rates, possibly by maintaining structural integrity, which in turn enhances flow rate and oral health, along with glycemic control. Vitamin E and β-carotene showed notable improvements in saliva production, while probiotics initially showed effectiveness that diminished over time. Furthermore, the benefits observed from extended olive oil supplementation merit further exploration. Additional research is needed to investigate the potential synergistic effects of combined supplementation and to establish optimal dosages, treatment duration, and side effects.

Disclosure

The authors report no conflicts of interest in this work.