Camel Milk-Based Fermented Product “Inullact-Fito” Ameliorates Metabolic and Immunological Disturbances in Alloxan-Induced Diabetes

Abstract

Background/Objectives: This study examined the metabolic, oxidative, immunological, and histomorphological effects of the multicomponent fermented biological product derived from camel milk, Inullact-Fito, in comparison to metformin in a rat model of alloxan-induced diabetes resulting from insulin insufficiency. The model was chosen as an experimental system that replicates pancreatic β-cell damage induced by oxidative stress rather than insulin resistance. Methods: Alloxan-induced diabetes was used to evaluate metabolic, oxidative, immunological, and histomorphological alterations. Metformin was utilized as a pharmacological comparator. Blood glucose levels, circulating insulin concentrations, markers of oxidative stress and lipid peroxidation, immunoglobulin levels, CD4⁺/CD8⁺ T cell balance, and pancreatic histostructure were assessed. Results: Alloxan administration led to substantial hyperglycemia, oxidative stress, immunological imbalance, and structural damage to pancreatic tissue. Following therapy with Inullact-Fito, blood glucose levels reduced dramatically (from 21.9 ± 0.22 to 9.85 ± 0.10 mmol/L, p < 0.05), circulating insulin concentrations were largely corrected, oxidative stress and lipid peroxidation markers decreased. Immunological evaluation revealed decreased serum immunoglobulin M and IgG levels (p < 0.05) and partial normalization of the CD4⁺/CD8⁺ T cell balance. Metformin showed comparative effects; however, its activity in this model is limited by its primary mechanism related to insulin resistance. Conclusions: Overall, the data reveal that Inullact-Fito combines metabolic, antioxidant, and immunomodulatory actions under experimental oxidative and metabolic stress conditions. Further research using models of insulin resistance and type 2 diabetes, as well as long-term clinical trials, is needed to fully evaluate the therapeutic potential, safety profile, and translational importance of this fermented dairy product as a functional nutritional intervention.

1. Introduction

Diabetes mellitus (DM) is a common metabolic disorder that is of increasing concern to healthcare systems worldwide due to its increasing prevalence. Type 2 DM accounts for over 90% of cases [1,2,3]. The physiological and molecular mechanisms, as well as the adverse consequences of diabetes, are currently being actively studied worldwide. There is growing scientific interest in bioactive substances that can safely regulate the metabolic disorders associated with diabetes.

In this regard, the identified antioxidant, bactericidal, immunomodulatory, adaptogenic, and hypoglycemic properties of new bio-preparations have attracted the interest of specialists.

One of the challenges in treating patients with diabetes is the inadequate control of high blood glucose levels with exogenous insulin and other antidiabetic drugs. This necessitates the search for additional ways to optimize glycemic control [4,5,6,7]. For this purpose, not only drugs but also various natural compounds that affect carbohydrate metabolism are often used. Several studies, including that by Abdelaz et al. (2022), examined the potential of post-biotic compounds, such as gamma-aminobutyric acid (GABA), in camel milk for their antioxidant, immunomodulatory, and hypoglycemic properties [1]. Their results showed that these biologics, especially when combined with conventional treatments, can significantly improve metabolic regulation and reduce hyperglycemia and hyperlipidemia in experimental models of streptozocin-induced diabetes [8].

Functional foods, especially those derived from fermented milk probiotics, have attracted the attention of scientists and consumers due to their role in maintaining metabolic health and overall well-being [9,10,11].

It has been shown that these products, which contain microbes and microbial compounds, enhance metabolic processes, microbiological status, and physiological functions [12].

The incorporation of both lactic acid bacteria and milk-derived yeasts into fermented products has emerged as a promising strategy for formulating nutritionally balanced functional foods [13,14]. Given that the main goal of the state policy of the Republic of Kazakhstan in the field of technology is to transition to an innovative path of development based on selected priorities, this study is highly relevant.

The dairy industry in the 21st century, in accordance with the state concept of healthy nutrition, faces three scientific and technical challenges:

• Achieving a new qualitative level in the production and use of probiotics, symbiotics, and prebiotics;
• Developing biotechnologies and nanotechnologies for FNPs;
• Developing theoretical and applied bases for producing biologically active substances for creating biologics and parapharmaceutical products from dairy raw materials [15,16].

The definition of “food safety” refers to the condition of food products indicating the absence of unacceptable risk associated with harmful effects on humans and future generations, as regulated by the law “On Food Safety” [17]. Thus, special-purpose dairy products are crucial for ensuring human health.

Fermented milk-based products have gained recognition for their beneficial effects on gastrointestinal function and their potential contribution to preventive healthcare [18].

In this context, the task of scientifically and practically substantiating the use of camel milk to expand raw materials and create products that meet rational nutrition requirements is highly urgent. Traditionally, camel milk is consumed fresh or fermented. The importance and future potential of modern fermented dairy products are also noted in biotechnology and bioengineering [12,19,20].

Given the vast variety of plant material, the need to consider its complex effects, the individual approach required for individuals with diabetes, and the presence of possible contraindications or complications, careful verification in model experiments is essential. Therefore, searching for and pre-clinically testing plant material, followed by clinical studies, is highly relevant for creating sound recommendations for the pharmacological industry.

The alloxan-induced diabetes model was selected due to its reproducibility, cost-effectiveness, and selective toxicity toward pancreatic β-cells mediated by reactive oxygen species. While this model is widely used for preclinical screening of antidiabetic agents, it primarily reflects insulin-deficient diabetes and does not fully mimic insulin resistance, dyslipidemia, or chronic metabolic complications characteristic of type 2 diabetes. Therefore, the present study should be considered a proof-of-concept evaluation, and future investigations using insulin-resistant models (e.g., high-fat diet/streptozotocin or genetic models) are warranted [21,22].

Based on the above, a new multicomponent specialized fermented milk bioproduct, “Inullact-Fito” (a conditional name), was developed as a functional nutritional intervention with potential antidiabetic properties [23]. The product is based on camel milk combined with sugar-lowering extracts from medicinal plants. In the present study, its biological effects were evaluated in an alloxan-induced insulin-deficient diabetes model as a proof-of-concept, while recognizing that extrapolation of the findings to insulin-resistant type 2 diabetes requires additional experimental models.

The hypoglycemic agent metformin, a representative of the biguanide class, is widely used in the management of type 2 diabetes mellitus. Its pharmacological action includes enhancement of peripheral glucose utilization and inhibition of hepatic gluconeogenesis [24]. Metformin has also demonstrated efficacy in alloxan-induced diabetes models, which primarily reflect insulin deficiency rather than insulin resistance, and its effects may involve activation of I2-imidazoline receptors [25,26]. Therefore, metformin was selected as a reference comparator in the present study to assess and contextualize the metabolic, immunological, and histomorphological effects of the fermented milk bioproduct “Inullact-Fito” under conditions of experimental alloxan-induced diabetes.

The aim of this study was to compare the metabolic, oxidative, immunological, and histomorphological effects of the multicomponent fermented camel milk product “Inullact-Fito” with metformin in a rat model of alloxan-induced insulin resistance. Due to the mechanistic limitations of the model and the need for further research in insulin resistance and type 2 diabetes models, the primary focus was on changes in carbohydrate and lipid metabolism, indicators of oxidative stress, immunological responses, and pancreatic histomorphology.

2. Materials and Methods

2.1. Materials

2.1.1. Preparation and Ingredients for “Inullact-Fito”

The following ingredients were used in the production of Inullact-Fito, a fermented dairy product specially formulated for patients with diabetes: camel milk; bacterial starter cultures, including pure cultures of Thermophilic streptococci (Streptococcus thermophilus) and Lactobacillus bulgaricus; the low-calorie natural sweetener stevioside; and hypoglycemic plant extracts. The technological process involved controlled mixing and fermentation, with the addition of formulation components at appropriate concentrations and temperature.

A comprehensive evaluation was conducted to assess the organoleptic properties, physicochemical characteristics, microbiological quality, and vitamin content of the experimental samples. Product quality was assessed using standard analytical methods. Organoleptic properties were evaluated by a panel of qualified specialists experienced in the assessment of fermented dairy products and focused on key sensory attributes, including appearance, consistency, taste, aroma, and overall acceptability. Physicochemical parameters, including pH and titratable acidity, were determined using conventional methods for fermented milk products. Microbiological quality was assessed using standard microbiological techniques, including determination of the total viable cell count and the number of lactic acid bacteria. Vitamin content was analyzed using standardized biochemical methods. All physicochemical, microbiological, and biochemical analyses were conducted in accordance with internationally recognized standard procedures recommended by the International Organization for Standardization (ISO-16140-3-2021; ISO-6887-1) [27,28], and the Association of Official Analytical Chemists (AOAC) [29], using established laboratory protocols commonly applied for fermented dairy products.

Camel milk used for the preparation of the fermented product was obtained from Peasant Farm “Gulmayra” (Otyrar District, Turkestan Region, Republic of Kazakhstan), which also served as the supplier of the raw material. The bacterial starter culture consisted of Streptococcus thermophilus and Lactobacillus bulgaricus (starter culture code YO 20, 10 units), manufactured by microMilk S.r.l. (Brescia, Italy) and supplied by LLP “Vernal” (Almaty, Kazakhstan).

The plant-derived components included stevioside (LLC “Vitachai”, Moscow, Russia), Jerusalem artichoke extract containing 90% inulin (LLC “Ecodate”, Perm, Russia), chicory extract (Ecologica—Healthy Nutrition, Moscow, Russia), blueberry extract (“Opt Group”, Saint Petersburg, Russia), and ginseng extract standardized to 10% ginsenosides (World-Way Biotech Inc., Changsha, Hunan Province, China). The ginseng extract was supplied by Individual Entrepreneur “ProPorcia” (Shymkent, Kazakhstan).

2.1.2. Design of Study Groups

The experiment was conducted on 46 mature rats of both sexes, weighing 180–210 g, housed in a vivarium with a standard diet at the B. Atchabarov Research Institute of Fundamental and Applied Medicine of the Kazakh National Medical University, named after S.D. Asfendiyarov. Animals were housed under standard vivarium conditions with controlled temperature, humidity, a 12 h light–dark cycle, and free access to a standard laboratory diet and water ad libitum. This study was approved by the Ethics Committee of the Akhmet Yassawi International Kazakh-Turkish University (protocol No. 1 from 23 June 2017), and was conducted at the B. Atchabarov Research Institute of Fundamental and Applied Medicine. The rats were divided into four groups:

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Group 1—intact animals (n = 8);

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Group 2—control pathology: a model of diabetes without treatment (n = 14);

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Group 3—animals with diabetes that received Inullact-Fito at an experimental therapeutic dose of 500 mg/kg once per day throughout the experiment (n = 12);

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Group 4—animals receiving metformin at a dose of 100 mg/kg (n = 12).

The number of animals per group was determined based on an exploratory preclinical study design, ethical considerations following the 3R (Replacement, Reduction, Refinement) principles, and consistency with comparable experimental diabetes studies. Control animals were studied on a normal diet without the introduction of alloxan. All drugs were administered via a stomach probe daily, once per day, throughout the experiment. The maintenance and care of the animals, as well as their removal from the experiment, were performed in accordance with the “Rules for Carrying Out Work Using Experimental Animals”. The fermented milk bioproduct used in our experiments, under the provisional name “Inullact-Fito”, was developed within the framework of the implementation of the state scientific program of the Republic of Kazakhstan under No. 5570/GF4.

Animals were randomly assigned to treatment groups using a simple randomization method. Researchers responsible for biochemical, immunological, and histological studies avoided assigning groups during the evaluation of the results, in accordance with ARRIVE guidelines. The dose of Inullact-Fito (500 mg/kg) was selected as the experimental treatment dose based on preliminary studies and nutritional value. Metformin was administered at a dose of 100 mg/kg, which is commonly used as the reference antidiabetic dose in experimental rat models. All study drugs were administered orally once daily via gavage to ensure accurate dosing and sustained release.

2.2. Methods

2.2.1. Diabetes Model Induction and Laboratory Analysis Techniques

An experimental diabetes model was created in animals through intraperitoneal injection of a 5% alloxan monohydrate solution (100 mg/kg body weight, dissolved in 0.9% sodium chloride solution). Animals were fasted for 24 h with free access to water before alloxan administration.

Hyperglycemia was confirmed by measuring fasting blood glucose levels using a glucometer. Animals that exhibited a sustained increase in blood glucose levels compared to intact control animals were considered diabetic and included in the experimental groups.

The total duration of the experiment and experimental treatment was 28 days; assessments were conducted on days 7, 14, and 28 after diabetes induction. Treatment began after confirmation of hyperglycemia and was administered once daily.

This model was chosen due to its reproducibility and widespread applicability in preclinical screening of antidiabetic drugs. At the end of the experiment (day 28), two hours after the final administration of the test substances, animals were euthanized under deep anesthesia induced by thiopental sodium administered intraperitoneally at a dose of 150 mg/kg, which represents a commonly accepted overdose for terminal anesthesia in rats. Adequate depth of anesthesia was confirmed by the absence of pedal and corneal reflexes prior to tissue collection. The abdominal cavity was opened with a midline incision, and the pancreas and liver were carefully excised, rinsed in cold physiological saline to remove blood residues, blotted dry, and processed for biochemical and histological analyses. Serum insulin was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (IBL International GmbH, Hamburg, Germany). Blood glucose levels were measured using a Bionime Rightest GM300 glucometer (Bionime Corporation, Taichung City, Taiwan (R.O.C.)). Parameters of lipid and nitrogen metabolism were assessed using standard biochemical methods with Lachema reagent kits ( Lachema s.r.o., Brno, Czech Republic).

Lipid peroxidation intensity was assessed by measuring thiobarbituric acid reactive substances (TBARS), expressed as malondialdehyde equivalents, according to the method of Ohkawa et al. [30]. Serum catalase activity was measured spectrophotometrically using the Aebi method based on the rate of hydrogen peroxide dissociation [31]. Serum urea concentration was measured colorimetrically using diacetyl monoxime [32]. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Reitman–Frankel method based on transamination reactions [33].

2.2.2. Assessment of T-Cell Subpopulations and Immunoglobulin Levels in Alloxan-Induced Diabetes

Assessment of immunological reactivity in alloxan-induced diabetes involves the determination of T cell subsets in whole blood. T cell subsets were assessed by using indirect surface immunofluorescence with monoclonal antibodies from the ICO series (Russia), specific to rat CD4⁺ and CD8⁺ antigens. A simple antibody panel was used to detect CD markers: CD4⁺ (helper T lymphocytes) and CD8⁺ (suppressive/cytotoxic T lymphocytes) [34]. Fluorescent signals were assessed using a Zeiss Axiolab A1 fluorescence microscope (ZEISS Scope.A1, Carl Zeiss Microscopy GmbH, Jena, Germany), and the results were expressed as relative percentages (%) of lymphocyte subsets. Immunophenotyping was performed according to established immunological methods for the detection of surface antigens described in standard protocols [34]. The concentrations of immunoglobulins M and G in blood serum were determined using ELISA kits (CellTrend GmbH, Luckenwalde, Germany; Cat. No. 52500-1 and 52300-1, respectively), according to the manufacturers’ instructions.

2.2.3. Histostructure Visualization

The histostructure of the pancreas was examined with optical microscopy (ZEISS Scope.A1, Carl Zeiss Microscopy GmbH, Jena, Germany). Pancreatic tissues were fixed in 10% formalin, paraffin-embedded, sectioned at 4–5 μm using a standard rotary microtome, and stained with hematoxylin and eosin (BioVitrum, Saint Petersburg, Russia) according to standard protocols. Histological analysis and image acquisition were performed using a Granum light microscope (Granum Laboratory Ltd., Kharkiv, Ukraine) equipped with a Granum DSM 310 digital camera, and images were processed using ToupView software (version 3.7).

2.3. Statistical Analysis

Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., USA) (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation (SD). The normality of data distribution was assessed using the Shapiro–Wilk test. Given the exploratory nature of this study and the limited sample size, homogeneity of variances was not formally tested using Levene’s test. Between-group comparisons at each experimental time point (days 7, 14, and 28) were conducted using one-way analysis of variance (ANOVA) when parametric assumptions were considered acceptable. For data that did not meet assumptions of normality or when variance homogeneity could not be assured, the nonparametric Kruskal–Wallis test was applied. Repeated-measures or two-way ANOVA was not performed due to sample size limitations. A p value of less than 0.05 was considered statistically significant.

3. Results

3.1. Effects of Inullact-Fito on Biochemical Parameters in Alloxan-Induced Diabetes

Administration of Allexan to induce diabetes in laboratory animals resulted in significant disturbances in carbohydrate, lipid, and nitrogen metabolism.

Table 1. Effect of Inullact-Fito and metformin on biochemical blood parameters in alloxan diabetes in experimental rats.

Indicators	Animal Groups (n = 46)
	Intact Control	Control Pathology	“Inullact-Fito”	Metformin
7th day
Glucose, mM/L	6.01 ± 0.33	9.63 ± 0.10	6.81 ± 0.58 *	7.52 ± 0.09 *
Insulin, μIU/mL	2.94 ± 0.18	0.89 ± 0.20	1.35 ± 0.25 *	1.10 ± 0.10 *
ALT, mE/L	131.3 ± 42.5	191.1 ± 79.2	162.4 ± 12.6 *	170.0 ± 30.2 *
AST, mE/L	618.5 ± 179.0	1076.6 ± 446.5	915.1 ± 102.3 *	936.6 ± 93.2 *
Cholesterol, mM/L	0.63 ± 0.03	2.73 ± 0.14	1.43 ± 0.16 *	1.45 ± 0.06 *
Urea, mM/L	4.43 ± 0.07	12.63 ± 1.09	7.91 ± 0.65 *	6.92 ± 0.68 *
Creatinine, mM/L	73.15 ± 1.22	173.27 ± 9.50	129.02 ± 5.20 *	113.17 ± 7.45 *
14th day
Glucose, mM/L	6.01 ± 0.33	12.40 ± 0.14	8.06 ± 0.08 *	9.92 ± 0.09 *
Insulin, μIU/mL	2.94 ± 0.18	0.68 ± 0.28	1.70 ± 0.35 *	1.35 ± 0.15 *
ALT, mE/L	131.3 ± 42.5	175.2 ± 69.6	142.9 ± 11.2 *	154.7 ± 29.3 *
AST, mE/L	618.5 ± 179.0	966.0 ± 444.6	814.4 ± 98.6 *	852.3 ± 86.1 *
Cholesterol, mM/L	0.63 ± 0.03	2.36 ± 0.25	1.38 ± 0.11 *	1.30 ± 0.2 *
Urea, mM/L	4.43 ± 0.07	10.65 ± 1.7	5.42 ± 0.7 *	4.53 ± 0.36 *
Creatinine, mM/L	73.15 ± 1.22	149.35 ± 7.60	86.36 ± 4.83 *	92.6 ± 5.3 *
28th day
Glucose, mM/L	6.48 ± 0.64	21.9 ± 0.22	9.85 ± 0.10 *	13.9 ± 0.14 *
Insulin, μIU/mL	2.96 ± 0.16	0.48 ± 0.08	2.01 ± 0.10 *	1.60 ± 0.40 *
ALT, mE/L	131.3 ± 42.5	171.3 ± 100.3	132.8 ± 9.6 *	143.8 ± 22.9 *
AST, mE/L	618.5 ± 179.0	778.6 ± 375.9	757.3 ± 80.6 *	770.1 ± 92.3 *
Cholesterol, mM/L	0.63 ± 0.03	1.99 ± 0.67	1.13 ± 0.08 *	1.15 ± 0.13 *
Urea, mM/L	4.43 ± 0.07	8.90 ± 0.53	3.18 ± 0.18 *	3.56 ± 0.15 *
Creatinine, mM/L	73.15 ± 1.22	130.16 ± 8.50	77.60 ± 4.12 *	83.80 ± 4.50 *

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; mmol/L, millimoles per liter; mE/L, milli-enzyme units per liter; μIU/mL (mcME/mL), micro-international units per milliliter. Statistical notation: * p ≤ 0.05 indicates a statistically significant difference compared with the control pathology (alloxan-induced diabetes) group.

shows that blood glucose levels in the control group were significantly higher than in healthy animals at every measurement point (p < 0.05). Furthermore, cholesterol, urea, creatinine, ALT, and AST levels increased, while serum insulin concentrations decreased significantly.

Application of Inullact-Fito to diabetic rats resulted in a significant decrease in blood glucose levels on days 7, 14, and 28 compared to the control group (p ≤ 0.05). On day 28, glucose concentrations decreased from 21.9 ± 0.22 mmol/L in the control group to 9.85 ± 0.10 mmol/L in the Inullact-Fito-treated group. Serum insulin levels were significantly higher in the treatment group compared to untreated diabetic animals at all measurement points (p ≤ 0.05).

Treatment with Inullact-Fito resulted in significant reductions in serum cholesterol, urea, creatinine, ALT, and AST levels compared to the control group throughout all observation periods (p ≤ 0.05).

Metformin administration caused similar changes in biochemical parameters; however, blood glucose and insulin levels in the metformin-treated group differed significantly from those in the control group throughout the experiment (Table 1).

3.2. Lipid Peroxidation and Antioxidant Activity

As shown in

Table 2. The effect of Inullact-Fito and metformin on the processes of LPO and the activity of blood catalase in alloxan diabetes in experimental rats.

Indicators	Animal Groups (n = 46)
	Intact Control	Control Pathology	“Inullact-Fito”	Metformin
7th day
MDA in blood serum, μmol/mL	2.35 ± 0.10	6.72 ± 0.33	4.52 ± 0.52 *	5.72 ± 0.48 *
MDA in the pancreas, nmol/g	5.53 ± 0.60	8.62 ± 0.80	6.00 ± 0.60 *	7.20 ± 0.20 *
Catalase, mcat/L	21.32 ± 2.01	14.00 ± 1.32	18.2 ± 1.81 *	16.52 ± 1.63 *
14th day
MDA in blood serum, μmol/mL	3.27 ± 0.15	10.28 ± 0.11	3.95 ± 0.55 *	4.92 ± 0.65 *
MDA in the pancreas, nmol/g	5.53 ± 0.60	9.85 ± 0.10	8.00 ± 0.80 *	8.85 ± 0.90 *
Catalase, mcat/L	21.32 ± 2.01	12.01 ± 0.80	18.62 ± 1.81 *	15.41 ± 1.54 *
28th day
MDA in blood serum, μmol/mL	3.85 ± 0.40	15.4 ± 0.16	4.86 ± 0.50 *	7.47 ± 0.75 *
MDA in the pancreas, nmol/g	5.55 ± 0.60	8.90 ± 0.90	6.02 ± 0.60 *	7.80 ± 0.80 *
Catalase, mcat/L	21.35 ± 2.01	9.6 ± 0.85	17.08 ± 0.2 *	13.2 ± 0.13 *

Abbreviations: LPO, lipid peroxidation; MDA, malondialdehyde; μmol/mL, micromoles per milliliter; nmol/g, nanomoles per gram of tissue; mcat/L, microkatal per liter. Statistical notation: * p ≤ 0.05 indicates a statistically significant difference compared with the control pathology group.

, alloxan-induced diabetes was associated with a significant increase in serum and pancreatic malondialdehyde (MDA) concentrations on days 7, 14, and 28 compared to the control group (p ≤ 0.05). Serum catalase activity significantly decreased in the control group at all measurement points.

Administration of Inullact-Fito significantly reduced MDA concentrations in both serum and pancreatic tissue compared to untreated diabetic animals at all observation points (p ≤ 0.05). On day 28, serum MDA concentrations decreased from 15.4 ± 0.16 to 4.86 ± 0.50 μmol/mL, and pancreatic tissue MDA concentrations decreased from 8.90 ± 0.90 to 6.02 ± 0.60 nmol/g tissue. Catalase activity was significantly increased in the treatment group compared to the control group (p ≤ 0.05).

Metformin treatment also resulted in a decrease in MDA levels and an increase in catalase activity; however, these changes were less pronounced than in the Inullact-Fito group (Table 2).

3.3. Immunological Parameters

The immunological reactivity parameters are presented in

Table 3. Parameters of immunological reactivity in experimental animals with alloxan diabetes and its phytopharmacological correction.

Experiment Day	Group of Animals	CD4+, ×09/L	CD8+, ×109/L	IgM, g/L	IgG, g/L
7th day	Intact	1.71 ± 0.21	1.02 ± 0.20	0.50 ± 0.01	10.05 ± 0.11
	Control (Alloxan)	2.29 ± 0.23 *	0.69 ± 0.71 *	0.72 ± 0.08 *	12.51 ± 0.13 *
	Alloxan + Metformin	2.06 ± 0.22 *	0.76 ± 0.07 *	0.68 ± 0.07 *	11.75 ± 0.12 *
	Alloxan + “Inullact-Fito”	1.95 ± 0.17 *	0.79 ± 0.08 *	0.64 ± 0.06 *	10.37 ± 0.20 *
14th day	Intact	1.71 ± 0.21	1.02 ± 0.20	0.50 ± 0.01	10.05 ± 0.11
	Control (Alloxan)	2.13 ± 0.20 *	0.76 ± 0.65 *	0.95 ± 0.01 *	12.06 ± 0.12 *
	Alloxan + Metformin	1.81 ± 0.19 *	0.84 ± 0.08 *	0.71 ± 0.07 *	10.85 ± 0.11 *
	Alloxan + “Inullact-Fito”	1.74 ± 0.16 *	0.87 ± 0.10 *	0.61 ± 0.06 *	10.25 ± 0.12 *
28th day	Intact	1.71 ± 0.21	1.02 ± 0.20	0.50 ± 0.01	10.05 ± 0.11
	Control (Alloxan)	2.04 ± 0.19 *	0.81 ± 0.08 *	1.10 ± 0.01 *	12.06 ± 0.12 *
	Alloxan + Metformin	1.73 ± 0.17 *	0.97 ± 0.92 *	0.77 ± 0.08 *	10.80 ± 0.11 *
	Alloxan + “Inullact-Fito”	1.53 ± 0.16 *	1.05 ± 0.12 *	0.57 ± 0.05 *	9.85 ± 0.25 *

Abbreviations: CD4⁺, T-helper lymphocytes; CD8⁺, cytotoxic/suppressor T lymphocytes; IgM, immunoglobulin M; IgG, immunoglobulin G; ×10⁹/L, cells per liter. Statistical notation: * p ≤ 0.05 indicates a statistically significant difference compared with the control pathology (alloxan-treated) group.

. Alloxan-induced diabetes resulted in significant changes in T cell subsets characterized by an increase in CD4⁺ cell counts and a decrease in CD8⁺ cell counts compared to healthy animals (p ≤ 0.05). Serum immunoglobulin M (IgM) and immunoglobulin G (IgG) levels were also significantly increased in the disease control group at all observation points. Inullact-Fito treatment resulted in a significant normalization of CD4⁺ and CD8⁺ cell counts compared to untreated diabetic animals (p ≤ 0.05). Serum IgM and IgG concentrations were significantly reduced in the treatment group on days 7, 14, and 28 compared to the disease control group (p ≤ 0.05). Metformin treatment showed similar trends in immunological parameters. However, the values remained higher than those observed in healthy animals (Table 3).

3.4. Histological Findings

Histological analysis of pancreatic tissue from healthy mice revealed prominent islets of Langerhans with centrally located β cells and typical exocrine acinar architecture (Figure 1A). In the control group with pathology, structural abnormalities, decreased cell density, and smaller islets were observed in the pancreatic tissue (Figure 1B).

In pancreatic sections from rats treated with metformin, islet structure was partially preserved, but islet morphology was heterogeneous (Figure 1C). In the group treated with Inullact-Fito, improved islet architecture with higher cell density in pancreatic tissue was observed compared to untreated diabetic animals (Figure 1D).

4. Discussion

The current study looked at the metabolic, oxidative, immunological, and histomorphological effects of the multicomponent fermented camel milk-based product “Inullact-Fito” in an alloxan-induced insulin-deficient diabetes animal model. Interpretation of the obtained results should be considered in the context of the specific pathophysiological mechanisms underlying alloxan-induced pancreatic β-cell damage.

Alloxan-induced diabetes is characterized by selective reactive oxygen species-induced death of pancreatic β-cells, leading to severe insulin deficiency and persistent hyperglycemia. Oxidative stress plays a central role in the development of metabolic disorders in this model, as an imbalance between pro- and antioxidant systems leads to excessive production of free radicals and accumulation of lipid peroxidation products. According to Nakamura et al. (2006), persistent disruption of redox homeostasis can lead to multi-organ dysfunction, leading to structural and functional damage to various organs, as well as pancreatic β-cell death [25].

The administration of “Inullact-Fito” resulted in considerable improvements in carbohydrate and lipid metabolism, as demonstrated by large blood glucose reductions, partial restoration of circulating insulin concentrations, and normalization of cholesterol, urea, and creatinine levels. These effects were more dramatic than metformin and served as a pharmacological comparator; however, its efficacy in insulin-deficient models may be limited, and conclusions regarding superiority were avoided [35].

The observed metabolic benefits suggest that Inullact-Fito may have an insulin-sparing and insulin-independent effect.

The antioxidant properties of Inullact-Fito appear to be the main reason for its cellular protection. The biological product significantly reduced the concentration of malonaldehyde in serum and pancreatic tissues and restored catalase activity. This indicates a reduction in oxidative stress and an increase in endogenous antioxidant defense systems. These studies are consistent with recent publications showing that oxidative damage plays an important role in alloxan-induced β-cell toxicity [36]. In contrast, metformin showed significantly lower antioxidant activity under the same experimental conditions.

In addition to metabolic and oxidative disorders, alloxan-induced diabetes is associated with significant immunological changes, such as an increase in the CD4⁺/CD8⁺ ratio and increased serum immunoglobulin levels. This also indicates an imbalance of the immune system and chronic inflammatory activity. Similar immune dysregulations have also been observed in experimental diabetes models [34]. Treatment with Inullact-Fito partially normalized these parameters and demonstrated an immunomodulatory effect that contributed to the stabilization of overall metabolism and tissue protection. Overall, the observed metabolic, antioxidant, and immunological changes indicate a multifactorial biological activity of Inullact-Fito in the alloxan-induced diabetes model. A schematic summary of these interrelated effects is shown in Figure 2.

Histological examination of pancreatic tissue confirmed the results of the biochemical studies. Alloxan-induced diabetes resulted in significant structural damage to the endocrine pancreas; this damage included a decrease in islet size, a decrease in the number of β-cells, and an increase in connective tissue. Animals treated with Inullact-Fito showed partial preservation of islet architecture and improved cellular organization, indicating a protective or regenerative effect on β-cells. Although metformin showed some improvement in islet morphology, its effect was less consistent and heterogeneous compared to the fermented biological product. Some limitations of this study should also be noted. The alloxan-induced diabetes model primarily reflects diabetes caused by insulin deficiency and does not fully reproduce the pathophysiology of insulin resistance and type 2 diabetes. Furthermore, this study lacked quantitative morphometric analysis of β-cell mass and an assessment of long-term outcomes. Thus, although the studies conducted confirm the biological activity of Inullact-Fito, further studies using insulin resistance models and clinical trials are needed to confirm its therapeutic potential and suitability for clinical use.

5. Conclusions

This study demonstrated that the multicomponent fermented camel milk bioproduct Inullact-Fito exhibited positive metabolic, antioxidant, immunological, and histomorphological effects in a rat model of alloxan-induced insulin-deficient diabetes. Administration of the bioproduct resulted in a significant reduction in hyperglycemia, partial recovery of circulating insulin levels, reduction in lipid peroxidation, normalization of antioxidant defense markers, and modulation of immune parameters. Histological data also confirmed partial preservation of pancreatic islet architecture in the treated animals.

Importantly, this effect was observed in the context of an alloxan-induced model that primarily demonstrates β-cell damage and insulin deficiency, rather than insulin resistance. Therefore, these results should be considered as proof of concept for the biological activity of Inullact-Fito in conditions of experimental oxidative and metabolic stress. Although metformin served as a pharmacological comparator, its effects were less pronounced in this model, highlighting the importance of considering model-specific mechanisms when interpreting comparative efficacy studies. The observed benefits of Inullact-Fito may be due to its multicomponent composition and combination of antioxidant, metabolic, and immunomodulatory properties.

Overall, the results highlight the need for further investigation of Inullact-Fito in models of insulin resistance and type 2 diabetes, as well as in long-term and clinical trials, to determine its therapeutic potential, safety profile, and use as a functional nutritional intervention.