Twelve (12) healthy adult female Wistar rats weighing 150 to 180 g were used for this study. They were obtained from the Central Animal Facility of the Federal University of Mato Grosso, and were maintained under standard housing conditions in the animal section of Laboratory of Biological Evaluation of Food of the Department of Food and Nutrition, Federal University of Mato Grosso, Mato Grosso State, Brazil. The animals were adapted for two weeks preceding initiation of experimental regimen, received fed commercial (Labine chow) and clean tap water acessive ad libitum, exposed to clean tap water throughout the period of the study. The room temperature was maintained at approximately 25˚C, relative humidity at 55%, with a light cycle of 12 hours light and 12 hours dark. All animal experimental protocols were permitted by the Ethics Committee on Animal Research of the Federal University of Mato Grosso (Protocol No. 23108.039877/2021-21), and were carried out by its guidelines for animal use.

The animals were allotted into two groups of six (6) rats and kept in groups of three (3) animals per standard plastic rat cage. The groups were named Control and AEOM, consisting of female rats that received 0.9% saline solution and AEOM (100 mg/kg/day), respectively, that were administered by oral gavage at 3 mL/kg body weight for 15 days.

Twenty four (24) h after the last oral dose the AEOM, the animals were narcotized in a CO₂ and blood were obtained through heart punction into ordinary sample bottles. The blood samples were made to stand for 20 min for coagulation to occur, and afterwards centrifuged at 2000 rpm for 10 min and the supernatant (serum) collected and kept at 4˚C prior to biochemical assay. The pancreas was quickly excised, weighed, and immediately fixed in 4% neutral buffered formalin for histopathological examination.

Commercial kits (Wiener Lab. Group, Brazil®) were used for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine, urea, uric acid, total proteins (TP), albumin (ALB), glucose (GLU) and lipid profile [total cholesterol (TC), triglycerides (TG) and HDL-c]. The LDL value was calculated according to the Friedewald equation: LDL-c = total cholesterol − (HDL + VLDL); VLDL-c = triglycerides/5. Analyzes were carried out in the State Department of Health of Mato Grosso, Blood Center, Cuiabá, State of Mato Grosso, Brazil. All chemicals/reagents used in this investigation were of purest analytical grade.

Rats in all groups were weighed on the first day and at the completion of the treatment protocols. The percentage change in body weight was calculated using:

% changeinbody   weight = final   body   weight − initialbodyweight × 1 00 initialbodyweight

Pancreas weight was presented in absolute and relative values. Relative pancreas weight was calculated using the expression below:

Pancreas   ( g / 1 00 g   BW ) = weightofpancreas   ( g ) × 1 00 bodyweight   ( g )

Four rats from each group were used for the histological studies. The pancreatic tissue was fixed in 4% paraformaldehyde solution for 24 hours at 4˚C, dehydrated in increasing concentrations of ethanol, clarified in xylene and embedded in paraffin using a histological processor (MTP 100, Slee, Mainz, Germany). The tissue was serially sectioned at 3 µm thickness using a microtome (RM2125, Leica Biosystems Nussloch, Germany) and then mounted on slides with an adhesive surface. Ten sequential sec-tions of the pancreas were deparaffinized in an oven at 60˚C for 2 h, followed by im-mersion in xylene and decreasing concentrations of ethanol. These slides were rehy-drated and stained with hematoxylin and eosin for determination of the pancreatic regions under a light microscope at 40× magnification (Axio Scope A1, Zeiss, Ober-kochen, Germany).

The results were presented as mean ± standard deviation (S.D.) and analyzed using an unpaired Student’s t-test. The significance level was set at p < 0.05. For the analysis of the results, the program “Statistic for Windows”, (version 4.3, StatSoft, Inc., Tulsa, OK, USA).

The body and pancreas weights of the rats are presented in Table 1. Initial and final body weights and absolute and relative weights of pancreas did not differ between the groups.

The oral administration of AEOM for 15 days as shown in Table 2 caused a signifycant decrease (p < 0.05) in the levels of total cholesterol and HDL-c. AEOM did not alter TG and LDL-c levels.

Values expressed as mean ± SD (n = 6 per group).

Values expressed as mean ± SD (n = 6 per group). *Indicates difference statistical (t Student test, p < 0.05).

The oral administration of AEOM for 15 days as presented in Table 3 caused a non-significant change (p ≥ 0.05) in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities in female rats.

The oral administration of AEOM for 15 days as presented in Table 4 caused a significant decrease (p < 0.05) in the level of uric acid. However not caused a significant change (p ≥ 0.05) in the levels of creatinine and urea concentrationin the female rats.

The oral administration of AEOM for 15 days as presented in Table 5 did not cause significant change (p ≥ 0.05) in the levels of TP and GLUC, but reduced the serum albumin concentration in the female rats.

The histopathological examination of the control group showed physiological organization of the pancreas with no morphological alterations (Figure 2(A)). After 15 days of oral dosing of AEOM (Figure 2(B)) showed increasement in the pancretic islets size (H&E).

AEOM is an extract based on P. ostreatus, an edible mushroom important source of bioactive compounds [1] . For this reason, edible mushroom extracts have been used as dietary supplements and recommended for the prevention and treatment of various diseases [45] .

In the present study, we evaluated the use of AEOM by healthy adult rats and found similar body weight gain in both evaluated groups. However, we observed a significant reduction in serum albumin concentrations, a condition that can

Values expressed as mean ± SD (n = 6 per group).

Values expressed as mean ± SD (n = 6 per group). *Indicates difference statistical (t Student test, p < 0.05).

Values expressed as mean ± SD (n = 6 per group). *Indicates difference statistical (t Student test, p < 0.05).

result from reduced hepatic synthesis, increased catabolism and vascular permeability, and intestinal and renal loss [46] . Malnutrition, inflammation, liver disease are some situations that can contribute to the reduction of albumin synthesis [46] . Considering preserved body weight and unaltered total serum protein concentration, it is reasonable to assume that treatment with AEOM did not contribute to the deterioration of the nutritional status of the animals. Body weight fluctuations serve as a sensitive indicator of the general health status of animals [47] and normal serum total proteins are indicators of preserved nutritional status [48] . Additionally, unaltered values of AST, ALT, ALP and LDH are indicative that the AEOM did not cause liver damage [21] [22] [23] [44] . Renal loss of albumin is also an unlikely hypothesis, in view of the preserved renal function, judging by the similar serum concentrations of urea and creatinine in the evaluated groups. The reduction in serum uric acid seen in rats treated with AEOM and also observed in other studies with rodents treated with P. ostreatus, Agrocybe aegerita and Ganoderma applanatum [36] [37] [38] [39] has been associated with increased urinary acid excretion uric acid [36] [37] , which is negatively related to albuminuria [49] .

In the present study, treatment with AEOM reduced total cholesterol, as in murine models of hypercholesterolemia and diabetes [50] [51] [52] . The hypocholesterolemic effect of P. ostreatus has been attributed to the presence of polyunsaturated fatty acids, mainly eicosapentaenoic and docosahexaenoic acids [53] and polysaccharides that positively modulate the serum lipid profile [24] , as well as the stimulating effects of intestinal cholesterol excretion [50] [51] . Interestingly, in the present study, a reduction in HDL-c was observed, as opposed to reports of the positive modulatory effect of P. ostreatus or some of its components, such as polysaccharide residues, on HDL-c in models of diet-induced hypercholesterolemia [26] [54] , in mice with alloxana-induced diabetes [28] and in HIV-infected humans on antiretroviral therapy [55] . Epidemiological studies show a strong inverse correlation between HDL-c and cardiovascular disease risk [56] [57] , suggesting that in this study P. ostreatus had a detrimental effect on cardiovascular health. However, the reduction in HDL-c observed here may not be a cause for concern, considering the antioxidant properties of P. ostreatus [12] [13] [14] [15] .

The histomorphological evaluation of the pancreas showed larger pancreatic islets in the AEOM group compared to the control group. Despite the hypertrophy of the islets, the glycemia of the animals treated with P. ostreatus did not differ from that of the control animals. This result was not surprising, since the size of the islets does not seem to be related to the increase in their functional capacity or with the hormonal content. It has been shown that insulin secretion from small islets is greater compared to large islets, with a correlation with greater insulin content/area, greater density of insulin-secreting granules and greater insulin content/volume. Central β cells of large islets appear to contain less insulin/cell with a lower insulin granule density than peripheral β cells [58] [59] [60] .