SCREENING STUDY OF THE ANTIHYPERGLYCEMIC ACTION OF NEW SOLID QUERCETIN DISPERSIONS

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Introduction
Diabetes mellitus, as defined by the World Health Organization, is a state of prolonged increase in blood sugar levels, which can be caused by a number of external and internal factors with the development of absolute or relative insulin deficiency, which leads to a violation of carbohydrate, fat and protein metabolism. Over the past decades, the rate of spread of diabetes mellitus has accelerated significantly. According to the International Diabetes Federation (IDF), the number of patients with diagnosed diabetes mellitus among the adult population (20-79 years) in the world in 2045 will amount to 629 million [1].
Despite the constant improvement of directions in the treatment of type 2 diabetes, which prevails in the population of patients with diabetes, the achievement of normoglycemia and the prevention of chronic complications (such as retinopathy, angiopathy, neuropathy) remains a challenge, which indicates the need for the use of multicomponent method, which corresponds to the combination therapy using various groups of pharmacological agents [2].
The most used are combinations of oral antihyperglycemic drugs of various groups (for example, metformin in combination with sulfonylurea, sulfonylurea in combination with exenatide, etc.), which reduce the side effects of the components by reducing the doses of substances included in composition of the combination. However, drug combinations with antihyperglycemic action do not always provide prevention of complications of type 2 diabetes mellitus, which indicates the relevance of the development of new combined drugs.
Potential components of combined antidiabetic drugs can be herbal substances that are effective due to a wide range of pharmacological action and are quite safe with prolonged use. It should be noted that today in different countries of the world a large number of herbal substances are being studied in order to create effective antidiabetic drugs and available literature data on the experience of experimental and clinical use of phytopreparations in the complex therapy of type 2 diabetes mellitus [3,4]. Possible mechanisms of the therapeutic action of plant components include inhibition of the activity of α-glucosidase and α-amylase, the effect on glucose uptake by peripheral tissues and glucose transporters GLUT4, increased insulin secretion and proliferation of β-cells of the pancreas, inhibition of protein tyrosine phosphatase [3]. It has now been proven that most of these mechanisms, due to the pronounced antioxidant properties of phytopreparations, which are able to weaken the oxidative stress developing in patients with type 2 diabetes mellitus, combine the main links of glucose toxicity and cause severe chronic complications of the disease [5,6]. One of the phytopreparations of interest as a potential antidiabetic drug is the well-known and sufficiently studied flavonol quercetin -2(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromene-4-OH, which exhibits anti-inflammatory, antispasmodic, antihistamine, diuretic effect, has powerful antioxidant, radioprotective and antitumor properties [7,8].
Due to the insufficient solubility of quercetin and poor absorption in the gastrointestinal tract, scientists are constantly trying to improve the solubility and bioavailability, which will significantly expand the list of medical indications for the use of this natural bioflavonoid [9,10].
The aim of this study was to conduct pharmacological screening of new solid dispersions of quercetin for antihyperglycemic action and to identify substances that are promising for the creation of antidiabetic drugs.

Planning (methodology) of research
At present, a standardized solid dispersion of quercetin with polyethylene oxide and neusilin has already been obtained at the National University of Pharmacy, which, together with voglibose, is included in the composition of a new combined antidiabetic agent under the code name "Glikverin". The pronounced antihyperglycemic and antidiabetic effect of "Glikverin" has been experimentally proven in models of insulin resistance, type 2 diabetes mellitus and metabolic syndrome [11].
Considering previous experience in creating a solid dispersion with quercetin [12] at the Department of Industrial Technology of Drugs of the National University of Pharmacy (NUPh). Using a new technological approach, new samples of hard quercetin dispersions based on various carriers were obtained. It is planned to conduct their pharmacological screening for antihyperglycemic action, which will justify the creation of a drug for the treatment of type 2 diabetes mellitus.
Considering previous experience in creating a solid dispersion with quercetin [12] at the Department of Industrial Technology of Drugs of the NUPh by Doctor of Pharmacy Kovalevska Inna under the guidance of Doctor of Pharmacy, professor Ruban Olena, using a new technological approach, new samples of hard quercetin dispersions based on various carriers were obtained.
Since the main criterion for compensating for diabetes mellitus is a decrease in the level of hyperglycemia, two experimental models with high reproducibility and a short course were selected for screening: a model of impaired glucose tolerance induced by dexamethasone and a model of type 2 diabetes induced by dexamethasone [13].
Stages: 1. Study of the antihyperglycemic effect of solid dispersion samples on the model of impaired glucose tolerance.
2. Study of the antihyperglycemic effect in type 2 diabetes mellitus.
3. Determination of the most promising solid dispersion of quercetin for the development of new antidiabetic drugs.
4. Identification of promising directions for further research.

Materials and methods
Studies of drugs were carried out at the Central research laboratory of the National University of Pharmacy in 2021.

1. Study subjects
The objects of the research were 4 samples of solid dispersion of quercetin. Hydroxypropyl methylcellulose (HPMC) or polyvinylpyrrolidone (PVP) was used as a carrier in a ratio of 1:1 and 1:2 (Table 1). Solid dispersions were obtained by the liquid-phase method [14]. The technology for obtaining a solid dispersion of quercetin with carrier consists of several stages: obtaining a solution of API in ethanol 96 % (a portion of quercetin is dissolved in ethanol 96 % in a ratio of 1 to 3 with constant stirring), the resulting solution is mixed with a portion of the carrier (HPMC or PVP) with constant stirring at least 200 vol/min for 20 minutes until a homogeneous mass of a slightly yellow color is obtained. Microcrystalline cellulose is added to the mass of quercetin with carrier (HPMC or PVP) with constant stirring at 50 rpm until a fluffy homogeneous mass is obtained, dried in a dryer with air circulation at a temperature of 60 °C±5 until the final mass is not more than 3 %. Calibration takes place through a 1 mm sieve.

2. Research methods
The study of the hypoglycemic properties of the solid dispersion of quercetin was carried out in accordance with the guidelines for the experimental study of new antidiabetic agents [13].
Experiments were performed on white nonlinear male rats weighing 130-410 g raised in the National University of Pharmacy vivarium nursery. The animals were kept in standard vivarium conditions: at a temperature of 20-22 °C, humidity no more than 60-70 %, air exchange volume (extract-inflow) 8/10, day/night light mode in standard aluminium cages, no more than 5 animals in each.
The work with animals was carried out in accor- The studies were carried out in comparison with antidiabetic means from a group of biguanides -metformin tablets, 500 mg (produced by Lek S. A., Poland, an enterprise of the Sandoz company). Metformin was used at a dose of 60 mg/kg of body weight in rats, which was calculated using the coefficient of species sensitivity [16,17].
The experimental dose of the investigated solid dispersions of quercetin was selected on the basis of previously obtained results in the study of solid dispersions of quercetin with polyethylene oxide and neusilin and amounted to 50 mg/kg [11].
Solid dispersions of quercetin and the reference drug metformin were administered to animals in the form of solutions intragastrically in the morning on an empty stomach using a metal probe.
Reproduction of the experimental model of impaired glucose tolerance was carried out on 3-month-old male rats weighing 130-160 g using dexamethasone [13]. Dexamethasone (dexamethasone-KRKA, solution for injection 4 mg/ml amp., 1 ml (KRKA, Slovenia)) was injected subcutaneously at a dose of 0.125 mg/kg for 13 days.
Animals were divided into the following groups: intact control; control pathology, 4 groups of rats receiving samples of solid dispersion of quercetin No. 1-4 at a dose of 50 mg/kg; a group of rats receiving metformin at a dose of 60 mg/kg. At 14 on day, the animals were measured basal blood glucose and an oral glucose tolerance test (OGTT) was performed.
The blood glucose levels of the animals were determined on an empty stomach and at time points using the glucose oxidase method using a OneTouch Select glucometer (LifeScan Scotland Limited, UK). For this purpose, the animals were punctured along the tail vein, brought the device with a test strip inserted, and the device automatically took 1.5 μl of blood. The glucose in the blood reacts electrochemically with the reagents in the test strip, producing a weak electrical current. The strength of the current changes in proportion to the blood glucose content. Linear measurement ranges 1.1-33.3 mmol/L.
An oral glucose tolerance test was performed as follows. In rats after an overnight fast (16-18 hours) on an empty stomach, the initial blood glucose levels were determined, and a glucose solution was injected intragastrically at a rate of 3 g/kg. Further, the concentration of glucose in the blood was measured 30, 60, 120 minutes after the carbohydrate load [13].
Antihyperglycemic activity was evidenced by the ability of solid dispersions of quercetin to reduce the level of glycemia at the 30th minute of OGTT (the time of maximum rise in blood glucose in experimental rats in response to intragastric carbohydrate load). Additionally, according to the results of OGTT, the value of the integral indicator of the plane under the glycemic curves (glicemic area under curve, AUCglu (mmol/L min) was calculated.
Type 2 diabetes mellitus was modelled on 18-month-old male rats weighing 320-410 g by subcutaneous administration of dexamethasone (Dexamethasone-KRKA, solution for injection 4 mg/ml amp., 1 ml (KRKA, Slovenia)) at a dose of 0.125 mg/kg for 13 days [13]. Rats were divided into experimental groups as in the previous experiment.
The antihyperglycemic activity of solid dispersions of quercetin was assessed by the dynamics of basal blood glucose: baseline data, on days 7 and 14.

Statistical analysis
Statistical processing of the obtained results was carried out according to the generally accepted methods of variation statistics with the determination of the arithmetic mean and average error (M±m) by Student's t-test using the standard computer program "Statistica 6.0". The planes under the glycemic curves were calculated using the statistical software package "MedCalk, v. 9.3.7.0".

Results
The results of studying the antihyperglycemic effect of samples of solid dispersions of quercetin with impaired glucose tolerance are shown in Table 2.
Conducting an oral glucose tolerance test allows to reproduce in intact animals the state of postprandial hyperglycemia with an increase in blood glucose at the 30th minute of the experiment ( Table 2).
On the 14th day in animals with control pathology, at the 30 th minute of the glucose tolerance test, glycemia significantly exceeded the initial values by 2,2 times (p<0.001), after 60 minutes it gradually decreased, but after 120 minutes of the experiment it did not normalize to physiological parameters (Table 2). At the same time, the area under the glycemic curves was the highest among all experimental groups and significantly differed from the value of the intact control, which indicated impaired glucose tolerance in rats of control pathology.
The use of solid dispersions of quercetin showed that samples No. 1, 3 and 4 reliably prevent the development of hyperglycemia caused by carbohydrate load (Table 2). At the same time, samples No. 3 and 4 (PVP : quercetin, ratio 1:1 and 1:2) reduced blood glucose levels by 19 and 21 %, respectively, while sample No. 1 (HPMC: quercetin, 1:1) showed distinctly antihyperglycemic effect -the glucose level 30 minutes after glucose load significantly decreased by 28 % (p<0.05) compared with the indicator of the control pathology group and did not differ from the effect of metformin ( Table 2).
The use of sample No. 2 (HPMC : quercetin, 1:2) in this model turned out to be ineffective, the dynamics of changes in the concentration of glucose in the blood of rats at all-time intervals and AUCglu did not differ significantly from the control pathology.
Comparative analysis of the planes under the AUCglu glycemic curves, which is an integral parameter for assessing glucose load, confirmed the OGTT data.
Thus, the results obtained showed the antihyperglycemic properties of samples No. 1, 3, 4, which suggests an increase in the degree of release and absorption of quercetin due to the constituent components of these solid dispersions.
At the next stage, the antihyperglycemic properties of solid dispersions of quercetin were investigated in a model of type 2 diabetes mellitus caused by dexamethasone.
In our experiment, subcutaneous administration of dexamethasone to rats of the control group on the 7th day led to statistically significant moderate basal hyperglycemia, which persisted and significantly increased by 26 % compared to the initial values on the 14th day of the experiment (Table 3). Thus, the results obtained showed the development of experimental type 2 diabetes mellitus in animals. At the first observation period, the administration of all samples of solid dispersions of quercetin practically did not restrain the growth of hyperglyce-mia, in contrast to metformin, which, although not significantly, reduced the glucose level by 15 % compared to the control group (Table 3).
On the 14th day of the experiment, in the group of rats receiving sample No. 1 (HPMC:quercetin, 1:1), a significant antihyperglycemic effect was established (decrease in glycemia by 17 %, p<0.05), which is comparable to the effect the reference drug metformin (Table 3).
Test samples No. 3 and No. 4 showed only a positive trend towards lowering blood glucose levels. Perhaps, with a longer period of use, these investigated substances will exhibit an antihyperglycemic effect.
Sample No. 2, as in the model of impaired glucose tolerance, did not affect the glucose level at the end of the experiment, which allows us to conclude that it is inexpedient to use this solid dispersion of quercetin as a component of a combined antidiabetic drug.

Discussion
The experimental model of impaired glucose tolerance induced by dexamethasone was characterized by an excessive increase in the level of glycemia after carbohydrate loading and indicated the development of prediabetes due to the formation of insulin resistance and secondary or primary dysfunction of β-cells [18,19].
Testing of rats treated with solid dispersions of quercetin with HPMC and PVP made it possible to establish the ability of samples 1, 3, 4 to provide a sufficient balance between the flow of exogenous glucose into the blood and the processes of its utilization by peripheral tissues and to state the presence of antihyperglycemic action with almost complete normalization glycemia to the level of intact animals after 120 minutes.
Further, it was important to confirm the results of the previous experiment and to evaluate the antihyperglycemic properties of solid dispersion samples of quercetin in a model of type 2 diabetes mellitus induced by dexamethasone. It is known that excessive doses of glucocorticoids can lead to impairment of the secretory function of β-cells of the pancreas and the development of insulin resistance as a result of a direct effect on the expression of glucose transporters GLUT1 and GLUT4 [20].
During the experiment, a significant antihyperglycemic effect was revealed only in sample No. 1 (solid dispersion of quercetin with HPMC, 1:1) at the level of metformin, which indicates the maximum transition of Taking into account the wide range of pharmacological properties of quercetin, including stimulation of insulin secretion, increased glucose uptake by peripheral tissues, pronounced anti-inflammatory and antioxidant properties [21,22], The results obtained indicate the prospects for further research for in-depth pharmacological study of the solid dispersion of quercetin with HPMC in a ratio of 1: 1.
Study limitation. To expand the understanding of the antihyperglycemic properties of a solid dispersion of quercetin with HPMC, it would be advisable to study the level of insulin in the blood and calculate the HOMA index.
Prospects for further research. In the future, it is planned to study the antidiabetic properties and mechanisms of the therapeutic action of a solid dispersion of quercetin with HPMC on experimental models of diabetes mellitus and metabolic syndrome.

Conclusions
The solid dispersion of quercetin with HPMC in a 1:1 ratio was found to have a significant antihyperglyce-mic effect. The new substance of quercetin is effective at the metformin level in experimental insulin resistance and type 2 diabetes mellitus.
Solid dispersion of quercetin with HPMC in a 1:1 ratio is a promising substance for further in-depth pharmacological study for the purpose of to create a new antidiabetic agent.

Conflict of interests
The authors declare that they have no conflicts of interest.

Financing
This study is funded by the Ministry of Health of Ukraine at the expense of the state budget within the framework of program No. 2301020 «Research, scientific and scientific-technical development, implementation of work on state target programs and state orders, training and advanced training of scientific personnel in the field of health care, financial support for the development of scientific infrastructure and objects of national heritage» on the topic «Development of a complex drug for the treatment of type II diabetes» (Order of the Ministry of Health of Ukraine dated November 17, 2020 No. 2651).