The quercetin and dihydroquercetin effect on small intestine enzymes in case of hypothyroidism

ABSTRACT

This study examined the quercetin and dihydroquercetin features, which improved the carbohydrate activity in the small intestine and the T3, T4, frT3, frT4 hormone activity in blood in case of hypothyroidism caused by iodine deficiency, which is widespread throughout the world. For the hypothyroidism treatment, a solution of mercazolil 10 mg/kg is used for 14 days. The quercetin and dihydroquercetin effect on enzyme activity in the small intestine was organized based on healthy and experimental hypothyroidism. These flavonoids increase the activity of thyroid hormones in blood in case of iodine deficiency and improve the digestive enzyme activity in the small intestine.

АННОТАЦИЯ

В этом исследовании были рассмотрены свойства кверцетина и дигидрокверцетина, которые улучшили активность углеводов в тонком кишечнике и активность гормонов T₃, T₄, свT₃, свT₄ в крови во время гипотиреоза, вызванного дефицитом йода, которые широко распространены во всем мире.  По лечении гипотиреоза используется раствор мерказолила 10 мг / кг в течение 14 дней. Влияние кверцетина и дигидрокверцетина на активность ферментов в тонком кишечнике было организовано на основе здорового и экспериментального гипотиреоза. Эти флаваноиды повышают активность гормонов щитовидной железы в крови при дефиците йода и улучшают активность пищеварительных ферментов в тонком кишечнике.

Keywords: quercetin, dihydroquercetin, reactive oxygen species (ROS), reactive nitrogen species (RNS), mercazolil.

Ключевые слова: кверцетин, дигидрокверцетин, Реактивные формы кислорода, Реактивные формы азота, мерказолил.

Introduction

Flavonoids belong to a class of polyphenolic compounds found in plants. They have assured growth and development processes in plants. [3, p. 398] It has been established that plants contain more than 8000 flavonoids of various chemical structures. [22, p. 1220]

Flavonoids are not synthesized in human and animal cells; they enter the body while consuming plants. [14, p. 520]. Quercetin, the most abundant of the flavonoids (the name comes from the Latin –quercetum, meaning oak forest, Quercus oak) consists of 3 rings and 5 hydroxyl groups (Figure-1) Quercetin acts as a building block for other flavonoids. [15, p. 316]

Figure-1

Quercetin is found in a variety of foods including apples, berries, Brassica vegetables, capers, grapes, onions, shallots, tea, and tomatoes, as well as many seeds, nuts, flowers, barks and leaves. [7, p. 143]

The best-described property of quercetin is its ability to act as an antioxidant. Quercetin seems to be the most powerful flavonoid for protecting the body against reactive oxygen species, produced during the normal oxygen metabolism or are induced by exogenous damage. [1, p. 20, 4, p. 80] One of the most important mechanisms and the sequence of events by which free radicals interfere with the cellular functions seems to be the lipid peroxidation leading eventually the cell death. To protect this cellular death to happen from reactive oxygen species, living organisms have developed an antioxidant line of defense systems. [20, p. 35] Quercetin has been used in cancer prevention and to prevent the spread of various cancers, such as lung, prostate, liver, breast, colon, and cervical cancers. Its anticancer properties are mediated by various mechanisms involving cell signaling pathways and enzymatic activities that inhibit carcinogens. High levels of ROS induce oxidative stress, which in turn causes the over-activation of signal transduction pathways and promotes cell proliferation, as well as survival and metabolic adaptation to the tumor microenvironment. In this way, ROS promotes tumorigenesis. Quercetin regulates both internal and external pathways of ROS-mediated protein kinase C (PKC) signaling. [11, p. 1422, 12, p. 815]

Dihydroquercetin was discovered in 1936, by Albert, an American biochemist. More active than tocopherol, carotene, and also more stable. Dihydroquercetin belongs to a limited flavanone class of flavonoid compounds. It is a fine crystalline or amorphous powder of white to white-cream color depending on the method of preparation and the presence of other substances. Evergreen trees, especially those from the family of Pinaceae are considered rich sources of dihydroquercetin. [5, p. 62]

By substantial efficiency of the hydrogen atom and electron transfer and by keeping metal ions tightly sequestered (metal-chelating agent), dihydroquercetin brings fundamental means of antioxidant defense against free radical-mediated tissue damage.  The proven biological effects of dihydroquercetin, i.e. antioxidative activity, vitamin P activity, capillary protection, improving microcirculation and blood flow at the capillary level, promoting the development of new capillaries, helping to maintain blood pressure at physiological levels, providing a mild hypotension effect, improvement of the elasticity of red blood cells, reduction of blood viscosity, anti-ischemic effect, antidepressant effect, reduction of the low-density lipoprotein levels in blood plasma. Dihydroquercetin supports the cellular structure and cell metabolism. The simple monomer of dihydroquercetin is highly effective in both intracellular and extracellular environments. Studies in erythrocytes, mast cells, leucocytes, macrophages, and hepatocytes have shown that dihydroquercetin renders cell membranes more resistant to lesions. [19, p. 622]

The effect polyphenols include inhibition of carbohydrate digestion and glucose absorption in the intestine, stimulation of insulin secretion from the pancreatic b-cells, modulation of glucose release from the liver, activation of insulin receptors, and glucose uptake in the insulin-sensitive tissues, and modulation of intracellular signaling pathways and gene expression. Food and beverages high in available carbohydrates such as starch or sucrose induce postprandial hyperglycemia, hyperinsulinemia, and other hormonal and metabolic disturbances. The rapid absorption of glucose challenges the regulatory mechanisms of glucose homeostasis, and habitual consumption of high-glycemic diets may therefore increase the risk for obesity, type 2 diabetes, and cardiovascular disease [10, p. 2417]. Maintenance of glucose homeostasis is of utmost importance to human physiology, being under strict hormonal control. Failure of this control can result in metabolic syndrome, a multi-symptom disorder of energy homeostasis encompassing obesity, hyperglycemia, impaired glucose tolerance, hypertension, and dyslipidemia. [2, p. 1417]

Materials and methods. Two-month-old weighing 100–120 g male mice were kept in plastic boxes (5 animals/box). The size of the plastic boxes is 50 × 30 × 28 cm. Indoor temperature 22-24 ° C. Food and drinking water were provided in unlimited and the diet consisted of wheat, sunflower seeds, dairy products, meat products, wheat bread, herbs, vegetables, soups, and mixed feed. The animals were divided into experimental and control groups. The animals of the experimental group were received with mercazolil at a dose of 10 mg/kg for 14 days, and the animals of the control group were received with 1.0 ml of diluted drinking water. On day 15 of the experiment, blood was taken from the tails of control and experimental animals. [24, p. 27] A group of animals received from experimental hypothyroidism was given 20 mg/kg of quercetin and dihydroquercetin flavonoids for 14 days after 8-10 hours in the morning. From 8 to 10 am the animals were decapitated and the abdominal cavities were opened immediately, the small intestines were removed and placed in a container with water and ice for further experiments. During the experiments, the small intestine was separated from the mesentery and its cavity was washed with 20-30 ml of Ringer’s solution (pH-7.4). Intestinal mass was measured on an electronic scale. The intestinal mucosa was mixed with chilled Ringer’s solution 1/10 and homogenized using a tissue homogenizer for 1–1.5 min. The 2% solutions of lactose, sucrose and maltose were used as substrates for determining the activity of intestinal enzymes. According to the Dahlqvist method (Dahlqvist, 1984), lactase, sucrase, and maltase of the small intestine were determined using a glucose oxidase (Human Germany). The activity of lactase, sucrase, and maltase was calculated based on 1 g of tissue per 1 μm of the decomposition product formed in 1 minute. Statistical processing of the results was carried out according to the Student-Fisher method. The arithmetic average (M), the average error (± m), and the reliability index (R) were determined. P <0.05 is considered reliable.

Results and discussion

The digestive system plays an important role in providing the body with plastic and energetic material. Most dietary carbohydrate is digested in the upper gastrointestinal tract to monosaccharides which are then absorbed to the circulation. Thyroid hormone deficiency is one of the most serious health problems in the world today. The deficiency of the thyroid gland in the body leads to a decrease in metabolism. This leads to insufficient oxygen supply to the tissues or disruption of oxygen utilization, resulting in various pathological changes. Hypothyroidism is now widespread in the world and the World Health Organization recommends using various preparations to prevent and treat this disease.  Diets based on flavonoids are considered highly effective for the body. Indigestion and diarrhea, bloating and heaviness in the stomach are common symptoms of hypothyroidism. And so, obesity is common in this condition. These symptoms are directly related to the activity of enzymes in the small intestine.

Oxidative stress results are an imbalance between the antioxidant defense systems and the rate of production of reactive oxygen species (ROS). It not only leads to lipid peroxidation and oxidative DNA damage but also interferes with physiologic adaptation and intracellular signal transduction. The resulting change in the intracellular redox status leads to the activation of protein kinases, for example, tyrosine kinase, protein kinase C, and the mitogen-activated protein kinase cascade leading to altered cellular functions. [22, p. 273] The thyroid hormones tetraiodothyronine (thyroxin, T4) and a much smaller proportion of triiodothyronine, exert actions at the cellular level by binding to a set of specialized receptors that couple to both genomic and nongenomic signaling pathways. They are subjected to transformations in the peripheral tissues, mainly in the form of deiodination. The general metabolic effect of the thyroid hormones is a relative acceleration of the basal metabolism that includes an increase in the rate of both catabolic and anabolic reactions. This results in increased energy expenditure, oxygen consumption, respiratory rate, and heat production. While ROS production depends largely on the mitochondria, the thyroid hormones do not directly determine the respiratory state of the mitochondria; they also affect the cell antioxidant status. [18, p. 944] Hypothyroidism-associated ROS is the consequence of both increased productions of free radicals and reduced capacity of the antioxidative defense. Hypothyroidism-induced dysfunction of the mitochondrial respiratory chain can lead to the accelerated production of free radicals. Lipid peroxidation is reported to be high in hyperlipidemia, which is a consistent biochemical feature in hypothyroidism. The presence of oxidative stress in hypothyroidism correlates with the lipid risk factors of atherosclerosis. [16, p. 104] Metabolic disorder from autoimmune-based hypothyroidism can also increase oxidative stress. [18, p. 945]

Due to the strong antioxidant properties of quercetin and dihydroquercetin, flavonoids restore mitochondrial dysfunction caused by hypothyroidism and, as can be seen, improve the number of hormones in the blood by combining the free radicals.

In a healthy body, the amount of thyroxin (T4) was 5.38 ± 0.05 ng / mL, in hypothyroidism, this rate decreased by 29.0%, with the receiving of dihydroquercetin the amount of this hormone increased by 83%. Quercetin is more effective than dihydroquercetin, its share increased to 93.4%. The amount of triiodothyronine (T3) in a healthy body was 1.74 ± 0.04 ng / mL, dysfunction of the thyroid gland decreased to 71.3%, and this share increased by 87.4% with the use of dihydroquercetin. When corrected with quercetin, the amount of this hormone in the blood increased by 83.3%. If in a healthy organism the frT4 value was 1.68 ± 0.05 pg / mL, after hypothyroidism, this number decreased by 25.6%. Accordingly, correction with dihydroquercetin this amount increased to 90.4% and approached the norm. In experiments treated with quercetin, this figure reached 94.0%. Initially, the frT3 value was 4.73 ± 0.06 pg / mL, which was relatively low in experimental hypothyroidism (54.3%). Under the influence of the dihydroquercetin, it increased by 89.0%. The biological activity of quercetin was higher than dihydroquercetin, which increased the amount of free triiodothyronine in the blood by 94.5%.

Table 1.

Effect of quercetin and dihydyrdoquercetin polyohenols on the amount of T₃, T₄, frT₃, frT₄ hormones in rat serum in experimental hypothyroidism

In all cases n=5, p<0,05;p<0,01

The most characteristic abnormality in the metabolic syndrome is insulin resistance, which results from interactions between genetic and environmental factors, including diet and sedentary lifestyle [13, p. 34; 25, p. 227]. Metabolic syndrome is the major predisposing factor to type 2 diabetes, where defects in both insulin action and insulin secretion are present, but their relative contribution varies individually. The disturbance of glucose metabolism is often related to the increase of fat mass, especially in the abdominal area and ectopically, to the tissues where fat is not stored in normal energy homeostasis [8, p. 340]. This, in turn, results in inflammation and exacerbated oxidative stress at the whole-body level, and malfunction in several organs including the pancreas, liver, muscle, and adipose tissue [9, p. 258]. Digestion of carbohydrates and absorption of glucose allows for better glycemic control after a high-carb meal. The secreted glucose is absorbed through the intestinal enterocytes through certain carriers. There were decreases in the activities of enzymes in the small intestine with the receiving of quercetin and dihydroquercetin in a healthy body. Inhibition of digestive enzymes can prevent postprandial hyperglycemia, as glucose carriers can reduce glucose excretion and the rate of digestion in the small intestine. Digestive absorption processes are more active in the upper part of the small intestine. In a healthy body, the amount of lactase in the jejunum part of the small intestine is 12.7 ± 0.2 μmol/min/g of tissue, and in the ileum part of the small intestine - 10.9 ± 0.17 μmol/min/g of tissue (figure 2) .

Figure 2. Effect of quercetin and dihydyrdoquercetin polyohenols on the amount of lactase activity in rat small intestine in case of hypothyroidism

In all cases n=5, p<0,05;p<0,01

With the appointment of quercetin, this number decreased by 73.2% in the jejunum section and by 72.5% in the ileum section. Under the influence of dihydroquercetin, there was a decrease of 77.9% in the jejunum part and 74.3% in the ileum zone. This shows that the decrease in the activity of enzymes in the intestine under the action of flavonoids, and an increase in glucose in the body accelerates the processes of catabolism. This prevents the accumulation of excess weight in the body. In hypothyroid rats, the enzyme activity was relatively increased to 14.4 ± 0.7 μmol / min / g in the jejunum part of the small intestine. The enzyme activity improved by 96.0% in the jejunum part and by 97.5% in the medial part, respectively, when quercetin was administered after hypothyroidism. Under the influence of dihydroquercetin, the enzyme activity was similar to the healthy intestine enzymes (94.6%). In the small intestine, the enzyme activity decreased along the proximo-distal gradient. Correction with dihydroquercetin from the flavonoids used was more effective than quercetin.

The activity of the sucrose enzyme in the small intestine is, respectively, 51.03 ± 1.2 μmol / min / g in the proximal part of a healthy organism, the activity of this enzyme in the duodenum part is 64.0% (figure 3).

Figure 3. Effect of quercetin and dihydyrdoquercetin flavanoids on the amount of sucrose activity in rat small intestine in case of hypothyroidism

In all cases n=5, p<0,05;p<0,01

After receiving quercetin the activity of this enzyme in the jejunum part was 58.2%, in the duodenal region decreased by 78.0%. Under the influence of dihydroquercetin, the medium part of the small intestine decreased by 56.2%, and the distal part declined by 62.4%, respectively. Flavonoids limit the absorption of carbohydrates from food by reducing the activity of disaccharidases in the small intestine. This is achieved by normalizing glucose hemostasis. With the exclusion of experimental hypothyroidism, the activity of sucrose increased by 51.67 ± 1.3 μmol / min / g in the proximal part of the small intestine. This indicates a change in catabolic processes associated with energy catabolism in the body, along with metabolism in hypothyroidism. When adjusted with quercetin, sucrose activity showed good results in the jejunum part up to 94.7%, in the medium and ileum parts up to 94.0% and 96.0%. Under the influence of dihydroquercetin, it climbed to 96.8% in the jejunum intestine and 94.5% in the medium section approached the results of a healthy organism. This suggests that flavonoid-based diets are very effective in improving metabolism while digesting nutrients in the small intestine. Dihydroquercetin polyphenol substrates also had a relatively high effect on sucrose enzyme activity.

Maltase is the most active enzyme in the small intestine. This suggests that large amounts of enzyme substrates enter the small intestine and that most of the oligosaccharides formed during the initial hydrolysis of carbohydrates in the small intestine are substrates for the maltase enzyme. Initially, the enzyme maltase was found to be 221.8 µmol / min / g in the duodenum part of the intestine and it was 282.5 ± 8.9 µmol / min / g in the jejunum part of the intestine (figure 4).

Figure 4. Effect of quercetin and dihydyrdoquercetin polyohenols on the amount of maltase activity in rat small intestine in case of hypothyroidism and intact

In all cases n=5, p<0,05, p<0,01

Under the influence of quercetin, the activity of this enzyme decreased by 73.8% in the initial section of the intestine and by 74.1% in the jejunum section, respectively. With the introduction of the dihydroquercetin, this share decreased by 78.0% in the jejunum part and by 74.7% in the ileum part. The content of maltose substrate in the food consumed is high, and a decrease in the enzyme maltase, which is involved in their absorption by flavonoids, prevents postprandial hyperglycemia. When hypothyroidism was induced, the activity of this enzyme also increased by 296.41 ± 10.4, and it improved by 98.5% in the jejunum intestine and 92% in the ileum part when corrected with quercetin. Under the influence of dihydroquercetin, the result of a healthy organism approached 95.0% in the jejunum part of the small intestine and 88.5% in the medium part. Flavonoids are mainly digested in the lower intestine, so their effect is higher in the upper part of the small intestine, and its effect is reduced in the ileum part of the small intestine. Quercetin has a relatively good effect on the activity of the enzyme maltase, the difficulty of absorption of conjugates of quercetin in the small intestine enhances its effect.

Conclusion

In hypothyroidism, the activity of carbohydrates in the small intestine increases with metabolic disorders. Quercetin and dihydroquercetin flavonoids improve the activity of small intestinal enzymes while restoring the levels of the hormones T3, T4, frT3, frT3 in the blood.

In a healthy body, the activity of small intestinal disaccharides is reduced under the influence of quercetin and dihydroquercetin flavonoids. This is explained by that flavonoids prevent postprandial glycaemia and regulate the amount of insulin in the blood.

Dihydroquercetin is better than quercetin in improving the levels of triiodothyronine and free triiodothyronine in the blood. Dihydroquercetin was highly effective in the activities of the enzymes lactase and sucrose in the small intestine, and quercetin was highly effective in the activity of the maltase. These data increase the field of application of these flavonoids.

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