KR102659091B1 - Composition for detoxificating lead and improving sperm motility - Google Patents

Abstract

The present invention contains catechin, epicatechin, and 3,4-Dyhydroxybenzoic acid as active ingredients, and reduces lead concentration in the body exposed to lead (Pb). It relates to a composition that has the effect of improving sperm activity by suppressing the decline in sperm function caused by lead.

Description

Composition for preventing absorption of lead into the body and improving sperm activity {COMPOSITION FOR DETOXIFICATING LEAD AND IMPROVING SPERM MOTILITY}

The present invention relates to a composition for preventing the absorption of lead into the body and improving sperm activity, and more specifically, to a composition for preventing the absorption of lead into the body and improving sperm activity. More specifically, the present invention relates to a composition that effectively contains catechin, epicatechin, and 3,4-dihydroxybenzoic acid. It relates to a composition that inhibits the increase in lead concentration in the body exposed to lead (Pb) and improves sperm activity by suppressing the decrease in sperm function caused by lead by including it as an ingredient.

Exposure to heavy metals in the human body is known to be the cause of most unexplained male infertility. In particular, lead (Pb) is known to be a heavy metal that interferes with the male reproductive system. When 50 mg/l of lead acetate mixed with drinking water is fed to rats once a day, sperm count and motility decreases, the weight of the testes, spermatozoa, and epididymis decreases, and serum test sterone and β-estradiol (E2) levels decrease. It is reported that the level decreases and shows degenerative changes in the seminiferous tubules. Additionally, lead exposure resulted in down-regulation of p450 aromatase gene expression (Cyp19) as well as decreased activity of superoxide dismutase (SOD), glutathione ferrooxydiase (GPX), and catalase (CAT) enzymes in rat testicular tissue. It is reported to cause testicular oxidative stress (Eman Hassan et al., 2019).

In rats exposed to lead for 60 days, a decrease in body weight and testicular weight was confirmed, and the number and motility of sperm were reduced, sperm cell concentration, sperm ratio, significant sperm abnormalities, testicular oxidative stress, and histopathological changes were observed. caused it In addition, serum testosterone, LH, and FSH levels were significantly decreased, and the induction of testicular oxidative stress and lipid peroxidation was induced (Amany behairy et al., 2022).

Human exposure to lead mainly occurs from occupational or non-occupational sources and enters the body through inhalation, ingestion, and absorption (Sanders et al., 2009). Anthropogenic or occupational activities that involve the combustion of lead-based materials, such as smelting and recycling (Miler and Gosar, 2019), lead-based gases, or lead-based paint delaminated using lead-based aircraft fuel, generate lead particles that escape into the environment. The next individual is inhaled (Mani et al., 2020).

In addition, as it is recently known, interest in lead exposure is increasing because lead is constantly flowing into the body through the respiratory tract, as fine dust containing a large amount of heavy metals flowing into Korea from China contains lead.

Long-term exposure to lead affects male infertility due to changes in the testicles, that is, changes in the form of sperm, so various research and treatments are being developed to suppress this, including EDTA treatment to excrete lead in the blood. Efforts to reduce lead in the blood using natural products are also continuing.

In Republic of Korea Patent Publication No. 10-2101919, a mixture of red ginseng hydrogen water is used to improve sperm activity, and in Korean Patent Publication No. 10-2601506, Lactobacillus rhamnosus and Bifidobacterium longum are used. ) is obtaining the effect of improving sperm function through a formulation containing a combination of, and in Republic of Korea Patent Publication No. 10-2021-0137338, the effect of improving sperm function is being obtained by using Lactobacillus rhamnosus as an active ingredient.

Most epidemiological studies of the health effects of lead have focused on children. This is because children are more vulnerable to lead than adults. In children, it has been reported that as blood lead (B-Pb) increases, IQ (Intelligence Quotient) scores decrease. The reported paper summarizes the current opinion on health risk assessment associated with environmental exposure to lead in children: the B-Pb concern level of 100 μg/l suggested by the Centers of Disease Control in 1991; has long been accepted as a guideline value. Meanwhile, worldwide, B-Pb levels in children have decreased significantly, and the current geometric mean value in European countries is 20-30 μg/l. A recent analysis of the association of B-Pb levels with intelligence test scores suggests that the steepest decline in IQ occurs at blood levels below 100 μg/l, and that thresholds below which lead does not cause neurodevelopmental toxicity are not protective. revealed that Based on the results of the benchmark dose (BMD) analysis in 2010, the European Food Safety Authority (EFSA) concluded that IQ scores can decrease by 1 point if B-Pb increases to 12㎍/ℓ (BMDL ₁ ). It appears that this value can be used as a “unit risk” to calculate the impact of low levels of lead exposure (<100 μg/l) on the possible reduction in IQ and consequently on the health and socioeconomic status of the exposed population (Jakubowski M,2011).

The present invention was developed in consideration of the above-described prior art, and contains catechin, epicatechin, and 3,4-dihydroxybenzoic acid as active ingredients, thereby improving the absorption of lead into the body. The purpose is to provide a composition that has the effect of preventing and suppressing the decline in sperm function caused by lead.

The composition for preventing lead absorption into the body and improving sperm activity of the present invention to solve the above problems effectively contains catechin, epicatechin, and 3,4-Dyhydroxybenzoic acid. It is characterized by containing it as an ingredient.

In particular, the composition may include 20 to 30% by weight of catechin, 40 to 50% by weight of epicatechin, and 25 to 35% by weight of 3,4-dihydroxybenzoic acid.

The composition according to the present invention contains catechin, epicatechin, and 3,4-dihydroxybenzoic acid as active ingredients, thereby preventing the absorption of lead into the body and suppressing the decrease in sperm function caused by lead.

Figure 1 shows a control group (a), a group of rats that consumed drinking water containing lead (b), a group of rats that consumed drinking water containing catechin (c), and a group of rats that consumed drinking water containing epicatechin (d), 3 ,Lead in the blood of the group that consumed drinking water containing 4-dihydroxybenzoic acid (e), the group that consumed drinking water containing a low concentration composition (f), and the group that consumed drinking water containing a high concentration composition (g) This is the result of measuring the concentration.
Figure 2 shows a control group (a), a group of rats that consumed drinking water containing lead (b), a group of rats that consumed drinking water containing catechin (c), and a group of rats that consumed drinking water containing epicatechin (d), 3 , The sperm count of the group that consumed drinking water containing 4-dihydroxybenzoic acid (e), the group that consumed drinking water containing a low concentration composition (f), and the group that consumed drinking water containing a high concentration composition (g) This is the result of measurement.
Figure 3 shows a control group (a), a group of rats that consumed drinking water containing lead (b), a group of rats that consumed drinking water containing catechin (c), and a group of rats that consumed drinking water containing epicatechin (d), 3 , Mobility of sperm in the group that consumed drinking water containing 4-dihydroxybenzoic acid (e), the group that consumed drinking water containing a low concentration composition (f), and the group that consumed drinking water containing a high concentration composition (g) This is the result of measurement.
Figure 4 shows a control group (a), a group of rats that consumed drinking water containing lead (b), a group of rats that consumed drinking water containing catechin (c), and a group of rats that consumed drinking water containing epicatechin (d), 3 , Androgen receptor protein of the group that consumed drinking water containing 4-dihydroxybenzoic acid (e), the group that consumed drinking water containing a low concentration composition (f), and the group that consumed drinking water containing a high concentration composition (g) This is the result of measuring the level of expression.
Figure 5 shows a control group (a), a group of rats ingesting drinking water containing catechin (b), a group ingesting drinking water containing epicatechin (c), and a group of rats ingesting drinking water containing 3,4-dihydroxybenzoic acid. Testicular internal measurements of the group (d), the group of rats that consumed drinking water containing lead (e), the group that consumed drinking water containing a low concentration composition (f), and the group (g) that consumed drinking water containing a high concentration composition. This is the result of histological comparison of tubules.

Hereinafter, the present invention will be described in more detail. Terms or words used in this specification and claims should not be construed as limited to their common or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his or her invention in the best way. It must be interpreted with meaning and concept consistent with the technical idea of the present invention based on the principle that it is.

The composition for preventing lead absorption into the body and improving sperm activity according to the present invention is characterized by containing catechin, epicatechin, and 3,4-Dyhydroxybenzoic acid as active ingredients. do.

Catechin and epicatechin, which constitute the composition of the present invention, are flavonoid components and are known as compounds that exhibit antioxidant functions.

Flavonoids, one of the natural polyphenols with a benzo-γ-pyrone structure present in plants, are widely known as antioxidant active substances. Flavonoids have the ability to chelate metal ions. Plants have been shown to use chelation properties to detoxify heavy metals. It has been reported that a flavonoid component (morin) facilitates and efficiently excretes lead in rats (Aniruddha Adhikari et al. 2018).

Catechin increases viability in PC12 cells exposed to lead, and lead significantly reduces glutathione (GSH/oxidized glutathione (GSSP)) levels and significantly reduces glutathione reductase activity. Exposure to cadmium and lead significantly reduces the activity of antioxidant enzymes. This means that humans are exposed to the effects of toxic metals such as cadmium and lead in food, as they are present in the environment, including food. As there are no effective ways to reduce their concentrations, research is underway to develop ways to reduce their toxic effects on the body by chelating these metals (Anna Winia-Mieczan, 2018).

Heavy metal ions Cd ²⁺ , Hg ²⁺ , and Pb ²⁺ bind to bovine serum albumin (BSA), and catechin has an effect. Binding is reduced at low concentrations, but at high concentrations of lead, the binding affinity of catechin for BSA increases. was shown to increase by 9.2% (Mijun peng et al. 2011).

In addition, epigallocatechin is found in the liver to express the inflammatory signaling pathway gene TNF-α, which improves the subacute liver inflammatory state associated with obesity and improves insulin resistance in liver tissue (Huimin Hou et al ., 2020).

Lead induces the production of reactive oxygen species (ROS) and reduces cellular antioxidant defense systems, causing oxidative stress, which is why antioxidants play an important role in the treatment of lead poisoning. In PC12 cells exposed to lead, cell viability is reduced and a rapid rise in calcium (Ca) is induced, ROS accumulation and mitochondrial membrane potential are reduced. Catechin treatment significantly increases cell viability and intracellular calcium levels. and reduced ROS formation and improved mitochondrial membrane potential (MMP). In other words, it has the ability to scavenge ROS (liuji chen, 2003).

When epicatechin was administered to rats, it reduced visceral fat mass and reduced the size and TG of adipocytes as well as insulin resistance in insulin, leptin, and homeostasis (sergio de santos et al., 2019).

Recently, we have begun to understand how phytochemicals can affect metabolism, cell signaling, and gene expression. Hydroxybenzoic acid is related to salicylic acid and salicin, which was the first isolated compound with pharmacological activity. We investigated how a number of hydroxyphenols have the potential to improve age-related cardiovascular problems such as hypertension, atherosclerosis, and dyslipidemia, including 2,3-dihydroxybenzoic acid (pyrocatechuic acid); , Contains 5-dihydroxybenzoic acid (gentisic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid), 3,5-dihydroxybenzoic acid (α-Resorcylic acid), and 3-monohydroxybenzoic acid. do. The latter two compounds activate hydroxycarboxylic acid receptors, resulting in a reduction in lipolysis with a potential improvement in blood lipid profile. Some of the other compounds may reduce problems associated with oxidative stress, reducing generalized inflammation that can lead to problems such as atherosclerosis, as well as endothelial dysfunction that leads to high blood pressure. It has been known for years that increased intake of fruits and vegetables improves health. We are beginning to understand how specific phytochemicals are responsible for such therapeutic effects. The Hippocratic maxim, 'Let food be your medicine, and let medicine be your food' can now be tested experimentally, and the results of such experiments will improve the ability of nutritionists to design specific health-promoting diets ( Bernhard H J Juurlink, et al,, 2014).

In addition, 3,4-Dyhydroxybenzoic acid (3,4-DHBA) is an antioxidant active substance that removes hydroxyl radicals and hydrogen peroxide, and some dietary flavonoids prevent chronic diseases including lifestyle diseases. It exhibits antioxidant, anti-inflammatory and anti-osteoporosis activities. Dietary flavonoids (glycosidic form) are enzymatically hydrolyzed, absorbed in the intestine, and combined into the glucuronide/sulfate form by phase II enzymes in epithelial cells and liver. Gut microorganisms play an important role in the metabolism of flavonoids found in food. Certain products of some bacterial transformations, such as ring fission products and reduced metabolites, show improved properties. Studying the metabolism of flavonoids by gut microorganisms is important to understand the role of these compounds and their impact on health. This review focuses on the metabolic pathways, biological availability and physiological roles of flavonoids, especially the metabolites of quercetin and isoflavones produced by intestinal microorganisms (Kaeko Murota, 2018).

By containing the above-mentioned active ingredients, the composition of the present invention has the effect of suppressing the absorption of lead into the body, thereby suppressing the decline in sperm function caused by lead and improving sperm activity.

Specifically, the composition may include 20 to 30% by weight of catechin, 40 to 50% by weight of epicatechin, and 25 to 35% by weight of 3,4-dihydroxybenzoic acid. The content range of each ingredient is a range derived by experimentally confirming the effect of inhibiting lead absorption and improving sperm activity. It was found that if it is outside the above range, the improved effect seen when combining the three ingredients cannot be obtained.

The composition of the present invention can be diluted in water and consumed, and can be contained in food compositions and pharmaceutical compositions to obtain the desired effect.

Additionally, the composition can be powdered and used as a raw material for various functional foods. In this case, foods such as tablets, capsules, beverages, syrups, powders, and gelatin can be manufactured depending on the manufacturing method. In addition, the preferred intake of the composition may vary depending on the subject's body weight and the type and period of the drug, but it may be diluted in water and consumed at any time as described above. Additionally, the composition of the present invention is not toxic even when ingested at any time, so it can be safely taken for a long period of time.

Since the composition of the present invention inhibits the accumulation of heavy metals, especially lead components, in the body and is effective in improving sexual function and reproduction, it can be mixed with vitamins or taken with health supplements. In addition, when mixed in food or beverages, the composition of the present invention can be mixed at 0.01 to 15% by weight of the total food weight, and in the case of beverages, it is advisable to mix at a ratio of 0.02 to 5% by weight based on 100% by weight of the beverage. desirable. In addition, it can be added to carbonated drinks, natural fruit juices, vegetable drinks, etc., in which case it can be mixed in the range of 0.1 to 20% by weight based on 100% by weight of the beverage.

In addition, when a treatment for patients with lead toxicity or low sperm count is manufactured in the form of a pharmaceutical preparation, the active ingredients of the composition of the present invention are mixed with a pharmaceutically acceptable carrier and purified according to the purpose of administration. It can be formulated in a form for oral administration, such as hard, powder, or suspension. In this case, the single administration dose is 1 to 1,000 mg/kg, preferably 10 to 500 mg/kg, and more preferably 10 to 100 mg/kg, and can be administered orally 1 to 3 times a day.

The effects of the composition of the present invention on inhibiting lead absorption and improving sperm activity were confirmed through the following experiments.

3-week-old Sprague Dawley rats were purchased and stabilized for 7 days to adapt to the breeding room before performing the experiment. The breeding environment was maintained at a temperature of 23℃, humidity of 65%, and sunlight for 12 hours, and lead exposure was conducted by diluting Lead(II) Nitrate (L6258-Sigma Aldrich (St. Louis, MO, USA) in drinking water to a concentration of 500PPM). There were no restrictions on the diet, and the test substances were 50 mg/kg of catechin ((+)-Catechin hydrate) (C1251-Sigma Aldrich (St. Louis, MO, USA), epicatechin ( (-)-Epicatechin) 50 mg/kg (E1753-Sigma Aldrich (St. Louis, MO, USA), 3,4-Dihydroxybenzoic acid) 50 mg/kg (37580-Sigma Aldrich) (St. Louis, MO, USA) was supplied with water, and the composition was composed of 27% by weight of catechin, 42% by weight of epicatechin, and 31% by weight of 3,4-dihydroxybenzoic acid.

The low-concentration-composition (Com-Low) was made of a combination of 14 mg/kg of catechin, 21 mg/kg of epicatechin, and 15 mg/kg of 3,4-dihydroxybenzoic acid for a total of 50 mg/kg, and the high-concentration-composition (Com-High) prepared a composition with a total of 150 mg/kg of catechin 41 mg/kg, epicatechin 63 mg/kg, and 3,4-dihydroxybenzoic acid 46 mg/kg, and 1 ml of drinking water every day. It was diluted and administered orally. The experiment continued for a total of 4 weeks, and the animals were sacrificed after inducing fasting 12 hours before the end.

Blood was collected from the heart to check the lead concentration in the blood, and the genital area was also collected for testicular tissue and sperm analysis.

In the sperm analysis method, the unit of measured sperm count was calculated as 1×10 ⁶ /ml, and the % activity was calculated by counting 100 random sperm extracted from the tail of the epididymis.

To measure sperm motility, rat testes were evaluated using a method previously described by Behairy et al with some modifications. The epididymis of one testis was cut with sterilized scissors in a Petri dish, and the sperm were dispersed in 2 ml of physiological saline solution preheated at 37°C to evaluate individual sperm motility. A drop of epididymal suspension was transferred to a clean glass slide preheated to 37°C, covered with a glass cover slide preheated to 37°C, and observed under an optical microscope. Microscopic observation was made within 2 to 4 minutes after extraction from the epididymis, and approximately 100 sperm were analyzed. The proportion of motile sperm cells was assessed by manual calculation using a subjective score between 0 and 100%. Total sperm count/ml was determined using a sperm counting chamer.

Figure 1 shows a group of rats that consumed drinking water containing lead, a group of rats that consumed drinking water containing catechin, a group that consumed drinking water containing epicatechin, and a group of rats that consumed drinking water containing 3,4-dihydroxybenzoic acid. This is the result of measuring lead concentration in the blood of a group, a group that consumed drinking water containing a low concentration composition, and a group that consumed drinking water containing a high concentration composition.

Looking at the results in Figure 1, based on the group exposed to lead (Pb), the lead concentration in the blood of the group not exposed (Control) was close to 0%. At this time, in the group that consumed polyphenol substances, the lead concentration in the blood was confirmed to be lower than in the group exposed to lead. In particular, in the group that consumed the high-concentration composition, a statistically significant reduction of lead of 58% was confirmed, showing that lead It has been identified as a substance that prevents absorption into the body.

In addition, Figure 2 shows the group of rats that consumed drinking water containing lead, the group of rats that consumed drinking water containing catechin, the group that consumed drinking water containing epicatechin, and the drinking water containing 3,4-dihydroxybenzoic acid. This is the result of measuring the number of sperm in the group that consumed drinking water containing a low-concentration composition, and the group that consumed drinking water containing a high-concentration composition.

Looking at the results in Figure 2, it was confirmed that the sperm count, which was reduced by 21% due to lead exposure, was 100% for catechin and 118% for epicatechin, but recovered and increased to 104% in the high-concentration-composition group.

In addition, Figure 3 shows the group of rats that consumed drinking water containing lead, the group of rats that consumed drinking water containing catechin, the group that consumed drinking water containing epicatechin, and the drinking water containing 3,4-dihydroxybenzoic acid. This is the result of measuring the motility of sperm in the group that consumed drinking water containing a low-concentration composition, and the group that consumed drinking water containing a high-concentration composition.

Looking at the results in Figure 3, it was confirmed that the group that consumed all polyphenol substances recovered from 88% to 108%.

In addition, Figure 4 shows the group of rats that consumed drinking water containing lead, the group of rats that consumed drinking water containing catechin, the group that consumed drinking water containing epicatechin, and the drinking water containing 3,4-dihydroxybenzoic acid. This is the result of measuring the expression level of androgen receptor protein in the group that consumed drinking water containing a low-concentration composition, and the group that consumed drinking water containing a high-concentration composition.

Looking at the results in Figure 4, it was confirmed that the expression, which was reduced by 77% due to lead exposure, was recovered in all groups that consumed polyphenol substances, and in particular, the expression was confirmed to be increased by 104% in the high concentration-composition group.

In addition, Figure 5 shows the group of rats that consumed drinking water containing lead, the group of rats that consumed drinking water containing catechin, the group that consumed drinking water containing epicatechin, and the drinking water containing 3,4-dihydroxybenzoic acid. This is the result of a histological comparison of the seminiferous tubules in the testes of the group that consumed drinking water containing a low-concentration composition, and the group that consumed drinking water containing a high-concentration composition.

The results of Figure 5, which compares the morphological differences in testes in relation to the production of sperm, showed that the cell density of the entire seminiferous tubule was higher in the high-concentration-composition group compared to the lead exposure group in which the sperm cell density in the center of the seminiferous tubule was reduced. . This is a result that systematically confirms that sperm production activity is increased more than the control group.

The rights of the present invention are not limited to the embodiments described above but are defined by the claims, and those skilled in the art can make various changes and modifications within the scope of the claims. This is self-evident.

Claims (2)

For preventing lead absorption into the body and improving sperm activity, comprising 27% by weight of catechin, 42% by weight of epicatechin, and 31% by weight of 3,4-dihydroxybenzoic acid. Composition.
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