KR20220130966A - Composition for Prophylaxis or Treatment of Osteoporosis or Menopause-Related Decreased Motility Comprising Fermented Antler Extract - Google Patents

Abstract

The present invention relates to a composition effective for preventing or treating/alleviating symptoms of osteoporosis or female menopausal-related decreased motility that can be safely taken without side effects, and more particularly, to a composition for preventing or treating/alleviating symptoms of osteoporosis or female menopausal-related decreased motility, which contains a fermented antler extract fermented by lactic acid bacteria.

Description

Composition for Prophylaxis or Treatment of Osteoporosis or Menopause-Related Decreased Motility Comprising Fermented Antler Extract comprising fermented antler extract

The present invention relates to a composition effective for preventing or treating/improving symptoms of osteoporosis or female menopause-related reduced mobility, which can be safely taken without side effects.

A woman's menopause is a period of 1 to 2 years from the abolition of menstruation, when the female reproductive function is lost, and it is a period of transition from sexual maturity to old age. The age of menopause varies from person to person depending on constitution, nutritional status, and frequency of childbirth, and menopause tends to be slightly delayed due to the extension of life expectancy, but statistically it is considered to be 45 to 55 years old.

Before menopause, the secretion of estrogen and progesterone in the human body gradually decreases, and the symptoms of menopause appear due to the lack of hormones. One of the most common symptoms of menopause in women is osteoporosis, which occurs because bone formation is almost stopped while bone erosion continues due to abnormal tissue metabolism. Osteoporosis is most common in postmenopausal women, but can also be caused by malnutrition or reduced activity. Other symptoms of menopause include hot flashes, tachycardia, sweating, fatigue, anxiety, depression, memory loss, insomnia, and muscle weakness.

Hormone replacement therapy is the most effective way to relieve menopausal symptoms. It is known that the administration of female hormones increases bone density and reduces fractures, and it is also effective in maintaining the elasticity and thickness of the skin after menopause. However, if there is a history of hormone-dependent tumors such as endometrial cancer or breast cancer, hormone replacement therapy increases the risk of recurrence, and there is a risk of causing hypertension, thrombosis, biliary stones, and abnormal uterine bleeding.

Because of these problems, studies using natural plant-derived estrogen without side effects while exhibiting the same activity as estrogen are being actively conducted. In general, phytoestrogens, called phytoestrogen, are substances having a structure and function very similar to estrogen. Soybean isoflavone and flax seed lignan are known. However, the direct effect of these phytoestrogens in relieving menopausal symptoms has not yet been clearly elucidated, and phytoestrogens have also been reported to have potential for stroke, heart disease, and breast cancer if taken for a long time.

The deer's antlers fall off and reappear every year around the breeding season. Antler antlers are antlers that have been removed from the mouth in spring and summer, and are dried after collecting and drying those that have blood vessels inside and are flexible because keratin has not yet been formed. As the horn grows, the blood dries up and the calcium inside is hardened to ossification is called antler.

The nature of antler is warm and non-toxic, and the taste is sweet and salty. It is known to mainly act on the liver, kidney, heart, and pericardium to supplement basic physiological functions such as reproduction and growth. In oriental medicine, deer antler has been regarded as the best blood tonic since ancient times, and as recorded in Donguibogam et al. In addition, various effects such as recovery from fatigue, enhancement of physical vitality, inhibition of bone resorption and strengthening of renal function have been proven. However, deer antlers have side effects such as diarrhea, and are used for the prevention and treatment of various diseases as herbal ingredients with an astringent action in oriental medicine and as a prescription for abdominal medicine, but in this case, the expected effect may be lost or reduced. Accordingly, there is a need for a treatment method capable of increasing functionality while facilitating absorption of the deer antler extract.

In modern society, the standard of living is improving along with the continuous economic growth, but various diseases are increasing due to stress and dietary imbalances caused by complex social life, and the demand for preventing and managing them through health functional foods is increasing. to be. In recent years, health functional foods have been developed and distributed products that are convenient to take and have improved sensuality beyond simply emphasizing functionality. Deer antler is an animal material, high in protein and ash, and has limited product development due to the peculiar taste of animal origin. In addition, the high molecular weight protein contained in antlers is extracted with hot water or alcohol during extraction and concentrated to form a precipitate, which has emerged as a major obstacle to the development of liquid products of antler.

Registered Patent No. 10-0397220

In order to solve the problems of the prior art as described above, the present invention provides a composition effective for preventing or treating/improving symptoms of osteoporosis and women's menopausal-related reduced mobility without being a hormone agent or a hormone mimic that has a risk of hormone-dependent tumors. aim to

Another object of the present invention is to provide a composition containing a fermented antler ferment extract having excellent organoleptic properties by helping intestinal absorption, removing odor and taste peculiar to animals, and suppressing the formation of precipitates even in liquid products.

The present invention for achieving the above object relates to a pharmaceutical composition for preventing or treating osteoporosis or decreased female menopausal motility comprising a fermented antler fermented extract by lactic acid bacteria.

In the examples below, it was confirmed that the antler extract increases the expression of marker genes expressed in the process of bone formation, and the effect on the increase in the expression of the gene is further increased by fermentation of the antler extract. Also, in the mouse model in which menopausal was induced by ovarian extraction, the movement distance and exercise time of the mice were significantly reduced compared to the control group, but the decrease was somewhat alleviated by the administration of the antler extract. level was restored. In the menopause-induced mice, the body weight ratio of heart, liver and spleen weights increased compared to the control group, but the antler extract and the antler ferment extract restored the body weight ratio of various organ weights to the level of the control group in the menopause model mice, resulting in metabolism due to menopause. It has been shown to have the effect of mitigating changes in the process.

As the lactic acid bacteria used in the present invention, one or more strains selected from the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Bifidobacterium may be used. The incubation period is preferably 1 to 3 days, but it is not a problem if it is outside the above range. However, if the culture period is too short, the lactic acid bacteria fermentation effect cannot be sufficiently obtained, and if the culture period is too long, the economic feasibility is lowered.

In particular, in the fermented antler extract of the present invention, it is more preferable to treat the antler extract with a proteolytic enzyme before fermentation of lactic acid bacteria. Specifically, the fermented antler extract of the present invention comprises the steps of: A) extracting the antler using one or more solvents selected from solvents consisting of C1-C3 alcohols and water; B) hydrolyzing the antler extract obtained in step A) with a proteolytic enzyme; And C) the step of fermenting the hydrolysis product of step B) with lactic acid bacteria; can be prepared including.

Step A) is a step of extracting the hydrophilic useful component of the antler. The hydrophilic useful component may be extracted using one or more solvents selected from solvents consisting of C1-C3 alcohols and water as in conventional extraction of natural products. For extraction, any method used for extracting natural products in the art may be used, for example, cold extraction, heat extraction, ultrasonic extraction, etc. may be used, but the present invention is not limited thereto. The extraction time may be appropriately adjusted by those skilled in the art according to the extraction temperature and extraction method.

Step B) is a step of hydrolyzing the antler extract obtained in step A) with a proteolytic enzyme. Before hydrolysis with a proteolytic enzyme, the extract of step A) may be subjected to a concentration step. In particular, when the extraction solvent contains C1-C3 alcohol, it is preferable to remove the C1-C3 alcohol from the solvent for the enzymatic reaction.

As the proteolytic enzyme, either a purified form of an enzyme or a bacterial proteolytic enzyme may be used, but it is preferable to use one approved for use in food. Proteolytic enzymes include, for example, trypsin (Tyrpsin), neutrase, pepsin, α-chymotrypsin, in addition to the bacterial proteolytic enzyme Maxazyme ( Bacillus amyloliquefaciens ) used in the Examples below. , Papain, Protamex, Flavozyme, Alcalase, Pancrease, or various bacterial proteolytic enzymes recognized as food additives can be used, Also, the present invention is not limited thereto. It goes without saying that the treatment conditions for the proteolytic enzyme may be optimal depending on the enzyme used.

In this step, the high molecular protein contained in the extract obtained in step A) is hydrolyzed to facilitate absorption into the body, and the characteristic flavor of animal material is removed, and the functionality of the extract can be greatly increased by preventing the formation of precipitates upon concentration. have.

Since the composition is more easily absorbed in the intestine by the fermentation of lactic acid bacteria in step C), it does not cause diarrhea even when ingesting antler. In addition, the lactic acid bacteria fermentation proceeds more actively by the treatment of proteolytic enzymes, and as described above, it acts more effectively to increase the expression of osteogenic genes and prevent deterioration of menopausal motility.

The composition of the present invention may be prepared by a method known in the pharmaceutical field for use as a medicament for the prevention or treatment of osteoporosis or decreased female menopausal mobility, itself or a pharmaceutically acceptable carrier (carrier), excipient (forming agent), a diluent, etc. may be mixed and used. The composition of the present invention may be formulated and used as a formulation for oral or parenteral administration. The dosage of the active ingredient according to the present invention may be appropriately selected according to the absorption of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the severity of symptoms, etc., and the administration may be administered once a day. , may be administered in several divided doses. A typical dosage is 0.001 mg/kg·day to 10 g/kg·day.

The present invention also provides a health functional food composition for preventing or improving symptoms of osteoporosis or decreased female menopausal motility comprising a fermented antler extract fermented by lactic acid bacteria. The health functional food composition can be formulated and used in the form of tablets, capsules, pills or liquids as health functional food, or as additives of various foods, beverages, gum, tea, sweets, alcoholic beverages, chocolate, and vitamin complexes. Can be used. The fermented antler extract may be added to the food composition of the present invention in an amount of preferably 0.01 to 100% by weight.

As described above, the present invention can effectively prevent or treat/improve symptoms of osteoporosis or decreased menopausal motility without fear of hormone-related tumors caused by taking hormone preparations or hormone analogues by using the antler extract, which has long-term stability in alternative medicine. Therefore, it can contribute to the improvement of the quality of life in an aging society due to the increase in life expectancy.

In addition, the present invention prevents diarrhea by facilitating intestinal absorption by hydrolyzing the antler extract with proteolytic enzymes and then fermenting lactic acid bacteria, reduces the unique flavor and taste of animal foods, and suppresses the formation of precipitates in liquid products Therefore, it is possible to effectively utilize the useful ingredients of antler by providing a fermented deer antler extract with superior sensory properties and enhanced efficacy compared to the antler extract.

1 is a graph showing the cytotoxicity of the antler extract and the fermented antler extract.
Figure 2 is a graph showing the effect of the antler extract and the fermented antler extract on the expression of bone formation marker mRNA.
Figure 3 is a graph showing the effect of antler extract and fermented antler extract on the motility of menopause model mice.
Figure 4 is a graph showing the effect of the antler extract and the fermented antler extract on the metabolism of the menopause model mouse.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these embodiments are merely examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. It will be natural for those skilled in the art that various modifications and changes can be made within the scope of the technical spirit of the present invention based on these examples.

[Example]

Example 1: Preparation of deer antler extract (DA)

Deer antlers collected from Korean deer were divided into upper, middle, and lower antlers, cut into thin slices with a thickness of 5 mm or less, and dried in a weight ratio of 1: 1: 1 and used. . Table 1 below shows the content ratio (w%) of the results of analysis of general components of the mixed antler slices.

509.7 g of mixed antler slices and 4 L of water were placed in a stainless steel extractor and extracted at 100° C. for 24 hours. The extract was filtered through a filter cloth and concentrated under reduced pressure at 70°C. Purified water was added to the concentrate, diluted with 5 Brix to prepare an extract of 2,940 mL, and 500 mL each was transferred to a polypropylene (PP) container and stored in a freezer at -20°C. When used in the following examples, the frozen extract was taken out and melted in a water bath at 50 ° C.

Example 2: Preparation of Fermented Deer Antler Extract (FDA)

In order to hydrolyze the antler protein in the antler extract prepared in Example 1, Maxazyme NNP DS among the products approved by the Ministry of Food and Drug Safety was added at a concentration of 2% (w/v), followed by enzymatic reaction at 50° C. for 24 hours. Thereafter, lactic acid bacteria were added at a concentration of 2% (w/v) and cultured for 2 days at 37° C. in BCP petri dish. As lactic acid bacteria, Mixture of 8 lactobacilli (8 types of alpha mixed lactic acid bacteria-L(L)2, Mediogen) was used among the products approved by the Ministry of Food and Drug Safety;

For the lactic acid bacteria fermented product, the number of lactic acid bacteria was measured according to the method for measuring the number of lactic acid bacteria in the functional food industry. First, when distilled water was used as a substrate, lactic acid bacteria were not detected after fermentation. However, when the antler extract was fermented after protein hydrolysis in Preparation Example, the number of lactic acid bacteria after culturing was 2.04×10 ⁸ cfu/mL, and the antler extract was used as a substrate. In the case of lactic acid fermentation, it was 5.00×10 ⁷ cfu/mL. It is believed that the deer antler extract is used as a suitable nutrient source for culturing lactic acid bacteria, and the protein contained in the deer antler extract is converted into water-soluble substances or amino acids by proteolytic enzymes, thereby showing a more favorable effect for culturing lactic acid bacteria. It also suggests that it will help digestion and absorption when ingested by humans.

Example 3: Cytotoxicity evaluation

The cytotoxicity of antler extract (DA) and fermented antler extract (FDA) to U2OS, a human osteoblast, and RAW264.7, a mouse-derived macrophage, was measured through MTS assay. To this end, U2OS and RAW264.7 cells were aliquoted into a 96-well plate, and the antler extract or antler ferment extract was added at a concentration of 0, 0.13, 0.25, 0.50, 1.00 or 2.00 mg/mL and treated for 24 hours, followed by the MTS assay method. was used to measure cell viability.

Figure 1 shows the results, and as a result of treating the antler extract and the fermented antler extract to a concentration of 2.0 mg/mL, no significant change in cell activity was confirmed, so it could be confirmed that it could be safely used without cytotoxicity.

Example 4: Evaluation of the effect of fermented deer antler extract on bone formation

Human osteoblast U2OS was treated with the antler extract and antler ferment extract prepared in Examples 1 and 2 at a concentration of 1.0 or 2.0 mg/mL, cultured for 48 hours, and a marker gene expressed in the osteoblastic process of osteoblasts. Expression levels of RUNX2, SP7 and OCN were quantified by rtPCR. The expression level was measured as the mRNA expression level relative to the expression level of GAPHD, and the sequences of the primers used for rtPCR are described in Table 2.

2 is a result showing the expression level of the osteogenic differentiation marker gene. RUNX2 mRNA did not change or showed a tendency to decrease slightly by the fermented deer antler extract, but the expression level of the SP7 and OCN genes was greatly increased. Specifically, OCN mRNA was not significantly different from that of the control group when the antler extract was administered, but the fermented antler extract showed a significant change even when administered at 1.0 mg/mL. increased. SP7 mRNA was significantly increased by the administration of both the antler extract and the fermented antler extract, 1.52 times when treated with 2.0 mg/mL of the antler extract, and 2.31 times when treated with 2.0 mg/mL of the antler fermented extract. It was confirmed that the embryo expression increased.

This result indicates that the deer antler extract has the effect of promoting bone formation, and this effect was enhanced through fermentation.

Example 5: Evaluation of the effect of fermented deer antler extract on motility

The effect of the antler extract and the fermented antler extract on the motility of mice was evaluated by the treadmill test. Mice were acclimatized to ~week-old C67BL/6 mice for 1 week and then induced menopause through ovariectomy (OVX).

Thereafter, after having a recovery period for one week, the antler extract or fermented antler extract was mixed with the formulated feed in an environment providing a 12-week day/night cycle at 25° C. for 12 weeks, and it was fed ad libitum with sufficient water. After 12 weeks, the treadmill test was conducted until the speed was gradually increased from 10 cm/sec to 45 cm/sec at a 0° inclination and lagged behind by more than 10 seconds. Mobility was compared based on the exercise distance and exercise time, and FIG. 3 shows the results. The movement distance of the ovariectomized group was 346.6±70.7 m, which decreased compared to the control group that moved 476.6±76.3 m, but it was 417.4±90.8 m in the group taking the antler extract and 453.6±62.6 m in the group taking the antler ferment extract. It shows that mobility is restored. Exercise time was also significantly decreased by ovariectomy, but it was recovered by taking the antler extract, and the fermented antler extract showed exercise time similar to that of the control group.

That is, it was confirmed that the antler extract restores the decrease in motility of mice due to ovariectomy, and this effect is enhanced by the fermentation of the antler extract, similar to the bone formation promoting effect.

After the treadmill test was completed, mice were sacrificed, and the heart, liver and spleen were removed and weighed to determine the effect of the antler extract and the fermented antler extract on metabolism. As a result, as shown in FIG. 4 , the weight-to-weight ratio of the heart, liver, and spleen increased by ovariectomy was restored to the level of the control group by taking the antler extract or fermented antler extract.

<110> SEOWON UNIVERSITY INSTITUTE OF INDUSTRY-ACADEMY COLLABORATION <120> Composition for Prophylaxis or Treatment of Osteoporosis or Menopause-Related Decreased Motility Comprising Fermented Antler Extract <130> P0321-155 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tggacgaggc aagagtttca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcccgaggtc catctactgt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gatctggtgc ctagaagccc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 caagctccag cggctttaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gctccagggg atccgggta 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagcccagcg gtgcagagt 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ttgaggtcaa tgaaggggtc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gaaggtgaag gtcggagtca 20

Claims (4)

A pharmaceutical composition for preventing or treating osteoporosis or decreased female menopausal mobility, comprising a fermented antler ferment extract fermented by lactic acid bacteria.
The method according to claim 1,
The lactic acid bacteria is Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, a pharmaceutical composition for the prevention or treatment of osteoporosis or reduced female menopausal mobility, characterized in that at least one selected from the strains of the genus Bifidobacterium.
The method according to claim 1 or 2,
A) extracting the deer antler using one or more solvents selected from solvents consisting of C1-C3 alcohols and water;
B) hydrolyzing the antler extract obtained in step A) with a proteolytic enzyme; and
C) fermenting the hydrolysis product of step B) with lactic acid bacteria;
A pharmaceutical composition for the prevention or treatment of osteoporosis or decreased female menopausal motility, characterized in that it is prepared including a.
A health functional food composition for preventing or improving symptoms of osteoporosis or decreased female menopausal motility, comprising a fermented antler extract fermented by lactic acid bacteria.

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