KR20200064912A - Composition for Preventing or Treating for Metabolic Bone Disease or Climacteric Disease - Google Patents

Abstract

The present invention relates to a composition for preventing or alleviating metabolic bone diseases or climacteric symptoms, containing a complex extract of Schisandra chinensis and Platycodon grandiflorum, as an active component. When using the present invention, metabolic bone diseases or climacteric symptoms can be effectively prevented or alleviated without side effects.

Description

Composition for Preventing or Treating Metabolic Bone Disease or Climacteric Disease

The present invention was made by task number HI16C0275 under the support of the Ministry of Health and Welfare of the Republic of Korea, and the research institute specialized in research and management of the above task is the Korea Institute of Health Industry Promotion, and the research project name is “Korean Standard Clinical Clinical Guideline Development Project Group (Industrialization stage of the Chinese medical treatment technology public resource project) Research)”, the title of the research is “Optimization Study through Establishment and Validation of Quality Indicators of Saengmaek-gagam for the Development of Herbal Medicines for the Treatment of Menopausal Syndrome”, Organized by Viromed Co., Ltd., Research Period is 2018.07.02-2018.11.30.

This patent application claims priority to Korean Patent Application No. 10-2018-0150174 filed with the Korean Intellectual Property Office on November 28, 2018, and the disclosure of the patent application is incorporated herein by reference.

The present invention relates to a composition for preventing or treating metabolic bone disease or climacteric symptoms, comprising the complex extract of Omija and Gilkyung as an active ingredient.

Metabolic bone disease is caused by the loss of harmony in the activity between osteoblasts (osteoblasts), which serve to produce bones in the body, and osteoclasts, which serve to destroy bones. Osteoclasts are large multinucleated cells that destroy or absorb bone tissue that has become unnecessary in the course of bone growth. Mature osteoclasts are multinuclear cells and originate from hematopoietic stem cells. Osteoblasts differentiated from mesenchymal stem cells survive for about 34 months and make new bones where activated osteoclasts degrade old bones. A number of osteoblasts make bone matrix, and as the matrix gradually becomes mineralized, bone formation ends. After that, about 70% or more of osteoblasts die, and some of them survive as differentiated into osteoblasts and bone lining cells. Since the amount of bone is maintained by the balance of osteoclasts and osteoblasts, it is important to develop a therapeutic agent targeting molecules that play an important role for osteoclasts. In other words, when the activity of osteoclasts that absorb bone increases, the decomposition of bones accelerates, and diseases such as osteoporosis, which makes bones thinner and easily breaks, cause factors that can regulate the activity of osteoclasts to treat bone disease. Is being studied as.

For example, osteoopenia refers to all stages of osteoporosis, and it is known that the cause of the occurrence is due to excessive resorption and formation of osteoclasts. Bone atrophy in rheumatism is also associated with excessive osteoclast uptake. Fibrous dysplasia is caused by active osteoclasts. Paget disease and hypercalcemia are used as therapeutic agents by suppressing osteoclast function. Inhibition of excessive osteoclast production and/or activity can inhibit neoplastic bone destruction, and osteolysis and osteoarthritis increase osteoclast uptake or osteoclast differentiation. Is caused by.

On the other hand, it is known that cancer cells entering the bone proliferate in the microenvironment around the bone and stimulate osteoclast or osteoblast activity to determine whether to proceed to osteolytic bone metastasis or osteoblastic bone metastasis. Cancer cells that settle in the bones while circulating along the blood vessels secrete osteolytic factors (PTHrP (parathyroid hormonerelated protein), interleukin (IL)-1, IL-6, IL-8 and IL-11) . The secreted factors reduce the expression of osteogroterin (OPG) in osteoblasts and increase the expression of the receptor activator of NF-kB ligand (RANKL). Increased RANKL combines with RANK of osteoclast precursor cells to mature a large number of osteoclast precursor cells and eventually cause bone destruction by excessive bone absorption.

In addition, women's menopause (climacteric) is a kind of endocrine syndrome, which means a transitional period in which physiological and sexual functions are reduced or lost due to a decrease in estrogen, a female hormone caused by overall and gradual aging of ovarian function. Even in menopausal cases, the same symptoms appear in patients with estrogen deficiency due to other causes, such as ovarian resection and hypoovarian function. Symptoms of menopause include symptoms caused by vascular changes such as hot flashes, tachycardia, sweating or headaches, and musculoskeletal changes such as muscle pain, joint pain and back pain. In addition, there are symptoms due to urogenital changes such as urination or urinary incontinence, symptoms due to changes in the nervous system, such as decreased memory, depression, decreased concentration and dizziness, as well as symptoms such as decreased vision and changes in skin and hair In some cases, fatal diseases occur in women's health, such as menopausal osteoporosis or cardiovascular disease due to hormonal changes.

In order to improve the physical and mental health and quality of life of middle-aged women, there is a need to develop a therapeutic agent that can improve menopausal symptoms, and drugs such as hormone replacement therapy and non-steroidal drugs have been developed to improve menopausal symptoms. There is a bar. However, most of these drugs are known to have side effects such as headache and weight gain. Especially, in the case of estrogen replacement therapy, hormones are artificially administered to the body, and thus, with rejection, uterine bleeding, stroke, and heart It is known that the risk of seizures, breast cancer, and uterine cancer may increase.

Due to these problems, interest in replacing estrogen therapy with natural methods taken in the form of food or additives is increasing, and there is a need to develop a new menopausal treatment that has excellent effects of alleviating symptoms of menopause without side effects. This is true.

Accordingly, the present inventors completed the present invention by conducting many studies to find a substance capable of preventing or improving metabolic bone disease or menopausal symptoms.

The present inventors have made extensive efforts to develop a substance capable of preventing or improving metabolic bone disease or menopausal symptoms derived from natural products having excellent effects without side effects. As a result, the present invention has been completed by clarifying that the composition comprising the extract of Omija and Gilkyung complex has excellent efficacy in preventing, improving or treating metabolic bone disease or menopausal symptoms.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic bone disease or menopausal symptoms.

Another object of the present invention is to provide a food composition for preventing or improving metabolic bone disease or menopausal symptoms.

Another object of the present invention is to provide a method for preventing, improving, or treating metabolic bone disease or menopausal symptoms.

According to one aspect of the invention, the present invention (a) Omija (Schisandra chinensis) and Gilgyeong (Platycodon grandiflorum) complex extract as an active ingredient; And

(b) provides a pharmaceutical composition for preventing or treating metabolic bone disease or climacteric symptoms, including a pharmaceutically acceptable carrier.

The present inventors have made extensive efforts to develop a substance capable of preventing, improving or treating metabolic bone disease or menopausal symptoms derived from natural products, which have excellent effects without side effects. As a result, it was found that the composition comprising the complex extract of Omija and Gilkyung has excellent efficacy in preventing, improving or treating metabolic bone disease or menopausal symptoms.

The'Platycodon grandiflorum' in the present invention is the root of the bellflower (Platycodon grandiflorum A. DC.), a plant of the Campanulaceae family, and is usually Gilkyoung in Korea, Jigeng in China, and Japan. It is called Kikyo and has been used as a traditional oriental medicine. Gilkyung has been mainly used as a treatment for expectorant, antitussive, cough and bronchitis.

The'Omija (Schisandrae Fructus)' in the present invention is a plant having a dark red fruit with a diameter of about 1 cm in the shape of a ball, in which the red juice and reddish brown seeds are contained 1-2. Types include North Omija (S. chinensis), South Omija (S. sphenanthera), and Black Omija (S. nigra). It is known that it contains ingredients such as cyzandrine, gomisin, citral, malic acid, and citric acid, which can be used as a tonic agent by strengthening the heart, lowering blood pressure, and increasing immunity.

The composition of the present invention includes Omija and Gilkyung complex extracts as active ingredients. The extract of Omija and Gilgyeong of the present invention is prepared through (i) a single extraction process of extracting a mixture of Omija and Gilgyeong as an extraction solvent, or (ii) after preparing an extract of a single ingredient of Omija and Gilgyeong, respectively. It can be prepared by mixing the extract.

The term'extract' used in the present specification has a meaning commonly used as a crude extract in the art as described above, but broadly also includes a fraction in which an extract is additionally fractionated. That is, the complex extract of Omija and Gilkyung includes not only obtained using the above-described solvent, but also obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (made for different separation in size, charge, hydrophobicity or affinity), etc. Fractions obtained through the purification method are also included in the extract of Omija and Gilkyung of the present invention.

In one embodiment of the present invention, the extract of the present invention is a polar organic solvent extract. The term'polar organic solvent' used herein refers to (a) water, (b) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (eg methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol) Etc.), (c) acetic acid, or a mixture of polar organic solvents.

In one embodiment of the present invention, the polar organic solvent of the present invention is water, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, acetic acid or a mixture of two or more components selected from the aforementioned components.

In one embodiment of the present invention, the anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms of the present invention is any one selected from the group consisting of methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol. That's it.

The concentration of the organic solvent is 1 to 100% (v/v), specifically 10 to 100% (v/v), 20 to 100% (v/v), 30 to 100% (v/v)), 40 to 100% (v/v), 50 to 100% (v/v), 60 to 100% (v/v), 70 to 100% (v/v), 80 to 100% (v/v) days May be, and more specifically, may be 25% (v/v), 50% (v/v), 75% (v/v), or 95% (v/v), but is not limited thereto. In order to prepare an extract according to an embodiment of the present invention, the amount of the solvent used for extraction may be appropriately selected depending on the amount of herbal ingredients used. Specifically, for example, a volume of 1 to 20 times the weight of Omija and a long diameter used for preparing the extract, more specifically a volume of 2 to 20 times, more specifically a volume of 5 to 15 times, even more Specifically, it may be 7 to 12 times the volume, but is not limited thereto.

The extraction temperature of the extract of the present invention is not particularly limited, and may be, for example, 0°C to 120°C, and specifically 15°C to 95°C.

In one embodiment of the present invention, when the extraction solvent of the present invention is water, the extraction temperature is preferably 80°C or higher, and when the extraction solvent is a lower alcohol having 1 to 4 carbon atoms, it is preferable that it is 15°C to 30°C.

The extraction time of the extract of the present invention is not particularly limited, and may be, for example, 1 hour to 10 days, 1 hour to 120 hours, specifically 1 hour to 72 hours, 1 hour to 48 hours, and 1 hour to 36 hours. , 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 6 hours, but is not limited thereto.

The extract used in the present invention can be extracted through hot water extraction, cold needle extraction, reflux cooling extraction, ultrasonic extraction, or conventional extraction methods known in the art. In an embodiment of the present invention, the extract of the present invention may be prepared through hot water extraction or cold acupuncture extraction method using lower alcohol, and may be repeatedly extracted 1 to 10 times.

The Omija and Gilkyung complex extract of the present invention can be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In one embodiment of the present invention, the compounding weight ratio (w/w) of Omija and Gilkyung, which are active ingredients, is 1:20 to 20:1. In another embodiment, the compounding weight ratio of Omija and the long diameter is (w/w) is 1:15 to 15:1, 1:12 to 12:1, 1:10 to 10:1, 1:10 to 5:1, 1 :10 to 3:1, 1:10 to 1:2, 1:10 to 1:1, 1:8 to 8:1, 1:8 to 5:1, 1:8 to 3:1, 1:8 To 1:2, 1:8 to 1:1, 1:6 to 6:1, 1:6 to 5:1, 1:6 to 3:1, 1:6 to 2:1, 1:6 to 1 :1, 1:5 to 5:1, 1:5 to 3:1, 1:5 to 2:1, 1:5 to 1:1, 1:4 to 4:1, 1:4 to 3:1 , 1:4 to 2:1, 1:4 to 1:1, 1:3 to 3:1, 1:3 to 2:1, 1:3 to 1:1, 1:2 to 2:1, 1 :2 to 1:1, 1:1.5 to 1.5:1 or 1:1.

In a specific embodiment of the present invention, the compounding weight ratio of the Omija and the long diameter is 1:1 to 1:8, and more specifically 1:1, 1:2, 1:4, 1:8, 2:1, 4:1, or 8:1, and most specifically 1:2, but is not limited thereto.

The term'compound weight ratio (w/w)' in this specification refers to the weight ratio of each component before going through the extraction process. For example, when the composite extract of the present invention is prepared through a single extraction process for extracting a mixture of Schisandra chinensis and Gilkyung with an extraction solvent, it indicates the weight ratio of each single ingredient of Schisandra chinensis and Gilkyung contained in the mixture. In addition, when the composite extract of the present invention is prepared by mixing the extracts of a single component of Omija and Gilgyeong each, and then mixing the extracts of the two components, the weight ratio between the'single component reference weights' calculated by the following calculation formula is shown. :

[formula]

Weight per single component = weight of single component used to prepare a single extract x (weight of single extract used for final composite extract preparation/weight of prepared single extract).

The composition of the present invention can be prepared as a pharmaceutical composition.

In the present invention, the term'prevention' refers to all actions of inhibiting or delaying the onset of metabolic bone disease or menopausal symptoms by administration of a composition comprising the extract of Schisandra chinensis and Gilkyung according to the invention as an active ingredient.

In the present invention, the term'treatment' refers to all actions in which the symptoms of metabolic bone disease or menopausal symptoms improve or are advantageously changed by administration of a composition comprising the compound of Omija and Gilkyung according to the present invention as an active ingredient.

The pharmaceutical composition of the present invention includes a pharmaceutically effective amount of Omija and Gilkyung complex extract. The'pharmaceutical effective amount' means an amount sufficient to achieve the efficacy or activity of the above-mentioned Omija and Gilkyung complex extract, and is not particularly limited as long as the object of the present invention is achieved.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, and mineral oil, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remignton's Pharmaceutical Sciences (19th ed., 1995).

Suitable dosages of the pharmaceutical compositions of the present invention are variously prescribed by factors such as formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, administration route, excretion rate and response sensitivity. Can be. The typical dosage of the pharmaceutical composition of the present invention is in the range of 0.001-1000 mg/kg on an adult basis. In addition, the dose in the human body can be converted based on animal experiments.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

The term'metabolic bone disease' in the present invention means a bone-related disease caused by an imbalance between osteoblasts and osteoclasts. A specific example is a disease that promotes bone destruction due to pathological bone diseases such as osteoprosis, arthritis, periodontal disease, fracture or Paget disease due to excessive osteoclast bone absorption. This includes, but is not limited to the bone metabolic disease.

The above-described "osteoporosis" refers to a state in which the amount of bone is reduced and the strength of the bone is weakened due to a qualitative change, so that fracture is likely to occur. Bone protects various organs in the human body and functions as a reservoir to store necessary substances in the body, such as calcium, and the balance between osteoclasts that break down bones present in bone tissue and osteoblasts that produce bones Following this, homeostasis is maintained. In the case of osteoporosis, the balance of the activity of the two cells is broken, and excessive bone destruction by osteoclasts occurs, resulting in development and progression. A major molecule in osteoporosis is the receptor activator of nuclear factor kappa-B ligand (RANKL). RANKL binds to its receptor to activate various transcription factors to promote osteoclast differentiation.

The term "arthritis" refers to a joint disease involving inflammation in one or more joint sites. The general form of the arthritis is osteoarthritis (osteoarthritis), in which inflammation and pain occur due to damage to the bones and ligaments constituting the joints due to gradual damage or degenerative changes of cartilage protecting the joints. For the purposes of the present invention, the arthritis is a disease involving bone loss at the joint site, but the type is not particularly limited, but may be osteoarthritis or rheumatoid arthritis.

The "periodic disease" refers to the inflammatory state of the dental support tissue caused by bacteria, and can be divided into gingivitis and periodontitis. The cause of the disease lies in the formation of a bacterial membrane when oral bacteria are caused by poor oral hygiene. Tooth bacterial membrane refers to a lump of bacteria that grows after the bacteria stick to the surface of the tooth using the sticky substance in the saliva as an adhesive. If you leave the germ membrane on when you leave it, it will become inflamed, sometimes causing bleeding from the gums and bad breath. These symptoms are called gingivitis. As the gingivitis progresses further, the gap between the teeth and gums deepens, resulting in periodontal pockets, in which bacteria causing periodontal disease multiply, causing periodontitis. When periodontitis develops, the swelling and swelling of the gums may occur even with mild stimuli such as brushing, and it often turns into acute inflammation, causing pain. This inflammation decreases the function of making bones, and the action of absorbing bones increases, so that the alveolar bones are gradually lowered, the alveolar bones are destroyed and eventually the teeth are lost.

The "fracture" refers to a fracture of the bone in a state in which the continuity of the bone, bone end plate, or joint surface is abnormally broken. Causes of fracture include trauma such as traffic accidents, safety accidents occurring in industrial disorders, bone changes caused by diseases such as osteoporosis, bone cancer, and metabolic disorders, and stress on repetitive bones due to sports or loads. In addition, the fracture state is based on the fracture line (line along the end of the bone generated by the bone fracture), crack fracture, greenstick fracture, transverse fracture, sacrum fracture, spiral fracture, segmental fracture, fracture fracture, ruptured fracture , Compression fracture, depression fracture, etc.

The "paget disease" refers to a topical bone disease in which bone remodeling is excessively enhanced and the skeletal system of a wide area is affected. It is known that the pathological mechanism of Paget's disease is a combination of an excessive increase in bone resorption by osteoclasts having the function of cleaning bones and an increase in new bone formation by osteoblasts having a function of making bones as a compensation action. In addition, bones newly formed in bone Paget's disease are known to be structurally disordered and very vulnerable to bone deformation and fracture.

The term'menopausal symptoms' in the present invention includes symptoms caused by vascular changes such as hot flashes, tachycardia, sweating or headaches, and symptoms caused by musculoskeletal changes such as muscle pain, joint pain and back pain. In addition, there are symptoms due to urogenital changes such as urination or urinary incontinence, and symptoms caused by changes in the nervous system, such as decreased memory, depression, decreased concentration and dizziness, as well as symptoms such as decreased vision and changes in skin and hair. And include diseases that are fatal to women's health, such as menopausal osteoporosis or cardiovascular disease due to hormonal changes.

As demonstrated in the following examples, the complex extracts of Omija and Gilkyung of the present invention significantly increased the activity of TRAP in the osteoclast differentiation experiment of RANKL treatment on mouse macrophage cell lines compared to the negative control and Omija and Gilja individual extract treatment groups. By reducing the osteoclast differentiation, osteoporosis treatment and improvement efficacy was confirmed.

In addition, the complex extract of Omija and Gil-kyung of the present invention exhibits a vasodilating effect in the cardiovascular disease model using a thoracic aortic vascular segment compared to the negative control group and the individual extract-treated group of Omija and Gil-kyung, and thus climacteric cardiovascular disease. The efficacy of treatment and improvement has been confirmed.

In addition, the complex extract of Omija and Gilkyung of the present invention significantly improves biochemical indicators for osteoporosis such as ALP, Ca, Pi, MMP-9, and Osteocalcin compared to negative control and individual extracts in ovarian resected osteoporosis mouse model. The efficacy of treating and improving osteoporosis has been confirmed.

The effect of the complex extract of Omija and Gilkyung is synergistic for each indicator compared to the individual extract of Omija and Gilkyung.

According to another aspect of the present invention, the present invention provides a food composition for preventing or improving metabolic bone disease or climacteric symptoms, which contains a complex extract of Omija and Gilkyung as an active ingredient.

In the present invention, the term'improvement' refers to all acts of at least reducing the degree of metabolic bone disease or menopausal symptoms by administration of a composition comprising the extract of Schisandra chinensis and Gilkyung according to the present invention as an active ingredient.

When the composition of the present invention is prepared as a food composition, it contains components that are conventionally added at the time of food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents and synthetic flavoring agents can be used. For example, when the food composition of the present invention is prepared as a drink agent, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc. may be further included in addition to the active ingredient of the present invention.

In the case of the food composition of the present invention, the complex extract of Omija and Gilkyung contained in the pharmaceutical composition, which is one aspect of the present invention described above, can be used as it is, and the description of overlapping contents is omitted to avoid excessive complexity of the present specification Do it.

In addition, according to another aspect of the present invention, the present invention is a metabolic process comprising the step of administering a pharmaceutical composition or food composition comprising a complex extract of the above-described Schisandra chinensis and gills of the present invention as an active ingredient It provides a method of preventing, treating or improving bone disease or menopausal symptoms.

Metabolic bone disease and menopausal symptoms, which are target diseases of the treatment method or improvement method of the present invention, are as defined in relation to the target diseases of the pharmaceutical composition or food composition.

The term “administration” or “administer” as used herein refers to the same amount in a subject's body by administering a therapeutically or ameliorating effective amount of a composition of the invention directly to a subject (individual) suffering from the subject disease. It means to be formed.

The "therapeutically effective amount" of the composition means a content of a composition sufficient to provide a therapeutic or prophylactic effect to a subject who intends to administer the composition, and is meant to include a "prophylactically effective amount". Also, as used herein, the term "subject" is a mammal including humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons, rhesus monkeys, and the like. Most specifically, the subject of the invention is a human.

The method for preventing, improving or treating metabolic bone disease or menopausal symptoms of the present invention is a method comprising administering a pharmaceutical composition or a food composition of one aspect of the present invention. The description is omitted to avoid excessive redundancy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating metabolic bone disease or climacteric symptoms.

(b) The present invention provides a food composition for preventing or improving metabolic bone disease or climacteric symptoms.

(c) The present invention provides a method for preventing, improving or treating metabolic bone disease or menopausal symptoms.

(d) When using the pharmaceutical composition or food composition of the present invention, metabolic bone disease or climacteric symptoms can be effectively prevented, improved or treated without side effects.

Figure 1 shows the effect of reducing the TRAP activity of the complex extract of Omija and Gilkyung on differentiated osteoclasts according to Experimental Example 1. ### P <0.001 vs. Omija group; *** P <0.001 vs. Gil-Kyung group (one-way ANOVA, with Bonferroni's multiple comparison test).
Figure 2 shows the effect of reducing the TRAP activity of the complex extract for each hot water and ethanol concentration for differentiated osteoclasts according to Experimental Example 1. *** P <0.001 vs. The negative control group (one-way ANOVA, with Bonferroni's multiple comparison test).
Figure 3 shows the vasodilator effect of the complex extract of Schisandra chinensis and Gilkyung on the thoracic aorta section according to Experimental Example 2. # P <0.05 vs. Omija group; * P <0.05 vs. Gil Kyung group (one-way ANOVA, with Bonferroni's multiple comparison test).
4 to 8 are ALP (FIG. 4), Ca (FIG. 5), Pi (FIG. 6), MMP-9 (FIG. 7), and Osteocalcin in each blood compared to the ovarian ablation group (OVX+Vehicle) according to Experimental Example 3. (FIG. 8) The effects of Gilkyung, Omija individual and complex extracts on concentration are shown. * P <0.05, **** P <0.0001 vs. OVX+Vehicle group (one-way ANOVA, with Dunnett's multiple comparison test).

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Manufacturing example 1: Omija ( Schisandrae Fructus ) And Road ( Platycodon grandiflorum ) Preparation of complex extracts

Washed and dried Schizandra chinensis and Platycodon grandiflorum by weight ratio (w/w) of 1:1, 2:1, 4:1, 8:1, 1:2, 1:4 and 1:8 After mixing with 10% 70% aqueous ethanol solution, the mixture was stirred with stirring at a temperature of 20°C for 72 hours, filtered, concentrated under reduced pressure at 45-50°C, and lyophilized to obtain a complex extract powder. The yield was as shown in Table 1 below.

division Types of herbal medicine yield(%) Preparation Example 1-1 Omija and Gilkyung (1:1) 27.74 Preparation Example 1-2 Omija and Gilkyung (1:2) 25.21 Preparation Example 1-3 Omija and Gilkyung (1:4) 23.16 Preparation Example 1-4 Omija and Gilkyung (1:8) 21.08 Preparation Example 1-5 Omija and Gilkyung (2:1) 29.54 Preparation Example 1-6 Omija and Gilkyung (4:1) 30.93 Preparation Example 1-7 Omija and Gil Kyung (8:1) 32.16

Comparative example 1: Preparation of a single extract

Washed and dried Omija and Gilkyung were added with 10 times 70% ethanol aqueous solution to the weight of each crude drug, stirred well at 72°C for 72 hours, extracted, filtered and concentrated under reduced pressure at 45-50°C, then freeze-dried To obtain a total of two single extract powder, the yield was as shown in Table 2 below.

division Types of herbal medicine yield(%) Comparative Example 1-1 Schisandra 32.50 Comparative Example 1-2 Road 21.56

Manufacturing example 2: Omija ( Schisandra chinensis ) And Road ( Platycodon grandiflorum ) Preparation of complex extracts with different extraction solvents

Washed and dried Omija (Schisandra chinensis) and Gilkyung (Platycodon grandiflorum) were mixed at a weight ratio of 1:2 (w/w), and in the case of hot water extract, reflux was extracted at 90±5° C. for 3 hours, and ethanol extract In the case, 10 times of 0, 25, 50, 75, 95% ethanol aqueous solution was added and extracted with stirring at a temperature of 20° C. for 72 hours. The extracted hot water extract and ethanol extract were filtered, concentrated under reduced pressure at 45-50° C., and lyophilized to obtain a composite extract powder. The yield of the extract according to the extraction solvent was as shown in Table 3 below.

division Types of herbal medicine Extraction solvent yield(%) Preparation Example 1-8 Omija and Gilkyung (1:2) Distilled water (hot water) 37.57 Preparation Example 1-9 Omija and Gilkyung (1:2) 0% ethanol (cold cooling) 31.17 Preparation Example 1-10 Omija and Gilkyung (1:2) 25% ethanol (cold) 32.44 Preparation Example 1-11 Omija and Gilkyung (1:2) 50% ethanol (cold cooling) 29.55 Preparation Example 1-12 Omija and Gilkyung (1:2) 75% ethanol (cold) 21.94 Preparation Example 1-13 Omija and Gilkyung (1:2) 95% ethanol (cold) 7.91

Experimental Example One: RANKL (receptor activator of NF - kappaB ligand , Sigma- Aldrich , US) induced osteoclasts Of complex extracts against differentiation Inhibitory efficacy

TRAP (Tartrate-resistant acid phosphatase) is an enzyme used as an indicator of osteoclast differentiation, and a decrease in TRAP activity indicates inhibition of osteoclast differentiation. In this experiment, individual and complex extracts were derived from osteoclasts derived from mouse macrophage cell line RAW264.7 cells by osteoclasts derived and differentiated by RANKL (receptor activator of NF-kappaB ligand, Sigma-Aldrich, US). The effect on differentiation and the effect on the differentiation of osteoclasts of complex extracts by extraction solvent were confirmed.

1-1. Cell line culture

First, RAW264.7 cells were treated with 5% CO₂ at 37°C using 10% charcoal-stripped fetal bovine serum (GIBCO)-added phenol-red free α-MEM (GIBCO, US) medium. Cultured in an incubator. After dispensing RAW264.7 cells in a 96-well-plate to a 1x10 ³ cell number per well and stabilizing for 24 hours, RANKL at 50 ng/ml, Omija, individual extracts of pathogens and complex extracts at 600 ug/ml TRAP activity analysis was performed after incubating for 72 hours by treating as such or treating 600-ug/ml concentration of Omija, Gilkyung hot water complex extract and ethanol concentration-specific complex extract.

1-2. TRAP activity measurement

Specifically, 30 ul of TRAP activity evaluation solution (sodium acetate 600 mM, pH 5.5, L-ascorbic acid 17.6 mg/ml, sodium-tartrate dehydrate 9.2 mg/ml, 4-nitrophenylphosphate Na 3.6 mg/ml, Triton X-100 0.3%, EDTA 6 mM, NaCl 600 mM) and reacted in a 37° C. incubator for 30 minutes. After the reaction, the reaction was stopped with 300 mM NaOH, and absorbance was measured at a wavelength of 450 nm.

TRAP activity was expressed as a ratio compared to the RANKL treated group (negative control group).

In the case of the comparative experiment of the individual extract and the complex extract, as shown in Table 4 and FIG. 1, as a result of treating RANKL on RAW264.7 cells, the activity of TRAP was higher than that of the untreated group, which is all of the extracts. Decreased by treatment group. Particularly, compared to the individual extract-treated group with Omija and Gil-kyung, the superior effect of the complex extract compared to the individual extract was confirmed for the inhibition of osteoclast differentiation through a high decrease in TRAP activity in the experimental group treated with the complex extract.

Experimental group Omija: Gilkyung ratio TRAP activity (%) Untreated - 14.24± 1.49 RANKL treatment group - 100± 7.03 Omija extract (Comparative Example 1-1) - 63.23± 4.85 Gilkyung Extract (Comparative Example 1-2) - 22.98± 1.04 Complex extract (Production Example 1-1) 1:1 20.66± 1.68 Complex extract (Production Example 1-2) 1:2 7.54± 6.53 Complex extract (Production Example 1-3) 1:4 6.53± 0.06 Complex extract (Production Example 1-4) 1:8 6.20± 0.05 Complex extract (Production Example 1-5) 2:1 29.12± 1.26 Complex extract (Production Example 1-6) 4:1 64.39± 3.64 Complex extract (Production Example 1-7) 8:1 88.45± 5.78

As shown in Table 5 and FIG. 2, the hot water extract (Preparation Example 1-8) and the cold acupuncture extract according to the ethanol concentration (Preparation Example 1-9, 1-10, 1-11, 1-12, 1-13) It was confirmed that TRAP activity was significantly reduced in all the treated experimental groups.

Experimental group Omija: Gilkyung ratio TRAP activity (%) Untreated - 23.75± 2.42 RANKL treatment group - 100± 14.72 Hot water extract (Production Example 1-8) 1:2 31.79± 0.48 0% ethanol cold acupuncture complex extract (Production Example 1-9) 1:2 22.78± 1.90 25% ethanol cold acupuncture complex extract (Production Example 1-10) 1:2 22.06± 0.52 50% ethanol cold acupuncture complex extract (Production Example 1-11) 1:2 18.93± 0.92 75% ethanol cold acupuncture complex extract (Production Example 1-12) 1:2 18.95± 1.92 95% ethanol cold acupuncture complex extract (Production Example 1-13) 1:2 18.44± 0.92

In addition, as a result of correcting the result values of Tables 4 and 5, it was confirmed that Preparation Examples 1-2 of Table 4 and Production Examples 1-12 of Table 5 have similar TRAP activity.

Experimental Example 2: Miozami and Roadside Measurement of vasodilator efficacy of complex extracts

Estrogen deficiency causes vasoconstriction, causing blood pressure rise in postmenopausal women, and cerebral vasoconstriction causes stroke, so vasodilator efficacy is a major evaluation index for prevention and treatment of menopausal cardiovascular disease. Is being used. The present inventors to confirm the vasodilating effect of the extract of Schisandra chinensis and Gilkyung complex extracted in Preparation Example 1, of the thoracic aorta section of rats induced contraction with Phenylephrine (PE) using an Organ Bath test The relaxation reactivity was evaluated.

2-1. Production of thoracic aortic angiography

SD (Sprague Dawley) Rat 8-week-old males were used as the experimental animals, and after aspiration anesthesia with SD rats with Ethylether, the thoracic aorta was excised by excising the chest immediately. The extracted blood vessel was immediately supplied with a gas mixed with 95% O2 and 5% CO2 and maintained at 37°C, a Krebs-Henseleit solution (KH solution, composition, mM: NaCl, 118.0; KCl, 4.7; MgSO₄, 1.2; KH₂PO₄, 1.2; CaCl₂, 2.5; NaHCO₃, 25.0; and glucose, 11.1; pH 7.4) were removed, and tissues and fats around the blood vessels were removed, and then cut into a ring shape of about 2 mm in length to produce vascular fragments.

2-2. Isotonic shrinkage measurement

The produced vascular segments are tungsten rings at both ends, and the bottom is connected to a ring mounted at the bottom of an organ bath with a capacity of 10 ml, and the top is a physiograph, AD Instrument Co. , Australia), and the change in isotonic contraction through an isometric force transducer (AD Instrument Co., Australia) was continuously recorded with a PowerLab (ADLab Co., Australia) program. The blood vessel fragments were stabilized in an organ bath for 15 minutes, then loaded with 1 g of driven tension and stabilized for another hour, and then the experiment was conducted. During stabilization, the Krebs-Henseleit solution in the organic bath was replaced with a fresh solution every 20 minutes.

In order to confirm the vasodilatory activity of the vasculature, 1 μM of phenylephrine was treated in an organ bath in which the vasculature is located, and then contraction of the vasculature was induced for 40 minutes. Omija dissolved in Krebs-Henseleit solution in sufficiently contracted blood vessels, and individual extracts and their complex extracts were treated in an organ bath to make 30 μg/ml to compare the vasodilatory activity, and the negative control group was Krebs-Henseleit solution Only. Vascular relaxation rate (%) was calculated using the following equation.

expression

As shown in Table 6 and Figure 3, as a result of treating phenylephrine on the vascular segment, the contraction of the vascular segment was induced, and the vasodilation rate was about 3%. In contrast to the negative control group, the group treated with individual extracts of Schisandra chinensis and Gilkyung had no significant effect on vasodilation, but the rate of vasodilation was increased by about 7.58% when the complex extract was treated. Therefore, it was confirmed that the composite extract of the present invention has superior vasodilator efficacy compared to the negative control and individual extracts.

Experimental group Vascular section contraction value (g) Vascular relaxation rate (%) PE shrink
Before induction PE shrink
After induction After treatment with K-H solution or extract Eumseong Control 1.13±0.02 3.38±0.11 3.31±0.13 3.00±1.54 Omija extract (Comparative Example 1-1) 1.14±0.03 3.62±0.07 3.54±0.05 3.00±1.30 Gilkyung Extract (Comparative Example 1-2) 1.20±0.03 3.25±0.09 3.16±0.10 4.55±1.52 Complex extract (Production Example 1-2) 1.18±0.03 3.63±0.19 3.37±0.19 10.58±1.36

Experimental Example 3: Mioja and Roadside Measuring the efficacy of improving the bone loss index of the complex extract

Estrogen deficiency, characteristic of menopausal women, induces osteoclast differentiation that breaks down bones, resulting in osteoporosis due to bone loss. Currently, the animal model that best reflects the symptoms of bone loss due to estrogen deficiency in menopausal women is the ovariectomy (OVX) rat model. In the ovarian ablation rat model, the blood levels of calcium (Ca) and inorganic phosphate (Pi), which make up the bone, are reduced during the ablation of the ovary, and the major enzymes that break down the bone, matrix metallopeptidase-9 (MMP-9) and bone turnover ( It has been found that the amount of bone turnover (ALP) markers ALP (Alkaline phosphatase) and Osteocalcin increases, and these markers are used as a key indicator for bone loss evaluation. The present inventors performed biochemical analysis of blood in female rats excised from the ovaries in order to confirm the improvement effect on the bone loss markers of the extract of Schisandra chinensis and Gilkyung complex extracted in Preparation Example 1.

3-1. OVX Rat model production

SD (Sprague Dawley) Rat Female 6 weeks of age (about 160 g ± 20% in weight) was worn in Orient (Orient Bio, Gapyeong-gun, Gyeonggi-do) and purified for 14 days before use. Animals were anesthetized using Zoletil 50 (VIRBAC, France) and xylazine (Rompun®, Bayer AG, Germany) for ovarian resection. After epilating both sides of the abdomen and disinfecting the surgical site, incising the muscles and peritoneum, exposing the uterus and ovaries to determine the location of the ovaries, revealing the fallopian tubes and ovarian arteriovenous veins, resecting the ovaries using a conduit, and wounding Suturing was carried out. In the case of test substance administration, each extract of Omija (Comparative Example 1-1), Gilkyung (Comparative Example 1-2), and extract of Omija, Gilkyung complex (Preparation Example 1-2) once per day at a concentration of 200 mg/kg, It was administered orally daily for 7 days/week, 12 weeks. In the normal group and the negative control group, distilled water was administered.

3-2. Blood biochemical test

After 12 weeks of dosing, blood collected from the posterior vena cava and separated and analyzed by using a blood biochemical analyzer (7180 Hitachi, Japan) and an automatic electrolyte analyzer to analyze Alkaline phosphatase (ALP), Calcium, Inorganic phosphorus (Pi) Also, the osteolysis markers MMP (Matrix metallopeptidase)-9 and Osteocalcin were measured by ELISA.

As shown in Table 7 and FIGS. 4 to 8, as a marker of osteoblasts during ovarian ablation, blood levels of ALP and osteocalcin, which increase upon bone loss, and MMP-9, which directly degrade bone, increase, and constitute bone. The concentrations of the inorganic components Ca and Pi decreased. When the test substance was administered, the concentrations of ALP, MMP-9, and Osteocalcin decreased in the negative control group and the complex extract group compared to the individual drug extracts, and the Ca and Pi concentrations increased significantly. Therefore, it was confirmed from the above results that the mixed extract of the present invention has better efficacy in improving the index related to bone loss compared to the negative control and individual extracts.

Experimental group Ovarian ablation
(OVX) Test substance ALP
(U/L) Ca
(mg/dL) Pi
(mg/dL) MMP-9
(ng/mL) Osteocalcin
(ng/mL) Normal X Distilled water 782.01± 89.86 10.02
± 0.15 5.42
± 0.32 7.65
± 0.61 260.85
± 23.94 Eumseong Control O Distilled water 825.61± 76.23 9.74
± 0.16 4.75
± 0.29 9.81
± 0.73 332.96
± 37.91 Omija extract (Comparative Example 1-1) O Comparative Example 1-1 836.38
± 40.47 10.02
± 0.17 5.32
± 0.18 9.44
± 0.76 274.68
± 28.54 Gilkyung Extract (Comparative Example 1-2) O Comparative Example 1-2 841.26
± 56.21 9.89
± 0.14 5.78
± 0.12 12.38
± 1.86 299.93
± 22.68 Complex extract (Production Example 1-2) O Preparation Example 1-2 702.68
± 38.22 10.42
± 0.19 7.17
± 0.25 7.59
± 0.59 222.67
± 18.43

Claims (8)

(a) complex extract of Omija (Schisandrae fructus) and Gilty (Platycodon grandiflorum) as active ingredients; And
(b) A pharmaceutical composition for preventing or treating metabolic bone disease or climacteric symptoms, including a pharmaceutically acceptable carrier.
The composition of claim 1, wherein the compound weight ratio (w/w) of the omija and the long diameter is 1:20 to 20:1.
The composition of claim 1, wherein the extract is a polar organic solvent extract.
The composition according to claim 3, wherein the polar organic solvent is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetic acid or a mixture thereof.
Food composition for preventing or improving metabolic bone disease or menopausal symptoms, including omija and Gilkyung complex extract as an active ingredient.
The composition according to claim 5, wherein the compounding weight ratio (w/w) of the omija and the long diameter is 1:20 to 20:1.
The composition of claim 5, wherein the extract is a polar organic solvent extract.
The composition according to claim 7, wherein the polar organic solvent is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetic acid or a mixture thereof.

Images