KR20200043783A - A composition for improving, preventing and treating bone-related disease comprising polysaccharide fraction isolated from persimmon leaf - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, and treating bone-related diseases, which contains a polysaccharide fraction derived from persimmon leaves as an active component, thereby being able to alleviate, prevent, or treat diseases that can occur due to bone-related diseases. In addition, the composition has no toxicity, thereby being able to be consumed in the form of food.

Description

A composition for improving, preventing and treating bone-related disease comprising polysaccharide fraction isolated from persimmon leaf}

The present invention relates to a composition capable of improving, preventing or treating diseases that may be caused by bone-related diseases by containing the polysaccharide fraction derived from persimmon leaves as an active ingredient.

Bone (bone, bone) supports the soft tissues and weight of the human body and surrounds the internal organs, thereby protecting the internal organs from external shocks. It is also one of the important parts of the human body that structurally supports muscles and organs and stores other essential mineral substances such as calcium, phosphorus or magnesium in the body.

The bones of the grown-up adult do not stop and maintain the balance by removing and regenerating old bones until the day of death and replacing and replacing old bones with new bones. This is called bone remodeling, and the circulation of the bone that removes old bones and replaces them with new bones is essential to restore the microscopic damage caused by growth and stress and to maintain proper bone function. It is known that two types of cells are largely involved in the remodeling of the bone. One of the two types of cells is Osteoblast, which produces bone, and the other, Osteoblast, which destroys bone. .

The osteoblast produces Receptor activator of nuclear factor kappa-B ligand (RANKL) and its inducing receptor Osteoprotegerin (OPG). When the RANKL binds to RANK, a receptor on the surface of the osteoclast precursor cells, the osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption. However, when osteoprotegerin (OPG) binds to RANKL, the binding between RANKL and RANK is blocked, and osteoclast formation is suppressed and bone absorption does not occur.

Osteoporosis, which is a typical disease of bone-related diseases, refers to a condition in which the amount of bones decreases and the strength of bones weakens due to qualitative changes, and thus fractures are likely to occur. Osteoporosis, which has been defined as a low bone mass (Mass) state, is one of the important social problems, and in the United States, it affects about 260,000 women each year, of which about 12 to 24% die. In a situation where society becomes more and more aging and women's participation in society becomes active, osteoporosis and osteoporosis fractures in the elderly and postmenopausal women cause serious problems.

The causes of low bone density are genetic factors, early menopause, medications such as steroids, comorbidities, smoking and alcohol, etc., and the cause of fractures is low bone density, low weight, past fracture power, parent or brother fracture power, smoking and alcohol Etc. In other words, the minerals (particularly calcium) and substrate constituting the bone are in a reduced state, and the balance of bone remodeling is broken, so that the osteoclast action is increased than the osteoclast action. The inside of a normal bone has a dense structure like a mesh, but in the case of osteoporosis, the gap between the bone microstructures is widened and the microstructure is thinned and weakened, thereby increasing the risk of easily fractured bones even with a small impact.

The osteoporosis is a disease characterized by deterioration of the micro-architectural structure of bone tissue, which leads to an increase in bone fragility and an increased risk of fracture in the elderly. As such, more than 50% of women over 50 and 30% of men over 50 are known to be affected by osteoporosis, and the rate of bone loss accelerates in women in the postmenopausal period or in the years following. Most of them have no symptoms, but pain occurs when a fracture occurs, and various symptoms can occur depending on the area where the fracture occurs. Fractures can occur in all areas, but fractures often occur in the wrist, spine, and hip (femur). In addition, regardless of age, it can be classified as secondary osteoporosis caused by diseases, drugs, alcohol, smoking, or accidents.

Substances currently used as a therapeutic agent for osteoporosis are female hormone preparations (Estrogen), androgenic anagolic steroids, bisphosphonate preparations, parathyroid hormones, calcium preparations, phosphates and fluoride, which are traditionally used in postmenopausal women. Formulations, ipriflavone, vitamin D3, and the like. The female hormone has an effect of reducing fractures, but should be used with caution because it increases the risk of thrombosis and breast cancer, and estrogen suppresses cell death of osteoblasts, thereby increasing the survival time of cells and promoting the cell death of osteoclasts. Although it is a more or less effective method for treating menopausal symptoms and maintaining bone density by reducing the survival period, it has side effects that cause endometrial hyperplasia. Selective estrogen receptor modulators (SERMs) have been developed to compensate for these shortcomings. A drug of this class is raloxifene, and it has been reported that it not only prevents spinal fractures but also lowers the incidence of breast cancer in some. In addition, the bisphosphonate preparation is the most widely used osteoporosis treatment agent, which decreases the function of osteoclasts and reduces the number to prevent bone destruction. In addition, strontium (Strontium ranelate) as a therapeutic agent for osteoporosis to eat, while increasing bone formation, there is a problem that bone absorption is suppressed.

 Therefore, there is a need for a composition made of natural materials that can prevent, improve and treat bone-related diseases and have no side effects.

Republic of Korea Registered Patent No. 1468015 Republic of Korea Registered Patent No. 1827326

An object of the present invention is to provide a pharmaceutical composition capable of preventing or treating diseases that may be caused by bone-related diseases.

In addition, another object of the present invention is to provide a food composition capable of improving or preventing diseases that may be caused by bone-related diseases.

The pharmaceutical composition for preventing or treating bone-related diseases of the present invention for achieving the above object may contain a polysaccharide fraction derived from persimmon leaves as an active ingredient.

The persimmon leaf-derived polysaccharide fraction is 60 to 80% by weight of neutral sugar compared to the total polysaccharide fraction, 18 to 39% by weight of uronic acid, 3-deoxy-D-lyxo-2-heptulosaric acid (DHA), and KDO (3-deoxy-D-manno-2- octulosonic acid) KDO-like material may comprise 0.5 to 10% by weight.

The persimmon leaf-derived polysaccharide fraction may further include 0.5 to 20% by weight of protein.

The uronic acid is made of galacturonic acid and glucuronic acid; The neutral polysaccharide may include arabinose, rhamnose, galactose, fucose, glucose, and rhamnogalacturonan-II indicator polysaccharide.

The polysaccharide fraction is (a) processing the pectin hydrolase to persimmon leaf powder; And (b) recovering the fraction of the molecular weight 3 to 300 kDa from the enzyme-treated persimmon leaf powder; may be prepared by a manufacturing method comprising a.

The bone-related diseases include osteoporosis, osteoarthritis, degenerative arthritis, knee joints, hip joints, systemic lupus erythematosus (SLE), osteoarthritis, rheumatoid polymyalgia, ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, enteroarthritis, rheumatoid arthritis , Neuropathic arthrosis, acute rheumatic fever, gout, cartilage calcification, periodontal disease caused by alveolar bone destruction, inflammatory alveolar bone absorption disease, inflammatory bone absorption disease, calcium hydroxyapatite crystal sedimentation disease, and one or more selected from the group consisting of Lyme disease have. That is, the persimmon leaf-derived polysaccharide fraction of the present invention inhibits RANKL, an osteoclast inducer, and effectively inhibits differentiation into osteoclasts that cause joint destruction (suppresses the activity of TRAP, an early marker of osteoclast differentiation). It can be used as a material for preventing, improving or treating bone-related diseases, particularly osteoporosis, osteoarthritis, degenerative arthritis, rheumatoid arthritis, and the like.

In addition, the food composition for improving or preventing bone-related diseases of the present invention for achieving the other object described above may contain persimmon leaf-derived polysaccharide fraction as an active ingredient.

The composition for preventing, improving and treating bone-related diseases of the present invention inhibits osteoclast differentiation and reduces increased protein expression by RANKL treatment, as well as bone density (BMD), total volume (TV), and bone volume (BV) ), Bone surface area (BS), Bone surface area ratio (BS / TV), Bone shochu average thickness (Tb.Th), Average number per unit length (Tb.N) are improved.

In addition, the composition for preventing, improving and treating bone-related diseases of the present invention can be effectively used for the prevention and treatment of bone-related disease diseases, and can be consumed in the form of food because it has no toxicity.

1 is a microscopic image of the effect of inhibiting osteoclast differentiation by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention.
2 is a view measuring the activity of the RANKL signaling system induced by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention.
Figure 3a is a diagram measuring the protein expression activity of c-Fos and NFATc1 by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention, Figure 3b is in Example 1 of the present invention C-Fos mRNA expression by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to the numerical value, Figure 3c is an enzyme-treated crude polysaccharide fraction prepared according to Example 1 of the present invention (PLE-0) It is a graph showing the expression of NFATc1 mRNA by.
Figure 4a is a photograph taken with a microcomputer tomography system of the femur of the mouse divided into groups 1 to 6; Figure 4b is a graph showing the bone volume (BV / TV) of the mouse femur divided into groups 1 to 8; Figure 4c is a graph showing the thickness of the primary femur bone (Tb.Th) of the mouse femur divided into groups 1 to 8; 4D is a graph showing the number of minor bones (Tb.N) of the mouse femur divided into groups 1 to 8; Figure 4e is a graph showing the bone density (BMD) of the mouse femur divided into groups 1 to 8.
5 is a graph showing CTX in the blood of the mouse femur divided into groups 1 to 8.

The present invention relates to a composition capable of improving, preventing or treating diseases that may be caused by bone-related diseases by containing the polysaccharide fraction derived from persimmon leaves as an active ingredient.

Hereinafter, the present invention will be described in detail.

Persimmon leaves used as raw materials for persimmon leaf-derived polysaccharide fractions capable of improving, preventing or treating diseases caused by bone-related diseases of the present invention contain 100 mg / 100 g of vitamin C. It is known to have a pronounced effect on scurvy, hypertension, and anemia, about 20 times as much.

The persimmon leaf-derived polysaccharide fraction of the present invention is 60 to 80% by weight of neutral sugar, 18 to 39% by weight of uronic acid, KDO (3-deoxy-D-manno-2-octulosonic acid) compared to the total polysaccharide fraction ) 0.5 to 10% by weight of similar material. In addition, the protein may further include 0.5 to 20% by weight.

The uronic acid is made of galacturonic acid and glucuronic acid; The neutral polysaccharide includes arabinose, rhamnose, galactose, fucose, glucose, and Rhamnogalacturonan-II indicator polysaccharide.

Preferably, the neutral polysaccharide is composed of 20 to 40 mole% of arabinose, 10 to 40 mole% of rhamnose, 10 to 40 mole% of galactose, 1 to 10 mole% of fucose, compared to total neutral polysaccharide constituting the polysaccharide fraction derived from the persimmon leaf, Glucose from 1 to 10 mole% and rhamnogalacturonan-II indicator polysaccharide from 0.4 to 26 mole% polysaccharide.

Moreover, the rhamnogalacturonan-II indicator polysaccharide is 0.1 to 8 mole% of 2-methylfucose, 0.1 to 8 mole% of 2-methyl xylose, and apiose 0.1 compared to the total neutral polysaccharide constituting the polysaccharide fraction derived from the persimmon leaf. It is composed of polysaccharides of 5 to 5 mole% and 0.1 to 5 mole% aceric acid.

In addition, the KDO-like substance is made of 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) and 3-deoxy-D-manno-2-octulosonic acid (KDO).

Persimmon leaf-derived polysaccharide fraction of the present invention comprises the steps of: (a) treating pectin hydrolase to persimmon leaf powder; And (b) recovering a fraction having a molecular weight of 3 to 300 kDa from the enzyme-treated persimmon leaf powder. In addition, (c) recovering the fraction having a molecular weight of 5 to 30 kDa from the fraction recovered in step (b); may further include.

First, in step (a), pectin hydrolase is treated with persimmon leaf powder.

The pectin hydrolase is added at 1 to 20% by weight based on the weight of the persimmon leaf powder, preferably 5 to 15%, and more preferably 10% by weight.

In the present invention, persimmon leaves may be dried or dried, and pulverized or powdered may be used. Preferably, the persimmon leaf powder that treats the enzyme can be used by suspending it in distilled water about 5 to 15 times (w / v).

The hydrolase treatment is preferably treated for 1 to 5 days, more preferably 2 to 4 days.

The step (a) may further perform a process of inactivating the enzyme by heating the remaining pectin hydrolase at 90 to 110 ° C. for 10 to 60 minutes after enzyme treatment. The heating increases the elution of the soluble polysaccharide component, and denaturates and precipitates the polymer protein contained as some impurities to improve the polysaccharide extract obtained by centrifugation and purity.

In addition, the step (a) may include a process of decoloring persimmon leaf powder before enzyme treatment. The solvent for decolorization is not particularly limited as long as it is a decolorizing solvent that is harmless to the human body and is permitted to be used, but preferably includes at least one selected from the group consisting of ethanol, potassium sulfite, sodium sulfite, sulfur dioxide, and benzoic peroxide. , Preferably ethanol.

Next, in step (b), the residue is removed from the enzyme-treated persimmon leaf by centrifugation, solvent fractionation, or filtration to obtain a polysaccharide extract, and then fractionated with a molecular weight of 3 to 300 kDa to recover the fraction.

The method for obtaining the fraction is not particularly limited as long as it is a purification method based on molecular weight, but preferably includes ultrafiltration, solvent fractionation, or gel filtration chromatography, and more preferably ultrafiltration. In addition, the form of the final polysaccharide fraction can be an extract, concentrate or powder.

Specifically, the present invention removes those having a molecular weight of less than 3 kDa and those having a molecular weight exceeding 300 kDa by using the same method as the persimmon leaf-derived fraction obtained by treatment with the pectinase (PLE-). 0) was obtained.

 As a result of analyzing the constituent sugar of the obtained persimmon leaf-derived crude polysaccharide fraction (PLE-0) by gas chromatography, neutral polysaccharide compared to total polysaccharide 71.3 wt%, uronic acid 26.2 wt%, protein 0.7 wt%, KDO similar material 1.8 wt %; As a result of analyzing the components of the neutral polysaccharide, 2-methylfucose 1.6 mole%, rhamnose 15.6 mole%, fucose 2.4 mole%, 2-methylxylose 2.0 mole%, arabinose 26.3 mole compared to total neutral polysaccharide %, Xylose 6.7 mole%, apiose 4.5 mole%, aceric acid 1.4 mole%, mannose 2.1 mole%, galactose 32.3 mole%, glucose was 5.1 mole%. Among the neutral polysaccharides, 2-methylfucose, 2-methylxylose, apiose, and aceric acid are indicators of Rhamnogalacturonan-II (RG-II) (see Table 2). In addition, KDO-like substances 3-deoxy-D-manno-2-octulosonic acid (KDO) and 3-deoxy-D-lyxo-2-heptulosaricacid (DHA) are also indicators of RG-II.

Next, in step (c), a fraction having a molecular weight of 5 to 30 kDa is recovered from the fraction recovered in step (b).

That is, the crude polysaccharide fraction derived from persimmon leaf (PLE-0) recovered in step (b) is ultrafiltered to recover the fraction having a molecular weight of 5 to 30 kD again (PLE-II). At this time, the obtained fraction (PLE-II) is slightly less effective for bone-related diseases than the crude polysaccharide fraction derived from persimmon leaf (PLE-0) obtained in step (b).

When the constituent sugars of the recovered fraction (PLE-II) were analyzed by gas chromatography, neutral polysaccharides compared to total polysaccharides were 69.5% by weight, uronic acid 27.2% by weight, KDO-like substance 3.3% by weight; As a result of analyzing the components of the neutral polysaccharide, 2-methylfucose 4.0 mole%, rhamnose 27.9 mole%, fucose 4.9 mole%, 2-methylxylose 4.7 mole%, arabinose 28.2 mole% compared to the total neutral polysaccharide , Apiose 2.9 mole%, aceric acid 3.5 mole%, mannose 0.4 mole%, galactose 19.6 mole%, glucose was confirmed to be 3.9 mole%. Among the neutral polysaccharides, 2-methylfucose, 2-methylxylose, apiose, and aceric acid are indicators of Rhamnogalacturonan-II (RG-II). In addition, KDO-like substances 3-deoxy-D-manno-2-octulosonic acid (KDO) and 3-deoxy-D-lyxo-2-heptulosaricacid (DHA) are also indicators of RG-II.

It is reported that pectic substances are polysaccharides mainly present in the primary cell wall and middle lamella of higher plants and have a very complex structure. The pectin is composed of homogalacturonan, a large part of the entire molecule (Kwon MH et al., Food Sci. Ind .; 30: 30-43, 1997), but various oligo- (oligo) It is known that rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) branched with polysaccharide and polysaccharide are covalently bound. (Kim JM et al., Biochem. Bioph. Res. Co.; 344: 765-771, 2006).

In addition, a polysaccharide purification step of removing low-molecular substances and impurities by adding 50 to 100% of alcohol having 1 to 4 carbon atoms may be added to the final polysaccharide fraction.

The term 'fraction' used in referring to persimmon leaves herein includes not only the fraction obtained by treating the extraction solvent, but also the processed product of the polysaccharide fraction derived from persimmon leaves. For example, the persimmon leaf-derived polysaccharide fraction may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the persimmon leaf-derived polysaccharide fraction of the present invention has the meaning of including persimmon leaf polysaccharide processed, for example, persimmon leaf polysaccharide powder, which is formulated to administer persimmon leaves to animals in a broad sense. Although the experiment was conducted with the polysaccharide fraction derived from persimmon leaves in the present invention, it would be predictable to those skilled in the art that the desired effect can be achieved in the same form as the persimmon leaf polysaccharide processed product.

Meanwhile, in the present specification, the term 'containing as an active ingredient' means to include an amount sufficient to achieve the efficacy or activity of the persimmon leaf-derived polysaccharide fraction. In one example, the persimmon leaf-derived polysaccharide fraction is used at a concentration of 10 to 1500 µg / ml, preferably 100 to 1000 µg / ml. Since the persimmon leaf-derived polysaccharide fraction is a natural product, there is no side effect to the human body even if it is excessively administered, and thus the upper limit of the quantitative amount of the persimmon leaf-derived polysaccharide fraction contained in the composition of the present invention can be carried out by selecting within an appropriate range by those skilled in the art.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants, A lubricant or flavoring agent may be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectables. For example, for formulation in the form of tablets or capsules, the active ingredient can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and colorants can also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trachocanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Furthermore, it can be preferably formulated according to each disease or component using methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by appropriate methods in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration. .

Suitable dosages of the pharmaceutical compositions of the present invention vary by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity, Usually, a skilled physician can easily determine and prescribe a dose effective for the desired treatment or prevention. According to a preferred embodiment of the invention, the daily dosage of the pharmaceutical composition of the invention is 0.001-10 g / kg.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and / or excipient, or may be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, 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.

In addition, the present invention provides a food composition for improving, preventing or treating bone-related diseases containing persimmon leaf-derived polysaccharide fraction as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food, or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice cream, vitamin complexes, and health supplements And so on.

The food composition of the present invention may include, as an active ingredient, a polysaccharide fraction derived from persimmon leaves, as well as ingredients commonly added in food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. do. 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 a flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of a drink and a beverage, in addition to the persimmon leaf-derived polysaccharide fraction of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, and various plant extracts may be additionally included. have.

The present invention provides a health functional food comprising a food composition for improving, preventing or treating a bone-related disease comprising the persimmon leaf-derived polysaccharide fraction as an active ingredient. Health functional foods are foods made by adding persimmon leaf-derived polysaccharide fractions to food materials such as beverages, teas, spices, gums, and confectionery, or by encapsulation, powdering, suspension, etc. It means to come, but unlike general medicines, it has the advantage of not having side effects that can occur when taking medicines for a long time using food as a raw material. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. The amount of polysaccharide fraction derived from persimmon leaves in such a dietary supplement is not uniformly defined depending on the type of dietary supplement, but may be added in a range that does not impair the original taste of the food. It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the dietary supplement of the present invention may be in the form of pills, tablets, capsules or beverages.

In addition, the present invention provides the use of persimmon leaf-derived polysaccharide fractions for the manufacture of a medicament or food for the improvement, prevention or treatment of bone-related diseases. As described above, the persimmon leaf-derived polysaccharide fraction can be used for improvement, prevention or treatment of bone-related diseases.

In addition, the present invention provides a method for improving, preventing or treating bone-related diseases, comprising administering an effective amount of a polysaccharide fraction derived from persimmon leaves to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

As used herein, the term "effective amount" refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as considered by a researcher, veterinarian, physician or other clinician, as appropriate It includes an amount that induces relief of symptoms of a disease or disorder. The effective amount and the number of administrations for the active ingredient of the present invention can be changed according to the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health It can be adjusted according to various factors, including condition, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously. In the prevention, treatment, or improvement method of the present invention, in the case of adults, it is preferable to administer the persimmon leaf-derived polysaccharide fraction at a dose of 0.001 g / kg to 10 g / kg when administered once to several times a day.

In the treatment method of the present invention, the composition comprising the persimmon leaf-derived polysaccharide fraction as an active ingredient is conventional through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, sternum, transdermal, topical, intraocular or intradermal routes. Can be administered in a manner.

Hereinafter, a preferred embodiment is provided to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it is apparent to those skilled in the art that various changes and modifications are possible within the scope and technical scope of the present invention. It is no wonder that such variations and modifications fall within the scope of the appended claims.

Example 1. Persimmon leaf-derived crude polysaccharide fraction (PLE-0)

Persimmon leaf was used by powdering persimmon leaf (persimmon leaf, Gyeongbuk Yeongcheon) sold by Baekjangsaeng Co., Ltd. 90% ethanol was added to the dry powder to be 10 times (w / v) the dry powder weight, stirred for 48 hours, and filtered and dried to complete decolorization.

The decolorized dry powder was suspended in distilled water 10 times (w / v), pH 4, and pectinase (Rapidase C80MAX, Vision Biochem) was added at 10% by weight per raw material, followed by enzymatic treatment for 3 days in a 50 ° C incubator. Then, the enzyme-treated reaction solution was heated at 101 ° C for 30 minutes to extract soluble polysaccharide and inactivate the remaining pectinase.

After the enzyme treatment, the sample is centrifuged at 4,6 ° C for 6 minutes at 4 ° C. for 15 minutes to remove the residue, and filtered by ultrafiltration to remove fine substances with molecular weights less than 3 kDa and greater than 300 kDa. The crude polysaccharide fraction (PLE-0) was obtained.

Comparative Example 1. Persimmon leaf-derived polysaccharide fraction (PLW-0) _non-enzymatic treatment (hot water extraction)

The decolorized persimmon leaf dry powder used the same one as in Example 1 above. The bleached dry powder was suspended in distilled water (10 times (w / v)) and heated at 100 ° C. for 3 hours to obtain a hot water extract. After centrifuging the hot water extract at 4 ° C for 6,500 6g for 15 minutes, the supernatant was separated and 4 times the volume (v / v) of 80% ethanol was added to settle the polysaccharide by standing for 24 hours. The precipitated polysaccharide was dialyzed (molecular weight cut-off: 6,000 to 8,000) to obtain a hot water extracted crude polysaccharide fraction (PLW-0).

<Test Example>

Test Example 1. Analysis per composition of fractions

Constitutive sugar analysis was analyzed by GC (Gas Chromatography) by derivatizing each constituent sugar with alditol acetate after hydrolysis by partially modifying the method of Albersheim et al. After the polysaccharide sample of Example 1 was hydrolyzed by reacting in 2 M TFA (trifluoroacetic acid) at 121 ° C. for 1.5 hours, the hydrolyzate was separated into neutral sugar and acidic sugar by Dowex-1 (acetate form) resin. . It was dissolved in 1 mL of 1 M NH4OH (Ammoniasolution) and reduced with 10 mg of NaBH4 for 4 hours. Acetic acid was added to the appropriate amount to remove the residual NaBH4, and then methanol was repeatedly dried to remove excess acetic acid to convert to alditol corresponding to each constituent. Thereafter, 1 mL of acetic anhydride was added to each alditol and reacted at 121 ° C. for 30 minutes to convert to alditol acetate, which was extracted by separation with a chloroform / H 2 O 2 phase solvent system, The extract was dissolved in a small amount of acetone after drying and used as a sample for GC analysis.

GC analysis conditions of alditol acetate derivatives are as shown in [Table 1], and mole% of each constituent is the molecular response factor for the peak area, molecular weight, and flame ionization detector (FID) of each derivative. Was calculated by calculating each.

Apparatus GC ACME-6100 (Young-Lin Co. Ltd., Anyang, Korea) Detector Flame ionization detector (FID)
(Young-Lin Co. Ltd., Anyang, Korea) Column SP-2380 capillary column (Supelco, Bellefonte, USA) Column size 0.25 mm 30 m, 0.2 m film thicknes Oven temp. 60 (1 min) 220 (12 min) 250 (15 min)
30 / min 8 / min Injector temp. 250 Detector temp. 270 Carrier gas N2 (1.5 mL / min)

In Table 2 below, the KDO-liked material refers to 3-deoxy-D-manno-2-octulosonic acid (KDO) and 3-deoxy-D-lyxo-2-heptulosaricacid (DHA); Uronic acid refers to Galacturonic acid, Glucuronic acid. The sugars constituting the neutral sugar are the contents of the ingredients based on the total neutral polysaccharide.

Chemical composition (unit: weight%) Example 1 (PLE-O) Comparative Example 1 (PLW-O) Neutral sugar 71.3 60.4 Uronic acid 26.2 38.3 Protein 0.7 0.0 KDO-liked material 1.8 1.3 Component of neutral sugar (unit: mole%)
2-Mefuc 1.6 0.3 Rha 15.6 7.1 Fuc 2.4 1.2 2-Mexyl 2.0 0.5 Ara 26.3 33.6 Xyl 6.7 5.7 Api 4.5 3.9 Aceric acid 1.4 2.5 Man 2.1 1.2 Gal 32.3 28.5 Glc 5.1 15.5

As shown in Table 2 above, the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention contains proteins, unlike the hot-water extracted crude polysaccharide fraction (PLW-0) of Comparative Example 1 , It was confirmed that the content of the neutral polysaccharide was high.

Test Example 2. Osteoclast experiment

Cell culture

In order to investigate the effect of fractions on osteoclast differentiation, osteoclasts derived from bone marrow cells were obtained and used as osteoclast progenitor cells to determine the osteoclast number and TRAP activity according to the treatment concentration of each fraction prepared in Examples and Comparative Examples. Analysis. Bone marrow cells derived from bone marrow cells were cultured for 3 days in α-MEM medium containing macrophage colony stimulating factor (M-CSF) (60 ng / ml) and 10% FBS. Marrow Macrophage, BMM), which was used as osteoclast progenitor cells.

Test Example 2-1. Measurement of osteoclast viability

The obtained BMMs were mixed with M-CSF (60 ng / ml) in the medium and fractions of Examples and Comparative Examples for 2 days at various concentrations (0, 1.56, 3.125, 6.25, 12.5, 25, 50 μg / ml). It was added and cultured. Cell viability (%) was determined by measuring the absorbance at 450 nm using a cell counting kit-8 (Cell counting Kit-8, CCK-8, Dojindo Inc.), and the data are expressed as mean ± standard error. Did.

division Example 1 (PLE-O) Comparative Example 1 (PLW-O) Μg / ml 0 100 100 1.56 110.6 101.5 3.125 105.1 103.0 6.25 111.8 105.6 12.5 112.6 108.1 25 116.4 104.4 50 120.7 103.9 100 115.7 102.1 200 107.2 100.8

As shown in Table 3 above, the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention and the hot-water extracted crude polysaccharide fraction (PLW-0) of Comparative Example 1 have cell viability at all concentrations. It was confirmed that it was excellent.

Test Example 2-2. Analysis of effects on osteoclast differentiation

The obtained BMM was incubated for 4 days in a medium containing M-CSF (60 ng / ml) and RANKL (receptor activator of NF-κB ligand) (100 ng / ml), and BMM was cultured under the same culture conditions. And the comparative samples were treated with various concentrations (0, 1.56, 3.125, 6.25, 12.5, 25, 50 μg / ml). After fixing the cells with 10% formalin, the reaction was performed for 10 minutes using a TRAP enzymatic activity specific substrate (q-nitrophenyl phosphate), and then measured at 405 nm absorbance. After the reaction, TRAP (Tartrate-resistant acid phosphatase) staining is performed using naphthol AS-MS phosphate and Fast red violet LB salt for osteoclast staining, and TRAP positive with 3 or more nuclei Cells (TRAP + Multi-Nuclei Cells (MNCs)) were counted. Data are expressed as mean ± standard error, the following [Table 4] shows the TRAP activity, and [Table 5] shows the formation of multinuclear giant cells.

division Example 1 (PLE-O) Comparative Example 1 (PLW-O) Μg / ml 0 1.23 1.23 1.56 0.47 1.20 3.125 0.24 1.11 6.25 0.08 1.05 12.5 0.03 0.71 25 0.02 0.75 50 0.01 0.62

As shown in Table 4 above, the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention is an early marker of osteoclast differentiation compared to the hot-water extracted crude polysaccharide fraction (PLW-0) of Comparative Example 1 It was confirmed that the inhibitory effect of phosphorus TRAP appears.

division Example 1 (PLE-O) Comparative Example 1 (PLW-O) Μg / ml 0 158.8 158.8 1.56 112.4 153.5 3.125 58.9 151.7 6.25 9.6 148.0 12.5 0 144.9 25 0 140.6 50 0 135.4

As a result of confirming the effect of the fraction on the formation of multinuclear giant cells capable of confirming the final stage of osteoclast differentiation, the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention as shown in Table 5 above. It was confirmed that the inhibitory ability was excellent compared to the hot water extract crude polysaccharide fraction (PLW-0) of Comparative Example 1.

1 is a microscopic image of the effect of inhibiting osteoclast differentiation by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention.

As shown in Figure 1, it was confirmed that the higher the concentration of the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1, the better the effect of inhibiting osteoclast differentiation.

Test Example 2-3. Analysis of the effectiveness of RANKL signaling system

To investigate the effect of the fraction prepared according to Example 1 on the RANKL signaling system, the effect of the RANKL signaling system was analyzed. The BMM cells were pretreated with 25 μg / ml of distilled water (DW) as a carrier or a fraction of Example 1 for 1 hour and then treated with RANKL (100 ng / ml) for 0, 5, 15, 30, 60 minutes. Proteins were obtained and then Western blot analysis was performed. Protein activation was confirmed by Western blot analysis using antibodies selective for phosphorylation of specific amino acids.

2 is a view measuring the activity of the RANKL signaling system induced by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention.

As shown in Figure 2, p38, ERK and JNK proteins induced by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention was confirmed that the activity is inhibited.

Test Example 2-4. Analysis of the effects of osteoclast differentiation on the expression of key transcription factors

In order to confirm the effect of the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 on the expression of c-Fos and NFATc1, which are the key transcription factors of osteoclast differentiation, distilled water as a carrier in BMM cell culture Alternatively, 25 μg / ml of the fraction of Example 1 was pre-treated for 1 hour and then treated with RANKL (100 ng / ml) to obtain mRNA and protein on 0, 1, 2, 3, 4 days. The mRNA expression was expressed using relative real-time PCR and the results were expressed in terms of the standard ㅁ standard deviation by relative quantification using a 2 ^- oz ΔCT method normalized to 18S RNA, and protein expression was confirmed by Western blot analysis.

Figure 3a is a diagram measuring the protein expression activity of c-Fos and NFATc1 by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention, Figure 3b is in Example 1 of the present invention C-Fos mRNA expression by the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to the numerical value, Figure 3c is an enzyme-treated crude polysaccharide fraction prepared according to Example 1 of the present invention (PLE-0) It is a graph showing the expression of NFATc1 mRNA by.

As shown in Figure 3a, it was confirmed that the increase in protein expression of c-Fos and NFATc1 by RANKL treatment in BMM was reduced by the fraction of Example 1.

In addition, as shown in Figures 3b and 3c, c-Fos mRNA expression was 72.7%, 59.2%, 76% and 1, 2, 3 and 4 days, respectively, when compared to the control group when treating the fraction of Example 1 30% reduction and 79.1% and 88%, respectively, on days 1, 2, 3, and 4 for NFATc1 mRNA expression. It was confirmed that the reduction was 91.2% and 78.4%.

Test Example 3. Animal experiment

Experimental animals

28-30 g of 12 week old female Crl: CD1 (ICR) mice (Orient Bio Inc., Seoul, Korea) were used for the experiment. Before the experiment, the mice were used for the experiment after 7 days of purification, and during the purification, the animals were reared freely in a breeding room with a temperature of 22 ± 1 ° C and a humidity of 55 ± 10%, and the contrast cycle was adjusted to a period of 12 hours. Thereafter, sham-operate (Sham, n = 10) or ovarian resection (OVX, n = 50) was performed. Animal experiments were handled in accordance with the Animal Experiment and Operation Regulations of the Korean Institute of Oriental Medicine.

The ovarian excision (OVX) mice were divided into 10 animals each week after surgery, and the fractions of Example 1 and Comparative Example 1 were orally administered at two concentrations (0.10 g / kg and 0.20 g / kg) for 7 weeks. As a positive control used in this experiment, Baeksuo products sold by company B were used, and the dosage was determined based on the daily intake.

-Eight groups of mice-

Group 1 (Sham, Control): Drinking water instead of fractions

Group 2 (OVX): Drinking water instead of fractions after OVX

Group 3 (P2L): White sorghum 0.25 g / kg administered after OVX

Group 4 (P2H): 0.50 g / kg of white sorghum after OVX

Group 5 (PLE-O-L): 0.10 g / kg of enzyme-treated crude polysaccharide fraction (PLE-0) after OVX

Group 6 (PLE-O-H): 0.20 g / kg of enzyme-treated crude polysaccharide fraction (PLE-0) after OVX

Group 7 (PLW-O-L): 0.10 g / kg of hot water extracted crude polysaccharide fraction (PLW-0) after OVX

Group 8 (PLW-OH): 0.20 g / kg of hot water extracted crude polysaccharide fraction (PLW-0) after OVX

Test Example 3-1. Mitro-computed tomography (Micro-CT) analysis

For 3D skeletal structure analysis, it was photographed with a microcomputer tomography system (Quantum GX, PerkinElmer, USA). X-ray imaging conditions were set to 90 kV, 88 ㎂, and 10 mm viewing angle range (voxel size, 90 μm; scan time, 14 minutes). Computed tomography 3D images were imaged with a maximum resolution of 4.5 ㎛ with 3D Viewer, Quantum GX's built-in software. The microstructure analysis of the mouse bone was analyzed using the Analyze 12.0 analysis program (AnalyzeDirect, Overland Park, KS, USA); Bone density of the mouse femur was measured using a hydroxyapatite (HA) phantom (QRM-MicroCT-HA, Quality Assurance in Radiology and Medicine GmbH, Germany). In addition, bone density (BMD), total volume (TV), bone volume (BV), bone surface area (BS), bone surface area ratio (BS / TV), average thickness of bone shochu (Tb.Th), average number per unit length (Tb) .N) and other structural parameters were measured in the region of interest.

All experimental results were expressed as the mean ± standard error, and the scanner area in the femur was limited to the peripheral intercostal segment extending about 1.4 mm from the initial proximal tip on the sponge, and comparison between groups was performed by Duncan analysis.

Figure 4a is a photograph taken with a microcomputer tomography system of the femur of the mouse divided into groups 1 to 6; Figure 4b is a graph showing the bone volume (BV / TV) of the mouse femur divided into groups 1 to 8; Figure 4c is a graph showing the thickness of the primary femur bone (Tb.Th) of the mouse femur divided into groups 1 to 8; 4D is a graph showing the number of minor bones (Tb.N) of the mouse femur divided into groups 1 to 8; Figure 4e is a graph showing the bone density (BMD) of the mouse femur divided into groups 1 to 8.

As shown in Figure 4a, the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention was treated with a low concentration (PLE-0-L) and a high concentration (PLE-0-H) It was confirmed that the bone density of the silver mouse femur was superior to that of other groups.

In addition, as shown in Figures 4b to 4e, compared to the Sham group, the OVX group reduced the bone volume (percent bone volume, BV / TV) by 57.9%, the trabecular thickness (Tb.Th) decreased by 19.1%, It was confirmed that trabecular number (Tb.N) decreased by 48.2% and bone mineral density (BMD) decreased by 14.4%.

The bone loss induced after ovarian extraction is bone volume when the enzyme-treated crude polysaccharide fraction (PLE-0) prepared according to Example 1 is treated with low concentration (PLE-0-L) and high concentration (PLE-0-H). , It was confirmed that the thickness of the small bone, the number of small bones, and the bone density all significantly increased. Specifically, in the case of bone volume, 57.9% reduction compared to the Sham group after ovarian extraction was 10.6% and 23.3, respectively, when the low concentration (PLE-0-L) and high concentration (PLE-0-H) enzyme-treated crude sugar fractions were administered. % Level; In the case of the number of small bones, 48.2% reduction compared to the Sham group after ovarian extraction was 2.2% and 18.4%, respectively, when the low concentration (PLE-0-L) and high concentration (PLE-0-H) enzyme-treated crude sugar fractions were administered. Increased to; In the case of bovine bone thickness, 19.1% reduction compared to the Sham group after ovarian extraction was 9.4% and 5.9%, respectively, when the low concentration (PLE-0-L) and high concentration (PLE-0-H) enzyme-treated crude sugar fractions were administered. It was confirmed that the increase.

In addition, even when the bone density was reduced by 14.4% compared to the Sham group after ovarian extraction, the enzyme-treated crude polysaccharide fraction was orally administered at a low concentration (PLE-0-L) and a high concentration (PLE-0-H), similar to the normal group of the sham group. It was confirmed that can maintain the bone density of.

In addition, it was confirmed that the hot-water extracted crude polysaccharide fraction of low concentration (PLW-0-L) and high concentration (PLW-0-H) did not improve compared to after ovarian extraction in all cases.

Test Example 3-2. Blood bone metabolism marker analysis

In bone metabolism, CTX and PINP are representative blood indexes that can represent bone replacement rate. In the case of the carboxy terminal telopeptide (CTX), when the bone is absorbed by osteoclasts, type 1 collagen, a protein constituting bone, is decomposed and can be used as a blood index indicating bone absorption activity; The PINP (procollagen type I N-terminal propeptide) is an amino terminal site of type I procollagen, a precursor protein of type 1 collagen that constitutes bone, and is increased during bone formation to be used as a bone formation blood index.

5 is a graph showing CTX in the blood of the mouse femur divided into groups 1 to 8. It shows the effect on the bone loss in the femur of the ovarian ablation (OVX) mouse according to the sample administration of the 1 to 8 groups.

As shown in Figure 5, compared to the Sham group, the OVX group confirmed that the concentration of CTX in the blood increased by 38%. It was confirmed that the concentration of CTX in the blood increased after ovarian extraction was reduced to a level similar to that of the normal sham group in all concentration groups when orally administered the enzyme-treated crude polysaccharide fraction (PLE-0).

Test Example 4. Return mutation toxicity test

In order to confirm toxicity by the regression mutation test of the crude polysaccharide fraction (PLE-0) prepared according to Example 1, the method presented by Maron and Ames (1983) described in the literature was partially modified and tested ( Maron, DM and Ames BN (1983) Revised methods for the Salmonella mutagenicity test, Mutat.Res. 113: 173-215).

Strains (Salmonella typhimurium) TA98, TA100, TA1535, and TA1537 strains were used for mutagenic search for specific components. The treatment of the test substance was performed by a direct plate incorporation method in which a metabolic activity enzyme system (S-9 mix) was applied or not applied. The Salmonella typhimurium TA98 strain used for the return mutation test was purchased from Molecular Toxicology Inc. (USA). The strains were inoculated in 25 mL of liquid medium (2.5% Oxoid Nutrient broth No. 2) of 50 μL of the test strain solution stored in a frozen state using a shaking incubator (VS-8480SFN, Vision Science). It was used after incubation at ℃ for about 10 hours. The minimal medium (minimal glucose agar plate) was made with 1.5% Bacto agar (214010, BD Difco) and Vogel-Bonner medium E and 2% glucose, and the top agar was prepared with 0.6% agar and 0.5% NaCl. , Top agar was added with 0.05 mM histidine (43011, Fluka) -biotin (47868, Supelco).

Autoclave top agar is dispensed in 2 mL increments into a sterile tube preheated to 45 ° C in a dry bath (11-718-4, Fisher Scientific), and then 0.1 mL of the test substance solution and a culture medium. 0.1 mL was mixed with a top agar and immediately shaken with a vortex mixer (37600, Thermolyne) for 2-3 seconds, poured into a minimal glucose agar plate, tilted in various directions to spread evenly, and solidified. The excipient group (negative control group) was carried out by adding 0.1 mL of the excipient instead of the test substance solution and the positive control group by adding the positive control solution (2-Aminoanthracene (2AA), Sigma A1381). After the top agar was hardened, the plate was turned over with the plate lid closed, and colonies were counted after incubation at 37 ° C for about 48 hours.

Strain Metabolic activity
Enzyme system Return mutation number / plate 0 g / plate 500 g / plate 1000 g / plate TA98 -S9 19 ± 1 14 ± 2 23 ± 4 + S9 19 ± 2 16 ± 3 15 ± 5 TA100 -S9 86 ± 9 108 ± 13 93 ± 10 + S9 78 ± 11 84 ± 6 92 ± 3 TA1535 -S9 6 ± 1 8 ± 2 7 ± 1 + S9 8 ± 3 7 ± 1 8 ± 1 TA1537 -S9 2 ± 2 3 ± 1 5 ± 1 + S9 8 ± 3 7 ± 1 6 ± 2

As shown in Table 6 above, it was confirmed that the crude polysaccharide fraction (PLE-0) prepared according to Example 1 of the present invention did not cause a return mutation for the strain used under the test conditions.

Test Example 5. In vitro staining abnormality test using Chinese Hamster Lung (CHL) cells

As an experiment to confirm whether the crude polysaccharide fraction (PLE-0) prepared according to Example 1 induces structural or numerical abnormalities in chromosomes of Chinese Hamster Lung (CHL) cells cultured, Chinese hamster lung (CHL) cells are used. The chromosomal abnormality test was performed under the application and non-application of metabolic activity system. As a metabolic activity system, coroc was added to the liver homogenate of rats induced with Aroclor-1254.

The test substance was treated by suspending it in the physiological saline injection solution. The highest concentration was determined using Relative Increased Cell Count (RICC) as an indicator of cytotoxicity. Concentration groups were set as shown in the following table, including negative (excipient) and positive controls, and one flask per concentration group was used.

The actively proliferating cells were separated, 5 x 10 ⁴ cells were seeded in a 5 mL culture solution in a flask having a culture area of 25 cm2, and cultured for about 3 days, followed by treatment of the test material. Chromosomal specimens were prepared 24 hours after the start of treatment, and chromosomal abnormalities were counted from 150 metaphases per concentration group.

Treatment with metabolic active system for 6 hours

Relative Increased Cell Count (RICC) was calculated by the following formula as the number of cells obtained by separating and counting cells from the flask, and used as an indicator of cytotoxicity.

[Equation 1]

S9 density
(g / mL) Cell count RICC (%) Abnormal weather
(%) PP + ER (%) + 0 5063 ± 88 100 0.00 0.00 400 4333 ± 98 76 0.00 0.00 500 3968 ± 27 64 0.00 0.00 550 4037 ± 17 66 0.00 0.00 600 4007 ± 42 65 0.00 0.00 700 4034 ± 1 66 0.00 0.00 800 3334 ± 29 43 0.00 0.00 Benzo [a] pyrene 3682 ± 25 54 31.33 0.00

Turbidity was observed in the concentration group of 500 μg / mL or more.

As shown in Table 7 above, the frequency of structural abnormalities (except for the gap below) is 0.00, 0.00, 0.00, 0.00, 0.00, in the order of negative control, test substance 400, 500, 550, 600, 700 and 800 μg / mL. At 0.00 and 0.00%, all concentration groups treated with the test substance did not show a statistically significant increase compared to the negative control group, and there was no dose correlation. The frequency of the mid-phase with water abnormality was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase and no dose correlation in all concentration groups treated with the test substance compared to the negative control group.

In the positive control group, a statistically significant increase was observed in the mid-phase frequency (31.33%) with structural abnormalities (P <0.01).

Non-metabolized 6-hour treatment

S9 density
(g / mL) Cell count RICC (%) Abnormal weather
(%) PP + ER (%) + 0 8022 ± 23 100 0.00 0.00 200 6254 ± 33 70 0.00 0.00 250 5386 ± 50 56 0.00 0.00 300 5054 ± 13 50 0.67 0.00 330 4717 ± 7 45 0.00 0.00 350 4947 ± 124 49 0.00 0.00 400 4400 ± 81 39 0.00 0.00 4-Nitroquinoline-1-oxide 0.4 5942 ± 170 65 14.67 0.00

As shown in Table 8 above, the frequency of structural abnormality phase is 0.00, 0.00, 0.00, 0.00, 0.67, 0.00 and 0.00% in the order of negative control, test substance 200, 250, 300, 330, 350 and 400 μg / mL. , In all concentration groups treated with the test substance, there was no statistically significant increase compared to the negative control group, and there was no dose correlation. The frequency of the mid-phase with water abnormalities was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase and no dose correlation in all concentration groups treated with the test substance compared to the negative control group.

In the positive control group, a statistically significant increase was observed in the frequency of mid-phase with structural abnormality (14.67%) (P <0.01).

Non-metabolizing 24 hours treatment

S9 density
(g / mL) Cell count RICC (%) Abnormal weather
(%) PP + ER (%) - 0 7105 ± 8 100 0.67 0.00 50 5847 ± 6 75 0.67 0.00 80 5282 ± 70 64 0.00 0.00 100 4844 ± 66 55 0.00 0.00 120 4494 ± 94 48 0.00 0.00 130 4370 ± 109 46 0.00 0.00 150 3999 ± 8 39 0.00 0.00 4-Nitroquinoline-1-oxide 0.4 5829 ± 30 75 12.00 0.00

As shown in Table 9 above, the frequency of structural abnormality phase is 0.67, 0.67, 0.00, 0.00, 0.00, 0.00, 0.00 and 0.00% in the order of negative control, test substance 50, 80, 100, 120, 130 and 150 μg / mL. , In all concentration groups treated with the test substance, there was no statistically significant increase compared to the negative control group, and there was no dose correlation. The frequency of the mid-phase with water abnormalities was 0.00% in both the negative control group and all test substance treatment groups, and there was no statistically significant increase and no dose correlation in all concentration groups treated with the test substance compared to the negative control group.

In the positive control group, a statistically significant increase was observed in the mid-phase frequency (12.00%) with structural abnormalities (P <0.01).

As a result of counting the chromosomal abnormalities, the frequency of occurrence of the medium phase with chromosomal abnormalities in all test substance treatment groups, regardless of the treatment method, did not increase compared to the negative control group, and these results did not satisfy the positive judgment criteria.

Therefore, it was confirmed that the test substance crude polysaccharide fraction (PLE-O) did not cause chromosomal abnormality in CHL cells under the test conditions.

Hereinafter, a formulation example of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit it, but only to be specifically described.

Formulation Example 1. Preparation of powder

500 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight fabric to prepare a powder.

Formulation Example 2. Preparation of tablets

300 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsules

200 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into a gelatin capsule to prepare a capsule.

Formulation Example 4. Preparation of injection

600 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Mannitol 180 mg

Injectable sterile distilled water 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above-mentioned ingredient content per ampoule according to a conventional injection method.

Formulation Example 5. Preparation of liquid formulation

4 g of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Isomerized sugar 10 g

5 g mannitol

Purified water

Each component is added to the purified water according to the conventional method for dissolving it, dissolved in a proper amount of lemon flavor, mixed with the above ingredients, and then added to the purified water to adjust the total to 100g, and then sterilized by filling in a brown bottle. To prepare a liquid formulation.

Formulation Example 6. Preparation of granules

1,000 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

Vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ folic acid

Calcium Pantothenate 0.5 mg

Suitable amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively granule preparation in a preferred embodiment, the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional granulation method and then granulated. It can be prepared and used in the production of health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

 1,000 mg of crude polysaccharide fraction derived from persimmon leaves obtained in Example 1

1,000 mg citric acid

Oligosaccharide 100 g

2 g of plum concentrate

Taurine 1 g

900 mL total by adding purified water

After mixing the above ingredients according to a conventional health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered, obtained in a sterilized 2 L container, sealed and sterilized, then refrigerated and stored. It is used for preparing the functional beverage composition of the present invention.

Although the above composition ratio is a mixture of components suitable for a preference drink in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, country of demand, and usage.

Claims (13)

Pharmaceutical composition for the prevention or treatment of bone-related diseases, characterized by containing persimmon leaf-derived polysaccharide fraction as an active ingredient. According to claim 1, wherein the persimmon leaf-derived polysaccharide fraction is a neutral polysaccharide (neutral sugar) 60 to 80% by weight compared to the total polysaccharide fraction, uronic acid (uronic acid) 18 to 39% by weight, DHA (3-deoxy-D-lyxo- 2-heptulosaric acid) and KDO (3-deoxy-D-manno-2- octulosonic acid) containing a KDO-like substance comprising 0.5 to 10% by weight of a pharmaceutical composition for the prevention or treatment of bone-related diseases. [3] The pharmaceutical composition for preventing or treating bone-related diseases according to claim 2, wherein the persimmon leaf-derived polysaccharide fraction further comprises 0.5 to 20% by weight of protein. According to claim 2, wherein the uronic acid is galacturonic acid (galacturonic acid) and glucuronic acid (glucuronic acid) characterized in that it comprises a pharmaceutical composition for the prevention or treatment of bone-related diseases. [3] The prevention or treatment of bone-related diseases according to claim 2, wherein the neutral polysaccharide comprises arabinose, rhamnose, galactose, fucose, glucose and rhamnogalacturonan-II indicator polysaccharide. Pharmaceutical composition. The method of claim 1, wherein the polysaccharide fraction comprises: (a) treating pectin hydrolase on persimmon leaf powder; And
(B) recovering a fraction of a molecular weight of 3 to 300 kDa from the enzyme-treated persimmon leaf powder; pharmaceutical composition for the prevention or treatment of bone-related diseases, characterized in that produced by a manufacturing method comprising a. According to claim 1, wherein the bone-related diseases are osteoporosis, osteoarthritis, degenerative arthritis, knee joint, hip joint, systemic lupus erythematosus (SLE), spondylitis, rheumatoid multiple myalgia, ankylosing spondylitis, Reiter's syndrome, psoriatic arthrosis, Group consisting of enteroarthritis, rheumatoid arthritis, neuroarthritis, acute rheumatoid fever, gout, cartilage calcification, periodontal disease caused by alveolar bone destruction, inflammatory alveolar bone resorption disease, inflammatory bone absorption disease, calcium hydroxide apatite crystal precipitation disease and lime disease Pharmaceutical composition for the prevention or treatment of bone-related diseases, characterized in that at least one selected from. A food composition for improving or preventing bone-related diseases, comprising a persimmon leaf-derived polysaccharide fraction as an active ingredient. The method of claim 8, wherein the persimmon leaf-derived polysaccharide fraction is a neutral polysaccharide (neutral sugar) 60 to 80% by weight compared to the total polysaccharide fraction, uronic acid (uronic acid) 18 to 39% by weight, DHA (3-deoxy-D-lyxo- 2-heptulosaric acid) and KDO (3-deoxy-D-manno-2-octulosonic acid) consisting of a KDO-like substance comprising 0.5 to 10% by weight of bone-related diseases for improving or preventing food composition. 10. The method of claim 9, wherein the persimmon leaf-derived polysaccharide fraction is a food composition for improving or preventing bone-related diseases, characterized in that it further comprises 0.5 to 20% by weight of protein. The food composition for improving or preventing bone-related diseases according to claim 9, wherein the uronic acid comprises galacturonic acid and glucuronic acid. The improvement or prevention of bone-related diseases according to claim 9, wherein the neutral polysaccharide comprises arabinose, rhamnose, galactose, fucose, glucose and rhamnogalacturonan-II indicator polysaccharide. Dragon food composition. The method of claim 8, wherein the bone-related diseases include osteoporosis, osteoarthritis, degenerative arthritis, knee arthritis, hip joint, systemic lupus erythematosus (SLE), spondylitis, rheumatoid multiple myalgia, ankylosing spondylitis, Reiter's syndrome, psoriatic arthrosis, Group consisting of enteroarthritis, rheumatoid arthritis, neuroarthritis, acute rheumatoid fever, gout, cartilage calcification, periodontal disease caused by alveolar bone destruction, inflammatory alveolar bone resorption disease, inflammatory bone absorption disease, calcium hydroxide apatite crystal precipitation disease and lime disease Food composition for the improvement or prevention of bone-related diseases, characterized in that at least one selected from.

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