Composition for preventing, improving or treating osteoporosis comprising malve nut ferment with bacillus strain

Abstract

The present application relates to an osteoporosis prevention, improvement or treatment composition comprising a malve nut ferment fermented using a bacillus strain, and the malve nut ferment fermented using bacillus licheniformis CP6 strain has a high safety with a reduced cytotoxicity compared to a raw malve nut, and has an excellent effect of promoting differentiation and growth of osteoblasts, and thus the composition of the present application comprising the malve nut ferment can be used as a pharmaceutical composition for osteoporosis prevention or treatment and a food composition for osteoporosis prevention or improvement.

Description

Technical Field

[0001] This invention relates to compositions for the prevention, improvement, or treatment of osteoporosis comprising fermented products of Bacillus strains. Background Technology

[0002] Osteoporosis is a disease that weakens bones and increases the risk of fractures, with a tendency for a significant increase in incidence in old age or after menopause. Osteoporosis can be classified into three types: type I osteoporosis (of unknown cause), type I osteoporosis (caused by insufficient estrogen after menopause), and type II osteoporosis (found in older men and women). It is understood that type I and type II osteoporosis are mostly caused by endocrine disorders, but can also be caused by diseases, nutritional imbalances, or deficiencies in calcium, phosphate, and vitamin D due to reduced calcium absorption.

[0003] Osteoporosis is a disease that cannot be treated with short-term medication alone and requires long-term drug administration. Therefore, there is a current need to develop new substances with novel effects and bone structure, while also having low toxicity and side effects, and effectively preventing and treating osteoporosis. Furthermore, with the aging population, the prevalence of osteoporosis is increasing, and fractures lead to a decline in quality of life and enormous medical costs. Therefore, the importance of prevention and improvement in osteoporosis while treating the disease is becoming increasingly prominent. Thus, there is a need to develop natural raw materials for the prevention or improvement of osteoporosis that are non-toxic to the human body and can be easily taken through daily diets or functional foods.

[0004] Bacillus sp., a genus of microorganisms, are widely distributed aerobic or facultative anaerobic bacilli in nature, participating in processes such as denitrification, iron reduction, and manganese oxidation. Depending on environmental conditions, structural changes occur within the bacterial cells to form spores (terminus spores). Spores are highly resistant to heat, radiation, and chemicals, and contain a large amount of 2,6-pyridinedicarboxylate calcium salt, which is not present in vegetative cells.

[0005] Based on properties such as cell size, spore morphology and location within the cell, sugar utilization and products, nitrate reduction, and growth under high salt concentrations, bacteria are classified into various types. Representative species include Bacillus subtilis, anthrax bacteria, and Bacillus cereus (a pathogen causing food poisoning). In common terminology, "bacterium" also refers to either bacilli or the entire bacterium.

[0006] On the other hand, *Aurea helianthus*, an annual herb prized for its medicinal value, is rich in collagen in its flower stalks and roots, in addition to its flowers and leaves. This collagen is used to treat various types of severe pain, including those with antipyretic, detoxifying, anti-inflammatory, and analgesic effects. It is also rich in natural phytoestrogens, which may help alleviate menopausal symptoms. However, the uses of fermented products from its *Bacillus* spp. fermentation process have not yet been investigated.

[0007] Against this backdrop, the inventors identified a new strain of Bacillus and confirmed that the fermented product of the strain has a therapeutic effect on osteoporosis.

[0008] Existing technical documents

[0009] Patent documents

[0010] Korean Patent No. 1783469 Summary of the Invention

[0011] The problem to be solved

[0012] The purpose of this invention is to provide a composition for the prevention, improvement, or treatment of osteoporosis derived from natural substances, which can improve osteoporosis without the human toxicity caused by artificial compounds.

[0013] Solution to the problem

[0014] To address the aforementioned technical problems, in one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of osteoporosis is provided, comprising fermented Aurea helianthus using Bacillus sp. strains.

[0015] Furthermore, in one embodiment of the present invention, a food composition for the prevention or improvement of osteoporosis is provided, comprising a fermented product of sunflower using a strain of Bacillus.

[0016] In one instance, the aforementioned Bacillus strain may be a Bacillus strain of Bacillus licheniformis CP6 (accession number: KCTC18811P).

[0017] In one example, the fermentation product can be a fermentation product fermented at 30–40°C.

[0018] In one instance, the fermented material can be fermented for 0.5 to 10 days.

[0019] In one instance, the aforementioned fermentation product can be a fermentation extract from the fermentation product of *Hippophae rhamnoides*.

[0020] The effects of the invention

[0021] Osteoblast cell line experiments confirmed that the fermented product of *Hippophae rhamnoides* using the *Bacillus* strain of this invention significantly increased bone formation by affecting both early and late-stage osteoblast differentiation compared to unfermented *Hippophae rhamnoides*. Furthermore, cytotoxicity evaluation confirmed that the toxicity inherent in the natural product was reduced by the *Bacillus* strain. Therefore, *Hippophae rhamnoides* fermented product, without safety concerns, can be used as a treatment and health supplement for fractures or osteoporosis associated with decreased bone density.

[0022] The effects of this invention are not limited to those described above, but should be understood to include all effects that can be inferred from the composition of the invention as described in the description or claims. Attached Figure Description

[0023] Figure 1 To evaluate the cytotoxicity results of unfermented sunflower culture (C-1D) and sunflower ferment (CP6-1D).

[0024] Figure 2 The results of evaluating the differentiation-promoting ability of MC3T3-E1 cells in the control group (Con), the unfermented golden hibiscus culture treatment group (golden hibiscus C), and the golden hibiscus fermentation treatment group were obtained.

[0025] Figure 3a The results of evaluating ALP activity in the control group (Con), the unfermented golden hibiscus culture treatment group (golden hibiscus C), and the golden hibiscus fermentation treatment group using MC3T3-E1 cells were obtained.

[0026] Figure 3b The results of ALP staining using MC3T3-E1 cells in the control group (Con), the unfermented sunflower culture treatment group (Sunflower C), and the sunflower fermentation treatment group.

[0027] Figure 4 (a) The results of observing MC3T3-E1 cells under a microscope after staining for calcium with alizarin in the control group (Con), the unfermented sunflower culture treatment group (Sunflower C), and the sunflower fermentation treatment group. Figure 4 (b) To extract Figure 4 (a) The result of staining calcium by cell lysis. Figure 4 (c) For the effect at 550nm Figure 4 (b) is the result of absorbance measurement. Detailed Implementation

[0028] The present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis comprising a ferment of Aurea helianthus using a strain of Bacillus sp.

[0029] The Bacillus strain used in this invention can be Bacillus licheniformis CP6, which can be obtained through accession number KCTC18811P or by the method described in Korean Patent Application No. 10-2021-0028930. The aforementioned Bacillus licheniformis CP6 strain can survive at high temperatures and in high salt concentrations (e.g., NaCl), and exhibits high amylase and protease activity. When this strain is used to ferment sunflower, the cytotoxicity of the sunflower extract can be inhibited while enhancing the therapeutic efficacy for osteoporosis.

[0030] In this invention, the above-mentioned golden sunflower ferment can be fermented using Bacillus strains through conventional fermentation methods well known in the art, without any particular limitation. However, the strain can be inoculated into a culture medium containing 1% dried golden sunflower powder, 3% NaCl and 0.1% yeast extract, and then fermented by shaking.

[0031] More specifically, the above-mentioned fermentation material can be fermented at a temperature of 30–40°C, preferably at a temperature of 35–38°C, but is not particularly limited thereto.

[0032] More specifically, the aforementioned fermentable materials can be fermented at concentrations of 0.5 to 10, 0.5 to 9.5, 0.5 to 9, 0.5 to 8.5, 0.5 to 8, 0.5 to 7.5, 0.5 to 7, 0.5 to 6.5, 0.5 to 6, 0.5 to 5.5, 0.5 to 5, 0.5 to 4.5, 0.5 to 4, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, 0.5 to 2, 0.5 to 1.5, 4 to 10, and 4 to 9. 5, 4 to 9, 4 to 8.5, 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.5 or 4 to 6 days, preferably, fermenting golden sunflower effectively induces changes in the comparative components of the original substance and can be fermented for 0.5 to 7 days in terms of inhibiting the excessive decomposition of effective proteins in golden sunflower, more preferably, fermenting for 4 to 6 days in terms of inhibiting the cytotoxicity of the original golden sunflower substance while enhancing the therapeutic efficacy of osteoporosis, but not necessarily limited to this.

[0033] The fermentation product can be an extract of *Hippophae rhamnoides* fermented using the aforementioned strain. The extract can be a hot water extract, a cold soaking extract, a reflux extract, a solvent extract, a steam distillation extract, an ultrasonic extract, a dissolution, or a pressed extract; preferably, it can be an ethanol extract, but is not particularly limited thereto.

[0034] In this invention, the fermented product of *Hippophae rhamnoides* using the above-mentioned *Bacillus* strain can be included in a pharmaceutical composition for the prevention or treatment of osteoporosis. The pharmaceutical composition may contain the above-mentioned *Hippophae rhamnoides* fermented product alone, or may be provided as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers, excipients or diluents.

[0035] The term "pharmaceutically acceptable" means that the composition exhibits properties that are non-toxic to cells or humans exposed to it.

[0036] Furthermore, the above-mentioned pharmaceutical composition can be provided in combination with conventionally known osteoporosis treatments. That is, the above-mentioned pharmaceutical composition can be used in combination with known compounds that have therapeutic effects on osteoporosis.

[0037] The term "administration" as used above refers to the introduction of a prescribed substance into an individual using appropriate methods. "Individual" refers to all animals, including humans, mice, and livestock, for the purpose of treating osteoporosis. Specific examples include mammals, including humans.

[0038] The routes of administration of the above pharmaceutical compositions include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration.

[0039] The above-mentioned pharmaceutical composition can be administered orally or non-orally. When administered non-orally, it is preferred to choose the injection method of topical application, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but it is not limited to these. From the viewpoint of choosing a more effective absorption route, oral administration is preferred.

[0040] The preferred dosage of the above pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for optimal effect, the above composition is administered at a dose of 0.01–1000 mg / kg / day, preferably 0.1–500 mg / kg / day, but not limited thereto. The above administration can be once daily or divided into several doses. The above dosage is not limited in any way.

[0041] When formulating the above pharmaceutical compositions, commonly used diluents or excipients such as fillers, expanders, binders, wetting agents, disintegrants, and surfactants are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid dosage forms are formulated by mixing one or more excipients with the above extracts, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, and syrups. Besides water and liquid paraffin, which are commonly used simple diluents, they may also contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives. Dosage forms for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. As a non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a base material for suppositories, semi-synthetic fatty acid esters such as witepsol, polyethylene glycol, Tween 61, cocoa butter, glyceryl trilaurate, and glyceryl gelatin can be used.

[0042] The present invention also provides a food composition for the prevention or improvement of osteoporosis comprising a fermented product of golden hibiscus fermented using a strain of Bacillus.

[0043] The preparation method of the fermentation process of the above-mentioned golden sunflower ferment is as described above.

[0044] The aforementioned food composition further comprises one or more of a carrier, diluent, excipient, and additive, and can be dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, and liquids. Foods to which the above food composition can be added include various food products, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, vitamin complexes, and health foods.

[0045] As an additive that may also be included in the above-mentioned food composition, one or more ingredients selected from the group consisting of natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerol, ethanol, carbonating agents, and fruit pulp may be used.

[0046] Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as conventional sugars like dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents (such as semathymethane and stevia extracts, such as rebaudioside A and glycyrrhizic acid) and synthetic flavoring agents (such as saccharin and aspartame) can be beneficially used as flavoring agents. In addition, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, ethanol, and carbonating agents used in carbonated beverages can be included. Furthermore, the aforementioned health foods may contain fruit pulp used in the preparation of natural fruit juices and vegetable beverages. These ingredients can be used alone or in combination.

[0047] The specific examples of the carrier, excipient, diluent and additives mentioned above are not limited thereto, but preferably one or more are selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannitol, erythritol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, methylcellulose, water, syrup, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

[0048] When formulating the above food composition, commonly used fillers, extenders, binders, humectants, disintegrants, surfactants, and other diluents or excipients can be used for formulation.

[0049] The following detailed description includes exemplary embodiments for more specific illustration. However, these embodiments are merely illustrative, and the scope of the invention is not limited thereto.

[0050] Example 1: Preparation of fermented extract of golden sunflower

[0051] After removing the dried pistils and stamens of the golden hibiscus, the petals and receptacles were further dried in a desiccator for 48 hours, then pulverized using a bell-type mixer. The CP-6 strain was inoculated into a nutrient broth (NB) liquid medium containing 3% NaCl and shaken at 37°C and 180 rpm for 16 hours. A golden hibiscus medium containing 1% dried golden hibiscus powder (petals and receptacles), 3% NaCl, and 0.1% yeast extract was sterilized at 121°C for 15 minutes. The cultured strain was inoculated at an amount equivalent to 0.1 of the absorbance (optical density, OD) of the above golden hibiscus medium and fermented for 1 day at 37°C and 180 rpm. The unfermented golden hibiscus culture was designated "C-1D," and the fermented golden hibiscus culture was designated "CP-6-1D." Ten times the weight of 70% (v / v) ethanol was added to each culture, and the mixture was extracted with stirring every 12 hours at room temperature, for a total of three extractions. The extracts were filtered under reduced pressure using Whatman No. 2 filter paper (Whatman International Ltd., Maidstone, England), concentrated under reduced pressure using a rotary evaporator, freeze-dried, and stored in a deep freezer at -80°C for use in experiments.

[0052] Example 2: Cytotoxicity evaluation of the fermented extract of *Hippophae rhamnoides*

[0053] To confirm cytotoxicity, cell viability was determined by MTT assay. Cells were grown on Raw 264.7. The cells were stocked at 2 × 10⁶ cells per well. 4 Cells were seeded at a concentration of 100 μg / ml into 96-well plates and cultured for 24 hours. Unfermented *Hippophae rhamnoides* culture (C-1D) and fermented *Hippophae rhamnoides* culture (CP6-1D) were treated with 100 μg / ml and 500 μg / ml, respectively. After 20 hours, cell viability was determined by MTT assay. (The results are shown in the figures.) Figure 1 middle.

[0054] Neither the unfermented nor the fermented *Hippophae rhamnoides* culture at a concentration of 100 μg / ml exhibited cytotoxicity. However, at a concentration of 500 μg / ml, the cell viability of the unfermented *Hippophae rhamnoides* culture was approximately 93.08%, indicating some cytotoxicity. No cytotoxicity was detected in the fermented *Hippophae rhamnoides* culture. This suggests that strain CP-6 can reduce the toxicity of *Hippophae rhamnoides* as a natural product through fermentation.

[0055] Example 3: Evaluation of the anti-osteoporosis efficacy of MC3T3-E1 osteoblasts

[0056] 1. Culture and differentiation of MC3T3-E1 cell line

[0057] MC3T3-E1 cells, used as an osteoblastic cell line, were dispensed from ATCC for experimental purposes. For cell growth, α-minimum essential medium (MEM) containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 g / ml streptomycin was used. For differentiation, 10 mM β-glycerol phosphate and 50 μg / ml L-ascorbic acid were added to the basal medium.

[0058] 2. Evaluation of Differentiation Promotion Capacity

[0059] To confirm the differentiation-promoting activity of the CP-6 strain fermented *Hippophae rhamnoides* ferment, MC3T3-E1 cells were treated with the ferment and subjected to MTT assay.

[0060] Cells were seeded in 96-well plates and cultured for 24 hours. Differentiation induction medium was added, and differentiation was induced for 10 days. Afterward, unfermented *Hippophae rhamnoides* extract (*Hippophae rhamnoides* C) and *Hippophae rhamnoides* fermentation broth were processed. MTT assays were performed after 1 and 3 days, as shown in the figures. Figure 2 In this study, it was confirmed that the fermented golden hibiscus extract treatment group promoted osteoblast differentiation and induced an increase in the amount of osteoblasts compared to the unfermented golden hibiscus extract treatment group and the negative control group.

[0061] 3. Evaluation of alkaline phosphatase (ALP) activity

[0062] ALP is a marker factor for early osteoblast differentiation and plays a regulatory role in the transport of inorganic phosphate, cell division, and differentiation during calcification.

[0063] To confirm whether *Hippophae rhamnoides* fermentation product affects early osteoblast differentiation, ALP activity was measured. MC3T3-E1 cells were seeded in 6-well plates and cultured for 24 hours, followed by differentiation induction medium for 5 days. Afterward, unfermented *Hippophae rhamnoides* extract (*Hippophae rhamnoides* C) and *Hippophae rhamnoides* fermentation product were treated, and after 24 hours of culture, cells were collected and lysed in 0.2% Triton X-100 to extract cellular proteins. 100 mM p-nitrophe nylon phosphate (p-NPP) was added to the extracted proteins, and the mixture was incubated in a CO2 incubator for 30 minutes. The reaction was stopped by adding 3 M sodium hydroxide, and the absorbance was measured at 405 nm. The degree of ALP staining was confirmed using a TRAC P&ALP double staining kit (Takara Bio Inc., Shiga, Japan). (See figure). Figure 3a and Figure 3b In this study, it was confirmed that the fermented golden hibiscus extract treatment group showed higher ALP activity than the unfermented golden hibiscus extract treatment group and the negative control group, promoting osteoblast differentiation and growth.

[0064] 4. Evaluation of bone calcification degree

[0065] Calcified nodule formation refers to a marker of late-stage osteoblast differentiation. Alizarin stains calcium in the inorganic cellular matrix, thus it is used to confirm the degree of bone calcification.

[0066] MC3T3-E1 cells were seeded in 6-well plates and cultured for 24 hours, followed by differentiation induction medium for 10 days. Afterward, cells were treated with unfermented hibiscus extract (hibiscus C) and hibiscus ferment, and cultured for 24 hours. Cells were then fixed with 70% ethanol at 4°C for 1 hour. The concentration of alizarin-red (AR) solution was adjusted to 40 mM in 10 ml of distilled water, the pH was adjusted to 4.2, and the solution was filtered before use. Fixed cells were treated with AR solution, stained for 10 minutes, and then washed three times with distilled water. Nodule formation was confirmed by microscopy and shown below. Figure 4 In (a), cells were lysed by adding 10 mM Tris buffer (pH 7.0) containing 10% acetylpyridinium chloride, and the absorbance was measured at 550 nm to determine the degree of calcification, as shown in [the diagram]. Figure 4 (b) and Figure 4 (c) It was confirmed that the calcification point formation and bone calcification capacity were increased in the safflower fermentation treatment group compared with the unfermented safflower culture treatment group and the negative control group.

[0067] Through the above embodiments, it was confirmed that the fermentation products of *Hippophae rhamnoides* can affect the early and late differentiation indicators of osteoblasts and thus promote bone formation.

[0068] The above description of the invention is illustrative, and those skilled in the art will understand that it can be easily modified in other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, the individual structural elements described as a single type can be implemented separately; similarly, the dispersed structural elements can also be implemented in a combined form.

[0069] The scope of this invention is defined by the claims described below, and should be interpreted as including all modifications or variations derived from the meaning and scope of the claims and their equivalents.

[0070] Microbial Preservation Certificate

[0071] April 1, 2020

[0072] To: Lee Sang Jae

[0073] 140 Baekyang-daero, Sasang-gu, Busan, South Korea

[0074] We hereby notify you of the acceptance of your application for the preservation of microorganisms submitted on March 23, 2020, under application number 6967. The microorganism preservation number is as follows.

[0075] -as follows-

[0076] 1. Microbial preservation number: KCTC18811P

[0077] 2. Microbial name: CP6 (Bacillus licheniformis)

[0078] Korea Center for Type Culture Collection (printed)

Claims

1. A pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that, The fermentation product of *Aurea helianthus* using *Bacillus licheniformis* strain CP6 (accession number KCTC18811P) was used as the single active substance.

2. The pharmaceutical composition for the prevention or treatment of osteoporosis according to claim 1, characterized in that, The fermented product is a fermented product fermented at 30-40°C.

3. The pharmaceutical composition for the prevention or treatment of osteoporosis according to claim 1, characterized in that, The fermented material is fermented for 0.5 to 10 days.

4. The pharmaceutical composition for the prevention or treatment of osteoporosis according to claim 1, characterized in that, The fermented product is a fermentation extract of Aurea helianthus.

5. A food composition for the prevention or improvement of osteoporosis, characterized in that, The fermentation product of *Aurea helianthus* using *Bacillus licheniformis* strain CP6 (accession number KCTC18811P) was used as the single active substance.

6. The food composition for the prevention or improvement of osteoporosis according to claim 5, characterized in that, The fermented product is a fermented product fermented at 30-40°C.

7. The food composition for the prevention or improvement of osteoporosis according to claim 5, characterized in that, The fermented material is fermented for 0.5 to 10 days.

8. The food composition for the prevention or improvement of osteoporosis according to claim 5, characterized in that, The fermented product is a fermentation extract of Aurea helianthus.

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