Use of milk fruit extract to prepare a composition for improving bone and cartilage differentiation

Abstract

Use of a milk fruit extract for the preparation of a composition for improving bone quality and promoting chondrocyte differentiation. The milk fruit extract is extracted from early-harvested fruits of Chrysophyllum cainito using water as a solvent.

Description

Technical Field

[0001] This invention relates to the application of milk fruit extract, and particularly to the use of milk fruit extract, wherein the milk fruit extract has the effect of improving bone quality or promoting cartilage differentiation. Background Technology

[0002] Bone density is known to be a dynamic equilibrium. Osteophytes break down bone to release calcium into the bloodstream for utilization, while simultaneously metabolizing old bone tissue. Osteogenesis, on the other hand, is responsible for generating new bone. However, the decrease in hormones after middle age leads to a slowdown in bone formation and an increase in osteopetrosis, making osteoporosis more likely.

[0003] Milk apple is a perennial tree belonging to the Sapotaceae family, native to central Panama. Mature trees reach a height of about 10 meters. Its distinctive feature is the golden-yellow down on the underside of its leaves. The fruit resembles an apple in appearance and size. A cross-section of the fruit reveals star-shaped white flesh radiating outwards from the core, hence its common name, "star apple." When ripe, it comes in two varieties: purple and green. The purple-skinned variety has a thicker skin with flesh that transitions from purple to white, while the green-skinned variety has a thinner skin and white flesh. Summary of the Invention

[0004] In view of this, in order to further explore new applications of milk fruit, the present invention provides a composition for using milk fruit extract to prepare a composition that improves bone quality and promotes cartilage differentiation.

[0005] In some embodiments, a milk fruit extract is used to prepare a composition for improving bone health. The milk fruit extract is extracted with water from early-harvested milk fruit, wherein 0.1% of a complex fiber enzyme is added to the water.

[0006] In some embodiments, the weight ratio of early-harvested milk fruit to water is 1:5~10.

[0007] In some embodiments, the extraction includes an ice crystal breaking step, low-temperature extraction, and high-temperature extraction. The ice crystal breaking step involves placing early-harvested fruits at 0°C to 10°C for 7 days. The low-temperature extraction is performed at 45°C to 60°C for 50 to 70 minutes. The high-temperature extraction is performed at 85±5°C for 50 to 70 minutes.

[0008] In some embodiments, milk fruit extract is used to promote osteoblast production of osteocalcin.

[0009] In some embodiments, milk fruit extract is used to promote osteocalcin levels in the blood.

[0010] In some embodiments, milk fruit extract is used to increase lumbar spine bone mineral density (T score).

[0011] In some embodiments, the milk fruit extract is used to prepare a composition that promotes cartilage differentiation. The milk fruit extract is extracted with water from early-harvested milk fruit, wherein 0.1% of a complex fiber enzyme is added to the water.

[0012] In some embodiments, milk fruit extract is used to promote the secretion of glycosaminoglycans (GAGs) by chondrocytes.

[0013] In some embodiments, a method for preparing milk fruit extract is provided, wherein the milk fruit extract is extracted from early-harvested milk fruit with water, wherein 0.1% of compound fiber enzyme is added to the water, and the extraction includes an ice crystal breaking step, low-temperature extraction and high-temperature extraction, wherein the ice crystal breaking step involves placing the early-harvested fruit at 0°C to 10°C and letting it stand for 7 days, the low-temperature extraction is performed at 45°C to 60°C for 50 to 70 minutes, and the high-temperature extraction is performed at 85±5°C for 50 to 70 minutes.

[0014] In some embodiments, the milk fruit extract contains more than 30 ppm of myricetin.

[0015] In summary, the milk fruit extract of any embodiment can be used to prepare compositions that improve bone quality and promote cartilage differentiation. In some embodiments, the milk fruit extract has at least one of the following effects: promoting osteoblast production of osteocalcin, increasing blood osteocalcin levels, increasing lumbar vertebral bone density, or promoting chondrocyte secretion of polyglucosamine. Attached Figure Description

[0016] Figure 1 This is a graph showing the results of a relative osteocalcin test.

[0017] Figure 2 This is a graph showing the test results for polyglucosamine.

[0018] Figure 3 This is a graph showing the blood osteocalcin content in human subjects.

[0019] Figure 4 This is another blood osteocalcin content graph from a human experiment.

[0020] Figure 5 This is a graph showing the T-score results from human trials.

[0021] Figure 6 This is another T-score result graph from human trials.

[0022] Figure 7 This is the HPLC chromatogram of milk fruit extract.

[0023] Figure 8This is a graph showing the test results of the relative osteocalcin content of myricetin in bayberry flavonoids. Detailed Implementation

[0024] As used herein, the term "extract" refers to a product prepared by extraction. Here, an extract may be presented as a solution dissolved in a solvent, or as a concentrate or essence containing little or no solvent.

[0025] The term "receptor" as used in this article refers to humans or non-human mammals, preferably humans.

[0026] As used in this article, "milk fruit" refers to the whole fruit of the plant *Chrysophyllum cainito*, also commonly known as star apple. In some embodiments, milk fruit refers to the early-harvested fruit (unripe fruit) of a purple-skinned variety, which is the fruit when it is green. In some embodiments, the whole fruit includes the peel, pulp, and seeds.

[0027] In some embodiments, the milk fruit may be fresh, dried, or frozen. In some embodiments, drying may be air-drying, sun-drying, shade-drying, or freeze-drying. In some embodiments, the milk fruit may further comprise whole, chopped, diced, ground, milled, or otherwise processed to affect the size and integrity of the raw material. In some embodiments, the milk fruit used is milk fruit produced in Taiwan, China.

[0028] In some embodiments, a milk fruit extract is used to prepare a composition for improving bone health. The milk fruit extract is extracted with water from early-harvested milk fruit, wherein 0.1% of a complex cellulase is added to the water. In some embodiments, the complex cellulase refers to a general term for enzymes capable of degrading cellulose or glucosidic bonds. In some embodiments, the complex cellulase includes β-glucanase. Here, β-glucanase is an endoglucanase capable of hydrolyzing the (1,3)- or (1,4)- bonds in β-D-glucan. Here, the extraction involves soaking the milk fruit in water for a period of time.

[0029] In some embodiments, the weight ratio of water to early-harvested milk fruit is 5-10:1.

[0030] In some embodiments, the extraction includes an ice crystal disruption step, low-temperature extraction, and high-temperature extraction. In some embodiments, the ice crystal disruption step involves placing whole, early-harvested fruit at -10°C to 0°C for 7 days to break down cell walls and improve extraction efficiency. In some embodiments, low-temperature extraction is performed at 45°C to 60°C for 50 to 70 minutes. In some embodiments, high-temperature extraction is performed at 80°C to 90°C for 50 to 70 minutes. Here, extraction involves mixing milk fruit with water and maintaining the mixture at a specific temperature for a specific period of time.

[0031] In some embodiments, the milk fruit extract is extracted from early-harvested milk fruit that has undergone ice crystal cell wall breaking using water as a solvent, wherein 0.1% of a compound fiber enzyme is added to the water. In some embodiments, the milk fruit extract is extracted from early-harvested milk fruit that has undergone ice crystal cell wall breaking using water as a solvent, first at low temperature and then at high temperature, wherein 0.1% of a compound fiber enzyme is added to the water.

[0032] In some embodiments, milk fruit extract is used to promote the production of osteocalcin by osteoblasts. Osteocalcin, also known as Bone Gla Protein (BGP), is an indicator of bone formation rate. Osteocalcin is produced by osteoblasts, and its production level reflects osteoblast activity. Recently, osteocalcin production has also been used as an indicator of overall health and anti-aging.

[0033] In some embodiments, milk fruit extract is used to promote osteocalcin levels in the blood. Osteocalcin levels in the blood are positively correlated with bone formation.

[0034] In some embodiments, milk fruit extract is used to improve bone mineral density (T-score). Medically, bone mineral density test results are compared to the optimal bone mineral density of a healthy 30-year-old adult to calculate a comparison value called the T-score. A T-score of 0 indicates that your bone mineral density is equal to the average of a healthy young adult. A T-score < 0 is represented by a negative number; the larger the negative value (in standard deviation), the lower the bone mineral density.

[0035] In some embodiments, milk fruit extract is used to prepare compositions that promote chondrogenic differentiation. In some embodiments, milk fruit extract is used to promote the secretion of polyglucosamines (GAGs) by chondrocytes. Polyglucosamines are natural compounds that can increase collagen synthesis in chondrocytes, reduce the production of pro-inflammatory factors, and thereby reduce apoptosis.

[0036] In some embodiments, a method for preparing milk fruit extract is provided, including an ice crystal disruption step, low-temperature extraction, and high-temperature extraction. First, early-harvested milk fruit is left to stand at -10°C to 0°C for 7 days to form a cell-wall-breaking raw material (ice crystal disruption step). Next, the cell-wall-breaking raw material is mixed with water and then mixed at 55±5°C for 60 minutes to form a primary extract (low-temperature extraction), wherein 0.1% of a compound fiber enzyme is added to the water. Subsequently, the primary extract is heated to 85±5°C for 60 minutes (high-temperature extraction) to form the milk fruit extract.

[0037] In some embodiments, a crushing step is further included between the ice crystal breaking step and the low-temperature extraction, wherein the crushing step involves crushing the raw material using a pulverizer. In some embodiments, the pulverizer is set with a pulverizing aperture of approximately 30 mm. In some embodiments, the crushing step involves crushing water and the raw material together using a pulverizer. In some embodiments, the crushing step involves crushing water and the raw material together using a pulverizer, and then sieving the mixture through a sieve, which may have a mesh size of 400 mesh. In some embodiments, a filtration step is further included after high-temperature extraction, wherein the initial extract after high-temperature extraction is filtered through a 400-mesh filter to remove fine solids. In some embodiments, a concentration step is further included after high-temperature extraction, wherein the initial extract is concentrated under reduced pressure at 60°C ± 5°C until the Degrees Brix of the initial extract is 7.5 ± 0.5, at which point the concentration is stopped to obtain milk fruit extract.

[0038] In some embodiments, the milk fruit extract contains more than 30 ppm of myricetin.

[0039] In some embodiments, the composition is a food composition, and the composition contains at least a specific amount of milk fruit extract. In some embodiments, the specific amount of milk fruit extract is 2 grams per day.

[0040] In some embodiments, the above-described composition may be a pharmaceutical product. In other words, this pharmaceutical product contains an effective amount of milk fruit extract. In some embodiments, the effective amount of milk fruit extract is 2 grams per day.

[0041] The terms "specific dosage" or "effective content" used herein refer to the amount of a substance required in an individual to produce a specific effect. As will be recognized by those skilled in the art, the specific dosage or effective content will vary depending on the route of administration, the use of excipients, and the possibility of sharing with other substances.

[0042] In some embodiments, the aforementioned pharmaceutical products may be manufactured into a dosage form suitable for enteral or oral administration using techniques well known to those skilled in the art. The dosage form suitable for enteral or oral administration may be, but is not limited to: tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or similar substances.

[0043] In some embodiments, the aforementioned pharmaceutical product may be manufactured using techniques well known to those skilled in the art into a dosage form suitable for parenterally or topically administration. Dosage forms suitable for parenterally or topically administration may include, but are not limited to, injections, sterile powders, external preparations, and the like. In some embodiments, the pharmaceutical product may be administered via a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection.

[0044] In some embodiments, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technologies. For example, a pharmaceutically acceptable carrier may comprise one or more of the following agents: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these agents fall within the scope of professional competence and routine practice of those skilled in the art.

[0045] In some embodiments, a pharmaceutically acceptable carrier may further comprise a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0046] In some embodiments, the aforementioned composition may be an edible composition for non-medical purposes, and this edible composition contains at least a specific amount of milk fruit extract. In some embodiments, this edible composition may be made into a food product or may be a food additive. That is, the milk fruit extract of any of the foregoing embodiments may be added during the preparation of ingredients using existing techniques to obtain a food product, or the milk fruit extract of any of the foregoing embodiments may be added directly during the production of the food product. Here, the food product may be formulated with edible materials for human or animal consumption. In some embodiments, the edible composition may further include a food body, or may include a food body and a food additive.

[0047] In some embodiments, food products may be, but are not limited to: beverages, fermented foods, bakery products, health foods for non-medical purposes, or dietary supplements for non-medical purposes.

[0048] Example 1: Preparation of test samples

[0049] 1-1. Preparation of milk fruit extract in the experimental group

[0050] The product uses whole, early-harvested milk fruit (green on the outside) from Taiwan, China. The whole fruit includes the peel, seeds, and pulp. After being left to stand at -10°C for 7 days, the fruit is used to create a cell-wall breaking material (ice crystal breaking step). This material is then added to water at a weight ratio of 1:10. Next, the material and water are pulverized and mixed (using a SAMPO KJ-SD15G pulverizer with a 30mm coarse pulverization aperture).

[0051] After pulverizing and mixing the cell wall-breaking raw material, 0.1% of the compound fiber enzyme was added to water, and the mixture was heated to 55±5℃ and mixed for 60 minutes to form the initial extract (low-temperature extraction). The compound fiber enzyme was purchased from the brand Novozymes, and its composition included Water 59.9%, Sucrose 23%, Sodium chloride 10%, Beta-glucanase (endo-1,3(4)-) 7%, and Potassium sorbate 0.1%.

[0052] Next, the primary extract was heated to 85°C and maintained at that temperature for 1 hour to obtain the secondary extract. Subsequently, the secondary extract was filtered through a 400-mesh sieve to remove solids, and then concentrated under reduced pressure at 60±5°C until the Brix value of the solution reached 7.5±0.5, at which point the concentration was stopped to obtain the milk fruit extract. The brand / model of the concentrator used was BUCHI-Rotavapor R-100.

[0053] 1-2. Preparation of milk fruit extract for the control group

[0054] The raw milk fruit (purple on the outside) from fresh, ripe milk fruit produced in Taiwan, China, is used whole. The whole fruit includes the peel, seeds, and pulp. After being left to stand at -10℃ for 7 days, the raw material is broken down into a cell-wall-breaking material (ice crystal breaking step). This broken-down material is then added to water at a weight ratio of 1:10. Next, the broken-down material and water are pulverized and mixed (using a SAMPO KJ-SD15G pulverizer with a coarse pulverization aperture of 30mm).

[0055] After pulverizing and mixing the cell wall-breaking raw material, 0.1% of the compound fiber enzyme was added to water, and the mixture was heated to 55±5℃ and mixed for 60 minutes to form the initial extract (low-temperature extraction). The compound fiber enzyme was purchased from the brand Novozymes, and its composition included Water 59.9%, Sucrose 23%, Sodium chloride 10%, Beta-glucanase (endo-1,3(4)-) 7%, and Potassium sorbate 0.1%.

[0056] Next, the primary extract was heated to 85°C and maintained at that temperature for 1 hour to obtain the secondary extract. Subsequently, the secondary extract was filtered through a 400-mesh sieve to remove solids, and then concentrated under reduced pressure at 60±5°C until the Brix value of the solution reached 7.5±0.5, at which point the concentration was stopped to obtain the ripe fruit extract. The brand / model of the concentrator used was BUCHI-Rotavapor R-100.

[0057] Example 2: Test to promote osteocalcin secretion

[0058] 2-1. Materials and Instruments:

[0059] Experimental cell line: Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used. OP9 cells were purchased from the American Type Culture Collection (ATCC). ® OP9 cell line (ATCC CRL-2749™).

[0060] Cell culture medium: containing 90% MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, product number Cat.12000-022), 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, Cat.10437-028), and 1% penicillin-streptomycin (Penicillin-streptomycin, purchased from Gibco, Cat.15240-062).

[0061] Differentiation medium: Contains 90% DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, Cat. 12100-038), with additional components including 10% FBS (fetal bovine serum, purchased from Gibco, 10438-026), 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122), 50 μM ascorbic acid (purchased from Sigma), and 10 -7 M-dexamethasome (purchased from Sigma) and 10 mM β-glycerol (purchased from Sigma).

[0062] Buffered saline solution (DPBS solution): purchased from Gibco, product number 14200-75.

[0063] Osteocalcin Detection Kit (OC brand USCN, model SEA471Mu).

[0064] Flow cytometer: purchased from BD Pharmingen, model BDTM Accuri C6 Plus.

[0065] 2-2. Test Procedure:

[0066] OP9 cells were spaced at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 2 mL each in a 24-well culture dish and incubated in a CO2 incubator for 24 hours. Then, the cell culture medium was replaced with fresh differentiation medium.

[0067] The culture was continued for 7 days, with the differentiation medium being replaced with fresh medium every 3 days.

[0068] OP9 cells were divided into a blank control group, an experimental group, and a control group, and cultured for 7–10 days, with the differentiation medium replaced with fresh medium every 3 days. The experimental group's differentiation medium was supplemented with 0.03125 mg / mL of milk fruit extract prepared in Example 1. The blank control group received only differentiation medium without any additional test samples. The control group's differentiation medium contained 0.03125 mg / mL of ripe fruit extract prepared in Example 1.

[0069] Cell morphology was observed, confirming that OP9 cells had differentiated into osteocytes.

[0070] The supernatant is collected from each well of the culture tray into a microcentrifuge tube for subsequent analysis using the osteocalcin assay kit. The osteocalcin assay kit is then used to perform ELISA analysis according to the manufacturer's instruction manual.

[0071] The osteocalcin content in the supernatant of each well was measured by flow cytometry to facilitate osteocalcin secretion analysis. Each group underwent triple replication; therefore, the results of the triple replications were averaged to obtain the mean. The mean osteocalcin secretion of the blank group was then considered as 100%. The mean values ​​of the experimental and control groups were converted to relative osteocalcin secretion levels. Figure 1 As shown.

[0072] 2-3. Test Results:

[0073] The results were used to perform a student t-test using Excel software to determine whether there was a statistically significant difference between the two sample groups. Figure 1 As shown, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*" symbols there are, the more statistically significant the difference is relative to the control group.

[0074] See Figure 1 The experimental group showed a relative osteocalcin secretion level of 106.7%, indicating an effect of promoting osteocalcin secretion in bone cells. Conversely, compared to the control group, the osteocalcin secretion level in bone cells decreased to 98.3%. This demonstrates that milk fruit extract can enhance osteocalcin secretion in bone cells, while ripe fruit extract does not have this effect.

[0075] Example 3: Test to promote polyglucosamine secretion

[0076] Mouse chondrocyte precursor cells can differentiate into chondrocytes. This test uses microscopic observation of the appearance of Alsin blue stain and quantification with an ELISA reader to analyze the degree of differentiation of mouse chondrocyte precursor cells into chondrocytes, thereby assessing whether the test sample has the ability to promote chondrocyte differentiation. Alsin blue staining of polyglucosamine allows for observation of cell differentiation status by observing the amount of polyglucosamine.

[0077] 3-1. Materials and Instruments:

[0078] Cell line: Mouse cartilage precursor cells ATDC5 (purchased from Sigma, model 99072806).

[0079] ATDC5 medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, Cat. 12100-038) basal medium was mixed with Ham's F-12 basal medium at a 1:1 ratio, with additional ingredients added to contain 5% FBS (fetal bovine serum, purchased from Gibco, 10438-026), 2 mM L of glutamic acid (purchased from Gibco), and 1% penicillin / streptomycin (purchased from Gibco).

[0080] Differentiation medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, Cat. 12100-038) basal medium was mixed with Ham's F-12 basal medium at a 1:1 ratio, and additional ingredients were added to contain 5% FBS (fetal bovine serum, purchased from Gibco, 10438-026), 2 mM L of glutamine (purchased from Gibco), 1% penicillin / streptomycin (purchased from Gibco), and 1% insulin-transferrin-selenium.

[0081] Alcian blue, purchased from Sigma.

[0082] Buffered saline solution (DPBS solution): purchased from Gibco, product number 14200-75.

[0083] Formaldehyde: Purchased from Jingming, product number TG1794-4-0000-72NI.

[0084] Use 0.1 M HCl.

[0085] Trypsin: 10X Trypsin-EDTA (purchased from Gibco, product number 15400-054).

[0086] 3-2. Testing Procedure:

[0087] Mouse chondrocyte precursor cells were distributed at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were inoculated into 6-well culture dishes containing 2 mL of ATDC5 medium per well. The culture dishes were placed in 5% CO2 at 37°C and cultured until cells were observed to fill the dish. Fresh ATDC5 medium was replaced every 3 days during this period.

[0088] Mouse chondrocyte precursor cells were divided into a control group and an experimental group, and cultured for 35 days, with the differentiation medium being replaced with fresh medium every 3 days. The experimental group's differentiation medium was supplemented with 0.03125 mg / mL of milk fruit extract prepared in Example 1. The control group received only differentiation medium without any additional test samples.

[0089] Next, Alsin blue staining was performed, with the following steps: Remove differentiation medium and wash three times with 1X PBS. Add 0.5 mL of 10% formaldehyde and fix at room temperature for 30 minutes. Remove formaldehyde and wash three times with 1X PBS. Prepare 1% Alsin blue stain using HCl (pH=2.5). Add 0.5 mL of Alsin blue stain to each well and stain overnight at room temperature.

[0090] Finally, observation and quantification were performed. The processing steps were as follows: wash three times with 1XPBS. The blue signal of Alsin blue stained with polyglucosamine was observed under a microscope, photographs were taken, and color quantitative analysis was performed using FIJ software. The blue staining signal of Alsin blue turned red after color analysis. The results are as follows: Figure 2 As shown.

[0091] 3-3. Test Results:

[0092] Please see Figure 2 The red color was relatively sparse in the control group, while it was very dense in the experimental group, meaning that a greater amount of polyglucosamine was observed in the experimental group. Based on this, the test results show that early harvesting of milk fruit can promote the differentiation of precursor cells into chondrocytes and promote the production of more polyglucosamine by chondrocytes to protect joints.

[0093] Example 4: Human Experimentation

[0094] Sample: Milk fruit extract prepared in Example 1.

[0095] Participants: 9 participants. All participants were women aged 35 to 70. Generally, the medical reference range for osteocalcin is 11.0 ng / mL to 43.0 ng / mL for premenopausal women and 15.0 ng / mL to 46.0 ng / mL for postmenopausal women. Therefore, in the dynamic balance of bone, if the rate of bone formation is too slow, lower than the rate of bone loss (resorption rate), osteoporosis is likely to occur in the long term. Specifically regarding osteocalcin, a low concentration may indicate a slow rate of bone formation, while a high concentration indicates a rapid rate of bone replacement; both are signs of osteoporosis. The World Health Organization (WHO) classifies osteoporosis based on bone mineral density: a T-score of +1 to -1 is normal, a T-score of -1 to -2.5 indicates osteopenia, and a T-score of -2.5 or less indicates osteoporosis.

[0096] 4-1 Test Items

[0097] The tests include: blood osteocalcin concentration and bone density.

[0098] The osteocalcin concentration was measured using blood samples, and the testing was conducted by Harvard Healthcare.

[0099] Bone mineral density was measured using dual-energy X-ray absorptiometry (DXA), specifically the bone mineral density of the lumbar spine, which was then converted into a T-score. The average bone mineral density of the first to fourth lumbar vertebrae was used for the conversion.

[0100] 4-2. Testing Procedure:

[0101] Nine participants were instructed to ingest 2g of milk fruit extract daily for twelve weeks. Blood samples were collected and measured using the aforementioned equipment before ingestion (week 0 on the graph), after eight weeks of consumption (week 8 on the graph), and after twelve weeks of consumption (week 12 on the graph).

[0102] 4-3. Test Results:

[0103] Please see Figure 3The average blood osteocalcin level at week 0 was 15.93 ng / mL. After 8 weeks of daily intake of 2g of milk fruit extract, the average blood osteocalcin level of the 9 subjects increased to 16.96 ng / mL, and after 12 weeks of daily intake of 2g of milk fruit extract, the average blood osteocalcin level of the 9 subjects increased to 17.40 ng / mL. This means that after 8 weeks of using milk fruit extract, the blood osteocalcin level increased by 6.5%, and after 12 weeks, the blood osteocalcin level increased by 9.2%.

[0104] Please see Figure 4 Among the participants, six who had already gone through menopause were separately observed. At week 0, these six participants had an average blood osteocalcin level of 17.60 ng / mL. After 8 weeks of daily intake of 2g of milk fruit extract, their average blood osteocalcin level increased to 19.38 ng / mL. After 12 weeks of daily intake of 2g of milk fruit extract, their average blood osteocalcin level increased to 19.48 ng / mL. This means that after 8 weeks of using milk fruit extract, blood osteocalcin level increased by 10.1%, and after 12 weeks, it increased by 10.7%.

[0105] Please see Figure 5 The average T-score at week 0 was -1.30. After 8 weeks of daily intake of 2g of milk fruit extract, the average T-score of the 9 subjects improved to -1.29, and after 12 weeks of daily intake of 2g of milk fruit extract, the average T-score of the 9 subjects improved to -1.12. This means that bone mineral density increased by 13.9% after 12 weeks of using milk fruit extract.

[0106] Please see Figure 6 Among the participants, six who had already gone through menopause were separately examined. These six participants had an average T-score of -2.08 at week 0. After 8 weeks of daily intake of 2g of milk fruit extract, their average T-score improved to -2.04, and after 12 weeks of daily intake of 2g of milk fruit extract, their average T-score improved to -1.82. This means that bone mineral density increased by 12.5% ​​after 12 weeks of using milk fruit extract.

[0107] Example 5: HPLC analysis of the fingerprint spectrum of milk fruit extract

[0108] Therefore, high performance liquid chromatography (HPLC) was used to perform quantitative and qualitative analysis of the bioactive substances in the milk fruit extract prepared in Example 1.

[0109] The solvents used in this test were methanol and water, with 0.1% formic acid added to each. The flow rate was set to 1 ml / min, and the extraction conditions were set as follows: methanol:water ratio 2:98 at 0 minutes, methanol:water ratio 2:98 at 10 minutes, methanol:water ratio 70:water ratio 30 at 40 minutes, methanol:water ratio 100:water ratio 0 at 50 minutes, and methanol:water ratio 100:water ratio 0 at 60 minutes.

[0110] refer to Figure 7 The peak of the bioactive substance extracted at approximately 33 minutes was confirmed as myricetin after analysis and comparison. Simultaneously, HPLC quantitative analysis showed that the myricetin content in the milk fruit extract was 30.2 ppm.

[0111] Example 6: Test on the effect of myricetin flavonoids on osteocalcin secretion

[0112] 6-1. Materials and Instruments:

[0113] Experimental cell line: Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used. The OP9 cells were purchased from the American Type Culture Collection (ATCC®) OP9 cell line (ATCC CRL-2749™).

[0114] Cell culture medium: containing 90% MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, product number Cat.12000-022), 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, Cat.10437-028), and 1% penicillin-streptomycin (Penicillin-streptomycin, purchased from Gibco, Cat.15240-062).

[0115] Differentiation medium: containing 90% DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, Cat. 12100-038) with added components to include 10% FBS (fetal bovine serum, purchased from Gibco, 10438-026), 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122), 50 μM ascorbic acid (purchased from Sigma), 10⁻⁷ M dexamethasome (purchased from Sigma), and 10 mM β-glycerol (purchased from Sigma).

[0116] Buffered saline solution (DPBS solution): purchased from Gibco, product number 14200-75.

[0117] Osteocalcin testing kit (ELISA Kit for Osteocalcin (OC) brand USCN, model SEA471Mu).

[0118] Flow cytometer: purchased from BD Pharmingen, model BDTM Accuri C6 Plus.

[0119] 6-2. Test Procedure:

[0120] OP9 cells were spaced at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 2 mL each in a 24-well culture dish and incubated in a CO2 incubator for 24 hours. Then, the cell culture medium was replaced with fresh differentiation medium.

[0121] The culture was continued for 7 days, with the differentiation medium being replaced with fresh medium every 3 days.

[0122] After observing cell morphology and confirming that OP9 cells had differentiated into osteocytes, the osteocytes were divided into a control group and an experimental group, and cultured for 7 days, with the differentiation medium being replaced with fresh medium every 3 days. The differentiation medium for the experimental group was supplemented with 0.03125 mg / mL of myricetin. The control group received only the differentiation medium without any additional test samples.

[0123] The supernatant is collected from each well of the culture tray into a microcentrifuge tube for subsequent analysis using the osteocalcin assay kit. The osteocalcin assay kit is then used to perform ELISA analysis according to the manufacturer's instruction manual.

[0124] The osteocalcin content in the supernatant of each well was measured by flow cytometry to facilitate osteocalcin secretion analysis. Each group underwent triple replication; therefore, the results of the triple replications were averaged to obtain the mean. The mean osteocalcin secretion of the blank group was then considered as 100%, and the mean values ​​of the experimental groups were converted to relative osteocalcin secretion. Figure 8 As shown.

[0125] 6-3. Test Results:

[0126] The results were used to perform a student t-test using Excel software to determine whether there was a statistically significant difference between the two sample groups. Figure 8As shown, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*" symbols there are, the more statistically significant the difference is relative to the control group.

[0127] See Figure 8 The relative osteocalcin secretion level in the experimental group was 121.39%, indicating an effect of promoting osteocalcin secretion in bone cells. This shows that the bioactive substance myricetin in milk fruit extract can enhance and promote osteocalcin secretion in bone cells.

[0128] In summary, the milk fruit extract of any embodiment can be used to prepare a composition that improves bone quality and promotes cartilage differentiation. The milk fruit extract of any embodiment has at least one of the following effects: promoting osteocalcin production by osteoblasts, increasing blood osteocalcin levels, increasing lumbar vertebral bone density, and promoting the secretion of polyglucosamine by chondrocytes.

[0129] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. The use of a milk fruit extract for preparing a composition to improve bone health, wherein the milk fruit extract is extracted with water from early-harvested milk fruit (Chrysophyllum cainito), wherein 0.1% of a complex fiber enzyme is added to the water, and the weight ratio of water to the early-harvested fruit is 5-10:1, the extraction comprising an ice crystal breaking step, low-temperature extraction, and high-temperature extraction, wherein the ice crystal breaking step involves placing the early-harvested fruit at -10°C to 0°C for 7 days, the low-temperature extraction is performed at 45°C to 60°C for 50-70 minutes, and the high-temperature extraction is performed at 85±5°C for 50-70 minutes.

2. The use as described in claim 1, wherein the milk fruit extract is used to promote osteoblast production of osteocalcin.

3. The use as described in claim 2, wherein the milk fruit extract is used to promote osteocalcin levels in the blood.

4. The use as described in claim 1, wherein the milk fruit extract is used to increase lumbar spine bone mineral density (Tscore).

5. The use of a milk fruit extract for preparing a composition that promotes cartilage differentiation, wherein the milk fruit extract is extracted with water from early-harvested milk fruit (Chrysophyllum cainito), wherein 0.1% of a complex fiber enzyme is added to the water, and the weight ratio of water to the early-harvested fruit is 5-10:

1. The extraction includes an ice crystal breaking step, low-temperature extraction, and high-temperature extraction, wherein the ice crystal breaking step involves placing the early-harvested fruit at -10°C to 0°C for 7 days, the low-temperature extraction is performed at 45°C to 60°C for 50-70 minutes, and the high-temperature extraction is performed at 85±5°C for 50-70 minutes.

6. The use as described in claim 5, wherein the milk fruit extract is used to promote the secretion of polyglucosamines (GAGs) by chondrocytes.

7. A method for preparing a milk fruit extract, wherein the milk fruit extract is extracted from early-harvested milk fruit (Chrysophyllum cainito) with water, wherein 0.1% of a compound fiber enzyme is added to the water, and the weight ratio of water to the early-harvested fruit is 5-10:1, wherein the extraction includes an ice crystal breaking step, low-temperature extraction, and high-temperature extraction, wherein the ice crystal breaking step involves placing the early-harvested fruit at -10°C to 0°C for 7 days, the low-temperature extraction is performed at 45°C to 60°C for 50-70 minutes, and the high-temperature extraction is performed at 85±5°C for 50-70 minutes.

8. The preparation method according to claim 7, wherein the milk fruit extract contains more than 30 ppm of myricetin.

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