The use of mangosteen peel extract in the preparation of components for improving bone health.

Abstract

Invention details;

Description

Use of mangosteen shell extract in preparing bone health care composition Technical Field

[0001] The present invention relates to a use of a mangosteen shell extract, in particular to a mangosteen shell extract prepared from the mangosteen shell, and the use of the mangosteen shell extract in bone health care. Background Art

[0002] In modern society, approximately 200 million people worldwide are affected by osteoporosis. According to statistics, approximately 8.9 million fragility fractures occur each year, and approximately one-third of women and one-fifth of men over 50 years old experience a fragility fracture in their lifetime.

[0003] In addition, more than 500 million people worldwide suffer from degenerative arthritis every year. With the increase in the elderly population, the proportion of people suffering from arthritis has increased by 48% in the past 30 years from 2019. The symptoms of arthritis and soft tissue inflammation include cartilage wear between bones, structural deformation, or reduced synovial fluid secretion between joints, causing pain, swelling, fever, stiffness and other symptoms in the joints.

[0004] Orthopedic surgeons point out that osteoporosis is a largely irreversible symptom. Once bone loss occurs, subsequent treatment and prevention can only slow the rate of bone loss. However, treatments for arthritis primarily focus on relieving joint burden and pain, but are unable to restore previous bone conditions and joint mobility.

[0005] Therefore, bone health has gradually gained public attention in recent years. In addition to strengthening bones and maintaining joint flexibility, bone health also includes muscles that assist in bone and joint movement. To improve the above problems, technicians in this field urgently need to develop functional foods that can improve the above problems to benefit the vast population with such needs.

[0006] Invention Disclosure

[0007] In some embodiments, a mangosteen shell extract is used to prepare a bone health composition, wherein the mangosteen shell extract is prepared by extracting the fruit shell of Garcinia mangostana with an enzyme solution in two stages at different extraction temperatures.

[0008] In some embodiments, the extraction temperatures of the two-stage extraction are 45° C. to 65° C. and 75° C. to 95° C., respectively.

[0009] In some embodiments, the extraction time of each stage in the two-stage extraction is 60 minutes to 90 minutes.

[0010] In some embodiments, the aforementioned two-stage extraction step includes: performing a primary extraction on the mangosteen fruit shell with an enzyme solution at 45°C to 65°C for 60 minutes to obtain a primary extract, performing a secondary extraction on the primary extract at 75°C to 95°C for 60 to 90 minutes to obtain a secondary extract, and filtering and concentrating the secondary extract to obtain a mangosteen shell extract.

[0011] In some embodiments, the extraction temperature of the aforementioned primary extraction is 55±5°C, and the extraction temperature of the previous secondary extraction is 85±5°C.

[0012] In some embodiments, the enzyme solution is an aqueous solution containing 0.1 wt % to 1 wt % of saccharifying enzyme.

[0013] In some embodiments, the aforementioned saccharifying enzyme is glucoamylase, α-amylase, β-amylase, isoamylase, or a combination thereof.

[0014] In some embodiments, the mangosteen shell extract has a total flavonoid content of at least 19000 ± 5000 ppm.

[0015] In some embodiments, the bone health care mentioned above is strengthening bones, preventing joint wear and / or preventing bone loss.

[0016] In some embodiments, the mangosteen shell extract strengthens bones by inhibiting osteophage differentiation, increasing bone density, promoting intestinal calcium absorption, and promoting osteocalcin secretion.

[0017] In some embodiments, the mangosteen shell extract has at least one of the functions of promoting polyglucosamine expression and reducing inflammation to prevent joint wear.

[0018] In some embodiments, the mangosteen shell extract has at least one of the functions of inhibiting osteophage differentiation and promoting osteocalcin secretion to prevent bone loss.

[0019] In some embodiments, the mangosteen shell extract has the function of alleviating inflammation in the body.

[0020] In summary, the mangosteen shell extract of any embodiment has bone health benefits. Furthermore, the mangosteen shell extract is produced through a two-stage extraction process at different extraction temperatures to fully release the active ingredients in the mangosteen shell and increase the active ingredient content in the mangosteen shell extract. In some embodiments, the mangosteen shell extract has benefits such as strengthening bones, preventing joint wear, alleviating bone loss, improving calcium absorption, alleviating inflammation in the body, and preventing inflammation caused by joint wear.

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but this does not limit the present invention.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a preparation process of the mangosteen shell extract of any embodiment;

[0024] Figure 2 is a photo of the experimental group and the control group showing the inhibition of osteophage differentiation;

[0025] FIG3 is a photo of the polyglucosamine secretion experiment of the experimental group and the control group;

[0026] Figure 4 is a photo of the calcium absorption experiment of the experimental group and the control group;

[0027] FIG5 is a graph showing the results of a calcium absorption experiment;

[0028] FIG6 is a graph showing the average detection data of osteocalcin secretion of subjects at week 0 and week 12;

[0029] FIG7 is a graph showing the average detection data of in vivo inflammatory indicators of subjects at week 0 and week 12;

[0030] FIG8 is a graph showing the average bone density test data of subjects at week 0, week 8, and week 12; and

[0031] FIG9 is a graph showing the average bone density test data of subjects with low bone density at week 0, week 8, and week 12.

[0032] Wherein, reference numerals: S100~S400: steps

[0033] Best Mode for Carrying Out the Invention

[0034] The following describes some specific implementations of this invention. Without departing from the spirit of this invention, this invention may be implemented in a variety of different forms, and the scope of protection should not be limited to the conditions specifically stated in the specification.

[0035] Mangosteen (Garcinia mangostana), also known as mangosteen, mangosteen, mountain bamboo, mangosteen fruit, and phoenix fruit, is a species of the genus Garcinia in the family Garciniaceae. Mangosteen is a tropical evergreen tree native to the Sunda Islands and the Moluccas in the Malay Archipelago. When ripe, the mangosteen fruit is spherical, 4 to 8 cm in diameter, with a smooth skin, fleshy sepals, and remnants of the stigma within the outer pericarp. The peel of the mangosteen fruit is dark and bitter, and is inedible. The flesh is white and garlic-like, with a sweet and mellow aroma and taste.

[0036] Mangosteen extract is obtained by mixing the husk of the fruit of Garcinia mangostana (hereinafter referred to as mangosteen husk) with an extraction solvent in a certain proportion and subjecting it to two extraction stages at different extraction temperatures for a certain period of time. In some embodiments, the mangosteen husk used may be the husk of mature mangosteen (Garcinia mangostana).

[0037] In some embodiments, see Figure 1. First, the obtained mangosteen shells are coarsely crushed into mangosteen shells to obtain mangosteen shell fragments or particles (hereinafter referred to as mangosteen shell raw material) (step S100). In some embodiments, the mangosteen shell raw material is mangosteen shells that have been cut and coarsely crushed into mangosteen shell fragments approximately 12 mm in size. For example, the mangosteen shells are chopped and then passed through a sieve with a pore size of 12 mm to obtain the mangosteen shell raw material for extraction. Using a sieve with a specific pore size ensures that the mangosteen shell fragments meet the required size, thereby increasing the contact area with the solvent in the subsequent extraction step.

[0038] After step S100, the mangosteen shell raw material is then mixed with an enzyme solution to perform a primary extraction on the mangosteen shell raw material using the enzyme solution to obtain a primary extract (i.e., step S200). The enzyme solution is an aqueous solution containing 0.1% to 1% (e.g., 0.5% by weight) of a saccharifying enzyme. For example, the enzyme solution is a saccharifying enzyme dissolved in RO water. The saccharifying enzyme may be, but is not limited to, glucoamylase, α-amylase, β-amylase, isoamylase, or a combination thereof. In some embodiments, the weight ratio of the mangosteen shell raw material to the enzyme solution is 0.2-5:1-10 (e.g., 1:4). In some embodiments, the extraction temperature for the primary extraction is 45°C to 65°C (e.g., 55±5°C), and the extraction time for the primary extraction is 60 minutes to 90 minutes. For example, after the temperature of the enzyme solution is raised to the extraction temperature for the primary extraction, the mangosteen shell raw material is added for the primary extraction to obtain the primary extract.

[0039] After step S200, the primary extract is subsequently subjected to a secondary extraction to obtain a secondary extract (i.e., step S300). In some embodiments, the extraction temperature of the secondary extraction is 75°C to 95°C (e.g., 85±5°C), and the extraction time of the secondary extraction is 60 minutes to 90 minutes. For example, after the temperature of the primary extract is raised to the extraction temperature of the secondary extraction, a secondary extraction is performed to obtain a secondary extract. In some embodiments, the Brix sweetness (Degrees Brix, °Bx) of the secondary extract obtained after the secondary extraction is greater than 1.3°Bx (tested at 20°C). In some examples, the temperature of the primary extract is raised to 85±5°C and then extracted for 60 minutes, and the Brix sweetness of the secondary extract is tested at 60 minutes to see if it meets the specification (i.e., whether it is greater than 1.3). If it does not meet the specification, it can be extracted for another 30 minutes and confirmed again.

[0040] In this regard, a specific ratio of extraction solvent to extractant, a specific size of the extractant (e.g., crushed fruit shells), specific extraction temperatures and times at specific stages, or any combination thereof, can significantly improve extraction efficiency. Furthermore, a specific extraction temperature can more completely release the active ingredients in the raw material, while a specific extraction time can prevent the degradation of the active ingredients in the extract caused by prolonged extraction.

[0041] In some embodiments, after step S300, the secondary extract is filtered and concentrated to obtain a mangosteen shell extract (i.e., step S400). In some embodiments, the filtering step refers to filtering the secondary extract with a filter of a specific pore size to filter out the mangosteen shell fragments after extraction. In some embodiments, the concentration is to use steam at a specific temperature and pressure to reduce the pressure and concentrate the filtered secondary extract, and to confirm whether the mangosteen shell extract is obtained by testing whether the concentrated Brix sweetness meets the specification. In some embodiments, the Brix sweetness of the mangosteen shell extract is 6.0±0.5°Bx (tested at 20°C). In some examples, after the secondary extract is filtered with a filter of 400 mesh, it is concentrated at 55°C to 65°C (for example, 60±5°C) at 1±0.2kg / cm 2 The filtered secondary extract is concentrated by steam until its Brix sweetness reaches 6.0±0.5°Bx (measured at 20°C), and then the concentration is stopped to obtain the mangosteen shell extract.

[0042] In some embodiments, the mangosteen shell extract has a total flavonoid content of at least 19000 ± 5000 ppm.

[0043] In some embodiments, mangosteen shell extract has bone health benefits. For example, mangosteen shell extract can strengthen bones, prevent joint wear and / or alleviate bone loss.

[0044] In some embodiments, in addition to strengthening bones, preventing joint wear and / or preventing bone loss, the mangosteen shell extract can also increase joint mobility and assist muscles in bone and joint movement.

[0045] In some embodiments, the mangosteen shell extract has at least one of the functions of inhibiting osteoclast differentiation, increasing bone density, promoting polyglucosamine expression, promoting intestinal calcium absorption, promoting osteocalcin secretion, and reducing inflammation.

[0046] It should be understood that the recipients mentioned below refer to subjects to whom the mangosteen shell extract is administered invasively (e.g., by injection) or non-invasively (e.g., by consumption). In some embodiments, the recipient is a human.

[0047] In some embodiments, mangosteen shell extract can effectively inhibit the differentiation of osteophagocytes in the recipient's body and reduce the number of mature osteophagocytes, thereby strengthening bone and increasing bone density. In some embodiments, mangosteen shell extract can increase bone density to strengthen bones.

[0048] In some embodiments, mangosteen shell extract can increase the intestinal absorption rate of calcium in the recipient's body (e.g., by up to 10%), thereby strengthening bones and bone mass. Furthermore, it can prevent or reduce the chance of the body absorbing calcium from bones when calcium intake is insufficient.

[0049] In some embodiments, mangosteen shell extract can promote osteocalcin secretion, thereby increasing blood osteocalcin concentrations and supporting bone formation. Specifically, osteocalcin is primarily produced by osteoblasts, and its content is positively correlated with bone formation. Generally speaking, higher osteocalcin concentrations indicate better bone formation. Therefore, mangosteen shell extract promotes osteocalcin secretion in receptors, thereby supporting bone formation in these receptors.

[0050] For example, the mangosteen shell extract can strengthen bones by inhibiting osteoclast differentiation, increasing bone density, promoting intestinal calcium absorption, and promoting osteocalcin secretion. For example, the mangosteen shell extract can alleviate bone loss by inhibiting osteoclast differentiation and promoting osteocalcin secretion.

[0051] In some embodiments, the mangosteen shell extract can promote the expression of polyglucosamine in the recipient body to reduce cartilage wear and tear and prevent joint wear.

[0052] In some embodiments, mangosteen shell extract can help relieve inflammation in the body. For example, when a recipient takes mangosteen shell extract, the level of high-sensitivity C-reactive protein (hs-CRP) in the body is reduced. hs-CRP is a special protein produced by liver cells. When the body is injured, infected, or inflamed, its concentration in the blood increases and is one of the indicators of inflammation. When joint cartilage loss causes bone friction and inflammation, taking mangosteen shell extract can relieve inflammation in the body, thereby reducing joint pain. In addition, studies have found that during the process of bone metabolic conversion, inflammatory reactions have a significant impact on bone physiology and bone remodeling, thereby inducing osteoporosis. Therefore, mangosteen shell extract relieves inflammation in the body, thereby reducing the possibility of inducing osteoporosis.

[0053] For example, the mangosteen shell extract can prevent joint wear by promoting the expression of polyglucosamine and reducing inflammation.

[0054] In some embodiments, the mangosteen shell extract can be manufactured into a form suitable for enteral or oral administration using techniques well known to those skilled in the art.

[0055] In some embodiments, enteral or oral administration may be, but is not limited to, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or the like.

[0056] In some embodiments, any of the aforementioned compositions may be an edible product. In other words, the edible product comprises a specific amount of mangosteen shell extract. In some embodiments, the edible product may be a general food, a health food, or a dietary supplement.

[0057] In some embodiments, the aforementioned edible products can be manufactured into dosage forms suitable for oral administration using techniques well known to those skilled in the art. In some embodiments, the aforementioned general food can be the edible product itself. In some embodiments, the general food can be, but is not limited to, beverages, fermented foods, bakery products, or seasonings.

[0058] In some embodiments, the resulting mangosteen shell extract can be further used as a food additive to produce a food composition containing the mangosteen shell extract. In this case, the mangosteen shell extract of any embodiment can be added during raw material preparation or during food production using conventional methods, and then formulated with any edible material to form an edible product (i.e., a food composition) for consumption by humans and non-human animals.

[0059] In some embodiments, the aforementioned composition may be in liquid or solid form (eg, powder packets or tablets).

[0060] In some embodiments, the dosage of the aforementioned composition is 2 g / day of liquid mangosteen shell extract.

[0061] In some embodiments, the usage amount of the aforementioned composition is 0.2 g / day of solid mangosteen shell extract.

[0062] Example 1: Preparation of mangosteen shell extract

[0063] The mangosteen shells were roughly crushed into pieces of about 12 mm in size and then mixed with enzyme solution at a weight ratio of 1:4 for the subsequent two-stage extraction step. The enzyme solution was an aqueous solution containing 0.5% by weight of glucoamylase (purchased from TRUMP Chemical Corp.) dissolved in RO water. After the enzyme solution was heated to 55±5°C, the mangosteen shells were added to the enzyme solution at a temperature of 3.0±0.5 kg / cm 2 Extract for 60 minutes using a reflux pump to obtain a primary extract. Next, raise the extraction temperature to 85±5°C and subject the primary extract to a secondary extraction for 60 minutes to obtain a secondary extract. Confirm that the Brix sweetness of the secondary extract is greater than 1.3°Bx (measured at 20°C). If this does not meet the requirement, extract for an additional 30 minutes until the Brix sweetness meets the requirement.

[0064] The secondary extract, which has been confirmed to have reached the required Brix sweetness, was filtered through a 400-mesh filter to remove the mangosteen shell material and then heated at 60±5℃ at 1±0.2kg / cm 2 The filtered secondary extract is concentrated by steam until its Brix sweetness reaches 6.0±0.5°Bx (measured at 20°C), and then the concentration is stopped to obtain the mangosteen shell extract.

[0065] Example 2: Osteophage differentiation experiment

[0066] Osteophages are multinuclear cells formed by the fusion and differentiation of multiple mononuclear cells. Their primary function is to degrade and destroy bone tissue, and they are closely linked to osteoporosis. Therefore, by observing the ability of mangosteen shell extract to inhibit the differentiation of mononuclear cells into osteophages, we can infer its efficacy in improving osteoporosis.

[0067] The cells used here were peripheral blood mononuclear cells (PBMCs), obtained by centrifugation of peripheral blood from healthy donors. The cell culture medium used was α-MEM medium (Gibco; Cat. 12000-022) supplemented with 10% fetal bovine serum (Gibco; Cat. 10437-028) and 1X antibiotics (Gibco; Cat. 15240-062). The osteophage differentiation medium used was α-MEM medium (Gibco; Cat. 12000-022) containing 10% fetal bovine serum (Gibco; Cat. 10437-028), 1X antibiotics (Gibco; Cat. 15240-062), 40 ng / mL human RANKL (Peprotech; Cat. 310-01,) and 25 ng / mL human M-CSF (Peprotech; Cat. 300-25).

[0068] First, PBMC cells were cultured at 1×10 6 The cells were seeded at a density of 10 cells / well in a 24-well cell culture dish containing 500 μL of cell culture medium per well and cultured in a carbon dioxide incubator for 24 hours.

[0069] The cultured PBMCs were divided into a control group and an experimental group. The experimental culture medium was replaced with the corresponding experimental culture medium according to the group and cultured for 14 to 21 days, with the experimental culture medium replaced every three days. The experiment was terminated by observing osteophagocyte differentiation in the control group using a microscope (ZEISS; Cat. Vert. A1). The experimental culture medium for the control group consisted of osteophagocyte differentiation medium, while the experimental culture medium for the experimental group consisted of osteophagocyte differentiation medium supplemented with 0.03125 mg / mL of the mangosteen shell extract prepared in Example 1.

[0070] Add 1 drop of ActinRed TM 555 ReadyProbes TMReagent (Thermo; Cat. R37112) and Hoechst 33342 stain (Hoechst 33342; purchased from Thermo; Cat. 62249; 1:20,000 dilution) were added to the control and experimental groups in the dark for 15 minutes to stain myocytes and osteoblasts. Subsequently, the morphology and nuclei of osteophages were observed and photographed under a fluorescence microscope (inverted microscope with camera system; ZEISS; Cat. Vert. A1) in the dark to assess the sample's ability to inhibit osteophage differentiation. The photographic results are shown in Figure 2.

[0071] See Figure 2. The left image shows a fluorescence photograph of the control group, while the right image shows a fluorescence photograph of the experimental group. The blue circles represent cell nuclei, the orange represents actin, and the red arrows indicate osteophages. When PBMCs are undifferentiated, they are small, and actin is expressed throughout the cell, resulting in punctate fluorescence after staining, as shown in the experimental group on the right in Figure 2. However, when PBMCs are stimulated with osteoclast differentiation factors, they differentiate and fuse into larger, multinucleated osteophages. Actin forms distinct rings around the cell edges, resulting in visible rings of fluorescence around the cell edges after staining, as shown in the control group on the left in Figure 2. Therefore, Figure 2 shows that mangosteen shell extract can inhibit PBMCs from being stimulated by osteoclast differentiation factors, thereby inhibiting osteophage differentiation, reducing bone metabolism, strengthening bones, and preventing bone loss.

[0072] Example 3: Polyglucosamine secretion experiment

[0073] Here, the cells used were ATDC5 cells ( CRL-2846 TM) are chondrogenic cells. The cell culture medium used was DMEM:Ham's F12 (1:1) medium (purchased from Gibco) supplemented with 2 mM L-glutamine (purchased from Gibco), 5% fetal bovine serum (FBS, purchased from Gibco), and 1% penicillin / streptomycin combination antibiotics (purchased from Gibco). The chondrocyte differentiation medium used was DMEM:Ham's F12 (1:1) medium (purchased from Gibco) containing 1% insulin-transferrin-selenium (purchased from Gibco), 2 mM L-glutamine (purchased from Gibco), 5% fetal bovine serum (FBS, purchased from Gibco), and 1% penicillin / streptomycin antibiotic combination (purchased from Gibco).

[0074] First, 1×10 5 ATDC5 cells were cultured in a 6-well culture plate with 2 mL of cell culture medium per well and cultured in a 37°C constant temperature incubator for 24 hours, and then the cell culture medium was replaced every three days until the cells were confluent.

[0075] ATDC5 cells were divided into a control group and an experimental group. The cell culture medium was replaced with the experimental medium and cultured for 35 days, with the experimental medium replaced every three days. The experimental medium for the control group consisted of chondrocyte differentiation medium, while the experimental group consisted of chondrocyte differentiation medium supplemented with 0.03125 mg / mL of the mangosteen shell extract prepared in Example 1.

[0076] After differentiation, remove the experimental culture medium from both groups and rinse the cells three times with 1X PBS. Add 0.5 mL of 10% formaldehyde (Echo Chemical, Taiwan, Cat. TG1794-4-0000-72NI) and fix the cells at room temperature for 30 minutes. Then, remove the formaldehyde and rinse the cells three times with 1X PBS.

[0077] Next, 0.5 mL of 1% Alcian blue solution (pH 2.5; prepared with 0.1 M HCl) was added to each well of the culture plate and stained overnight at room temperature. After staining, the plates were rinsed three times with 1X PBS and observed under a ZEISS microscope to detect the blue signal produced by Alcian blue staining of glycosaminoglycans. The results are shown in Figure 3.

[0078] See Figure 3. The left side shows a photograph of the control group, while the right side shows a photograph of the experimental group. The blue signal between the background cells is polyglucosamine, and the red arrows indicate chondrocytes differentiated from ATDC5 cells. After chondrogenic cells differentiate into mature chondrocytes, the chondrocytes have the ability to produce polyglucosamine. Therefore, by comparing the blue background signals in the experimental and control groups in Figure 3, it can be seen that the addition of mangosteen shell extract promotes the differentiation of ATDC5 cells into chondrocytes and increases the production of polyglucosamine. Polyglucosamine is a major component of cartilage and synovial fluid. Aging or overuse of joints can lead to cartilage wear and insufficient synovial fluid, which in turn narrows the distance between joints. Furthermore, accumulated wear and tear can cause joint friction, which in severe cases can lead to joint inflammation, swelling, and pain. Because polyglucosamine is associated with joint health, consuming mangosteen shell extract can increase the production of polyglucosamine in the receptors, improving joint discomfort, preventing joint wear and pain, joint degeneration, and reducing the incidence of arthritis.

[0079] Example 4: Calcium absorption experiment

[0080] The cells used here were human intestinal cells (hereinafter referred to as C2BBe1 cells; ATCC CRL-2102), which are chondrogenic cells. The cell culture medium used was Dulbecco's Modified Eagle Medium (purchased from Gibco, Cat. 12100-038) supplemented with 10% fetal bovine serum (FBS, purchased from Gibco, Cat. 10438-026) and 1% penicillin / streptomycin antibiotic combination (purchased from Gibco, Cat. 15140-122).

[0081] First, 2×10 5 C2BBe1 cells were seeded into a 6-well culture plate with 2 mL of cell culture medium per well and cultured in a 37°C constant temperature incubator for 24 hours.

[0082] The cultured C2BBe1 cells were divided into a control group and an experimental group. The cell culture medium of each group was replaced with the experimental medium and cultured for 1 hour. The experimental medium of the control group was cell culture medium, while the experimental medium of the experimental group was cell culture medium supplemented with 0.0625 mg / mL of the mangosteen shell extract prepared in Example 1.

[0083] 2 μM Fluo-4 AM calcium indicator (purchased from Invitrogen) was added to each group and incubated at room temperature for 45 minutes. After washing once with 1X PBS, cell culture medium was added and the fluorescence of C2BBe1 cells in both groups was observed using a fluorescence microscope (Zeiss). Intensified fluorescence in C2BBe1 cells indicates a higher level of calcium ion in the cells. The results are shown in Figure 4.

[0084] See Figure 4. The left side of Figure 4 shows a fluorescence image of the control group, while the right side shows a fluorescence image of the experimental group. Here, green fluorescence represents calcium ions. Therefore, Figure 4 demonstrates that mangosteen shell extract can increase the calcium indicator level within intestinal cells.

[0085] Each C2BBe1 cell group was then trypsinized to harvest the cells. The pellets were then centrifuged at 300 x g for 5 minutes to collect the C2BBe1 cell pellets. The pellets were then resuspended in 500 μL of PBS containing 2% FBS to obtain the test samples. Each sample was then treated with Fluo-4 AM calcium indicator (purchased from Invitrogen) and the absorbance at excitation / scattering (Ex / Em) wavelengths of 494 / 506 nm was measured using a flow cytometer (BD Accuri C6 Plus) to measure intestinal calcium absorption. The control group values ​​were considered 100% relative calcium absorption, and relative calcium absorption was then estimated for the experimental groups. The results are shown in Figure 5. * indicates a p-value less than 0.05.

[0086] See Figure 5. The control group's relative calcium absorption was 100%, while the experimental group's was 110.4%. Furthermore, the experimental group's calcium absorption increased by approximately 10% compared to the control group. This indicates that mangosteen shell extract can increase relative calcium absorption within C2BBe1 cells. In other words, when recipients consume mangosteen shell extract, their intestinal cells absorb more calcium, which helps maintain normal blood calcium levels by preventing calcium from being absorbed from the bones. It also strengthens bone and bones, and prevents bone loss.

[0087] Example 5: Human trials

[0088] In this study, a self-controlled study was conducted to compare the bone health benefits of eight subjects before and after taking a composition containing mangosteen shell extract. The composition used was a 50mL bottle of mangosteen shell extract drink, containing 2 grams of mangosteen shell extract and 48 grams of water.

[0089] Testing Method: Eight subjects took one bottle of mangosteen shell extract daily for 12 weeks. Bone density was measured using dual-energy X-ray absorptiometry (DXA) at Cathay Health Checkup Center before (week 0), after 8 weeks (week 8), and after 12 weeks (week 12). Fasting blood samples were collected before (week 0) and after 12 weeks (week 12) and tested at Liren Medical Laboratory. Eight subjects were aged 25 to 55 and were frequent coffee and / or carbonated beverage consumers.

[0090] Blood test items: Blood bone formation marker (Liren Medical Laboratory, note HB0699) and blood inflammation marker (Liren Medical Laboratory, note HE0106). The blood bone formation marker refers to the level of osteocalcin; the blood inflammation marker refers to the level of high-sensitivity C-reactive protein (hs-CRP).

[0091] Bone density testing: This instrument measures bone density and displays a "T score." The "T score" is used as an indicator of bone density, with a normal T score between -1 and 1. A T score less than -1 indicates low bone mass, while a T score of -2.5 or less indicates osteoporosis.

[0092] Example 5-1: Blood osteocalcin test

[0093] Please see Figure 6. The average osteocalcin concentration in the blood of the eight subjects was 19.1 ng / mL at Week 0. After taking the mangosteen shell extract drink containing mangosteen shell extract for 12 weeks, the average osteocalcin concentration in the blood increased to 19.6 ng / mL. This means that after taking the mangosteen shell extract for 12 consecutive weeks, the average osteocalcin concentration in the blood of the eight subjects increased by 2.6%, and the improvement rate reached 62.5% (i.e., 5 subjects).

[0094] It can be seen that taking mangosteen shell extract can increase the concentration of osteocalcin in the blood, thereby helping bone formation, increasing bone density, strengthening bones and skeleton, and preventing bone loss.

[0095] Example 5-2: Detection of inflammatory markers in blood

[0096] Please see Figure 7. The average hs-CRP level in the blood of the eight subjects was 0.158 mg / dL at Week 0. After taking the mangosteen shell extract drink containing mangosteen shell extract for 12 weeks, the average hs-CRP level in the blood decreased to 0.048 mg / dL. This means that after taking the mangosteen shell extract for 12 consecutive weeks, the average hs-CRP level in the blood of the eight subjects decreased by 69.6%, and the improvement rate reached 75% (i.e., 6 subjects).

[0097] Therefore, it can be seen that taking mangosteen shell extract can relieve inflammation in the body and prevent inflammation caused by joint wear or prevent joint pain caused by inflammation caused by joint cartilage loss.

[0098] Example 5-3: Bone density test

[0099] See Figure 8. The average T score for the eight subjects was -0.16 at week 0, -0.10 at week 8, and 0.06 at week 12. This indicates that the bone density of these eight subjects at week 0 was between -1 and 1, placing them within the normal bone density range. After 12 consecutive weeks of taking mangosteen shell extract, the T scores of all eight subjects increased by 0.22, indicating an average improvement in bone mass, and therefore bone density. In other words, taking mangosteen shell extract can help increase bone density, prevent bone loss, and strengthen bones.

[0100] Four of the eight subjects had a T score less than 0.0 at week 0, indicating low bone density. The test results for these four subjects are summarized in Figure 9. See Figure 9. At week 0, the average T score for these four subjects was -0.95, which is between the normal bone density value of -1 and 1, approaching low bone density. At week 8, the average T score was -0.9, and at week 12, the average T score was -0.7. This indicates that after 12 consecutive weeks of taking mangosteen shell extract, the T scores of the four subjects increased by 0.25, indicating an average improvement in bone mass, and therefore bone density. In other words, taking mangosteen shell extract can help increase bone density and strengthen bones.

[0101] As shown above, continuous consumption of mangosteen shell extract for 12 weeks increased blood osteocalcin levels, alleviated inflammation in the subjects, and improved bone density. This demonstrates that mangosteen shell extract has the function of reducing inflammation and maintaining bone health.

[0102] In summary, the mangosteen shell extract according to any embodiment of the present invention is produced through a two-stage extraction process at different extraction temperatures and exhibits bone health benefits. This two-stage extraction process at different extraction temperatures fully releases and retains the active ingredients in the mangosteen shell, thereby increasing the active ingredient content in the mangosteen shell extract. Furthermore, the mangosteen shell extract exhibits benefits such as strengthening bones, preventing joint wear and bone loss, increasing bone density, enhancing calcium absorption, alleviating inflammation in the body, and preventing inflammation caused by joint wear.

[0103] Although the technical contents of the present invention have been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Any slight changes and modifications made by any person skilled in the art without departing from the spirit of the present invention should be included in the scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended claims.

[0104] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims of the present invention.

[0105] Industrial Applicability

[0106] The invention discloses a use of a mangosteen shell extract for preparing a bone health care composition, wherein the mangosteen shell extract is prepared by subjecting the fruit shell of Garcinia mangostana to two-stage extraction at different extraction temperatures in sequence using an enzyme solution.

Claims

DEPCT691. The use of mangosteen peel extract for the preparation of components for improving bone health, where the mangosteen peel extract was prepared by a two-step extraction process with mangosteen peel (Garcinia mangostana) using enzymatic solutions at different extraction temperatures, respectively.

2. The use according to claim 1, where the mangosteen peel extract contains at least 19000 ± 5000 ppm of total flavonoids.

3. The use according to claim 1, where the enzymatic solution is an aqueous solution.

3. Use according to claim 3, where the diastase is glucoamylase, alpha-amylase, beta-amylase, isoamylase, or a combination of any of those enzymes.

4. Use according to claim 1, where the extraction temperature for two-stage extraction is 45°C to 65°C and 75°C to 95°C, respectively.

5. Use according to claim 5, where the extraction temperature for two-stage extraction is 55±5°C and 85±5°C, respectively.

6. Use according to claim 5, where the extraction temperature for two-stage extraction is 55±5°C and 85±5°C, respectively. 7.

8. The use under claim 1 where the extraction time in each step of the two-step extraction process is 60 to 90 minutes.

9. The use under claim 1 where the two-step extraction process consists of the following steps: primary extraction of mangosteen peel with an enzymatic solution at 45°C to 65°C for 60 minutes to obtain the primary extract; secondary extraction of the primary extract at 75°C to 95°C for 60 to 90 minutes to obtain the secondary extract; and filtration and concentration of the secondary extract to obtain the mangosteen peel extract.

10. The use under claim 1 where bone health is defined as strengthening bones, preventing wear and tear of joints, and / or preventing bone mass loss.

11. The use under claim 1 where the mangosteen peel extract has at least one of the following functions: inhibition of osteoclast deformation, increasing bone density, promoting intestinal calcium absorption, promoting osteocalcin secretion, and anti-inflammatory effects to strengthen bones.Claim 1 states that mangosteen peel extract is used for at least one of the following functions: promoting polyglucosamine expression and reducing inflammation to prevent joint wear and tear.

12. Claim 1 states that mangosteen peel extract is used for at least one of the following functions: inhibiting osteoclast transformation and promoting osteocalcin secretion to prevent bone loss.

13. Claim 1 states that mangosteen peel extract has anti-inflammatory properties in subjects requiring such substances.

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