Application of giant bamboo shoot and melon juice in preparation of joint health care composition

Abstract

The invention relates to application of giant bamboo shoot and melon juice. The giant bamboo shoot and melon juice is used for preparing a joint health-care composition. Wherein the giant bamboo shoot melon juice is obtained by extracting giant bamboo shoot melon (Cucurbita maxima) by using water as a solvent, and the giant bamboo shoot melon juice is obtained by extracting the giant bamboo shoot melon (Cucurbita maxima) by using water as a solvent.

Description

TECHNICAL FIELD

[0001] The present invention relates to the application of giant squash, in particular, the use of giant squash juice for preparing joint health composition. BACKGROUND

[0002] Giant squash is a special variety from Alaska, from the Matanuska Valley in the northeast of Alaska Angola, which is also known as the "giant vegetable valley" because it has grown many giant varieties. Giant squash is different from ordinary squash and is usually used for ornamental rather than food. SUMMARY

[0003] In view of this, in order to develop more applications of giant squash, the use of giant squash juice for preparing joint health composition is proposed.

[0004] In some embodiments, the use of giant squash juice is for preparing joint health composition. The giant squash juice is extracted from giant squash (Cucurbita maxima) with water as the solvent.

[0005] In some embodiments, the giant squash juice is used to increase the bone quality of the joint to improve the support of the joint.

[0006] In some embodiments, the giant squash juice is used to increase the amount of osteocalcin secreted by bone cells.

[0007] In some embodiments, the giant squash juice is used to slow down bone loss.

[0008] In some embodiments, the giant squash juice reduces the concentration of beta-crosslaps (β-CTx) in serum.

[0009] In some embodiments, the giant squash juice is used to improve the mitochondrial activity of bone cells.

[0010] In some embodiments, the giant squash juice is used to improve bone density. In some embodiments, the giant squash juice is used to improve T-score or Z-score.

[0011] In some embodiments, the giant squash juice is used to inhibit the inflammatory response of the cartilage tissue of the joint. In some embodiments, the giant squash juice is used to improve the decrease in collagen content caused by the inflammatory response. In some embodiments, the giant squash juice is used to improve the decrease in hyaluronic acid content caused by the inflammatory response.

[0012] In some embodiments, the giant squash juice is used to improve the degree of joint stiffness. In some embodiments, the giant squash juice is used to improve the WOMAC score. In some embodiments, the giant squash juice is used to improve joint degeneration.

[0013] In summary, the giant squash juice of any of the embodiments can be used to prepare a composition for joint health. In some embodiments, the giant squash juice has at least one of the following effects: increasing bone mass, increasing osteocalcin secretion from bone cells, reducing bone loss, increasing mitochondrial activity of bone cells, increasing bone density, increasing T-score or Z-score, inhibiting the inflammatory response of cartilage tissue of the joint, improving the decrease in collagen content caused by the inflammatory response, improving the decrease in hyaluronic acid content caused by the inflammatory response, and reducing the concentration of beta-crosslaps (β-CTx) in serum, improving joint flexibility, and improving joint degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 is a graph of the results of testing the giant squash juice of one embodiment to increase the osteocalcin secretion from bone cells.

[0015] Figure 2 Figure 2 is a graph of the results of testing the giant squash juice of one embodiment to increase the mitochondrial activity of bone cells.

[0016] Figure 3 Figure 3 is a graph of the results of testing the giant squash juice of one embodiment to increase collagen content.

[0017] Figure 4 Figure 4 is a graph of the results of testing the giant squash juice of one embodiment to increase hyaluronic acid content.

[0018] Figure 5 Figure 5 is a graph of the average T-score results of a human experiment.

[0019] Figure 6 Figure 6 is a graph of the average Z-score results of a human experiment.

[0020] Figure 7 Figure 7 is a graph of the concentration of beta-crosslaps (β-CTx) in serum of a human experiment.

[0021] Figure 8 Figure 8 is a graph of the WOMAC score results of a human experiment.

[0022] Figure 9 Figure 9 is a graph of the degree of joint stiffness results of a human experiment.

[0023] Figure 10 Figure 10 is a graph of the overall satisfaction statistics results of a human experiment. DETAILED DESCRIPTION

[0024] As used herein, the term "juice" refers to a product prepared by extraction. Juice may be in the form of a solution dissolved in a solvent, or it may be a concentrate or essence containing little or no solvent.

[0025] As used in this article, "giant cucumber" refers to the complete fruit of the giant cucumber plant (scientific name: Cucurbita maxima). The complete fruit includes the pericarp, pulp, seeds (also called cucumber seeds), and flesh (also called cucumber flesh).

[0026] In some embodiments, the giant squash juice is obtained by extracting giant squashes with water as a solvent.

[0027] In some embodiments, the giant bamboo shoots used for extraction may be fresh giant bamboo shoots, dried giant bamboo shoots, or frozen giant bamboo shoots. In some embodiments, drying may be air-drying, sun-drying, shade-drying, or freeze-drying.

[0028] In some embodiments, the giant cucumbers used for extraction may be whole or giant cucumbers that have undergone physical processing procedures such as chopping, dicing, slicing, grinding, or other methods that alter the size and integrity of the original material.

[0029] In some embodiments, the giant cucumber juice may be a primary extract of giant cucumber formed after a water extraction step. In some embodiments, the water extraction step may include a soaking process. In the soaking process, the giant cucumber is soaked in water at a specific temperature for a specific time to release the active ingredients in the giant cucumber into the water to form the primary extract of giant cucumber.

[0030] In some embodiments, in addition to the soaking process, the water extraction step may also include a verification process and / or a re-soaking process. The verification process determines when to complete the soaking process by measuring the Brix (°Bx) value of the initial extract of giant cucumber obtained from the soaking process. When the Brix value reaches 0.5 ± 0.2°Bx, it indicates that the initial extract of giant cucumber has been obtained. If the measured Brix value is less than 0.3°Bx, the re-soaking process is then performed. The re-soaking process extends the specific time by another 30 minutes. That is, the initial extract of giant cucumber is accepted based on a Brix value of 0.5 ± 0.2°Bx.

[0031] In some embodiments, the water extraction step is performed at a temperature of 90 ± 5 °C. In some embodiments, the water extraction step is performed at a temperature of 90 °C. In some embodiments, the water extraction step is performed for a period of time of between 100 minutes and 140 minutes. In some embodiments, the water extraction step is performed for a period of time of 120 minutes.

[0032] In some embodiments, the giant squash juice can be a giant squash filtrate formed after the giant squash is sequentially subjected to a water extraction step and a filtration step. The filtration step is performed on the giant squash initial extract formed in the water extraction step to remove solid materials in the giant squash initial extract by using a sieve or centrifugation, for example. For example, the giant squash initial extract is filtered by using a 400 mesh sieve to obtain the giant squash filtrate.

[0033] In some embodiments, the giant squash juice can be a giant squash concentrate formed after the giant squash is sequentially subjected to a water extraction step, a filtration step and a concentration step. The concentration step can be performed before the filtration step, after the filtration step, or both before and after the filtration step. In this regard, the concentration step is performed to remove excess water in the solution formed in the previous step (e.g., the giant squash initial extract formed in the water extraction step, or the giant squash filtrate formed in the filtration step) by using a physical treatment method, so as to reduce the volume of the obtained giant squash juice. For example, the concentration step can be performed by using a concentration machine (brand / model: BUCHI - Rotavapor R-100) at a temperature setting of 60 ± 5 °C and a pressure setting of 1 ± 0.2 kg / cm 2 The concentration is stopped when the solution has a Brix value of 3.0 ± 0.2, to obtain a giant squash concentrate having a Brix value of 3.0 ± 0.2. 2 .

[0034] In some embodiments, the giant squash juice can also be a giant squash concentrate formed after the giant squash is sequentially subjected to a water extraction step and a concentration step (i.e., without being subjected to a filtration step).

[0035] In some embodiments, the giant squash juice has the ability to increase the mass of joint bone. In some embodiments, the giant squash juice has the ability to increase the secretion of osteocalcin by bone cells. Osteocalcin is the most abundant non-collagenous protein in bone, and is mainly produced by bone cells. The secretion of osteocalcin generally represents a pointer of the rate of bone formation and osteoblast activity. Specifically, the giant squash juice can be formulated into a composition for increasing the mass of joint bone or for increasing the secretion of osteocalcin by bone cells.

[0036] In some embodiments, the giant squash juice has the ability to enhance the mitochondrial activity of skeletal cells. Mitochondria are the powerhouses of cells, and play an important role in energy transfer. The higher the activity, the more vigorous the physiological activity of the cell. Therefore, increasing the activity of mitochondria in cells can prevent cell apoptosis and premature aging, and improve muscle endurance. Specifically, the giant squash juice can be made into a composition for enhancing the mitochondrial activity of skeletal cells.

[0037] In some embodiments, the giant squash juice has the ability to enhance bone density. Specifically, the giant squash juice can be made into a composition for enhancing bone density. In some embodiments, the giant squash juice has the ability to enhance T-score or Z-score. T-score or Z-score is a common bone density evaluation index, which is commonly used to diagnose whether there is osteoporosis.

[0038] In some embodiments, the giant squash juice has the ability to slow down bone loss. Specifically, the giant squash juice can be made into a composition for slowing down bone loss. In some embodiments, the giant squash juice can slow down bone loss by reducing the concentration of β-crosslaps (CTx) in serum. β-crosslaps (CTx) is the degradation product of type I collagen cross-linked telopeptide, which reflects the degree of bone resorption. The higher the concentration, the more serious the damage to bone tissue. Specifically, the giant squash juice can be made into a composition for slowing down bone loss.

[0039] In some embodiments, the giant squash juice has the ability to inhibit the inflammatory response of the cartilage tissue of the joint. Joint cartilage is mainly composed of proteoglycans, water, collagen, and hyaluronic acid. Once an inflammatory response occurs, the ability of chondrocytes to secrete collagen and hyaluronic acid will decrease. Specifically, the giant squash juice can be made into a composition for inhibiting the inflammatory response of the cartilage tissue of the joint. In some embodiments, the giant squash juice can improve the decrease in collagen content caused by the inflammatory response. In some embodiments, the giant squash juice can improve the decrease in hyaluronic acid content caused by the inflammatory response.

[0040] As mentioned above, the giant squash juice can improve or prevent osteoporosis by increasing the secretion of osteocalcin or / and increasing bone mass and bone density. Furthermore, the giant squash juice can also prevent osteoporosis by slowing down bone loss. In addition, the giant squash juice can also improve or prevent degenerative arthritis by inhibiting the inflammatory response. Among them, the decrease in joint flexibility, degenerative arthritis and osteoporosis are all unhealthy indicators.

[0041] Degenerative arthritis can be classified mainly according to the location of occurrence. If the degeneration occurs in the knee, it is called knee degenerative arthritis; if it occurs in the hip, it is called hip degenerative arthritis; if it occurs in the cervical spine, it is called cervical degenerative arthritis. The specific manifestations of degenerative arthritis are reduced joint flexibility. The initial degenerative arthritis usually causes inflammation, redness, tightness or mild pain due to the wear of the cartilage on the joint surface. The second stage of degenerative arthritis usually causes stiffness, weakness or moderate pain due to fibrosis of the cartilage on the joint surface. The third stage of degenerative arthritis usually causes inability to stand for a long time, high pain or difficulty walking due to fragmentation of the cartilage on the joint surface. The more serious fourth stage of degenerative arthritis usually causes joint adhesion, deformation, water accumulation and other conditions due to the wear of the cartilage on the joint surface, further causing joint noise or bone spur. In particular, the juice of Cucurbita maxima can be made into a composition for increasing bone mass or / and preventing degenerative arthritis.

[0042] Poor bone mass is specifically manifested in reduced bone density, bone loss, and destruction of bone microstructure, leading to fragile bones and increased risk of fractures. Poor bone mass in the joint causes reduced bone support in the joint, which in turn reduces the support of the joint, causing the cartilage on the joint surface to bear excessive pressure and cause inflammation.

[0043] In some embodiments, the aforementioned composition can be a health product, a food product or a food additive for non-medical purposes. In other words, the health product, food product or food additive contains a specific amount of Cucurbita maxima juice.

[0044] In some embodiments, the aforementioned health product, food product, or food additive can further comprise a food acceptable carrier that is widely used in food manufacturing technology. For example, the food acceptable carrier can comprise one or more of the following agents: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and amount of these agents are within the ordinary skill and routine techniques of those in the art.

[0045] In some embodiments, the aforementioned food acceptable carrier of the health product, food product, or food additive comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and aqueous solution containing alcohol.

[0046] In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements.

[0047] In some embodiments, the composition has a specific usage amount of the giant hyacinth squash juice. That is, the effective usage amount of the giant hyacinth squash juice is 2 grams per day. Specifically, assuming that one serving of the composition is administered per day and the giant hyacinth squash juice is a concentrated powder, the composition contains at least 2 grams of the giant hyacinth squash juice.

[0048] Example 1: Preparation of giant hyacinth squash juice

[0049] First, the raw material for giant squash is made by cutting whole fruits of giant squash produced in Taiwan, China into chunks.

[0050] Next, the water (RO water) is heated to 90±5℃, and then the raw material is soaked for 120 minutes to obtain the initial extract of giant cucumber. The weight ratio of giant cucumber raw material to water is 1:5.

[0051] Subsequently, after the initial extract of the giant cucumber cooled to room temperature, the extract was sieved through a 400-mesh sieve and the filtrate was collected.

[0052] Finally, using a concentrator (brand / model: BUCHI-Rotavapor R-100), the solution was concentrated under reduced pressure at a set temperature of 60±5℃ until the Brix value of the solution was 3.0±0.2. The concentrated liquid obtained is the giant cucumber juice.

[0053] Example 2: Giant Cucumber Juice Test Promotes Osteocalcin Secretion

[0054] Osteocalcin (OC) is the most abundant non-collagenous protein in bone, hence also known as Bone Gla protein. Osteocalcin is considered an indicator of bone formation rate, used to monitor osteogenic function and assess bone replacement rate. Osteocalcin is not only absorbed into the bone matrix but is also secreted into the bloodstream, reflecting osteoblast activity. In addition to its osteogenic function, osteocalcin released into the bloodstream also plays a role in regulating blood glucose, enhancing cognitive function, inhibiting fat accumulation, and regulating androgen levels. This assay uses ELISA to analyze the osteocalcin content secreted by OP9 cell lines when differentiating into osteoblasts, thereby assessing the sample's impact on bone formation rate.

[0055] 2-1. Material and Equipment Specifications:

[0056] 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 (ATC). OP9 cell line (ATCC CRL-2749) TM ).

[0057] Cell culture medium: contains 80% MEM AM (Minimum Essential Medium Alpha Medium, purchased from Gibco, Cat. 12000-022), 20% Fetal Bovine Serum (purchased from Gibco, Cat. 10437-028), and 1% Penicillin-streptomycin (purchased from Gibco, Cat. 15240-062).

[0058] Differentiation medium: contains 90% DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, Cat. 12100-038) with additional components to contain 10% FBS (fetal bovine Serum, purchased from Gibco, 10438-026) and 1% Penicillin-streptomycin (purchased from Gibco, Cat. 15140-122), 50 μΜ ascorbic acid (purchased from Sigma), 10 -7 M dexamethasome (purchased from Sigma), and 10 mM β-glycerol (purchased from Sigma).

[0059] Dulbecco's phosphate buffered saline (DPBS solution): purchased from Gibco, Cat. 14200-75.

[0060] Osteocalcin detection kit (ELISA Kit for Osteocalcin (OC) brand USCN, Cat. SEA471M).

[0061] Flow cytometer: purchased from BD Pharmingen, Cat. BD™ Accuri C6 Plus.

[0062] 2-2, Test procedure:

[0063] OP9 cells were seeded at a density of 2 x 10 4 cells per well in a 24-well culture plate containing 2 mL of cell culture medium per well and incubated in a CO 2 incubator for 24 hours. Subsequently, the cell culture medium was replaced with fresh differentiation medium.

[0064] Subsequently, the cells were incubated for 7 days with fresh differentiation medium being replaced every 3 days.

[0065] The OP9 cells were divided into a blank group and an experimental group and then cultured for 7-10 days, with fresh differentiation medium being replaced every 3 days. The differentiation medium of the experimental group was added with 0.125% of the prepared giant squash juice. The blank group was not added with any test sample, but only with the differentiation medium.

[0066] The cell morphology was observed to confirm that the OP9 cells had been differentiated into osteocytes.

[0067] The supernatant was collected from each well of the culture plate into a microcentrifuge tube for subsequent detection by the osteocalcin detection kit. Here, the osteocalcin detection kit was used to complete the ELISA analysis according to the manufacturer's provided operation manual.

[0068] The content of osteocalcin in the supernatant of each well was measured by flow cytometry to facilitate the analysis of osteocalcin secretion. Each group was repeated three times, so the results of the three repeated experiments of each group were averaged to obtain the average value, and then the average value of the osteocalcin secretion of the blank group was taken as 100%, and the average value of the experimental group was converted into the relative osteocalcin secretion amount, as shown in Figure 1

[0069] 2-3, Test results:

[0070] The results were analyzed by student t-test using Excel software to determine whether there was a statistically significant difference between the two sample populations, as shown in Figure 1 , where "*" 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 "*" is, the more significant the statistical difference relative to the blank group.

[0071] Referring to Figure 1 , the relative osteocalcin secretion amount of the experimental group was 148.5%, which had the effect of promoting the osteocalcin secretion of bone cells. It was shown that the giant squash juice could significantly promote the osteocalcin secretion of bone cells. Osteocalcin is an essential substance for bone matrix mineralization, and high osteocalcin can reflect the improvement of bone mass, the enhancement of bone microstructure, the reduction of fracture risk, the increase of bone resorption, the improvement of bone density, the slowing down of bone loss, and the increase of joint support.

[0072] Example Three: Test of Giant Squash Juice on Mitochondrial Activity

[0073] 3-1, Materials and instruments:

[0074] Cell strain: Mouse myoblast C2C12 (hereinafter referred to as skeletal muscle cell) was used. C2C12 cells were purchased from American Type Culture Collection, ​C2C12 cell line (ATCC CRL-1772 TM ).

[0075] Cell culture medium: Dulbecco's modified Eagle's medium (Gibco) containing 10 vol% FBS (Gibco) with additional ingredients to make it contain 1% antibiotic-antimycotic (Gibco; Cat. 15240-062).

[0076] Buffered salt solution (DPBS solution): Gibco, Cat. 14200-75.

[0077] Trypsin: 10X Trypsin-EDTA (Gibco, Cat. 15400-054).

[0078] Mitochondrial membrane potential detection kit (BD MitoScreen (JC-1) kit, Cat. BD 551302). The kit contains JC-1 dye (lyophilized) and 10X assay buffer. Prepare the JC-1 working solution before use. Dilute the 10X assay buffer 10-fold with 1X PBS to make 1X assay buffer. Add 130 μL DMSO to the JC-1 dye (lyophilized) to make the JC-1 stock solution. Then, dilute the JC-1 stock solution with 1X assay buffer to make the JC-1 working solution (i.e., JC-1 mitochondrial-specific dye). The dilution ratio is 1:100 of JC-1 stock solution to 1X assay buffer.

[0079] Flow cytometer: BD Accuri C6 Plus (BD Pharmingen).

[0080] 3-2. Test procedure:

[0081] Skeletal muscle cells were seeded at a density of 1 x 10 5 cells per well in a 6-well culture plate containing 2 mL of cell culture medium per well and incubated in an incubator (37°C, 5% CO 2 ) for 24 hours. The experimental group was added with cell culture medium containing 0.125% (m / v) of the giant serpent fruit juice prepared in Example 1. The blank group was added with simple cell culture medium (i.e., without giant serpent fruit juice).

[0082] The cell culture medium in the culture dish was removed and rinsed with 1 mL of 1X PBS solution twice. Then 200 μL of trypsin was added to each well and reacted for 5 minutes in the dark to detach the cells in each well and collected into the corresponding 1.5 mL centrifuge tube, and the centrifuge tube containing the cells was centrifuged at 400 x g for 5 minutes. After centrifugation, the supernatant was removed, and the cells were rinsed with 1X DPBS solution, and the centrifuge tube containing the cells was centrifuged at 400 x g for 5 minutes again. After the second centrifugation, the supernatant in each centrifuge tube was removed and 100 μL of JC-1 working reagent was added to each centrifuge tube and incubated for 15 minutes in the dark. After 15 minutes, each centrifuge tube was centrifuged at 400 x g for 5 minutes. After centrifugation, the supernatant in each centrifuge tube was removed, and the cells were rinsed with 1X assay buffer solution and centrifuged at 400 x g for 5 minutes, and this step was repeated twice. After the second centrifugation, the supernatant in each centrifuge tube was removed, and 200 μL of 1X DPBS solution (with 2% FBS) was added to each centrifuge tube to resuspend the cells to obtain the test cell solution.

[0083] The membrane potential of the mitochondria of the cells in the test cell solution in each well was measured by flow cytometry to analyze the mitochondrial activity. Each group was repeated three times, so the results of the three repeated experiments of each group were averaged to obtain the average value, and then the average value of the blank group was taken as 100% relative mitochondrial activity (JC-1 aggregation amount), and the average value of the experimental group was converted into relative mitochondrial activity (JC-1 aggregation amount), as shown in Figure 2

[0084] 3-3, Test Results:

[0085] The results were analyzed using student t-test with Excel software to determine whether there was a statistically significant difference between the two sample populations, as shown in Figure 2

[0086] Referring to Figure 2 , which is a comparison histogram of the relative mitochondrial activity of the experimental group treated with giant melon juice and the blank group without treatment. As can be seen from the graph, the relative mitochondrial activity of the experimental group is 111.2%. In other words, compared with the blank group, the mitochondrial activity of the skeletal muscle cells of the experimental group is increased by 11.2%. It shows that giant melon juice can improve the mitochondrial activity of skeletal muscle cells. Skeletal muscle cells with vitality can improve muscle endurance, and the improvement of muscle endurance can further increase joint support force to prevent wear and tear of articular cartilage.

[0087] Example Four: Test for Promoting Collagen Production​​

[0088] Interleukin-1beta (IL-1β) was used to induce inflammation in chondrocytes, and the effect of IL-1β on collagen content was further observed.

[0089] 4-1. Materials and instruments:

[0090] Cell line: chondrocyte precursor ATDC5 (purchased from Sigma, product number Cat. 99072806), hereinafter referred to as chondrocyte.

[0091] Cell culture medium: DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) (purchased from Gibco, product number 12500-062) with 5% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, 10438-026) and 1% antibiotic-antimycotic (Antibiotic-Antimycotic, purchased from Gibco; Cat. 15240-062).

[0092] Differentiation medium: DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) (purchased from Gibco, product number 12500-062) with 5% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, 10438-026), 2Mm L-glutamine (purchased from Gibco), 1% penicillin / streptomycin (purchased from Gibco, Cat. 15140122), and 1% insulin-transferrin-selenium.

[0093] Collagen detection kit (ELISA kit for Collagen, brand USCN).

[0094] ELISA reader (BioTeck brand).

[0095] 4-2. Test procedure:

[0096] First, chondrocytes were seeded in culture dishes at an appropriate density, and incubated in a CO 2Incubate overnight in an incubator. After the cells are attached, replace the medium with differentiation medium, and replace it every three days until the cells are fully differentiated.

[0097] The differentiated chondrocytes were divided into experimental, control, and blank groups. The test medium was added to each well, and then the cells were incubated at 37°C for 2 hours.

[0098] The test medium for the control group was cell culture medium containing 30 ng / mL of IL-1β, and the test medium for the experimental group was cell culture medium containing 30 ng / mL of IL-1β and 0.125% (m / v) of the giant caltrap juice prepared in Example 1. The test medium for the blank group was simply cell culture medium (i.e., without giant caltrap juice).

[0099] The supernatant of each group was collected, and enzyme-linked immunosorbent assay was performed according to the instructions in the ELISA kit. Then, the absorbance of each group at OD 450 nm was measured using a spectrophotometer, and the results are shown in Figure 3

[0100] 4-3. Test results:

[0101] Here, the amount of collagen secreted is shown in relative multiples. The standard deviation was calculated using the STDEV formula in Excel software, and T-test was used in Excel software to analyze whether there was a statistically significant difference. "*" represents a statistically significant difference relative to the blank group, and the more "*" there is, the more significant the difference. Where "*" represents a p-value of less than 0.05, "**" represents a p-value of less than 0.01, and "***" represents a p-value of less than 0.001. "#" represents a statistically significant difference relative to the control group, and the more "#" there is, the more significant the difference. Where "#" represents a p-value of less than 0.05, "##" represents a p-value of less than 0.01, and "###" represents a p-value of less than 0.001.

[0102] See Figure 3 ​The value of the blank group is taken as 100%, representing the collagen content of chondrocytes in a normal state. Compared with the blank group, the relative secretion amount of collagen in the control group was 48.18%, representing a 51.82% decrease in collagen in IL-1β chondrocytes in an inflammatory state. Compared with the blank group, the relative secretion amount of collagen in the experimental group was 62.84%. However, compared with the control group, the relative secretion amount of collagen in the experimental group increased by 14.66%. It can be seen that even in the case of chondrocytes stimulated by IL-1β to produce inflammation, the use of giant sponge gourd juice can improve the decrease in collagen content caused by the inflammatory response. Collagen is an essential substance for maintaining joint lubrication and movement, and an increase in collagen can improve / slow down degenerative joint inflammation, slow down damage caused by inflammation, improve mild joint inflammation in the early or second stage, and increase joint support and improve joint flexibility.

[0103] Example Five: Test for Promoting Hyaluronic Acid Production

[0104] Interleukin-1 beta (IL-1β) was used to induce chondrocytes to exhibit an inflammatory state, and the effect on the intracellular hyaluronic acid content was further observed.

[0105] 5-1. Materials and Instruments:

[0106] Cell strain: Chondrocyte precursor ATDC5 (purchased from Sigma, product number Cat. 99072806), hereinafter referred to as chondrocytes.

[0107] Cell culture medium: DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) (purchased from Gibco, product number 12500-062) with additional ingredients containing 5% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, 10438-026) and 1% antibiotic and antimycotic (Antibiotic-Antimycotic, purchased from Gibco; Cat. 15240-062).

[0108] Differentiation medium: DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12) (purchased from Gibco, product number 12500-062) with additional ingredients containing 5% Fetal Bovine Serum (purchased from Gibco, 10438-026), 2Mm of L-Glutamine (purchased from Gibco), 1% penicillin / streptomycin (purchased from Gibco, Cat. 15140122), 1% Insulin-transferrin-Selenium.

[0109] Hyaluronic Acid ELISA kit (brand USCN).

[0110] ELISA reader (BioTeck brand).

[0111] 5-2, Test procedure:

[0112] First, the chondrocytes were seeded into the culture dish at an appropriate density, and then incubated in a CO 2 incubator overnight. After the cells adhered, the differentiation medium was replaced, and the medium was replaced every three days until the differentiation was complete.

[0113] The differentiated chondrocytes were divided into experimental, control and blank groups. Each well was added with the test medium, and then incubated at 37°C for 2 hours.

[0114] Among them, the test medium of the control group was the cell culture medium containing 30 ng / mL of IL-1β, the test medium of the experimental group was the cell culture medium containing 30 ng / mL of IL-1β and 0.125% (m / v) of the giant caltrop juice prepared in Example 1. The test medium of the blank group was simply the cell culture medium (i.e. without giant caltrop juice).

[0115] Then, the supernatant of each group was collected, and the enzyme-linked immunosorbent assay was completed according to the operation method of the ELISA kit. Then, the spectrophotometer was used to measure the absorbance value of each group at OD value 450 nm, and the test results are shown in Figure 4 .

[0116] 5-3, Test results:

[0117] The relative secretion amount of hyaluronic acid is shown in the figure. The standard deviation is calculated by the STDEV formula of Excel software, and the statistical difference is analyzed by Student t-test in Excel software. "*" represents the statistical difference compared with the blank group, and the more "*" represents the more significant difference. "*" represents p value less than 0.05, "**" represents p value less than 0.01, and "***" represents p value less than 0.001. "#" represents the statistical difference compared with the control group, and the more "#" represents the more significant difference. "#" represents p value less than 0.05, "##" represents p value less than 0.01, and "###" represents p value less than 0.001.

[0118] Please refer to Figure 4 The value of the blank group is considered as 100%, representing the hyaluronic acid content of chondrocytes in normal state. Compared with the blank group, the relative secretion amount of hyaluronic acid of the control group is 90.58%, representing that the hyaluronic acid of IL-1β chondrocytes in inflammatory state decreases by 9.42%. Compared with the blank group, the relative secretion amount of hyaluronic acid of the experimental group is 96.52%. However, compared with the control group, the relative secretion amount of hyaluronic acid of the experimental group increases by 5.94%. It can be seen that even in the case of chondrocytes stimulated by IL-1β to produce inflammation, the use of giant cucurbit juice can improve the decrease of hyaluronic acid content caused by inflammation. Hyaluronic acid is an essential substance for maintaining joint lubrication and activity. The increase of hyaluronic acid can improve / slow down the inflammation of degenerative joints, slow down the damage caused by inflammation, improve the mild joint inflammation in the early or second stage, and increase the joint support force and improve the joint flexibility.

[0119] Example six: human test of giant cucurbit juice

[0120] 6-1, subjects: 8 subjects. Among them, each subject is a 20-year-old adult and self-reports joint discomfort, or a high-risk fracture (meets at least one of menopausal women, has been diagnosed with low bone density, or age >= 65 years old).

[0121] 6-2, test items: bone density evaluation index T score, bone density evaluation index Z score, β-crosslaps, questionnaire survey (joint stiffness degree, WOMAC score and overall satisfaction).

[0122] The bone density was measured by dual energy X-ray absorptiometry (DXA), and the bone density of the lumbar vertebrae was measured and converted into T-score and Z-score. In this embodiment, the average bone density of the first to fourth lumbar vertebrae was converted.

[0123] The beta-crosslaps (β-CTx) concentration was measured by the fasting blood of the subjects by the Harvard Medical Clinic. This is an important biochemical indicator of bone resorption.

[0124] The questionnaire was the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score scale, which is a widely used questionnaire for evaluating the symptoms of arthritis in patients. The evaluation items further include the degree of knee stiffness for self-evaluation. In addition, the satisfaction of the subjects with the product was investigated, including very dissatisfied, dissatisfied, ordinary, satisfied, and very satisfied.

[0125] 6-3. Test method:

[0126] The 8 subjects in the experimental group consumed 2000 mg of the giant squash juice prepared in Example 1 every day for 12 consecutive weeks. In this embodiment, 2000 mg of the giant squash juice prepared in Example 1 was added to 48 mL of water to form giant squash juice for the subjects to drink.

[0127] The data measured before consumption (i.e., at week 0) is referred to as the control group, and the data measured after 12 weeks of consumption (i.e., at week 12) is referred to as the experimental group.

[0128] 6-4. Test results:

[0129] The average values of all subjects are shown in the following figures. The standard deviation was calculated using the STDEV formula of the Excel software, and the T-test was used in the Excel software to analyze whether there was a statistically significant difference to obtain the p value. In the figures, “*” means that the p value is less than 0.05.

[0130] First, according to the World Health Organization (WHO) classification of osteoporosis based on bone density, T-score +1 to –1 is normal, T-score –1 to –2.5 is osteopenia, and T-score less than or equal to –2.5 is osteoporosis. The T-score results of the 8 subjects are shown in Table 1 below.

[0131] Table 1:

[0132] Control group (week 0) Experimental group (week 12) Subject 1 -0.1 0.6 Subject 2 -2.5 -2.3 Subject 3 -2.3 -2.1 Subject 4 -1.7 -1.7 Subject 5 -1.8 -1.5 Subject 6 0.3 0.3 Subject 7 -1.2 -0.9 Subject 8 -1.3 -1.2

[0133] Table 1 Figure 5 The average T-score of the 8 subjects was -1.33 at the 0th week, and the average T-score of the 8 subjects was -1.10 after 12 weeks of daily consumption of the giant pumpkin juice, meaning that the average bone density of the subjects was improved by 17% after 12 weeks of use of the giant pumpkin juice. Among the 8 subjects, 6 subjects had improved T-scores, meaning that the proportion of improved subjects was 75%. The higher the T-score, the better the bone absorption capacity, the better the bone density, the less the bone loss, and the more sufficient the joint support.

[0134] The Z-score is a comparison of the measured bone density of an individual with the average bone density of the same age group. The Z-score helps to find bone loss caused by certain underlying diseases and is commonly used to diagnose whether children, premenopausal women, or men under the age of 50 have osteoporosis. A Z-score equal to or greater than -1.5 is normal, and a Z-score less than -1.5 is low. The Z-score results of the 8 subjects are as follows in Table 2.

[0135] Table 2

[0136]

[0137]

[0138] Table 2 Figure 6 The average Z-score of the 8 subjects was -0.85 at the 0th week, and the average Z-score of the 8 subjects was -0.45 after 12 weeks of daily consumption of the giant pumpkin juice, meaning that the average bone density of the subjects was improved by 33.8% after 12 weeks of use of the giant pumpkin juice. Among the 8 subjects, 6 subjects had improved Z-scores, meaning that the proportion of improved subjects was 75%. The higher the Z-score, the better the bone absorption capacity, the better the bone density, the less the bone loss, and the more sufficient the joint support.

[0139] The β-crosslaps concentration mainly reflects the degree of bone absorption. The higher the concentration, the more severe the bone tissue damage, and patients with osteoporosis or myeloma often have elevated β-crosslaps concentrations in their serum. The reference value of β-crosslaps for premenopausal women is 0.136 to 0.689, and the reference value of β-crosslaps for postmenopausal women is 0.177 to 1.015. The reference value of β-crosslaps for men under the age of 30 is 0.148 to 0.967, the reference value of β-crosslaps for men between the ages of 30 and 70 is 0.132 to 0.936, and the reference value of β-crosslaps for men over the age of 70 is 0.118 to 0.776. The β-crosslaps concentration detection results of the 8 subjects are as follows in Table 3.

[0140] Table III:

[0141] Control group (week 0) Experimental group (week 12) Subject 1 0.354 ng / mL 0.189 ng / mL Subject 2 1.020 ng / mL 0.678 ng / mL Subject 3 0.602 ng / mL 0.441 ng / mL Subject 4 0.500 ng / mL 0.443 ng / mL Subject 5 0.549 ng / mL 0.734 ng / mL Subject 6 0.606 ng / mL 0.477 ng / mL Subject 7 0.350 ng / mL 0.324 ng / mL Subject 8 0.149 ng / mL 0.210 ng / mL

[0142] Please refer to Table III above and Figure 7 The average concentration at week 0 was 0.516 ng / mL, and after 12 weeks of daily ingestion of the giant pumpkin juice, the average concentration of the 8 subjects decreased to 0.437 ng / mL, that is, the improvement of the bone tissue damage of the subjects reached 15.35% after 12 weeks of using the giant pumpkin juice. Among the 8 subjects, 6 subjects had improved β bone collagen C-terminal peptide concentration, that is, the proportion of the number of improved persons was 75%. The higher the β bone collagen C-terminal peptide concentration, the more the collagen cross-linking increased, so that the bone mass was improved, the bone microstructure was enhanced, the risk of bone fracture was reduced, the bone resorption was increased, the bone density was improved, the bone loss was slowed down, and the joint support force was increased.

[0143] The WOMAC score was obtained using the WOMAC scoring scale, which mainly includes self-evaluation of two parts of "joint discomfort degree" and "physical activity", to understand the influence on joint degeneration, and each question can select one of the five options of no difficulty, slight, moderate, severe and very severe.

[0144] The "joint discomfort degree" questionnaire includes the following questions:

[0145] Question (1): How much pain do you have in your joints when walking on a flat road?

[0146] Question (2): How much pain do you have in your joints when going up and down stairs?

[0147] Question (3): How much pain do you have in your joints when sleeping at night?

[0148] Question (4): How much pain do you have in your joints when sitting or lying down?

[0149] Question (5): How much pain do you have in your joints when standing straight?

[0150] Question (6): How stiff are your joints when you first get up in the morning?

[0151] Question (7): How stiff are your joints if you sit or lie down for a while around evening time?

[0152] The "physical activity" questionnaire includes the following questions:

[0153] Question (8): How difficult do you feel when going down stairs?

[0154] Question (9): How difficult do you feel when going up stairs?

[0155] Question (10): How difficult is it for you to stand up from a chair?

[0156] Question (11): How difficult is it for you to stand still?

[0157] Question (12): How difficult is it for you to bend over?

[0158] Question (13): How difficult is it for you to walk on a flat surface?

[0159] Question (14): How difficult is it for you to get in and out of a car?

[0160] Question (15): How difficult is it for you to go shopping for groceries?

[0161] Question (16): How difficult is it for you to put on socks?

[0162] Question (17): How difficult is it for you to get up from a bed?

[0163] Question (18): How difficult is it for you to take off socks?

[0164] Question (19): How difficult is it for you to lie down on a bed?

[0165] Question (20): How difficult is it for you to go in and out of a bathroom to take a shower?

[0166] Question (21): How difficult is it for you to sit?

[0167] Question (22): How difficult is it for you to use the toilet (sitting position)?

[0168] Question (23): How difficult is it for you to do simple household chores (cooking, cleaning)?

[0169] Question (24): How difficult is it for you to do heavy household chores (moving furniture)?

[0170] In the case where the score of the 0th week is 100%, the score of the 12th week is converted, that is, the lower the score, the better the condition. The score conversion results are shown in Table 4 below.

[0171] Table 4:

[0172] Control group (week 0) Experimental group (week 12) Subject 1 100 78 Subject 2 100 96 Subject 3 100 75 Subject 4 100 112 Subject 5 100 75 Subject 6 100 112 Subject 7 100 73 Subject 8 100 76

[0173] Referring to Table 4 above, after taking the giant squash juice every day for 12 weeks, the WOMAC scores of 6 out of 8 subjects were reduced, which means that the proportion of improved persons was 75%. Referring to Figure 8The average of the WOMAC scores of the 8 subjects was reduced from 100% at the initial state (week 0) to 87.13% at week 12, a reduction of 12.87% in the WOMAC score. The WOMAC score represents improvement or reduction in the inflammation of the degenerative joint, improvement in the degree of joint stiffness, pain, and improvement in joint support and flexibility.

[0174] The above questions (5), (6), and (7) are the self-evaluation of the degree of joint stiffness in the WOMAC score scale. The score at week 0 is taken as 100%, and the score at week 12 is converted, i.e., the lower the score, the better the condition. The conversion results are shown in Table 5. Table 5:

[0175]

[0176]

[0177] Referring to Table 5, after 12 weeks of daily consumption of the giant pumpkin juice, 6 out of the 8 subjects self-evaluated that their joint stiffness was reduced, i.e., the proportion of improvement was 75%. Referring to Table 5, Figure 9 The average of the joint stiffness reduction of the 8 subjects was reduced from 100% at the initial state (week 0) to 62.96% at week 12, a significant reduction of 37.04% in the WOMAC score. The reduction in joint stiffness represents improvement or reduction in the inflammation of the degenerative joint, improvement in joint support and flexibility.

[0178] The overall satisfaction with the giant pumpkin juice was self-evaluated in the questionnaire at week 12. The results are shown in Table 6. Figure 10 .

[0179] Referring to Table 6, Figure 10 44% of the 8 subjects were very satisfied, 45% were satisfied, and 11% were ordinary. It can be seen that 89% of the subjects were satisfied with the overall performance of the giant pumpkin juice.

[0180] In summary, the use of the juice of the giant squash of any of the embodiments described herein for the preparation of a composition for joint health care. In some embodiments, the juice of the giant squash has at least one of the following effects: increasing bone mass, increasing the amount of osteocalcin secretion from bone cells, reducing bone loss, increasing mitochondrial activity in bone cells, increasing bone density, increasing T-score or Z-score, inhibiting the inflammatory response of the cartilage tissue of the joint, improving the decrease in collagen content caused by the inflammatory response, improving the decrease in hyaluronic acid content caused by the inflammatory response, and reducing the concentration of beta-crosslaps (β-CTx) in serum, increasing joint flexibility, and improving joint degeneration.

[0181] Of course, the present application can also have other various embodiments, and those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application. However, these corresponding changes and modifications should all belong to the protection scope of the claims of the present application.

Claims

1. A use for the juice of a giant winter melon, characterized in that, The giant cucumber juice, used to prepare a joint health care composition, is extracted from the giant cucumber (Cucurbita maxima) using water as a solvent.

2. The use as described in claim 1, wherein the giant cucumber juice is used to increase joint bone mass.

3. The use as described in claim 2, wherein the giant cucumber juice is used to increase the amount of osteocalcin secreted by bone cells.

4. The use as described in claim 2, wherein the giant cucumber juice is used to slow bone loss.

5. The use as described in claim 4, wherein the giant cucumber juice reduces the concentration of beta-crosslaps (β-CTx) in serum.

6. The use as described in claim 1, wherein the giant cucumber juice is used to enhance mitochondrial activity in bone cells.

7. The use as described in claim 1, wherein the giant cucumber juice is used to increase bone density.

8. The use as described in claim 7, wherein the giant cucumber juice is used to improve T-scores or Z-scores.

9. The use as described in claim 1, wherein the giant cucumber juice is used to improve joint flexibility.

10. The use as described in claim 9, wherein the giant cucumber juice is used to inhibit inflammatory responses in the cartilage tissue of joints.

11. The use as described in claim 10, wherein the giant cucumber juice is used to improve the decrease in collagen content caused by the inflammatory response.

12. The use as described in claim 10, wherein the giant cucumber juice is used to improve the decrease in hyaluronic acid content caused by the inflammatory response.

13. The use as described in claim 9, wherein the giant cucumber juice is used to improve joint stiffness.

14. The use as described in claim 9, wherein the giant cucumber juice is used to improve the Body Activity Scale (WOMAC) score.

15. The use as described in claim 9, wherein the giant cucumber juice is used to improve joint degeneration.

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