Composition comprising fermented morinda citrifolia extract having osteoarthritis alleviation effect

Abstract

The present invention relates to a composition comprising a fermented Morinda citrifolia extract and having an osteoarthritis alleviation effect, and a method for preparing the composition comprising the fermented Morinda citrifolia extract. The present invention provides a composition comprising a fermented Morinda citrifolia extract and having an osteoarthritis alleviation effect.

Description

Composition comprising fermented noni concentrate having an effect of improving osteoarthritis

[0001] The present invention relates to a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect and a method for producing a composition comprising a fermented noni concentrate.

[0002] Degenerative arthritis primarily occurs in weight-bearing joints like the knees and spine. The only fundamental treatment is surgery, and other options include anti-inflammatory medications for pain relief.

[0003] Acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) are used to relieve arthritis pain, but long-term use can cause digestive and blood clotting problems and have side effects such as nausea, vomiting, constipation, loss of appetite, and dizziness.

[0004] Although hyaluronic acid and glucosamine may help slow down joint degeneration as cartilage protectors, their effectiveness in promoting cartilage production and inducing regeneration is limited.

[0005] To date, no medication has been developed to prevent or treat degenerative arthritis. While there are various publicly notified and individually approved raw materials that are beneficial for joint health, the development of new materials is still necessary.

[0006] Noni is effective in anti-inflammatory, anti-cancer, anti-bacterial, diabetes, and cardiovascular diseases, and the iridoid substances contained in noni have also been reported to have effects such as antioxidant, anti-bacterial, improving liver function, and improving heart function. Fermented Morinda citrifolia extract (FME) obtained through fermentation technology is an extract with increased iridoid components, and previous research results have shown that fermented noni has an improving effect on inflammatory cytokines. Therefore, the present inventors attempted to confirm the osteoarthritis improving effect of fermented noni concentrate using an osteoarthritis test tube model in which an inflammatory response was induced by IL-1β treatment in SW1353 cells, a human chondrosarcoma cell line.

[0007] The purpose of the present invention is to provide a composition containing fermented noni concentrate having an osteoarthritis improvement effect and a method for producing the same.

[0008] To achieve the above-described purpose, the present invention provides a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect.

[0009] In one embodiment, the fermented noni concentrate may include diacetylasperulosic acid as an active ingredient.

[0010] In one embodiment, the active ingredient may comprise 5.76 to 8.64 mg per 1 g of fermented noni concentrate.

[0011] In one embodiment, the fermented noni concentrate can be prepared by inoculating noni with Lactobacillus plantarum NST 1805 (Accession No.: KCCM12833P) and then fermenting it.

[0012] In one embodiment, the fermented noni concentrate can be prepared by juicing the fermented noni and then concentrating the juice to 55 to 65 brix.

[0013] In addition, the present invention provides a food composition having an osteoarthritis improvement effect including a fermented noni concentrate.

[0014] In addition, the present invention provides a method for producing a composition including a fermented noni concentrate having an osteoarthritis improvement effect, the method comprising the steps of inoculating lactic acid bacteria into noni and fermenting it, preparing a fermented noni extract from the fermented noni obtained by the fermentation, and concentrating the fermented noni extract.

[0015] According to the present invention, fermented noni concentrate has an anti-inflammatory effect and a cartilage decomposition protein inhibition effect against osteoarthritis.

[0016] Figure 1 is a graph showing the effect of fermented noni concentrate according to the present invention on nitric oxide (NO) production.

[0017] Figures 2a, 2b, 3a, 3b and 4 are graphs showing the inhibitory effect of the fermented noni concentrate according to the present invention on cartilage degradation and inflammation-related protein expression.

[0018] Figures 5 and 6 are graphs showing the inhibitory effect of the fermented noni concentrate according to the present invention on cartilage degradation and inflammation-related mRNA expression.

[0019] Figure 7 is a graph showing the MMP-9 inhibitory effect of the fermented noni concentrate according to the present invention.

[0020] Hereinafter, embodiments disclosed in this specification will be described in detail with reference to the attached drawings. Regardless of the drawing numbers, identical or similar components will be given the same reference numbers and redundant descriptions thereof will be omitted. In addition, when describing embodiments disclosed in this specification, if it is determined that a detailed description of related known technology may obscure the gist of the embodiments disclosed in this specification, the detailed description thereof will be omitted. In addition, the attached drawings are only intended to facilitate easy understanding of the embodiments disclosed in this specification, and the technical ideas disclosed in this specification are not limited by the attached drawings, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention.

[0021] Terms that include ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another.

[0022] Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0023] In this specification, terms such as “include” or “have” are intended to specify the presence of a feature, number, step, operation, component or combination thereof described in the specification, but should be understood not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components or combinations thereof.

[0024] The present invention relates to a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect and a method for producing a composition comprising a fermented noni concentrate.

[0025] In this specification, ‘fermented noni’ means noni inoculated with lactic acid bacteria and then fermented and aged for a specified period of time.

[0026] In this specification, 'fermented noni concentrate' means a liquid obtained by performing at least one concentration process on the juice of the fermented noni.

[0027] In this specification, the term 'composition having an osteoarthritis improvement effect' refers to a food group or food composition that has been designed and processed to sufficiently express the body's regulation function for regulating the biological defense rhythm, disease prevention, and recovery, etc., by using physical, biochemical, or bioengineering techniques to give added value to the food so that the food's function can be performed and expressed for a specific purpose.

[0028] In the present specification, compositions having an osteoarthritis-improving effect include various foods, beverages, gum, tea vitamin complexes, functional foods, etc. Furthermore, the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., and the above components may be used independently or in combination. The above components may be added in an appropriate amount within a range that does not inhibit the osteoarthritis-improving effect according to the present invention. For example, the above components may be included in an amount of 0.1 to 1 wt% based on the total weight of the composition having the osteoarthritis-improving effect.

[0029] In addition, the composition having the osteoarthritis improvement effect may include a food additive acceptable from a food science perspective, and may further include an appropriate carrier, excipient, and diluent commonly used in the manufacture of a composition having the osteoarthritis improvement effect.

[0030] These immune-boosting compositions in the form of beverages, granules, tablets, powders, pills, and capsules are easy to carry and can be easily taken anytime, anywhere.

[0031] Hereinafter, a method for manufacturing a composition having an osteoarthritis improvement effect according to the present invention will be described in detail.

[0032] The manufacturing method according to the present invention may include (S1) a step of inoculating lactic acid bacteria into noni fruit and fermenting and maturing it, (S2) a step of preparing a fermented noni extract from the fermented noni obtained through the fermentation and maturation, and (S3) a step of concentrating the fermented noni extract.

[0033] (S1) Step

[0034] In the S1 stage, lactic acid bacteria can be inoculated into noni fruit to allow fermentation and maturation.

[0035] The above lactic acid bacteria may be Lactobacillus plantarum NST 1805 (Accession number: KCCM12833P).

[0036] The above lactic acid bacteria can be inoculated onto noni fruit in the form of a lactic acid bacteria solution, preferably a lactic acid bacteria aqueous solution. At this time, 0.1 to 0.5 parts by weight of the lactic acid bacteria solution can be inoculated per 100 parts by weight of the noni fruit. When the inoculation amount of the lactic acid bacteria solution is sufficient, the biotransformation described above can occur smoothly, resulting in fermented noni containing ingredients that exhibit a significant effect in improving osteoarthritis.

[0037] Meanwhile, the fermentation and maturation can be carried out at 35 to 40°C for 1440 hours or more. Specifically, the fermentation can be carried out for 1440 hours or more from the start of fermentation, thereby preventing the formation of mold and homogenizing the fermented liquid.

[0038] The above fermentation and maturation temperature may be 35 to 40°C. If the fermentation and maturation temperature is lower than 30°C, fermentation may not occur normally, and if it exceeds 40°C, the fermentation and maturation time may be prolonged, which may cause the lactic acid bacteria to die.

[0039] (S2) Step

[0040] In step S2, a concentrated fermented noni extract can be produced from the fermented noni obtained through the above fermentation.

[0041] First, the fermented noni obtained through the above fermentation can be juiced.

[0042] At this time, the fermented noni that has undergone the fermentation process may contain both noni fruit and fermented liquid. Meanwhile, prior to the juicing step, a step of preheating the fermented noni may be further included. The preheating may be performed by heating the fermented noni at a low temperature. Preheating softens the fibers of the fermented noni, thereby increasing the juice yield and facilitating the extraction of nutrients from the fermented noni.

[0043] At this time, the preheating temperature is preferably 70 to 80°C. If the preheating temperature is lower than 60°C, there will be no change in the state of the fibers in the fermented noni, and thus the effect of improving the juice yield cannot be expected. If the preheating temperature is higher than 80°C, the fermented noni may be denatured or the lactic acid bacteria in the fermented noni may be sterilized.

[0044] In particular, if the preheating temperature is 90℃ or higher, there is a problem that more than 90% of the lactic acid bacteria are sterilized. In addition, the preheating time is preferably 1 to 2 hours. If the preheating time is less than 30 minutes, there will be no change in the state of the fiber in the fermented noni, so the effect of improving the juice yield cannot be expected. If the preheating time exceeds 2 hours, the fermented noni may denature or the lactic acid bacteria in the fermented noni may die.

[0045] The above-mentioned juicing can be performed by extracting the fermented noni from the noni fruit and fermented liquid contained in the fermented noni to produce a fermented noni extract. At this time, the juicing can be performed by applying pressure to the fermented noni, and the juicing is preferably performed within a preset pressure range. If the juicing pressure is below the preset pressure range, the juicing may not be performed properly, resulting in a decrease in the yield. On the other hand, if the pressure exceeds the preset pressure range, byproducts of the noni fruit may also be mixed in, resulting in a decrease in the purity of the extract.

[0046] In addition, after the juicing, a further filtration process can be performed to improve purity and enhance product reliability.

[0047] In one embodiment, the filtration of the juice may be performed through an 80 mesh filter.

[0048] (S3) Step

[0049] In step S3, the fermented noni juice can be concentrated to obtain a fermented noni concentrate.

[0050] After juicing, the moisture contained in the fermented noni extract can be evaporated to concentrate it to 55 to 65 brix.

[0051] More specifically, the fermented noni extract can be concentrated to a Brix of 60 (±5) at a temperature of 60 to 65°C to produce a concentrate. At this time, the Brix value can be measured using a saccharometer (PAL-3) from ATAGO.

[0052] Afterwards, the fermented noni concentrate can be sterilized. The sterilization can be performed using a wide range of sterilization methods common in the art without limitation.

[0053] In one embodiment, the fermented noni concentrate can be sterilized at 65°C to 95°C for 30 minutes.

[0054] Fermented noni concentrate contains deacetylasperulosidic acid as an active ingredient, and may contain 5.76 to 8.64 mg per 1 g of fermented noni concentrate.

[0055]

[0056] Example

[0057] Noni fruit was washed, frozen at -27°C to remove bacteria, and then thawed at 37°C for 2 days. Afterwards, it was fermented with a 2% aqueous solution of Lactobacillus plantarum NST 1805 (Accession No.: KCCM12833P) at 37°C for 1440 hours.

[0058] The above fermented noni was preheated at 78°C for 1 hour and 30 minutes and then juiced using a juice extractor.

[0059] Afterwards, it was filtered using an 80 mesh filter, concentrated to produce a fermented noni concentrate of 60 brix, and then sterilized at 65°C to 95°C for 30 minutes.

[0060]

[0061] Experimental Example 1: Effect on nitric oxide (NO) production

[0062] Excessive amounts of NO are produced at sites of inflammation, promoting necrosis of cell tissue. Therefore, measuring the amount of NO produced at sites of inflammation can reveal anti-inflammatory effects.

[0063] To evaluate the osteoarthritis improvement effect of fermented noni concentrate, human chondrosarcoma SW1353 cells (hereinafter referred to as SW1353 cells) were used.

[0064] Fermented noni concentrate was treated to SW1353 cells and treated with IL-1β, a pro-inflammatory cytokine, to create an environment similar to osteoarthritis, and the anti-inflammatory effect was confirmed compared to the osteoarthritis-induced test group.

[0065] SW1353 cells were treated with fermented noni concentrate at various concentrations and cultured for 24 hours after treatment with 10 ng / mL of IL-1β. Afterwards, 100 μL of the supernatant was mixed with an equal volume of Griess reagent, and the cells were incubated at room temperature for 10 minutes. The absorbance was measured at 540 nm using an ELISA reader.

[0066] The lower the absorbance, the less NO production there is, and the lower the NO production, the better the anti-inflammatory effect.

[0067] Referring to Figure 1, it was confirmed that the amount of NO production increased when IL-1β was treated, and that the amount of NO production decreased in the test group treated with fermented noni concentrate. This indicates that fermented noni concentrate significantly inhibited NO production.

[0068] Experimental Example 2. Inhibitory Effect of Fermented Noni Concentrate on Cartilage Degradation and Inflammation-Related Protein Expression

[0069] Collagen, gelatin, and other major proteins that make up cartilage are known to promote cartilage destruction and induce osteoarthritis when an inflammatory response is induced in the joints. In this experimental example, we aimed to confirm the inhibitory effect of fermented noni concentrate when the protein expression of MMP-1, -3, and -13 increased in an osteoarthritis-induced cell model.

[0070] The experimental method is to seed SW1353 cells in a 6-well plate at 5.0 X 10 5Cells were seeded per well and cultured in a CO2 incubator for 24 h. Fermented noni concentrate was pretreated at various concentrations, and IL-1β (10 ng / m) was treated and cultured for 24 h. After washing twice with phosphate-buffered saline (PBS), the cells were dissolved in Radioimmunoprecipitation assay (RIPA) buffer (Rockland Inc., Gilbertsville, PA, USA) containing protease inhibitor cocktail and EDTA solution, and incubated at 4°C for 10 min.

[0071] Cell lysates were centrifuged at 4°C and 15,000 rpm for 15 minutes, and protein was quantified in the supernatant using Bradford reagent (Bio-rad, Hercules, CA, USA). Samples were separated by electrophoresis on a 10% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and blocked (5% blocking reagent in TBST) for 1 hour at room temperature.

[0072] The primary antibody was treated overnight at 4°C, washed three times with 0.1% TBST, and then reacted with the secondary antibody for 1 hour. After washing with 0.1% TBST and reacting with the enhanced chemiluminescence western blotting detection kit (Bio-rad), protein bands were identified using chemiDoc (Bio-rad) equipment, and the band density was measured using Image J software.

[0073] Referring to Figures 2a and 2b, the effect of fermented noni concentrate on iNOS and COX-2 expression in an osteoarthritis-induced cell model was confirmed to be increased by IL-1β treatment due to an inflammatory response, and the expression level was inhibited by fermented noni concentrate treatment. iNOS showed statistical significance from 10 ng / mL, and inhibition of COX-2 expression showed statistical significance at 100 ug / mL treatment.

[0074] Referring to Figures 3a and 3b, it was confirmed that when IL-1β was treated in the osteoarthritis-induced cell model, the protein expression of MMP-1, -3, and -13 increased, and in the test group treated with fermented noni concentrate, the expression of MMP-1 and -3 significantly decreased.

[0075] Referring to Figure 4, it was confirmed that when IL-1β was treated in the osteoarthritis-induced cell model, the mRNA expression of MMP-1, -3, and -13 increased, and in the test group treated with fermented noni concentrate, the mRNA expression of MMP-1, -3, and -13 significantly decreased.

[0076] Experimental Example 3. Inhibitory Effect of Fermented Noni Concentrate on Cartilage Degradation and Inflammation-Related mRNA Expression

[0077] When osteoarthritis is induced, chondrocytes are stimulated by pro-inflammatory cytokines, and the expression of MMPs and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, -5, which degrade collagen and proteoglycan, which are proteins that constitute the extracellular matrix of cartilage, is promoted, and the expression of inflammatory cytokines also increases. In this experimental example, we tried to confirm the inhibitory effect of fermented noni concentrate when the expression of MMP-1, -3, -13 and ADAMTS-4, -5, which are proteins that degrade ECM, and the expression of inflammatory cytokines (TNF-α, IL-6) increased in an osteoarthritis-induced cell model.

[0078] The experimental method is to seed SW1353 cells in a 6-well plate at 5.0 X 10 5 Cells were seeded per well and cultured in a CO2 incubator for 24 hours. Fermented noni concentrate was pretreated at various concentrations, and IL-1β (10 ng / m) was treated and cultured for 24 hours. Total RNA was obtained using Nucleozol (Macherey-Nagel, Germany), and the purified RNA was quantified and then one-step real-time PCR was performed using RNA-directTM SYBR® Green Real-time PCR Master Mix (QRT-201, TOYOBO, Osaka, Japan).

[0079] Referring to Figure 5, when IL-1β was treated in SW1353 chondrosarcoma cells, the expression of ADAMTS-4, -5, an enzyme that decomposes ECM, increased, and in the test group treated with fermented noni concentrate, the mRNA expression of ADAMT-4, -5 significantly decreased.

[0080] Referring to Figure 6, when IL-1β was treated in SW1353 chondrosarcoma cells, mRNA expression of inflammatory cytokines (TNF-α, IL-6) increased. It was confirmed that mRNA expression of IL-6 was inhibited in the test group treated with fermented noni concentrate.

[0081] Experimental Example 4. MMP-9 Inhibitory Effect of Fermented Noni Concentrate

[0082] The experimental method is to seed SW1353 cells in a 6-well plate at 5.0 X 10 5Cells were seeded per well and cultured in a CO2 incubator for 24 hours. Fermented Morinda citrifolia extract (FME) was pretreated at various concentrations, and IL-1β (10 ng / m) was treated and cultured for 24 hours. After culture, the expression level of MMP-9 was measured using the ELSIA Kit using the cell supernatant.

[0083] In the same way, the expression level of MMP-9 in unfermented noni concentrate (Morinda citrifolia extract, ME) was measured.

[0084] Referring to Figure 7, the IL-1β treatment group showed a significant increase in the expression of MMP-9 compared to the normal group, and it was confirmed that it was decreased by noni concentrate and fermented noni concentrate treatment. In addition, in the unfermented noni concentrate treatment group, the 200 μg / mL treatment group showed a significant decrease compared to the osteoarthritis-induced group (Control group), but in the fermented noni concentrate treatment group, the 100 and 200 μg / mL treatment groups showed a significant decrease compared to the osteoarthritis-induced group (Control group). Through this, it was confirmed that the fermented noni concentrate effectively inhibited the mRNA expression of MMP-9 even at a relatively low concentration.

[0085] While the embodiments of the present invention have been described above with reference to the attached drawings, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without altering the technical concept or essential features thereof. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

[0086] [Accession number]

[0087] Name of depositor: Korea Center for Microbiological Conservation (KCCM)

[0088] Accession number: KCCM12833P

[0089] Date of acceptance: 20201117

[0090] The best mode for carrying out the invention as described above has been described.

[0091] The present invention can provide a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect and a method for producing a composition comprising a fermented noni concentrate.

Claims

1. A composition comprising fermented noni concentrate having an effect of improving osteoarthritis.

2. In paragraph 1, A composition characterized in that the above fermented noni concentrate contains diacetylasperulosic acid as an active ingredient.

3. In paragraph 2, The above active ingredients are, A composition characterized in that it comprises 5.76 to 8.64 mg per 1 g of fermented noni concentrate.

4. In paragraph 3, The above fermented noni concentrate is, A composition characterized by being manufactured by fermenting noni after inoculating it with Lactobacillus plantarum NST 1805 (Accession No.: KCCM12833P).

5. In paragraph 4, The above fermented noni concentrate is, A composition characterized in that it is manufactured by juicing the fermented noni and concentrating the juice to 55 to 65 brix.

6. A food composition having an osteoarthritis improvement effect, comprising a fermented noni concentrate according to any one of claims 1 to 5.

7. Step of inoculating lactic acid bacteria into noni and fermenting it; A step of preparing a fermented noni extract from the fermented noni obtained by the above fermentation; and A method for producing a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect, comprising a step of concentrating the above fermented noni extract.

8. In paragraph 7, The above lactic acid bacteria are, A method for producing a composition comprising a fermented noni concentrate having an osteoarthritis improvement effect characterized by Lactobacillus plantarum NST 1805 (Accession No.: KCCM12833P).

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