JP5992691B2 - Hyaluronic acid production promoter and type II collagen production promoter - Google Patents

Description

  The present invention relates to a joint function improving agent and food and drink. Specifically, the hyaluronic acid production promoting effect, the type II collagen production promoting effect, and the joint function improving agent having the tumor necrosis factor (TNF-α) production inhibitory effect, and the amount of hyaluronic acid production in synovial cells can be increased. The present invention relates to a hyaluronic acid production promoter, a type II collagen production promoter, a TNF-α production inhibitor, and a food or drink containing these.

It is known that synovial fluid (joint fluid) secreted from cartilage and synovial tissue covering the surface of joints functions to reduce the impact of joint loads and smooth joint movements. Yes. Hyaluronic acid which is a main component of the synovial fluid plays a role of a lubricant and a cushion for smoothly moving the joint.
The concentration of hyaluronic acid in the synovial fluid is about 3.4 mg / mL to 5 mg / mL in a normal person, but in the case of osteoarthritis, the concentration decreases by about 30% to 50%. It is known to do. Symptoms such as cartilage wear, joint pain, and movement disorders appear due to a decrease in the amount of hyaluronic acid in the synovial fluid or a decrease in its viscosity.

  In the above diseases, it is conceivable to increase the amount of hyaluronic acid in the joint fluid in order to improve the lubrication function, cover (protect) the articular cartilage, suppress pain, and improve or normalize the pathological joint fluid. For example, when rheumatoid arthritis patients are treated with joint injection of sodium hyaluronate, it has been reported that the above symptoms are improved (see Non-Patent Document 1).

  However, treatment of the above diseases is long-lasting and requires a doctor's prescription. Examples of drugs that produce hyaluronic acid in human cells include cytokines such as insulin-like growth factor-1, epidermal growth factor (see Non-Patent Document 2) and interleukin-1 (see Non-Patent Document 3), or phorbol. Esters (see Non-Patent Document 4) and the like are known, but none of them can be used simply and safely as a pharmaceutical or a food or drink.

In addition, articular cartilage rich in viscoelasticity is present on the surface of the joint where the bone is in contact, protecting the joint and smoothing the joint surface to make the joint move smoothly. It is known that when the articular cartilage is worn with intense exercise or aging, it is likely to shift to osteoarthritis. The articular cartilage has a problem that it has a poor self-repairing ability and is difficult to heal naturally.
Thus, for wound healing of articular cartilage and maintenance of a healthy joint, it is required to have an effect of inducing differentiation of cartilage progenitor cells into chondrocytes and promoting production of type II collagen, which is a major component of cartilage.

  It is also known that when joint function is reduced and arthritis is occurring, macrophages are activated to actively produce cytokines such as TNF-α. When the cytokine accelerates the induction of neutrophils and monocytes and the activation of macrophages themselves and helper T4 cells, inflammation expands and pannus (the surface cells of the inflamed synovium proliferate, A state in which it spreads like a carpet and infiltrates cartilage and bone). The pannus and cartilage are stimulated by TNF-α to produce extracellular matrix proteolytic enzyme (MMP-3), which is thought to cause symptoms such as joint deformation as a result of destroying cartilage and bone. . In order to prevent entry into such a spiral of inflammation, it is required to suppress cytokine production in macrophages.

  Therefore, increasing the amount of hyaluronic acid produced by synovial cells in vivo, supplementing the joint with the produced hyaluronic acid, promoting the production of type II collagen by inducing differentiation from cartilage progenitor cells to chondrocytes, And exerts an effect on osteoarthritis by inhibiting the destruction of cartilage by inhibiting the production of cytokines in macrophages, that is, maintaining smooth movement of joints, preventing joint deformation, and deformability A simple and highly safe material having an effect of preventing and improving pain due to arthropathy, movement disorders, and the like is awaited.

On the other hand, lotus has been eaten from ancient times or has been taken as a herbal medicine, and its safety has been established.
As an example of using this lotus, for example, an extract obtained by extracting a lotus seed germ using water or a water-miscible organic solvent has a blood pressure lowering effect (see Patent Document 1). The extract extracted from the germ has an effect on cardiovascular diseases such as hypertension, respiratory diseases such as bronchial asthma, and central nervous diseases such as insomnia (see Patent Document 2), also extracted from lotus germ That the extracted extract has an anti-aging action on the skin (see Patent Document 3), and that the extract extracted from the lotus germ also has an action of promoting metabolism of the epidermis layer of the skin (Patent Document 4) Each) is disclosed.

  However, for the lotus (seed) embryo, only the above-mentioned effects are known. Regarding hyaluronic acid production promoting action, type II collagen production promoting action, TNF-α production inhibiting action, joint function improving action, etc. It was not known.

JP 63-66126 A JP-A 64-90129 JP 2002-29980 A JP 2002-68993 A

"Inflammation", Japanese Society for Inflammation, 11, 16, 1991 “Biochema Biophysica Acta”, 1014, p. 305 (1989) “Japan Society of Obstetrics and Gynecology” magazine, 41, 1943, 1989 “Experimental Cell Research”, vol. 148, p. 377 (1983)

  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention provides osteoarthritis by increasing hyaluronic acid production in synoviocytes, increasing type II collagen production in chondrocytes, and reducing TNF-α production in macrophages. An object of the present invention is to provide a highly safe novel joint function improving agent capable of improving joint dysfunction due to the above, and food and drink.

  As a result of earnest research to achieve the above-mentioned object, the present inventors have been eating since ancient times or used as a traditional Chinese medicine, and the lotus germ that is a part of the lotus fruit whose safety has been established. It was found that the lotus germ extract extracted using the above has hyaluronic acid production promoting action, type II collagen production promoting action, and TNF-α production inhibiting action.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A joint function improving agent comprising a lotus germ extract.
<2> The joint function improving agent according to <1>, wherein the joint function improving agent is any one of a hyaluronic acid production promoter, a type II collagen production promoter, and a tumor necrosis factor (TNF-α) production inhibitor. It is.
<3> A food or drink comprising the joint function improving agent according to any one of <1> to <2>.
<4> A skin external preparation comprising the joint function improving agent according to any one of <1> to <2>.

  According to the present invention, the above-mentioned problems can be solved and the object can be achieved, increasing hyaluronic acid production in synovial cells, increasing type II collagen production in chondrocytes, and By reducing the amount of TNF-α produced in macrophages, a highly safe novel joint function improving agent capable of improving joint dysfunction due to osteoarthritis and the like, and food and drink can be provided. .

FIG. 1 is a graph showing the collagen production promoting action in Reference Example 1. FIG. 2 is a graph showing the collagen production promoting action in Example 2. FIG. 3 is a graph showing the collagen production promoting action in Example 3.

(Joint function improving agent)
The joint function improving agent of the present invention contains a lotus germ extract, and further contains other components as necessary. The joint function improving agent may be any one of a hyaluronic acid production promoter, a type II collagen production promoter, and a tumor necrosis factor (TNF-α) production inhibitor. The hyaluronic acid production promoter referred to in the present invention has an action of promoting the production of hyaluronic acid in synoviocytes as described above, and its action is confirmed by the test methods of Examples described later. Means a substance that can. The type II collagen production promoter has an action of promoting type II collagen production in chondrocytes, and the TNF-α production inhibitor suppresses TNF-α production in macrophages. It has an action. The joint function improving agent has at least one of the hyaluronic acid production promoting action, the type II collagen production promoting action, and the TNF-α production inhibiting action.

In the present invention, an embryo (embryo) in the seed of a lotus of the genus Nymphalidae ( Nelumbo lucifera Gaertn.) Is used. Embryos are usually green and stick-shaped, and those that are taken out from the collected seeds and dried raw or immediately are preferably used.

  The lotus germ extract is obtained by mixing the germ with water and various organic solvents, for example, lower aliphatic alcohols such as methanol and ethanol, polar solvents such as acetone and 1,3-butylene glycol, and intermediates such as chloroform and ethyl acetate. It can be obtained by extraction using a solvent or a mixture thereof, and further using a mixed extraction solvent of water and these organic solvents.

  In the extraction, the germ obtained from the lotus seed can be used as it is, but in order to increase the extraction efficiency, it is preferably used after pulverization. In addition, when a degreasing treatment is performed in advance using a non-aqueous solvent (non-polar solvent) such as hexane, the impurity content is reduced, and a more effective extract can be obtained.

  The extraction can be performed using an arbitrary apparatus depending on the extraction solvent at room temperature or under heating. For example, the ground lotus germ is introduced into a treatment tank filled with the extraction solvent, and the soluble components are eluted with occasional stirring. Then, it can filter and remove an extraction residue and can obtain an extract. The amount of the solvent used at this time is preferably about 1: 5 to 1:20 in mass ratio with respect to the lotus germ.

  The extract (liquid) thus obtained can be used as it is as a joint function improving agent as it is, but as long as the action of these agents is not hindered, a concentrated solution obtained by concentrating the extract or a further concentrated extract or extraction. It can also be provided as a dried product obtained by distilling off the solvent and the like.

  In addition, the lotus germ extract has a characteristic odor and taste, and therefore it is preferable to purify it for the purpose of deodorization, decolorization and the like to such an extent that its action is not reduced. Of course, depending on the purpose of use, for example, when it is contained in a food or drink according to the present invention, since it is not used in large quantities, it can be used as it is.

  Furthermore, in the joint function improving agent of the present invention, various sugars such as sucrose and lactose, excipients such as protein and starch, etc., in order to facilitate storage and handling of the extract as necessary Any auxiliary agent may be used. Moreover, it can also be provided by appropriately formulating, and for example, it can also be provided as an arbitrary dosage form such as powder, granule, tablet.

<Usage>
The joint function improving agent of the present invention is a hyaluronic acid production promoter that increases the amount of hyaluronic acid produced by synovial cells and exhibits effects on osteoarthritis by supplementing the joint with the produced hyaluronic acid. Applicable. Further, the joint function improving agent of the present invention induces differentiation of cartilage progenitor cells into chondrocytes, increases the production amount of type II collagen, which is a main component of cartilage, and improves wound healing of articular cartilage and healthy joints. It can be applied as a type II collagen production promoter that is effective for maintenance. In addition, the joint function improving agent of the present invention can be applied as a TNF-α production inhibitor having an effect of suppressing destruction of cartilage and suppressing inflammation by suppressing cytokine production in macrophages. Therefore, the hyaluronic acid production promoter, type II collagen production promoter and TNF-α production inhibitor of the present invention maintain smooth joint movement, prevent joint deformation, pain due to osteoarthritis, and movement disorders It can be used as a joint function improving agent that is safe for the human body and has an effect of preventing and improving the above. As the form, it can mix | blend and use for food-drinks or a skin external preparation, or can administer as a pharmaceutical formulation. These agents can be applied not only to humans but also to animals.

  When blending the joint function improving agent in the external preparation for skin, the blending amount (dry mass) of the lotus germ extract is preferably 0.0001% by mass to 70% by mass, and 0.001% by mass in the total amount of the external preparation. -30 mass% is more preferable.

  When the joint function improving agent is applied to an external preparation for skin, in addition to the above components, if necessary, components that are usually used in external preparations for skin such as cosmetics and pharmaceuticals, as long as the effects of the present invention are not impaired, for example, Antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder components, color materials, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  In addition, disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, sequestering agents such as gluconic acid, preservatives such as methylparaben, ethylparaben, butylparaben, caffeine, tannin, Verapamil, tranexamic acid or derivatives thereof, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid or derivatives thereof, salts thereof, vitamin C, magnesium ascorbate phosphate, Whitening agents such as ascorbic acid glucoside, arbutin, and kojic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose can also be appropriately blended.

  The external preparation for skin can be widely applied to cosmetics, quasi-drugs, etc., particularly preferably cosmetics applied to the outer skin, and the dosage form can also be applied to the skin. Any type of solution, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. can be used. Applied.

  The use form is also arbitrary, and for example, it can be used for facial cosmetics such as lotion, emulsion, cream and pack, makeup cosmetics such as foundation, lipstick and eye shadow, aromatic cosmetics, bath preparations and the like.

  Moreover, as a toiletry product, it can apply widely, for example to forms, such as a body soap and a soap. Furthermore, if it is a quasi-drug, it can be widely applied to various ointment forms. And the form which the joint function improving agent of this invention can take is not limited to these dosage forms and forms.

  When the joint function improving agent of the present invention is used as a pharmaceutical preparation, the preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.). The dosage form is also arbitrary, for example, any form such as oral solid preparations such as tablets, granules, powders, capsules, oral liquid preparations such as internal liquids and syrups, or parenteral liquid preparations such as injections. Moreover, it can prepare suitably by a well-known method. For these pharmaceutical preparations, commonly used binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonicity Excipients such as agents, stabilizers, pH adjusters and the like may be used as appropriate.

(Food)
The joint function improving agent of the present invention can be used orally by adding it to any food or drink, or as a dietary supplement based on these.
The food and drink is not particularly limited, for example, various soft drinks, fruit juice drinks, Japanese and Western sweets, dairy products and other livestock processed products, processed fruit products, processed vegetable products, processed grain products, processed fish products, There are many foods and drinks, such as so-called various health foods mainly composed of seasonings and vitamins. Moreover, as a dietary supplement, it can also provide as a thing of the form of a liquid agent or a tablet etc., for example. The addition rate in this case varies depending on the form of the extract such as liquid or solid, taking into account the general intake of food and drink to be added, but generally the lotus germ extract intake per day for adults As for about 1 mg to 1,000 mg, it is appropriate to add so as to be about 3 mg to 6,000 mg in terms of the dried lotus germ as a raw material.

  In addition, as long as the hyaluronic acid production promoting action, the type II collagen production promoting action, and the TNF-α production inhibiting action are not hindered, various raw materials usually used in the production of food and drink can be used for the food and drink according to the present invention. .

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1: Evaluation of hyaluronic acid production promoting action)
The lotus germ extract was tested for hyaluronic acid production promoting action in human synovial cells. Specifically, first, human synovial cells (HFLS-RA) derived from patients with rheumatoid arthritis were cultured for 7 days using a human synovial cell growth medium (manufactured by Synoviocyte Basal Medium, CELL APPLICATIONS, INC.). As a culture apparatus, a CO ₂ incubator manufactured by Sanyo Electric Co., Ltd. was used, and the culture conditions were a CO ₂ concentration of 5% and an incubation temperature of 37 ° C.
Thereafter, trypsin treatment was performed for 3 minutes using Trypsin / EDTA solution, and the cells were collected.
Next, the collected cells were mixed with 1% by weight fetal bovine serum (FBS) -containing human cell synovial cell growth medium (Synoviotic Basal Medium, CELL APPLICATIONS, 1.25 × 10 ⁵ cells / mL). After dilution with INC., 100 μL was seeded per well in a 96-well plate and cultured for 24 hours. After culture, 100 μL of lotus germ extract dissolved in 1% by mass FBS-containing human cell synovial cell growth medium was added to each well and cultured for 24 hours.

In addition, as the lotus germ extract, lotus germ extract powder MF (manufactured by Maruzen Pharmaceutical Co., Ltd.) was used. As the concentration of the lotus germ extract, three types of final concentrations in the medium of 25 μg / mL, 100 μg / mL, and 400 μg / mL were tested.
Subsequently, after culture, the amount of hyaluronic acid in the medium of each well was measured by a sandwich method using hyaluronic acid binding protein (HABP). Specifically, the culture supernatant was diluted 11-fold with AssayBuffer (0.5 M NaCl, 0.02% (v / v) Tween-20, 1% (w / v) BSA / PBS), and then applied to the HABP plate. Applied. After standing at room temperature for 2 hours, after washing with Washing Buffer (1.5M NaCl, 0.05% (v / v) Tween-20, dH ₂ O), a B-HABP solution (manufactured by Seikagaku Biobusiness Co., Ltd.) is used. 100 μL was added and allowed to react for 1 hour. Thereafter, color was developed with Streptavidin, Peroxidase Conjugate (manufactured by CALBIOCHEM), and the absorbance at 415 nm was measured. The amount of hyaluronic acid was converted from the standard curve of the sample, which is the absorbance measured for the sample obtained from each well. The results are shown in Table 1.
The calculation method of the hyaluronic acid production promotion rate is as follows.
Hyaluronic acid production promotion rate (%) = A / B × 100
However, A represents the amount of hyaluronic acid when the lotus germ extract is added, and B represents the amount of hyaluronic acid when the lotus germ extract is not added.

  As a control test for the above test, human synovial cells (HFLS-RA) derived from patients with rheumatoid arthritis were changed to neonatal skin fibroblasts (NB1RGB, RIKEN GENE BANK), and the medium was changed from human synovial cell growth medium to fibroblasts. The hyaluronic acid production promoting action of the lotus germ extract in neonatal skin fibroblasts was examined in the same manner as described above except that the medium was changed to the Medium Essential Medium Alpha Medium (GIBCO). The results are shown in Table 1.

From Table 1, it was found that the lotus germ extract has an excellent hyaluronic acid production promoting effect on human synovial cells. Moreover, since the lotus germ extract has no hyaluronic acid production promoting action on neonatal skin fibroblasts, it was found that the action is specific to synovial cells.

(Evaluation of type II collagen production promoting action: Reference Example 1 and Examples 2 and 3)
As a test sample, lotus germ extract powder MF (manufactured by Maruzen Pharmaceutical Co., Ltd.) was used, and the type II collagen production promoting action in chondrocytes was determined according to the methods described in JP2010-189315A and JP2011-213694A, The test was conducted as follows. The cartilage progenitor cell ATDC5 used in this test is a cell that differentiates into a chondrocyte after becoming confluent, and is known to produce type I collagen when undifferentiated and type II collagen when differentiated. (See “The Journal of Cell Biology”, vol. 133, No. 2, p. 457 (1996)). Therefore, the culture conditions were changed, and a test for confirming the action of the test sample during undifferentiation and during differentiation was performed.

(Reference Example 1)
Mouse-derived cartilage progenitor cells ATDC5 (obtained from RIKEN BioResource Center) are precultured using Dulbecco's MEM / Ham F-12 medium (mass ratio 1: 1, manufactured by Nissui Pharmaceutical Co., Ltd.) containing 5% by mass of FBS. Thereafter, the cells were collected by trypsin treatment. The collected cells were diluted with 5% FBS-containing Dulbecco's MEM / Ham F-12 medium to a concentration of 5.0 × 10 ⁴ cells / well, and then seeded at 500 μL per well in a 24-well plate. Cultured for days. After completion of the culture, the medium was removed, and 500 μL of a test sample dissolved in Dulbecco's MEM / Ham F-12 medium (without FBS) containing 0.1% by mass BSA was added to each well and cultured for 3 days. After completion of the culture, the medium was removed, the cells were fixed to the plate, and the collagen in the well was stained with Sirius Red (Wako Pure Chemical Industries, Ltd.) and the non-collagen protein was stained with Fast Green (Wako Pure Chemical Industries, Ltd.). After dyeing, the mixture was extracted with a mixed solution of methanol and 1N NaOH (mass ratio 1: 1), and the absorbance at a wavelength of 540 nm was measured. At the same time, the absorbance at a wavelength of 605 nm was measured as the amount of non-collagen protein, and the amount of collagen was calculated from the difference between the two.
In addition, the calculation method of a collagen production promotion rate is as follows.
Collagen production promotion rate (%) = A / B × 100
A: Collagen production when test sample is added B: Collagen production when no test sample is added (negative control)

  In addition, it confirmed by microscope observation that the cell which added the negative control which does not add a test sample, and a lotus germ extract powder MF is undifferentiated. Therefore, this test corresponds to the evaluation of the type I collagen production promoting action. In addition, as a positive control induced to differentiate, magnesium ascorbate phosphate was used instead of the lotus germ extract powder MF, and the same evaluation was performed. The results are shown in Table 2 and FIG.

  From Table 2 and FIG. 1, in the undifferentiated cartilage progenitor cells, lotus germ extract powder MF did not have a collagen production promoting effect. Therefore, it is considered that there is no type I collagen production promoting action.

(Example 2)
In Reference Example 1, the collagen production promotion rate was evaluated in the same manner as in Reference Example 1, except that the culture time after addition of the test sample was changed from 3 days to 14 days. The results are shown in Table 3 and FIG. It was confirmed by microscopic observation that the negative control cells to which no sample was added were undifferentiated. In addition, it was confirmed by microscopic observation that some of the cells added with lotus germ extract powder MF were differentiated into chondrocytes. In addition, as a positive control induced to differentiate, magnesium ascorbate phosphate was used instead of the lotus germ extract powder MF, and the same evaluation was performed.

  From Table 3 and FIG. 2, the collagen production promotion effect was recognized in the lotus germ extract powder MF. In Example 2, it is considered that the ability to promote collagen production of both type I and type II is detected, but when combined with the results of Reference Example 1, lotus germ extract powder MF induces differentiation to induce II. It is thought that the production of type collagen is promoted.

(Example 3)
In Reference Example 1, cells were fixed and stained in the same manner as in Reference Example 1 except that the cell culture conditions were changed as follows, and the collagen production promotion rate was calculated. The results are shown in Table 4 and FIG. The culture conditions in this example were conditions for differentiating cartilage progenitor cells into chondrocytes with magnesium ascorbate phosphate, and it was confirmed by microscopic observation that the cells were differentiated.
<Culture conditions>
Mouse-derived cartilage progenitor cells ATDC5 (obtained from Riken BioResource Center) were precultured using Dulbecco's MEM / Ham F-12 medium containing 5% by mass FBS, and then cells were collected by trypsin treatment. Differentiation induction medium [alpha-MEM culture medium (Nissui Pharmaceutical Co., Ltd. make, FBS is added to which 50 micrograms / mL magnesium ascorbate phosphate is added so that the collect | recovered cell may become a density | concentration of 5.0 * 10 < ⁴ > cells / well. No)), and then seeded in a 24-well plate at 500 μL per well and cultured for 7 days. After completion of the culture, the medium was removed, and 500 μL of a test sample dissolved in MEM / Ham F-12 medium (without FBS) containing 0.1% by mass BSA was added to each well and cultured for 1 day.

  From Table 4 and FIG. 3, the collagen production promotion effect by the lotus germ extract powder MF was recognized. Therefore, it is considered that the lotus germ extract powder MF has a type II collagen production promoting action on differentiated chondrocytes.

  From the results of Reference Example 1 and Examples 2 and 3, it is considered that the lotus germ extract has a type II collagen production promoting action. Further, from Example 2, it is possible to promote the production of type II collagen by inducing differentiation of undifferentiated cartilage progenitor cells, and from Example 3, it is possible to promote the production of type II collagen by differentiated chondrocytes. It was suggested. Accordingly, it is considered that the lotus germ extract has an effect of treating cartilage damage and maintaining healthy cartilage.

( Reference Example 4: Evaluation of tumor necrosis factor (TNF-α) production inhibitory effect)
Using lotus germ extract powder MF (manufactured by Maruzen Pharmaceutical Co., Ltd.) as a test sample, the tumor necrosis factor (TNF-α) production inhibitory action was tested by the following test method.

Mouse macrophage cells (RAW264.7, manufactured by Dainippon Pharmaceutical Co., Ltd.) were cultured using 10% by mass FBS-containing Dulbecco MEM (manufactured by Nissui Pharmaceutical Co., Ltd.), and the cells were collected using a cell scraper. The collected cells were diluted with Dulbecco's MEM containing 10% by mass FBS so as to have a concentration of 1.0 × 10 ⁶ cells / mL, and then seeded at 100 μL per well in a 96-well plate, at 37 ° C., 5% CO _2. The cells were cultured for 4 hours. After completion of the culture, the medium is removed, 100 μL of a test sample dissolved in 10% by mass FBS-containing Dulbecco MEM containing DMSO at a final concentration of 2% is added to each well, and the final concentration is 1 μg / mL to 10% by mass FBS-containing Dulbecco MEM. Dissolved lipopolysaccharide (LPS, E. coli
0111; B4, manufactured by DIFCO) was added and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After completion of the culture, the amount of TNF-α in the culture supernatant of each well was measured using the following sandwich ELISA method.
A rat anti-mouse TNF-α monoclonal antibody (manufactured by Endogen Inc.), which is a primary antibody, is dissolved in 50 mM sodium carbonate buffer (pH 9.6) to a concentration of 2.5 μg / mL, and 100 μL of the solution is dissolved in a 96-well microwell. In addition to the plate, it was coated overnight at 4 ° C. Subsequently, each well was washed with a washing solution (phosphate buffer containing 0.05% by mass of Tween 20) and then blocked with a phosphate buffer containing 1% by mass of BSA.
After washing each well with a washing solution, the culture supernatant was diluted with a test medium, and 100 μL thereof was added to each well and incubated at 37 ° C. for 120 minutes. After washing each well, a rabbit anti-mouse TNF-α polyclonal antibody (manufactured by Endogen Inc.) dissolved in a phosphate buffer containing 0.3% by mass BSA as a secondary antibody at a concentration of 2.5 μg / mL. 100 μL was added and incubated at 37 ° C. for 60 minutes before washing.
Next, 100 μL of 1,000-fold diluted alkaline phosphatase-labeled anti-rabbit IgG antibody (manufactured by CHEMICON Inc.) was added and incubated at 37 ° C. for 60 minutes. After washing each well, 150 μL of a substrate solution prepared by dissolving 50 mg of p-nitrophenyl phosphate in 100 mL of a color-development buffer (20 mM magnesium sulfate-containing Tris-HCl buffer, pH 8.0) was added to the well, and the mixture was incubated at 37 ° C. The color was developed by performing an enzyme reaction for 20 minutes to 30 minutes, and the absorbance at 405 nm was measured. From the standard curve prepared from the standard solution of recombinant mouse TNF-α (manufactured by Endogen Inc.), TNF- The α amount (pg / mL) was determined.
The inhibition rate of TNF-α production was calculated by the following formula.
TNF-α production inhibition rate (%) = {(BA) / B} × 100 (formula)
A: TNF-α amount when test sample is added B: TNF-α amount when test sample is not added (control)

In addition, the said TNF- (alpha) production suppression rate is measured by making the density | concentration of a test sample into 25 microgram / mL, 50 microgram / mL, and 100 microgram / mL, TNF- (alpha) is calculated | required by the interpolation method from these TNF-alpha production suppression rates. The test sample concentration IC ₅₀ (μg / mL) at which the production inhibition rate was 50% was determined. The results are shown in Table 5.

  From Table 5, it was shown that the lotus germ extract has a TNF-α production inhibitory action. Therefore, it is considered that the lotus germ extract suppresses the destruction of cartilage by suppressing the production of cytokines in macrophages and suppresses arthritis.

  According to the joint function improving agent of the present invention, joint dysfunction due to osteoarthritis and the like can be improved by hyaluronic acid production promoting action, type II collagen production promoting action and TNF-α production inhibiting action, It can be applied as a novel and highly functional joint function improving agent. Moreover, since the production amount of hyaluronic acid in synovial cells can be increased, it can be applied as a hyaluronic acid production promoter. Moreover, since the production amount of type II collagen in chondrocytes can be increased, it can be applied as a type II collagen production promoter. Moreover, since the production amount of TNF-α in macrophages can be reduced, it can be applied as a TNF-α production inhibitor. Moreover, it can contribute to the symptom relief of osteoarthritis by adding the said joint function improving agent to arbitrary food-drinks, or as a dietary supplement which has these as a main component. Specifically, the smooth movement of the joint can be maintained, the deformation of the joint can be prevented, and the prevention and improvement of pain due to osteoarthritis, movement disorder, and the like can be achieved.

Claims (2)

  Hyaluronic acid production promoter characterized by containing a lotus germ extract (except for food and drink use). Type II collagen production promoter characterized by containing a lotus germ extract (except for the purpose of improving inflammation due to osteoarthritis and the use of food and drink).