JP5194253B2 - Novel pharmacological use of proteoglycan contained in salmon cartilage - Google Patents

Description

  The present invention relates to a novel pharmacological use of proteoglycan contained in salmon cartilage.

Proteoglycan is a main component constituting animal cartilage together with collagen and hyaluronic acid, and has been known for a long time to have excellent water retention. In recent years, a technique for separating and purifying proteoglycan from salmon nasal cartilage safely and at low cost using only acetic acid, sodium chloride and ethanol has been established (Patent Document 1), and a large amount of proteoglycan can be supplied today. The research and development has been carried out energetically. However, the full pharmacological action of proteoglycan has not been clarified yet.
Japanese Patent No. 3731150

  Therefore, an object of the present invention is to provide a novel pharmacological use of proteoglycan contained in salmon cartilage.

  As a result of intensive studies in view of the above points, the present inventors have found that proteoglycans contained in salmon cartilage have a cell growth promoting action and a hyaluronic acid synthesis promoting action.

The present invention made on the basis of the above findings is a skin fibroblast proliferation promoter comprising, as an active ingredient, proteoglycan contained in salmon cartilage .
Also, the present invention is as claimed in claim 2 wherein a promoter of hyaluronic acid synthesis in dermal fibroblasts as an active ingredient proteoglycan contained in salmon cartilage.

  ADVANTAGE OF THE INVENTION According to this invention, the cell growth promoter and hyaluronic acid synthesis promoter as a novel pharmacological use of proteoglycan contained in salmon cartilage can be provided.

  The present invention is a cell growth promoter and hyaluronic acid synthesis promoter containing proteoglycan contained in salmon cartilage as an active ingredient. Proteoglycan contained in salmon cartilage, for example, according to the method described in Patent Document 1, after eluting crude proteoglycan from minced salmon nasal cartilage using acetic acid as an elution solvent, and then filtering the resulting eluate The semi-solid precipitate containing the crude proteoglycan obtained by centrifuging, adding salt-saturated ethanol to the supernatant and centrifuging is dissolved in acetic acid, and then dialyzed and separated and purified. The proteoglycan obtained from salmon nasal cartilage in this way is highly purified having a molecular weight of about 300 to 400 kDa, and is suitable as an active ingredient of the cell growth promoter and hyaluronic acid synthesis promoter of the present invention. is there. Note that proteoglycans obtained from salmon nasal cartilage according to the method described in Patent Document 1 are already commercially available as lyophilized powders.

  The cell growth promoter of the present invention has, for example, an excellent growth promoting action on dermal fibroblasts, and therefore can be applied as a pharmaceutical to wound healing, activation of skin metabolism, and the like. The administration can be performed by oral administration or parenteral administration (for example, subcutaneous administration, rectal administration, transdermal administration, etc.). What is necessary is just to formulate in the dosage form suitable for each administration method in the case of administration. Examples of the dosage form include tablets, capsules, granules, powders, fine granules, pills, troches, sublingual tablets, suppositories, ointments, emulsions, suspensions, syrups, and the like. Preparation of non-toxic excipients, binders, lubricants, disintegrants, preservatives, isotonic agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, buffering agents, etc. It can carry out by a method known per se using an additive. Non-toxic additives include, for example, starch, gelatin, glucose, lactose, fructose, maltose, magnesium carbonate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose, gum arabic, polyethylene glycol, propylene glycol, petrolatum, glycerin, ethanol Syrup, sodium chloride, sodium sulfite, sodium phosphate, citric acid, polyvinylpyrrolidone, water and the like. The content of the active ingredient in the preparation varies depending on the dosage form, but it is generally desirable that the concentration is 0.01 to 100% by weight. The dosage of the preparation can be widely adjusted according to the gender, age and weight of the administration subject, the severity of symptoms, the diagnosis of a doctor, etc., but can generally be 0.01 to 300 mg / Kg per day. The above dose may be administered once or divided into several times a day. The cell growth promoting agent of the present invention can be applied to the above-mentioned uses in the form of cosmetics such as creams to which an effective amount sufficient to exert a cell growth promoting effect is added, and various forms of foods (including supplements). You can eat as well. Moreover, since the hyaluronic acid synthesis promoter of the present invention promotes the synthesis of hyaluronic acid in vivo, it can be applied to prevent skin aging by activating cells and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to the following description and is not interpreted.

Example 1: Human skin fibroblast proliferation promoting effect of proteoglycan isolated and purified from salmon nasal cartilage (experimental method)
Human skin fibroblasts (HDF: Cascade Biologics, Portland, OR, USA) in a 100 mm dish, 5% CO ₂ , 37 ° C in a minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) After culturing until confluent under the above conditions, the cells were washed with Ca 2 ⁺ -, Mg ²⁺ -free phosphate-buffered saline (CMF-PBS). Next, lyophilized powder of proteoglycan (PG) separated and purified from salmon nasal cartilage in MEM containing 2% FBS (molecular weight 344 kDa, hexosamine: uronic acid: sulfuric acid = 1.00: 0.99: 0.67, Wako Pure Chemical Industries, Ltd.) Was added to a 35 mm dish and seeded with 3.0 × 10 ⁴ cells / ml and cultured. As a comparative control, HDF was cultured only in MEM containing 2% FBS without adding PG. At the start of culture and on the 2nd, 4th, 6th, and 8th days after the start of culture, the cells were treated with 0.25% trypsin, centrifuged to collect the cells, 1 ml of MEM was added to suspend the cells, trypan The cells were stained with blue and 1: 1, and the number of cells was counted with a Burker-Turk calculator.

(Test results)
The results are shown in Figure 1. By adding PG to the medium, the proliferation rate of human skin fibroblasts was significantly increased and the number of cells increased. Therefore, PG was found to have a cell growth promoting action.

Reference Example 1: Influence of PG on matrix metalloproteinase (MMP) activity of HDF
Gelatin zymography and casein zymography were performed to examine the effect of PG on MMP activity of HDF.
(experimental method)
A medium in which HDF was cultured under the conditions described in Example 1 was collected and used as a sample. SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 0.147) at a constant current of 15 mA / gel (gel size: 9 mm x 8 mm x 1 mm). 10% acrylamide gel containing 1% gelatin or casein). The gel after electrophoresis was gently shaken in 50 mM Tris-HCl buffer (0.1 M NaCl, 2.5% Triton X-100, pH 7.5) at room temperature for 1.5 hours to regenerate the denatured protein by SDS. The regenerated gel was immersed in 50 mM Tris-HCl buffer (0.1 M NaCl, 10 mM CaCl ₂ , pH 7.5) and incubated overnight at 37 ° C. Then, after staining with Coomassie Brilliant Blue R-250, decolorized in ethanol: acetic acid: H ₂ O (25: 8: 65), and further in ethanol: acetic acid: H ₂ O (10: 15: 175) Decolorized until appropriate. Enzyme activity was detected as a transparent band on a blue background.

(Experimental result)
The result is shown in figure 2. In gelatin zymography, a band showing enzyme activity was detected at a molecular weight of 67 kDa, but the enzyme activity did not differ depending on the presence or absence of PG (A: lane 1 was cultured in the absence of PG, lane 2 was Culture results in the presence of 0.1% PG). In casein zymography, bands showing enzyme activity were detected at positions of 45 kDa and 47 kDa, and this band was remarkably enhanced by PG (B: lane 1 was cultured in the absence of PG, lane 2 was 0.1%) Results of culture in the presence of PG).

Reference Example 2: Identification of MMP produced by HDF and the effect of PG Anti-human MMP-1, anti-human MMP-2, and anti-human MMP were identified to identify the MMP that showed activity in the zymography performed in Reference Example 1. Western blotting was performed using the -3 antibody.
(experimental method)
After collecting the medium in which HDF was cultured under the conditions described in Example 1, 0.25 units chondroitinase AC I Flavo ( Flavobacterium heparinum, Seikagaku Corporation) was added to 2 ml of the collected medium, and enzymatic digestion was performed at 37 ° C. for 2 hours. Then, centrifugal filtration with an inverted centrifugal filtration tube ARTKISS MWCO10000 (ADVANTEC) was performed for 1 hour, and ammonium sulfate (80% saturation, 560 mg / ml ammonium sulfate) was further added, and the mixture was allowed to settle overnight and concentrated. After conducting SDS-PAGE (10% acrylamide) at a constant current of 15 mA / gel (gel size: 9 mm x 8 mm x 1 mm), the protein in the gel was Hybond-ECL nitrocellulose membrane (7 cm x 8 cm, Amersham Biosciences, Piscataway) , NJ, USA) in transbuffer (containing 192 mM glycine, 25 mM tris aminometane, 20% methanol) using a semi-drive lotter AE-6677S (ATTO Corporation). The transferred membrane was blocked in a blocking solution (Tris buffered saline containing 0.1% Tween 20 and 3% skim milk) at room temperature for 1 hour, and then dissolved in TBST solution (Tris buffered saline containing 0.1% Tween 20). Anti-human MMP-1 antibody, anti-human MMP-2 antibody and anti-human MMP-3 antibody (all of which are Fuji Pharmaceutical Co., Ltd.) were reacted overnight at 4 ° C. After washing the membrane, it was reacted with ECL anti-mouse IgG horseradish peroxidase-linked whole antibody from sheep (Amersham Biosciences) for 1 hour at room temperature. After the membrane was washed, color was developed by adding ECL western blotting detection reagents (Amersham Biosciences), and chemiluminescence was detected with Chemi Doc XRS (Bio-Rad Laboratories, Hercules, CA, USA).

(Experimental result)
The results are shown in Figure 3. In the case of using the anti-human MMP-1 antibody, a band indicating a protein amount was detected at a molecular weight of 45 kDa, and the protein amount was increased by PG (lane 1 was cultured in the absence of PG, lane 2 was 0.1 in lane 2). Results of culture in the presence of% PG). In the case of using anti-human MMP-2 antibody, a band indicating the amount of protein was detected at a molecular weight of 67 kDa, but the amount of protein was not different depending on the presence or absence of PG (lane 3 was cultured in the absence of PG). As a result, lane 4 is a culture result in the presence of 0.1% PG). In the case of using the anti-human MMP-3 antibody, a band indicating the amount of protein was not detected (lane 5 was cultured in the absence of PG, and lane 6 was cultured in the presence of 0.1% PG).

Reference Example 3: Effects of PG on gene expression of MMP and hyaluronic acid synthase (HAS) produced by HDF
The expression of MMP and HAS mRNA produced by HDF was examined by RT-PCR.
(experimental method)
The cell layer of HDF cultured under the conditions described in Example 1 was washed with CMF-PBS, treated with 0.25% trypsin, centrifuged to collect cells, and RNeasy Mini Kit (QIAGEN, Japan) and QIAshredder (QIAGEN) were used. To extract total RNA. Total RNA was reverse transcribed into cDNA using Omniscript RT kit (QIAGEN) and Oligo (dT) 15 primer at 37 ° C for 60 minutes and 95 ° C for 3 minutes. This cDNA was amplified by PCR. Amplification was performed in a reaction solution containing 10 × PCR buffer, 5 × Q solution, dNTP mixture, sense and antisense primer, and HotStarTaq DNA Polymerase (QIAGEN). PCR primers are sense, 5'-CACAGCTTTCCTCCACTGCTGCTGC-3 '(SEQ ID NO: 1) for detecting MMP-1 gene; antisense, 5'-GGCATGGTCCACATCTGCTCTTGGC-3' (SEQ ID NO: 2), sense for detecting MMP-2 gene , 5'-ATGGCAAGGAGTACAACAGC-3 '(SEQ ID NO: 3); antisense, 5'-GCTGGTGCAGCTCTCATATT-3' (SEQ ID NO: 4), sense for detecting MMP-3 gene, 5'-ATGAAGAGTCTTCCAATCCTACTGT-3 '(SEQ ID NO: 5) Antisense, 5'-CATTATATCAGCCTCTCCTTCATAC-3 '(SEQ ID NO: 6), sense, 5'-GTGAGTGGCTGTACAACGCG-3' (SEQ ID NO: 7) for detecting the HAS1 gene; antisense, 5'-AGAGGGACGTAGTTAGCGGC-3 '(SEQ ID NO: 8) , Sense, 5'-TGGCATCACACCTCATCATC-3 '(SEQ ID NO: 9) for detecting the HAS2 gene; antisense, 5'-ACCAATTGCGTTACGTGTTG-3' (SEQ ID NO: 10), sense, 5'-TTGGCTGTGTGCAGTGTATTAGT-3 'for detecting the HAS3 gene (SEQ ID NO: 11); antisense, 5′-GGTCTCTGTGAGGCACTTGG-3 ′ (SEQ ID NO: 12) was used. Moreover, sense, 5′-CCACCCATGGCAAATTCCATGGCA-3 ′ (SEQ ID NO: 13); antisense, 5′-TCTAGACGGCAGGTCAGGTCCACC-3 ′ (SEQ ID NO: 14) was used as a control GAPDH gene detection primer. PCR cycle is 1 cycle (95 ° C 15 minutes), 25 cycles (94 ° C 1 minute, 54 ° C 1 minute, 72 ° C 1 minute), 1 cycle (72 ° C 10 minutes) for MMP-1, MMP-2 and GAPDH For MMP-3, 1 cycle (95 ° C for 15 minutes), 30 cycles (94 ° C for 1 minute, 54 ° C for 1 minute, 72 ° C for 1 minute), 1 cycle (72 ° C for 10 minutes), and HAS1 and HAS2 For HAS3, one cycle (95 ° C for 15 minutes), 35 cycles (94 ° C for 1 minute, 61 ° C for 1 minute, 72 ° C for 1 minute), and 1 cycle (72 ° C for 10 minutes) were used.

(Experimental result)
The results are shown in FIG. The gene expression level of MMP-1 and MMP-3 was significantly increased by PG, but the gene expression level of MMP-2 was not different depending on the presence or absence of PG (A: culture results in the absence of PG in lane 1) , Lane 2 is a culture result in the presence of 0.1% PG). In addition, the gene expression level of HAS2 was significantly increased by PG, but the gene expression level of HAS1 and HAS3 was not different depending on the presence or absence of PG (B: lane 1 was cultured in the absence of PG, lane 2 was Culture results in the presence of 0.1% PG).

Example 2: Promoting effect of PG on hyaluronic acid synthesis of HDF From Reference Example 3, it was found that the gene expression level of HAS2, which is the main enzyme of hyaluronic acid synthesis, was increased by PG. The effect of PG was examined by observation of hyaluronic acid coat using formalin-immobilized horse erythrocytes and measurement of [ ³ H] glucosamine incorporated into hyaluronic acid.
(experimental method)
(1) Hyaluronic acid coat observation
Glutaraldehyde stabilized horse red blood cells (Cosmo Bio) were fixed overnight with formalin neutral buffer solution diluted to 1.5% with CMF-PBS, washed 3 times with CMF-PBS, then 1.0 × 10 ⁸ cells / cell with CMF-PBS. Diluted to ml to form formalin-fixed horse erythrocytes. Using the medium described in Example 1, HDF was seeded in a 35 mm petri dish at 3.0 × 10 ⁴ cells / ml and cultured for 3 days. After removing the medium, formalin-immobilized horse erythrocytes were applied to the cells and examined with a phase contrast microscope at a magnification of 100 to observe a hyaluronic acid coat covering the periphery of the cells.
(2) Measurement of [ ³ H] glucosamine uptake into hyaluronic acid
HDF was cultured for 3 days in MEM containing 2% FBS supplemented with various concentrations of PG and 5μCi / ml [ ³ H] glucosamine (specific activity 40 Ci / mmol, ICR Radiochemicals, Irvine, CA, USA) Thereafter, the medium was collected, and actinase was added to each medium so that the final concentration was 0.1%, followed by incubation at 45 ° C. for 3 hours. Thereafter, 50% trichloracetic acid was added in an amount of 1/5 of the reaction mixture, mixed and allowed to stand at 0 ° C. This was deproteinized by centrifugation, and 4 times the amount of salt-saturated ethanol was added to the supernatant. The 80% ethanol-insoluble fraction was precipitated at -20 ° C, and the washing operation to obtain the precipitate by centrifugation was repeated three more times. It was. The obtained 80% ethanol-insoluble fraction was divided into two, 6.25TRU Streptomyces hyaluronidase (Seikagaku Corporation) dissolved in 0.25M sodium acetate buffer (pH 6.0) on one side and 0.25M sodium acetate buffer (pH 6) on the other side. .0) alone was added and allowed to react overnight at 37 ° C. Perform 4-fold sodium chloride saturated ethanol precipitation and mix the obtained supernatant with liquid scintillator EMULSIFIER-SAFE (Packard, MERIDEN, CT, USA) 1: 4, and the radioactivity is liquid scintillation counter LSC-5100 (ALOKA Co Ltd.) and measured one sample for 4 minutes.

(Experimental result)
(1) Observation of hyaluronic acid coat The results are shown in FIG. As indicated by the arrows, the amount of hyaluronic acid around the cells not covered by red blood cells was increased by PG.
(2) Measurement of [ ³ H] glucosamine uptake into hyaluronic acid is shown in FIG. The radioactivity of [ ³ H] Glucosamine increased with increasing PG concentration, indicating that PG promotes hyaluronic acid synthesis of HDF in a concentration-dependent manner.

Reference Example 4: Effect of enzyme-digested PG on MMP-1, MMP-3 and HAS2 gene expression produced by HDF
In order to investigate whether the active site exists in the PG core protein or the sugar chain, the core protein obtained by degrading the PG sugar chain with chondroitinase and the sugar obtained by decomposing the PG core protein with actinase The effects of MMP-1, MMP-3 and HAS2 on gene expression were examined for the chains.
(experimental method)
Add 0.4units chondroitinase ABC protease free ( Proteus vulgaris, Seikagaku Corporation) and 0.5M Tris-HCl buffer (pH 8.0) to 2.5% PG solution, and incubate at 37 ° C for 1 hour to remove PG sugar chains. Disassembled. In addition, 1% actinase and 0.5 M Tris-HCl buffer (10 mM CaCl ₂ , pH 8.0) are added to a 2.5% PG solution and incubated overnight at 45 ° C. to degrade the PG core protein. Deproteinization was performed using chloracetic acid, and the pH was brought close to 8.0 with buffer. HDF was cultured using MEM containing 2% FBS to which the core protein and sugar chain of PG thus obtained were added in an amount corresponding to 0.1% as PG, respectively. Total RNA was extracted from the cell layer and RT-PCR was performed.

(Experimental result)
The results are shown in FIG. The core protein of PG increased all the gene expression levels of MMP-1, MMP-3 and HAS2 (lane 1), and the extent was almost the same as when 0.1% PG was added (lane 4). On the other hand, even when PG sugar chains were added, the expression levels of these genes did not increase (lane 2), and were the same as the culture results in the absence of PG (lane 3).

Summary of Examples and Reference Examples (Discussion)
PG is organically incorporated into the cellular environment as an extracellular matrix component, and increases hyaluronic acid synthesis by increasing the HAS2 gene expression level in the cell, thereby contributing to the formation of an environment suitable for cell growth. When it promotes the production of MMP-1 and MMP-3 in cells that play an important role in the turnover of structural proteins that form the cellular environment and securing the space for cell growth, and as a result, it exerts a growth promoting effect on cells it was thought.

Formulation Example 1: Tablets Proteoglycan freeze-dried powder separated from salmon nasal cartilage, lactose, starch, carboxymethylcellulose, talc, and magnesium stearate were mixed well and then tableted.

Formulation Example 2: Capsule A capsule was prepared by thoroughly mixing each component of freeze-dried proteoglycan powder separated from salmon nasal cartilage, lactose, starch, and magnesium stearate, and then filled into capsules.

Formulation example 3: Cream preparation Proteoglycan lyophilized powder isolated and purified from salmon nasal cartilage, stearyl alcohol, stearic acid, hydrogenated lanolin, squalane, octyldodecanol, propylene glycol, polyoxyethylene cetyl ether, glyceryl monostearate A cream was prepared from preservatives, fragrances and purified water according to a conventional method.

  The present invention has industrial applicability in that it can provide a cell growth promoter and a hyaluronic acid synthesis promoter as novel pharmacological uses of proteoglycans contained in salmon cartilage.

3 is a graph showing the HDF growth promoting action of PG in Example 1. 3 is a zymography result showing the effect of PG on the HDF MMP activity in Reference Example 1. FIG. It is the result of the Western blot which shows the identification of MMP which HDF in the reference example 2 produces, and the influence of PG. FIG. 6 is a result of RT-PCR showing the effect of PG on the gene expression of MMP and HAS produced by HDF in Reference Example 3. FIG. FIG. 3 is a phase-contrast microscope photograph showing the promoting action of PG on the hyaluronic acid synthesis of HDF in Example 2 (by observation of hyaluronic acid coat). ³ is a graph showing the action of PG to promote hyaluronic acid synthesis by HDF (measured by [ ³ H] glucosamine incorporation into hyaluronic acid). It is the result of RT-PCR which shows the influence of enzyme digestion PG on the gene expression of MMP-1, MMP-3 and HAS2 which HDF produces in the reference example 4.

Claims (2)

A skin fibroblast proliferation promoter comprising proteoglycan contained in salmon cartilage as an active ingredient . An agent for promoting hyaluronic acid synthesis in dermal fibroblasts, which contains proteoglycan contained in salmon cartilage as an active ingredient.