JP7193069B2 - Iron salt of proteoglycan, proteoglycan, method for producing the same, cell growth promoter, and preparation for external use - Google Patents

Description

TECHNICAL FIELD The present invention relates to an iron salt of proteoglycan, a novel proteoglycan having a specific infrared absorption peak, a method for producing the same, and a cell growth-promoting agent and an external preparation containing them.

Proteoglycans are present in animals and form the matrix in the extracellular matrix of connective tissue. Proteoglycan is a general term for covalently bound products of glycosaminoglycan and protein, and is characterized by extremely high sugar content compared to general glycoproteins. Proteoglycan is a naturally occurring high-molecular compound, and its molecular weight and the types, amounts, and ratios of amino acids and sugars (neutral sugars, uronic acids, amino sugars, etc.) it contains differ depending on the source material and extraction/manufacturing conditions. There are various molecular species.
It is known that alkali metal salts of proteoglycans have high water retention capacity (eg, Patent Document 1).
On the other hand, proteoglycans have been known to have a skin fibroblast proliferation-promoting activity, and one of the active mechanisms is known to be via an increase in phosphorylated Erk1/2 expression (e.g., Non-Patent Document 1).
Thus, compounds derived from proteoglycans and compounds derived from proteoglycans (hereinafter also simply referred to as "proteoglycan compounds") can have beneficial physiological activities, and therefore novel proteoglycan compounds are desired. was

JP 2016-29150 A

Sano M et al, Biosci Biotechnol Biochem 81(7): 1369-78, 2017

The present invention has been made in view of the above-mentioned problems of the prior art, and includes iron salts of proteoglycans, novel proteoglycans having a specific infrared absorption peak, methods for producing them, and cell growth promoting agents and topical preparations containing them. for the purpose of providing

The present inventors have found that iron salts of proteoglycans or novel proteoglycans having specific peaks in the infrared absorption spectrum have stronger cell growth-promoting activity than conventional proteoglycans, especially transforming known as a growth factor involved in cell growth. It was found to exhibit the activity of promoting the expression of the growth factor TGF-β1. The present invention has been completed based on this finding. That is, the present invention is as follows.

(1) Iron salts of proteoglycans.

(2) A proteoglycan having an absorption peak at wavenumbers of 2800 cm ⁻¹ to 2930 cm ⁻¹ in an infrared absorption spectrum measured by the KBr disk transmission method.

(3) The proteoglycan or iron salt thereof according to (1) or (2) above, which has an iron content of 1% by mass or more and a calcium content of 1% by mass or less per dry weight.
(4) The proteoglycan or iron salt thereof according to any one of (1) to (3) above, which is a colloidal particle.
(5) The proteoglycan or iron salt thereof according to any one of (4), wherein the colloidal particles have an average particle size of 500 nm to 1500 nm.
(6) A method for producing the proteoglycan or iron salt thereof according to any one of (1) to (5) above, which method comprises reacting the raw material proteoglycan and an iron compound.

(7) A cell proliferation promoting agent comprising the proteoglycan or iron salt thereof according to any one of (1) to (5) above.
(8) An external preparation containing the proteoglycan or iron salt thereof according to any one of (1) to (5) above.

The proteoglycan or iron salt thereof of the present invention is a novel substance, and can have, for example, a stronger cell proliferation activity than conventional proteoglycans, and particularly can have TGF-β1 expression promoting activity.
The production method of the present invention can produce the proteoglycan or its iron salt.
According to the present invention, it is possible to provide a cell growth-promoting agent and an external preparation containing the proteoglycan or its iron salt.

FIG. 4 is a diagram showing quantitative RT-PCR test results of TGF-β1 mRNA expression levels in the Fe-PG-applied group and the PG-applied group. FIG. 10 is a diagram showing the results of evaluating Fe-PG concentration-dependent NHDF proliferation promotion.

Embodiments of the present invention will be described in detail below, but the present invention is by no means limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention. .
In addition, in this specification, unless otherwise specified, "-" represents from the above to the following.

<Proteoglycan or iron salt of proteoglycan>
The present invention relates to iron salts of proteoglycans.
The present invention also relates to a proteoglycan having an absorption peak at a wave number of 2800 cm ⁻¹ to 2930 cm ⁻¹ in an infrared absorption spectrum measured by a KBr disk transmission method.
The proteoglycan of the present invention preferably has two absorption peaks at wave numbers of 2800 cm ⁻¹ to 2930 cm ⁻¹ , one absorption peak at wave numbers of 2830 cm ⁻¹ to 2880 cm ⁻¹ and a wave number of 2890 cm ⁻¹ to 2930 cm ⁻¹ . It is more preferable to have one absorption peak at wavenumbers of 2840 cm ^-1 to 2870 cm ^-1 and one absorption peak at wavenumbers of 2900 cm ^-1 to 2925 cm ^-1 , It is particularly preferable to have one absorption peak at wave numbers of 2845 cm ⁻¹ to 2860 cm ⁻¹ and one absorption peak at wave numbers of 2905 cm ⁻¹ to 2920 cm ⁻¹ .
The above absorption peak preferably has a transmittance of 50% or less at a wave number of 2830 cm ^-1 to 2880 cm ^-1 when the transmittance at 1850 cm ^-1 to 1900 cm ^-1 is 100%. may be 30% or more, but may be 30% or less.
Also, the transmittance of the peak between 2890 cm ⁻¹ and 2930 cm ⁻¹ is preferably 30% or less.
The above absorption peak may occur due to proteoglycan forming an iron salt.
Hereinafter, the proteoglycan of the present invention and the iron salt of the proteoglycan of the present invention are collectively referred to as "proteoglycan or its iron salt".
The proteoglycan or iron salt thereof of the present invention may have absorption peaks similar to those of the raw material proteoglycan described later (particularly, peaks with a transmittance of more than 50%), in addition to the absorption peaks described above.

In the proteoglycan or its iron salt of the present invention, the iron content per dry weight is not particularly limited as long as it does not impair the effects of the present invention, but it is preferably 1% by mass or more, and 2% by mass or more. is more preferable, 3% by mass or more is still more preferable, and 4% by mass or more is particularly preferable.
The upper limit of the iron content is not particularly limited, and is, for example, 10% by mass or less, typically 8% by mass or less.

The proteoglycan or iron salt thereof of the present invention may or may not contain components other than iron and the proteoglycan that forms the iron salt (hereinafter also simply referred to as "other components"). is preferably less than the content of iron per dry weight (if more than one other component is present, the total content).
Various minerals etc. are mentioned as said other component.
Examples of various minerals include calcium, magnesium, potassium, sodium, phosphorus, chlorine and the like.
In the proteoglycan or iron salt thereof of the present invention, the content of other components (e.g., calcium) per dry weight is preferably 1% by mass or less, more preferably 0.7% by mass or less, and 0 0.5% by mass or less is more preferable.

The proteoglycan or iron salt thereof of the present invention may or may not be colloidal particles, but is preferably colloidal particles.
The average particle diameter of the colloidal particles is preferably 500 nm to 1500 nm, more preferably 700 nm to 1200 nm, even more preferably 800 nm to 1000 nm.
The average particle size can be measured using a dynamic light scattering particle size distribution analyzer (eg, LB-550, manufactured by Horiba Ltd.).

The method for producing the proteoglycan or its iron salt of the present invention is not particularly limited as long as the iron salt of the proteoglycan is formed. is preferred.

The proteoglycan of the present invention or proteoglycan, which is a raw material for the iron salt thereof, is a general term for covalently bound products of glycosaminoglycans and proteins, and is characterized by an extremely high sugar content compared to general glycoproteins. Proteoglycan is a naturally occurring high-molecular compound, and its molecular weight and the types, amounts, and ratios of amino acids and sugars (neutral sugars, uronic acids, amino sugars, etc.) it contains differ depending on the source material and extraction/manufacturing conditions. There are various molecular species. Molecular weights of proteoglycans are generally several hundred thousand to several million. Proteoglycans are derived from mammalian cartilage such as cows, chickens, and whales, and fish cartilage such as salmon, sharks, and rays. In addition, there are various proteoglycan extraction agents such as organic acids such as acetic acid, acids, alkalis, and guanidine hydrochloride, but the present invention does not limit extraction and production conditions such as extraction agents and temperature and time.

The raw material proteoglycan may or may not contain other components, and the content of other components (e.g., calcium) per dry weight (when multiple other components are present, total content) is, for example, 10% by mass or less, preferably 8% by mass or less, and more preferably 6% by mass or less.
The lower limit of the content of other components (such as calcium) is not particularly limited, and is, for example, 0.3% by mass or more, typically 0.5% by mass or more.

<Method for producing proteoglycan or its iron salt>
The method for producing proteoglycan or its iron salt according to the present invention includes reacting the aforementioned proteoglycan as a raw material with an iron compound.
The above reaction is not particularly limited as long as it can form an iron salt. A nucleophilic reaction to an iron atom by a functional group possessed by is preferably a salt exchange reaction by an iron ion contained in the iron compound.
Examples of the iron compound include any inorganic iron salts (e.g., iron sulfate, iron chloride, etc.) or organic iron salts (e.g., iron acetate), with inorganic iron salts being preferred, iron (II) sulfate heptahydrate is more preferred.

The reaction conditions between the proteoglycan as a raw material and the iron compound are preferably 0° C. or higher and 20° C. or lower, more preferably 0° C. or higher and 10° C. or lower, and more preferably 0° C. or higher and 5° C. or lower. More preferred.
The reaction time is preferably 1 hour or longer, more preferably 10 hours or longer, and even more preferably 20 hours or longer.

The method for producing proteoglycan or its iron salt of the present invention preferably includes purifying the obtained proteoglycan or its iron salt after the above reaction.
The purification method preferably includes at least one selected from the group consisting of dialysis and filtration, and more preferably includes both dialysis and filtration.
The dialysis method is not particularly limited, and is preferably carried out at 0°C or higher and 20°C or lower, more preferably at 0°C or higher and 10°C or lower, and even more preferably at 0°C or higher and 5°C or lower.
The filtration method is also not particularly limited, and any filter paper can be used.
If necessary, freeze-drying can be performed to obtain the proteoglycan or iron salt thereof of the present invention.

<Cell proliferation promoting agent>
The cell growth promoting agent of the present invention contains the proteoglycan or its iron salt.
Since the cell proliferation promoting agent of the present invention has, for example, an excellent proliferation promoting effect on skin fibroblasts, it can be applied as a pharmaceutical for wound healing, activation of skin metabolism, and the like. The administration is preferably transdermal administration.
For administration, it may be formulated into a dosage form suitable for each administration method. Formulations include, for example, ointments, emulsions, suspensions, etc., and preparation of these formulations includes non-toxic excipients, binders, lubricants, disintegrants, preservatives, isotonic agents, Additives such as stabilizers, dispersants, antioxidants, coloring agents, corrigents, and buffering agents can be used in a manner known per se. Examples of non-toxic additives include 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 formulation varies depending on its dosage form. Conventional proteoglycans generally require a concentration of 0.01 to 100% by mass, whereas the cell growth promoting agent of the present invention Even at a concentration of 0.0001 to 1% by mass, it is possible to exert a cell growth-promoting effect.
The dosage of the formulation can be adjusted widely according to the sex, age and weight of the subject to be administered, severity of symptoms, diagnosis by a doctor, etc., but conventional proteoglycans are generally 0.01 to 300 mg/kg per day. As required, the cell proliferation-promoting agent of the present invention can exhibit cell proliferation-promoting action even at 0.0001 to 3 mg/kg per day.
The above dosage may be administered once or divided into several times a day. The cell proliferation-promoting agent of the present invention may be applied in the form of cosmetics to which an effective amount sufficient to exhibit cell proliferation-promoting action is added for the above uses.

<External agent>
The external preparation of the present invention contains the proteoglycan or its iron salt.
The external preparation of the present invention is not particularly limited as long as it is applied to the skin, and examples thereof include medical external preparations for skin and cosmetics. In addition to the above-mentioned proteoglycan or its iron salt, the topical preparation of the present invention includes ingredients that are usually used in skin topical preparations such as pharmaceuticals, quasi-drugs, and cosmetics, such as aqueous ingredients, oily ingredients, powder ingredients, and alcohols. , moisturizing agents, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, pigments, and the like can be blended as needed. Such formulation can be carried out according to the method described in the General Rules for Pharmaceutical Preparations of the Japanese Pharmacopoeia.

The "dosage form" of the external preparation of the present invention is not particularly limited as long as it is a dosage form that can be applied to the skin. , adhesive patches such as patches, and the like. In addition, the external preparation of the present invention can be used as pharmaceuticals, quasi-drugs, cosmetics, and the like.
When the topical preparation is a topical preparation, the concentration of the proteoglycan or its iron salt in the topical preparation is not particularly limited as long as it does not impair the effects of the present invention. .4 to 400 μg/ml is preferred.
When the external preparation is a topical preparation, the method of use is to take an appropriate amount (e.g., several grams (1 g, etc.), several tens of μl (50 μl, etc.)) and apply it once to several times a day (e.g., 2 times). or 3 times) can be applied to the skin.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the scope of the present invention is not limited to these Examples.

<Example 1>
[Preparation of iron salt of proteoglycan]
Commercially available salmon-derived proteoglycan (manufactured by Kakuhiro Proteoglycan Research Institute) was used as the raw material proteoglycan.
First, 100 mg of the raw material proteoglycan was dissolved in 50 mL of deionized water to obtain liquid A, and then 0.2 M iron (II) sulfate heptahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) Liquid B was obtained by dissolving, and liquids A and B were precooled to 4°C.
After adding B liquid while stirring A liquid with a stirrer at 4°C, the mixture was stirred with a stirrer overnight at 4°C.
The resulting reaction solution was placed in a dialysis cellulose tube (manufactured by EIDIA) and dialyzed against deionized water in a low temperature room at 4°C. Water was exchanged 3 times a day for 3 days.
The precipitate generated in the dialysis cellulose tube after dialysis was filtered using a glass filter paper (GA100, manufactured by Advantech Toyo Co., Ltd.).
After the above filtration, the filtrate was collected, concentrated under reduced pressure with a rotary evaporator (manufactured by Tokyo Rikakikai Co., Ltd.), and freeze-dried (manufactured by Tokyo Rikakiki Co., Ltd.) to obtain an iron salt of proteoglycan as a flocculent light brown substance.

[Particle size distribution measurement of iron salt of proteoglycan]
The particle size distribution of the obtained iron salt of proteoglycan was measured using a dynamic light scattering particle size distribution analyzer (LB-550, manufactured by Horiba, Ltd.).
The iron salt of the proteoglycan was adjusted to 0.2% (W/V) using deionized water, and the result of measurement was that the colloidal particles had an average particle size of 952.1 nm.

[Measurement of iron content and calcium content]
The iron content and calcium content of the obtained iron salt of proteoglycan were measured using an energy dispersive X-ray fluorescence analyzer (EDX-800HS, manufactured by Shimadzu Corporation).
Iron (III) oxide (manufactured by Wako Pure Chemical Industries, Ltd.) and calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) are sufficiently mixed with corn starch (manufactured for exclusive use, manufactured by Wako Pure Chemical Industries, Ltd.) to a predetermined concentration. , a calibration curve of the iron content and calcium content and the fluorescence intensity was prepared.
As a result of the above measurement, the iron content of the obtained iron salt of proteoglycan was 4.8% by mass based on the dry weight. The calcium content was 0.5% by mass or less, which is the lower limit of the calibration curve.
The calcium content of proteoglycan as a raw material was analyzed by chelate titration and found to be 5.3% by mass per dry weight.
The iron content of the starting proteoglycan was 0.5% by mass or less, which is the lower limit of the calibration curve.

[Measurement of infrared absorption spectrum of iron salt of proteoglycan]
The infrared absorption spectrum of the iron salt of proteoglycan obtained above was measured by the KBr disk transmission method using a Fourier transform infrared spectrophotometer (FT/IR-420, manufactured by JASCO Corporation). Measurement was performed under conditions of a measurement range of 4000 to 400 cm ⁻¹ , a resolution of 4 cm ⁻¹ , an accumulation number of 54, and a scan speed of 2 mm/sec.
As a result of the above measurement, two peaks 2919 cm ^-1 (transmittance 24%) and 2851 cm ^-1 were found in the iron salt of proteoglycan, which were not found in the starting proteoglycan and iron (II) sulfate heptahydrate.
(41% transmittance) was observed.

<Example 2>
[Evaluation of proteoglycan iron salt promoting expression of TGF-β1]
7-week-old female ICR mice (manufactured by CLEA Japan) were divided into the following Fe-PG application group, PG application group and control group with 8 mice in each group, and kept in a breeding room with a constant temperature and humidity constant environment. After 8 days of breeding, left auricle and back skin were collected. The handling of experimental animals was approved by the Hirosaki University Animal Care and Use Committee and complied with the Hirosaki University Animal Experiment Regulations.

Fe-PG application group: A group in which the iron salt of proteoglycan obtained in Example 1 (hereinafter also simply referred to as "Fe-PG") was applied to the back in an amount of 0.01 mg/back for 7 consecutive days.
PG application group: A group in which commercially available salmon-derived proteoglycan (manufactured by Kakuhiro Proteoglycan Laboratory Co., Ltd.) (hereinafter also simply referred to as "PG") was applied to the back in an amount of 0.01 mg/back for 7 consecutive days, and a control group: A group in which neither Fe-PG nor PG was included in the drinking water administered for 7 days, and neither Fe-PG nor PG was applied to the back for 7 consecutive days.

Using the skin of each group collected above, total RNA was extracted, and quantitative RT-PCR was performed using the cDNA obtained by the reverse transcription reaction and using GAPDH expression as an internal standard.

FIG. 1 shows quantitative RT-PCR test results of TGF-β1 mRNA expression levels in the Fe-PG-applied group and the PG-applied group.
As is clear from the results shown in FIG. 1, compared to the PG-coated group, the Fe-PG-coated group showed a higher TGF-β1 mRNA expression level with a significant difference of less than 0.05 (P value (Tukey method)). I understand.

<Example 3>
[Evaluation of Proteoglycan Iron Salt Proliferation Promotion of Human Skin Fibroblasts]
The proteoglycan iron salt (Fe-PG) concentration-dependent growth-promoting effect on human dermal fibroblasts (NHDF) was evaluated using Fe-PG or PG as a control at 0.049, 0.098, 0.195, 0. 391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, 800 μg/ml in medium supplemented with WST-8 assay after 72 hours. evaluated. Specifically, it is as follows.

(Culture of NHDF)
NHDF was purchased from Kurabo and cultured at 37°C in 5% _CO2 , 95% air. The medium used was DMEM medium containing 10% FBS (fetal bovine serum; product name SH30910.03; manufactured by Thermo Fisher Scientific), 50 units/ml penicillin and 50 mg/ml streptomycin (product name 08456-36; manufactured by Nacalai Tesque). board.

(WST-8 assay)
A cell counting kit (trade name CK04; manufactured by Dojindo) containing WST-8 [3-(4,5-dimethyl 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] was used.
NHDF cells were plated on a 96-well culture plate at a concentration of 4×10 ⁴ cells/ml, 100 μl per well, and cultured for 24 hours. Each well was replaced with 100 μl of DMEM medium containing penicillin/ml and 50 mg/ml streptomycin and cultured for 72 hours.
After that, 10 μl of WST-8 was added to each well and cultured for 1 hour. The absorbance of the 96-well culture plate was measured at 450 nm using a microplate reader (trade name TriStar LB 941; manufactured by Berthold Technologies). The results are shown in FIG. In FIG. 2, the values are expressed as % of PG as a control.

As is clear from the results shown in FIG. 2, Fe-PG has the effect of significantly promoting NHDF proliferation at concentrations of 0.391 to 400 μg/ml compared to control PG.

Claims (7)

Iron salts of proteoglycans derived from iron(II) sulfate heptahydrate. 2. The iron salt of proteoglycan according to claim 1, wherein the iron content per dry weight is 1% by mass or more and the calcium content is 1% by mass or less. A cell growth promoting agent comprising the proteoglycan iron salt according to claim 1 or 2. An external preparation comprising the proteoglycan iron salt according to claim 1 or 2. 5. A cell growth-promoting agent or external preparation containing the iron salt of proteoglycan according to claim 3 or 4, which is a colloidal particle. 6. The cell growth-promoting agent or external preparation containing the iron salt of proteoglycan according to claim 5, wherein the colloidal particles have an average particle size of 500 nm to 1500 nm. 3. A method for producing the iron salt of proteoglycan according to claim 1 or 2, comprising reacting the raw material proteoglycan and the iron compound at 0°C or higher and 10°C or lower.

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