Process for the production of proteoglycans

Abstract

The present invention relates to a method for producing a plant-derived proteoglycan, for example, including a process of extracting a proteoglycan from a plant, and the like. The present invention provides a method for producing a plant-derived proteoglycan having a molecular weight of more than 100,000 and less than 5,000,000, including a process of dissolving ghatti gum using water as a solvent, and a process of fractionating the molecular weight of a solution obtained in the dissolving process.

Description

[0001] Cross-references

[0002] This application claims priority based on Japanese Patent Application No. 2021-178577, filed on November 1, 2021, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to a method for manufacturing plant-derived proteoglycans, including, for example, a process for extracting proteoglycans from plants. Background Technology

[0004] Proteoglycans (hereinafter sometimes referred to as "PGs") are glycoproteins with several to dozens of glycosaminoglycan chains such as chondroitin sulfate and keratin sulfate covalently bound to a core protein. They are widely distributed in the body, including skin and cartilage, as part of the extracellular matrix. In cartilage, PGs form aggregates with collagen and hyaluronic acid; a representative cartilaginous PG is called agglutinin aggregates. Agglutinin aggregates have numerous glycosaminoglycan chains bound to the core protein and possess hyaluronic acid and linker protein binding regions at their N-terminus.

[0005] Glycosaminoglycans possess a long, straight-chain structure without branches. Due to the presence of multiple sulfate and carboxyl groups, they carry a negative charge and exhibit an elongated shape due to electrostatic repulsion. Furthermore, due to their hydrophilicity, sugars retain a large amount of water, fulfilling the unique cartilage function of resisting elasticity and impact. Consequently, PGs are known to possess various physiological functions, including anti-inflammatory effects, hyaluronic acid synthesis promotion, and epidermal growth factor (EGF)-like effects, promising applications in food and cosmetics. As proteoglycans derived from animals, examples include those derived from salmon nasal cartilage and squid head cartilage.

[0006] However, since animal-derived proteoglycans are present in trace amounts in cartilage, their manufacturing cost is high. Furthermore, in the cosmetics and food markets, plant-derived ingredients have a superior image compared to animal-derived ingredients, thus there is a demand to replace animal-derived proteoglycans with plant-derived ingredients (Patent Document 1).

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Application Publication No. 2019-172718

[0010] Patent Document 2: WO2018 / 062554

[0011] Non-patent literature

[0012] Non-Patent Document 1: Journal of Japanese Biochemical Society 89(4):498-507(2017) Contents of the Invention

[0013] Technical Problems to Be Solved by the Invention

[0014] The present invention has been completed under such circumstances, and an object thereof is to provide a method for producing a plant-derived proteoglycan having an excellent physiological action from a plant, and the like.

[0015] Solutions to the Technical Problems

[0016] In order to solve the above problems, the present inventors have found that a plant-derived proteoglycan containing a predetermined amount of arabinogalactan protein can be efficiently produced at a predetermined purity from ghatti gum, thereby completing the present invention.

[0017] That is, the present invention includes the following embodiments.

[0018] (1) A method for producing a plant-derived proteoglycan having a molecular weight of more than 10,000 and not more than 5,000,000, comprising the following steps:

[0019] a step of dissolving a part of a Combretaceae plant using water as a solvent; and

[0020] a step of subjecting the solution obtained in the dissolving step to molecular weight fractionation.

[0021] (2) The method according to (1), wherein the plant-derived proteoglycan having a molecular weight of more than 50,000 and not more than 1,000,000 is produced. The lower limit of the molecular weight is more preferably 100,000, still more preferably 200,000, and still more preferably 300,000. The upper limit of the molecular weight is more preferably 500,000, still more preferably 400,000.

[0022] (3) The method according to (1) or (2), wherein a microfiltration membrane having a nominal pore size of 0.1 to 250 μm (more preferably 0.1 to 10 μm) is used.

[0023] Effects of the Invention

[0024] The method for producing a plant-derived proteoglycan of the present invention can produce a plant-derived proteoglycan having an excellent physiological action from a plant. Brief Description of the Drawings

[0025] Figure 1 The results obtained by analyzing the composition of Production Example 1 by HPLC are shown. Detailed Implementation

[0026] The present invention will be described according to embodiments.

[0027] (Source:)

[0028] In the specification of this application, the use of the phrase “derived from” is intended to include: the following (1) to (3).

[0029] (1) Refined;

[0030] (2) Separated; and / or

[0031] (3) Modified [including low molecular weight treatment and high molecular weight treatment (polymerization)] or modified.

[0032] (Plant-derived proteoglycans)

[0033] In this invention, "plant-derived proteoglycans" refers to those obtained through extraction and other processes from parts of plants in the Combretaceae family (including sap such as Dawa gum). The inventors believed that by using these plant parts, compositions containing arabinogalactan protein (AGP) could be manufactured with high purity and / or high efficiency, thus inventing this invention.

[0034] Plants of the Combretaceae family (Quisqualis indica L.) are distributed in southern China and Southeast Asia. They are evergreen vines cultivated for medicinal and ornamental purposes. In their early growth stages, they are shrub-like, and later, they twine around other objects while extending to about 10 meters in length.

[0035] Broadleaf elm mainly grows naturally in the forests of central India and is a deciduous tree characterized by its smooth white bark.

[0036] Daiwa gum is formed by the drying and solidification of sap secreted from cracks in the trunk of the broadleaf elm (Anogeissus latifolia Wallich) in the Combretaceae family. It is also known as Indian gum. The main components of daiwa gum are water-soluble polysaccharides composed of arabinose, galactose, mannose, xylose, and glucuronic acid, naturally existing primarily as Ca, Mg, and K salts. Daiwa gum typically dissolves in water to approximately 30% by weight at room temperature or above. Daiwa gum contains about 3% protein, similar to gum arabic, forming a "polysaccharide-protein complex" (Patent Document 2).

[0037] The "plant-derived proteoglycans" in this invention also include arabinogalactan protein (AGP). AGP is a proteoglycan widely distributed in plant tissues, primarily found locally in the cell wall (extracellular matrix). AGP typically consists of a hydroxyproline-rich (Hyp) core protein bound to an arabinogalactan (AG) sugar chain rich in galactose (Gal) and L-arabinose (L-Ara). The types of core proteins are diverse, and the structures of the sugar chains are complex and varied (Non-Patent Literature 1).

[0038] The solvents used in this invention may be, for example, acetic acid, water, citric acid, alkaline solutions, surfactant solutions, etc.

[0039] By using powdered cellulose and / or oil-absorbing pads on proteoglycan extracts, it is also possible to easily adsorb and remove impurities such as lipids. Examples of powdered cellulose include cellulose fibers (Nippon Paper Corporation: KCFLOCK W-50(S), KC FLOCK W-50, KC FLOCK W-100, KC FLOCK W-100G, KC FLOCK W-200, KCFLOCK W-200G, KC FLOCK W-250, KC FLOCK W-300G, KC FLOCK W-400G, NP Fiber W-100F, NP Fiber W-300F, NP Fiber W-10MG2, NP Fiber W-06MG, KC FLOCK-50GK, KC...). FLOCK-100GK; Rettenmaier (manufactured models: VITACEL-L10, VITACEL-L20, VITACEL-L600-30, VITACEL-L90, ARBOCEL-BMW40, VITACEL-L500, etc.). For oil-absorbing pads, non-woven fabrics primarily made of polypropylene are preferred. Examples include: Maeda Koken Co., Ltd.'s oil-absorbing sheets SP-1300N(DX) and SP-1100N; Nippon Paper CRECIA Co., Ltd.'s oil-absorbing pads PP-100 series; and Tanaka Sangyo Co., Ltd.'s Luclean A-50 and Luclean B-50. Furthermore, as for nonwoven fabrics made from polyolefins and polyesters, Nippon Paper CRECIA Co., Ltd.'s Powerful ECO series can be cited. And as for oil-absorbing pads primarily made from cotton, Daio Paper Co., Ltd.'s Prowipe Cotton Oil Mat can be cited.

[0040] The following examples illustrate the invention in further detail, but the invention is not limited to these examples. It should be noted that in the following examples, the unit % representing the amount of various ingredients added refers to mass %.

[0041]

Example

[0042] (Manufacturing Example 1: Manufacturing of a composition containing proteoglycans derived from dawa gum)

[0043] The composition is manufactured in the following manner.

[0044] 1. Dissolving process

[0045] Dissolve 2.5 kg of Daiwa resin in 20 times its volume of purified water (50 kg).

[0046] 2. Ultrafiltration process

[0047] Purified water was added to the filtrate obtained through a membrane filter with a pore size of 0.45 μm to prepare a solution for use in the ultrafiltration process. This prepared solution was then subjected to ultrafiltration to remove solutions with a molecular weight below 50,000, and to recover solutions with a molecular weight exceeding 50,000 (39 kg).

[0048] 3. Filtration process

[0049] The solution (39 kg) with a molecular weight exceeding 50,000 recovered through step 2 is passed through a membrane filter with a pore size of 0.45 μm, and the filtrate is recovered.

[0050] 4. Freeze-drying

[0051] The filtrate obtained in step 3 is freeze-dried to obtain a dried product (1.4 kg of a composition containing proteoglycans derived from Daiwa gum).

[0052] It should be noted that 10g of this dried substance dissolves in 100mL of water at 20°C. The resulting solution is transparent.

[0053] (Analysis of the composition of Manufacturing Example 1)

[0054] The composition of Manufacturing Example 1 was subjected to the following analyses, etc.

[0055] (1) Qualitative analysis of AGP

[0056] (2) Sugar composition analysis

[0057] (3) Quantitative analysis of protein content based on BCA protein concentration assay (BCA Protein Assay)

[0058] (4) Analysis of amino acids using an automated amino acid analyzer

[0059] (5) HPLC analysis.

[0060] (1) Qualitative analysis of AGP

[0061] To confirm whether the composition of Manufacturing Example 1 contains arabinogalactan protein (AGP), this was confirmed by precipitation using Yariv reagent (Biosupplies Australia Pty Ltd, 100-2), which specifically binds to AGP. The property of precipitation as an AGP-Yariv reagent complex was utilized in an aqueous sodium chloride (NaCl) solution. The composition of Manufacturing Example 1 was dissolved in a 0.15M aqueous sodium chloride (NaCl) solution. The Yariv reagent was also dissolved in a 0.15M aqueous sodium chloride (NaCl) solution. Equal volumes of the prepared solutions were mixed and allowed to stand overnight at 4°C. After standing, the precipitation was observed, and a red precipitate was observed. This confirmation confirmed that the composition of Manufacturing Example 1 contains arabinogalactan protein (AGP). This confirmation also confirmed that the composition of Manufacturing Example 1 contains a plant-derived proteoglycan.

[0062] (2) Sugar composition analysis

[0063] Sugar composition analysis was performed, and the composition of Manufacturing Example 1 contained approximately 83% sugar.

[0064] The details of the sugar and protein content in the composition of Manufacturing Example 1 are shown below.

[0065] • Arabinose (39.3%), galactose (33.6%), glucuronic acid (4.5%), mannose (2.5%), xylose (1.6%), rhamnose (1%), protein (2%), other (15.5%)

[0066] (3) Quantitative analysis of protein content based on BCA protein concentration assay (BCA Protein Assay)

[0067] The composition was confirmed to contain approximately 2% protein by quantitative determination of protein content based on the BCA Protein Assay.

[0068] (4) Analysis of amino acids using an automated amino acid analyzer

[0069] Amino acid analysis confirmed that the protein in this composition contains approximately 0.5% hydroxyproline (Hyp). Hydroxyproline is the binding site of the sugar chains in plant proteoglycans.

[0070] (5) HPLC analysis

[0071] The composition of Manufacturing Example 1 was analyzed by HPLC. Approximately 1 g of the product obtained from the composition (dried product) of Manufacturing Example 1 was accurately weighed and added to phosphate buffer (pH 6.8) to prepare 10 mL of solution, which was used as the sample solution. Each sample was passed through a 0.45 μm membrane filter and subjected to HPLC under the following operating conditions. The molecular weight was determined from the following standard curve.

[0072] In addition, as a molecular weight marker, the molecular weight at the peak was determined based on a standard curve prepared using Shodex STANDARD P-82 (manufactured by Showa Denko Corporation). It should be noted that a standard curve (standard pullulan) was prepared for a molecular weight range of 5,000 to 800,000.

[0073] Operating conditions

[0074] Analyzer: HPLC analytical device

[0075] Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation)

[0076] Column: Gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation)

[0077] Column temperature: 40℃

[0078] Sample injection volume: 50 μL

[0079] Mobile phase: Phosphate buffer (pH 6.8)

[0080] Flow rate: 0.5 mL / min.

[0081] Figure 1 The results of HPLC are shown. Figure 1 In the results shown, the molecular weight of the peak calculated from the column retention time is 300,000 to 400,000.

[0082] [Experimental Example 1] Human Epidermal Keratinocyte Proliferation Assay

[0083] Human epidermal keratinocytes NHEK (adult epidermal keratinocytes, KURABO, KK-4109) were prepared into 3 × 10⁻⁶ cells. 3Human epidermal keratinocytes were seeded at a rate of 1 cell per well in the prepared 96-well plates and cultured for 24 hours in basal epidermal keratinocyte proliferation medium (KBM medium, KURABO) supplemented with a proliferation additive at 37°C and 5% CO2. Then, the medium was replaced with basal epidermal keratinocyte proliferation medium excluding FGF and BPE (bovine pituitary extract) as the proliferation additive, and the following samples were injected into each well, and cultured for 3 days at 37°C and 5% CO2. After 3 days of culture, the number of human epidermal keratinocytes in each group (samples 1 to 5) was determined using a Cell Counting Kit-8. This determination was performed with 5 samples in each group (samples 1 to 5). Table 1 below shows the relative values ​​when the average value of the 5 samples in each group is set to 100, using the results calculated from the average value of the 5 samples in each group. In Table 1, "**" indicates a significant difference based on Dunnett's test compared to the values ​​of group 1 (p < 0.05).

[0084] • Sample 1 group: As a control, a specified amount of purified water was added.

[0085] • Sample 2 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 200 μg / mL.

[0086] • Sample 3 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 400 μg / mL.

[0087] • Sample 4 group: A commercially available composition containing gum arabic was added to achieve a final concentration of 200 μg / mL.

[0088] • Sample 5 group: A commercially available composition containing gum arabic was added to achieve a final concentration of 400 μg / mL.

[0089] It should be noted that, as a commercially available composition containing gum arabic, the gum arabic used is (Fujifilm and Kazumitsu Pure Chemical Industries, Ltd., 016-00025, CAS RN). TM (9000-01-5).

[0090] [Table 1]

[0091] Sample 1 100.0 Sample 2 126.0(**) Sample 3 127.3(**) Sample 4 115.6(**) Sample 5 119.0(**)

[0092] The results shown in Table 1 confirm that the groups with the composition obtained in Manufacturing Example 1 (samples 2 and 3) have a higher human epidermal keratinocyte proliferation capacity compared with the groups with the commercially available composition containing gum arabic (samples 4 and 5).

[0093] [Experimental Example 2] Human fibroblast proliferation assay

[0094] The presence or absence of human fibroblast proliferation capacity of the composition obtained in Manufacturing Example 1 was evaluated. 96-well plates were prepared, each well containing 5% FBS (manufactured by Thermo Trace) in DMEM medium. Normal human skin fibroblasts (KURABO) were added to each well at a concentration of 4 × 10⁶ cells / well. 3 Cells were seeded into the prepared 96-well plates at a rate of 1 cell per well. After seeding, the normal human skin fibroblasts were cultured for 24 hours at 37°C and 5% CO2. Then, the DMEM medium containing 5% FBS (manufactured by Thermo Trace) was replaced with DMEM medium containing 0.25% FBS. Following the replacement, the following samples were injected into each well, and the normal human skin fibroblasts were cultured for 3 days at 37°C and 5% CO2. After 3 days of culture, the number of normal human skin fibroblasts in each group (samples 1 to 5) was determined using a Cell Counting Kit-8 (DOJINDO). This determination was performed with 5 samples in each group (samples 1 to 7). Table 2 below shows the relative values ​​when the average value of the 5 samples in each group is set to 100. In Table 2, "**" indicates a significant difference (p < 0.05) based on the Tukey-Kramer test compared to the values ​​of group 1. Table 2 This indicates a significant difference based on the Tukey-kramer test compared to the value of sample 7 (p < 0.05).

[0095] • Sample 1 group: As a control, a specified amount of purified water was added.

[0096] • Sample 2 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 10 μg / mL.

[0097] • Sample 3 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 100 μg / mL.

[0098] • Sample 4 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 1000 μg / mL.

[0099] • Sample 5 group: A commercially available composition containing gum arabic was added to achieve a final concentration of 10 μg / mL.

[0100] • Sample 6 group: A commercially available composition containing gum arabic was added to achieve a final concentration of 100 μg / mL.

[0101] • Sample 7 group: A commercially available composition containing gum arabic was added to achieve a final concentration of 1000 μg / mL.

[0102] It should be noted that, as a commercially available composition containing gum arabic, the gum arabic used is (Fujifilm and Kazumitsu Pure Chemical Industries, Ltd., 016-00025, CAS RN). TM :9000-01-5).

[0103] [Table 2]

[0104]

[0105] The results shown in Table 2 confirm that the groups containing the composition obtained in Manufacturing Example 1 (samples 2 and 3) exhibited higher human fibroblast proliferation capacity compared to the groups containing the commercially available composition containing gum arabic (samples 4 and 5). Furthermore, it was confirmed that the human fibroblast proliferation capacity was significantly higher in sample 4 compared to sample 7.

[0106] [Experimental Example 3] Type I collagen production-promoting activity (ELISA-based detection)

[0107] The composition obtained in Manufacturing Example 1 was evaluated by ELISA for its ability to promote type I collagen production.

[0108] Prepare 24-well plates with each well containing 5% FBS (Thermo Trace) in DMEM medium. Normal human skin fibroblasts (KURABO) are added to each well at a ratio of 2 × 10⁶ cells / well. 4 Cells were seeded into the prepared 24-well plates at a rate of [number] cells / well. After seeding, the normal human skin fibroblasts were cultured for 24 hours at 37°C and 5% CO2 until confluence. Then, the DMEM medium containing 5% FBS (Thermo Trace) was replaced with DMEM medium containing 0.25% FBS. Following the replacement, the following samples were injected into each well, and the normal human skin fibroblasts were cultured for 1 day at 37°C and 5% CO2. The production of type 1 collagen (ng / mL) in each group was evaluated using a PIP EIA KIT (Takara Bio) on the cell culture supernatant. This assay was performed with three samples in each group (samples 1–3). The results using the average of the three samples calculated for each group are shown in Table 4 below. "**" in Table 3 indicates a significant difference (p < 0.05) based on Dunnett's test compared to the average of sample 1.

[0109] • Sample 1 group: As a control, a specified amount of purified water was added.

[0110] • Sample 2 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 10 μg / mL.

[0111] • Sample 3 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 100 μg / mL.

[0112] As shown in Table 3 below, it can be confirmed that the composition obtained by the drug manufacturing example 1 improves the type I collagen production promoting activity.

[0113] [Table 3]

[0114] Sample 1 780.4 Sample 2 952.5(**) Sample 3 1070.0(**)

[0115] [Experimental Example 4] Type I collagen gene expression (detection based on qRT-PCR)

[0116] The expression of type I collagen genes in the composition obtained in Manufacturing Example 1 was evaluated by qRT-PCR.

[0117] Normal human skin fibroblasts (KURABO) were divided into 2×10⁻⁶ cells in each well. 4 Cells were seeded into the prepared 24-well plates at a rate of [number] cells / well. After seeding, the normal human skin fibroblasts were cultured at 37°C and 5% CO2 for 24 hours until confluence. Then, the DMEM medium was replaced with experimental DMEM medium (a serum-free medium different from that in Example 2), and cultured for another 24 hours. After culture, the following sample was administered to each well, and the normal human skin fibroblasts were cultured at 37°C and 5% CO2 for 24 hours. The cultured cells were recovered according to the RNeasy Mini Kit (QIAGEN) protocol, and mRNA was purified. The purified mRNA was reverse transcribed according to the Prime Script RT Master Mix (TaKaRaBio) protocol, and the reaction products were subjected to RT-PCR using TB Green Premix EX Taq II (TaKaRaBio). RT-PCR was performed using a LightCycler96 system (Roche), and the expression level of the target gene was quantified using the accompanying software. The determination was performed with three samples in each group (groups of samples 1 through 3). Table 4 below shows the relative values ​​when the mean of the three samples in each group is set to 100, using the results of calculating the mean of the sample 1 group. "**" in Table 4 indicates a significant difference (p < 0.05) based on Dunnett's test compared to the value of the sample 1 group.

[0118] • Sample 1 group: As a control, a specified amount of purified water was added.

[0119] • Sample 2 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 10 μg / mL.

[0120] • Sample 3 group: The composition obtained in Manufacturing Example 1 was added to a final concentration of 100 μg / mL.

[0121] As shown in Table 4 below, it can be confirmed that the composition obtained by the drug manufacturing example 1 also enhances the type I collagen production promoting activity not only in test example 3, but also in test example 4.

[0122] [Table 4]

[0123] Sample 1 100 Sample 2 144.7 Sample 3 163.0(**)

[0124] [Experiment 5] Evaluation of the skin condition improvement effect based on human monitoring trials

[0125] During the period from December 2021 to January 2022, 12 healthy men and women aged 30 years and older (mean age 49.8 years) were included as subjects in this monitoring trial. It should be noted that for the lotions shown in Table 5 below, a solution was prepared by dissolving the composition obtained in Manufacturing Example 1 in 30% 1,3-butanediol to a final concentration of 1% by mass (the solution of the composition obtained in Manufacturing Example 1 is shown in Table 5).

[0126] (Experimental Methods)

[0127] The lotion with the composition shown in Table 5 (Table 5 lotion) was applied to the left half of the subject's face at prescribed intervals, and the lotion shown in Table 6 (Table 6 lotion) was applied to the right half of the subject's face at prescribed intervals. The prescribed intervals were twice daily (morning and evening) for 4 weeks. After 4 weeks, each subject had applied 24g of both Table 5 and Table 6 lotion. Measurements (such as the presence or absence of wrinkles) were then performed on day 0 before application and at week 4 after application.

[0128] [Table 5]

[0129] Detergent containing the composition obtained in Manufacturing Example 1

[0130]

[0131] [Table 6]

[0132] Placebo lotion (containing no composition obtained in Manufacturing Example 1)

[0133]

[0134] (Determination of the presence or absence of wrinkles)

[0135] Using ANTERA 3D TM (Miravex) measured the presence or absence of wrinkles at the corners of the eyes and the presence or absence of nasolabial folds in 12 subjects. The average values ​​(average values ​​of the moisture distribution measurements) of these 12 subjects are recorded below in the sections on "Measurement of Wrinkles at the Corners of the Eyes" and "Measurement of Wrinkles on the Nasolabial Folds".

[0136] (Measurement of wrinkles around the eyes)

[0137] With the values ​​of day 0 (before application) of the detergent application groups in Table 5 and Table 6 set to 100, after 4 weeks, the values ​​of the detergent application group in Table 6 were 103.99 and the values ​​of the detergent application group in Table 5 were 96.59 (p < 0.05 compared with the values ​​of the detergent application group in Table 5 on day 0 (Wilcoxon test)).

[0138] (Determination of nasolabial folds)

[0139] With the values ​​of day 0 (before application) of the detergent application groups in Table 5 and Table 6 set to 100, after 4 weeks, the values ​​of the detergent application group in Table 6 were 99.69 and the values ​​of the detergent application group in Table 5 were 98.10 (p < 0.05 compared with the values ​​of the detergent application group in Table 5 on day 0 (Wilcoxon test)).

[0140] (Determination of the presence or absence of spots)

[0141] The presence or absence of freckles is determined by counting the number of freckles on half of the face (from the lower eyelid to the entire cheek). This measurement uses VISIA. TM The experiment was conducted using Evolution (VISIA-Evo, Canfield Scientific). With the values ​​for day 0 (before coating) of both the detergent-coated groups in Table 5 and Table 6 set to 100, after 4 weeks, the values ​​for the detergent-coated group in Table 6 were 103.78 and for the detergent-coated group in Table 5 were 97.79.

[0142] (Determination of the presence or absence of porphyrins)

[0143] The presence or absence of porphyrin-induced morphology is determined by counting the number of spots on half of the face (from the lower eyelid to the entire cheek). This assay uses VISIA. TM The experiment was conducted using Evolution (VISIA-Evo, Canfield Scientific). With the values ​​for day 0 (before application) of both the detergent application groups in Table 5 and Table 6 set to 100, after 4 weeks, the values ​​for the detergent application group in Table 6 were 100.03 and those for the detergent application group in Table 5 were 83.96 (p < 0.01 compared to the values ​​for the detergent application group in Table 5 at day 0 (Wilcoxon test)).

[0144] (Determination of dermal collagen density)

[0145] Dermal collagen density was measured on the lateral aspect of the nose using DermaLab (registered trademark) Combo. With the values ​​at day 0 (before application) of both the Table 5 and Table 6 lotion application groups set at 100, after 4 weeks, the values ​​for the Table 6 lotion application group were 94.07 and for the Table 5 lotion application group were 97.22 (p < 0.01 compared to the value of the Table 5 lotion application group at day 0 (Wilcoxon test)).

Claims

1. A method for manufacturing a proteoglycan derived from Daiwa gum, characterized in that, The process includes the following steps: The process of dissolving parts of the Daiwa resin using water as a solvent; and Ultrafiltration is used to classify the solution obtained in the dissolution process by molecular weight to remove solutions with a molecular weight below 50,000. Use a nominal aperture of 0.1 to 10 mm. μ m microfiltration membrane The molecular weight of the proteoglycan derived from Dawa gum is 300,000 to 400,000, and the molecular weight is the peak molecular weight converted from pullulan polysaccharide.

2. The method according to claim 1, wherein, The nominal pore size of the microfiltration membrane is 0.

45. μ m.

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