KR20040094955A - Isolation of MMP-1 secretion inhibitor from Eucommia ulmoides and its purification method - Google Patents

Abstract

PURPOSE: Provided are an isolation of MMP-1 synthesis inhibitor from Eucommia ulmoides and its purification method by ethanol extract, fractioning, silicagel column chromatography, HPLC and the like. The MMP-1 synthesis inhibitor is useful for functional foods, cosmetics and drugs for preventing skin aging. CONSTITUTION: The MMP-1 synthesis inhibitor is characterized by containing, as an active ingredient, aucubin extracted from Eucommia ulmoides by: extracting Eucommia ulmoides with methanol at room temperature, followed by concentration at 40 deg.C; fractioning the extract with hexane to give hexane fractions; fractioning the remnant with chloroform to give chloroform fractions; and fractioning the remnant with ethylacetate to give ethylacetate fractions; and concentrating the remnant to give methanol fractions.

Description

Isolation of MMP-1 secretion inhibitor from Eucommia ulmoides and its purification method}

The present invention relates to a dermal cell MMP-1 synthesis inhibitor isolated from a worm and a method for purifying the same. More specifically, the present invention relates to aucubin ([1S- (1α, 4aα, 5α, 7aα)]-1, 4a, 5, 7a-tetrahydro-5-hydroxy-7- (hydr The present invention relates to an extract for inhibiting MMP-1 (matrix metaloproteinase-1) synthesis of dermal cells containing oxymethyl) cyclopenta [c] pyran-1-yl-β-D-glucopyranoside) as an active ingredient.

The skin is directly exposed to the external environment of the body tissues and can be most sensitively affected by biological, chemical and physical stimuli. As it is easily exposed to the outside, external environmental factors have been thought to affect aging in addition to the aging process caused by physiological and biochemical changes in the body.

Skin aging is divided into chronologic aging process over time and extrinsic aging process by external factors (Claude, S. et al., Free Radical Biol. & Med. 26). : 174 (1999)). External factors affecting skin aging are caused by wind, temperature, humidity, tobacco smoke, pollution, ultraviolet rays, etc. In particular, aging caused by ultraviolet rays is called photoaging. Sunlight can be divided into gamma rays, X rays, ultraviolet rays, visible rays, and infrared rays. Double ultraviolet rays (UV rays) are ultraviolet rays A (UVA, 320 to 400 nm) and ultraviolet rays B (UVB, 290 to 320) depending on the wavelength. nm), ultraviolet C (UVC, 200-290 nm) (fisher, GJ New Engl. J. Med. 337: 1419 (1997)), and double ultraviolet B has been reported as a major factor of photoaging (see reference). : Kang, S. et al., Arch.Drmatol. 133: 1280 (1997)).

Symptoms in chronic sun-damaged skin include abnormal elastotic deposition of the upper collagen in the dermis, increased proteoglycans, and markedly reduced collagen, the main protein of the dermis (see Yaar M. et. al., J Investig. Dermatol. Symp. Proc. 3: 47 (1998), Li, JJ et al., Cancer Research 56: 483 (1996)). Generally, the dermis is composed of the majority of type I collagen and some type III collagen, elastin, proteoglycan, fibronectin (Bailly, C. et al., J. Invest. Dermatol. 94: 47 (1990)). . In addition, collagen gives skin strength and tension, which protects the skin from external irritation or force, accounting for 90%, and the reduction of collagen is closely related to aging of the skin (see Huang C). et al., Proc. Natl. Acad. Sci. USA.94: 5826 (1997).

Matrix metalloproteinases (MMPs) are a family of enzymes that are involved in the degradation of extracellular matrix (ECM) and basement membrane (BM), depending on their structure and functional properties, resulting in interstitial collagenase, stromelysin, gelatinase, and membrane-type MMP. (MT-MMP) and four subfamily (see Fisher, GL et al., J. Clin. Invest. 101: 1432 (1998)). MMP is a metalloproteinase having zinc in its active center, and is secreted in the form of latent zymogen in vivo. In order to have enzymatic activity, structural modifications occur, resulting in cleavage and activation of the amino terminus. Cells secrete MMP (see Fisher, GJ and Voorhees JJ Photochem. Photobiol. 69: 154 (1999)). Fisher et al. Reported that MMP activity in the skin is increased even with a single UV irradiation and that the collagen in the skin is significantly disrupted, thereby affecting collagen breakdown of the dermal layer and playing a very important role in photoaging. , KS et al., J. Dermatol. Sci. 17: 182 (1998)).

The improvement of quality of life and the abundance of materials have increased the women's desire for aesthetic pursuit, and accordingly the cosmetic industry has grown rapidly. Recently, functional products using natural materials, but there are still few studies, retinoic acid has been reported to have MMP-1 inhibitory activity (see Fisher, GJ and Voorhees JJ Photochem. Photobiol. 69: 154 (1999), Lee, KS et al., J. Dermatol. Sci. 17: 182 (1998)).

Eucommia Cortex is a deciduous tree belonging to the plant family Eucommia, which dried the skin of Eucommia ulmoides Olive . Only medicinal bark bark, which is over 15 years old, is used for medicinal purposes. It has no toxic toxin. The surface is grayish brown, and the inside is dark purple and smooth. Beetle bark has been used as an herbal medicine since ancient China, and it has been shown to have high blood pressure, pain relief, neuralgia, prevention of miscarriage, cholesterol lowering effects, and according to the herbaceous tree, it works on the kidneys and liver to relieve back and knee pain, Although it is known to strengthen bones, remove residual urine, lighten the body and prevent aging, it has not been reported that the extract of the larvae itself is an effective inhibitor of dermal cell MMP-1 synthesis.

Against this background, the present inventors primarily studied the micromaterials having various physiological activities from edible biological sources, and firstly confirmed that Methanol extracts containing MMP-1 were inhibited by Methanol extracts. 1 The present invention was completed by separating aucubin, which is an iridoid-based substance having a synthetic inhibitory activity.

An object of the present invention is to solve the conventional problems as described above, and an object of the present invention is to provide an MMP-1 synthesis inhibitor which inhibits MMP-1 synthesis of dermal cells. It is still another object of the present invention to provide a method for separating MMP-1 synthesis inhibitory substance from larvae. In order to achieve the above object, the present invention is characterized by separating the MMP-1 synthesis inhibitor separated from the two larvae from the larvae. In order to achieve the above object, dermal cell MMP-1 synthesis inhibitor and the purification method of the present invention isolated from the worms of the present invention and pulverized worms, extracted with methanol at room temperature, the residue in the order of nucleic acid, chloroform, ethyl acetate Fractionation is performed by a series of purification steps, such as silica gel column chromatography, HPLC, characterized in that the separation from the larvae.

1 is a diagram showing the step of purifying material from the larvae.

Figure 2 is a diagram showing the fractional MMP-1 inhibitory activity.

3 is a chromatogram showing the result of HPLC-C ₁₈ column chromatography.

Figure 4 is a diagram showing the structure of aquabin identified by ¹ H-NMR.

5 is a chart showing aquabin cytotoxicity in dermal cells.

Figure 6 is a diagram showing the MMP-1 inhibitory activity of the concentration of aquabin.

In the present invention, acubin ([1S- (1α, 4aα, 5α, 7aα)]-1, 4a, 5, 7a-tetrahydro-5-hydroxy-7- (hydroxymethyl) cyclo extracted from the larvae It is an extract for inhibiting the synthesis of matrix metaloproteinase-1 (MMP-1) of dermal cells containing penta [c] pyran-1-yl-β-D-glucopyranoside) as an active ingredient.

Hereinafter, with reference to the accompanying drawings the technical details of the dermal cell MMP-1 synthesis inhibitor and the purification method isolated from the present invention configured as described above in detail as follows. The present inventors found that the methanol extract of the two insects of the extract extracted from the edible biological resources has a relatively high MMP-1 inhibitory activity. In order to separate the MMP-1 synthesis inhibitors contained in the two worms, extracted with 100% methanol at room temperature, and then fractionated in the order of nucleic acid, chloroform, ethyl acetate, and then silica gel column chromatography with methanol fraction having high inhibitory activity. As a result, obtained the purified fractions and subjected to HPLC collected fractions with high MMP-1 inhibitory activity, the structural analysis by ¹ H-NMR and HPLC the peak suppression as compared with the ¹ H-NMR of aucubin standard (Wako Co.) It was confirmed that the active substance was acubin.

Next will be described in more detail with reference to the embodiment of the present invention configured as described above. These examples are only for illustrating the present invention in detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Selection of Extraction Conditions and Confirmation of Inhibitory Activity for MMP-1 Synthetic Inhibitor

While searching for extracts extracted from various edible biological resources, it was confirmed that the larvae extract exhibited the inhibitory activity of MMP-1 synthesis. In order to isolate the MMP-1 synthesis inhibitor contained in the larvae, the larvae were blanched at 100 ° C. for 5 minutes to inactivate and pulverize enzymes in living cells, and extracted with 100% methanol at room temperature. The residue was extracted with nucleic acid and chloroform. , Ethyl acetate was partitioned in the order of methanol fraction and each was filtered through a filter cloth, and the residue was concentrated under reduced pressure and dried to obtain an extract (FIG. 1).

In order to perform MMP-1 synthesis inhibition experiment on each fraction extract, HS68 (normal human skin fibroblast), a dermal cell, was cultured on 12 well-plates at a concentration of 5x10 ⁴ cells / well, and reached about 80% confluency. After removing the medium and washed with PBS to remove the serum components in the medium, the sample was added to a new serum free DMEM at a concentration of 0.1 mg / ml and incubated in 37 ℃ CO ₂ , incubator for 20 hours. After incubation, the cells were washed with PBS, and then irradiated with UV B (UVB) at a dose of 35 mJ / cm ² in a PBS locked cell and incubated for 48h. The control was left at the same time under fluorescent light without UVB irradiation. After 48h incubation, the supernatant medium was taken and measured as follows using the ELISA method. 100 μl (10 μl / ml DW) of glutaraldehyde was dispensed into the wells in an immuno-well plate and reacted at 37 ° C. for 1 hour, followed by washing the well-plate with distilled water and removing water. Dispense 100 μl of the culture medium and react at 37 ° C. for 1 hour, dilute MMP-1 antibody (mouse) in a washing solution (0.5% Tween 20 in PBS) at 1: 1,000, and dispense 100 μl each at 37 ° C., The reaction was carried out for 1 hour. The anti-mouse IgGperoxidase conjugated was diluted to 1: 30,000 in a washing solution and reacted at 37 ° C. for 1 hour, followed by the substrate [TMB 10 mg / mL DMSO, 3% H ₂ O ₂ , 50 mM sodium acetate buffer (pH 5.1)]. 200 μl per well was added and reacted with the light blocked for 15 minutes. After the reaction was completely stopped by adding 50 µl of 1 MH ₂ SO ₄ , absorbance was measured at 450 nm using a micro plate reader (Model 550, BIO-RAD Laboratories, USA). MMP-1 inhibitory activity by the sample was converted according to the following equation.

Inhibitory activity of MMP-1 secretion (%) =

[1- (As-Ab / Ac-Ab)] × 100

Ac: absorbance of mock irradiated medium

Ab: Absorbance of medium not irradiated with UVB and not sampled

As: absorbance of medium treated with UVB sample

As a result of measuring the MMP-1 inhibitory activity according to the fraction, it was confirmed that the methanol fraction exhibits high activity (Fig. 2). After loading the methanol fraction as a sample on the silica gel 60 column activated with chloroform, and developing 5 bed volumes with a mixed solution of chloroform and methanol (CHCl ₃ : MeOH = 100: 0 ~ 0: 100), the MMP-1 inhibitory activity was examined. Small fractions having MMP-1 inhibitory activity were isolated at chloroform-methanol 80:20 and 70:30. The separated fractions were injected into HPLC (u-Bondapak C _18, 7.8 × 300 mm), and the flow rate was 3 ml / min under water and methanol gradient (H ₂ O: MeOH = 100: 0 ~ 0: 100) solvent conditions. Eluted to concentrate only the active fraction. Using the u-Bondapak C ₁₈ (3.9 × 150 mm) column as an active fraction, analytic HPLC was performed under water: acetonitrile = 97: 3 solvent conditions to obtain peaks showing MMP-1 inhibitory activity (FIG. 3). Concentrated MMP-1 synthesis inhibitory peaks and structural analysis using ¹ H-NMR (500 MHz, 11.4 TeslaAvance-500, Bruker, German) showed HPLC peak and ¹ H-NMR of aucubin standard (Wako Co.) As a result of this agreement, the inhibitory active substance is acubin ([1S- (1α, 4aα, 5α, 7aα)]-1, 4a, 5, 7a-tetrahydro-5-hydroxy-7- (hydroxymethyl) Cyclopenta [c] pyran-1-yl-β-D-glucopyranoside) (FIG. 4).

Example 2: Cytotoxicity test of aucubin isolated from larvae

In order to investigate the cytotoxicity of aucubin isolated by MMP-1 synthetic inhibitor, HS68, a normal human dermal dermal cell, was cultured in 12 well-plate at the concentration of 5x10 ⁴ cells / well, and reached to about 80% confluency. Remove the serum component in the medium by washing with PBS. Samples were added to fresh serum free DMEM at a concentration of 10 ⁻¹ to 10 ⁻¹⁰ mg / ml and incubated in a CO ₂ incubator at 37 ° C. for 20 hours. After incubation, the cells were washed with PBS and then irradiated with UVB at a dose of 35 mJ / cm ² in a PBS locked cell and incubated for 48h. The control was left at the same time under fluorescent light without UVB irradiation. After 48h incubation, MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide: 5 mg / ml] was added and reacted for 3 hours at 37 ° C. with light blocking. After melting with DMSO, absorbance was measured at 570 nm using a microplate reader.

Figure 5 shows the cytotoxicity of the concentration of aucubin isolated from the larvae extract, the cell viability was more than 100% compared to the control distilled water did not appear cytotoxicity.

Example 3 Comparison of MMP-1 Synthesis Inhibition Rate of aucubin Isolated from Tobacco

Inhibition rate of MMP-1 synthesis was measured for each concentration of aucubin isolated from the worms by the method of Example 1. As a comparative example, vitamin C (10 ^-5 mg / ml), which is widely known as an antioxidant, was used. At almost all concentrations of aucubin, MMP-1 inhibitory activity was higher than or similar to that of vitamin C. In particular, aucubin showed the highest MMP-1 synthesis inhibitory activity of 72% at 10 ^-5 mg / ml (Fig. 6).

As described above, the synthesis inhibitory effect and purification method of the aucubin MMP-1 synthesis inhibitor isolated from the two insects of the present invention, and after the blanching and pulverization, extracted with methanol to fractionate the residue in the order of nucleic acid, chloroform, ethyl acetate It was obtained by performing a series of purification steps, such as silica gel column chromatography, HPLC, and the effect that can be used in functional foods, functional cosmetics or drugs for preventing skin aging.

Claims (1)

Aucubin ([1S- (1α, 4aα, 5α, 7aα)]-1, 4a, 5, 7a-tetrahydro-5-hydroxy-7- (hydroxymethyl) cyclopenta [c] ] An extract for inhibiting MMP-1 (matrix metaloproteinase-1) synthesis of dermal cells, comprising pyran-1-yl-β-D-glucopyranoside) as an active ingredient.