KR101689156B1 - Health functional composition for skin anti-aging extracted from Rhizomes of Cyperus rotundus - Google Patents

Abstract

The present invention relates to a health functional composition for preventing skin aging from extracting fragrance. More particularly, the present invention relates to a health functional composition for preventing skin aging of ultraviolet rays which inhibits the activity of transient receptor potential vanilloid channel 1 (TRPV1) composed of an extract extracted with methanol, a fraction isolated from an extract, and an active ingredient contained in a fraction, And a process for producing the same.

Description

{Health functional composition for skin anti-aging extracted from Rhizomes of Cyperus rotundus}

The present invention relates to a health functional composition for preventing skin aging from extracting fragrance. More particularly, the present invention relates to a health functional composition for preventing skin aging of ultraviolet rays which inhibits the activity of transient receptor potential vanilloid channel 1 (TRPV1) composed of an extract extracted with methanol, a fraction isolated from an extract, and an active ingredient contained in a fraction, And a process for producing the same.

Cyperus rotundus has been reported as an analgesic or antispasmodic in oriental medicine. Recently, it has been reported that the herbal extract has an anti-inflammatory effect.

In addition, the present inventors have disclosed a composition for preventing or treating sepsis in a composition for preventing or treating septicemia containing an ethanol extract of a herbal supplement as described in Korean Patent Laid-Open Publication No. 10-2011-134552, As shown in Fig.

Transient receptor potential Vanilloid channel 1 (TRPV1) is a non-selective cation channel that detects various toxic chemicals and thermal stimuli. Recently, however, the expression of TRPV1 in skin has been reported to be involved in heat or ultraviolet induced skin aging.

TRPV1, known as the capsaicin receptor, is a non-selective cation channel with high Ca ^{2 +} permeability. TRPV1 is a well-known TRP channel that senses temperatures in excess of 43 ° C and also plays an important role in signal transduction at the sensory nerve endings in the peripheral nervous system. TRPV1 is localized in myelin C fibers and small diameter neurons and has been studied extensively in neurosensory sensory neurons such as the posterior ganglion. Recently, TRPV1, which is distributed in cells constituting the skin such as keratinocytes and mast cells, It has been reported that it plays a functional role in the skin tissue.

The epidermal keratinocytes form a skin barrier as a major component of the skin barrier and maintain the shape and function of the epidermis. In order for keratinocytes to maintain the stratum corneum, proliferation and apoptosis must be balanced to protect the skin from noxious stimuli and excessive moisture loss.

If by a certain stimulus, such as UV TRPV1 it is activated Ca ^{2 +} concentration increases, which inhibits the growth of cells and induce cell death. Topical application of the capsaicin can be active in the mouse experimental model in which the TRPV1 through a tape peeling method damage the skin barrier and the skin barrier recovery, while delay TRPV1 inhibitors capsazepine facilitates recovery of the film (Yun JW et al., J. Dermatol Sci 2011; 62: 8-15).

On the other hand, it has been reported that, during ultraviolet irradiation, calcium influx through TRPV1 plays an important role in the secretion and activity of UV-induced matrix metalloproteinase-1 (MMP-1), which degrades skin collagen and causes deep skin wrinkles (Lee YM et al., J. Dermatol Sci 2012; 65: 81-85). In this article, it is disclosed that intracellular calcium influx through the activity of TRPV1 inhibits the recovery of skin barrier and photoaging.

Therefore, development of an effective TRPV1 inhibitor has been required to treat ultraviolet ray-induced skin aging and skin-membrane related diseases, and a natural component having such a utility has been required.

Therefore, the inventors of the present invention have found that when using the rootstocks of various herbals having various analgesic, antioxidant and anti-inflammatory effects as a raw material for natural materials and measuring the anti-aging effect of the herbal extracts on the various ingredients contained in the raw materials, Component is effective for preventing skin aging by inhibiting TRPV1 activity, and thus completed the present invention.

The problem to be solved by the present invention is to measure the anti-aging effect of the herbal composition through the ingredients in the natural product by using the rootstock of the herbal product having various analgesic, antioxidant and anti-inflammatory effects as a raw material for natural products. In addition, the oleanolic acid component in the herbal extracts is effective in preventing skin aging by inhibiting TRPV1 activity, thereby developing a skin anti-aging health functional composition.

The object of the present invention is achieved by the steps of: (i) adding 3 to 7 parts by weight of methanol to 1 part by weight of dried pulverized rootstock root to obtain an extract; And

Ii) adding n-hexane, dichloromethane, ethyl acetate and n-butanol to the methanol extract to obtain a n-hexane fraction and an ethyl acetate fraction;

And a method for producing the TRPV1 inhibitor composition.

The active ingredient of the n-hexane fraction and the ethyl acetate fraction is an oleanolic acid.

On the other hand, the above-mentioned oleanolic acid is characterized in that it does not inhibit TRPV1 activity induced at a low concentration of about 10 micromolar in vitro, but has a dual property of inhibiting TRPV1 activity at a high concentration of 90 micromolar or higher.

It is still another object of the present invention to provide an ultraviolet antioxidant containing an extract of nerve root and n-hexane and an ethyl acetate fraction obtained according to the above method as an active ingredient.

It is still another object of the present invention to provide a health functional composition for preventing aging of ultraviolet skin comprising the root extract root extract n-hexane fraction and ethyl acetate fraction obtained as described above as an active ingredient.

Therefore, the effect of the present invention can be achieved by measuring the anti-aging effect of the herbal composition through the ingredients in the natural product using the rhizome of the herbal product having various analgesic, antioxidant, anti-inflammatory effects as a raw material of natural products, And to provide a skin anti-aging health functional composition having oleic acid.

Figure 1 shows the structural formula of the chemicals separated in the perfume.
Figure 2 shows the capsaicin-induced human TRPV1 current (I _TRPV1 ).
Figure 2a is a representative trace of capsaicin-induced I _TRPV1 produced by 1 μM capsaicin using whole cell patch clamp technology. FIG. 2B shows a current-voltage (IV) relationship curve between the starting point (1) and the steady state peak I _TRPV1 (2) in human TRPV1 overexpressing HEK293T cells.
Figure 3 shows the effect of the fragrance ethyl acetate fraction on the TRPV1 capsaicin-induced current (I _TRPV1 ).
Figure 3A shows a representative inhibition of I _TRPV1 by the ethyl acetate fraction of the _perfume . After confirming I _TRPV1 activity using 1 [mu] M capsaicin, the fractions were applied to human TRPV1 overexpressing HEK293T cell lines at 10, 30 and 100 [mu] g / mL.
FIG. 3B shows the results of a comparison between the control peak currents (1), 10 (2), 30 (3), 100 μg / mL fraction (4) and 1 μM N- (4-tertiaryarylbutylphenyl) -4- (IV) relationship curve with a pyridine-2-yl-tetrahydropyrazine-1 (2H) -carboxamide (BCTC). This fraction specifically inhibited I _TRPV1 .
Figure 3c summarizes the inhibition of I _TRPV1 at +100 mV.
Figure 4 shows the effect of elastase and epicatechin on TRPV1 capsaicin-induced current (I _TRPV1 ).
Figures 4A and 4B are representative representations of the effect of elastase on I _TRPV1 . The corresponding current-voltage relationship curve is measured several times after medication. Figure 4c summarizes the change in I _TRPV1 induced by elastase at +100 mV. Figures 4d and 4e are representative representations of the effect of epicatechin on I _TRPV1 . The corresponding current-voltage relationship curve is measured several times after medication. Figure 4f summarizes the changes in I _TRPV1 induced by epicatechin at +100 mV.
Figure 5 shows the dual effect of oleanolic acid via TRPV1 capsaicin-induced current (I _TRPV1 ).
Figures 5a and 5b show the results of a comparison between the control peak currents (1), 10 (2), 30 (3), 100 μg / ml fraction (4) and 1 μM N- (4-tertiaryaryl butylphenyl) -4- (IV) relationship curve with a 3-chloropyridin-2-yl) -tetrahydropyrazine-1 (2H) -carboxamide (BCTC). The oleanolic acid showed a double effect on TRPV1 depending on the concentration and the inhibition effect was not significant at the relatively low concentration (30 μM) but was greatly inhibited at the high concentration (90 μM). Figure 5c summarizes the change in I _TRPV1 induced by oleanolic acid at +100 mV. Note that oleanolic acid inhibits I _TRPV1 only at a concentration of 90 μM.

The rootstock rootstock was extracted with methanol, and then dissolved in n-hexane, dichloromethane, and ethyl acetate to sequentially obtain fractions. Finally, n-butanol fractions were obtained. HTRPV1 was expressed in HEK293T cells and the total current was measured in order to confirm whether methanol extract or the like could cause a change in capsaicin-induced TRPV1 current (I _TRPV1 ). After confirming that the initial current value is close to zero, we applied 1 μM of capsaicin in aqueous solution.

Figure 2a shows a typical chart record of capsaicin-induced I _TRPV1 in hTRPVl expressing HEK293T cells. The current-voltage relationship (IV) at initial (1) and peak current I _TRPV1 (2) shows that capsaicin application produces a typical strong cationic outgoing current for hTRPV1 (Figure 2b). After confirming the constant state of I _TRPV1 , the present inventors applied a sample to hTRPV1 expressing HEK293T cells. For the remaining I _TRPV1 confirmation, we applied a selective TRPV1 antagonist, 1 μM BCTC (≥98% purity) and used as a positive control at the end of each experiment (FIG.

Table 1 summarizes the inhibition rate of hTRPV1 using various fractions. The clamp current voltage +100 mV was used to generalize the extract-processing current (Iext / Icon × 100%).

Effect of the fragrant extract fraction on capsaicin-induced TRPV1 current (I TRPV1 ) (I / Icon) x 100% TRPV1 @ + 100mV Fraction 10 μg / mL 30 μg / mL 100 μg / mL Control group 100 100 100 Methanol extract (n = 9) 111 ± 9 128 ± 14 105 ± 15 The hexane fraction (n = 4) 105 ± 6 92 ± 1 50 ± 2 The dichloromethane fraction (n = 5) 131 ± 20 153 ± 35 147 ± 19 Ethyl acetate fraction (n = 6) 79 ± 3 66 ± 3 51 ± 5 Butanol fraction (n = 6) 111 ± 7 104 ± 6 102 ± 7

Methanol extract and dichloromethane and butanol fractions did not inhibit capsaicin-induced I _TRPV1 at 100 μg / mL. Only the hexane and ethyl acetate fractions all inhibited I _TRPV1 to about 50%. Although low doses of hexane fraction (10 μg / mL) did not inhibit I _TRPV1 , I _TRPV1 was sensitively inhibited by the 10 μg / mL ethylacetate fraction.

Figure 3 shows the I _TRPV1 response to the ethyl acetate fraction. As shown in FIG. 3A, the ethyl acetate fraction significantly inhibited I _TRPV1 in a dose-dependent manner at 10, 30 and 100 μg / mL. Figure 3b shows the IV relationship curves between the ethyl acetate fraction and control (1), 10 (2), 30 (3), 100 (4) μg / mL and 1 μM BCTC (5). The ethyl acetate fraction showed 21 ± 3, 34 ± 3 and 49 ± 5% inhibition at 10, 30 and 100 μg / mL, respectively.

In order to determine what exhibits the I _TRPV1 inhibitory effect on the constituents of the acetate fraction, the inventors have isolated the three components of oleanolic acid, elastase and (-) - epicatechin in the fraction. Was measured by the above-described cell patch clamp method. As shown in Fig. 3, the current of capsaicin-induced I _TRPV1 was inhibited only by the oleanolic acid. Elagic acid increased the current at 90 mM and epicatechin also slightly increased the current. However, this current increase was only 12 ± 2.8%.

As shown in FIG. 5, oleanolic acid had double characteristics in a concentration-dependent manner. At a concentration as low as 10 μM, the current was not inhibited as compared with the control, but the current was inhibited at a concentration of 90 μM. Figure 5 shows the dual effect of oleanolic acid via TRPV1 capsaicin-induced current (I _TRPV1 ).

Figures 5a and 5b show the results of a comparison between the control peak currents (1), 10 (2), 30 (3), 100 μg / ml fraction (4) and 1 μM N- (4-tertiaryaryl butylphenyl) -4- (IV) relationship curve with a 3-chloropyridin-2-yl) -tetrahydropyrazine-1 (2H) -carboxamide (BCTC). The oleanolic acid showed a double effect on TRPV1 depending on the concentration and inhibition was not significant at the relatively low concentration (30 μM) but was inhibited at the high concentration (90 μM). Figure 5c summarizes the change in I _TRPV1 induced by oleanolic acid at +100 mV. Note that oleanolic acid inhibits I _TRPV1 only at a concentration of 90 μM.

TRPV1 is sensitive to temperatures higher than 43 ° C. It is distributed mainly in neuronal and non-neuronal cells in small diameter Aδ and C fiber-sensitized neurons, but plays an important role in TRPV1 pain detection and neurogenic inflammation.

The present inventors measured the effect of the fraction component extracted from the herbal extract on TRPV1. Did not completely inhibit TRPV1 but efficiently inhibited capsaicin-induced I _TRPV1 in the hTRPV1 trait HEK293T cells in the ethyl acetate fraction.

Hereinafter, the present invention will be described in more detail by way of examples. However, the scope of the present invention is not limited to these embodiments.

(Example 1) Materials and methods

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). (3-chloropyridin-2-yl) -tetrahydropyrazin-1 (2H) -carboxamide (BCTC) was added to a concentration of 10 mM capsaicin and 10 mM N- A stock solution prepared using dimethylsulfoxide (DMSO) as a solvent was prepared. All stock solutions were stored at -20 ° C.

Dried rootstocks were purchased from Gyeongju herbal medicine room. ¹ H-NMR (500 MHz) and ¹³ C-NMR (125 MHz) spectra were recorded on a Varian Unity INOVA 500 spectrometer. ESI-MS data were obtained using an Agilent 1100 LC / MSD Trap Classic.

(Example 2) Extraction and Separation

600 g of dried root powder was extracted twice with 3 L of methanol to obtain 64.8 g of methanol extract. Sequential extraction with 300 mL of n-hexane, 300 mL of dichloromethane, 200 mL of ethyl acetate and 200 mL of n-butanol successively yielded 6.9 g, 2.1 g, 1.7 g and 2.6 g of each fraction, respectively.

A portion of the ethyl acetate fraction was dissolved in a mixed solution (650 mL) of dichloromethane-methanol-distilled water 10: 1: 0.1 using silica gel column chromatography and chromatographed. 52.5 mg of oleanolic acid were obtained in the ethyl acetate fraction. The ethyl acetate fraction is further fractionated into three sub-fractions with methanol using a Sephadex LH-20 column. The first fraction is passed through a silica gel column again to give 25.8 mg of elastase and 30.2 mg of epicatechin is obtained in the last fraction.

(Example 3) Cell culture

HEK293T cells (ATCC, Manassas, VA, USA) were cultured in DMEM medium in a humidified incubator in the presence of 20% O ₂ /10% CO ₂ . Jurkat T cells (clone E6-1, ATCC) are cultured in RPMI 1640 medium in a humidified incubator in the presence of 20% O ₂ /5% CO ₂ . All cells were subcultured once every 2-3 days.

(Example 4) Transformation of human TRPV1

For patch clamp (patch clamp) experiment, trans faction transferred the day before HEK293T cells in 25-cm ² cell culture dish were subcultures. Lipofectamine Plus reagent (Life technologies) was used to overexpress the human TRPV1 (hTRPV1) ion channel in the HEK293T cell line. A mammalian expression vector (pcDNA5 / FRT) containing the hTRPV1 gene was transfected. Transfection methods were performed according to the manufacturer's protocol. To confirm that the transfection was successful, the pEGFP-N1 vector capable of expressing the green fluorescent protein (GFP) was transfected as hTRPV1. In the experiment, only cells that emit light due to GFP were selected using a fluorescence microscope. The amounts of hTRPV1 and pEGFP-N1 genes used in the transfection were 0.9 mg and 0.1 mg, respectively. Experiments were carried out within 24 to 36 hours after transfection.

(Example 5) Electrophysiology

To measure the activity of hTRPV1, experiments were performed at room temperature using a cell patch clamp technique. A glass electrode having a resistance of 2 to 4 M was used. Analog signals from a patch clamp amplifier (Molecular Devices, Sunnyvale, Calif., USA) were converted to digital signals through a digital converter (Digidata 1440A, Molecular devices) and stored on a personal computer with an operating clock of 3.2GHz. We used pClamp software 10.4 to record and store the current response at fixed voltage and excitation voltage. The results obtained for the membrane voltage clamp were analyzed and processed using clampfit v10.4 (Molecular devices), origin 8.0 (Microcal, Northampton, MA, USA). The perfusion of the extracellular solution was maintained at a rate of 3 mL per minute. The composition of the extracellular fluid was 140 mM NaCl, 4 mM KCl, 1 mM MgCl ₂ , 1 mM EGTA, 5 mM D-glucose and 10 mM HEPES, and the pH was maintained at 7.4 using NaOH. The composition of the intracellular fluid was made with 140 mM CsCl, 10 mM NaCl, 5 mM EGTA, 3 mM MgATP, 10 mM HEPES, and the pH was maintained at 7.2 using CsOH.

Claims (5)

I) adding 3 to 7 parts by weight of methanol to 1 part by weight of the dry powdered rootstock root to obtain an extract; And
Ii) obtaining an ethyl acetate fraction obtained by further adding ethyl acetate to the methanol extract;
Wherein the composition comprises an ethyl acetate fraction as an active ingredient,
The ethyl acetate fraction was incubated in a dose dependent manner with transient receptor potential vanilloid channel 1 (TRPV1) Wherein the composition is applied to the external surface of the skin.
2. The process of claim 1, wherein the active ingredient of the ethyl acetate fraction is an oleanolic acid. delete delete delete

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