KR20160081083A - Antioxidative or cytoprotective composition comprising new hydroquinone compounds isolated from Rhus verniciflua extract - Google Patents

Abstract

The present invention relates to an antioxidative or cytoprotective composition containing a novel hydroquinone compound isolated from an raspberry extract wherein the novel compound quinoline A or quinoline B isolated from the raspberry extract has an increased Nrf2 / ARE expression A composition containing them as an active ingredient or a natural substance derived from Rhus verniciflua containing them can be effectively used for protecting cells against oxidative stress and can be effectively used for the protection of cells from the ricotta extract- HPLC peak identification information can be useful for predicting the standard and efficacy of RVSB extracts or natural products derived from L. lucidum.

Description

[0001] The present invention relates to an antioxidative or cytoprotective composition comprising a novel hydroquinone compound isolated from a raspberry extract and a novel hydroquinone compound isolated from Rhus verniciflua extract.

The present invention relates to an antioxidative or cytoprotective composition containing a novel hydroquinone compound isolated from raspberry extract.

The scientific name of Rhus is Rhus As a verniciflus, lacquer, which is a sap of lacquer tree, has been used in traditional medicines such as new medicines, new medicinal herb , and herbal medicinal herbs , and has been used for gastrointestinal diseases and the like. It stimulates the circulation of the human body, Has been used. In recent years, studies on anticancer activity, antioxidant activity, and antimicrobial activity of lacquer leaf, bark and stem extracts have been actively conducted as well as lacquer sap. The urushiol extract contains a large amount of urushiol, which has a catechol nucleus in which the alkyl side chain is replaced by 15 to 17 carbon atoms, and these urushiol compounds are known to be the main cause of allergy have. In recent years, the development of techniques for reducing the content of urushiol and expanding the application range of rubmak extract has been actively carried out. In addition to urushiol, the lacquer shells contain antioxidants such as Fustin, Fisetin, Sulfuretin, Butein, Vutin, Kaempferol-3-O-rhamnoside, , Anti-inflammatory and neuro-protective flavonoids or phenolic materials. However, lacquer, which is a sap of lacquer tree, contains urushiol components. Among the lacquer ingredients other than urushiol ingredient, there is no known quinone substance exhibiting antioxidative or antiinflammatory or neuroprotective action.

The nerve cell protection, antioxidative and anticancer effects of the hydroquinone substances having an alkyl side chain such as the quinoline A and the quinoline B of the present invention are already known. However, even though the hydroquinone core itself is toxic to humans (chemical information system, ncis.nier.go.kr), the efficacy or toxicity varies depending on the alkyl substituent of hydroquinone. Since hydroquinone having an alkyl side chain such as the quinoline A and the quinoline B of the present invention is not known at all to date, it is possible to predict the degree of efficacy of the natural substance containing them and to set the component standard.

In the present invention, the two kinds of new hydroquinone compounds and urushiol derivatives were separated from domestic rhubarb extracts, urushiol derivatives were separated from the imported rhubarb extracts, and their activation activities of the Keap1 / Nrf2 / ARE pathway were measured. As a result, And quinoline B alone showed physiological activities. Since the hydroquinone derivative components have not been reported from natural substances derived from lacquer or lacquer tree, it is possible to differentiate the quality standards of natural substances derived from Rhus verniciflua, and in particular to protect the cell protection And can be useful for the development of natural materials derived from Japanese lacquer tree.

Nrf2 is a transcription factor capable of inducing cytoprotective action in response to oxidative stress. It binds to Keap1 (Kelch-like ECH-associated protein 1) in the cytoplasm and migrates to the nucleus when it falls apart from Keap1. When Nrf2 moves to the nucleus, it binds to the antioxidant response element (ARE) and ultimately induces antioxidant production, thereby protecting the cell from oxidative stress. Therefore, a substance that activates a cell metabolic pathway leading to Keap1 / Nrf2 / ARE in a specific cell or tissue can be used as a functional food or drug for protecting or preventing oxidative damage of each tissue. In particular, the search for a Keap1 / Nrf2 / ARE pathway activator has been actively conducted for each target cell in order to identify substances that prevent skin, liver, kidney and nerve cell damage which may be highly oxidative damage. Sulfolafane, a representative natural product-derived cell protection substance, is known to protect dopaminergic neurons from oxidative stress by inducing expression of NQO1 (NAD (P) H / quinine oxidoreductase 1) through the above mechanism. However, there is a disadvantage in that when the sulforaphane is administered into the body, it is degraded within 1 hour and the blood-brain barrier permeability is low.

KP 1306863 (March 03, 2014) KP 1320149 (Dec. 14, 2014) KP 1265879 (May 31, 2013) KP 1270841 (March 05, 2013)

Song, Chi-Hyun et al., Korean J. Environ Agric. 2012, 31 (1), 60-67 Joon Nam et al., Food and Chemical Toxicology, 2013, 58, 355-361 Choi, YJ et al., Journal of the Korean Society for Applied Biological Chemistry, 2014, 57 (1), 27-30 Joon Nam et al., Food and Chemical Toxicology, 2012, 50 (6), 1940-1945 Appleton et al. Journal of Organic Chemistry, 2009, 74 (23), 9195-9198 Groweiss et al. Journal of Natural Products, 1997,60 (2), 116-121 Goldring et al. Toxicology 2008, 246, 24-33 Eggler et al. Proc. Natl. Acad. Sci. USA. 2005, 102, 10070-10075 Kobayashi et al. Mol Cell. Biol. 2006, 26, 221-229 Tong et al. Biol. Chem. 2006, 387, 1311-1320 Han et al. J. Pharmacol. Exp. Ther. 2007, 321, 249-256

The present invention provides an antioxidative or cytoprotective composition containing a novel hydroquinone compound isolated from raspberry extract.

The inventors of the present invention isolated the major components contained in the Raji extract of Wonju and the imported Raji extract of the foreign country and analyzed the nuclear magnetic resonance spectroscopy spectrum to investigate the component differentiation of the domestic lacquer of domestic lacquer in comparison with the imported lacquer product, And two new hydroquinone components, quinolone A and quinoline B, were identified in the extracts. In addition, Nrf2 / ARE activating activity of all isolated components was measured and it was revealed that only quinolin A and quinolin B act on Nrf2 / ARE activating activity, and the components of imported raloxifene and Korean raspberry extracts were analyzed by high performance liquid chromatography The present inventors completed the present invention by confirming that a relatively large amount of columnarquinol A or columnarquinol B was selectively contained in the domestic ricotta extract. Also, through the present invention, the two new compounds are present only in a specific raspberry extract, and as a Nrf2 / ARE activating substance, raspberry extract containing them and natural substances derived from Rhus verniciflua, cosmetics containing them as active ingredients, The composition was found to be more useful for protecting human cells against oxidative stress.

Accordingly, one example of the present invention is a method for producing a plant extract, which can be prepared by isolating from at least one selected from the group consisting of sap of lacquer tree, ruminal lacquer extract, extract of lacquer tree-derived material, dried extract of said sap or extract, and concentrate of said sap or extract There is provided an antioxidative or cytoprotective composition comprising a peripheral quinol A represented by Formula 1 and a quinoline B represented by Formula 2 below.

≪ Formula 1 >

(2)

The novel compound, quinolin A or quinoline B, isolated from ralgason's extract in the present invention has a physiological activity of increasing Nrf2 / ARE expression. Therefore, a composition containing these compounds as an active ingredient, or a natural substance derived from a lacquer containing them, Lt; RTI ID = 0.0 > cells. ≪ / RTI >

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the relationship between the concentration of quinoline A of the formula 1 and the concentration of the quinoline B of the formula 2 separated from the examples 1 to 2 of the present invention, The relative expression level of ARE-Luc was shown when 7 kinds of urushiol derivatives were treated with HepG2 cell line containing ARE-Luc plasmid at a concentration of 5 μM. At this time, the treatment group of the reference drug, sulfurazine, was set to 100%, and the relative expression level of ARE-Luc by the test group was expressed as the expression ratio of ARE-Luc to the reference drug.
FIG. 2 is a chromatogram (below) of a domestic rhubarb extract and an overseas rhubarb extract (below) under the conditions of Example 3 of the present invention and Test Example 1, The HPLC chromatograms of the components and the respective peaks were labeled with the numbers of the separated materials, and the negative chromatograms showed relative ABTS antioxidant.
FIG. 3 is a chromatogram comparing the domestic rhizome extract and the overseas rhizome extract (below) under the conditions of Test Example 2 of the present invention (below). HPLC The HPLC chromatogram of the components detected at 210 nm, with each peak labeled by the number of the isolated material.
FIG. 4 is a chromatogram obtained by comparing and analyzing the contents of two kinds of Korean raspberry raspberry extracts under the conditions of Test Example 3 of the present invention. HPLC The HPLC chromatogram of the components detected at 210 nm, with each peak labeled by the number of the isolated material.
FIG. 5 is a chromatogram comparing the contents of two kinds of raspberry extracts derived from other regions imported from China under the conditions of Test Example 3 of the present invention. HPLC The HPLC chromatogram of the components detected at 210 nm, with each peak labeled by the number of the isolated material.

The present invention relates to an antioxidative or cytoprotective composition containing a hydroquinone compound of formula (I)

(I)

More specifically, the present invention relates to an antioxidant or cell protective composition containing a hydroquinone compound represented by the following general formula (1) or (2).

≪ Formula 1 >

(2)

In one embodiment of the present invention, the hydroquinone compound may be separated from the raspberry extract. The separation process is as follows:

Lacquer is collected to produce lacquer concentrate. The concentrate means the one obtained by drying or concentrating the lacquer sap or Rhus verniciflua extract by a conventional method.

In one embodiment of the present invention, the rumba extract is used as a concept including both a crude extract of a natural product derived from lacquer and a crude extract as a solvent fraction or a fraction obtained through column chromatography.

In one embodiment of the present invention, the raspberry extract according to the present invention may be prepared by drying the raspberry sap or drying a natural substance derived from a lacquer tree with water, a linear or branched C ₁ to C ₄ alcohol, dichloromethane, Lacquer extracts obtained by extracting with one or more extraction solvents selected from the group consisting of ether, acetone and the like; And

Lacquer fractions obtained by adding one or more fraction solvents selected from the group consisting of hexane, chloroform, ether, dichloromethane, ethyl acetate, butanol, water and the like to the lacquer extract to perform solvent fractionation; The lacquer extract is applied to column chromatography using charcoal, celite or polystyrene or a polyamide or polymer adsorbent resin to prepare water, C ₁ to C ₄ linear or branched alcohols, dichloromethane, ethyl acetate, ether and acetone And lacquer fractions obtained by using at least one solvent selected from the group consisting of lacquer fractions.

The lacquer or the lacquer tree may be wild or cultivated. In particular, the lacquer is generally referred to as a sap of lacquer or juice, but in the production of the lacquer extract, roots, stalks or shells containing sap or juice thereof may be used. It can be used in dry or dry conditions.

After the production of the rumba extract or crude extract or fraction, the substance obtained is mixed with 50% (v / v) to 100% alcohol / water mixture of 1 to 4 carbon atoms or 60% (v / v) acetonitrile / Solution to 100% acetonitrile to separate and purify the compounds of Formulas 1 and 2 above. Column chromatography can use silica gel, Sephadex, RP-18, RP-8, RP-4, polyamide, polystyrene or polymer based reversed phase or adsorption filler. The column chromatography can be carried out several times by selecting an appropriate filler as necessary. In addition to the above-mentioned solvent composition, it is also possible to use a dichloromethane-alcohol, a chloroform-alcohol (for example, methanol), a hexane-ethylacetate ), Ethyl acetate-alcohol, acetonitrile-water or acetone-water.

In the embodiment of the present invention, the elution conditions of the column chromatography for separating and purifying the compounds of the formulas (1) and (2) were acetonitrile and water, and 10% acetonitrile / Starting with water, the acetonitrile composition was continuously adjusted to 60% (v / v) acetonitrile / water for 10 minutes by continuously increasing the acetonitrile content and then continuously increased to 100% acetonitrile content over 60 minutes, 100% acetonitrile was used as the final effluent solvent and a 20 mm x 250 mm RP-18 column was used as the column. 4 mg of the cyclic quinoline A of the formula 1 was obtained from 15 g of the rquach extract and 15 mg of the cyclic quinoline B of the formula 2 was obtained through the separation process.

The separated compounds were identified by chemical analysis using a molecular ion analyzer (MS) and a nuclear magnetic resonance spectroscopy (NMR). The chemical structure of the separated compounds was identified. Wonjuquinol A had a molecular weight of 332 and a molecular formula of C ₂₁ H ₃₂ O ₃ (Wonjuquinol B) was a colorless goggle material having a molecular weight of 314 and a molecular formula of C ₂₁ H ₃₀ O ₂ .

In the present invention, the urushiol derivatives contained in the ruby extracts except for the quinolin A and the quinolin B were identified, and the structure was identified through instrumental analysis. By establishing the HPLC analysis method for the separated components, the extracts of the rubmak extract or the lacquer- It provides a technique that can analyze the contained columnar quinol A or columnar quinol B or urushiol components at once by HPLC.

In the embodiment of the present invention, from the raspberry extracts collected in the vicinity of Wonju, Gangwon province through the separation process of the cyclic quinone A and the cyclic quinone B, the compounds of the following formulas (3), (4) , 20 mg, 52 mg and 42 mg, respectively, were separated from 15 g of the extract. Three kinds of urushiol derivatives represented by the formula (3) were separated by MS and NMR to obtain the 3 - ((11E, 13Z) -8 -hydroxypentadeca-11,13-dien-1-yl) benzene-1,2-diol, 3 - ((8Z, 11E, 13Z) -pentadeca-8,11,13-trien- benzene-1,2-diol of Formula 5 and (Z) -3- (pentadec-8-en-1-yl) benzene-1,2-diol of Formula 5.

(3)

≪ Formula 4 >

≪ Formula 5 >

Further, from the 13 g of the raspberry extracts imported from China through the above separation process, the above-mentioned Formulas (1) and (2) were not detected or almost not detected, 15>, 38 mg and 10 mg were separated from 13 g of the imported rhubarb extracts, respectively, and the three kinds of base urushiol derivatives represented by <Formula 6>, <Formula 7>, <Formula 8> 4 urushiol derivatives represented by the formula (9) were separated from 13 g of the extract, respectively, and 2 mg, 3 mg, 11 mg and 7 mg of the extract were separated. 11E) -pentadeca-8,11-dien-1-yl) benzene-1,2-diol, 3 - ((8Z, 11Z) -pentadeca-8,11- -1,2-diol, 3 - ((8Z, 12Z, 15Z) -heptadeca-8,12,15-trien-1-yl) benzene- 3 - ((10Z, 13E) -heptadeca-10,13-dien-1-yl) benzene-1,2-diol.

(6)

&Lt; Formula 7 >

(8)

&Lt; Formula 9 >

As the HPLC analysis conditions used for comparative analysis of HPLC peaks, production regions and parts of Rhus verniciflua extract components of the separated components of the present invention by liquid chromatography (HPLC), the elution conditions of the mobile phase were Trifluoracetic acid water containing 0.02% of acetonitrile and 0.02% of trifluoroacetic acid (TFA) containing 0.02% of acetone, acid, TFA) and starting with 10% acetonitrile / water at a flow rate of 1.0 mL / min and continuously containing acetonitrile , The composition of acetonitrile was adjusted to 100% (v / v) in 30 minutes, 100% acetonitrile was used as a final distillation solvent for 5 minutes, and then adjusted to 10% acetonitrile / water for 5 minutes Respectively. A 4.6 mm x 150 mm column for the RP-18 HPLC column was used and the analytical HPLC was performed on an Agilent 1200 series model equipped with an Agilent 6120 MS and a UV / VIS (DAD) detector or a Waters 1525 model, and the content of the ralgastic herbal extracts of the present invention could be easily comparatively analyzed by an HPLC chromatogram of UV 210 nm (FIG. 2). In addition, a separate reagent injection pump and a UV / VIS detector were installed so that the sample passed through the UV / VIS detector of HPLC and immediately moved to another UV detector simultaneously with the above HPLC analysis, and 2,2'-azino-bis -3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reagent, the relative antioxidant activity of each HPLC peak was measured simultaneously. In the HPLC chromatograms of FIG. 2 of the present invention, the larger the intensity of the signal shown below the peak of each peak, the greater the antioxidant activity with respect to the ABTS reagent.

As a result of analyzing the ricotta extract by the HPLC analysis conditions, it was found that the domestic ricotta extract of the present invention contained the cyclic quinoline A of Formula 1 or the cyclic quinoline B of Formula 2, In the extract, the above-mentioned columnar quinoline A or columnar quinole B was not detected or almost not detected. On the other hand, the components represented by the following formulas (8) to (9) were not detected or almost not detected in the domestic raspberry extract. In addition, it was confirmed that the quinol A and the quinoline B of the uriciol derivative had a relatively strong ABTS antioxidative activity compared to the urushiol derivative.

In addition, in order to comparatively analyze the relative content or the content of the components of the compounds of the formula (I) to the compound of the formula (I) with liquid chromatography (HPLC), the elution condition of the mobile phase is Trifluoracetic acid (TFA) Of water containing 0.02% of methanol and 0.02% of trifluoroacetic acid (TFA) can be used. As an example of the present invention, the methanol content was continuously increased at a flow rate of 0.7 mL / min to 70% methanol / water, and the methanol composition was adjusted to 100% (v / v) in 30 minutes. 100% And maintained for 10 minutes. A 4.6 mm x 150 mm column for the RP-18 HPLC was used as the column. Analytical HPLC was performed on the Agilent 1200 series model and Agilent 6120 MS and UV / VIS detector were used as the detector. 3).

In the embodiment of the present invention, in order to verify that the quinoline A or the quinoline B is contained in the domestic raspberry extract of the present invention, it is not contained in the raspberry extracts imported from China, 4] and [5]. In domestic rattan lavoxy extracts, the concentration of quinoline A or quinolone A in the domestic ricotta extract , But it was not contained in the extracts of Chinese rattan saplings or was rarely contained. Among the urushiol derivatives, the urushiol derivatives represented by the formulas (8) to (9) are contained in the Chinese lacquer, but are not contained in the domestic lacquer extract of the present invention. Therefore, the inclusion of the quinoline A or the quinoline B can provide important information for predicting the standard and efficacy of the raspberry extract or the natural substance derived from the lacquer. The analysis method of the raspberry extract of the present invention, And imported lacquer products.

The present invention also provides a method of measuring the Nrf2 / ARE activating activity of seven urushiol derivatives represented by the above Formulas 3 to 9 including the above-mentioned columnar quinolone A and the above-mentioned columnar quinoline B, , Or an Nrf2 / ARE activating cell protective composition containing at least one of them as an active ingredient.

In an embodiment of the present invention, a cell line (purchased from BPS bioscience) containing an ARE-Luc plasmid is added to HepG2 cells in MEM (HyClone) medium, 10% FBS (Invitrogen), non-essential amino acid (HyClone) 1X, PS ) 1X culture medium, cultured under a 5% carbon dioxide gas flow, and cultured by splitting every 3 days. Then, the above-mentioned columnar quinolone A and the quinoline B and 7 kinds of urushiol derivatives were treated at a concentration of 10 μM, and after 24 hours, ARE-Luc expression levels were measured. Sulforaphane (SFN), a natural substance whose activity was already reported for NRF2, was used as the reference drug. As a result of the measurement of the ARE-Luc expression intensity of each component, the seven kinds of urushiol components showed little or no activity, and the amount of ARE-Luc expression increased by 39.28% in the case of quinolin A and 18.08% in case of quinoline B (Fig. 1). Since rhizome quinolines A and B have a strong Nrf2 / ARE activating action compared to urushiol among the main components of raspberry extract, extracts of saponins, rhizome extracts and Rhus verniciflua extracts containing rhizome quinolone A or quinoline B, A composition comprising the dried product of the extract and the concentrate of the sap or extract may exhibit cytoprotective action by Nrf2 / ARE activation.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Isolation of Compounds from Korean Raspberry Extracts

15 g of domestic lacquer was applied to a chromatographic fractionation method using water or methanol as an eluent and using a Diaion HP20 resin (Mitsubishi Chemical, Japan) column to obtain 12.0 g of a fraction eluted physically and 2.9 g of a methanol elution fraction. 2.9 g of the methanol fraction obtained through HP20 column chromatography was injected into the HPLC equipped with a Phenomenex Luna C18 (2) 10 쨉 m (21.2 x 250 mm) column three times, at a flow rate of 10.0 ml / min and 10% v / v) Starting with acetonitrile / water, the composition of acetonitrile was increased with 60% (v / v) acetonitrile / water for 10 minutes, followed by continuously increasing the composition of acetonitrile for 60 minutes to yield 100% acetonitrile To be an eluent.

4 mg of the cyclic quinoline A of Formula 1 was obtained through the separation process and 15 mg of the quinoline B of Formula 2 was obtained. Further, 20 mg of 3 - ((11 E , 13 Z ) -8-hydroxypentadeca-11,13-dien-1-yl) benzene- 4> of 3 - ((8 Z, 11 E, 13 Z) -pentadeca-8,11,13-trien-1-yl) benzene-1,2-diol and 52 mg of the <formula 5> (Z) (Pentadec-8-en-1-yl) benzene-1,2-diol.

The ¹ H NMR and ESI-MS data of the separated compounds are as follows.

Circumferential quinol ^{A (Wonjuquinol A): 1 H} NMR (400 MHz, CDCl3) δ 6.56 (d, J = 8.5 Hz, 1H), 6.54 (d, J = 2.9 Hz, 1H), 6.45 (dd, J = 8.5, 3.0 Hz, 1H), 6.31 ( dd br q, J = 15.0, 11.0, 1.2 Hz, 1H), 5.91 (t br q, J = 11.0, 1.2 Hz, 1H), 5.61 (dt, J = 15.0, 7.0 Hz , 1H), 5.33 (dq, J = 11.0, 7.0 Hz, 1H), 3.53 (m, 1H), (br dd, J = 8.0, 7.5 Hz, 2H), 2.25-2.07 (m, 2H), 1.68 ( dd, J = 7.0, 1.2 Hz, 3H), 1.56-1.43 (m, 4H), 1.40-1.33 (br m, 2H), 1.31-1.21 (br m, 8H); ESI-MS m / z 331 [MH] ^- ; Molecular formula C ₂₁ H ₃₂ O ₃ .

Circumferential quinol ^{B (Wonjuquinol B): 1 H} NMR (400 MHz, CDCl 3) δ 6.64 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 2.9 Hz, 1H), 6.54 (dd, J = 8.5 , 3.0 Hz, 1H), 6.35 (dd br q, J = 15.0, 11.0, 1.2 Hz, 1H), 5.98 (t br q, J = 11.0, 1.2 Hz, 1H), 5.64 (dt, J = 14.0, 6.6 J = 6.6, 2H), 2.54 (br dd, J = 8.0, 7.5 Hz, 2H), 2.04 (br dt, J = 6.6, 6.6 Hz, 2H), 1.74 (dd, J = 7.0, 1.2 Hz, 3H), 1.59 (m, 2H), 1.39-1.28 (br m, 8H); ESI-MS m / z 313 [MH] ^- ; Molecular formula C ₂₁ H ₃₀ O ₂ .

3 - ((11 E, 13 Z) -8-Hydroxypentadeca-11,13-dien-1-yl) benzene-1,2-diol < Formula ^{3>: 1 H NMR (400} MHz, CDCl 3) δ 6.73- J = 10.1, 1.1 Hz, 1H), 5.97 (t brq, J = 10.9,1.1 Hz, 1H), 5.66 (dt, J = 14.0, 6.6 Hz, 1H), 5.40 (dq , J = 11.0, 7.0 Hz, 1H), 3.65 (m, 1H), 2.60 (br dd, J = 8.0, 7.5 Hz, 2H), 2.21 (m, 2H), 1.74 ( dd, J = 7.1, 1.6 Hz, 4H), 1.63-1.50 (m, 4H), 1.48-1.41 (br m, 2H), 1.31-1.21 (br m, 8H); ESI-MS m / z 331 [MH] ^- ; Molecular formula C ₂₁ H ₃₂ O ₃ .

3 - ((8 Z, 11 E, 13 Z) -Pentadeca-8,11,13-trien-1-yl) benzene-1,2-diol < Formula ^{4>: 1 H NMR (400} MHz, CDCl 3) δ 6.76-6.68 (m, 3H), 6.37 (dd br q, J = 15.1, 11.0, 1.2 Hz, 1H), 6.00 (t br q, J = 11.0, 1.2 Hz, 1H), 5.66 (dt, J = 14.0, 6.6 Hz, 1H), 5.50-5.38 (m, 3H), 2.87 (t, J = 6.7 Hz, 2H), 2.61 (br dd, J = 8.0, 7.5 Hz, 2H), 2.06 (br dt, J = 6.6, 6.6 Hz, 2H), 1.75 (dd, J = 7.0, 1.2 Hz, 3H), 1.62 (m, 2H), 1.40-1.30 (br m, 8H); ESI-MS m / z 313 [MH] ^- ; Molecular formula C ₂₁ H ₃₀ O ₂ .

(Z) -3- (Pentadec-8 -en-1-yl) benzene-1,2-diol < Formula ^{5>: 1 H NMR (400} MHz, CDCl 3) δ 6.75-6.68 (m, 3H), 5.40 J = 6.6, 6.6 Hz, 4H), 1.61 (m, 2H), 1.39-1.25 (br dd, J = m, 16H), 0.89 (t, J = 6.5 Hz, 3H); ESI-MS m / z 317 [MH] ^- ; Molecular formula C ₂₁ H ₃₄ O ₂ .

< Example  2> Separation of Compounds from Extracts from Rumi

13 g of ricin-extract obtained from China was applied to a chromatographic fractionation method using water or methanol as an eluent and using a Diaion HP20 resin (Mitsubishi Chemical, Japan) column to obtain 12.0 g of a fraction eluted physically and 0.6 g of a fraction eluted with methanol Respectively. 2.9 g of the methanol fraction obtained through HP20 column chromatography was injected into HPLC equipped with a Phenomenex Luna C18 (2) 10 mu m (21.2 x 250 mm) column, and the HPLC separation conditions were the same as in Example 1 Was started at 10.0 ml / min and the composition of acetonitrile was increased with 60% (v / v) acetonitrile / water for 10 minutes starting with 10% (v / v) acetonitrile / water, Lt; / RTI &gt; to 100% acetonitrile as the final eluent.

Three kinds of base urushiol derivatives represented by Formulas (3), (4) and (5) were separated by 15, 38 and 10 mg, respectively. (8 Z, 11 E) -pentadeca -8,11-dien-1-yl) benzene-1,2-diol to 2 mg, <formula 7> of 3 - ((8 Z, 11 Z) -pentadeca-8 (8 Z , 12 Z , 15 Z ) -heptadeca-8,12,15-trien-1-yl) benzene-1,2- -yl) benzene-1,2-diol of Formula 9 and 3 - ((10 Z , 13 E ) -heptadeca-10,13-dien- 7 mg.

3 - ((8 Z, 11 E) -Pentadeca-8,11-dien-1-yl) benzene-1,2-diol < Formula ^{6>: 1 H NMR (400} MHz, CDCl 3) δ 6.75-6.68 ( m, 3H), 5.46-5.33 (m , 4H), 2.72 (t, J = 5.5 Hz, 2H), 2.59 (br dd, J = 8.0, 7.5 Hz, 2H), 2.01 (br dt, J = 6.5, 6.5 Hz, 2H), 1.96 (br dt, J = 6.5, 6.0 Hz, 2H), 1.61 (m, 2H), 1.41-1.27 (br m, 10H), 0.88 (t, J = 7.4 Hz, 3H); ESI-MS m / z 315 [MH] ^- ; Molecular formula C ₂₁ H ₃₂ O ₂ .

3 - ((8 Z, 11 Z) -Pentadeca-8,11-dien-1-yl) benzene-1,2-diol < Formula ^{7>: 1 H NMR (400} MHz, CDCl 3) δ 6.75-6.68 ( m, 3H), 5.47-5.30 (m , 4H), 2.77 (t, J = 6.0 Hz, 2H), 2.60 (br dd, J = 8.0, 7.5 Hz, 2H), 2.07-2.01 (br m, 4H) , 1.61 (m, 2H), 1.44-1.27 (br m, 10H), 0.91 (t, J = 7.4 Hz, 3H); ESI-MS m / z 315 [MH] ^- ; Molecular formula C ₂₁ H ₃₂ O ₂ .

¹ H NMR (400 MHz, CDCl ₃ ):? - (8 Z , 12 Z , 15 Z ) -Heptadeca-8,12,15-trien- (m, 2H), 2.80 (t, J = 6.6 Hz, 2H), 2.60 (m, 2H), 5.70-5. (dd, J = 8.0, 7.5 Hz, 2H), 2.03 (br dt, J = 6.6 Hz, 2H), 1.73 (d, J = 6.5 Hz, 3H), 1.61 (m, 2H), 1.39-1.30 br m, 5H), 1.30-1.24 (br m, 7H). ESI-MS m / z 341 [MH] ^- ; Molecular formula C ₂₃ H ₃₄ O ₂ .

3 - ((10 Z, 13 E) -heptadeca-10,13-dien-1-yl) benzene-1,2-diol < Formula ^{9>: 1 H NMR (400} MHz, CDCl 3) δ 6.75-6.68 ( J = 6.5, 2H), 2.60 (br dd, J = 8.0, 7.5 Hz, 2H), 2.03 (br dt, J = 6.5, 6.5 Hz, 2H), 1.96 ( br dt, J = 6.5, 6.0 Hz, 2H), 1.61 (m, 2H), 1.41-1.31 (br m, 7H), 1.31-1.24 (br m, 7H), 0.88 ( t, J = 7.4 Hz, 3H); ESI-MS m / z 343 [MH] ^- ; Molecular formula C ₂₃ H ₃₆ O ₂ .

< Example  3> Sample pretreatment and analysis for analysis of components containing raspberry extract

10 mg of the methanol eluted fraction obtained through HP20 column chromatography in Examples 1 to 2 was dissolved in 10 ml of methanol and filtered using a 0.45 μm syringe filter. 10 ml of the filtrate was concentrated under reduced pressure at 30 ° C using a rotary vacuum concentrator to obtain a dried sample of 9.8 mg and again dissolved in methanol to prepare an analytical sample having a concentration of 1 mg / ml. The prepared samples were injected in a Phenomenex Luna C18 (2) (particle size 5 μm) 4.6 mm × 150 mm column with 10 μl, and analytical HPLC was performed on the Agilent 1200 series model equipped with a UV / VIS (DAD) detector. The composition of the eluent was the same as that of Experimental Example 1 described below. In addition, a separate reagent injection pump and a UV / VIS detector were installed so that the sample was passed through the UV / VIS detector of the HPLC and immediately moved to another UV detector simultaneously with the above HPLC analysis. In the separately mounted mixer, 2,2'-azino -bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reagent, the relative antioxidant capacity of each HPLC peak was measured simultaneously at 734 nm. In the HPLC chromatograms of FIG. 2 of the present invention, it was confirmed that the larger the intensity of the signal below the peak of each peak is, the greater the antioxidant activity with respect to the ABTS reagent (see FIG. 2).

In addition, in order to find a usable solvent for HPLC analysis other than acetonitrile, the analytical conditions of the above Experimental Example 2 were analyzed. When the methanol / water mixed solution was used as the eluent, (See Fig. 3).

< Example  4> Sample pretreatment and analysis of ingredients of Reticulatad herb extract

Rat lacquer extracts collected from Wonju, Gangwon-do, Wonju, Gangwon-do, Wonju, Shinpyeong, Gangwon province, and two kinds of Chinese liquorice extracts from China were dissolved in methanol at a concentration of 1 mg / ml without any separate column chromatography. After filtration using a filter, 10 μl was injected into the HPLC. The analytical HPLC was a Waters 1525 model equipped with a UV / VIS (PDA) detector, and the conditions of the assay were as follows. Four kinds of analytical samples were obtained through the sample pretreatment, and the results of analysis of the four kinds of samples are shown in FIG. 4 and FIG.

< Experimental Example  1> Acetonitrile / Water mixture solution As an eluent  One HPLC  analysis

The concentration of the sample was 1 mg / ml, and 10 μl of the sample was injected into the HPLC. The column used was a Phenomenex Luna C18 (2) (particle size 5 μm) 4.6 mm x 150 mm HPLC column. Analytical HPLC was performed on a Waters 1525 model equipped with an Agilent 1200 series model equipped with a UV / VIS (DAD) detector or a UV / VIS (PDA) detector. The elution conditions of the mobile phase were acetonitrile containing 0.02% of trifluoroacetic acid (TFA) and water containing 0.02% of trifluoroacetic acid (TFA), and a 10% acetonitrile / Water, the acetonitrile content was continuously increased to 100% (v / v) in 30 minutes, 100% acetonitrile was used as a final effluent solvent for 5 minutes, Min with 10% acetonitrile / water (Table 1).

Table 1. HPLC solvent composition with acetonitrile / water mixed solution as eluent

< Experimental Example  2> Mix the methanol / water mixture As an eluent  One HPLC  analysis

The concentration of the sample was 1 mg / ml, and 10 μl of the sample was injected into the HPLC. The column used was a Phenomenex Luna C18 (2) (particle size 5 μm) 4.6 mm x 150 mm HPLC column. Analytical HPLC Analytical HPLC was performed on an Agilent 1200 series model equipped with an Agilent 6120 MS and a UV / VIS (DAD) detector. The elution conditions of the mobile phases were as follows: methanol containing 0.02% of trifluoroacetic acid (TFA) and water containing 0.02% of trifluoroacetic acid (TFA) were used and eluted with 70% methanol / water at a flow rate of 0.7 mL / The methanol content was continuously increased to 100% (v / v) in 30 minutes. Methanol% acetonitrile was used as a final distillation solvent for 10 minutes (Table 2).

Table 2. HPLC solvent composition with methanol / water mixture solution as eluent

< Experimental Example  3> Nrf2 / ARE   Activity measurement

The expression level of ARE-Luc was measured in order to evaluate the physiological activity of inducing Nrf2-ARE expression in cells against oxidative stress caused by active oxygen generated in the cells by toxic substances and carcinogens Was used. Cell lines (purchased from BPS bioscience) containing ARE-Luc plasmid were added to HepG2 cells (human liver cancer cell line) in MEM (HyClone) medium, 10% FBS (Invitrogen), non-essential amino acid (HyClone) 1X, PS (Invitrogen) 1X medium, 5% CO ₂ air, and cultured with splitting every 3 days. As a control drug, 20,000 cells were prepared per well in a 96-well plate using sulfurazine (SFN), a natural substance already reported for NRF2, and treated with sulfolane (SFN) for 24 hours. The expression level of ARE-Luc Respectively. To measure the physiological activity of the compound, 20,000 cells were injected into a 96-well microwell plate, cultured in a 5% CO ₂ incubator for 24 hours, treated with 500 nL (10 mg / mL) 40 microliters were injected and observed with a luminometer. As a result of measurement of the relative amount of ARE-Luc expression, urushiol derivatives did not show any activity, and quinolone A and quinoline B showed 39.28% and 18.08% increase of ARE-Luc expression level compared to the control drug, respectively ].

Claims (2)

An antioxidant or cytoprotective composition comprising a compound of formula (I)

.
The method according to claim 1,
Wherein the compound of formula I is resistant to Nrf2 / ARE activation or oxidative stress.

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