KR100782443B1 - Novel flavonoid compound and preparation method thereof - Google Patents

Abstract

The present invention relates to a novel flavonoid compound and a method for preparing the same, and specifically, the method comprising extracting blue water ( Artemisia apiacea Hance) with an organic solvent or the like to obtain an organic solvent fraction and purifying it by chromatography again. It relates to a method for preparing a flavonoid compound represented by the formula (1) and a novel compound of the formula (1) which can be produced by the above method. The compounds according to the invention can be used as one component of pharmaceutical compositions having various activities as novel flavonoids.

<Formula 1>

Flavonoids, purpura, apicin

Description

Novel flavonoid compound and preparation method

1 is a result of ¹ H-NMR spectrum analysis of the flavonoid compound of the present invention isolated from blue water.

Fig. 2 shows the results of ¹³ C-NMR spectral analysis of the flavonoid compound of the present invention isolated from blue water.

Figure 3 shows the results of DEPT 90 spectral analysis of the flavonoid compound of the present invention isolated from blue water.

4 shows the results of DEPT 135 spectral analysis of the flavonoid compound of the present invention isolated from blue water.

Figure 5 shows the HOMO COSY spectrum analysis of the flavonoid compound of the present invention isolated from blue water.

Fig. 6 shows the results of HMQC spectrum analysis of flavonoid compounds of the present invention isolated from clear water.

Fig. 7 shows the results of HMBC spectrum analysis of flavonoid compounds of the present invention isolated from clarification.

8 is an IR spectrum analysis result of the flavonoid compound of the present invention isolated from blue water.

Fig. 9 shows the MS spectrum analysis of the flavonoid compound of the present invention isolated from blue water.

The present invention relates to novel flavonoid compounds and methods for their preparation. More specifically, a novel method for preparing a flavonoid compound, which comprises extracting an organic solvent or the like from Cheongho ( Artemisia apiacea Hance) and purifying it through chromatography, etc. It relates to a flavonoid compound.

Flavonoids are water-soluble pigments widely distributed in plants such as vegetables and fruits such as green tea, legumes, grapes, garlic and onions. Flavonols, flavones, isoflavones, flavonones, flavan-3-ols and anthocyanidins Plants classified into Dean subgroups and containing these various forms of flavonoids have long been used to treat a variety of diseases. Recently, flavonoids have been reported to play the role of natural antioxidants in removing free radicals from cells and tissues (Kris-Etherton PM, et al., Curr Opin Lipidol ., 2002, 13, 41-49: Nijveldt RJ, et al., Am J Clin Nutr ., 2001, 74, 418-425; Braca A, et al., J Ethnopharmacol, 2002, 79, 379-381), also reported as anti-inflammatory substances that inhibit CAMs and matrix protease (Bito T, et al., FEBS Lett ., 2002, 520, 145-152; Sartor L., et. al., Biochem Pharmacol , 2002, 64, 229-237; Middleton E. Jr, et al., Rev. , 2000, 52, 673-751). In addition, flavonoids have attracted much attention for their utility in the prevention and treatment of circulatory diseases (Cotelle N., Curr Top Med). Chem ., 2001, 1, 569-590; Hirano R, et al., J Nutr Sci Vitaminol ( Tokyo ), 2001, 47, 357-362). Therefore, techniques for obtaining flavonoids having various functions as described above are being developed. In the related art, a method of separating high-purity flavonoid components from ginseng leaves (Korea Patent Publication No. 1992-0005995), Method of extracting and purifying flavonoid compounds from ginkgo leaves containing various herbal ingredients (Korean Patent Publication No. 1994-0006596) and extracting flavonoid-containing substances from safflower leaves to produce safflower leaf tea (Korea Patent 0465778). In addition, studies on various pharmaceutical compositions containing flavonoids are also actively conducted. The conventional techniques for this include anti-atherosclerosis compositions containing flavonoids that can suppress the progression of arteriosclerosis (Korea Patent Publication No. 2004-0108265), a composition containing a soft water extract having antioxidant activity in the prevention and treatment of various diseases caused by oxidative stress, and a flavonoid compound isolated therefrom (Korean Patent No. 0514916) and the dolmul extract isolated from There is a pharmaceutical composition (Korean Patent Publication No. 2005-0074753) for preventing and treating hypertension, including a flavonoid compound.

On the other hand, Cheongho ( Artemisia apiacea Hance) is a kind of wormwood belonging to the chrysanthemum family and is known in Korea as Gaesae . It is known to be native to Korea, China, Japan, rivers, riversides and coastal areas, and it is known to have more than 350 species, and it has traditionally been known to be effective in eczema, jaundice, fever, geothermal and analgesic (Yook, CS) "Coloured medicinal plants of Korea". Seoul: 1989, p: 5). Therefore, in recent years, researches on the components of Chung Ho, which are effective in various diseases, are being actively conducted. The components contained in the clarifications to date are campesterol, stigmasterol, beta-sitosterol, 7-methoxycoumarin, 7,8-dimethoxycoumarin (7). , 8-dimethoxycoumarin, 7,8-methylenedioxycoumarin, scopoletin, protocatechualdehyde, ethyl and methyl caffeate, daphnetin ), 7-hydroxy-8-methoxycoumarin, 7-isopentenyloxy-8-methoxycoumarin and alpha pinene of volatile components α-pinene, artemisia ketone, artemicapin C, apigenin, daucosterol, dacosterol, cacticin, hyperin, alpha amino Α-amyrin, β-amyrin and 5,6,7-trimethoxycoumarin are known (Shimom ura et al., Phytochemistry , 1979, 18, 1761-17; Shimomura et al., Chem . Pharm . Bull. , 1980, 28, 347-3; Shimomura, H. et al., Yakugaku Zasshi , 1980, 100, 1164-116; Yano, K. Flavor Ind ., 1970, 1, 328-3; Kim, OC et al., Hanguk Nonghwa Hakhoechi , 1994, 37, 37-42; Lee, S. et al., Arch. Pharm . Res., 2002, 25, 285-2; Lee, S. et al., Arch. Pharm . Res., 2003, 26 , 902-9; Kim, KS et al., Nat. Prod. Sci ., 2005, 11, 10-12).

Accordingly, the present inventors completed the present invention by identifying and purifying novel flavonoid compounds and identifying them through their physicochemical and spectral analysis, while studying to find a substance having physiological activity from Cheongho.

It is therefore an object of the present invention to provide novel flavonoid compounds.

Another object of the present invention is to provide a method for preparing the compound.

In order to achieve the object of the present invention as described above, the present invention provides a novel flavonoid compound.

Moreover, in order to achieve the other objective of this invention, this invention provides the manufacturing method of the said compound.

Hereinafter, the present invention will be described in more detail.

The present invention is characterized by providing a novel flavonoid compound represented by the following formula (1).

[Formula 1]

The compound of Formula 1 is provided for the first time by the present inventors.

The present invention also provides salts, preferably pharmaceutically acceptable salts of the flavonoid compounds represented by the formula (1). The term “pharmaceutically acceptable salts” means salts suitable for use in contact with tissues of humans and lower animals without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of pure medical judgment. Such pharmaceutically acceptable salts are well known in the art. See, eg, SM Berge et al., J. Parmaceutical Sciences , 66, 1, 1977. Salts can be prepared in situ during the final separation and purification of the compounds of the invention or separately by reaction with inorganic or organic bases.

In addition, the present invention may include derivative compounds such as glycosides in which hydrates of the flavonoid compounds represented by the formula (1) or compounds such as sugar (glucose) are bonded to each side chain.

Flavonoid compounds according to the invention can be isolated from nature or prepared by chemical synthesis of flavonoid compounds known in the art.

Preferably the flavonoid compounds of the invention can be isolated and purified from natural plants. That is, it can be obtained from a plant or part of a plant using a method of extracting and separating conventional materials. Stems, roots or leaves are appropriately dried and macerated to obtain the desired extract, or only dried and extracted with a suitable organic solvent, and the desired extract can be purified using methods known to those skilled in the art. Can be purified. Preferably the flavonoid compounds of the invention can be isolated and purified from clarification.

Most preferably, the flavonoid compounds of the present invention can be isolated and purified by a method comprising the following steps:

(a) extracting Artemisia apiacea Hance with 1 to 4 carbon atoms;

(b) fractionating the extract obtained in step (a) with n-hexane, dichloromethane, ethyl-acetate and n-butanol;

(c) separating and purifying the dichloromethane fraction, ethyl-acetate fraction and n-butanol fractions obtained in the step (b) by concentration gradient chromatography, respectively.

In the step (a), chungho can use the whole plant as it is or the dry body, it can be used to crush the dry body of chungho in a grinder to increase the extraction efficiency. As a drying method, both dry, shade, hot air drying, freeze drying and natural drying methods can be used. Preferably hot air drying and lyophilization methods can be used.

The organic solvent used for the clarification extraction is not particularly limited, but preferably an organic solvent having 1 to 4 carbon atoms may be used. Examples of the organic solvent include, but are not limited to, various solvents such as methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, dichloromethane and petroleum ether. May be used alone or in combination. In one embodiment of the present invention, the clarification is extracted with methanol.

In the step (b), the extract obtained in the step (a) is fractionated and extracted by using n-hexane, dichloromethane, ethyl-acetate and n-butanol. Preferably, in step (b), the extract of Chungho obtained using methanol in step (a) is concentrated under reduced pressure, the concentrate is suspended in water, and then the mixture is fractionated by mixing n-hexane first. Obtain n-hexane fraction. Dichloromethane is mixed with the remaining aqueous layer and fractionated to obtain a dichloromethane fraction. Ethyl acetate was mixed with the remaining aqueous layer and fractionated to obtain an ethyl-acetate fraction. Then, the remaining aqueous layer was mixed with n-butanol and fractionated to obtain an n-butanol fraction.

In step (c), the dichloromethane fraction, ethyl-acetate fraction and n-butanol fraction obtained in step (b) are separated and purified by concentration gradient chromatography, respectively. As the chromatography, column chromatography filled with various synthetic resins such as silica gel or activated alumina, high performance liquid chromatography (HPLC), etc. may be used alone or in combination. Preferably, the ethyl-acetate fraction is applied to the silica gel column among the fractions obtained in step (b), and various fractions can be obtained while gradually increasing the polarity by adjusting the composition of the eluting solvent. Among the fractions obtained in the above process, the fraction having active activity can be carried out in a concentration gradient silica gel chromatography to gradually increase the polarity by adjusting the composition of the eluting solvent. However, extraction and separation and purification of the compounds are not necessarily limited to the above-described methods.

In general, flavonoid compounds are known to have excellent antioxidant activity. Therefore, the novel flavonoid compounds of the present invention are expected to have antioxidant activity and other useful physiological activities. Furthermore, it can be developed as a medicine for treating diseases of animals including humans based on specific physiological activity.

 Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

< Example  1>

From clearing  extraction

Cheongho ( Artemisia apiacea Hance) purchased from Gyeongdong Market was taken as an emotional sample and deposited in the Plant Collection of the Department of Plant Applied Science, Chung-Ang University (No. LEE 2005-01). The process of extracting 5 kg of dry powder of Cheongho with 10 L of methanol was repeated three times. The resulting extract was concentrated under reduced pressure to give 225 g of the extract, which was suspended in water and 40 g in a volume of 1000 ml n-hexane fraction, 38 g in dichloromethane fraction, 56 g in ethyl acetate fraction and in n-butanol fraction. 30 g of extract were obtained sequentially.

< Example  2>

Purification of the Compound

Silica gel column chromatography (200-400 mesh ASTM, Merck) was performed on 50 g of ethyl acetate fraction in the n-hexane fraction, dichloromethane fraction, ethyl acetate fraction and n-butanol fraction obtained in Example 1. At this time, a mixed solvent of chloroform and methanol was used as the eluent, and the concentration ratio chromatography of chloroform and methanol was gradually increased in polarity to 0, 10, 30, 50, 70 and 100%. . As a result, six aliquots of EA1 6g, EA2 8g, EA3 7.5g, EA4 7.5g, EA5 7.5g and EA6 8.5g were obtained, and silica gel column chromatography was again performed on 7.5g of EA3 aliquots. 2 mg of the compound represented by Formula 1 was isolated. At this time, a solvent of methanol and ethyl acetate was mixed at a ratio of 70:30 as an eluent, and concentration gradient chromatography was performed.

< Example  3>

Identification of Compounds of Formula 1

In order to identify the structure of the compound separated and purified in Example 2, IR spectra, NMR analysis, and EI-MS mass spectrometry were performed in the following manner. IR spectra were performed with Jasco's FT-IR-300E spectrometer, and ¹ H NMR (400 MHZ) and ¹³ C (100 MHZ) spectra were performed with a Varian, USA 400 NMR spectrometer. For mass spectrometry, JMS-AX505WA (JEOL, Japan) was used. The result of analyzing the compound purified by the same method as described above is as in <3-1>.

<3-1> Result of Compound Analysis of Chemical Formula 1

The analysis results of the compound of formula 1 of the present invention are as follows.

Appearance: Yellow crystals (methanol)

IR (KBr) v max: 3383, 1615, 1015 cm ^-1

¹ H-NMR (400 MHz, DMSO- d6 ): δ 13.03 (s, 5-OH), 7.44 (s, H-6 '), 7.09 (s, H-2'), 6.95 (s, H-8 ), 6.55 (s, H-4 '), 3.92 (s, 7-OMe), 3.80 (s, 5'-OMe), 3.71 (s, 6-OMe)

¹³ C-NMR (100 MHz, DMSO- d6 ): δ 183.1 (C-4), 162.8 (C-2), 161.6 (C-5), 159.3 (C-7), 154.1 (C-3), 153.5 (C-9), 152.8 (C-3 ') , 142.5 (C-5 '), 132.5 (C-6), 112.8 (C-1', C-6 '), 107.6 (C-2'), 105.7 (C-10), 105.2 (C-4 ' ), 92.5 (C-8), 60.9 (6-OMe), 57.6 (5'-OMe), 57.3 (7-OMe)

EIMS (70 eV, rel. Int.,%): M / z 360 [M] ⁺ (100), 345 (67), 331 (12), 314 (16), 285 (6), 181 (21), 165 (11), 153 (9), 137 (5)

As a result, the compound of the present invention showed yellow crystals, and the molecular ion peak at m / z 360 in EI-MS was consistent with the molecular structure of C ₁₈ H ₁₆ O ₈ . In particular, retro Diels-Alder fragmentation of flavonoids was observed at m / z 153 (see FIG. 9). Of the hydroxy absorption at the ^first band, 1615 cm ^{- -} results of IR spectrum 3383 cm ¹ in the α, β- unsaturated are C = 0 absorption band, 1015 cm ^{- 1} In the CO absorption band it is observed (see Fig. 8). Typical flavonoid signals were observed in the ¹ H NMR spectrum, with aromatic 5-OH at δ 13.03 and 3-OMe signals at δ 3.91, 3.80 and 3.71. Further, hydrogen-8, -2 ', -4' and -6 'were observed at δ 6.95, 7.09, 6.55 and 7.44, respectively (see FIG. 1). In the ¹³ C NMR spectrum of the compounds of the present invention C = O was observed at δ 183.1 and -OMe at δ 57.3, 57.6 and 60.9 (see FIG. 2). The analysis results of the ¹ H-NMR and ¹³ C-NMR signals for the direct and long-range correlation of the heteronuclear nucleus are shown in Table 1 below.

NMR Chemical Transitions and Associations of Compounds of Formula 1 location δ _H δ _C DEPT HMBC 2 - 162.8 C - 3 - 154.1 C - 4 - 183.1 C - 5 - 161.6 C - 6 - 132.5 C - 7 - 159.3 C - 8 6.95 (s) 92.5 CH C-6, C-9, C-10 9 - 153.5 C - 10 - 105.7 C - One' - 112.8 C - 2' 7.09 (s) 107.6 CH C-2 3 ' - 152.8 C - 4' 6.55 (s) 105.2 CH C-3, C-2 ', C-3' 5 ' - 142.5 C - 6 ' 7.44 (s) 112.8 CH C-2, C-3, C-3 ', C-5' 5-OH 13.03 (s) - - - 6-OMe 3.71 (s) 60.9 CH ₃ C-6 7-OMe 3.92 (s) 57.3 CH ₃ C-7 5'-OMe 3.80 (s) 57.6 CH ₃  C-5 '

Based on the above results, the compound of Formula 1 was prepared as 3,5,3'-trihydroxy-6,7,5'-trimethoxyflavone (3,5,3'-trihydroxy-6,7,5'- trimethoxyflavone), which was the first novel flavonoid isolated from nature that has not been reported yet, and was named by the inventors as apicin.

As discussed above, the compounds of the present invention are novel flavonoids isolated and purified for the first time in nature by the present inventors, and can be usefully used for research of antioxidant activity and specific biological activity, and are used in diseases of animals including humans. It will be an important leader in the development of therapeutics. It can also be used to screen for new bioactivity of flavonoids.

Claims (4)

Flavonoid compounds represented by the following formula (1) and salts thereof. <Formula 1>

(a) extracting Artemisia apiacea Hance with 1 to 4 carbon atoms; (b) fractionating the extract obtained in step (a) with n-hexane, dichloromethane, ethyl-acetate and n-butanol; (C) The method for producing a flavonoid compound of claim 1, comprising the step of separating and purifying the dichloromethane, ethyl- acetate and n-butanol fractions obtained in step (b), respectively, by concentration gradient chromatography. The preparation according to claim 2, wherein the organic solvent of step (a) is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, chloroform, ethyl acetate, methylene chloride and dichloromethane. Way. The method of claim 2, wherein the chromatography in step (c) is silica gel chromatography.

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