KR20030022620A - Novel chlorogenic acid methyl ether compounds isolated from Phyllostachys edulis leaf and a use thereof - Google Patents

Abstract

PURPOSE: Provided are 3-oxy-caffeoyl-1-methyl-quinic acid and 5-oxy-caffeoyl-4-methyl-quinic acid, which are novel chlorogenic acid derivatives compounds extracted from a leaf of Phyllostachys Pubescens, and its use. CONSTITUTION: The 3-oxy-caffeoyl-1-methyl-quinic acid is represented by formula 1. The 5-oxy-caffeoyl-4-methyl-quinic acid is represented by formula 2. The compounds are prepared by the steps of (i) washing, lyophilizing and then crushing a leaf of Phyllostachys Pubescens, and extracting the obtained dry powder under reflux, (ii) precipitating the extract with ethanol to remove polymer compounds, and sequentially fractionating the extract with hexane, chloroform, ethylacetate, and butanol, which have different polarities respectively, (iii) purifying the fraction by column chromatography and thin layer chromatography, and (iv) separating the purified extract by using high speed liquid chromatography.

Description

Novel chlorogenic acid methyl ether compounds isolated from Phyllostachys edulis leaf and a use approximately

The present invention relates to a novel chlorogenic acid methyl ether-based compound extracted from the dead bamboo leaf and its use. More specifically, chlorogenic acid methyl ether-based compound and its antioxidant activity, immunopotentiation, and hypoglycemic lowering were extracted using an organic solvent, extracted with column chromatography, thin layer chromatography, and HPLC. will be.

All aerobic organisms, including humans, survive through energy metabolism using oxygen (O ₂ ). However, when oxygen in vivo is subjected to various physical, chemical and biological stresses, superoxide anion radical (O ^2- ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (OH), etc. It turns into a harmful active oxygen species, causing fatal physiological disorders in the human body and, in severe cases, causing disease and losing life. In particular, if the harmful effects of free radicals and free radicals continue to accumulate as the cells age, diseases related to various adult diseases such as carcinogenesis, arteriosclerosis, heart disease, and skin aging, as well as the overall aging of cells It has been suggested as a cause of human disease occurrence and aging (Harman, D., Free Radicals in Biology, Academic Press, New York, 255-275 (1982)).

As a result, research into the development of antioxidants known to control free radicals and reactive oxygen species, which have been identified as the cause of aging and geriatric diseases, has been actively conducted, and superoxide dismutase, peroxidase, and catalase have been developed. ), Many studies have been conducted on antioxidants such as glutathione peroxidase and low molecular weight antioxidants derived from natural products such as tocopherol, ascorbate, carotenoid, and glutathione. (Chang, SS, et al., J. Food Sci. 42: 1102, 1977; Hammerschmidt, PA and Pratt, DE, J. Food Sci. 43: 556, 1977; Pratt, DE and Watts, BW, J. Food Sci. 29: 17, 1964), 2,6'-di-tert-butyl-4-hydroxy toluene (2,6'-di-tert-butyl-4-hydroxytoluene, BHT), 2,6'- Di-tert-butyl-4-hydroxyanisole (2,6-di-tert-butyl-4-hydroxyanisole, BHA), etc. Many synthetic antioxidants have been developed and used in medicine and food (Kitahara, K., et al., Chem. Pharm. Bull. 40: 2208, 1992; Hatano, T., Natural Medicines 49: 357,1995; Masaki , H., et al., Biol. Pharm. Bull. 18: 162, 1995).

Butyl hydroxy anisole (BHA) and butyl hydroxy toluene (BHT), the synthetic antioxidants are excellent in antioxidant power, but the usage and use are strictly limited due to human toxicity. Therefore, there is an active attempt to find an antioxidant that is high in natural products and does not harm the human body. For example, catechin family compounds of green tea leaves (Nanjo, F .; Goto, K .; Seto, R .; Suzuki, M .; Hara, Y. Free Radic. Biol. Med. 21: 895-902, 1996), carrots Beca carotene-based compounds of the well-known, in the case of domestic flavone-based antioxidants isolated from the was studied by the present inventors (Kweon, MH; Kwak, JH; Sung, HC; Yang, HCJ Biochem. Mol. Chem. 29: 423-428, 1996).

On the other hand, a complement system consisting of about 20 kinds of serum proteins is a classical pathway that is activated from the C1 protein to the immune complexes of antigens and antibodies, and zymosan or beta-glucan (β-glucan). ) Is divided into alternative pathways where C3 activation, a complement-centric pathway, occurs directly. This complement system plays an important role in the host defense mechanism against external infectious agents such as bacteria, fungi, viruses. However, fragments such as C3a, C4a, and C5a, which are released during abnormal complement system activation, are anaphylatoxins and are a major cause of various inflammatory diseases including asthma, allergies, and atopic dermatitis (Bellanti, TA). (1985) Saunders, Philadelphia, pp. 106., Miyagawa, S., Hirose, H. Shirakura, R. Nakata, Y. Kawashima, Y. Seya, T. Matsumoto, M. Uenaka, A. and Kitamura, H (1988), 825., Zvaifler NH (1989), S17., Sabharwal UK, Vaughan JH, Fong S, BennettPH, Carson DA, Curd JG. (1982), 161., Murray JF, Matthay Ma, Luce Jm, Flick Mr. (1988), 720)

Therefore, when the complement system is excessively activated, autoimmune disease, exacerbation of inflammation, and severe rejection in transplantation are caused. Therefore, complement inhibitors can be used as anti-inflammatory or autoimmune diseases (Arvind Sahu et. Al., Immunopharmacology, 49 (2000) 133-148), Arvind Sahu et. al., Biochemical pharmacology, Vol. 57.pp. 1439-1446, 1999).

Xanthine oxidase is an enzyme involved in purine metabolism, which oxidizes xanthine or hypoxanthine in human cells to produce uric acid and reactive oxygen species. When uric acid increases in the blood, it may accumulate in the blood or fracture due to its low solubility, causing gout or depositing in the kidney, causing kidney disease. Therefore, inhibiting excessive activity of xanthine oxidase is known to inhibit the production of harmful oxygen species in the human body and to treat gout (Tandtaka Noro et. Al., Chem. Pharm. Bull). , 31 (11) 3984-3987 (1983), studies to isolate new xanthine oxidase inhibitors from natural products are also underway worldwide (Hao Li et al. J. Natural Product, 62, 1053-1055 (1999). )

Alphaglucosidase acts as a final hydrolysis of disaccharides in the small intestine of the animal and releases glucose into the small intestine mucosa, thus inhibiting the activity of this enzyme can prevent the increase of blood sugar by inhibiting the absorption of carbohydrates after meals. It is very important for diabetic patients to control the rapid post-prandial blood sugar rise in diabetic patients, especially non-insulin-dependent diabetes mellitus (NIDDM) patients who have excessive insulin resistance. (Chiasson, Jean-Louis MD et. Al., Diabetes Care, 21 (10) 1720-1725. October (1998), Martin, Ann E. et. Al., American Journal of Health-system Pharmacy. 53 (19) 2277-2290, October (1996))

Bamboo is widely distributed in subtropical and temperate regions. Especially, bamboo is an evergreen tree belonging to the genus Japonica, which is common in Asian monsoon zones. Bamboo is mainly cultivated in the southern part of Korea. . Kings and cotton rods are used more often than for food. Mengjongjuk (孟宗竹) is the thickest among bamboos, with a diameter of about 20cm in Japan. It is also used for making furniture, but it is commonly called edible bamboo because it is thick and can be eaten. In addition to the parts used for wood, all the dead bamboo leaves are being discarded. In addition, they have been used as a treatment for vomiting, anti-inflammatory, miscarriage, sweating, and intermediates (consent of consent, Chinese herbal medicine dictionary) and other substances that exhibit antimicrobial properties. It has been used as a bamboo container in Japan since ancient times, and has been used in food storage for meat and dumplings in Korea.

Most studies on bioactive substances of bamboo including bamboo shoots have been reported in Korea and Japan mainly on antibacterial activity. Already, 2,6-dimethylbenzoquinone and benzoic acid, which are antimicrobial substances in bamboo leaves, have been identified by Japanese researchers. In Korea, the antimicrobial activity of bamboo leaf extracts (Kim, Mi-Jung et al., Korean Journal of Nutrition and Nutrition, Vol. 25 No. 1, p.135-142, 1996) and the general bamboo shoots Very limited studies have been reported, including reports on component analysis (Jung Jong Sung et al., Korean Journal of Forestry, vol. 78, No. 1, p. 55-60, 1989).

There have been no reports on the active substance having antioxidant activity in the dead leaves, but the present inventors have specified that the extract of the dead leaves in the domestic application No. 2001-55000 has antioxidant activity.

The present inventors have isolated two novel methyl chlorogenic compounds from the extracts of the dead bamboo leaf, and the new substance has antioxidative and complement inhibitory activity, xanthine oxidase inhibitory activity, and alpha-glucosidase inhibitory activity. It confirmed and completed this invention.

It is therefore an object of the present invention to provide novel 3-chloro-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid, which are novel chlorogenic acid derivatives extracted from the dead leaves. . It also provides the use of the novel 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid as antioxidants.

The present invention is freeze-dried after lyophilization and then pulverized and then powdered and extracted with ethanol, using hexane, chloroform, ethyl acetate, butanol sequentially to partition the organic solvent fraction, column chromatography and thin layer chromatography, and Purified by HPLC, two novel methyl chlorogenic acid compounds were isolated, and the antioxidant activity was determined by measuring the free activity scavenging activity, the determination of free radical scavenging activity, and the lipid peroxidation inhibitory activity of the isolated new material. It was completed by confirming the alpha-glucosidase and xanthine oxidase inhibitory activity.

Hereinafter, the configuration and operation of the present invention.

Figure 1 shows the results of electron spin resonance spectroscopy (ESR) spectra of the DPPH free radicals treated with the compound and alpha-tocopherol separated from the present bald bamboo leaves.

The manufacturing method according to the present invention comprises the steps of reflux extraction at 80 ± 2 ° C. using 70% ethanol after lyophilizing or drying and crushing the dead bamboo leaves after crushing; Ethanol precipitation of the extract to remove the polymeric compound, and then sequentially partitioning fractions using hexane, chloroform, ethyl acetate, butanol with different polarities; Dissolving the extract in distilled water to purify the column with four steps of adsorptive HP-20 column, polar silica gel column, LH-20 gel filtration column, ODS reversed phase chromatography; The column purification active fractions are dissolved in methanol and finally purified by thin phase chromatography followed by normal and reverse phases. Separation of pure water using high performance liquid chromatography using a reversed phase hydrophobic column.

As a result of pure separation by the above method, three compounds were separated. As a result of identifying the molecular structure, one was known compound, 3-feruloylquinic acid, which was separated from raw coffee beans. Compounds (Corse, J .; Sondheimer, E .; Lundin, R. Tetrahedron, 18: 1207-1210, 1962).

The other two compounds have been recognized by the American Chemical Society as novel chlorogenic acid derivatives of the formulas (1) and (2).

[Formula 1]

The compound of Formula 1 is a chlorogenic acid methyl ether compound. The compound is a kind of ester compound composed largely of methylquinic acid on the left side and caffeic acid on the right side. On the one hand, it is a chlorogenic acid derivative having a methyl group in oxygen connected to carbon 1 and consisting of ester 3. The compound is composed of 19 carbons, 20 hydrogens and 9 oxygens, and has 4 free hydroxyl groups, that is, hydroxyl groups at 4, 5, 3 'and 4' carbons, respectively. I have one each. In particular, the dihydroxyl groups at the 3 ′ and 4 ′ positions of the caffeic acid site have an ortho type of catecol structure. In addition, methoxyl and carboxyl groups are present on the quinic acid No. 1 carbon, respectively, and oxygen linked to the third carbon of the quinic acid and 9 ＇carbon of caffeic acid are bonded as esters. This was named 3-oxy-cafeoyl-1-methyl-quinic acid.

[Formula 2]

The compound represented by Chemical Formula 2 is a chlorogenic acid methyl ether compound similar to 3-oxy-cafeoyl-1-methyl-quinic acid, which is Compound 1, methylquinolinic acid on the right side and caffeic acid on the left side It is a kind of ester compound. Unlike 3-oxy-cafeoyl-1-methyl-quinic acid, it is a chloroacid derivative having a methyl group in oxygen linked to carbon 4 and consisting of ester 5. The compound is a structural isomer of 3-oxy-cafeoyl-l-methyl-quinic acid, consisting of 19 carbons, 20 hydrogens and 9 oxygens, with 4 free radicals at positions 3, 5, 3 'and 4' Each has one hydroxyl group (free hydroxyl). The dihydroxyl groups at the 3 'and 4' positions of the caffeic acid site, as in compound 1, have an ortho-type catecol structure, but also methoxyl to quinine acid 4 carbon composed of a carboxyl group at position 1 There is a practical group, and the structure linked to the ester of the oxygen linked to the 5th carbon of quinic acid and 9 ＇carbon of caffeic acid. This was named 5-oxy-cafeoyl-4-methyl-quinic acid.

Scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, xanthine and xanthine oxidase for each compound represented by Chemical Formulas 1 and 2 The scavenging activity of the active oxygen, ie superoxide, and the lipid peroxidation inhibitory activity in the microsomes isolated from rat liver were measured, and the scavenging function of free radicals was directly measured by electrospin resonance (ESR). It was confirmed that the antioxidant activity of the compound.

In addition, it was confirmed that the hemolysis inhibitory activity of the complement to the sensitized amount of red blood cells for each compound represented by the above formulas (1) and (2) has a complement inhibitory activity, by measuring the degree of inhibition of the oxidation of xanthine It was confirmed that there is an oxidase inhibitory activity, and by inhibiting alpha-glucosidase by the colorimetric determination of the colorant produced by the reaction of hydrogen peroxide produced by adding glucose oxidase reagent with orthodianisidine (ο-dianisidine) It was confirmed that there is activity.

The novel 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid extracted from the seedlings of the present invention are superior to the conventional natural antioxidant alpha-tocopherol. appear. Therefore, the antioxidant composition containing the novel 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid, respectively, according to the present invention can prevent and treat aging and adult disease. It can be used as a pharmaceutical composition or a nutraceutical and cosmetics. In addition, since the dead bamboo leaf has been used for a long time as a herbal medicine and edible extract of the present invention can be expected that there will be no problems such as toxicity and side effects.

In addition, 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have complement inhibitory activity, and the complement activity regulators of the present invention are both in vitro and in vivo. Has the ability to modulate complement activity. Therefore, the complement activity modulator of the present invention can be used to treat asthma, allergy, arthritis, rheumatism, systemic lupus erythematosus, retinitis, hepatitis, enteritis, pancreatitis, nephritis, rhinitis, atopic dermatitis related to complement activity In addition, it can be useful for suppressing the transplant rejection reaction caused by organ transplantation.

In addition, 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have xanthine oxidase inhibitory activity, and thus the xanthine oxidase inhibitor composition of the present invention It can be usefully used to treat diseases of gout and kidney stones.

In addition, 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have alpha-glucosidase inhibitory activity, and thus the alpha-glucosidase inhibitor of the present invention. The composition can be usefully used for the treatment of diseases associated with obesity, diabetes, hyperlipidemia associated with it.

Hereinafter, the specific configuration and operation of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these embodiments.

Example 1 Separation and Purification of Pure Substances from

Bamjongjuk leaf used in the present invention is a leaf of Bamjongjuk inhabiting Hacheongmyeon, Geoje-do, Korea, collected and washed with water, lyophilized or crushed after drying, and used as a pure material.

Step 1: Extraction of Ethanol from the Blind Bamboo Leaf

500 g of the dried bamboo leaf powder was placed in a 5 L volume round bottom flask, and 3 L of 70% ethanol was added to the mixture at 80 ± 2 ° C. for 6 hours at reflux for 3 to 4 times to extract about 10 L of the extract. After extraction, the residue was removed by centrifugation (4 ° C., 5000 rpm, 25 minutes) and a reduced pressure filter. The filtrate was removed using a rotary evaporation pressure reducer and lyophilized to obtain about 90 g of antioxidant extract.

Stage 2: Distribution of Organic Solvents from

After dissolving the extract in step 1 in 1 L of distilled water, 3L of cold 95% ethanol was added thereto, stirred and left to stand at 4 ° C for 12 hours. The precipitate produced in this process is removed by centrifugation (4 ° C., 6000 rpm, 30 minutes) and reduced pressure filter filtration, and the filtrate is dried under reduced pressure and redissolved in 2 L of distilled water. In addition, 2 L of n-hexane was mixed in a 5 L volumetric funnel and fractionated twice into a hexane layer and an aqueous layer. 2L of chloroform was added to the aqueous layer, and the mixture was partitioned twice into a chloroform layer and an aqueous layer, and ethyl acetate was further added to the aqueous layer, and twice separated into an ethyl acetate layer and an aqueous layer. Finally, 2L of n-butanol was added to the aqueous layer to add a butanol layer. Fractions were twice with and the aqueous layer.

Each fraction obtained above was dried under reduced pressure to remove each organic solvent, followed by lyophilization. The hexane fraction, the chloroform fraction, the ethyl acetate fraction, the butanol fraction, and the final aqueous layer fraction were 15.2 g, 6.9 g, 2.1 g, 9.3 g, and 16.1 g, respectively. I got it.

Step 3: Purification of Column Chromatography

The n-butanol fraction of step 2 was dissolved in 50 mL of alkaline water (pH 10), adsorbed onto the top of the first column (4 x 28 cm) filled with adsorptive diion HP-20 resin, and eluted with 30% methanol. 2.2 g of active fractions are eluted in a second column (5 x 12 cm) filled with polar silica gel using a chloroform solvent with increasing methanol concentration. The active fraction was dried under reduced pressure and dissolved in 1 mL of methanol, and introduced into a third column (2 x 95 cm) filled with low molecular gel filtration Sephadex LH-20. Fractions were made in 4 mL portions per minute. The active fractions (tubes 48-60) were mixed and concentrated in a fourth column (1.5 x 8.5 cm) filled with hydrophobic octadecyl silane (ODS), and the flow rate was 0.1 with a concentration gradient of 30-70% methanol. Fractionation of 2 mL under mL / min yielded 357 mg of strong antioxidant activity fraction (tubes 10-18).

Step 4: Thin Layer Chromatography Purification

After dissolving the active fraction of step 3 in a small amount of methanol and dropping onto a thin layer chromatography plate (20 x 20 cm) coated with polar silica gel, a mobile solvent consisting of n-butanol: methanol: water = 4: 1: 2 Explode using and scrape off the active band at the Rf value of 0.7. This component was eluted with methanol and redeposited on a reversed phase plate (20 x 10 cm) coated with hydrophobic octadecsilane (ODS) silica to develop 30% methanol as a mobile phase solvent, and the final purified dead leaf leaf antioxidant at the Rf 0.3 position. 108 mg of the composition was obtained.

Step 5: pure separation by HPLC

70 mg of the antioxidant composition obtained in step 4 was dissolved in 10 mL of methanol for HPLC, and then passed through a 0.2 μm sample filter to isolate the antioxidant single substance using stabilized high performance liquid chromatography. High-speed liquid chromatography uses Waters Co Model 2690 alliance series equipped with US Waters Co SimmertryPrep column (SymmertryPrep, 7μm, 21. x 150 mm), acetonitrile and 1 Tertiary distilled water containing 3% trifluoroic acid (TFA) was mixed at a ratio of 17 to 83, and the degassed mobile phase solvent was flowed at a flow rate of 1.5 mL / min, and Waters 996 photodiode array, PDA) Purified compounds using the UV absorbance pattern of the material and the absorbance of the two wavelength ranges (254 and 300 nm), resulting in compound 1 (7.0 mg) with retention time of 16 minutes and retention time of 20 minutes Compound 2 (5.2 mg) and Compound 3 (8.5 mg) with a retention time of 28 minutes were obtained. Of the three compounds, Compound 1 was identified as a known substance 3-feruloylquinic acid isolated from raw coffee beans, but Compounds 2 and 3 were recognized by the American Chemical Society as a novel chlorogenic acid derivative. received.

Example 2 Identification of Structure of New Compound

In order to measure the maximum ultraviolet absorption bands of Compounds 2 and 3 separated from Example 1, that is, the novel Compounds 1 and 2 of the present invention, each sample was dissolved in methanol at a concentration of 1 mg / mL, and was then irradiated with an ultraviolet-visible spectrometer. (Shimadzu), UV-2401) and measured in the 190-500nm range. AlCl ₃ , NaOH, NaOAC, and the like were used to observe the change in the ultraviolet absorption band according to the addition of the reagent.

To prepare the infrared absorption region, 2-3 mg of each compound was well mixed with potassium bromide (KBr) and made into a disk of 0.5 cm in diameter, and the maximum IR absorbance was measured on an infrared spectrometer (Jasco FTIR-430).

The molecular weight of the compound was determined by the high resolution of the glycerol matrix using an electron foam ionization (ESI) mass spectrometer (Micromass Quattro II) and a high resolution fabmass mass spectrometer (JMS-700 Mstation mass spectrometer). MS was measured.

Nuclear Magnetic Resonance (NMR) analysis showed that pure purified compounds (5-7 mg) were completely dried, dissolved in CD ₃ OD (0.5 mL), injected into a 5 mm NMR tube, and brewed with a Bruker model (Bruker AMX-500). NMR analysis was performed, ¹ H-NMR was measured at 500 MHz, and ¹³ C-NMR was measured at 125 MHz. For other melting point measurement, DSC 2010 model was used, and optical activity measurement was performed using AUTOPOL III polarimeter.

As a result of the measurement, the physical and chemical properties of the novel compound 1 of the present invention are as follows.

(1) Molecular Formula: C ₁₇ H ₂₀ O ₉

(2) Melting Point: 202-204 ℃

(3) Optical activity: [α] ²⁵ _D -16.9 (water, c 0.35)

(4) Appearance: White powder

(5) Solubility: Soluble in water, methanol, ethanol, butanol and insoluble in chloroform, hexane, etc.

(6) TLC positive color development: FeCl _3, bromocresol green

(7) ultraviolet absorption band (methanol, nm): 245 sh, 300, 329; (+ NaOH) 266 sh, 310, 374; (+ AlCl ₃ ) 355; (+ AlCl ₃ + HCl) 245sh, 299, 328; (+ NaOAc) 338, 377 sh; (+ NaOAc + H ₃ BO ₃ ) 254, 306, 351

(8) Infrared absorption band (KBr, cm ^-1 ): 3423, 2957, 1734, 1686, 1631, 1522, 1443

(9) Molecular weight: 368: ESI-MS, m / z 369 [M + H] ⁺ , FAB-MS m / z 369 [M + H] ⁺ , m / z 391 [M + Na] ⁺

(10) ¹ H and ¹³ C-NMR

¹ H NMR: (CD ₃ OD, 500 MHz) 7.51 (1H, 15.9 Hz, H-7), 7.04 (1H, d, 2.0 Hz, H-2), 6.94 (1H, dd, 8.2 Hz, 2.0 Hz, H-6), 6.77 (1H, d, 8.4 Hz, H-5), 6.20 (1H, d, 15.9 Hz, H-8) 5.26-5.30 (1H, m, H-3), 4.12-4.15 (1H , m, H-5), 3.71 (1H, dd, 7.5 Hz, 3.1 Hz, H-4), 3.70 (OCH ₃ ), 1.93-2.23 (4H, m, H-2, H-6);

¹³ C NMR: (CD ₃ OD, 125 MHz) 175.4 (COO-), 168.3 (C-9), 149.7 (C-3), 147.2 (C-4), 146.8 (C-7), 127.7 (C- 6), 123.0 (C-1), 116.5 (C-5), 115.1 (C-2, C-8), 75.8 (C-4), 72.6 (C-1), 72.1 (C-3), 70.3 (C-5), 53.0 (OCH ₃ ), 38.1 (C-6), 37.8 (C-2)

(11) chemical structural formula

[Formula 1]

(12) Chemical name: 3-O-caffeoyl-1-methylquinc acid

Physical and chemical characteristics of the novel compound 2 of the present invention are as follows.

(1) Molecular Formula: C ₁₇ H ₂₀ O ₉

(2) Melting Point: 200-201 ℃

(3) Optical activity: [α] ²⁵ _D -39.2 (ethanol, c 0.30)

(4) Appearance: White powder

(5) Solubility: Soluble in water, methanol, ethanol, butanol and insoluble in chloroform, hexane, etc.

(6) TLC positive color development: FeCl _3, bromocresol green

(7) ultraviolet absorption band (methanol, nm): 244 sh, 296, 328; (+ NaOH) 261 sh, 308, 372; (+ AlCl ₃ ) 263sh, 306, 351; (+ AlCl ₃ + HCl) 244sh, 296, 328; (+ NaOAc) 274, 334, 375; (+ NaOAc + H ₃ BO ₃ ) 252sh, 303, 348

(8) Infrared absorption band (KBr, cm ^-1 ): 3433, 2950, 1735, 1680, 1631, 1519, 1445

(9) Molecular weight: 368: ESI-MS, m / z 369 [M + H] ⁺ , FAB-MS m / z 369 [M + H] ⁺ , m / z 391 [M + Na] ⁺

(10) ¹ H and ¹³ C-NMR

¹ H NMR: (CD ₃ OD, 500 MHz) 7.61 (1H, 15.9 Hz, H-7), 7.06 (1H, d, 2.0 Hz, H-2), 6.95 (1H, dd, 8.2 Hz, 2.0 Hz, H-6), 6.78 (1H, d, 8.2 Hz, H-5), 6.35 (1H, d, 15.9 Hz, H-8) 4.23-4.30 (3H, m, H-3, 4, 5), 3.75 (OCH 3), 1.99-2.21 (4H, m, H-2, H-6);

¹³ C NMR: (CD ₃ OD, 125 MHz) 176.5 (COO-), 168.9 (C-9), 149.5 (C-3), 146.9 (C-4), 146.8 (C-7), 127.9 (C- 6), 123.0 (C-1), 116.5 (C-5), 115.8 (C-8), 115.1 (C-2), 74.3 (C-4), 73.9 (C-1), 71.3 (C-3) ), 70.6 (C-5), 52.9 (OCH ₃ ), 40.8 (C-2), 36.4 (C-17)

(11) chemical structural formula

[Formula 2]

(12) Chemical Name: 5-O-caffeoyl-4-methylquinic acid

Example 3 Investigation of Antioxidant Activity of Novel Compounds According to the Present Invention

In order to investigate the antioxidant activity of the novel compound of Example 2, the scavenging activity, free radical scavenging activity, lipid peroxidation inhibitory activity, and free radical scavenging function of free radicals were measured, respectively.

Experimental Example 1 Measurement of Free Radical Scavenging Activity

The free radical scavenging activity was measured using 1,1-diphenyl-2-picryl hydrazyl (1,1-diphenyl-2-picryl hydrazyl, DPPH) according to the method of Bullos. That is, 20 mg of DPPH was dissolved in 150 mL of ethanol to make a DPPH solution, and then a sample dissolved in 0.5 ml of methanol was added at a concentration of 0-100 μg / mL, immediately shaken for 5 seconds, and then 10 to 30 minutes later. , The absorbance decrease for the control group without the sample at 517 nm is shown as free radical scavenging activity. The concentration of material required to eliminate 50% free radicals is expressed as an IC ₅₀ value. The lower this value, the stronger the antioxidant activity.

Free radical scavenging activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid and free radical scavenging activity of alpha-tocopherol, a conventional antioxidant, according to the present invention The results measured as described above are shown in Table 1 below.

Free Radical Scavenging Activity of the Novel Compound of the Invention and Alpha-tocopherol compound DPPH scavenging activity (IC ₅₀ value, μM) 3-Oxi-cafeoyl-1-methyl-quinic acid 6.9 5-Oxy-caffeoyl-4-methyl-quinic acid 8.8 Alpha-tocopherol 40.6

In the results of Table 1, the free radical scavenging activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid, which are novel compounds of the present invention, is widely known. Compared with alpha-tocopherol, a natural antioxidant vitamin, it showed significantly stronger activity. That is, in the case of 3-oxy-cafeoyl-1-methyl-quinic acid represented by Formula 1, DPPH scavenging activity was about 5.8 times compared to alpha-tocopherol, and 5-oxy-cafeoyl-4-methyl represented by Formula 2 -Quinic acid showed slightly lower activity than 3-oxy-cafeoyl-1-methyl-quinic acid, but DPPH scavenging activity was about 4.5 times stronger than alpha-tocopherol.

Experimental Example 2 Measurement of Active Oxygen Scavenging Capacity

Superoxide anion scavenging activity was measured by photooxidation of nitroblue tetrazolim (NBT) by active oxygen using an active oxygen generator using xanthin / xanthin oxidase enzyme reaction. It was measured using a change in absorbance (530 nm). The concentration of the substance required to eliminate 50% free radicals is expressed as an IC ₅₀ value.

The active oxygen scavenging activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid and the active oxygen scavenging activity of alpha-tocopherol, which are conventional antioxidants, The measurement results are shown in Table 2 below.

Free radical scavenging activity of novel compounds and alpha-tocopherols according to the present invention compound Free radical scavenging activity (IC ₅₀ value, μM) 3-Oxi-cafeoyl-1-methyl-quinic acid 1.2 5-Oxy-caffeoyl-4-methyl-quinic acid 2.8 Alpha-tocopherol > 50

From the results of Table 2, the active oxygen scavenging activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid, which are novel compounds of the present invention, is widely known. Compared with the alpha-tocopherol antioxidant vitamin, it showed a much stronger activity. In the case of 3-oxy-cafeoyl-1-methyl-quinic acid represented by the formula (1), the active oxygen scavenging activity is 50 times or more as compared to alpha-tocopherol. 5-oxy-cafeoyl-4-methyl-quinic acid represented by the formula (2) showed slightly lower activity than 3-oxy-cafeoyl-1-methyl-quinic acid, but active oxygen scavenging activity was 20 than alpha-tocopherol. It showed more than twice as strong activity.

Experimental Example 3 Measurement of Lipid Peroxidation Inhibitory Activity

Lipid peroxidation inhibitory activity was determined by inducing lipid peroxidation by Fe ²⁺ / ascorbate system using microsomes obtained by ultracentrifugation (77000g, 60 minutes, Hitachi RP 30) of isolated rat hepatocytes. Malondialdehyde (malonaldehyde, MDA) was reacted with thiobarbituric acid (TBA) to quantify the amount of MDA reduced by the sample. 200 μL of the liver microsome solution containing the sample and 225 μL of the sodium dodesyl sulfate (SDS) solution were added to the test tube, shake-mixed for 5 seconds, 20% acetic acid, 75 μL distilled water, and 1 mL of 1.2% TBA solution were added thereto. Warmed in water bath for 30 minutes. After cooling at room temperature for 30 minutes, the supernatant was measured for absorbance at 532 nm by centrifugation at 3000 rpm for 20 minutes to determine lipid peroxidation inhibitory activity (%) against the control group without the sample. The concentration of the substance required to eliminate 50% free radicals is expressed as an IC ₅₀ value.

Lipid peroxidation inhibitory activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid according to the present invention and lipid peroxidation inhibitory activity of alpha-tocopherol, a conventional antioxidant, The measurement results are shown in Table 3 below.

Lipid Peroxidation Inhibitory Activity of the Novel Compounds of the Present Invention and Alpha-tocopherol compound Lipid peroxidation inhibitory activity (IC ₅₀ value, μM) 3-Oxi-cafeoyl-1-methyl-quinic acid 14.6 5-Oxy-caffeoyl-4-methyl-quinic acid 19.2 Alpha-tocopherol 50.1

In the results of Table 3, natural antioxidants, which are widely known to inhibit lipid peroxidation activity of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid, which are novel compounds of the present invention Compared with the alpha-tocopherol vitamin, it showed a very strong activity. In the case of 3-oxy-cafeoyl-1-methyl-quinic acid represented by Formula 1, lipid peroxidation inhibitory activity was about 3.2 times stronger than that of alpha-tocopherol, and 5-oxy-cafeoyl-4- represented by Formula 2 Methyl-quinic acid showed slightly lower activity than 3-oxy-cafeoyl-1-methyl-quinic acid, but lipid peroxidation inhibitory activity showed about 2.6 times stronger antioxidant activity.

Experimental Example 4 Measurement of the Clearing Function of Free Radicals by ESR

In order to directly check the scavenging function of free radicals, 1,1-diphenyl-2-picryl hydrazyl (1,1-diphenyl-), a stable free radical compound, was prepared using an electron spin resonance spectroscopy (ESR). 2-picryl hydrazyl (DPPH) solution was added to the compound of the present invention and alpha-tocopherol to directly determine whether the radical scavenging. The measurement conditions of the electron spin resonance spectrometer (Bruker model ESP-300s ESR, Silberstreifen, Germany) were 25 ℃, 10 mW microwave power, 41.9 s sweep time, microwave frequency 9.78 GHz and 200 μM DPPH and 0.1 mM samples. Used.

By the method described above, the phenomenon of 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid directly eliminating free radicals was tested under the same conditions as alpha-tocopherol. The results are shown in FIG. As shown in FIG. 1, three types of anti-oxidant compounds of P. oleracea containing 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid as compared to the control group (1) (3, 4, 5) completely erased DPPH radicals, but alpha-tocopherol (2) did not completely erase under the same experimental conditions.

Example 4 Investigation of Biological Activity of Novel Compounds According to the Present Invention

Complement inhibitory activity, xanthine oxidase inhibitory activity, and alpha-glucosidase inhibitory activity were measured to investigate the physiological activity of the novel compound of Example 2.

Experimental Example 5: Complement Inhibitory Activity

Anti-complement activity was observed in normal human serum (NHS) and GVB⁺⁺(gelatin veronal buffered saline, pH 7.2) 50 μl of each sample (100 μg / mL) dissolved in buffer and tertiary distilled water was mixed and reacted at 37 ° C. for 30 minutes, and then GVB was added to the reaction solution.⁺⁺350 μl each was added and it was serially diluted from 10-fold to 160-fold. 750 μl GVB here⁺⁺Sensitized red blood cells (IgM-hemolysin-sensitized sheep erythrocyte, 10⁸cells / mL) were added and reacted for 1 hour, followed by centrifugation with 2.5 mL of PBS (phosphate buffered saline, pH 7.4) buffer, and the absorbance of the supernatant was measured at 412 nm. Complement inhibitory activity is ITCH₅₀, Inhibition of 50% Total Complement Hemolysis. Complement inhibitory activity is shown in Table 4, expressed as a percentage (%).

Physiological Activity of Novel Oxy-Capeoyl-1-Methyl-Quinic Acid and 5-Oxi-Capeoyl-4-Methyl-Quinic Acid compound Complement inhibitory activity Xanthine oxidase inhibitory activity Alphaglucosidase inhibitory activity 3-Oxi-cafeoyl-1-methyl-quinic acid 66.3% 88.6% 52.7% 5-Oxy-caffeoyl-4-methyl-quinic acid 52.5% 80.1% 48.3% *: Concentration of treated material in each bioactivity test is 0.1 mg / mL.

As shown in Table 4, 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have complement inhibitory activity. Severe rejection of autoimmune diseases, exacerbations of inflammation, and transplantation when the complement system, a group of proteins in the serum that is responsible for part of the human immune system and destroys foreign substances or pathogens infiltrated from outside with antibodies, is excessively activated. 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid of the present invention, which are complement inhibitory activities, result in a reaction, respectively, asthma, allergy, arthritis, It can also be used to treat inflammatory diseases including rheumatoid, systemic lupus erythematosus, retinitis, hepatitis, enteritis, pancreatitis, nephritis, rhinitis, atopic dermatitis and to inhibit organ transplant rejection.

Experimental Example 6: Xanthine oxidase inhibitory activity

Xanthine oxidase activity is carried out with some modification of Noro et al. (Noro, T; Oda, Y .; Toxhio, M; Ueno, A. Chem. Pharm. Bull. 31: 3984-3987, 1983). It was. A test tube containing 0.1 mg sample solution (0.1 mL), 50 mM phosphate buffer (pH 7.5, 0.3 mL) and 0.1 enzyme solution (0.5 unit / mL, xanthine oxidase) was mixed at 25 ° C. for 15 minutes. After standing, 0.15 mM xanthine substrate solution (0.2 mL) was added thereto, followed by reaction at 25 ° C. for 30 minutes. The reaction was stopped with 0.1 mL HCl solution, and the ultraviolet absorbance at 290 nm was measured. Xanthine oxidase inhibitory activity was expressed as a percentage compared to the control without the sample, and the results are shown in Table 4 above.

As shown in Table 4, 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have xanthine oxidase inhibitory activity. Excessive activity of xanthine oxidase accumulates uric acid, which causes gout or kidney stones, and free radicals that are harmful to the body. Accordingly, the 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid of the present invention having xanthine oxidase inhibitory activity are respectively referred to as gout or kidneystone. It can be used as a disease treatment.

Experimental Example 7: Alpha-glucosidase inhibitory activity

Alpha-glucosidase is a method of Ove (Ove, N .; Sjostorm, H .; Cowell, GM; Tranum-Jenser, J .; Hansen, O .; Welinder, KGJ Biol. Chem. 261: 12306-12309, 1986) was used as a modifier from the pig small intestine, the substrate necessary for the reaction was prepared by reacting for 30 minutes at 37 ℃ by preparing 1% starch, 50 mM maltose, 100 mM sucrose in 0.1 M phosphate buffer Glucosidase activity was measured by colorimetric determination of the colorant produced by reacting hydrogen peroxide with o- dianisidine by adding glucose oxidase reagent to the reaction solution (0.1 mg / mL) at 540 nm. The results are shown in Table 4 above.

As shown in Table 4, 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid were found to have alpha-glucosidase inhibitory activity.

In the case of inhibiting alpha glucosidase, it is possible to inhibit the absorption of carbohydrates after a meal, thereby preventing excessive blood sugar increase. Therefore, the present invention 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy which have alpha glucosidase inhibitory activity Caffeoyl-4-methyl-quinaconic acid can be used to treat diabetes, hyperlipidemia, or to control obesity.

As described above through the above examples and experimental examples, the present invention is a novel compound 3-oxy-cafeoyl-1-methyl-quinic acid and 5-oxy-cafeoyl-4 that have been separated and purified from the dead bamboo leaves. -Methyl-quinic acid can be used as an antioxidant derived from natural products because it has excellent antioxidant activity, especially active oxygen scavenging function, and simultaneously shows complement inhibitory activity, xanthine oxidase and alpha glucosidase inhibitory activity. Treatment of inflammatory diseases including allergic, arthritis, rheumatoid, systemic lupus erythematosus, retinitis, hepatitis, enteritis, pancreatitis, nephritis, rhinitis, atopic dermatitis and inhibitors for organ transplant rejection, gout associated with xanthoxidase activity It can be developed for medicines such as diabetes, hyperlipidemia, and obesity inhibitors related to the treatment of stones, alpha-glucosidase activity And cosmetics using the above-mentioned effects, it is also applicable to functional foods biomedicine field is industrially very useful invention.

Claims (12)

3-O-caffeoyl-1-methyl-quinic acid (3-O-caffeoyl-1-methylquinc acid) compound represented by Formula 1 below. [Formula 1] 5-O-caffeoyl-4-methylquinic acid compound represented by the following formula (2). [Formula 2] Antioxidant composition, characterized in that the compound of claim 1 or claim 2 as an active ingredient. A cosmetic composition comprising the compound of claim 1 or 2 as an active ingredient. A functional food comprising the compound of claim 1 or 2 as an active ingredient. Complement activity inhibitory agent comprising the compound of claim 1 or 2 as an active ingredient. The method of claim 6, wherein the complement inhibitor is composed of asthma, allergies, arthritis, rheumatoid, systemic lupus erythematosus, retinitis, hepatitis, enteritis, pancreatitis, nephritis, rhinitis, atopic dermatitis and organ transplant rejection associated with complement activity Complement activity modulator, characterized in that useful for the treatment of inflammatory diseases. A xanthine oxidase inhibitor composition comprising the compound of claim 1 or 2 as an active ingredient. The xanthine oxidase inhibitor composition according to claim 8, wherein the xanthine oxidase inhibitor composition is useful for the treatment of diseases of gout and kidney stones associated with the inhibitory activity of xanthine oxidase. An alpha- glucosidase inhibitor composition comprising the compound of claim 1 or 2 as an active ingredient. The alpha-glucosidase inhibitor composition according to claim 11, wherein the alpha-glucosidase inhibitor composition is useful for the treatment of diabetes, hyperlipidemia and obesity-related diseases associated with alpha-glucosidase inhibitory activity. Washing the seedling leaves with lyophilization and then crushing the dried powder to reflux with ethanol; Ethanol precipitation of the extract to remove the polymeric compound, and then sequentially partitioning fractions using hexane, chloroform, ethyl acetate, butanol with different polarities; Purifying the organic solvent fractions using column chromatography and thin layer chromatography; And New compounds 3-oxy-cafeoyl-l-methyl-quinic acid and 5-oxy-cafeoyl-4-methyl-quinic acid are extracted from the dead bamboo leaves, which comprises the step of purely separating the purified extract using high-performance liquid chromatography. How to manufacture.