KR20040058736A - Gallotannin compounds from Juglans mandshurica with anticomplement activity - Google Patents

Abstract

PURPOSE: Provided is Mandshurica walnut extract which characteristically has anticomplement activity. In particular, gallotannin compound isolated from the Mandshurica walnut extract is useful for therapeutics for diseases caused by abnormal increase and activation of complements. Also, provided are medicaments and food additives containing gallotannin compounds. CONSTITUTION: A therapeutic for allergic diseases is characterized by containing Mandshurica walnut extract, wherein the active ingredient of the Mandshurica walnut extract is selected from compounds represented by the formulae(1,2) or their derivatives.

Description

Extract of Sap Tree with Anti-complement Activity {Gallotannin compounds from Juglans mandshurica with anticomplement activity}

The present invention relates to a sputum bark extract having an anti-complement activity, and more particularly, to an anti-complement activity from a sputum bark extract, and also less toxic, as a therapeutic agent and a therapeutic agent for diseases caused by abnormal increase and activation of complements. The present invention relates to a method for separating a galoteannin compound which can be usefully used, and for separating an active ingredient from pharmaceuticals and food additives and phlegm containing the same.

Complement system consisting of about 20 kinds of serum proteins is divided into classical pathways activated by immune complexes and non-traditional pathways activated by cell components. This complement system plays an important role in the host defense mechanism against external infectious agents such as bacteria, fungi and viruses, but C3 is released during abnormal complement activation._aC4_aC5_aFragments, such as anaphylatoxin, have become a major cause of various inflammatory diseases including asthma, allergies, atopic dermatitis, etc. [Bellanti T.A., 1985,In Immunology III, pp. 106; Miyagawa S., Hirose H., Shirakura R., Nakata Y., Kawashima Y., Seya T., Matsumoto M., Uenaka A., Kitamura H., 1988,Transplation46, 825; Zvaifler N.J., 1989,Arthritis care res. 2, S17; Sabharwal U.K., Vaughan J.H., Fong S., Bennett P.H., Carson D.A., Curd J.G., 1982,Arthritis Rheum. 25, 161]. Therefore, as a search for substances to prevent and treat this has been attempted, lipopolysaccharide (lipopolysaccharide),β(1 → 3) glucan, inulin, 6-branchβ(1 → 3) Polysaccharides of polymers such as glucan and arabinogalactan were mainly isolated from fungi, bacteria, and herbal medicines [Muschel L.H., Schmoker K., Webb P.M., 1964,Proc. Soc. Exp. Biol. Med.117, 63; Okuda T., Yoshioka Y., Ikekawa T., Chihara G., Nishioka K., 1972,New Biol.238, 59; Hamuro J., Hadding U., Bitter-Suermann D., 1978,Immunology34, 695; Yamada H., Nagai T., Cyong T-C., Otsuka Y., Tomoda M., Shimizu N., Shimada K., 1985,Carbohydr. Res.144, 101], The activity was not strong, and low molecular weight phytosterol has also been reported as an anti-complement active [Tsuyoshi E., Tomoko K., Masanori K., Seigo F., Akira U., 1991,Yakugaku zasshi111 (6), 299; Hiruki Y., Nasami Y., Tsukasa M., Takayuki N., Horoaki K., Jong-Chol C., Akira N., Haruo T., Satoshi O., 1987,Chem. Pharm. Bull. 35 (12), 4851].

On the other hand, Juglans mandshurica Maximowicz is a deciduous arboretum of Dicotyledoneae, Juglandales, which grows in the north of Korea. 1996, Flora of Korea, Kyo-Hak Publishing Co., Ltd., Seoul, pp. 43], roots as a cancer treatment in civilian [Soon JK, 1995, Arch. Pharm. Res . 18, 203-205], fruit as nuclear antler, gastritis and abdominal pain, bark as nuclear antler, is used to treat hari, red eye [Bae KH, 2000, Medicinal Plants of Korea, Kyo-Hak Publishing Co., Ltd., Seoul, pp. 50.]. Component of Azusa, and is mainly composed of phenolic compounds, naphthoquinone paddy his allocated stay (naphthoquinone, naphthoquinonyl glucosides), naphthalene glycosides (naphthalenenyl glucosides), alpha-Te Neutral Theory glycoside (α -tetralonyl glucosides), diallyl heptane glycosides (diarylheptanoyl glucosides) and gallotannin [Soon JK, 1995, Arch. Pharm. Res . 18, 203-205; Joe YK, Son JK, Park SH, Lee IJ, Moon DC, 1996, J. Nat. Prod . 59, 159-160; Kim SH, Lee KS, Son JK, Je GH, Lee JS, Lee CH, Cheong CJ, 1998, J. Nat. Prod. 61, 643-645; Min BS, Nakamura N., Miyashiro H., Kim YH, Hattori M., 2000, Chem. Pharm. Bull . 48, 194-200], studies of the efficacy of cytotoxicity on HT-29 (human colon carcinoma) and A549 (human lung carcinoma) [Kim SH, Lee KS, Son JK, Je GH, Lee JS, Lee CH, Cheong CJ, 1998, J. Nat. Prod. 61, 643-645], inhibitory action of reverse transcriptase of HIV-1 virus [Min BS, Nakamura N., Miyashiro H., Kim YH, Hattori M., 2000, Chem. Pharm. Bull . 48, 194-200] and the proliferative inhibitory effect of HIV-1 virus [Min BS, Lee HK, Lee S. M., Kim YH, Bae KH, Otake T., Nakamura N., Hattori M., 2002, Arch. Pharm . Res . 25, 441-445], but studies on anti-complement activity using sputum have not been reported.

Accordingly, the present inventors, while studying a substance having an anti-complementary effect, the gallotannin compound obtained from the sputum tree exhibits anti-complementary activity, and also has low toxicity and various refractory properties including immune system diseases related to abnormality increase and activation of complements. The present invention was completed by revealing that it can be used as a food additive for treating and assisting treatment of chronic diseases.

Accordingly, it is an object of the present invention to provide a use as a food and therapeutic agent for diseases caused by abnormal increase and activation of complement containing a sputum bark extract, its gallotannin compound as an active ingredient and derivatives thereof.

Another object of the present invention is to provide a method for separating galoteannin compounds exhibiting anti-complement activity from sputum trees.

The present invention is characterized by its use as an agent for treating diseases and food additives caused by abnormal increase and activation of complements containing extracts, galoteannin compounds and derivatives thereof as sputum.

In addition, a method for separating the gallotannin compound exhibiting anti-complement activity from the sputum tree is another feature.

Referring to the present invention in more detail as follows.

The present invention relates to the use as a food and therapeutic agent for diseases caused by abnormal increase and activation of the complement containing the sputum bark extract and its active ingredient, the gallotannin compound represented by the formula (1) to (2) and derivatives thereof.

More specifically, the present invention is 1,2,6-trigalloylglucoside represented by the following formula (1,2,6-trigalloylglucoside), 1,2,3,6-tetragallo represented by the following formula (2) It includes a disease therapeutic agent and a food additive caused by the abnormal increase and activation of complements that use monoglucoside (1,2,3,6-tetragalloylglucoside) or derivatives thereof.

The gallotannin compounds represented by the above formulas (1) and (2) may also be used as derivatives thereof, and preferably esters containing one, two or five galloyl groups in the glocos.

The gallotannin compounds represented by Chemical Formulas 1 to 2 may be prepared by various methods, and specifically, may be obtained by chemical synthesis or extraction from plants.

The gallotannin compound of the present invention has shown activity against anticomplement. In the following example, to investigate the anti-complement activity, the anti-complement activity by the classical pathway test method was examined.

As a result, gallotannin compounds include asthma, arthritis, allergies, rhinitis, atopic dermatitis, lupus, Crohn's disease, and immune system disorders such as oxidative burst, which are associated with increased abnormality and activation of complement. It can be used for the prevention and treatment of various refractory and chronic diseases.

In particular, proteins such as C3 _a , C4 _a and C5 _a caused by an increase in complement abnormality expressed as part of humoral immunity act as anaphylatoxin to secrete histamine and serotonin, resulting in asthma and allergies. , Atopic dermatitis, and other major inflammatory diseases. It is expected to be very useful in treating asthma and rhinitis, which are allergic diseases including immune system diseases by suppressing the complement generated by the immune response.

The gallotannin compounds and derivatives thereof of the present invention can be administered orally or parenterally during clinical administration and can be provided in the form of general pharmaceutical preparations.

The gallotannin compound and derivatives thereof of the present invention may be administered in various oral or parenteral formulations during actual clinical administration. When formulated, the gallotannin compound and its derivatives may be used in formulating a filler, extender, binder, humectant, disintegrant, surfactant, and the like. Prepared using diluents or excipients.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in the gallotanine compound and derivatives thereof. Mixed with gelatin. In addition to simple excipients, lubricants such as magnesium styrene and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous and suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The content in the preparation of the active ingredient according to the present invention can be appropriately selected depending on the absorbency, inactivation rate, excretion rate, age, sex and condition of the user in the body. In the case of the gallotannin compound of this invention, it is 0.01-100 mg / kg, Preferably it is 0.1-10 mg / kg, and can be administered 1-3 times a day.

The gallotannin compound of the present invention did not show a toxicity change upon oral administration of 100 mg / kg in experimental mice, and the minimum oral dose of love (LD ₅₀ ) was 100 mg / kg or more, indicating a very high bio stability. The gallotannin compound of the present invention can be administered stably to a living body.

In addition, the present invention is a 1,2,6-trigalloyl glucoside represented by the formula (1), 1,2,3,6-tetragalloyl glucoside represented by the formula (2) or derivatives thereof as an active ingredient It can be used as a food supplement for the treatment of diseases caused by the abnormal increase and activation of complements, including functional foods, dietary supplements or special nutritional products.

As used herein, the term "functional food" means a food product having improved functionality of a general food product by adding the gallotannin compound and derivatives thereof to the general food product.

Different from functional foods, 'health supplement' or 'special dietary supplement' in the present specification is a health food prepared by adding the gallotannin compound to a general food, or by encapsulating, powdering, and suspension, and ingesting the same. If it means to bring a specific effect on health, but unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for a long time.

The content of the functional food, dietary supplements and specialty health foods containing the galotenin compound and derivatives thereof according to the present invention can be varied according to the food group used, the content of which is measured when used in the pharmaceutical composition. It is performed within the specified toxicity range.

In addition, the present invention

1) extracting pulverized juglans mandshurica by dissolving in methanol or a mixed solvent of methanol and water, and then concentrated under reduced pressure and fractionated with hexane and ethyl acetate to obtain a hexane fraction and an ethyl acetate fraction; And

2) The ethyl acetate fraction was subjected to adsorption chromatography (eluent: chloroform-methanol, 10: 1) and partition chromatography (eluent: 60% aqueous methanol solution and 20% acetonitrile aqueous solution). Obtaining a galoteannin compound.

Step 1) first extract the sputum tree three times in succession using methanol at room temperature to obtain a methanol extract, and then concentrate under reduced pressure to obtain a sputum methanol extract. Suspended by adding water to the methanol extract of sputum, and then hexane Fractions and ethyl acetate to obtain the respective fractions.

In step 2), the ethyl acetate fraction is separated into respective gallotannin compounds by performing adsorption and partition chromatography, and the adsorption and partition chromatography may use any conventional method used by researchers in the art. Preferably silica gel and reversed phase C-18 silica gel column chromatography are used. The elution solvent used is a mixed solvent of chloroform-methanol, aqueous methanol solution and acetonitrile aqueous solution.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Gallotannin Compounds from Sputum Trees

About 1 kg of dried barley crushed bark crushed to a size of about 1 mm was extracted three times with 2 L of methanol at room temperature, and the extracts were collected and concentrated under reduced pressure to obtain 100 g of sputum methanol extract. The obtained methanol extract was suspended in 1 L of distilled water, and then hexane (1 L 3 times) and ethyl acetate (1 L 3 times) were fractionated, respectively.

After obtaining an ethyl acetate fraction (30 g) obtained by concentrating the ethyl acetate layer under reduced pressure, silica gel column chromatography was fractionated with an elution solvent (chloroform / methanol = 20/1 → 5/1). The ethyl acetate layer was separated into six fractions, and part of the third fraction (eluent: chloroform: methanol = R _f = 0.45 at 10: 1) was again subjected to silica gel column chromatography (eluent: chloroform). / Methanol = 9/1), reverse phase C-18 silica gel (elution solvent: 60% methanol and 20% acetonitrile aqueous solution) of the compound of formula 1 (1,2,6-trigalloylglucoside) and Compounds (1,2,3,6-tetragalloylglucoside) were obtained, respectively.

Example 2: Structural Analysis of Lactone Compounds

(1) 1,2,6-trigalloylglucoside compound of formula 1

The 1,2,6-trigalloylglucoside compound of Chemical Formula 1 obtained in Example 1 is a white amorphous powder that is well soluble in methanol, but is not soluble in hexane.

The infrared absorption spectra showed peaks at 3420, 1710, 1610, 1540, 1525, 1450, 1310, and 1240 cm ⁻¹ , and absorption peaks at 216 and 278 nm with methanol as the solvent in the ultraviolet absorption spectrum.

Mass spectrometry showed a molecular weight of 636.

The structural formula was found to be C ₂₇ H ₂₄ O ₁₈ using mass spectrometry and nuclear magnetic resonance spectra. The hydrogen nuclear magnetic resonance spectrum analysis results are shown below. In addition, the structure of the 1,2,6-trigalloylglucoside was determined by instrumental analysis of ¹³ C nuclear magnetic resonance spectra.

¹ H NMR (500 MHz)

δ 7.07, 7.02, 6.93 (2H, s, H-galloyl, respectively), 5.91 (1H, d, J = 7.4 Hz, H-1-glucose), 5.21 (1H, t, J = 7.4, H-2-glucose ), 4.54 (1H, d, J = 12.0, H-6-glucose), 4.42 (1H, dd, J = 12.0, 2.1, H-6-glucose), 3.80 (1H, dd, J = 10.0, 7.4, H-3-glucose), 3.65 (2H, m, H-4,5-glucose).

(2) 1,2,3,6-tetragalloylglucoside compound of formula (2)

The 1,2,3,6-tetragalloylglucoside compound of Formula 2 obtained in Example 1 is a white amorphous powder that is well soluble in methanol, but is not soluble in hexane.

In the infrared absorption spectrum, peaks were observed at 3400, 1700, 1610, 1540, 1455, and 1355 cm ⁻¹ , and absorption peaks were observed at 216 and 278 nm as methanol solvents in the ultraviolet absorption spectrum.

Mass spectrometry showed a molecular weight of 788. The structural formula is C ₃₄ H ₂₈ O ₂₂ as a result of measuring the number of hydrogen and carbon using mass spectrometry and nuclear magnetic resonance spectra. The hydrogen nuclear magnetic resonance spectrum analysis results are shown below. In addition, the structure of the 1,2,3,6-tetragalloylglucoside was determined by instrumental analysis of ¹³ C nuclear magnetic resonance spectra.

¹ H NMR (500 MHz)

δ 7.15, 7.06, 7.04, 6.92 (2H, s, H-galloyl), 6.11 (1H, d, J = 7.6, H-1-glucose), 5.40 (1H, t, J = 8.4, H-3- glucose), 5.43 (1H, t, J = 8.4, H-2-glucose), 4.60 (1H, d, J = 12.1, H-6-glucose), 4.52 (1H, dd, J = 12.1, 2.1, H -6-glucose), 4.02 (1H, m, H-5-glucose), 3.95 (1H, t, J = 8.4, H-4-glucose).

Example 3: Anticomplement Activity of the Galotenine Compounds

Anti-complement activity test of 1,2,6-trigalloyl glucoside and 1,2,3,6- tetragalloyl glucoside was performed by the conventional method. That is, anticomplementary activity against the traditional pathway is described by Mayer method [Kabat EA, Mayer MM, 1961, In 'Experimental Immunochemistry' 2nd ed. Charles and Thomas, USA] and complement response of modified Klerx [Klerx JPAM, Beukelman CJ, Dijk HV, Willers JMN, 1983, J. Immunol. Methodes 63, 215] and the Englberger method [Bitter-Suermann D., Hadding U., Melchert F., Wellensiek HJ, 1970, Immunochemistry , especially for the complement components C ₃ and C ₅ convertase. 7, 955] by adding 1,2,6-trigalloylglucoside and 1,2,3,6-tetragalloylglucoside obtained in Example 1, thereby complementing the sensitized amount of red blood cells. Hemolysis inhibition was tested. The results are shown in Table 1 below. Control drugs include tiliroside isolated from Magnolia fargesii [Jung KY, Oh SR, Park SH, Lee IS, Ahn KS, Lee JJ, Lee HK, Biol. Pharm. Bull ., 21, 1077].

Effect of Gallotannin Compounds on Anticomplement Activity compound IC ₅₀ ( μ M) 1,2,6-trigalloylglucoside 136 1,2,3,6-tetragalloylglucoside 34 Tyrilloside 101

As shown in Table 1, showed the two types of Gallo tannin compounds wherein both complement activity, and especially glucosides one to 1,2,3,6-tetra-go control is a IC ₅₀ value of 34 μ M Stronger activity was shown than the tyrrilloside used as the drug.

Example 4: Acute Toxicity Test

Acute toxicity experiments were performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were orally administered once at a dose of 100 mg / kg / ml of the two kinds of gallotannin compounds of the present invention. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were examined. Hematological and hematological examinations were performed. Necropsy was performed to visually observe the abnormalities of the tonic and thoracic organs.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, all of the gallotannin compounds of the present invention did not show toxicity changes in rats up to 100 mg / kg / ml, respectively, and the oral administration minimum dose (LD ₅₀ ) was determined to be a safe substance having 100 mg / kg.

Preparation Example 1 Preparation of Powder and Capsule

10 mg of the gallotannin compound were mixed with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable apparatus.

The components of the powder and capsules are as follows.

Gallotenine Compounds 10 mg

Lactose ·························· 14.8 mg

Crystalline cellulose 3 mg

Magnesium Stearate 0.2 mg

Preparation Example 2 Preparation of Injection Solution

An injection was prepared by mixing 10 mg gallotanine compound, 180 mg mannitol, 26 mg Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water. The solution was placed in a bottle and heated at 20 ° C. for 30 minutes for sterilization.

Gallotenine Compounds 10 mg

Mannitol 180 mg

Na ₂ HPO ₄ · 12H ₂ O ·············· 26

Distilled water ··················· 2974 mg

Preparation Example 3 Preparation of Beverage

0.1 g of gallotanine compounds, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ were prepared by mixing.

The components of the beverage are as follows.

Galotenine Compounds 0.1 g

15 g of vitamin C

Powdered vitamin E 7.5 g

Ferrous iron ···························· 19.75 g

Zinc Oxide ... 3.5 g

Nicotinic acid amide ... 3.5 g

0.2 g of vitamin A

Vitamin B ₁ .0.25 g

0.3 g of vitamin B ₂

·······························

Preparation Example 4 Preparation of Health Supplement

0.1 g of gallotanine compound, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ were prepared by mixing.

The components of the beverage are as follows.

Lactone Compounds 0.1 g 0.1 g

15 g of vitamin C

Powdered vitamin E 7.5 g

5.75 g of ferric nitrate ·················

Zinc Oxide ... 3.5 g

Nicotinic acid amide ... 3.5 g

0.2 g of vitamin A

Vitamin B ₁ .0.25 g

0.3 g of vitamin B ₂

Starch ···················· 18.0 g

·······························

As described above, the gallotannin compound obtained from the sputum tree of the present invention is excellent in anti-complement activity and is treated for various refractory and chronic diseases, including diseases related to complements abnormally proliferated in vivo, that is, immune system diseases, or the like. It can be used for therapeutic support purposes and has low toxicity and can be very useful for the pharmaceutical or functional food industry.

Claims (5)

A therapeutic agent for allergic diseases, characterized by containing an extract of Juglans mandshurica . According to claim 1, The active ingredient of the extract of phlegm is an allergic disease therapeutic agent, characterized in that the compound represented by the following formula (1), the compound represented by the following formula (2) or derivatives thereof. [Formula 1] [Formula 2] A food additive, comprising a extract of Juglans mandshurica . The food additive according to claim 3, wherein the active ingredient of the sputum tree extract is a compound represented by the following Chemical Formula 1, a compound represented by the following Chemical Formula 2, or a derivative thereof. [Formula 1] [Formula 2] 1) extracting pulverized juglans mandshurica by dissolving in methanol or a mixed solvent of methanol and water, and then concentrated under reduced pressure and fractionated with hexane and ethyl acetate to obtain a hexane fraction and an ethyl acetate fraction; And 2) The ethyl acetate fraction was subjected to adsorption chromatography (eluent: chloroform-methanol, 10: 1) and partition chromatography (eluent: 60% aqueous methanol solution and 20% acetonitrile aqueous solution). A method for separating a gallotannin compound from a sputum tree comprising the step of obtaining a gallotannin compound. [Formula 1] [Formula 2]