KR101153870B1 - Effective Perilla extract for inhibiting neuraminidase - Google Patents

Abstract

The present invention relates to extracts of perilla plants having neuraminidase inhibitory activity and compositions for preventing or treating influenza comprising the same as an active ingredient. According to the present invention, the polyphenol-based compounds included in the perilla extract can provide a composition, health food, livestock feed additives and the like that can inhibit and treat influenza by inhibiting neuraminidase.

Perilla, luteolin, rosmarinic acid, apigenin, neuraminidays, influenza, extract

Description

Effective Perilla extract for inhibiting neuraminidase

The present invention relates to extracts of perilla plants having neuraminidase inhibitory activity and compositions for preventing or treating influenza comprising the same as an active ingredient.

Influenza virus is a disease that causes acute respiratory illness. It is a highly contagious virus that causes general physical symptoms such as high fever, headache, muscle pain, and cough. There are three types of influenza virus, A, B, and C, but it is type A and B that cause human illness. Influenza type A exists in various types according to the types of viral surface proteins hemagglutinin (HA) and neuraminidase (NA), depending on the variation of HA type H1-H15, NA According to variation, it is classified into N1-N9 type. Influenza virus type B is less severe than type A, and human infection by influenza virus type C is rare. However, although the HA and NA antigens of recent birds do not usually cause disease in humans, they are easily caused in humans by exchanging genes with the kinds of antigens that cause mutations in the virus or disease in humans. It is changing and is emerging as an international social problem. In particular, in early 2009, the prevalence of swine flu infections in Mexico and the U.S. increased the number of cases of swine flu infection worldwide. According to the WHO report as of July 31, 2009, patients infected with swine flu 162,380 patients and 1,154 deaths from infections were reported. Therefore, a lot of research is being carried out worldwide in the development of influenza virus treatment and prevention. As a result, sales of Tamiflu (Roche Holding), a neuraminidase inhibitor used to treat swine flu, reached $ 12.2 billion by the end of September 2009.

Currently, neuraminidase inhibitors inhibitors are drugs used to treat influenza viruses. Neuraminidase inhibitors are influenza virus surface antigens and play an important role in releasing newly produced viruses out of cells. Therefore, there is a demand for the development of a substance for suppressing neuraminidase, which is a major target for developing influenza virus therapeutics.

In the present invention, as a result of repeated studies to solve the above problems, the present invention was completed. Accordingly, it is an object of the present invention to provide a perilla extract having a neuraminidase inhibitory activity and a composition for preventing or treating influenza comprising the same as an active ingredient.

According to one aspect of the present invention, a perilla plant extract having neuraminidase inhibitory activity is provided.

According to one embodiment of the present invention, the perilla plant (Perilla frutescens var. Frutescens), perilla (Perilla frutescens var. Crispa), perilla citrus (Perilla citriodora), Perilla hirtella and Perilla It may be at least one selected from the group consisting of Setillaensis (Perilla setoyensis).

According to an embodiment of the present invention, the perilla plant extract may be a crude perilla extract extracted from perilla plants with a lower alcohol having 1 to 6 carbon atoms or an aqueous solution thereof.

According to one embodiment of the present invention, the perilla plant crude extract may be an ethanol extract of the perilla plant.

According to an embodiment of the present invention, the perilla plant extract may be a solvent-soluble extract obtained by sequentially fractionating perilla plant crude extracts having different polarities, n-hexane, ethyl acetate, and methanol.

According to one embodiment of the present invention, the perilla plant extract may be a polyphenol-based compound separated from the solvent soluble extract.

According to an embodiment of the present invention, the separation method may be silica gel column chromatography.

According to an embodiment of the present invention, the polyphenol-based compound is hydroxyflavone, 3 ', 4'5,7-tetrahydroxyflavone and 2R-2 [[(2E) -3- (3,4- Dihydroxyphenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating influenza comprising the perilla plant extract as an active ingredient.

According to another aspect of the present invention, there is provided a food composition for preventing or treating influenza, comprising the perilla plant extract as an active ingredient.

According to another aspect of the invention, there is provided a livestock feed for the prevention or treatment of influenza comprising the perilla plant extract as an active ingredient.

According to the present invention, the polyphenol-based compounds included in the perilla extract can provide a composition, health food, livestock feed additives and the like that can inhibit and treat influenza by inhibiting neuraminidase.

Perilla frutescens var. frutescens ) is a herbaceous annual herb from Labiatae, native to Asia. It is a crop that has been cultivated in Korea, India, and northeastern China, including Korea, and has a fat content of 44% and an omega-3 fatty acid linolenic acid content of 63%. Also perilla perilla related to perilla ( Perilla) frutescens var. crispa ) has been cultivated for a long time as a medicinal or coloring plant. There are many high farms and names recorded for the tea plant, and the color book describes the tea plant as a medicinal plant or element, and it is dealt with as a letter in the thaw farm, haengpoji, and the executive economic magazine. The leaves of the perilla vary from dark purple to green and purple. The fragrance also differs from perilla. Perilla Perilla plants other than in a wild diploid citriodora , Perilla hirtella and perilla There is setoyensis .

In Korea's first farming farm, 1429, the perilla was recorded as a yuma and a commissioner, and the seeds and seeds of perilla and leaves were eaten mainly. According to the agreement, perilla is known to heat up the body and taste very much, to be poisonous and to soothe, to stop coughing and thirst, to soothe the liver, to nourish the inner essence, or to fill the bone marrow. Perilla leaves have also been used to treat the sea water (上 氣 咳嗽), to remove the odor and even the odor, and to bite or boil. In addition, linolenic acid, an omega-3 fatty acid in perilla, has anticancer effects such as inhibiting cancer cell proliferation. In particular, it inhibits the occurrence of breast and colon cancers. It is an essential fatty acid of the nervous system, which affects the optic nerve and improves learning ability. And secondary metabolites, rosmarinic acid, luteolin, apigenin, chrysoeriol, caffeic acid, and antioxidants, which have antioxidant, anti-inflammatory and anticancer activity. Compounds such as anthocyanin are contained in perilla. In particular, due to the economic growth and the diversification of people's dietary consumption, they prefer foods that contribute to nutrition and health. In this regard, perilla is a crop genetically possessing various biologically active substances, and scientifically proves its new biological activity, which has great agricultural and industrial value.

Hereinafter, the present invention will be described in detail.

The present invention provides a perilla plant extract having an inhibitory activity of neuraminidase involved in releasing the influenza virus extracellularly.

Preferably the plant is deulkkaegwa perilla (Perilla frutescens there is . frutescens ), Perilla frutescens there is . crispa), lemon perilla (Perilla citriodora), lobular common bistort (Perilla hirtella ) and perilla cetoensis setoyensis ) may be one or more selected from the group consisting of. In addition, all of the perilla, including the outposts of the seeds and roots, stems, leaves and the like of these perilla crops can be used in the present invention.

Perilla plant extract of the present invention may be a polyphenolic compound.

Preferably, the polyphenolic compound is hydroxyflavone, 3 ', 4', 5,7-tetrahydroxyflavone and 2R-2 [[(2E) -3- (3,4-dihydroxyphenyl ) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid.

Table 1 shows the structure and chemical formula of the polyfemol compound.

compound rescue Chemical formula One

4 ', 5,7-trihydroxyflavones; Apigenin 2

3 ', 4'5,7-tetrahydroxyflavones; Luteolin 3

2R-2 [[(2E) -3- (3,4-dihydroxyphenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid ; Rosemarine acid

The polyphenol-based compound may be used in the form of a pharmaceutically acceptable salt, and all salts, hydrates, and solvates prepared by conventional methods, commercially available reagents may be used, and in the present invention, extract from perilla seed, Used by separation and purification.

Extraction, separation and purification methods of the polyphenol-based compounds according to the present invention can be used by selecting a method well known in the art or those extracted according to the present invention as described below, synthesized or commercially available. Preferably, the polyphenol-based compound may be extracted from perilla seed.

In the extraction process, the seeds or outposts of perilla and pulverized plants are ground and extracted by stirring with a lower alcohol having 1 to 6 carbon atoms or an aqueous solution thereof, and then the crude extract is filtered and the supernatant is reduced and concentrated to prepare a powder. The powder is subjected to silica gel column chromatography under organic solvent conditions. The supernatant is a clear liquid present on top of the precipitate or precipitate, also called the supernatant.

Specifically, the perilla seed is washed, dried and pulverized in a pulverizer and placed in an extraction container in a suitable amount of an organic solvent having 1 to 6 carbon atoms or an aqueous solution thereof, preferably an aqueous 80% ethanol solution for 3 to 7 days. Allow to extract for a while. The extraction process can be repeated several times, and then additionally subjected to methods such as concentration or freeze drying. It may also be removed using n-hexane or ether to remove the oil component of the perilla. The lower alcohol or an aqueous solution thereof may be 10 to 20 times the weight of the perilla plant seed or starch pulverized powder, the stirring may be performed at 20 ~ 30 ℃. The extract extracted in this way corresponds to the crude extract.

Next, water is added to 80% ethanol extract of perilla seed and suspended, followed by fractionation using n-hexane and ethyl acetate to obtain a soluble extract. The yield varies depending on the extraction solvent conditions, preferably 80% ethanol solvent can be used.

 At this time, a conventional fractional extraction method can be used, and preferably a fractional funnel can be used. The extract can obtain n-hexane soluble extract, ethyl acetate soluble extract, methanol soluble extract. However, when extracting oil from the perilla seed and using the remaining perilla gourd sample, n-hexane fractionation process can be omitted.

Next, the ethyl acetate soluble extract can be separated and purified using silica gel chromatography in reverse or normal phase to obtain polyphenol compounds 1 and 2.

In silica gel chromatography, silica gel, which is about 20 to 30 times the amount of a sample, is filled in a column, and a sample is added to adsorb the sample onto the silica gel, followed by flowing the non-polar solvent and the polar solvent in varying concentrations. The way it goes. The most frequently used solvent systems include chloroform and methanol mixtures, n-hexane and ethyl acetate mixtures. Basically, thin layer chromatography (TLC) examines various kinds of developing solvents to determine a developing solvent having good resolution. When chloroform is used as a developing solvent, care should be taken because the specific gravity is higher than that of other solvents, so the outflow rate tends to be faster. Finally, the substance adsorbed on silica gel is eluted completely as a polar solvent methanol. When performing effective silica gel chromatography, the amount of silica gel, the developing solvent and the flow rate are important.

Silica gel has a disadvantage in that a natural product having various polar functional groups is easily bonded due to its high reactivity and polarity. In this case, reverse polarity chromatography is used to chemically treat the polar surface of silica gel to combine functional groups such as C18, C8, alkyl groups, aromatic phenyl groups, and cyan groups, and to use them as stationary phases. When the non-polar functional group is bonded to the silica gel, the surface of the particle is changed to non-polar, so that the material is separated between the stationary phase and the mobile phase by hydrophobic interaction. In this case, unlike silica gel chromatography, a polar solvent such as methanol or water is used as the mobile phase. In general, when the stationary phase has a greater polarity than the mobile phase, such as silica gel chromatography, the case where the mobile phase is greater than the stationary phase is called reverse phase chromatography. In reverse phase chromatography, the less polar components in the sample are eluted later than the more polar samples. Water, methanol, acetonitrile, tetrahydrofuran (THF), buffers, and mixed solutions thereof are used as mobile phases.

In the silica gel chromatography for separating the compounds, preferably, n-hexane, n-hexane-ethyl acetate mixed solvent and methanol may be sequentially used, and in the additional chromatography, n-hexane-acetone mixed solvent may be used. . N-hexane used at this time? The volume ratio of the ethyl acetate mixed solvent is preferably 99: 1 to 1: 2, and in the case of n-hexane-acetone mixed solvent, the volume ratio of 70: 1 to 1: 2 is preferable. The chromatography may be performed once or several times until a single compound is purified, and may be concentrated and recrystallized as necessary. The separated compound can be analyzed by conventional methods such as molecular weight measurement and NMR measurement.

By the above method, the compound 4 ', 5,7-trihydroxyflavone (4', 5,7-trihydroxyflavone: apigenin), 3 ', 4', 5,7-tetrahydroxyflavone (3 ', 4 ', 5,7-tetrahydroxyflavone (luteolin) can be obtained.

In addition, the methanol soluble extract may be separated and purified by compound 3 using silica gel chromatography. In the silica gel chromatography for separating the compounds, chloroform, a chloroform-methanol mixed solvent and methanol may be sequentially used as a mobile phase, and the volume ratio of the chloroform-methanol mixed solvent used at the time is preferably 99: 1 to 1: 2. Do. When the separation is to be performed using reverse phase column chromatography (RP-C18), water, water, methanol solvent and methanol may be sequentially used to separate the solvent using a volume ratio of 99: 1 to 1: 2. In addition, the chromatography may be performed once to several times until a single compound is purified, and may be concentrated and recrystallized as necessary. The separated compound can be analyzed by conventional methods such as molecular weight measurement and NMR measurement.

Antiviral activity experiments using perilla extract or polyphenol-based compounds isolated therefrom may use the virus H1N1 neuraminidase inhibitory activity assay. To evaluate the virus H1N1 neuraminidase inhibitory activity, the sample and neuraminidase were placed in a 3 mL cuvette and added to 50mM Tris buffer (including pH 7.5, 5mM CaCl ₂ and 200mM NaCl) to allow the mixture to react well for 10 minutes at 32 ° C. Phosphorescent 2 '-(4-methylumberlyryl) -alpha-dn-acetylneuraminic acid [2'-(4-methylumbellifery) -α-DN-acetylneuraminic acid] was added to fluorescence spectrophotometer (excitation: 365 nm; emission). : 445 nm). The degree of neuraminidase inhibitory activity can be compared with the background experiment by enzymatic activity of 100% (based on the experiment) and the treatment of the extract in the experiment with the same volume of solvent instead of the extract.

Specifically, 4 ', 5,7-trihydroxyflavone, 3', 4 ', 5,7-tetrahydroxyflavone, and 2R-2 [[(2E) -3- (3,4-di Hydroxyphenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid compounds have a compound concentration that inhibits the activity by 50% against virus H1N1 neuraminidase. Were 80 μM, 8.5 μM and 47 μM, respectively. Among the polyphenol-based compounds of perilla seed, 3 ′, 4 ′, 5,7-tetrahydroxyflavone, that is, luteolin component showed the strongest effect. The perilla extract of the present invention or the polyphenol-based compound isolated therefrom can be said to have been identified for the first time that it has a strong inhibitory effect on the virus H1N1 neuraminidase.

The composition of the present invention has an influenza virus inhibitory effect can be used as a pharmaceutical composition for preventing and treating influenza virus, algae, swine influenza virus, health functional food composition or functional concentrate feed. In addition, the oil can be used more useful by extracting the remaining perilla persimmon foil.

The formulation of the medicament prepared using the composition of the present invention is not limited, and may be prepared in tablets, capsules, powders, granules, liquids, pills, and the like.

There is no particular limitation on the kind of food to which the composition of the present invention is applied. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage of the present invention may contain various flavors or natural carbohydrates and the like as additional ingredients, as in general beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, natural sweeteners, such as tautin and a stevia extract, or synthetic sweeteners, such as saccharin and aspartame, can be used. The proportion of the natural carbohydrate is generally 0.01 to 0.04 g, preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above ingredients, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited or limited by the following examples.

Example  1: Preparation of Perilla Extract

Perilla Seed  Extraction, Separation and Purification

Perilla seed (1.0 kg) was washed clean, dried and crushed with a grinder and put in an extraction container and shaken for 3 days using n-hexane or ether to remove the perilla oil component. The oil-free sample was extracted with reciprocating shaking for 2 days at room temperature in an aqueous 80% ethanol solution (3 L). The extracted sample was filtered through a filter paper, and then concentrated under reduced pressure to give a light brown extract (100 g). 500 ml of purified water was added thereto, and the mixture was suspended, and then fractionated in the order of n-hexane, ethyl acetate, and methanol to obtain n-hexane fraction (5 g), ethyl acetate fraction (30 g), and methanol fraction (65 g), respectively. The obtained ethyl acetate fraction (30 g) was used for silica gel column chromatography (500 g, 70-230 mesh, Merk), and as a mobile phase solvent, a mixed solvent of n-hexane to ethyl acetate (99: 1 to 1: 2) and methanol Were used sequentially to obtain seven fractions (fraction.E1 to fraction.E7). Fraction. E3 (2 g) was adsorbed on silica gel (5 g, 70-230 mesh), and silica gel column chromatography (20 g, 230-400 mesh) was used. The mobile phase solvent was n-hexane: ethyl acetate mixed solvent. To give a yellow precipitate which was purified to give compound 1 (40 mg).

Fraction. E5 (3 g) was adsorbed onto silica gel (10 g, 70-230 mesh), and silica gel column chromatography (60 g, 230-400 mesh) was used. The mobile phase solvent was n-hexane: ethyl acetate mixed solvent ( 50: 1 ~ 1: 2) was used to obtain another yellow precipitate which was purified to give Compound 2 (50 mg).

In addition, the methanol fraction (65 g) was used for silica gel column chromatography (700 g, 70-230 mesh, Merk), and as a mobile phase solvent, chloroform, methanol (99: 1 to 1: 2 mixed ratio) mixed solvent and methanol were sequentially 5 fractions (fraction. M1 -M. M5) were obtained. Fraction. M4 (5 g) was adsorbed onto silica gel (6 g, 70 to 230 mesh), and silica gel column chromatography (100 g, 230 to 400 mesh) was used. The mobile phase solvent was grayish brown using a chloroform: methanol mixed solvent. A precipitate was obtained and purified to give compound 3 (80 mg). Silicakel TLC pattern of the isolated polyphenol-based compound is shown in Figure 1a.

Perilla Seed  Polyphenolic Compound Analysis

The material obtained in Example 1-1 was determined by molecular weight and molecular formula using HPLC-MS analyzer (High-performance liquid chromatography mass chromatography), ¹ H NMR, ¹³ by nuclear magnetic resonance analysis (Bruker AMX 500) C-NMR, HOMO-COSY, ¹ H-Detected heteronuclear Multiple-Quantum Coherence (HMQC), Heteronuclear Multiple-Bond Coherence (HMBC), and Distortionless Enhancement by Polarization (DEPT) spectra were obtained to determine molecular structure.

The measurement results are as follows. Compound 4 ', 5,7-trihydroxyflavone (4', 5,7-trihydroxyflavone: apigenin), compound 2'3 ', 4', 5,7-tedra Hydroxyflavones (3 ', 4', 5,7-tetrahydroxyflavone; luteolin), 2R-2 [[(2E) -3- (3,4-dihydroxyphenyl) -1-oxo, a polyphenolic compound 3 -2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid ((2R) -2 [[(2E) -3- (3,4-dihydroxyphenyl) -1-oxo 2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid; rosmarinic acid). The structure of the isolated polyphenol-based compound is shown in Figure 1b.

[Compound 1]: 4 ', 5,7-trihydroxyflavone

1) Physical property: yellow amorphous crystal

2) Molecular Weight: 270

3) Molecular formula: C ₁₅ H ₁₀ O ₅

4) ¹ H-NMR (500 MHz, DMSO- d ₆ ) δ 7.91 (d, J = 8.8 Hz, H-2 ', 6'), 6.92 (d, J = 8.8 Hz, H-3 ', 5') , 6.76 (s, H-3), 6.47 (d, J = 2.1 Hz, H-8), 6.18 (d, J = 2.1 Hz, H-6).

5) ¹³ C-NMR (125 MHz, DMSO- d ₆ ) δ 182.0 (C-4), 164.5 (C-5), 164.1 (C-2), 161.8 (C-7), 161.5 (C-4 ' ), 157.7 (C-9), 128.8 (C-2 ', 6'), 121.5 (C-1 '), 116.3 (C-3', 5 '), 104.1 (C-10), 103.2 (C- 3), 99.2 (C-6), 94.3 (C-8).

[Compound 2]: 3 ', 4'5,7-tetrahydroxyflavone

1) Physical property: yellow amorphous crystal

2) Molecular Weight: 360

3) Molecular formula: C ₁₅ H ₁₀ O ₆

4) ¹ H-NMR (500 MHz, DMSO- d ₆ ) δ 7.42 (dd, J = 8.1, 2.3 Hz, H-6 '), 7.40 (d, J = 2.3 Hz, H-2'), 6.90 (d , J = 8.1 Hz, H-5 '), 6.66 (s, H-3), 6.45 (d, J = 2.1 Hz, H-8), 6.20 (d, J = 2.1 Hz, H-6).

5) ¹³ C-NMR (125 MHz, DMSO- d ₆ ) δ 182.0 (C-4), 164.5 (C-5), 164.3 (C-2), 161.9 (C-7), 157.7 (C-9) , 150.1 (C-4 '), 146.1 (C-3'), 121.9 (C-1 '), 119.3 (C-6'), 116.4 (C-5 '), 113.8 (C-2'), 104.1 (C-10), 103.2 (C-3), 99.2 (C-6), 94.2 (C-8).

[Compound 3]: 2R-2 [[(2E) -3- (3,4-dihydroxyphenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl Propanoic acid

1) Physical property: gray brown amorphous crystal

2) Molecular Weight: 286

3) Molecular formula: C ₁₈ H ₁₆ O ₈

4) ¹ H-NMR (500 MHz, Methanol- d ₄ ) δ 7.57 (d, J = 15.9 Hz, H-7), 7.06 (d, J = 2.0 Hz, H-2), 6.95 (dd, J = 8.1 , 2.0 Hz, H-6), 6.79 (d, J = 8.2 Hz, H-5 '), 6.77 (d, J = 2.0 Hz, H-2'), 6.72 (d, J = 8.2 Hz, H- 6 '), 6.63 (dd, J = 8.1, 2.0 Hz, H5), 6.27 (d, J = 15.9 Hz, H-8), 5.22 (dd, J = 8.3, 4.4 Hz, H-8'), 3.10 (dd, J = 13.8, 8.8 Hz, H-7'α), 3.04 (dd, J = 13.8, 8.8 Hz, H-7 'β).

5) ¹³ C-NMR (125 MHz, Methanol- d ₄ ) δ 173.4 (C-9 '), 168.4 (C-9), 149.6 (C-4'), 147.6 (C-7), 146.6 (C- 3 '), 146.0 (C-4), 145.1 (C-3), 129.2 (C-1'), 127.6 (C-1), 123.1 (C-6), 121.8 (C-5), 117.5 (C -2 '), 116.4 (C-5'), 116.2 (C-6 '), 115.2 (C-2), 114.3 (C-8), 74.5 (C-8'), 37.8 (C-7 ') .

The ¹ H-NMR and ¹³ C-NMR spectra of 3 ', 4', 5,7-tetrahydroxyflavone, the compound 2 having the highest neuraminidase inhibitory activity among the functional components of perilla seed, are shown in FIGS. 2A and 2B. Indicated.

Perilla Seeds  Extraction of compounds Neuraminidays  Inhibitory activity test

In order to determine the viral H1N1 neuraminidase inhibitory activity of the perilla seed extract or polyphenol-based compound isolated from the perilla seed of the present invention was carried out as follows.

Neuraminidase is an influenza virus surface antigen and is known to play an important role in releasing the newly generated virus out of cells, and has been used as a major target for the development of existing influenza virus therapeutics. Thus, in the present invention, virus-derived H1N1 neuraminidase was purchased from R & D Systems, and the substrate 2 '-(4-methylumberlyryl) -alpha-dn-acetylneuraminic acid [2'-(4-methylumbellifery) -α-DN-acetylneuraminic acid] was purchased from Sigma. First, perilla seed extract or polyphenol-based compound separated therefrom is prepared by diluting by a certain concentration. After the sample was prepared, put a certain amount of sample and H1N1 neuraminidase into 3mL cuvette, add 50 mM Tris buffer (including pH 7.5, 5mM CaCl ₂ , 200mM NaCl), mix well, and incubate at 32 ° C for 10 minutes. 2 '-(4-methylumberlyryl) -alpha-dn-acetylneuraminic acid was added and analyzed with a spectroMax M2e (Molecular Divices, USA), a fluorescence spectrometer, with an excitation wavelength of 365 nm and an emission wavelength of 445 nm. The final concentration of H1N1 neuraminidase used in the experiment was 2.5 ng, the final concentration of the substrate was 200 μM and the reaction temperature was 32 ° C.

Inhibition rate was calculated from the difference in fluorescence intensity from the reaction solution into which ethanol was added instead of the extract, and the results are shown in FIG. 3. In addition, Equation 1 applied to the activity evaluation of the enzyme is as follows.

[Equation 1]

Neuraminidase activity (%) = 100 [1 / (1 + ([sample concentration] / IC ₅₀ ))]

The IC ₅₀ indicates an extract concentration that inhibits alpha-glucosidase activity by 50%.

The H1N1 neuraminidase inhibitory activity of the perilla seed extract and the isolated polyphenol-based compound is shown in Table 2 below.

compound Compound name IC ₅₀ ([mu] M) Perilla 80% Ethanol
extract Ethanol extract 200 μg / mL Polyphenolic
compound One 4 ', 5,7-trihydroxyflavones 80 μM 2 3 ', 4'5,7-tetrahydroxyflavone 8.5 μM 3 2R-2 [[(2E) -3- (3,4-dihydroxyphenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid 47 μM

As shown in Table 2, it can be seen that the perilla seed extract of the present invention or a compound isolated therefrom inhibits the virus H1N1 neuraminidase. In particular, Compound 2 was the best among the compounds that had an antiviral inhibitory effect. Neuraminidase inhibition rate according to the sample concentration of the compound 2 and 3 is shown in FIG.

Toxicity Test of Perilla Extract

Toxicity experiments were performed on perilla extract, the active ingredient of the present invention as follows.

Perilla extract was dissolved in 1% sodium carboxymethyl cellulose, diluted with water, and orally administered to mice (10 per group) at 0, 4, 20, 100, 500 mg / kg, respectively, and then for 7 days. Observed. No rats died due to oral administration of perilla extract, and no rats died for 7 days.

Example 2 : Preparation of Tablets Containing Perilla Extract

A tablet containing 15 mg of perilla extract as an active ingredient is prepared by the following method.

250 g of the active ingredient was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Example 3 : Preparation of Capsule Containing Perilla Extract

A capsule was prepared by filling a hard gelatin capsule with 500 mg of wild dog extract as an active ingredient.

Example 4 : Preparation of Beverage Containing Perilla Extract

After dissolving 500 mg of wild dog extract or active ingredient in an appropriate amount of water, add vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharide as an auxiliary component, add an appropriate amount of sodium benzoate as a preservative, and then add water to make 100 Made with ml prepared a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined in the appended claims. It will be understood that the invention may be varied and varied without departing from the scope of the invention. Many embodiments other than the above-described embodiments are within the scope of the claims of the present invention.

1A is a TLC developed picture of perilla extract and the active single component isolated.

1B is a structure of a compound identified from perilla seed

2a and 2b are ¹ H-NMR and ¹³ C-NMR spectra of perilla perilla 3 ', 4', 5,7-tetrahydroxyflavones.

3 is a graph showing the effect of neuraminidase inhibitory activity on the main components of perilla.

Figure 4a is a graph showing the change in the neuraminidase inhibitory effect according to the concentration of compound 1 in perilla active ingredient with a fluorescence spectrometer.

Figure 4b is a graph showing the neuraminidase inhibitory effect of the concentration changes of Compound 2 and Compound 3 components in perilla active ingredient.

Claims (11)

A composition for preventing or treating influenza comprising as an active ingredient a solvent soluble extract of seeds of perilla plants obtained by sequentially dividing n-hexane, ethyl acetate and methanol, which are solvents having different polarities. According to claim 1, wherein the perilla plant ( Perilla frutescens var. Frutescens ), perilla ( Perilla frutescens var. Crispa ), Perilla citriodora , Perilla hirtella and Perilla cetoensis ( Perilla setoyensis ) composition, characterized in that at least one selected from the group consisting of. delete delete delete The composition of claim 1, wherein the perilla plant extract is a polyphenol-based compound isolated from a solvent soluble extract. The composition of claim 6, wherein the separation method is silica gel column chromatography. The compound according to claim 6, wherein the polyphenolic compound is selected from hydroxyflavones, 3 ', 4'5,7-tetrahydroxyflavones and 2R-2 [[(2E) -3- (3,4-dihydroxy. And phenyl) -1-oxo-2-propenyl] oxy] -3- (3,4-dihydroxyphenyl) propanoic acid. delete delete delete

Images