KR101457520B1 - Composition containing neorhodomela extracts or polybromocatechol from neorhodomela extracts having antiviral activity - Google Patents

Abstract

The present invention provides a composition containing red ginseng extract having antiviral activity as an active ingredient. Also, the step of extracting the red nectar with an organic solvent and distillation under reduced pressure to obtain an extract; Extracting the organic solvent extract obtained in the above step by using a difference in polarity of the solvent to obtain a fraction extract; And separating and purifying the fraction extract obtained in the above step by chromatography. The present invention also provides a method for separating pellibromocatechol. According to the present invention, it is possible to provide an antiviral substance which is excellent in the inhibitory effect on human rhinovirus and is safe and low toxicity since it is derived from a natural product. In addition, it is possible to easily isolate polyketone from the red nectar and to provide a pharmaceutical composition which can be used as a preventive and therapeutic agent for viral diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition having an antiviral activity containing a red nigella extract or a polyribomacetecol isolated therefrom. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a composition having an antiviral activity containing a red nectar extract or a polyribomacetecol isolated therefrom.

Various diseases caused by bacteria and bacteria are prevented and treated by the development of preventive agents and antibiotics. However, many diseases caused by viruses have difficulties in treatment and prevention due to the evolution of various viruses and the emergence of variants.

In particular, there are a number of diseases caused by influenza viruses, rhinoviruses, rhinosinusitis and stomatitis that cause flu, and herpes virus-induced avian influenza (AI), severe acute respiratory syndrome The emergence of new viruses that cause SARS has heightened worldwide interest in the prevention and treatment of various viral diseases, threatening human and other ecosystems. Most commonly used antiviral agents are mostly iododeoxyuridine (IDU) or acyclovir (ACV), azidothymidine (IFN), nucleoside derivatives or interferon (IFN) , AZT), etc., and these substances exhibit various side effects such as cytotoxicity, hepatotoxicity and over-tolerance. As a representative example, side effects such as vomiting, bronchitis, headache, cough, and dizziness have been reported in the case of oseltamivir phosphate (so-called Tamiflu), which has been developed as a therapeutic agent against human influenza viruses A and B and avian influenza virus . In addition, it has been reported that the resistance of resistant strains to existing drugs is increasing, and there are limitations to the effective treatment of viral diseases due to such side effects. Therefore, in order to improve the side effects of conventional chemical synthetic drugs, studies are being conducted actively to search for substances having antiviral activity from safe natural substances with few side effects.

Among seaweeds, seaweeds have been ingested as food for a long time, so they are highly safe and highly likely to develop new drugs derived from natural products. In particular, seaweeds are known to have excellent physiological activities, such as antioxidant, anti-cancer and anti-diabetic effects, and the extracts isolated from various seaweeds have been reported to have immune enhancement, antibacterial and anticancer activities.

However, the antiviral active substances obtained from seaweeds have been reported to be mainly active as mixed extracts or regulated products and have been reported as antiviral substances of plants and fishes which are not viruses applied to humans. Therefore, studies on the effect of compounds isolated from seaweeds on human viral pathology have been continuously carried out, but no examples have been developed in practice.

Meanwhile, red nectar ( Neorhodomela aculeata ) are red algae and red algae, which are native to Japan and China, including the Yellow Sea and the East Sea of Korea. There are patented antioxidant activity using red nectar (Japanese Patent Laid-open No. 20247901) and hair tonic (US Patent No. 20060165636), but there is no patent for antiviral activity of red nectar.

Japanese Patent Laid-Open No. 20247901 U.S. Published Patent Application No. 20060165636

In order to solve the above-mentioned problems, the present invention provides a safe antiviral substance derived from a natural product, which comprises a red nectar extract, a fraction thereof, and a polyribomacetecole isolated from the fraction, And it is an object of the present invention to provide a method for easily separating mocha teques.

In order to solve the above-mentioned problems, the present invention provides a composition containing a red nectar extract having antiviral activity as an active ingredient.

In one embodiment of the present invention, the red-green algae extract may be a fractional extract obtained by using a difference in polarity of a solvent.

In one embodiment of the present invention, the solvent may be at least one member selected from the group consisting of hexane, dichloromethane, ethyl acetate, n-butanol and distilled water, and in particular, may be hexane or ethyl acetate.

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In one embodiment of the present invention, the red ginseng extract comprises 2,3-dibromo-4,5-dihydroxybenzyl alcohol, 2,3-dibromo-4,5-dihydroxybenzylaldehyde, 2,3-dibromo-4,5-dihydroxybenzyl methyl ether, 2,5-dibromo-3,4-dihydroxybenzyl methyl ether, 2,2 ', 3,3'-tetrabromo 4,4 ', 5,5'-tetrahydrodiphenylmethane, 2,2', 3-tribromo-3 ', 4', 5-tetrahydroxy-6'-methoxymethyldiphenyl Methane, and salts thereof. ≪ RTI ID = 0.0 > [0040] < / RTI >

In one embodiment of the present invention, the composition may be one having antiviral activity against human rhinovirus.

In one embodiment of the invention, the composition may be a pharmaceutical composition.

In addition, the present invention provides a method for producing an extract, comprising: extracting red nectar from an organic solvent and distillation under reduced pressure to obtain an extract;

Extracting the organic solvent extract obtained in the above step by using a difference in polarity of the solvent to obtain a fraction extract; And

And separating and purifying the fraction extract obtained in the above step by chromatography. The present invention also provides a method for separating polyribomacetecol.

In one embodiment of the present invention, the organic solvents from acetone, acetonitrile, methanol, ethanol, isopropanol, butanol, and the group consisting of carbon tetrachloride (CCl _4), chloroform (CHCl ₃₎ and dichloromethane (CH ₂ Cl ₂₎ It may be more than one selected.

In one embodiment of the present invention, the fraction comprises a hexane layer fraction, a dichloromethane fraction, an ethyl acetate layer fraction, an n-butanol layer, and a hexane layer fraction obtained by fractionating a red nugget extract with hexane, dichloromethane, ethyl acetate, n- And one or more of the fraction and the aqueous layer fraction.

In addition, the present invention provides a composition having antiviral activity comprising the above-mentioned polyabomycetecole as an active ingredient.

According to the present invention, it is possible to provide an antiviral substance which is excellent in the inhibitory effect on human rhinovirus and is safe and low toxicity since it is derived from a natural product. In addition, it is possible to easily isolate polyketone from the red nectar and to provide a pharmaceutical composition which can be used as a preventive and therapeutic agent for viral diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically illustrating a process from preparation of an extract having antiviral activity from a red nectar extract according to an embodiment of the present invention and separation of the extract from a polyribomacetecol compound. FIG.
FIG. 2 is a graph showing the results of chromatographic analysis of the fractions (F2 to F7) obtained by dividing the ethyl acetate layer fraction extract of the red-green algae extract according to an embodiment of the present invention into small fractions by silica gel column chromatography, to be.
FIG. 3 is a photograph showing the results of staining the survival of cells appearing by treating a compound according to an embodiment of the present invention (inducing human lyn virus 2 in HeLa cells).
FIG. 4 is a photograph showing the results of staining the survival of cells appearing by treating a compound according to an embodiment of the present invention (inducing human lynovirus 3 in HeLa cells).

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate the present invention by those skilled in the art.

The present invention provides a composition containing red ginseng extract having antiviral activity as an active ingredient.

In the present invention, the red nigella refers to the algae of the red nematode and the red nematode (Neorhodomela ), and preferably refers to the red nematode and the red nematocide nematode ( Neorhodomela aculeata ). Red nodules and seaweeds contain terpenoids or phenols. In particular, bromine compounds have been reported to exhibit antimicrobial activity, but there has been no study of antiviral activity. The present invention has been completed.

In the present invention, the red nectar extract is not particularly limited as long as the red nectar is extracted by a known extraction method. Preferably, it may be an extraction using an organic solvent.

For example, the collected red nectar samples are washed once or twice with salty water on a surface, dried on a shade, and extracted with an organic solvent. Examples of the organic solvent include acetone, acetonitrile, alcohol such as methanol, ethanol, isopropanol, butanol, noncovalent organic solvent such as carbon tetrachloride (CCl ₄ ), chloroform (CHCl ₃ ), dichloromethane (CH ₂ Cl ₂ ) Or a mixture of two or more kinds of solvents may be used, but the present invention is not limited thereto. Particularly preferred solvents are acetone, methanol, ethanol, dichloromethane or a mixture of methanol and dichloromethane.

The extraction time may vary depending on the amount of the sample to be used and the type of the solvent, but is usually from 6 hours to 48 hours, preferably from 12 hours to 24 hours, but is not limited thereto.

After extraction with an organic solvent, the red nectar extract can be obtained by removing the organic solvent by distillation under reduced pressure. The decompression distillation temperature may vary depending on the solvent used, but it is preferably carried out at 10 to 40 ° C, preferably at 20 to 30 ° C, but is not limited thereto.

In addition, the red nectar extract may be a fraction extract obtained by using the difference in polarity of the solvent.

In the present invention, the solvent may be at least one selected from the group consisting of hexane, dichloromethane, ethyl acetate, n-butanol and distilled water, and may be hexane or ethyl acetate, but is not limited thereto.

For example, the red nugget extract is dissolved in methanol or ethanol and extracted with hexane to obtain a fraction. Only the alcohol layer is distilled under reduced pressure, and the residue is dissolved again in distilled water. The residue is extracted with ethyl acetate to obtain a fraction layer. Finally, n -Butanol, and finally separated into a hexane layer, an ethyl acetate layer, an n-butanol layer and an aqueous layer. From each of the obtained fraction layers, the solvent was removed under reduced pressure to obtain a fraction extract.

As another method, there may be a method of dividing the red nugget extract into distilled water and an organic solvent. The organic solvent used is usually ethyl methyl ketone, ethyl acetate, carbon tetrachloride, chloroform, dichloromethane, among which ethyl acetate, chloroform and dichloromethane are preferable, but not limited thereto.

In the present invention, the red nugget extract may comprise polybromocatechol and its salts and derivatives thereof

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Specifically, the polybromocatechol may be 2,3-dibromo-4,5-dihydroxybenzyl alcohol, 2,3-dibromo-4,5-dihydroxybenzylaldehyde, 2,3-di Bromo-4,5-dihydroxybenzyl methyl ether, 2,5-dibromo-3,4-dihydroxybenzyl methyl ether, 2,2 ', 3,3'-tetrabromo- ', 5,5'-tetrahydrodiphenylmethane, 2,2', 3-tribromo-3 ', 4', 5-tetrahydroxy-6'-methoxymethyldiphenylmethane and its salts May be selected from the group consisting of The salt of polyribomacetecol may be a pharmaceutically acceptable salt.

The composition according to the present invention may have anti-viral properties by containing a red nectar extract and specifically have antiviral activity against human rhinovirus. Human renovirus may be HRV2 and HRV3. Therefore, it is possible to provide a pharmaceutical composition for use as a therapeutic agent or a preventive agent for a viral disease caused by human rhinovirus.

In addition, the present invention provides a method for easily separating polyribomacetecol from red nectar extract.

Specifically, a step of extracting red nectar from an organic solvent and distillation under reduced pressure to obtain an extract; Extracting the organic solvent extract obtained in the above step by using a difference in polarity of the solvent to obtain a fraction extract; And separating and purifying the fraction extract obtained in the above step by chromatography. The present invention also provides a method for separating pellibromocatechol.

The step of extracting red nectar from an organic solvent and then distillation under reduced pressure to obtain an extract is the same as the above-mentioned method for preparing an extract of red nectar.

The step of fractionating the organic solvent extract by using the polarity of the solvent to obtain the fraction extract is also the same as the method of preparing the fraction extract of the red nectar described above.

The fraction may be a hexane layer fraction, a dichloromethane fraction, an ethyl acetate layer fraction, an n-butanol layer fraction and an aqueous layer fraction obtained by fractionating a red nigra extract with hexane, dichloromethane, ethyl acetate, n-butanol or distilled water.

In the step of separating and purifying the fraction extracts by chromatography, the chromatography can be carried out by, for example, normal phase silica gel column-chromatography, reverse phase preparative high performance liquid chromatography , Prep-HPLC) and reverse phase semi-preparative high performance liquid chromatography (Semi-Prep-HPLC).

The normal-size silica gel tube-chromatographic separation method is a separation method used in a general method in a laboratory. The diameter of a glass tube used and the size of normal-size silica gel (stationary phase: Merk silica gel, Size 5 ~ 40 ㎛ or 35 ~ 70 ㎛) can be used, but in the case of a glass tube, a tube having an inner diameter of 2 to 5 cm and a length of 30 to 60 cm and a tube height of 50 to 70% It is preferable to use a normal-phase silica gel tube.

The composition of the eluent used is somewhat different depending on the amount of the sample and the silica gel tube, but preferably the mixed solvent of hexane: ethyl acetate: methanol = 10: 1: 0 to 0: 1: 1 by volume is added sequentially Is preferably used.

Reverse phase preparative-HPLC separation conditions vary depending on the amount of the sample and the size of the tube to be used, but usually, reversed phase preparative-HPLC (stationary phase: Phenomenex Luna C18 (2) Column, particle size 10 μm, length 250 x 21.20 mm) was prepared in a liquid chromatograph (Gilson Companion) instrument, the sample was adsorbed on C-4 resin and placed in a sample expansion vessel. The eluent was diluted with 10% acetonitrile And the mixture is separated by increasing to acetonitrile: water 10% to 100% (v / v TFA 0.02% added) for 90 to 120 minutes.

Reverse phase quasi-fractionation-HPLC separation conditions may vary depending on the amount of the sample and the size of the tube to be used, but usually the reverse phase preparative-HPLC (stationary phase: Phenomenex Luna C18 (2) Column, particle size 10 μm, length 250 x 10.00 mm) was prepared in a liquid chromatography (Gilson Companion) instrument and the sample was dissolved in the initial eluent and injected. The eluent was then eluted with acetonitrile: water 40:60 (v / v TFA 0.02%), and the mixture is separated by proceeding for 60 to 90 minutes.

The present invention also provides a composition having an antiviral activity containing, as an active ingredient, polyribomacetecol isolated from red nectar by the above-described method for separating polyribomacatole. The composition containing polyribomacetecol as an active ingredient may have an antiviral activity, specifically, an antiviral activity against human lynovirus. Human renovirus may be HRV2 and HRV3. Therefore, it is possible to provide a pharmaceutical composition for use as a therapeutic agent or a preventive agent for a viral disease caused by human rhinovirus.

The pharmaceutical composition may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances, Can be formulated in the form of an administration agent or a parenteral administration agent.

Examples of the oral administration agent include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, powders, granules, granules and pellets, , Diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols) . The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be prepared by conventional mixing, granulating or coating methods. In addition, it may be a transdermal dosage form in the form of parenteral administration. For example, it may be in the form of injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, But is not limited thereto.

The pharmaceutical composition according to one embodiment of the present invention may be administered by parenteral, rectal, topical, transdermal, subcutaneous, and the like. The pharmaceutical composition according to one embodiment of the present invention can be administered topically, for example, to the scalp.

The dosage of the active ingredient is within the level of those skilled in the art, and the daily dose of the drug depends on various factors such as the degree of progress of the subject to be administered, the age of onset, age, health condition, As a standard, it is generally possible to administer the composition at a dose of 1 / / kg to 200 mg / kg, preferably 50 / / kg to 50 mg / kg, once or twice a day, Are not intended to limit the scope of the present invention.

The extract of red nectar obtained through the method according to the present invention and the polybromocatechol compounds of formulas (1) to (3) isolated therefrom show similar activity to ribavirin, which is a conventional antiviral agent, Is a safe natural antiviral agent. In particular, the polyribomacetecol compound of the present invention exhibits excellent antiviral activity as a single component.

The present invention will be described in more detail through the following examples. However, the examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Example 1] Extract of red nigga

The red nectar used in this example was collected from the East Coast of Korea, washed with distilled water, dried on a shade, and extracted with an organic solvent. Dried red nectar 3kg was immersed in 9 liters of 100% methanol and left to stand for 24 hours at room temperature. The solution was extracted twice, and the solution was filtered with filter paper to distill off the solvent, and 16 g of crude extract was obtained.

[Example 2] Extraction of hexane layer fraction of reddish black quail extract

3.3 g of 16 g of the red ginseng extract obtained in Example 1 was dissolved in 500 mL of water and extracted three times with 500 mL of hexane. The obtained hexane layer was distilled under reduced pressure to obtain 1.0 g of a hexane layer extract.

[Example 3] Extraction of ethyl acetate layer fractions of reddish black crude extract

After fractionating the hexane in Example 2, the remaining aqueous layer was extracted three times with 500 mL of ethyl acetate, and the resulting ethyl acetate layer was distilled under reduced pressure to obtain 2.2 g of an ethyl acetate layer extract.

[Example 4] Extraction of n-butanol layer fractions of red nectar extract

The ethyl acetate layer fraction of Example 3 was fractionated and the remaining water layer was extracted twice with 60 mL of n-butanol. The n-butanol layer thus obtained was distilled under reduced pressure to obtain 293 mg of n-butanol layer extract.

[Example 5] Extraction of aqueous layer of red nectar extract

The n-butanol layer of Example 4 was fractionated and the remaining aqueous layer was distillated under reduced pressure to obtain 664 mg of an aqueous layer extract.

[Example 6] Isolation of polybromocatechol

2.2 g of the ethyl acetate layer fraction extract of the red ginseng extract according to Example 3 was adsorbed onto 4 g of silica gel and purified by normal phase silica gel column chromatography (Merk EM9385, 230 to 400 mesh, 650 mm (inner diameter height)), (EA) and methanol (n-Hx: EA = 10: 1 -> -> EA: MeOH = 0: 100) to obtain 7 small fractions (F1-F7 ).

HPLC (fixed phase: Phenomenex Corp. Luna C18 (2)) was used to try to separate the active components from these small fractions, while the F4 fraction (n-Hx: EA = 0: 100, 722 mg) (V / v TFA 0.02%) at a flow rate of 10 ml / min for 90-120 minutes, eluting the eluent from 10% of the initial acetonitrile through a column (column, particle size 10 μm, length 250 x 21.20 mm) ) To obtain 18 peaks at UV 280 nm. Among them, peak P5-1 was purified by reversed-phase quasi-preparative HPLC with 40% acetonitrile (v / v 0.02% TFA addition) to give compound 1a in the form of a linear crystal (53.8 mg). Peaks P9-2 and P14-2 were prepared by reverse phase quasiprep HPLC using 30% and 40% acetonitrile (v / v 0.02% TFA added), respectively, in a single solvent at a flow rate of 4 ml / min. Compound 1b (0.8 mg) and compound 2b (47.0 mg). Peak P1518 (58.6 mg) was re-run on reversed phase preparative-HPLC under the condition of acetonitrile: water (40-> 43% gradient, v / v 0.02% TFA addition, 30 min) (6.4 mg). Compound 1b and compound 2b were further fractionated in F5 fraction (EA: MeOH = 10: 1, 377 mg) and purified by HPLC with acetonitrile: water 10% in F6 fraction (EA: MeOH = 5: The mixture of Compound 1c and Compound 1d was separated through the addition of ~100% (0.02% v / v TFA added, 120 min). A flow chart of the above process is shown in FIG.

The six compounds were identified by NMR, FR-IR, mass spectroscopy, and the like.

As a result of confirming the structure of the compound with ¹ H-NMR and ¹³ C-NMR (Varian 500 MHz spectrometer, Varian, USA), the compounds were 2,3-dibromo-4,5-dihydroxybenzyl alcohol , Lanosol, lanosol), 2,3-dibromo-4,5-dihydroxybenzylaldehyde (1b), 2,3-dibromo-4,5-dihydroxybenzylmethyl ether (1c) 2,5-dibromo-3,4-dihydroxybenzyl methyl ether (1d), 2,2 ', 3,3'-tetrabromo-4,4', 5,5'-tetrahydroxy Phenylmethane (2a), 2,2 ', 3-tribromo-3', 4,4 ', 5-tetrahydroxy-6'-methoxymethyldiphenylmethane (2b).

The chemical structures of the six separated compounds (1a to d, 2a, 2b) are as follows.

^{The results of 1} H-NMR and ¹³ C-NMR are shown below.

Compound (1a)

¹ H NMR (500 MHz, acetone- d ₆ )? 7.2 (s, 1H, H-6), 4.6

^{13 C NMR (125 MHz, acetone-} d 6) δ 144.8 (C-5), 143.1 (C-4), 134.1 (C-1), 113.7 (C-6), 112.6 (C-2), 112.4 ( C-3), 64.2 (C-7)

ESIMS m / z 310.9 [MH] ^- .

Compound 1b Compound

¹ H NMR (500 MHz, acetone- d ₆ )? 10.2 (s, 1H, H-7), 7.4

^{13 C NMR (125 MHz, acetone-} d 6) δ 190.4 (C-7), 157.0 (C-4), 150.3 (C-5), 144.4 (C-1), 127.4 (C-2), 120.7 ( C-3), 113.1 (C-6)

ESIMS m / z 296.9 [M + H] < ⁺ >.

Compound (1c)

^{1 H NMR (500 MHz, acetone-} d 6) δ 7.07 (s, 1H, H-6), 4.41 (s, 2H, H-7), 3.39 (s, 3H, H-8)

^{13 C NMR (125 MHz, acetone-} d 6) δ 115.4 (C-6), 75.1 (C-7), 58.4 (C-8)

ESIMS m / z 310.9 [MH] ^- .

Compound (1d)

^{1 H NMR (500 MHz, acetone-} d 6) δ 6.71 (s, 1H, H-6), 4.43 (s, 2H, H-7), 3.34 (s, 3H, H-8)

^{13 C NMR (125 MHz, acetone-} d 6) δ 109.1 (C-6), 75.8 (C-7), 57.9 (C-8)

ESIMS m / z 310.9 [MH] ^- .

Compound (2a)

¹ H NMR (500 MHz, acetone- d ₆ )? 6.58 (s, 1H, H-6,6 '), 4.04

^{13 C NMR (125 MHz, acetone-} d 6) δ 145.4 (C-5, 5 '), 143.8 (C-4, 4'), 132.0 (C-1, 1 '), 116.5 (C-6, 6 , 116.4 (C-2, 2 '), 113.6 (C-3, 3'), 44.5 (C-7)

ESIMS m / z 546.7 [MH] ^- .

Formula 2b Compound

^{1 H NMR (500 MHz, acetone-} d 6) δ 8.73 (s, 1H, OH-4), 8.71 (s, 1H, OH-3 '), 8.17 (s, 1H, OH-4'), 8.08 ( 1H, H-5), 7.00 (s, 1H, H-5 '), 6.08 (s, 1H, H-6), 4.21 -7), 3.24 (s, 1H, H-9)

^{13 C NMR (125 MHz, acetone-} d 6) δ 144.5 (C-4 '), 144.0 (C-5), 142.6 (C-4, 3'), 131.5 (C-1), 129.8 (C-6 ), 128.6 (C-1 '), 115.4 (C-2, C-5'), 114.0 -9), 38.4 (C-7)

ESIMS m / z 510.6 [MH] ^- .

[Reference Example 1] Culture of virus

The virus used for measuring the antiviral activity of the red nectar extract obtained from Example 1 and the polyribomacetecol compound isolated therefrom was a lynovirus, and the lynovirus was infected with HeLa cells and proliferated. HeLa cells were cultured in MEM medium containing 10% fetal bovine serum in a 32 ° C incubator. The culture media used for virus growth and antiviral activity were cultured in 2% serum containing 20 mM MgCl ₂ Respectively.

[Experimental Example 1] Measurement of the inhibitory effect of the red nectar extract on the viral growth of rhinovirus

In order to measure the inhibitory effect of each of the extracts obtained in Examples 1 to 5 on the viral growth inhibition of lynovirus, 2 × 10 ⁴ cells of HeLa cells were placed in each well of 96-well plates and cultured for 24 hours. After 24 hours, the culture supernatant of each well was removed and a lynovirus infectious solution was added to each well and then administered to each well to be a polyribomacetecol compound of 25 占 퐂 / ml media. The viability of each extract was measured by SRB assay after 48 hours. 100 μl of 10% TCA (trichloroacetic acid) was added to each well, followed by incubation at 4 ° C for 1 hour and washing with distilled water several times. After drying at room temperature, 100 μl of 0.4% (w / v) SRB (sulforhodamine B) solution dissolved in 1% (v / v) acetic acid was added and stained for 30 minutes. The SRB staining solution not bound to the cells was washed several times with 1% (v / v) acetic acid and dried again. To each well, 100 μl of a 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the stain bound to the cells, and then the absorbance at 560 nm was measured to compare the inhibitory effects. At this time, the inhibitory effect of the compound of the present invention compared with the cells treated with DMSO alone, DMSO, HRV (human lynovirus) 2 and HRV3 treated cells was determined and the results are shown in the following Table 1 .

Comparison of Human Rhinovirus Inhibitory Effects of Red Native Extracts on HeLa Cell Lines Low performance (TI = CC ₅₀ / IC ₅₀ ) Extract classification HRV2 HRV3 Methanol extract
(Example 1) 1.14 {CC ₅₀ / IC ₅₀ => (20 μg / ml) /
(17.58 +/- 0.59 [mu] g / ml)} 1.09 {CC ₅₀ / IC ₅₀ => (20 μg / ml) /
(18.27 占 2.22 占 퐂 / ml)} Hexane extract
(Example 2) 50% inhibition at maximum capacity
Failed to display 50% inhibition at maximum capacity
Failed to display Ethyl acetate extract
(Example 3) 1.29 {CC ₅₀ / IC ₅₀ => (20 μg / ml) /
(15.50 占 4.17 占 퐂 / ml)} 1.21 {CC ₅₀ / IC ₅₀ => (20 μg / ml) /
(16.50 占 4.17 占 퐂 / ml)} n-butanol extract
(Example 4) 50% inhibition at maximum capacity
Failed to display 50% inhibition at maximum capacity
Failed to display Water extract
(Example 5) 1.21 {CC ₅₀ / IC ₅₀ => (20 μg / ml) /
(16.50 占 2.76 占 퐂 / ml)} 50% inhibition at maximum capacity
Failed to display

The test was carried out at a concentration of 2 ㎍ / ml. As shown in the above Table 1, the inhibitory activity index (TI = CC ₅₀ / IC ₅₀ ; CC ₅₀ , concentration required to exhibit 50% cytotoxicity, The concentration required for IC ₅₀ , 50% virus inhibition) was 1.14 for HRV2 and 1.09 for HRV3, 1.29 for HRV2 and 1.25 for HRV3, respectively, and the methanol extract Among the extracts classified according to the degree of polarity, it was confirmed that the ethyl acetate extract had an inhibitory activity against viruses.

[Experimental Example 2] Determination of the inhibitory effect of the extract of red nectar organic solvent fraction on the viral growth inhibition against the rhinovirus

The antiviral activity of the fraction extract of the ethyl acetate extract according to the concentration gradient obtained in Example 3 against the lynovirus was measured in the same manner as in Experimental Example 1,

Comparison of Human Rhinovirus Inhibitory Effect of Red Native Ethyl Acetate Fraction Extract in HeLa Cell Lines HRV2 HRV3 Fraction CC ₅₀ (쨉 g / mL) IC ₅₀ (쨉 g / mL) TI CC ₅₀ (쨉 g / mL) IC ₅₀ (쨉 g / mL) TI F1 > 20 11.38 ± 3.01 1.76 > 20 ND - F2 > 20 8.36 + - 2.42 2.39 > 20 ND - F3 22.93 ND - 25.80 7.69 ± 0.45 2.60 F4 > 20 18.55 + - 0.51 1.08 > 20 18.52 + 0.49 1.08 F5 24.29 ND - 18.98 ND - F6 > 20 ND - > 20 ND - F7 > 20 10.69 ± 1.34 1.87 > 20 ND - Ribavirin > 20 17.14 + 1.48 1.17 > 20 14.25 + - 2.20 1.40

As described in Experimental Example 1, TI = CC ₅₀ / IC ₅₀ ; The concentration required to exhibit CC ₅₀ , 50% cytotoxicity, the IC ₅₀ , the concentration required to exhibit 50% viral inhibitory effect, and the ND indicating that the IC ₅₀ value can not be measured. As shown in the above Table 2, F1, F2, F4 and F7 showed 11.38 ± 3.01, 8.36 ± 2.42, 18.55 ± 0.51 and 10.69 ± 1.34 ㎍ / mL of virus growth inhibition ability in HRV2 and F3 and F4 in HRV3 were 7.69 ± 0.45 and 18.52 ± 0.49 ㎍ / mL, respectively. For HRV2, F1, F2 and F7 fractions showed better antiviral inhibitory ability than ribavirin used as an antiviral agent. However, a single compound separation experiment was carried out on fraction F4 which shows inhibitory effect on virus replication in HRV2 and HRV3 at the same time.

[Experimental Example 3] Measurement of the inhibitory effect of the polyribomacetecol compound isolated from the red nectar to the viral growth inhibition against the lynovirus

The antiviral activity of the polyribomacate compounds isolated from the ethyl acetate fraction F4 according to Example 6 was measured in the same manner as in Experimental Example 1 and shown in Table 3. Ribavirin was used as a positive control.

Comparison of human rhinovirus inhibitory effect of polyribomacetecol compound isolated from red nectar in HeLa cell line HRV2 HRV3 CC ₅₀ IC ₅₀ TI CC ₅₀ IC ₅₀ TI 1a > 20 2.50 + - 0.66 8.00 > 20 ND - 1b > 20 ND - > 20 10.3 ± 3.92 1.93 2a 30.5 7.58 ± 0.59 2.88 > 20 5.27 ± 2.22 3.80 2b > 20 7.11 + - 0.64 2.81 > 20 4.69 ± 0.44 4.26 Ribavirin > 20 2.15 ± 0.51 9.30 > 20 5.09 ± 0.60 3.93

The test was carried out at a concentration of 20 μg / ml. However, the 1c and 1d complexes were not suitable for confirming the antiviral activity of a single compound and were excluded from the experimental results. The antiviral efficacy of the polyribomacortecol compounds isolated from the red nectar was determined to be 1a and 2b in the case of HRV2 at the same concentration. In HRV3, the 2b compound showed similar activity to ribavirin, while 2a , Similar activity to 2b, but cytotoxicity was also confirmed. 1a, 1c, 2a and 2b have been reported to exhibit antibacterial activity against bacteria, but they have not been used in antiviral therapy.

Therefore, in the present invention, it has been found that the compounds 1a and 2b and the ethyl acetate extract have a novel inhibitory effect on the rhinovirus, and compared with the efficacy of ribavirin used as an antiviral agent, These extracts and compounds have industrially useful value.

The composition according to the present invention can be widely applied for prevention and treatment of a cold, which is a disease caused by a virus. Industrialization is also possible by providing a simple separation method.

Claims (16)

The present invention relates to a pharmaceutical composition containing, as an active ingredient, an extract of red nematodes having an antiviral activity against human rhinovirus and red nematodes ( Neorhodomela aculeata )
The extract is prepared by dissolving 2,3-dibromo-4,5-dihydroxybenzyl alcohol, 2,3-dibromo-4,5-dihydroxybenzylaldehyde, 2,2 ', 3,3'-tetra Bromo-4,4 ', 5,5'-tetrahydrodiphenylmethane, and salts thereof. The method according to claim 1,
Wherein the extract is a fractional extract that is fractionated using a difference in polarity of the solvent. 3. The method of claim 2,
Wherein the solvent is at least one selected from the group consisting of hexane, dichloromethane, ethyl acetate, n-butanol and distilled water. The method of claim 3,
Wherein the solvent is hexane or ethyl acetate. delete delete delete delete A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 4,
Red nigra and red nigrometer ( Neorhodomela aculeata ) were extracted with an organic solvent and then distilled under reduced pressure to obtain 2,3-dibromo-4,5-dihydroxybenzyl alcohol, 2,3- Dibromo-4,5-dihydroxybenzylaldehyde, 2,2 ', 3,3'-tetrabromo-4,4', 5,5'-tetrahydrodiphenylmethane and salts thereof ≪ RTI ID = 0.0 > 1, < / RTI >
Fractionating the extract obtained in the above step using the difference in polarity of the solvent to obtain a fraction extract; And
And separating and purifying the fraction extract obtained in the above step by chromatography. delete delete 10. The method of claim 9,
Wherein the organic solvent is at least one selected from the group consisting of acetone, acetonitrile, methanol, ethanol, isopropanol, butanol, carbon tetrachloride (CCl ₄ ), chloroform (CHCl ₃ ) and dichloromethane (CH ₂ Cl ₂ ). 10. The method of claim 9,
The fraction is obtained by preparing at least one of a hexane layer fraction, a dichloromethane fraction, an ethyl acetate layer fraction, an n-butanol layer fraction and an aqueous layer fraction obtained by fractionating an extract with hexane, dichloromethane, ethyl acetate, n-butanol or distilled water ≪ / RTI > delete delete delete

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