KR101116748B1 - Flavonoid comprising anti-virus activity - Google Patents

Abstract

The present invention relates to a flavonoid compound having an antiviral activity, and relates to a use as an antiviral agent containing a flavonoid compound obtained by methanol separation of banaba ( Lagerstroemmia speciosa Pers) and purified by purification using chromatography.

Antiviral, Flavonoid Compounds, Banaba

Description

Flavonoid comprising anti-virus activity

The present invention relates to flavonoid compounds having antiviral activity.

The following antiviral drugs are widely used as drugs to treat viral infections. Ribavirin is known as Hepatitis C [Foster GR 2004. Semin Liver Dis. 24 supple 2: 97-104], SARS, used to treat Ebola virus infection [Gunther S, Asper M, Ropser C, Luna LK, Drosten C, Becker-Ziaja B, Borowski P, Chen HM, Hosmane RS. 2004. Antiviral Res. 63 (3): 209-215], acyclovir has been used to treat herpesviruses (Dewhurst 2004. Herpes, 11 (supple 2): 105A-111A), and Izideoxy cyme Dean (azidodeoxy thymidine) has been used to treat AIDS [Boyer PL, Sarafiano SG, Clark PK, Arnold E, Hughes SH. 2006. PLoS Pathog 2 (2): 0101-0111]. Colds are mainly caused by rhinovirus infection, but there are no commercialized drugs that can suppress rhinovirus infection, but compounds that inhibit the proliferation of rhinoviruses have been promoted in several reports [De Palma AM, Vliegen I, Clercq ED]. , Neyts J. 2008. Med Res Rev. 28 (6): 823-884; Collett MS, Neyts J, Modlin JF. 2008. Antiviral Res 79: 179-187; Grassauer A, Weinmuellner R, Meier C, Pretsch A, Grassauer EP, Unger H. 2008. Virology J 5: 107-123; Schmidtke M, Wutzler P, Zieger R, Riabova OB, Makarov VA. 2008. Antiviral Res. doi: 10.1016; Maugeri C, Alisi M, Apicella C, Cellai, Dragone P, Fioravanzo E, Florio S, Furlotti G, Mangano G, Ombratto R, Luisi R, Pompei R, Rincicotti V, Russo V, Vitiello M, Cazzolla N. 2008. Biooragnic Med Chem 16 (8): 23091-3107].

For literature on aurobol 7-glucosides see Anhut S et al., Anhut S, Zinsmeister D, Mues R, Barz W, Mackenbrock K, Koster J, Markham KR. 1984 Phytochemistry 23 (5): 1073-1975] and the like were first isolated from selected plants and have also been found in lupine plants (Marczak L, Wijtaszk P, Stobiecki M. 2008. J Plant Physiol 165: 239-250). No biological activity is known. However, antiviral activity of flavonoids or glycated flavonoids with similar structures has been reported in several articles [Journal of antimicrobial chemotherapy, L. C. Chiang, W. Chiang, M. C. Liu and C. C. Lin, Vol. 52 (pp 194-198, 2003), Flavonoids have been reported to have antiviral effects against herpes virus, adenovirus, etc., and biflavonoid compounds such as robustaflavone have been reported to have inhibitory effects against influenza virus. I [Planta medica, Yuh-Meei Lin, Vol. 65 (pp120-125)], the antiviral effect was not excellent.

Regarding the antiviral activity of banaba, in the patent filed by Kwon et al. (Korean Patent No. 668689), the banaba extract exhibits anti-rhinovirus activity. The present invention isolates an active substance having anti-rhinovirus activity from the extract of Banaba and confirms that the active substance is orobol-7-glucoside. Patent [Korean Patent Registration No. 668689] filed by the present inventors described that Banaba extract inhibits the activity of rhinovirus (type 3) at a concentration of 50 μg / ml, but in the present invention, the same type 3 rhinovirus Orobol-7-gluconoside showed 50% antiviral activity even at a concentration of 0.18 ㎍ / ml, showing more than 200-fold efficacy. . Ribavirin also contains enterovirus 71 [Bible JM, Pantelidis P, Chan PKS, Tong CYW. 2007. Rev Med Virol. 17: 371-379] [medicines showing inhibitory effect against Li ZH, Li CM, Ling P, Shen FH, Chen SH, Liu CC, Yu CK, Chen SH. 2008. J Infect Dis. 197 (6): 854-857], but in the comparative experiments in the present invention, the inhibitory effect of ribavirin was less than 50% even at the concentration of 100 μg / ml, but the aurobol-7-glucoside was 50% inhibitory concentration value. (IC ₅₀ ) was found to be 1.56 ± 0.47 μg / ml, indicating that the virus had very superior antiviral activity compared to ribavirin. Also, aurobol-7-glucoside has IC ₅₀ values of 4.61 ± 1.37 µg / ml (A / WS / 33) and 8.58 ± 1.15 µg / ml (A / PR) for influenza viruses A and B. / 8/34), 32.68 ± 5.60 μg / ml (B / Lee / 40), and oseltamivir (Tamiflu), 29.68 ± 15.20 μg / ml (A / WS / 33). The present invention was completed by confirming that it exhibits significant antiviral activity when compared with that indicated by.

Accordingly, an object of the present invention is to provide an antiviral composition containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are each H or OH.

The present invention is characterized by an antiviral composition containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are each H or OH.

Hereinafter, the present invention will be described in detail.

The present invention relates to the use as an antiviral agent containing the compound of Formula 1 as an active ingredient obtained by methanol extraction of Lagerstroemmia speciosa Pers and purified by chromatography.

As mentioned above, the compounds according to the invention can be synthesized by organic synthesis or can be extracted and separated from vanabar.

The physicochemical properties of the materials separated from Banaba were analyzed using electron spray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance spectra.

1) Compound of Formula 1

i) Material Properties: Powder ii) Molecular Weight: 448

iii) Molecular formula: C ₂₁ H ₂₀ O ₁₁ iv) Mass spectrometry (MH) ^- : 448 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra were measured using a methanol solvent as a dew, it was found to be a compound represented by the following formula (1a).

[Formula 1a]

On the other hand, as an active ingredient of the composition of the present invention can be used in the form of a pharmaceutically acceptable salt thereof instead of the compound of Formula 1, wherein the salt is an acid addition salt formed by a pharmaceutically acceptable free acid (free acid) This is useful. The compound of Formula 1 may form an acid addition salt which is pharmaceutically acceptable according to a conventional method in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

Finally, the present invention includes an antiviral composition containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Since the pharmaceutical composition contains a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, it inhibits the propagation of rhinoviruses and thus is useful for treating or preventing symptoms such as diarrhea and dehydration due to rhinovirus infection. Can be used. In addition, by inhibiting the proliferation of influenza virus, it can be usefully used to treat or prevent the flu or cold caused by the RNA rule genome, such as rhinovirus or influenza virus.

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration, and may be used in the form of general medicines and health foods.

Pharmaceutical compositions of the invention may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs ( formulated into elixirs). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection. To formulate into a parenteral formulation, the compound of Formula 1 is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

In addition, the effective dose of the compound represented by Formula 1 is generally 1 to 100 mg / day, preferably 5 to 20 mg / day, per 1 kg of adult patient weight, at regular intervals according to the judgment of the doctor or pharmacist In several times a day, preferably two to three times daily.

In addition, the health food is a food prepared by adding the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof to food materials such as beverages, teas, spices, chewing gum, confectionary, or the like, encapsulated, powdered, suspension, etc. As such, ingesting it means that it brings a specific effect on health, but unlike general medicines, there is no side effect that can occur when taking long-term use of the drug as a food raw material.

As described above, the compound according to the present invention or a pharmaceutically acceptable salt thereof may be usefully used for treating or preventing a disease caused by a viral infection.

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 and Purification of Rhinovirus Proliferation Inhibitors

In order to search for compounds that can inhibit the propagation of rhinoviruses, the inventors have isolated and purified the compounds from natural plants using extraction and chromatography.

First, 1 kg of Banaba purchased from USE TECHNO CORPORATION (KOREA) was extracted with 100% methanol for 48 hours, concentrated under reduced pressure, and extracted with ethyl acetate. The ethyl acetate layer was concentrated, and the concentrated material was dissolved in 2 ml of methanol, followed by C18 silica gel column adsorption chromatography using a mixed solvent of methanol and water (20 → 100) as an eluting solvent. The active fractions were concentrated by adsorption chromatography. High performance liquid chromatography (Column: ODS-MA, Flow rate: 2 mL / min, Dissolving the concentrated active fraction in methanol and using acetonitrile (20-40% acetonitrile, 22 minutes) as an eluting solvent. 212 nm detection) to separate the active fraction of the holding time of 22 minutes [Fig. 1], and freeze-dried the residue (residue) obtained by removing the solvent with a reduced pressure dryer (Orobol 7- Glucoside) 15 mg.

[Formula 1a]

Example 2: Physicochemical Characterization of Active Substances

In order to analyze the physicochemical properties of the active material, the inventors used methods such as electrospray ionization mass spectrometry (ESI-MS), Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectra. In the NMR experiment, each sample was dissolved in methanol in a solvent and measured in a 5 mm NMR tube. Chemical shifts were measured based on the peak of each solvent or based on the peak of TMS (tetramethylsilane). As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, it was confirmed that the compound of the present invention had the following physical and chemical properties.

As a result, the active substance had a powder form, a molecular weight of 448 and a molecular formula of C ₂₁ H ₂₀ O _11. The mass spectrometry (MH) ⁻ of the compound was 448 (m / z). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol measured as a solvent together with the physicochemical properties were found to be a compound exhibiting a virus inhibitory effect [FIG. 2].

<NMR data>

C ¹ H-NMR
(400 MHz, CD3OD) ¹³ C-NMR
(100 MHz, CD3OD) 2 8.54 (1H, s) 154.4 3 - 122.7 4 - 180 5 - 162.9 6 6.40 (1H, doublet, J = 2 Hz) 99.5 7 - 161.6 8 6.20 (1H, d, J = 2 Hz) 94.5 9 - 157.1 10 - 106.0 One' - 121.4 2' 7.84 (1H, doublet, J = 2.0 Hz) 116.5 3 ' - 144.9 4' - 145.6 5 ' 6.87 (1H, doublet, J = 8.4 Hz) 115.4 6 ' 7.6 (1H, doublet of doublets, J = 2.4, 8.4 Hz) 119.9 Glc 1 5.17 (1H, d, J = 7.6 Hz) 99.8 2 73.0 3 77.1 4 69.4 5 76.3 6 60.6

Example 3: Measurement of the inhibitory effect of aurobol 7-glucoside on rhinovirus

In order to measure the inhibitory effect of orobol 7-glucoside on rhinovirus, the present inventors performed the following in vitro experiment using HeLa cells, a human cervical cancer cell line.

A method for measuring the virus growth inhibition ability of aurobol-7-glucoside was determined by four methods of rhinovirus type A virus and four types of virus type B virus based on the method of patent application by Kwon Doo et al. (Korean Patent Registration No. 0682069). The inhibitory effect on virus activity was measured. HeLa cells were incubated in each well of a 96-well microplate until the bottom was completely covered with cells. Remove the existing culture, wash each well twice with phosphate buffer, put the rhinovirus solution prepared at the concentration of TCID50 in each well, and purchase ribavirin, an existing antiviral agent, from Aurobol-7-glucoside and Sigma. After dissolving each in dimethylsulfoxide (dimethylsulfoxide) each solution was administered in each well at a concentration of 0.01 ~ 100 ㎍ / ㎖ and incubated for 48 hours and observed the shape of HeLa cells under a microscope to determine the antiviral activity 1 It was determined next. 0.4% (w / v) sulforhodamine B (SRB) solution dissolved in 1% (v / v) acetic acid after adding to 100 μl of 70% acetone in each well, left for 1 hour at -20 ℃, dried in a dryer After 100 μl addition and staining for 30 minutes, the SRB staining solution which did not bind to the cells was washed four times with 1% (v / v) acetic acid and dried again. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well of the dye bound to the cells in each well to dissolve the dye, and the absorbance was measured at 562 nm to compare the virus inhibitory effect. At this time, the proliferation inhibitory effect of the rhinoviruses represented by the compound of the present invention was determined using the cells treated with DMSO alone and the cells administered with the DMSO and rhinovirus infection solution as a control group.

As shown in Table 1, orobol-7-glucoside shows a 50% inhibitory effect (IC ₅₀ ) at a concentration range of 0.5-8.8 ㎍ / ml for 8 rhinoviruses, showing a small difference depending on the virus type. In comparison, ribavirin showed a significant difference between virus types such as type 3, type 5, and type 15 with a 50% inhibitory effect value of 3.0 ㎍ / ml (type 14) or more than 100 ㎍ / ml, depending on the type of virus. In view of the wide variety of viruses, aurobol-7-glucoside was found to have a more effective antiviral effect in treating colds than ribavirin, a standard comparator. From the above results, it was confirmed that the aurobol-7-glucoside of the present invention is a compound derived from natural products that can inhibit rhinoviruses in vitro.

Example 4: Measurement of the inhibitory effect of orobol 7-glucoside on enterovirus

In order to measure the inhibitory effect of orobol 7-glucoside on enterovirus 71, the present inventors performed the following in vitro experiment using Vero cells, which are monkey kidney cell lines.

The method for measuring the virus growth inhibition ability of orobol-7-glucoside was measured by the method of patent application by Kwon Doo et al. (Korean Patent Registration No. 0682069) to determine the effect of inhibiting viral activity on enterovirus 7. Vero cells were incubated in each well of a 96-well microplate until the bottom was completely covered with cells. Existing cultures were removed, each well was washed twice with phosphate buffer, and the RV71 solution prepared at the concentration of TCID50 was placed in each well, and ribavirin, an antiviral agent, was purchased from aurobol-7-glucoside and Sigma. After dissolving in dimethylsulfoxide, each solution was administered to each well at a concentration of 0.01 to 100 ㎍ / ml, incubated for 48 hours, and observed for the antiviral activity by observing the shape of Vero cells under a microscope. Determined. 0.4% (w / v) sulforhodamine B (SRB) solution dissolved in 1% (v / v) acetic acid after adding to 100 μl of 70% acetone in each well, left for 1 hour at -20 ℃, dried in a dryer After 100 μl addition and staining for 30 minutes, the SRB staining solution which did not bind to the cells was washed four times with 1% (v / v) acetic acid and dried again. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well of the dye bound to the cells in each well to dissolve the dye, and the absorbance was measured at 562 nm to compare the virus inhibitory effect. At this time, DMSO-only cells and cells administered with DMSO and rhinovirus infection solution were used as controls to determine the proliferation inhibitory effect of enteroviruses represented by the compounds of the present invention.

Comparison of Growth Inhibitory Effects of Enterovirus 71 on Vero Cell Lines division CC ₅₀
(占 퐂 / ml) IC ₅₀
(占 퐂 / ml) Aurobol-7-Glucoside > 100 1.56 ± 0.47 Ribavirin > 100 > 100

As shown in Table 2, the 50% inhibitory efficacy value (IC ₅₀ ) of the virus of Vero cells infected with enterovirus 71 of orobol-7-glucoside was 1.56 ± 0.47 μg / ml, whereas ribavirin inhibited 50% of the virus. The concentration required to show the effect is more than 100 ㎍ / ml, it can be seen that aurobol-7-glucoside has a better virus inhibitory effect than ribavirin standard reference material. From the above results, it was confirmed that the aurobol-7-glucoside of the present invention is a compound derived from a natural product capable of inhibiting enteroviruses in vitro.

Example 5 Measurement of Inhibitory Effect of Aurobol 7-Glucoside on Influenza Virus

In order to measure the inhibitory effect of orobol 7-glucoside on influenza virus, the present inventors performed the following in vitro experiment using MDCK cells, which are dog kidney cell lines.

A method for measuring the virus growth inhibition ability of aurobol-7-glucoside was based on the method of patent application by Kwon Doo et al. (Korean Patent Registration No. 0682069) (A / WS / 33, A / PR). / 8/34), the effect of inhibiting viral activity against one virus (B / Lee / 40) of type B virus was measured. MDCK cells were incubated in each well of a 96-well microplate until the bottom was completely covered with cells. Existing cultures were removed, each well was washed twice with phosphate buffer, and influenza virus solutions prepared at the concentration of TCID50 were placed in each well, and aurobol-7-glucoside and Tamiflu, an existing anti-influenza virus agent, were purchased. After dissolving in dimethylsulfoxide, each solution was administered to each well at a concentration of 0.01 to 100 μg / ml, incubated for 48 hours, and the primary determination of antiviral activity was observed by observing the appearance of MDCK cells under a microscope. It was. 0.4% (w / v) sulforhodamine B (SRB) solution dissolved in 1% (v / v) acetic acid after adding to 100 μl of 70% acetone in each well, left for 1 hour at -20 ℃, dried in a dryer After 100 μl addition and staining for 30 minutes, the SRB staining solution which did not bind to the cells was washed four times with 1% (v / v) acetic acid and dried again. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well of the dye bound to the cells in each well to dissolve the dye, and the absorbance was measured at 562 nm to compare the virus inhibitory effect. In this case, the proliferation inhibitory effect of the influenza virus represented by the compound of the present invention was determined using the cells treated with DMSO alone and the cells administered with the DMSO and influenza virus infection solution as a control group, and is shown in Table 3 below.

As shown in Table 3, two influenza type A viruses (A / WS / 33, A / PR / 8/34) and one influenza B virus in MDCK cells infected with orobol-7-glucoside influenza virus The 50% inhibitory potency values (IC ₅₀ ) for (B / Lee / 40) were 4.61 ± 1.37 μg / ml, 8.58 ± 1.37 μg / ml and 32.68 ± 5.60 μg / ml, respectively, compared to 50% of Tamiflu. The concentrations required to exhibit the virus inhibitory effects were 65.30 ± 15.20 ㎍ / ml and 100 ㎍ / ml or more, respectively, indicating that aurobol-7-glucoside had a better virus inhibitory effect than Tamiflu, a standard comparator. From the above results, it was confirmed that the aurobol-7-glucoside of the present invention is a compound derived from natural products that can inhibit influenza viruses in vitro.

Example 5: Toxicity Test

Toxicity experiments were performed on the orobol-7-rhamnoside of the present invention as follows.

Orobol-7-glucoside was dissolved in dimethylsulfoxide (DMSO), diluted with water, and then administered to each mouse (10 mice per group) at 10 g / kg and observed for 7 days, but no mice died.

Preparation Example 1 Preparation of Powder

10 g of active ingredients (aurobol-7-rhamnoside)

50 g of corn starch

40 g of carboxy cellulose

Total amount 100 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was added to the 5 times hard capsule to prepare a capsule.

Preparation Example 2 Preparation of Tablet

10 g of active ingredients (aurobol-7-rhamnoside)

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

Total amount 100 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet is 100 mg, of which the active ingredient content is 10 mg.

Preparation Example 3 Preparation of Injection

Active ingredient (Orobol-7-Rhamnoside) 10 mg

Sodium chloride 600 mg

Ascorbic Acid 100 mg

Water for injection

Total amount 100 ml

Injections were prepared in the same composition as above. This solution was placed in an ampoule and heat sterilized at 120 ° C. for 30 minutes.

Formulation Example 4 Beverage Preparation

After dissolving 500 mg of the active ingredient (Orobol-7-Rhamnoside) in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, oligosaccharides as an auxiliary agent, and an appropriate amount of sodium benzoate are added as a preservative. Water was added to make the whole amount to 100 ml to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

FIG. 1 shows the H ³ -NMR pattern of aurobol 7-glucoside isolated from banaba methanol extract.

Claims (7)

delete An antiviral composition comprising the compound represented by Formula 1a as an active ingredient. [Formula 1a]

3. The antiviral composition according to claim 2, wherein the compound is isolated from banaba extract. The antiviral composition according to claim 2 or 3, wherein the antiviral composition is active against rhinoviruses. The antiviral composition according to claim 2 or 3, wherein the antiviral composition is active against enteroviruses. The antiviral composition according to claim 2 or 3, wherein the antiviral composition has an activity against influenza virus. The anti-viral health food comprising a compound represented by the following Formula 1a or a pharmaceutically acceptable salt thereof as an active ingredient; [Formula 1a]

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