KR20070074833A - Anti-rsv composition comprising extract of rosmarinus officinalis - Google Patents

Abstract

An anti-RSV(respiratory syncytial virus) composition comprising the extract of Rosmarinus officinalis is provided to inhibit activity of respiratory syncytial virus without toxicity, and improve application convenience of the composition, so that the composition is useful for prevention and treatment of disease caused by the respiratory syncytial virus including cough, sneezing, fever, wheezing, bronchiolitis, pneumonia and respiratory failure. The anti-RSV composition comprises the extract of Rosmarinus officinalis which is prepared by extracting Rosmarinus officinalis with a solvent selected from water, organic solvent such as methanol or a mixture thereof, and further comprises at least one selected from vitamin B1, B2, B6, C and E, niacin, carnitine, betaine, folic acid, pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium and a mixture thereof.

Description

Anti-Respiratory Syncitium Virus Composition Comprising Rosemary Extracts {Anti-RSV composition comprising extract of Rosmarinus officinalis}

1 shows the results of an in vitro neutralization test of eight candidate extracts selected through primary screening. Candidate extracts used in the test are: A22 ( Melia azedarach var. japonica ; Mulberry), A47 ( Nymphoides peltata ; yellow lotus), A63 ( Ailanthus altissima ; Leather bark), A74 ( Chaenomeles sinensis ; Chinese quince), A76 ( Ailanthus altissima ; Leatherwood-stemwood ), B11 ( Glycyrrhizae Radix ; Licorice), C12 ( Cynanchum atratum ; White rice), C32 ( Rosmarinus officinalis ; Rosemary)

FIG. 2 shows the results of an in vivo neutralization test of three candidate extracts selected through in vitro neutralization test.

3 Rosmarinus Methanol extract of officinalis is extracted again by solvent and the results of in vitro neutralization tests on them are shown.

4 is Rosmarinus Methanol extract of officinalis is extracted again by solvent and the results of in vitro neutralization tests on them are shown.

Respiratory syncytial virus (RSV) is a genome containing non-segmented RNA and belongs to paramyxoviridae. Infected host cells synthesize 10 proteins such as NS1, NS2, P, N, M, SH, G, F, M2 and L. Surface antigens are F protein (fusion protein), G protein, and SH protein. G protein and F protein in the outer membrane of the virus with relatively high molecular weight are glycosylated glycosylated and plays the most important role in the immune and antigenic variation of RSV infection. G proteins are involved in attachment to host cells to be infected, F proteins are involved in the formation of giant cells and invasion and attachment of viruses, and F and G proteins cause neutralizing antibodies.

RSV is a major pathogen of pediatric lower extremity infections and is the major virus causing pneumonia and bronchitis (Brandt et al., 1973; Selwyn et al., 1990). In particular, pulmonary dysplasia, congenital heart disease, cystic fibrosis, infants with cancer or various immunodeficiencies, and adults with immunocompromised conditions before bone marrow transplantation are high-risk groups that can cause fatal infections (McDonald et al., 1982; Chandwani et al., 1990; Bruhn et al., 1977). In the elderly, the infection is similar to that of the influenza virus, and excess mortality is reported in the RSV epidemic than in the influenza epidemic (Lancet 1993 Flemming et al).

There are 100,000 to 200,000 high-risk patients in the United States, with hospitalizations of more than 90,000 RSV infections each year, with about 4,500 deaths reported. In Korea, RSV admitted to pediatric hospitals is prevalent every year, and 60% of all viral lower limb infections isolated from Seoul National University Pediatric Hospital are caused by RSV. In Korea, hospitalization and death due to RSV infection is estimated to be very high. do.

Despite these serious problems, no vaccines or specific therapies are currently available. In particular, formalin-killed vaccines were developed in the past and clinical trials were conducted. However, severe lower respiratory tract disease occurred when RSV was infected after vaccination. Currently, Ribavirin (Virazole) is used as an approved treatment, but its clinical efficacy is not high, and only a few of the infected patients are effective. In addition, it has high toxicity and has the inconvenience of spraying in the form of aerosol and is only used in some limited cases. Therefore, the development of therapeutic agents for RSV is very urgent.

To date, many plant extracts have been reported to have antiviral activity, especially Sophora flavescens , Scutellaria baicalensis , Selaginella sinensis , Eleutherococcus senticosus , Schefflera heptaphylla (RS et al., 2002; Ma et al., 2001; Glatthaar-Saalmuller et al., 2001; Li et al., 2005).

In order to obtain a substance having excellent RSV inhibitory activity in addition to the above-mentioned natural products, the present inventors completed the present invention by confirming that the rosemary extract has anti-RSV activity in the process of searching for an active substance that inhibits RSV.

One object of the present invention is a rosemary ( Rosmarinus officinalis ) to provide a composition having anti-respiratory syntosis virus activity comprising the extract.

Still another object of the present invention is to provide a method for preventing or treating respiratory synthium virus infection by administering a composition comprising a rosemary extract.

In one embodiment, the present invention provides a rosemary ( Rosmarinus) officinalis ) to provide a composition having anti-respiratory syntosis virus activity comprising the extract.

As used herein, the term “antiviral activity” refers to the ability to inhibit viral particle production, ie, the formation and / or replication of respiratory syncytial virus (RSV) in host cells, particularly humans. The composition provided by the present invention has antiviral activity by interfering with mechanisms such as viral replication and / or proliferation.

In the present invention, the term "extract" is prepared by an extraction process using an organic solvent such as water or alcohol or a mixed solvent thereof. Extracts include water or organic solvent extracts, dry powders thereof or any form formulated using the same.

In the present invention, the term “ Rosmarinus officinalis ) ”includes all stems, leaves and flowers of natural, hybrid rosemary. Rosemary has been widely used in cosmetic compositions but has not been reported for antiviral activity against RSV.

As a result of examining the anti-RSV activity of the rosemary extract in the mouse, it was confirmed that less than 10 plaques were observed in the rosemary-administered group compared to the control group having about 35-55 plaques. This is a result showing that rosemary extract has a high neutralization capacity. This is much higher than ribavirin, where about 35-40 plaques are observed.

Each composition comprising the rosemary extract of the present invention does not increase the efficacy, but may include additional ingredients that are commonly used in medicinal compositions to improve the smell, taste, time and the like. In addition, the composition of the present invention is inorganic, such as vitamins B ₁ , B ₂ , B ₆ , C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, mixtures thereof, and the like. It may further comprise organic additives.

The rosemary of the present invention can be used as it is powdered, but preferably extracted using a solvent. The liquid extracted with the solvent can be used directly, and the extract can be further filtered and / or dried.

As an extraction solvent, an organic solvent such as water, alcohol, or a mixed solvent thereof is used and the active ingredient of the herbal medicine is not destroyed or minimized, and the extract is heat or cold extracted. Filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon, etc., or freezing, centrifugal separation, etc. may be used, but is not limited thereto. Drying the filtrate includes, but is not limited to, lyophilization, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high frequency drying, infrared drying, and the like. The drying process can reduce the water content, and may also add a process to reduce the water content by placing the filtrate in the concentrator before drying. If desired, a process of grinding the final dried extract may be added.

When extracted using an organic solvent, methanol, ethanol, isoprotanol, butanol, ethylene, acetone, ether, hexane, chloroform, ethyl acetate, DMF (N, N-dimethylformamide), DMSO (dimethyl sulfoxide), etc. Can be extracted with an organic solvent at room temperature or by heating. Preferably at 10 ℃ to 50 ℃, extraction for 24 to 48 hours. In the present invention, rosemary was extracted by using methanol for 24 hours at room temperature. In addition, the methanol extract can be reextracted with ethyl acetate. Preferably at 10 ℃ to 50 ℃, extraction for 24 to 48 hours.

The composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, etc., in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be used in combination, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms.

In another aspect, the present invention provides a method for preventing or treating a respiratory sinitium virus infection by administering a composition comprising a rosemary extract.

As used herein, the term "prevention" means any action that inhibits or delays the onset of viral infection by administration of a composition. As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms caused by viral infection by administration of the composition. Symptoms caused by respiratory infections include coughing, sneezing, high fever, wheezing, bronchitis, pneumonia, and respiratory failure.

The route of administration of the composition may be administered via any general route as long as it can reach the desired tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level for the severity, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited by the following examples.

Example 1: Primary Plant Extract Screening

157 kinds of extracts prepared from 115 plants (prepared by extracting powder samples with methanol (MeOH) for 24 hours at room temperature) were prepared by the Korea Plant Research Bank of Korea Biotechnology Bank, and the primary screening was performed. The screening method used a microneutralization method. First, the extracted extract was dissolved in DMSO and diluted in PBS so that the final concentration of DMSO was 2.5%. The extract was step-diluted in DMEM medium, mixed with 1000 pfu of B-type RSV virus, mixed with 5 × 10 ⁴ Hep2 cells, and placed in a 96-well plate. After incubation in a 37% 5% CO ₂ incubator for 3 days, the medium was removed and fixed using 3.7% formaldehyde. After blocking using 5% skim milk, anti-F mouse monoclonal antibody was reacted. After washing three times with PBS, the secondary antibody anti-mouse IgG-HRP was reacted. Again washed with PBS and 0.2 M citrate-PO ₄ containing 0.04% ortho-phenylenediamine-dihydrochloride (OPD) and 0.012% H ₂ O ₂ Buffer (pH 5.0) Buffer was added to each well and developed for 5 minutes. 2.5 MH ₂ SO ₄ was added to stop the reaction and the absorbance at 492 nm was measured. After deriving 8 candidates through the primary screening, the neutralization ability of the 8 extracts was again assayed (FIG. 1).

Extract C32 ( Rosmarinus in Figure 1 Three extracts, including officinalis ), showed high neutralizing ability and were subjected to in vivo efficacy evaluation.

Example 2: In vivo efficacy evaluation

Three selected candidates (A63 [ Ailanthus altissima ; Leather-bark], C12 [ Cynanchum atratum ; White rice], C32 [ Rosmarinus officinalis ; Rosemary] was confirmed in vivo antiviral activity. First, the intraperitoneal administration of cyclophosphamide (cyclophosphamide) at 100 mg / kg to Balb / c mice was additionally administered 5 days later to inhibit immunity. Mice were anesthetized 24 hours after the last dose and the virus was inhaled intranasally. Infected with 1 × 10 ⁹ pfu of virus. Each extract was dissolved in 5% Tween 80 and then intraperitoneally administered at a dose of 50 mg / kg. Thereafter, the same amount was administered intraperitoneally every 12 hours. Four days after infection, mice were sacrificed and lungs were removed. The extracted lungs were crushed in ice using a Dounce Homogeneizer, centrifuged and the supernatant was recovered. The recovered supernatant was step-diluted to determine titer of virus by plaque assay. After infecting the supernatant for 200 hr in a 24-well plate in which Hep2 cells were cultured, the infection solution was removed, and 1 mL of methylcellulose media (2% FBS) was overlayed. After incubation for 6 days, the medium was removed and fixed using 3.7% formaldehyde. After blocking with 5% skim milk, anti-F mouse monoclonal antibody was reacted. After washing three times with PBS, the secondary antibody anti-mouse IgG-HRP was reacted. After washing with PBS again, it was developed with DAB coloring solution. After color development washed with distilled water again and dried. The number of colored plaques was counted to determine the titer of the virus.

Test results C32 ( Rosmarinus officinalis ) showed the best efficacy. As a result of testing with three mice, 35-55 plaques were observed in the control group, while less than 10 plaques were observed in the C32 (Rosmarinus officinalis) group. This shows a very high neutralization capacity compared to Ribavirin in which about 35-40 plaques were observed (FIG. 2).

Example 3: Rosmarinus Neutralization Analysis by Fraction of officinalis Extract

Rosmarinus Methanol extract of officinalis was further divided into hexane (Hexane), ethyl acetate (EtOAc), butanol (BuOH), water (H ₂ O) as a solvent. Hexane extract is dispersed in distilled water, rosemary methanol extract in distilled water according to a conventional method, and shaken with the same amount of hexane, the supernatant is separated and the extract is combined again, the combined hexane extract is concentrated under reduced pressure at 40 ℃ or less Prepared. Ethyl acetate extract, butanol extract was prepared in the same manner as described above using ethyl acetate, butanol instead of hexane extraction solvent. The remaining water layer is concentrated under reduced pressure at 50 ° C. or lower to obtain H ₂ O extract. Recovery results of the fractions are shown in Table 1.

Rosmarinus fractions of officinalis menstruum % Recovery MeOH 100 Hexane 12 EtOAc 40 BuOH 25 H ₂ O 23

5. Rosmarinus Fractional Neutralization of officinalis

Each obtained fraction was analyzed by the neutralization test (microneutralization) in the same manner as 2 (Fig. 3). However, considering the yield of each fraction, C32 ( Rosmarinus MeOH fraction of officinalis ) was diluted to contain the amount contained in the original MeOH fraction by multiplying the recovery rate (e.g. 50 X 0.12 = 6 ㎍ / ml with C32 at 50 μg / ml for the Hexane fraction). ml and expressed as 50 μg / ml, the concentration of C32)

As shown in Figure 3 by the EtOAc fractions indicates almost the same neutralization activity, and overall MeOH fraction fraction representing the actual neutralizing activity is Rosmarinus officinalis It was confirmed that the MeOH extract was present in the fraction dissolved in the EtOAc solvent.

In order to confirm this again in vivo neutralization test was carried out in the same manner as 3 (Fig. 4). In addition, the C32 fraction was administered at a dose of 50 mg / kg, and the EtOAc fraction was administered at a dose of 19.63 mg / kg in consideration of the yield. In fact, it was confirmed that the EtOAc fraction showed almost the same neutralizing capacity as the total MeOH fraction.

The results above are Rosmarinus It is strongly suggested that extracts of officinalis , especially EtOAc extracts, can be used as therapeutic agents for RSV infection inhibition.

As described above, the composition comprising the rosemary extract can effectively prevent and treat infection of respiratory sinusitis virus, which is the main cause of diseases such as cough, sneezing, high fever, wheezing, bronchitis, pneumonia, respiratory failure.

Claims (3)

Rosemary Extract Rosmarinus officinalis ) composition having anti-respiratory syncitium virus activity. The composition of claim 1, wherein the rosemary extract is extracted using water, an organic solvent or a mixed solvent thereof. The composition of claim 2, wherein the rosemary extract is extracted using methanol.

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