KR100810032B1 - Extract of Coptidis rhizma comprising antifungal protein - Google Patents

Abstract

The present invention relates to a rhubarb extract comprising a protein having antifungal activity and a preparation method thereof.

The extract of the present invention exhibits antifungal activity, and in particular, has excellent antifungal activity against Candida albicans, the main cause of infectious atopic dermatitis, and has low toxicity and side effects unlike conventional antifungal agents. It can be used for prophylactic skin protection as well as for treatment.

Candida albicans, antifungal, Candida albicans

Description

Extract of Coptidis rhizma comprising antifungal protein with antifungal activity             

1 is a schematic diagram of a method for extracting a yellow lotus extract comprising a protein having antifungal activity using a dialysis membrane.

Figure 2 is a schematic diagram of a method for extracting a rhubarb extract containing a protein having antifungal activity using an ultrafiltraion membrane.

3 is a graph of the antifungal efficacy of Candida albicans infected rats of the barberry extract.

The present invention relates to a extract of Coconutis rhizome comprising a protein having antifungal activity, and specifically, a method of preparing an extract comprising a protein having antifungal activity from a cousin, a extract of the coagulum obtained therefrom and such extract It relates to an antifungal composition comprising as an active ingredient.

Candida albicans is a polymorphic fungus that is the leading causative agent of infectious atopic dermatitis. This fungus causes the most problems in humans among pathogenic fungi. In addition to the above skin diseases, local infections cause vaginitis, and there are statistics that over 70% of all women are infected at least once in their lifetime.

Housewives and diaper rashes are also the main cause of Candida albicans. In comparison with Staphylococcus aureus, another causative agent of diaper rash, more than 80% of diaper rashes are attributed to Candida albicans, the remainder being caused by Staphylococcus aureus (Arch. Dermatol. 114: 56 , 1978). Another report shows that Candida species cause the most problems with diaper rash (Med. Cutan. Ibero. Lat. Am., 10 : 225, 1982; Int. J. Dermatol., 35 : 791,1996).

Recently, superficial cutaneous candidiasis has been known to be the most problematic infectious atopic skin disease (Hautarzt., 53: 724, 2002; Cli. Microb. Rev., 15 : 545, 2002). These results suggest that medications are important after onset but can be prevented by atopic dermatitis caused by Candida albicans. In other words, candida albicans belonging to the normal flora (normal flora) can be present in most normal people, so proper prevention will be able to prevent such skin diseases.

In case of such an infection, there is no life disruption, but it causes many problems in daily life. In addition, contagious diseases such as AIDS patients, cancer patients, and diabetics mainly cause systemic infections, leading to death.

Chemotherapeutic agents are used to treat such local and systemic candidiasis. Representative antifungal drugs currently in use include polyene-based amphotericin B, azole-based fluconazole, myconazole, and itraconazole. However, most of them have high toxicity and low permeability, and have many side effects. In addition, the high resistance rate has a fatal disadvantage of low cure rate, and the development of new antifungal agents is required. Recently, a lot of resistance has been reported for caspofungin developed by Merck Co.

For this reason, the treatment of fungal infections by new methods is being developed, and among them, the development of vaccines, which are immunological methods, is being actively conducted.

Another alternative is the development of antimicrobial peptides combined with the concept of toll-like receptors (TLR) through innate immunity. Most organisms (mainly mammals, amphibians, insects, and plants) have a mechanism of action against invading microorganisms. After recognition of bacteria through a common receptor (TLR) that recognizes these invading bacteria, they secrete peptide substances. Kills these invading bacteria through The peptidic antimicrobial agent, which is also called defensin due to its antimicrobial effect, is composed of amino acids in composition, unlike conventional antibiotics. Most of these substances are extracted from the skin of amphibians (eg frogs) and are known to be more effective against bacteria than against fungi. In some plants defensin has been isolated, but efficacy has been reported for fungi other than Candida albicans.

There is also a prophylactic method using the chemotherapeutic agent, but when using the chemotherapeutic agent as a therapeutic agent, there are many problems that tend to cause most side effects.

Considering these issues, it is required to develop antifungal agents that are different from conventional chemical antibiotics and are produced in nature and have no side effects.

Herbal medicine, Coptidis rhizoma, is known to be effective against bacterial infections. Its main substance contains alkaloids, protoberberine. This protoberberine is a generic alkaloid mixture, which contains berberine, coptisine, jatrorhizne, palmitine, among others. It is known to have anticandida activity (J. Antimicro.

Chemother., 47 : 513, 2001; J. Antimicro., 43 : 667, 1999).

In the experiments on extracts extracted by alcohol after extracting Korean herbal medicines such as Hwang, Hubak, Ho Jang-geun and spectators from Korean Patent Application No. 1995-016816 and Korean Patent Application No. 2000-0075149 It has been reported to have antimicrobial activity against the causing bacteria Streptococcus mutans. However, the antimicrobial activity was due to Burberry and palmitine, not the proteinaceous component of sulfur.

In light of these considerations, the development of fungal infections, which are different from conventional chemical antibiotics and are produced in nature, have no side effects and have a therapeutic effect and prophylatic skin care. There is a great demand.

Under these backgrounds, the present inventors have conducted many studies to develop antifungal active substances derived from natural products without side effects, and have completed the present invention by finding that the anti-fungal activity of rhubarb protein extract exhibits excellent antifungal activity.

It is an object of the present invention to provide a method for extracting rhubarb extract comprising a protein having antifungal activity.

Still another object of the present invention is to provide a rhubarb extract comprising a protein having antifungal activity.

Still another object of the present invention is to provide an antifungal composition containing a rhubarb extract comprising an protein having antifungal activity as an active ingredient.

Coptidis rhizome is a perennial herbaceous plant belonging to Ranunculus and is known to have pharmacological effects such as antimicrobial action, blood pressure lowering action and anti-inflammatory action. Prior to the present invention, the antimicrobial activity of the barberry was reported to be due to the component of protoberbourne, but it was not known that the extract including the protein derived from the barberry had antifungal activity. In addition, the Republic of Korea Patent Application No. 1999-0049296 reported that in the experiments of extracts extracted from hot water extracts of yellow lotus, bokbunja, clove, ume, quince, joiner, etc., the yellow lotus extract does not show antimicrobial activity against Candida albicans. By the way, the inventors confirmed that the extract of the barberry extract containing the protein isolated through a series of purification processes shows excellent anti-candida activity in vitro and in vivo and is also nontoxic. In particular, in vivo experiments showed superior anti-candida activity compared to the group that was previously known to have an anti-candida effect.

In one embodiment, the present invention relates to a method for preparing a sulfur extract comprising a protein having antifungal activity from Cooptidis rhizome.

In a preferred embodiment, the method comprises the step of extracting the yellow lotus powder with an organic solvent and filtering the organic solvent extract through a separator. The separator is preferably a separator having a pore size having a molecular weight cut-off of 3000 to 50000 Da, more preferably 10000 to 30000 Da.

Therefore, as a specific embodiment the present invention comprises the steps of (a) extracting the powder (Coptidis rhizome) with an organic solvent; And (b) filtering the rhubarb extract through a pore size membrane having a molecular weight cut-off of 3000 to 50000 Da, in a method of producing a rhubarb extract comprising a protein having antifungal activity. It is about.

Dialysis membrane, microfiltration membrane, ultrafiltration membrane or reverse osmosis membrane may be used as the separation membrane having the molecular weight cut-off, and more preferably, dialysis membrane or ultrafiltration membrane may be used.

In the extraction method of the present invention, the organic solvent may be used by itself or diluted with alcohols such as methanol, ethanol or butanol, hexane, ethyl acetate, chloroform, acetone and the like.

If necessary, the method may further include salting out the protein component by adding a salt for precipitating the protein to the organic extract of the yellow lotus before passing through the separator.

In another specific embodiment, the present invention comprises the steps of (a) extracting the sulfur powder in an organic solvent; And (b) relates to a method for producing a sulfur extract comprising a protein having antifungal activity, including the step (salting-out) of the protein component in the sulfur extract.

In this method, the salt that precipitates the protein can be used without limitation salts that can increase the ionic strength of the protein-containing aqueous solution to salt-out the protein, preferably the cation is NH ₄ ⁺ , K ^+, and is any one selected from the group consisting of Na ^+, this anion seat rate ^{_{(citrate), PO 4 3-,}} SO 4 2-, CH3COO -, Cl -, OH - and NO ₃ ^- in the group consisting of selected and any one of the coupling the positive and negative compound, and more preferably such as _{NH 4 OH, (NH 4)} 2 SO 4, KNO 3, NaNO 3, NaSO 4.

In still another aspect, the present invention relates to a rhubarb extract comprising a protein having antifungal activity prepared by the above method.

The rhubarb extract comprising the protein of the present invention is Candida albicans, Candida pseudotropicalis , Staphylococcus aureus, Bacillus pumilus, Staphylococcus schiuri ( Staphylococcus sciuri , Bacillus sphaericus , Pseudomonas aeruginosa , Propionibacterium acnes It can be used for the infection, etc., and particularly preferably used for the infection of Candida albicans.

Candida albicans is a genus of incomplete bacillus fungi and is a pathogen of candidiasis. It exists in the mouth or skin of the human body or animal, and it is harmless to the human body in normal condition, but when antibiotics are used for a long time or the human body has weakened resistance to immunity, it causes candidiasis by breeding abnormally in the body. In areas with high frequency of infection, itching and pain occur in the mucous membranes of the mouth and genital area. Therefore, the antifungal composition of the present invention can be usefully used for the treatment or prevention of atopic dermatitis, housewife eczema, diaper rash and the like caused by Candida albicans.

In still another aspect, the present invention relates to an antifungal composition containing as an active ingredient a rhubarb extract comprising a protein having antifungal activity prepared by the above method.

The composition of the present invention can be administered in a variety of dosage forms such as oral, parenteral, topical administration, oral administration, for example, oral solids with diluents (eg, lactose, dextrose, Sucrose, cellulose, corn starch, potato starch, etc.), suspending agents (e.g. silica, talc, stearic acid, magnesium or calcium stearate, polyethylene glycol, etc.), binders (e.g. starch, arabic gum, gelatin methylcellulose, carboxymethyl Cellulose, polyvinyl pyrrolidone, etc.), disintegrant (e.g., starch, alginic acid, alginate, sodium starch glycolate, etc.), formal mixtures, dyes, sweeteners, wetting agents (e.g., lecithin, polysorbates, lauryl sulfate Etc.), and other pharmacologically inactive substances generally used in medicine. When administered topically, excipients (e.g. lactose, starch, cellulose, lactose, polyethylene glycol, etc.), lubricants (e.g. magnesium stearate, stearic acid, glyceryl behenate, talc, etc.), preservatives (e.g. benzyl) Alconium chloride, etc.), and the like.

The composition of the present invention is a sugar coated tablet, film-coated tablet, enteric-coated tablet, peroral tablet, sublingual tablet, chewable tablet It can be formulated into various kinds of tablets, pill, hard or soft capsules, ointments, lotions, injections and the like.

In particular, when the composition of the present invention is used for the prevention of fungal infections, it may be formulated into cleaning agents such as creams, lotions, gels, water, foams, etc. using conventional techniques in the art.

The amount of the composition of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.01 g to 10 g per kg body weight is administered daily or every other day or divided several times a day, for example, once to three times. May be administered. The amount of use can be preferably adjusted by one of ordinary skill in the art. Since the dosage of the herbal composition may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage does not limit the scope of the present invention in any aspect.

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 Extraction and Separation of Laurel Protein

Example 1-1 Extraction of Laurel Protein

Rhubarb powder (100 g) was added to 80% methanol (500 ml) and extracted at room temperature for 3 days. The extracted solution was filtered through filter paper (Whatman, # 2, 110mm), and the filtrate was distilled under reduced pressure at 40 ° C. for about 60 minutes using a concentrator (Rotary vacumn evaporator: Eyela, N-1000, Japan) to remove methanol. It was.

Example 1-2 Separation of the Lactose Protein by Dialysis Membrane

The concentrated solution of Example 1-1 was placed in a dialysis membrane (Spectra / Por, MWCO 8000 Da, USA) and stirred in a cooled distilled water (2 L) at the lowest speed (0-10 rpm) in a 4 ° C. ice chamber. Dialysis at 5-6 days. The cooled distilled water was exchanged every 3 hours. The dialysate was classified into CRP-DD (precipitate) and CRP-DL (supernatant) and lyophilized to obtain a proteinaceous extract in powder form (FIG. 1).

Separately, the methanol extract concentrate was dissolved in distilled water, and then 60% ammonium sulfate (390 g / L) was added to form a precipitate, and centrifuged (5000 x g) to obtain pellets (CRP-DAs).

Example 1-3: Separation of Laurel Proteins by Ultrafiltration Membrane

The concentrate of Example 1-1 was subjected to a series of ultrafiltration membrane separations. First, the concentrate was separated into an ultrafiltration membrane (Millipore, MWCO = 30kDa, USA), and the remaining molecular weight of 30,000 or more in the membrane was dissolved in water, lyophilized and stored in powder form (CRP-U30), and the molecular weight passed through the membrane. Substances below 30,000 are separated into ultrafiltration membranes (Millipore, MWCO = 10kDa, USA). Substances with molecular weight of 10,000 or more remaining in the membrane are dissolved in water, lyophilized and stored in powder form (CRP-U10). Substances having a molecular weight of 10,000 or less were separated into an ultrafiltration membrane (Millipore, MWCO = 3kDa, USA), dissolved in water, and the substance having a molecular weight of 3,000 to 10,000 in the membrane was lyophilized and stored in powder form (CRP-U3).

Example 2 Identification of Proteins

Example 2-1 BCA Protein Essay

Identification of proteins isolated from L. was used for commercial BCA protein assay (BCA protein Assay, Pierce, USA). As a standard protein, BSA (Bovine serum albumin, Pierce, USA) was used to obtain a standard curve for comparative quantitation and quantification.

Protein assay was applied to each well in a 96-well plate using a microtiter method in a designated well, BCA reagent was added and allowed to stand at room temperature for 30 minutes. -Tek Instrument, USA). The standard curve quantified the protein content of the sample using BSA.

The extracts isolated in Example 1 (CRP-DD, CRP-DL, CRP-DAs, CRP-U30, CRP-U10, CRP-U3) all showed a positive reaction in protein qualitative reaction and the protein quantitative results are shown in Table 1 above. It was like Protein concentration was highest in CRP-DL, and ammonium sulfate treated CRP-DAs were twice as high as CRP-DD.

Example 2-2: electrophoresis

In Example 2-1, the presence of protein in the extract of L. was identified by qualitative and quantitative reaction, respectively, but the discontinuous SDS (Sodium dodecyl sulfate) PAGE method was used to visually confirm the presence of the protein. 10% or 20% -SDS PAGE purchased from Sigma was used and the concentration of protein extract was 10 μg / well, respectively. The band formed was weighed in comparison to the molecular weight marker (Sigma). Both CRP-DD and CRP-DAs were able to confirm the identity of the protein by the electrophoresis method. The molecular weight of this CRP was found to be less than 29 KDa when compared to the standard marker.

Example 3 In Vitro Anti-Candida Activity Investigation of Lactose Protein Extract

The anti-candida effect of the extract CRP isolated in Example 1 was investigated using Agar-diffusion susceptibility. Each CRP was dissolved in DPBS (Dulbecco's phosphate Buffer Solution; Sigma), filtered and sterilized, and serially diluted to make wells in solid media (Mycobiotic agar; Difco, USA) inoculated with Candida albicans, and then each 100ul was put in 37 ° C. The growth was confirmed by the growth of Candida albicans around the well. Candida albicans for inoculation was used for 24 hours culture in GYEP (Glucose, Yeast Extract, Pepton) liquid medium.

Each CRP was found to have a growth inhibitory effect on Candida albicans (Table 2). The best effect was confirmed with CRP-DD and CRP-DAs. Both CRP-DD and CRP-DL showed antifungal activity, and CRP-DD was more effective.

Example 4 In Vivo Anti-Candida Activity of the Lactobacillus Protein Extract

Candida albicans (5 × 10 ⁵ cells / mouse) was inoculated into BALB / c mice (Charles River Lab, USA) by tail vein and first infected, followed by intraperitoneal administration of CRP 1mg / mouse 3 hours later. The time was confirmed.

Control mice received DBPS (Dulbecco's Phosphate Buffer Solution; Sigma) instead of CRP. CRP was dissolved in DPBS and used after filtration-sterilization. Animal experiments were performed according to (Han and Cutler. 1995. Infect. Immun. 63, 2714-2719; Han et al., 2003. In: Candida and candidosis. ASM Press, Washington, D. C. pp 243-256).

To compare the therapeutic effects on Candida albicans-infected mice, fungi Fluconazole (azo antifungal agent) and DPBS were used as a control, and another substance extracted from rhubarb was Burberry.

In the 25-day observation, only two mice in the Fluconazole-administered group died, whereas all mice in the DPBS-administered group died in 11 days, while the Burberry-administered rats showed little difference from the mice in the DPBS group. The lethal velocity was shown. Husband protein extract-rats in the administration group died of two on the 12th day, one died on the 11th day, one on the 13th day, one on the 17th day (Fig. 3). The LSF protein-administered group was less effective than the Fluconazole-administered group, but was superior to the DPBS-administered group and the Burberry-administered group.

Example 5 Toxicity Test of Rhubarb Protein Extract

BALB / c mice were treated with 5 mg of CRP intraperitoneally to extracts of nasturtium protein and compared with the survival rate of the control group receiving only DPBS.

BALB / c mice receiving CRP did not die as 30 days as DPBS group. From these results, it was confirmed that rhubarb protein extract had no side effects in vivo.

The extract of Huangshan containing the protein having antifungal activity according to the preparation method of the present invention is obtained from a natural substance, Huangshan, and has excellent anticandida activity without toxicity, and thus can be usefully used for the prevention or treatment of fungal infections including Candida albicans. have.

All simple modifications or changes of the present invention can be easily implemented by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Claims (11)

(a) extracting Coptidis rhizome powder with a solvent selected from the group consisting of methanol, ethanol and butanol; And (b) filtering the rhubarb extract through a pore size separator having a molecular weight cut-off of 3000 to 50000 Da, thereby producing a rhubarb extract comprising a protein having antifungal activity. . The method of claim 1 wherein a pore size membrane is used having a molecular weight cut-off of 10000 to 30000 Da. The method of claim 1, wherein the separation membrane is a dialysis membrane, a microfiltration membrane, an ultrafiltration membrane, or a reverse osmosis membrane. The method of claim 3, wherein the separation membrane is a dialysis membrane or an ultrafiltration membrane. delete delete The method of claim 1, further comprising salting out the protein component by adding a salt that precipitates the protein to the sulfur extract of step (a). Prepared by extracting the powder of Colicidis rhizome with a solvent selected from the group consisting of methanol, ethanol and butanol and filtering the coarse extract through a pore size membrane having a molecular weight cut-off of 3000 to 50000 Da. Rhubarb extract comprising a protein having antifungal activity. Coconutis rhizome powder is extracted with a solvent selected from the group consisting of methanol, ethanol and butanol, and the extract is filtered through a membrane of pore size having a molecular weight cut-off of 3000 to 50000 Da. An antifungal composition comprising a rhubarb extract comprising a protein having an antifungal activity as an active ingredient. The antifungal composition of claim 9 for Candida albicans. The composition of claim 9 for treating or preventing atopic dermatitis, housewife eczema or diaper rash caused by Candida albicans.

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