KR100700911B1 - Acidic polysaccharide extracted from ginseng root and its composition for preventing human cellular interaction of Propionibacterium acnes and Staphylococcus aureus - Google Patents

Abstract

A composition comprising an acidic polysaccharide is provided to significantly prevent Propionibacterium acnes and Staphylococcus aureus from being cellular adhered to human skin without any inhibition effect on other skin flora, thereby being usefully used for preventing and treating acne and atopic skin diseases. The composition for preventing and treating acne and atopic skin diseases comprises an acidic polysaccharide which is prepared by hot-water extracting crushed root of Panax ginseng, precipitating the extract in ethanol and performing an ion-exchange chromatography thereon with NaCl concentration of 0.2-0.4M and is isolated by gel-permeation chromatography having a molecular weight of 10-15 kDa. In the composition, the acidic polysaccharide contains 20-35 wt.% of uronic acid.

Description

Acidic polysaccharide extracted from ginseng root and its composition for preventing human cellular interaction of Propionibacterium acnes and Staphylococcus aureus}

Figure 1 is a schematic diagram showing the separation and purification of the present invention acid polysaccharide from ginseng root.

Figure 2 is a graph showing the ion exchange, gel filtration chromatography effluent profile of the present acid polysaccharide.

Figure 3 shows the inhibitory activity of the acidic polysaccharide of the present invention. Hemagglutination caused by acne bacillus ( P. acnes ) and Staphylococcus aureus ( S. aureus ) is shown to inhibit the activity according to the concentration.

Figure 4 shows that the acidic polysaccharide of the present invention has no inhibitory activity with the aggregation reaction by the skin flora Staphylococcus epidermidis, Escherichia coli , Lactobacillus acidophilus ( Lactobacillus acidophilus ) By giving it specificity to acne bacilli ( P. acnes ) and Staphylococcus aureus ( S. aureus ).

The present invention is an acidic polysaccharide extracted from ginseng roots inhibiting human epithelial cell binding of acne bacilli ( Propionibacterium acnes ) and Staphylococcus aureus , which are recognized as a causative agent of acne (image acne) and atopic dermatitis and acne containing them And it relates to a composition for preventing and treating atopic skin diseases.

Acne (acne vulgaris, pimples) is one of the most common skin diseases from adolescence to middle age. Medically, acne is an inflammatory lesion of the hair follicles in the skin and the sebaceous gland, the oil glands of the skin. Internal causes of acne include sebaceous glands secreted by endocrine effects such as hormonal changes depending on mental and physical stress, abnormalities of their components, and blockage of sebaceous glands or hair follicles due to excessive sebum secretion. Cortisol caused by stress and a hormone called progesterone during women's menstrual periods are also closely linked (4). External causes include increased sebum and stimulating factors caused by propionibacterium acnes living in sebaceous glands, which stimulate the hair follicle epithelial wall, resulting in thickening of keratin in the epithelium and obstruction of abnormal hair follicles at this time. Occurs and decreases oxygen inflow into hair follicles. This phenomenon is a very good development condition in anaerobic P. acnes , the causative agent of acne, and promotes the growth in acne lesions (5). In general, healthy skin contains bacteria such as acne bacilli and staphylococci. Some humans have hundreds to hundreds of thousands of acne bacillus in 1cm ² of skin, and even more than the germ of the Demodex worms (a type of tick) is parasitic. Acne caused by Demodex insects are stronger than the acne caused by acne bacilli, and the inflammation is much stronger.

Atopic dermatitis is one of the most common skin diseases, a chronic inflammatory skin disease that occurs in the folds of the face or limbs of people with allergies or family history (6). Lesions of the skin vary depending on the age of the patient and the degree of acuteness of the disease. External factors of atopy include chemicals such as fragrances, preservatives, disinfectants, and solvents, as well as indoor dust and mite, mold, food, pollen, and dogs. In some cases, the intolerance of wool-like woolen fabrics can be a stigma. Inhaled antigens penetrate the damaged skin barrier of atopic dermatitis and cause contact dermatitis. The main cause of house dust is Dermatophagoides pteronyssinus , and the role of food allergy is still controversial, but it is generally known to affect children more than adults. Atopic dermatitis often coexists with other allergic diseases. External factors include increased serum IgE levels, particularly immune deficiency that is susceptible to bacteria, viruses, and fungi, and physiological changes in sympathetic and parasympathetic responses (11). Especially important is that 90% of atopy patients have Staphylococcus aureus accumulated in the skin and breed, and many studies show that staphylococcal infection of epithelial cells of atopy patients. However, the mechanism of infection and reproduction of staphylococci in epithelial cells is not clear for these atopic patients. The prevalence of atopic dermatitis is high in children with atopic dermatitis, suggesting a genetic predisposition, and the incidence of atopic dermatitis increases with increasing lactation period from mothers with atopic dermatitis. 12).

In general, acne is sebaceous glands present in the dermal layer of the skin is increased, sebum secretion increases, pores become clogged, or inflammation caused by acne-causing bacteria P. acnes , purulent bacteria S. aureus or folliculitis. In puberty, acne begins as the sebaceous gland matures due to hormonal stimulation of androgens, but after adulthood, sebum is not discharged by makeup, or the corticosteroids increase due to stress, lack of sleep, etc. Sometimes. In addition, secondary lesions occur when external stimulating factors such as ultraviolet rays, chemicals, climate change such as heat, dryness, humidity, and yellow breeze are given, or when life becomes irregular due to poor internal organ function. The lipase enzyme produced by P. acnes hydrolyzes sebum into triglycerides and free fatty acids, and free fatty acids appear to have comedogenic effects on hair follicles. The number of bacteria of P. acnes is in order of micro comedo, closed comedo, and opencomedo, and most of bacteria in P. acnes are P. acnes . In particular, P. acnes is involved in the formation of papules in the early stages of acne. Therefore, acne treatment is used antibiotics such as clindamycin, erythromycin, tetracycline to inhibit the growth of bacterial bacteria, benzoyl peroxide to remove the skin, or vitamin A agent compounds that inhibit sebum production.

There are many studies on the causes and pathophysiology of atopic dermatitis, but the exact mechanism of action has not been identified. Common factors that worsen atopic dermatitis are high temperature, humidity, heavy exercise, chemical fiber clothing, highly irritating soaps, chemical irritation, stress, mites, dust and the like. Eliminating aggravating factors can help reduce symptoms of atopic dermatitis. In patients with atopic dermatitis, an increase in the amount of serum antibody IgE, inhibition of the cellular immune system, an increase in the amount of neutrophil in the blood, and an increase in the production of interleukin-4 (IL-4) are commonly observed (8, 12). IL-4 stimulates IgE biosynthesis, interferon-gamma (INF-γ) mediates delayed hypersensitivity reactions, and other interleukin (IL-3, IL-13) are involved in the mechanism of atopic dermatitis. It is known to be (6). Patients with atopic dermatitis have a relatively small amount of INF-γ produced by monocytes in peripheral blood for stimulation compared to normal individuals. Indeed, several studies have shown that INF-γ has an effect on severe atopic dermatitis that is ineffective with conventional therapies (11). Exposure to external allergens increases the production of allergen-specific IgE, which binds to mast cells and causes the release of mast cells' inflammatory material. In this case, the released histamine directly stimulates the receptor causing the itch, and neuropeptide, protease, and cytokine cause itching by indirect action (10). In particular, bacterial gene nucleic acid material (CpG) and cell membrane material (LPS) such as Staphylococcus aureus are known to cause allergies. The skin of patients with atopic dermatitis has a higher frequency of detection of staphylococci in both affected and non-infected areas than normal skin. Staphylococcus aureus isolates from patients with atopic dermatitis are significantly higher than those found in normal humans, including the antibiotic-methicillin and strains of fusidic acid. Staphylococcus aureus is also a major cause of exacerbations during the acute exacerbation of atopy.

Systemic therapies for treating atopic dermatitis include antihistamines, antibiotics, and corticosteroids. Antihistamines are often tachyphylaxis in long-term use, and oral corticosteroids are rarely used except in the absence of external preparations. The treatment of secondary infections caused by atopic dermatitis is combined with oral treatment rather than external antibiotics alone. Use dicloxacillin, erythromycin, cephalexin to treat staphylococci (8, 11).

As described above, antibiotics are mainly used for the purpose of treating and protecting skin disease pathogens, especially acne and atopic dermatitis. However, many pathogens are resistant to antibiotics due to antibiotic misuse and abuse, and the lifetime of antibiotics is also shortened. Along with the emergence of antibiotic resistant bacteria, an important point is the fact that the balance of skin microorganisms when antibiotics are administered is compromised. Because gram-negative bacteria do not survive competition with gram-positive bacteria that adapt well to dry conditions such as skin, removing gram-positive bacteria from the skin with antibiotics begins to increase the pathogenic gram-negative bacteria. Therefore, efforts to replace antibiotics are needed, and resistant bacteria caused by the abuse of antibiotics for acne and atopy treatment used in dermatology in Korea may be a threat to the health of children and adolescents in the future. Recently, non-antibiotic therapies such as probiotics, phages and phytomedicines have been developed for the treatment and prevention of pathogens. In particular, phytochemicals extracted from plants are expected to replace antibiotics in the future.

Accordingly, the present inventors recognize the problem of inducing the resistant bacteria and destroying the beneficial bacteria of the antibiotics used in conventional skin diseases, and have a significant inhibitory effect on the adhesion of acne bacilli and staphylococci to human skin cells, and have an inhibitory effect on other dermatological bacteria. During the search for a natural bioactive substance with no specific effects, carbohydrate fractions were separated from Korean ginseng bioactive substances, and in particular, the physiological activity of the acidic polysaccharide sugar copolymer fraction was measured at a level below millimolar. It has a strong inhibitory effect on the erythrocyte agglutination reaction by P. acnes and Staphylococcus aureus , and it does not show any inhibitory effect on human beneficial bacteria, which has proved to be a high potential as a preventive and treatment agent for acne and atopic skin diseases. The present invention has been completed.

Therefore, the main object of the present invention is to provide a composition containing an acidic polysaccharide having a specific effect that has a significant inhibition on acne bacilli, Staphylococcus aureus adhesion to human skin cells, but does not inhibit other dermatological bacteria.

Another object of the present invention is to provide a composition useful for preventing and treating acne and atopic skin diseases containing the acidic polysaccharide of the present invention.

According to one aspect of the present invention, the present invention provides a cell-binding inhibitory composition of acne bacilli and atopy staphylococcus aureus containing acidic polysaccharides obtained by pulverizing ginseng roots and extracting hot water and then ethanol precipitation as an active ingredient.

In the present invention, preferably, the acidic polysaccharide is a fraction separated by ion exchange chromatography and gel filtration chromatography after dissolving and dialysis the ethanol precipitate in a phosphate buffer solution.

In the present invention, preferably, the fraction is separated at a sodium chloride concentration of 0.2-0.4 M in ion exchange chromatography, and separated at a molecular weight of 10-15 kDa size (Mw) of gel filtration chromatography. The fractions in the numerical range contained 20-35% by weight of uronic acid and showed the highest cell binding inhibitory activity of the present invention.

According to another aspect of the present invention, the present invention provides a composition for preventing and treating acne, atopic skin disease, containing the acidic polysaccharide of the present invention as an active ingredient.

The acidic polysaccharide of the present invention is understood to be effective in the prevention and treatment of various skin diseases such as acne, atopic dermatitis caused by human epithelial cell binding of acne bacilli or atopic staphylococcus aureus due to human cell cell binding inhibitory activity. Since the carbohydrate composition of human skin cells and carbohydrates of the red blood cells are the same or similar, the examples of the present invention tested the effects of the acidic polysaccharides of the present invention on the inhibition of hemagglutination induced by P. acnes and S. aureus . .

The acidic polysaccharide of the present invention may be prepared by a method known in the pharmaceutical field for use as the composition for preventing and treating such powders, powders, mixed with itself or pharmaceutically acceptable carriers, excipients, diluents, etc. It may be prepared and used in the form of granules, tablets, capsules, or injections. They can also be administered orally or parenterally.

The dosage for administering the acidic polysaccharide according to the present invention as an active ingredient may be appropriately selected according to the age, sex, condition, and symptoms of the disease of the patient, and preferably 100-500 mg of the acidic polysaccharide extract per day for adult and its It can be administered as a powder.

Hereinafter, the present invention will be described in more detail.

The present invention is performed by ion exchange chromatography and gel filtration chromatography in acidic polysaccharides inhibiting erythrocyte agglutination caused by P. acnes and Staphylococcus aureus and water-soluble extracts of Korean ginseng. It consists of a manufacturing method to separate. First, in order to separate the sugar mixture, extract with hot distilled water according to the general intake and extraction method of Korean ginseng. This is in contrast to the conventional method of extracting natural bioactive substances using organic solvents, which are difficult to apply mainly to cosmetics and medicines. 2) The isolated acidic polysaccharide has a high content of uronic acid and is very specific for inhibiting human skin cell recognition of P. acnes and S. aureus . Therefore, the technical problem to be achieved by the present invention is the step of extracting the ginseng root portion with hot distilled water, separating the bioactive acid polysaccharides by ion exchange and gel filtration chromatography using the latest carbohydrate engineering technology, and P. It consists of activity measurement step that inhibits erythrocyte agglutination by acnes, S. aureus, S. epidermidis, E. coli and L. acidophilus .

Existing ginseng bioactive substance extraction relies mainly on organic solvent extraction method, which is contrary to what we consume by boiling ginseng in water. In the present invention, the water-soluble fraction extracted by hot distilled water (60-100 ^o C) can be said to be a more practical approach to the search for ginseng bioactive substances. Euronic acid has a characteristic that the C ₆ alcohol carbon of the neutral monosaccharide is substituted with a carboxyl group and has a negative charge at neutral pH. In particular, the separated acidic polysaccharide fraction reached 20-30% of uronic acid content, and the highest content of uronic acid was higher than other polysaccharide fractions separated and extracted under given conditions. The carbohydrate composition of acidic polysaccharides extracted and purified from ginseng roots shows that the composition of gluturonic acid is close to 20% of the total sugar. In addition, the minimum effective amount of acidic polysaccharides inhibiting the hemagglutination by P. acnes and S. aureus was 0.5 mg / ml at low concentrations based on high molecular weight. Moreover, the application of carbohydrates specific for inhibiting cellular recognition of skin diseases is an environmentally friendly and novel approach, allowing the development and design of industrially effective sugar copolymers by studying the properties of acidic polysaccharides at the molecular level.

P. acnes Gram-positive bacteria do not form spores. They are rod-shaped anaerobic bacteria and are called acne bacilli (13). It is morphologically very similar to Corynebacterium and is generally non-toxigenic. Anaerobic but limited aerobic (microaerobic) proliferation is possible, it is a typical skin flora of the skin and hair follicles that cause acne.

Staphylococcus aureus ( Sphylococcus aureus ) is involved in purulent skin diseases, causes purulent wounds on the skin, and causes boils and boils on the surface of the skin. In particular, S. aureus is not only a primary cause of atopic dermatitis, but also a secondary cause of infections such as skin pores, clogged glands, or invasion (7). Within 10% of normal people, staphylococcus aureus is found in the skin in 90% of atopic patients. In addition, when atopic dermatitis develops, staphylococcal infections merge.

In general, the skin flora, which plays a role of mixing sweat and sebum, is beneficial to the skin as a beneficial flora, while too much harmful flora, such as acne bacilli and staphylococci, causes the balance of the skin to collapse.

Carbohydrates, such as glycoproteins or glycolipids on the cell surface, have the function of ligands that recognize proteins, providing a place for attachment of external pathogens, viruses, toxins, and hormones, in addition to cell-to-cell interactions (21). When a pathogen penetrates human cells and proliferates, it causes inflammation and causes a disease, and when the pathogens initially block the attachment of human cells to a pathogen, it plays a role of disease prevention. If developed as an anti-adhesive drug using human cell surface carbohydrates, which play an important role in the initial process of human pathogen infection, it can block the initial infection of the pathogen. Therefore, anti-adhesion treatment and prevention are developed as bioenvironmental therapies that maintain the skin's microbial balance by selectively reducing contact between human tissue and pathogens by eliminating or preventing the attachment of infectious agents and selectively allowing the attachment of beneficial bacteria. It is possible.

P. acnes occurs mainly in the sebaceous glands of skin follicles and survives. According to genome sequence studies, surface-associated proteins and 4-7 proline-threonine repetitive protein (PTRP) proteins are involved in the interaction with human cells. Reported (3). Recently, P. acnes produced a hyaluronate lyase enzyme to break down hyaluronan polysaccharides, a major constituent of the extracellular matrix of skin connective tissue, and this action has been reported to aid the adhesion of bacteria to skin cells (22). In particular, PTRP protein has been found to play an important role in human cell interaction as a cell surface protein of P. acnes (3). Thus, the study of the interaction and binding between carbohydrates of human cells and the bacterial surface proteins that recognize them will contribute to understanding the mechanism of adhesion of these cells to human skin cells. Bacterial cells generally respond to hemagglutination, a reaction that occurs when protein-adhesive receptors (adhesin) on the surface of bacteria recognize carbohydrates or proteins on the surface of human cells such as red blood cells. Especially for P. acnes and S. aureus , the supply of ham and hemoglobin is essential for cell growth for iron and other important nutrients. Therefore, hemocytic recognition is a mechanism of action necessary for cell growth for parasitic in vivo by P. acnes and S. aureus , thereby securing ham supply (23-25). Hemagglutination, which has been used for cell-to-cell recognition, has been widely applied to cell-to-cell recognition because of the property that the surface of red blood cells has the same or similar carbohydrate composition as human epithelial cells. Therefore, it is possible to study the interaction between bacterial surface proteins and human cells through the hemagglutination of P. acnes and S. aureus . The important point is that the carbohydrates of the skin cells and carbohydrates of the red blood cells are the same or similar, so that inhibiting the binding by interaction can be used for the treatment and prevention of skin diseases caused by P. acnes and S. aureus , especially acne and atopy. will be.

Therefore, it is important to develop a sugar compound using carbohydrates recognized by bacterial surface protein receptors (adhesin). When antibiotics or chemicals developed for the treatment of skin diseases are very limited, the emergence of resistant bacteria due to antibiotic abuse and the harmonious balance of beneficial / unproductive bacteria in the human body are destroyed. to be. The use of existing antibiotics or chemicals not only adversely affects the development but also cell destruction, while new substances that inhibit carbohydrate recognition and interaction on the surface of human cells inhibit the intercellular binding and block the symbiosis of skin disease cells. It is also important in that it is a non-destructive technique for minimizing skin diseases such as acne. Therefore, the development of carbohydrates that inhibit the recognition or adhesion of human cells will create new bio-environmental materials in the 21st century. A representative example of this non-destructive technology is breast milk or saliva. That is, since breast milk or saliva contains physiological glycopolymers, it is essentially performing a role of clearing in the digestive tract by inhibiting the attachment of ineffective or pathogenic bacteria in the human body. The present invention is a technology that detects and isolates biologically active substances from natural resources, which inhibits the adhesion, proliferation and activity of P. acnes and S. aureus , but does not selectively inhibit fungi and beneficial bacteria, especially in the field of biotechnology. It is a technology that uses newly recognized carbohydrate engineering research methods.

Conventionally, biological activity has been measured in crude extract fractions containing polysaccharides or carbohydrates from Korean ginseng. Thus, while molecular studies of which specific components in fractions have activity have hit technological limits, the use of carbohydrate engineering techniques that are currently being developed allows for the separation, purification, identification and structural analysis of polysaccharides. Development into value-added products is becoming possible. P. acnes and S. aureus bind to red blood cells to induce agglutination reaction. Among the water-soluble extracts of Korean ginseng, the acidic polysaccharide fraction separated by ion exchange resin and gel filtration resin is not only acne bacilli but also Staphylococcus aureus. It inhibits hemagglutination by S. aureus and has a relatively high inhibitory effect compared to other polysaccharide fractions. Ginseng has been used for a long time as a herbal medicine and food, acid polysaccharides extracted from the ginseng root of the present invention was also found to be nontoxic through animal experiments.

Q Sepharose Fast Flow ion exchange resins used in the present invention were purchased from Amersham Phamacia Biotech, Sweden.

Enzymes pectinase, trypsin, glucuronic acid, galacturonic acid, rhamnose, arabinose, xylose used in the present invention Glucose and galactose were purchased from Sigma, USA.

Type O erythrocytes required for erythrocyte aggregation reaction used in the present invention were secured in Korea University Medical School, and used after treatment with anticoagulant and trypsin enzyme. P. acnes (ATCC 11828) used in the present invention was incubated for 2 days or more under anaerobic conditions in brain-heart infusion medium, and S. aureus and S. epidermidis bacteria were distributed at Korea University Medical Center hospital. Liquid culture was performed under miracle conditions. All cells were centrifuged at 6,000 g for 30 minutes and stored in 170 ^o C liquid nitrogen for experiments.

Escherichia coli (BL21 (DE3), BL21 (DE3) / pLysS) used in the present invention was incubated for 6 hours at 37 ^o C in Luria-Bertani (LB) agar medium without antibiotic treatment, and the cultured E. coli was -70 after centrifugation. ^o L. acidophilus was stored in C and used for the experiment.

In the present invention, the total amount of carbohydrate, uronic acid and protein is phenol-sulfuric acid. Methods, carbazole, and Lowry methods were determined using glucose, galacturonic acid, and bovine serum albumin, respectively, as standard (26, 27).

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Isolation of Acidic Polysaccharides from Ginseng Roots

The method for preparing acidic polysaccharides is obtained by extracting and homogenizing the roots of Korean ginseng 6 years old with 50 mM phosphate buffer (pH 7.2) or water at 80 ° C for 3 hours, and then centrifuging to remove the precipitates. Precipitated with four volumes of ethanol. After drying the ethanol precipitate, it was dissolved in a minimum volume of 10 mM phosphate buffer solution (pH 7.5, 10 mM MgCl ₂ , 1 mM CaCl ₂ ) and treated with Rnase and Dnase at 37 ^° C. for 2 hours to stop the enzyme activity. After centrifugation of the enzyme-treated reaction solution at 20,000 g for more than 30 minutes, the supernatant was collected, and about 4 times the volume of cooled ethanol was added to the supernatant, and the resultant was processed to the final 80% of ethanol concentration, followed by centrifugation at 20,000 g for 30 minutes. The precipitate obtained by separating was used as a crude extract. Ginseng polysaccharide crude extract was dialyzed in water and separated by ion exchange chromatography. The acidic polysaccharide used a concentration gradient of 0-1 M sodium chloride solution, and the effluent fractions were collected and quantified for total sugars and acidic sugars, respectively, and the fractions having inhibitory activity against red blood cell aggregation against P. acnes and S. aureus It was confirmed that outflow was separated at 0.3 M. The separated polysaccharides were lyophilized after dialysis and then separated by polysaccharide size using sephacryl S200 gel filtration chromatography to obtain the largest and most active fractions (FIG. 2). Figure 1 shows the effluent profile of acidic polysaccharides derived from Korean ginseng root using Q sepharose ion exchange and Sephacryl S200 gel filtration chromatography. (A) Q ion exchange chromatography. Blue line shows UV 226nm, red line shows salt (NaCl) concentration change. Orange line shows the total equivalent profile of each fraction. (B) Gel filtration chromatography with Sephacryl S-200 FPLC. Blue line shows UV 226nm, orange line shows total equivalent weight, purple line shows acid equivalent weight profile. 1 shows the overall separation and purification process of the ginseng root sample separated by gel filtration chromatography. No protein was detected in the isolated acidic polysaccharide fraction. In addition, it was confirmed that the non-binding fraction mainly consists of neutral polysaccharides, and the binding fraction was an acidic polysaccharide, particularly an active fraction. The active single peak purified by gel filtration chromatography was found to be 10-15 kDa in size.

Example 2: Monosaccharide Carbohydrate Analysis of Acidic Polysaccharides

A) Hydrolysis: The samples were dissolved in 3M trifluoroacetic acid (trifluoroacetic acid) and hydrolyzed at 120 ^o C for 2 hours in a sealed screw cap reacti tube. After hydrolysis, the mixture was cooled to about 50 ^° C., and acetic acid was removed by trichloro by a concentration and drying method, and 0.5 ml of methanol was added for complete removal, followed by final concentration and drying.

B) Reduction: 1 M ammonia water containing 10 mg of sodium borohydride (NaBH ₄ ) was added to the sample and the standard sample, and reacted at room temperature for 1 hour. Glacial acetic acid was added to remove the sodium borohydride and boric acid remaining after the reduction reaction, and concentrated and dried while continuing to add methanol solution containing 10% acetic acid.

C) Separation of acidic sugars and neutral sugars: Acidic sugars and neutral sugars in the monosaccharides of the reduced sample were separated by using an ion exchange column. In other words, the reduced sample was dissolved in distilled water, and the non-binding fraction was neutral sugar through the ion exchange column, and the fraction bound and released by 1 N HCl was used as the acid sugar. Neutral and acidic sugar samples were removed by concentrating water and hydrochloric acid and drying under reduced pressure, respectively. The dried acidic sugar sample was once again reduced, concentrated and dried.

D) Acetylation Reaction: The reduced monosaccharide was sealed by adding equal amounts of pyridine and acetic anhydride, and then acetylated at about 95 ^° C. for one hour. At room temperature, the sample was concentrated to dryness, and toluene solution was added to completely evaporate and concentrate so that no pyridine remained in the sample. After acetylation, the solvent was completely removed and the remaining residue was dissolved in ethyl acetate / water, vortexed, and then centrifuged at 1,000 g to separate the organic solvent layer and the aqueous layer. Of these, an organic solvent layer was taken, and concentrated in vacuo again to be used as a sample for GC analysis.

E) Analysis by Gas Chromatography: Samples were measured by gas chromatography. Samples were separated using a DB-225 fused silica capillary column, and monosaccharide derivatives were measured using a flame-ionization detector. Oven temperature was analyzed at 220 ^° C. The monosaccharides of the sample were identified by comparing the retention time with the standard sample, which was pretreated, and the mol% of the monosaccharides was determined from the peak area and the molecular weight (Mw).

Mass spectra measurement: Identification of monosaccharides was confirmed by gas chromatography / mass spectra.

The isolated acidic polysaccharide fractions not only had a high content of uronic acid but also a very high content of arabinose and galactose compared to other sugar copolymer fractions (Table 1). In contrast, the neutral polysaccharide fraction obtained from the non-binding fraction was found to have a relatively low content of uronic acid, and the composition ratio of monosaccharide was mostly glucose. In particular, it was found that the content of uronic acid, which is represented by galacturonic acid and gluguronic acid, was relatively higher than that of other polysaccharide fractions. Among them, the content of gluguronic acid was about 18% as a specific sugar. Acidic polysaccharides purified from ginseng roots have already been shown to have high cell adhesion inhibitory activity against Helicobacter pylori gastrointestinal pathogens and Porphyromonas gingivalis oral disease (28, 29). Inhibitory activity of pathogen cell adhesion to ginseng root acidic polysaccharide has not been reported.

TABLE 1 Carbohydrate Composition of Acidic Polysaccharide Final Fraction F2 Isolated from Ginseng Root

Arabinose Xylose Rhamnose Mannose Galactose Glucose Galacturonic acid Glucuronic acid Polysaccharide Final Fraction 28.0 3.2 4.7 2.6 26.3 7.9 7.9 18.4

  NOTE Each value is in relative mole percent (mol%).

Example 3: inhibition of hemagglutination by acne bacilli and Staphylococcus aureus

The cultured P. acnes and S. aureus were diluted with phosphate buffer solution, mixed with the same amount of red blood cell solution and reacted with light stirring at room temperature for 30 minutes to 3 hours, or overnight. It was. In order to inhibit the coagulation reaction using the separated polysaccharide fractions, P. acnes , S. aureus solution and sugar mixture solution were respectively mixed, reacted at room temperature, and then reacted by mixing the red blood cell solution again. Was measured using a microscope.

As a result of activity inhibition of hemagglutination by P. acnes and S. aureus , the final fractions of acidic polysaccharides among the fractions purified from ginseng root showed inhibitory activity at low concentrations of 0.5 mg / ml respectively. In general, specific monosaccharides such as sialic acid (N-acetylneuraminic acid), glucuronic acid (glucuronic acid) is showing a cell adhesion inhibitory activity at a concentration of about 5 mg / ml or more, that is, 25 mM or more. On the contrary, it can be seen that the acidic polysaccharide of the present invention has a relatively high activity (inhibitory activity at a level of about 25 μM based on a molecular weight of about 10 kDa) (FIG. 3). Figure 3 is a micrograph (100-fold magnification) of the hemagglutination inhibitory activity by acne bacilli (A) and Staphylococcus aureus (B). PG F2, an acidic polysaccharide fraction of about 12kDa, isolated and purified from Korean ginseng root, showed inhibitory activity against acne bacilli and Staphylococcus aureus at concentrations above 0.5 mg / ml. Negative coagulation (NC; results only with red blood cells) and positive coagulation (PC; results with only red blood cells and acne bacilli or Staphylococcus aureus).

Example 4: S. epidermidis Escherichia coli, Lactobacillus  Inhibition of hemagglutination by bacteria

Cultured dermal bacteria S. epidermidis , Escherichia coli, and Lactobacillus were diluted with a buffer solution, mixed with an equal amount of red blood cell solution, and then reacted with a slight stirring at room temperature for 30 minutes to 3 hours, or overnight. Reaction. In order to inhibit the coagulation reaction using the isolated polysaccharide fractions, the fungal solution and the sugar mixture solution were mixed and reacted at room temperature, and then the red blood cell solution was mixed and reacted, and then the coagulation reaction was measured using a microscope. . S. epidermidis , a skin flora, showed no inhibitory activity of erythrocyte aggregation at high concentrations of 2.0 mg / ml of the final fraction of acidic polysaccharides purified from ginseng roots (FIG. 4). L. acidophilus also showed no inhibitory activity at a concentration of 0.5 mg / ml of the polysaccharide fraction (FIG. 4). In the case of E. coli, the activity of inhibiting the aggregation of red blood cells, the final fraction of acidic polysaccharides purified from ginseng root did not show inhibitory activity even when using a high concentration of 2.0 mg / ml (Fig. 4). Figure 4 is a micrograph (100-fold magnification) of the hemagglutination inhibitory activity by Lactobacillus (LA), Escherichia coli (EC), S. epidermidis (SE). No inhibitory activity from acidic polysaccharide fraction PG F2 (0.5-2.0 mg / ml) isolated and purified from Korean ginseng root. Negative coagulation reaction (NC; results when only red blood cells are the same as in Figure 3.) and positive coagulation reaction (PC; results when the red blood cells and bacteria together). Therefore, the acidic polysaccharide of the present invention shows a high inhibitory effect on the hemagglutination reaction induced by P. acnes and S. aureus bacteria, while no inhibition on hemagglutination by S. epidermidis , Escherichia coli, Lactobacillus bacteria, etc. By showing no effect, it was found that P. acnes and S. aureus bacteria are specific to human cell adhesion. As described above, the acidic polysaccharide of the present invention is determined to be a specific polysaccharide having a high content of gluguronic acid. There are no reports of purified polysaccharides that inhibit hemagglutination of P. acnes and S. aureus bacteria. Therefore, we have purified acidic polysaccharides with high content of specific uronic acid sugars from the native native plant of Korean ginseng, and using the purified acidic polysaccharides to inhibit human cell binding by the causative agents of skin diseases caused by P. acnes and S. aureus. The present invention has been achieved by confirming the activity.

As described above, according to the present invention, 1) an acidic polysaccharide fraction which inhibits the aggregation reaction between P. acnes and S. aureus bacteria, which cause the development of skin diseases, and human erythrocytes, and 2) ion exchange in water-soluble extracts of Korean ginseng An invention has been made for a process for separating acidic polysaccharide fractions by gel filtration chromatography. Through the present invention, it is expected that the development effect of the leading material for preventing and treating acne and atopic skin diseases will be obtained. In particular, in the case of conventional antibiotics or chemical drugs, the developmental technology as well as the side effects are cell-destructive, while new substances that inhibit carbohydrate recognition and interaction on the surface of human cells inhibit the cell-cell adhesion and binding, thereby minimizing skin diseases. In terms of destructive technology, it has the meaning of future-oriented cosmetics and medicines. In particular, in the cosmetics industry, where skin balance is important, the shift to a new concept of microbial balance in the skin expands wider marketability by using eco-friendly excellence of sugar blends to prevent or treat infections caused by bacterial invasion in the future. Is considered possible. Finally, acidic polysaccharides isolated from Korean ginseng, the foremost natural resource, are non-toxic as natural materials and have the effect of minimizing side effects. Since there is an excellent effect of the inhibitory activity according to the relative concentration of natural new substances, it is a very excellent invention in the cosmetic and pharmaceutical industries.

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Claims (5)

Pulverized ginseng roots were extracted with hot water, ethanol precipitated, and acidic polysaccharides isolated at 0.2-0.4 M sodium chloride in ion exchange chromatography and separated at molecular weight 10-15 kDa size (Mw) in gel filtration chromatography. A composition for preventing and treating acne and atopic skin diseases, which are contained as ingredients. delete delete The composition of claim 1, wherein the acidic polysaccharide contains 20-35% by weight of uronic acid. The composition of claim 1, wherein the acidic polysaccharide has a cell binding inhibitory activity between acne bacilli and atopic staphylococcus aureus.

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