ANTIMICROBIAL COMPOSITION CONTAINING NATURAL EXTRACT, NANO SILVER AND NATURAL ESSENTIAL OIL

Technical Field

[1] The present invention relates to a composition containing grapefruit, purslane (

Portulaca oleracea L.) and Houttuyniae Herba (Houttuynia cordata Thunb) extracts as natural extracts, nano silver and natural essential oils, and having excellent antimicrobial activity. Background Art

[2] A myriad of microorganisms inhabit the skin and cause various skin diseases.

Examples of such microorganisms include Staphylococcus aureus causing skin suppuration and resident skin flora such as Staphylococcus epidermis, Propioni- bacterium acnes and Pityrosporum ovale.

[3] These bacteria decompose sebaceous matter and sweat secreted from the skin, thus causing unpleasant odors and the resulting decomposed products irritate the skin, causing inflammation. In particular, Propionibacterium acnes decomposes sebum to produce free fatty acids, utilizing lipase which is a fatty acid-decomposing enzyme. The resulting free fatty acids not only irritate skin, but also cause inflammatory lesions such as red rashes including papules, pustules and nodules, as exhibited in acne.

[4] Dermatophytes invade the stratum corneum of hairs and skin, causing superficial mycoses. As examples of Dermatophytes causing superficial mycoses, mention may be made of Microsporum, Trichophyton and Epodermophyton which are pathogens causing trichophytosis.

[5] Candida albicans causes skin candidiasis and chronic skin/mucosal candidiasis.

Such fungi cause superficial mycoses leading to various lesions due to their parasitic nature and proliferation on corneous tissues of the skin. As a causative agent that causes granulomatous lesions on other skin and subcutaneous tissues, mention may be made of the pathogenic fungi Sporothrix schenckii .

[6] Meanwhile, among herbal medicines, there are known a large number of medicines that have been widely utilized as external preparations since ancient times, in treatment of skin diseases caused by bacteria and fungi.

[7] Recently, a great deal of attempts have been made to scientifically elucidate effectiveness of herbal medicines that have been conventionally used in folk remedies and passed on from generation to generation, and based on results therefrom, research to find active ingredients of herbal medicines having novel antimicrobial activity is being actively undertaken.

[8] As a relatively well-known herbal medicine having antimicrobial activity, berberine, which is an alkaloid contained in Phellodendron amurense, Coptis japonica, etc., is listed as berberine chloride in the Korean Pharmacopoeia and has a broad antimicrobial spectrum.

[9] In particular, berberine is widely used in treatment of skin diseases and is reported to have strong antimicrobial activity against Propionibacterium acnes that is a pathogenic causative agent of acne. Disclosure of Invention Technical Problem

[10] Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide an antimicrobial composition containing natural extracts, nano silver and natural essential oils, and having excellent antimicrobial activity against bacteria and fungi such as Staphylococcus aureus, resident skin flora and Dermatophytes.

[11] It is another object of the present invention to provide an antimicrobial composition containing natural extracts, nano silver and natural essential oils, suitable for use in formulations such as hand cleaning agents, aromatic fragrances and herbal gels. Technical Solution

[12] In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of an antimicrobial composition comprising 10 to 30% by weight of natural extracts containing a 1:1:1 mixture (w/w) of grapefruit, purslane (Portulaca oleracea L.) and Houttuyniae Herba (Houttuynia cordata Thunb) extracts, 1 to 5% by weight of nano silver having a concentration of 50 to 1,000 ppm, 5 to 30% by weight of a mixture of natural essential oils and the balance of ethanol.

[13] The antimicrobial composition in accordance with the present invention exhibits excellent antimicrobial activity against bacteria and fungi such as Staphylococcus aureus, resident skin flora and Dermatophytes, and is suitable for preparation of formulations comprising the antimicrobial composition and having antimicrobial activity, such as hand cleaning agents, aromatic fragrances and herbal gels. Best Mode for Carrying Out the Invention

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

[15] The present invention employs grapefruit, purslane (Portulaca oleracea L.) and

Houttuyniae Herba (Houttuynia cordata Thunb) as natural extracts. Grapefruit belongs to the Citrus family, and is slightly larger than an adult fist with a smooth and light yellow skin. Fruit flesh of grapefruit is very soft, succulent and white. Grapefruit tastes slightly bitter. Grapefruit seed extract (GSE) kills microorganisms by blockage of amino acid supply in cell tissues and decomposition and elution of low molecular

weight intracellular substances, and thus are receiving a great deal of attention as a multipurpose and non-toxic bacteriostatic agent from the medical world, for application thereof as sterile antimicrobial agents, natural antibiotics, and the like.

[16] According to the Korean Journal of Food Preservation, Vol. 1, 1-7 (1994), it is stated that grapefruit seed extract (GSE) exhibits antimicrobial activity on a wide variety of microorganisms.

[17] Minimal inhibitory concentrations of GSE on various microorganisms are, respectively, 10 to 500 ppm for bacteria, 250 to 1,000 ppm for fungi, and 100 to 250 ppm for yeast.

[18] According to the Journal of the Korean Fisheries Society, Vol. 23(4), 297 302

(1990), it is stated that grapefruit seed extract (GSE) exerts anti-proliferative activity on bacteria, for example, at a concentration of 50 ppm for the genus Vibrio, and at a concentration of more than 30 ppm for Bacillus cereus and Staphylococcus aureus, and exhibits sterilizing effects at a concentration of more than 50 ppm for Vibrio vulnificus.

[19] Further, according to the Journal of the Korean Society of Food and Nutrition. Vol.

24(5), 690-694 (1995), results of acute oral toxicity, skin irritation and acute ocular mucosal irritation tests on grapefruit seed extract (GSE) in order to examine stability of grapefruit seed extract (GSE), which is taking into consideration application thereof as a natural broad-spectrum antimicrobial agent, show that GST exhibits an acute LD value of 3.75 g/kg for laboratory white rats, and exhibits weak toxicity on the skin thus causing substantially no lesions such as edema, but displays eye irritability.

[20] According to the Korean Journal of the Food Hygiene and Safety, Vol. 10(4),

263-270 (1995), grapefruit seed extract (GSE) is reported to inhibit proliferation of En- terobacter pyrinus and Fusarium sp., linked to spoilage of fruits and vegetables, at a low treatment concentration of less than 500 ppm, thus demonstrating distinctive antimicrobial effects of GSE.

[21] Meanwhile, Houttuyniae Herba (Houttuynia cor data Thunb), which is an ingredient of natural extracts constituting an antimicrobial composition in accordance with the present invention, is a perennial herbaceous plant belonging to the Saururaceae family.

[22] The plant grows well in damp climes and smells of raw fish. Rhizomes of

Houttuyniae Herba are white and soft, and extend horizontally, while its main stem on the ground has no leaves or trichomes, grows to 20 to 50 cm tall and has several longitudinal cords.

[23] Houttuyniae Herba has a pungent fish odor when its leaves are crushed, so it is also named "Yu Xing Cao" in Chinese and oriental medicine (yu is fish, xing means fishy smell, and cao is the general term for herbs), and is also called a deca-medicine (multi-cure drug) because it is believed to cure 10 kinds of diseases.

[24] In Chinese medicine and folk remedies, the whole plant of Houttuyniae Herba including roots, leaves and stems thereof is utilized as a medicine for treating or alleviating dropsy, syphilis, cystitis, metritis, lumps in the breast, pulmonary abscesses, otitis media, palsy, pneumonia, dermatitis, hepatitis and hypertension, as a cardiotonic/ antipyretic drug, for promoting urination, and for ameliorating gonorrhea, urethritis and the like.

[25] Houttuyniae Herba is one of the most famous folk medicines and a juice of green leaves thereof is therapeutically effective for treatment of purulent inflammation, furuncles, wounds, and the like via application thereof to affected parts, and also effectively removes sore toxic pathogenic factors and promotes urination. 30 g of decocted Houttuyniae Herba is used internally for treatment of gonorrhea and urethritis. In addition, decocted Houttuyniae Herba is used in washing to treat hemorrhoids and scabies and is used for bathing (Korean Medicinal Plant Dictionary).

[26] In folk remedies, Houttuyniae Herba juice is applied to affected parts for treatment of athlete's foot, hemorrhoids, snake bites and lacquer poisoning. It is effective for alleviation of fever, detoxification, and treatment of dysuria and eczema. In addition, it is also good for healing wounds, cleaning the face, as an insecticide, and for treatment of skin diseases and gastric diseases.

[27] In addition, Houttuyniae Herba exhibits advantageous effects such as regeneration of skin tissues, healing of wounds, antibacterial and antifungal properties, and anti- oxidative effects and blood circulation promoting effects, thus preventing skin aging.

[28] Meanwhile, purslane (Portulaca oleracea L.) is non-toxic and non-irritative and thus displays excellent therapeutic effects for treatment of skin diseases such as furuncles and edema. Therefore, purslane has been widely used as a medicinal substance, and has been frequently ingested by ancient people as a non-toxic food for achieving longevity.

[29] In Oriental Medicine, purslane has been said to have medical effects such as dissipating heat and detoxifying, promoting pus discharge and inducing diuresis, and therefore has been decocted and taken for treatment of dysentery, enteritis and bacterial red dysentery.

[30] In addition, for treatment of acute skin infections, scabies and eczema, purslane is pounded and applied to affected parts, or affected parts are washed with juice thereof, for external use, or purslane is decocted for drinking. Purslane is also used as antivenin as well as for treatment of insect bites and also heals various diseases which are accompanied by formation of pus and festering of wounds.

[31] Purslane is edible in the form of cooked potherbs without adverse side effects even when large amounts thereof are ingested. Such medicinal effects of purslane enable to effectively remove adverse side effects which may result from skin diseases or

cosmetic-causing troubles due to use of cosmetic or other environmental factors.

[32] Meanwhile, purslane contains a large quantity of natural moisturizing factors, i.e., polysaccharides and amino acids and thus maintains high content of water in severe dry weather, thereby making it very difficult to dry. That is, purslane has anti- inflammatory and skin irritation-alleviating effects. In accordance with the present invention, the antimicrobial composition employs a 1:1:1 mixture (w/w) of grapefruit, purslane (Portulaca oleracea L.) and Houttuyniae Herba (Houttuynia cordata Thunb) extracts as the natural extracts. The amount of the natural extracts used herein is within the range of 10 to 30% by weight, and preferably 1 to 5% by weight.

[33] Herein, where the amount of the natural extracts is less than 10% by weight, it is impossible to obtain desired antimicrobial effects due to decreased efficiency of active ingredients. In contrast, where the amount of the natural extract is greater than 30% by weight, synergism between nano silver and natural essential oils is inhibited. Therefore, it is preferred to use the natural extracts within the above-mentioned range.

[34] Meanwhile, when it is formed into nano particles, silver displays strong sterilizing and antimicrobial properties. Research indicates that silver can sterilize about 650 species of bacteria and viruses and silver is also known to exert excellent effects on fungi. In addition, nano silver is totally harmless to humans while exhibiting sterilizing power several tens of times stronger than chlorine-based sterilizers. Generally, high- purity nano silver is accepted to be harmless to humans even when taken internally. It is hypothesized that such antimicrobial action of nano silver is effected by the following three action mechanisms.

[35] Firstly, positive ions of silver (Ag ⁺ ) strongly bind to -SH, -COOH and -OH groups, which are present on bacteria, thereby destroying cell membranes or disturbing cellular functions. Secondly, colloidal silver in the nano state catalyzes conversion of oxygen into active oxygen species (O ⁺ , O and O), thereby exerting sterilizing action. That is, the ground of this mechanism is inferred from the fact that addition of superoxide dismutase (SOD) to silver leads to loss of antimicrobial activity thereof when oxygen (bound oxygen, dissolved oxygen, etc.) is converted into active oxygen species by catalysis of positive ions of silver (Ag ⁺ ) thereby exerting sterilizing action. Thirdly, antimicrobial and sterilizing actions of silver as a metal may be mentioned. Unlike other metals or conventional silver, it can be assumed that nano silver, which has a small size of less than 10 nm, can be ingested by microorganisms having a size of about 100 nm to 200 nm and therefore bacteria die from respiratory disorder and metabolism disorder due to uptake of nano silver. Further, it is believed that, in antimicrobial agents utilizing any other inorganic carriers, antimicrobial metal ions (for example, Ag ⁺ , Zn ⁺ and Cu ⁺ ) dissociated from inorganic carriers bind to cell membranes and proteins such as enzymes, thereby inhibiting energy metabolism of cells. This may be

explained by the evidence such as transfer of positive ions of silver (Ag ⁺ ) into the bacterial body. Of course, it can be said that silver has sterilizing properties as a metal itself, since silver particles are between 2 nm and 10 nm in size and intake of silver particles leads to death of microorganisms by causing metabolic disruption.

[36] Preferably, nano silver used herein has a particle size of 5 nm to 20 nm. The antimicrobial composition of the present invention contains 1 to 5% by weight of nano silver at a concentration of 50 to 1,000 ppm and preferably 3 to 5% by weight. Where the content of nano silver is less than the above-mentioned range, antimicrobial effects are lowered. In contrast, where the content of nano silver is greater than the above- mentioned range, problems associated with compatibility such as precipitation of silver due to saturation will occur. Therefore, it is preferred to use nano silver within the above-mentioned range.

[37] Meanwhile, aromatherapy is a branch of various traditional therapies, originated and developed from the discovery of the fact that flowers, leaves, roots and stems of plants have beneficial effects on sprit and body of humans, in ancient Egypt, China, India, and the like countries. Essential oils play an important role in life cycles and germination processes of plants and have certain actions against diseases, and therefore have been believed as hormones or vitality necessary for survival of plants and have been used in religious rituals, wound healing and for cosmesis.

[38] Among natural essential oils, eucalyptus oil, German chamomile oil, cedar wood oil, cinnamon oil, bergamot oil, lemon oil, cypress oil, lemon grass oil, juniper berry oil, tea tree oil, peppermint oil, rosemary oil, sandalwood oil, ginger oil, lavender oil and thyme oil, known to have antimicrobial and anti-inflammatory effects, have been used in treatment of skin diseases in Western countries for a long time, due to their excellent antimicrobial and anti-inflammatory effects.

[39] Eucalyptus is a tall evergreen tree of the Myrtaceae family, a characteristic component of Australian flora. Oil extracted from leaves of the eucalyptus tree has analgesic, antimicrobial and antiviral effects.

[40] German chamomile is a plant of the Compositae family native to the Mediterranean coast. Oil extracted from flower petals of German chamomile has been used for more than 2,000 years as a household medicine in Europe. German chamomile oil contains azulene, a powerful antimicrobial and anti-inflammatory agent, and thus is effective in treating skin inflammatory conditions such as acne, eczema, diaper eczema and burn.

[41] Cedar wood is a coniferous tree of the family Cupressaceae. Oil extracted from cedar wood was used for preservation of corpses or as cosmetic in ancient Egyptian. In addition, the odor of cedar wood oil repels ants, moths and other harmful insects and has antimicrobial, insecticidal and disinfecting actions.

[42] Cinnamon is a tropical tree of the Lauraceae family, and oil extracted from leaves

and branches thereof has excellent antimicrobial activity effectively acting on most bacteria. [43] Bergamot is a citrus tree of the Rutaceae family, and oil extracted from peels thereof has disinfecting effects and therefore is therapeutically effective against eczema, psoriasis, acne, seborrhea of skin and scalp, varicella and shingles and serves to effectively repel insects. [44] Lemon is a tree of the Rutaceae family, and oil extracted from peels thereof has disinfecting, antimicrobial and anti-oxidative action and is known to inactivate Mycobacterium tuberculosis even with a 0.2 dilution thereof. [45] Cypress is an evergreen tree of the family Cupressaceae, and oil extracted from leaves thereof has insect-repelling and antimicrobial effects. [46] Lemon grass is an aromatic tropical grass of the Poaceae family,and oil extracted from leaves thereof has antimicrobial, anti-oxidative and antifungal action. [47] Juniper berry is a coniferous evergreen tree of the family Cupressaceae, and oil extracted from berries thereof has antimicrobial and disinfecting action and thus is used in after-shave lotions. [48] Tea tree is a shrub of the Myrtaceae family, and oil extracted from leaves and twigs thereof has excellent antimicrobial, antibacterial, antiviral and disinfecting action against infection with all species of bacteria. [49] Peppermint is an aromatic herb of the Lamiaceae family, and oil extracted from leaves and flowering branch tips thereof is therapeutically effective against bronchial diseases and has anti-inflammatory,antimicrobial, antiphlogistic and antiviral effects. [50] Rosemary is an evergreen perennial shrub belonging to the Labiatae family, and oil extracted from flowers thereof has antibacterial, antifungal, disinfecting and insect repellent action. [51] Sandalwood is a small evergreen tree belonging to the Santalaceae family, and oil extracted from dried duramen of sandalwood has antimicrobial and disinfecting action. [52] Ginger is a medicinal perennial herb belonging to the Zingiberaceae family, and oil extracted from dried roots thereof has bactericidal, antimicrobial and anti-oxidative effects. [53] Lavender is a shrub of the Lamiaceae family, and oil extracted from flowers thereof is known to have the most popular and broad antimicrobial, sedative and antiinflammatory action. [54] Finally, thyme is an evergreen tree of the Lamiaceae family, and oil extracted from leaves and flower thereof has antimicrobial, anti-oxidative, antiseptic, sterilizing, detoxifying and parasite-repelling effects. [55] The antimicrobial composition of the present invention contains, as mentioned above, at least one natural essential oil selected from the group consisting of

eucalyptus oil, German chamomile oil, cedar wood oil, cinnamon oil, bergamot oil, lemon oil, cypress oil, lemon grass oil, juniper berry oil, tea tree oil, peppermint oil, rosemary oil, sandalwood oil, ginger oil, lavender oil and thyme oil. The amount of the natural essential oil used herein is within the range of 5 to 30% by weight, and preferably 5 to 15% by weight. Where the content of the natural essential oil is less than 5% by weight, antimicrobial activity is disadvantageously decreased. In contrast, where the content of the natural essential oil is greater than 30% by weight, the strong odor of the essential oil may cause customer dissatisfaction. Therefore, it is preferred to add the natural essential oil within the above-mentioned range. Mode for the Invention

[56] Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

[57] Ex ample 1

[58] According to composition ratio of ingredients given in Table 1 below, an antimicrobial composition containing grapefruit, purslane (Portulaca oleracea L.) and Houttuyniae Herba (Houttuynia cordata Thunb) extracts, nano silver having a concentration of 1,000 ppm and an essential oil consisting of cinnamon oil, tea tree oil and lemon oil was prepared. As a base solution, a 1:2 mixture of 1,3-butylene glycol and ethanol was used.

[59] Antimicrobial potency of the thus-prepared composition was examined by Paper

Disc Test (Kirby-Bauer Antimicrobial Sensitivity Test). First, in order to induce activation, the respective bacterial strains were pre-cultured in suitable media for 48 to 72 hours. 0.1 mL of the bacterial cultures thus prepared were respectively smeared and dried on the respective corresponding media. 30 D of the antimicrobial composition was aliquoted and added dropwise to paper discs having a diameter of 8 mm, followed by incubation under conditions as set forth in Table 2 below. After completion of incubation, diameters of clear zones appearing on the paper discs were measured. The thus-obtained results (antimicrobial effects) are shown in Table 3 below.

[60] Comparative Example 1

[61] In order to examine antimicrobial effects exhibited by natural extracts alone, the same procedure was carried out as in Example 1, except that an antimicrobial composition was prepared according to the composition and ratio of constituent ingredients given in Table 1 below. Diameters of clear zones appearing on the paper discs were measured. The results (antimicrobial effects) are shown in Table 3 below.

[62] Comparative Example 2

[63] In order to examine antimicrobial effects exhibited by nano silver alone, the same procedure as in Example 1 was employed, except that an antimicrobial composition was prepared according to the composition and ratio of constituent ingredients given in Table 1 below. Diameters of clear zones appearing on the paper discs were measured. The results (antimicrobial effects) are shown in Table 3 below.

[64] Comparative Example 3 [65] In order to examine antimicrobial effects exhibited by natural essential oil alone, the same procedure as in Example 1 was employed, except that an antimicrobial composition was prepared according to the composition and ratio of constituent ingredients given in Table 1 below. Diameters of clear zones appearing on the paper discs were measured. The results (antimicrobial effects) are shown in Table 3 below.

[66] Table 1 (unit: wt%)

[67] Table 2

[68] SK RCM : Reinforced clostridial agar medium [69] Table 3 (Unit : mm)

[70] As can be seen from results of Table 3, Example 1 exhibited broader clear zones than Comparative Examples 1 through 3, thus representing that the antimicrobial composition of the present invention has antimicrobial activity superior to natural extracts alone, nano silver alone and essential oils alone, respectively. Therefore, it can be said that combination of these ingredients exerts superior antimicrobial effects via synergistic effects therebetween.

[71] Preparation Example 1 [72] According to the following composition, a hand cleaning agent was prepared. Antimicrobial effects of the thus-prepared antimicrobial hand cleaning agent were measured. The results are shown in Table 4 below.

[73]

[74] Comparative Preparation Example 1 [75] According to the following composition, a hand cleaning agent having antimicrobial properties was prepared. Antimicrobial effects of the thus-prepared antimicrobial hand cleaning agent were measured. The results are shown in Table 4 below.

[76]

[77] Comparative Preparation Example 2 [78] According to the following composition, a hand cleaning agent having antimicrobial properties was prepared. Antimicrobial effects of the thus-prepared antimicrobial hand cleaning agent were measured. The results are shown in Table 4 below.

[79]

[80] Comparative Preparation Example 3 [81] According to the following composition, a hand cleaning agent having antimicrobial properties was prepared. Antimicrobial effects of the thus-prepared antimicrobial hand cleaning agent were measured. The results are shown in Table 4 below.

[82]

[83] Table 4 (Unit : mm)

[84] When antimicrobial activity tests were carried out on the hand cleaning/ moisturizing agents prepared in Preparation Example 1 and Comparative Preparation Examples 1 through 3, the results showed that synergistic effects between natural extracts, nano silver and natural essential oils further increase antimicrobial activity.

[85] The above-mentioned antimicrobial composition of the present invention can be applied to a variety of products as follows.

[86] Preparation Example 2 [87] According to the following composition, anaromaticfragrance having antimicrobial properties was prepared:

[88]

[89] Preparation Example 3 [90]

[91] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.