[2] Background Art [3] Among scores of Ginkgo leaf compounds are Ginkgo flavon glycosides, ginkgolides and bilobalide which are all known as useful compounds. Recently, these compounds have been intensively studied for isolation and pharmaceutical efficacy.

[4] Particularly, ginkgolides and bilobalides, classified as terpenes which are a group of unsaturated aliphatic cyclic hydrocarbons derived from plants, are in practice used for treating neurological disorders. In Europe, annual sales of single or combined preparations of Ginkgo extracts, stch as tablets, solutions, suppositories, injections and the like, are reported to amount to as mulch as about five hundred million. Now, chemical, pharmacological and clinical research is extensively conducted on the terpenes over the world. Other effective pharmacological active substances existing in ginkgo leaves are also under intensive and extensive study for pharmacological effects stch as anticancer, antiviral and anti-feedant effects.

[5] Generally, dandruff is characterized by small, loose flakes of dead skin on the scalp and is sometimes associated with seborrhea where sebum production is excessive. In its mildest variety, dandruff affects the scalp and mainly causes shedding of scales with itching. More severe varieties, called seborrheic dermatitis, include a troublesome oiliness of the hair and scalp, sores, greasy scales, crusts, and pimples in the scalp.

[6] Dandruff may be caused by several different factors, but the exact underlying cause of dandruff is unknown or is at least not agreed upon by the medical establishment.

One possible cause of dandruff may be overactive oil glands on the scalp which cause an increase in the natural skin shedding process. Poor diet, poor hygiene, genetic disposition, hormonal imbalances and infections may all possibly trigger dandruff.

Dandruff may become worse with stress. A biochemical change in the epidermal tissue due to the overgrowth of scalp cells may give rise to dandruff. Another possible cause of dandruff may be sensitivity to, or overgrowth of, a fungus present on the scalp.

Some external contributors to dandruff may include infrequent shampooing of the hair or inadequate hair rinsing, improper use of hair-coloring products, cold weather, dry environment, polluted air, and tight fitting hats.

[7] Particularly, a small skin fungus, known as Pityrosporum ovale, is now recognized as the most common cause of dandruff. When the fungus grows too rapidly or is highly active, the natural renewal of cells is disturbed and dandruff appears with itching.

Thus, most of the dandruff treatment agents in current use pay attention to inhibiting the growth and activity of Pityrosporum ovale in addition to returning the metabolism of scalp cells to normal states.

[8] Over the past decades, many new antibacterial agents have been developed for treating dandruff. Of them, ketooonazole is reported to be very effective in treating pityriasis versicolor and dandruff (Rausch LJ, Jacobs PH. 1984, cutis 34: 470-471 ; Hay RJ, Midgeley G. 1984, Clinical and Experimental Dermatology 9: 571-573; Savin RC. 1984. Journal of the American Academy of Dermatology 10: 824-830). In practice, many of the dandruff treatment compositions in current use comprise ke- toconazole as an effective ingredient. Ebwever, ketoconazole suffers from the drawback of having a bad effect on the endocrine system when it is used for a long period of time.

[9] Itraoonazole is a triazole antifungal agent having more potent activity against various types of fungi and higher safety to the body than ketoconazole. With high affinity for keratotic tissues, triazoles are found to effectively inhibit the growth and activity of many kinds of fungi which are pathogenic to the human body. As with all triazole antifungal agents, itraoonazole binds to the fungal p450 enzymes and stops the cells from making ecrosterol, the main component of the cell wall. The enzyme p450 is in the sterol biosynthesis pathway that leads from lanosterol to ecrosterol. Ebwever, itraconazole is problematic in delivery into the body. Because of not being absorbed through sweat glands into the body, itraconazole takes an oral route for administration.

Another problem with itraconazole is that at least 1,000 mg of itraconazole must be ad- ministered a day for medicinal efficacy.

[10] Also, most patients with dandruff or seborrheic dermatitis are prescribed to take various medicines simultaneously, but administration of various medicines may cause serious side effects in the liver and the immune system. In addition, one ingredient may inhibit medicinal effects of other ingredients. Most of the shampoos or lotions in current use contain selenium sulfide or zinc pyrithione as an effective ingredient for the treatment of dandruff.

[11] Besides genetic disposition, depilation-causing factors include diet imbalances, excessive drinking and smoking, drugs, lack of sleep, stress, parturition, menopausal disorders, frequent hair permanent waving treatment, hair dyeing, and hormone imbalances. When depilation proceeds, hairs on the forehead and vertex become thin and feeble, with an increase of depilated hairs. General hair treatment shampoos may be also causative of depilation when used over a long time.

[12] Dandruff or depilation, though not dangerous, causes psychological stresses for most people. From old times, prevention or control of dandruff or depilation has been studied, but there continues to exist a need for a treatment agent which is highly effective in inhibiting dandruff-causing pathogens and preventing depilation as well as being environmentally friendly and safe for the body.

[13] In spite of the imminent necessity of environmental protection, the use of agricultural chemicals increases every year. Now, most commercially available in- secticides and herbicides are based on organic chlorine, organic phosphorus and carbamate compounds. In addition to being toxic, these compounds are of low biodegradability and increase in concentration through the food chain. Despite the advantages of chemical insecticides, the problems associated with their use have been well documented. These include the resurgence of pest populations after decimation of the natural enemies, development of insecticide-resistant populations, and negative impacts on non-target organisms within and outside the crop system. One of the more serious problems is the development of resistance. Many insect pest species now possess resistance to some or several types of insecticides, and few chemical control options exist for these pests. Additionally, pesticide residues which remain on foods may be harmful to the body.

[14] To overcome the problems associated with chemical pesticides, biological pest control agents are developed as the next generation agricultural medicines. Biological pest control agents take advantage of microorganisms, natural enemies and bio- chemicals extracted from natural substances. Ebwever, difficulty in handling mi- croorganism agents and natural enemies is a serious impediment in commercializing them. Hence, the development of natural materials which are friendly to the en- vironment, harmless to humans, animals and plants, easy to handle, and able to be produced at low cost, and able to kill pest effectively, is required. Fbwever, despite of many kinds of natural biomass having inhibitory activity against pests and bacteria, few of them have been developed as pesticides.

[15] Ginkgo biloba is extremely hardy, as it is resistant to many insects, diseases and pollution. The plant is mostly distributed in Far Eastern regions. Ebwever, basic studies on Ginkgo biloba leaf ingredients with pesticide and antibacterial activities have been made in countries other than Far East countries. Alcohol extracts of Ginkgo biloba roots are observed to potently inhibit the growth of Pyranstamubilatis larva, a starch moth found in Europe, by professor S. D. Berk of Wisconsin University.

According to the research results obtained by the lab of the professor R. T. Major of Virginia University, Japanese beetles do not gnaw Ginkgo bilova leaves, but instead, starve death. Acetone extracts of Ginkgo biloba leaves are found to cease the growth of various bacteria including Erwinia amylovora, E. coli, P. phaseolicola, Xanthomonas phaseoli, and B. Pumilus. J. W. Mitchell et al. revealed that the alcohol extracts of Ginkgo biloba leaves also can prevent the infection of southern bean mosaic virus and tobacco mosaic virus. As a result of resistance against the fungi, 2-hexenal and carbonyl groups as nonaromatic hydrocarbon is highly inhibitory against the growth of Monilinia fructicola. Further, wax materials obtained from the epidermal tissue of Ginkgo biloba leaves are found to restrain the spore germination of fungi.

[16] The present inventors have been intensive and thorough research on natural materials which are preventive of dandruff and depilation and useful as antibacterial agents, pesticides and insect repellents without harm to the body and the environment.

And the present inventors have completed this invention by confirming that com- positions mixing in appropriate ratios of ginkgolide A, ginkgolide B and bilobalide which are the terpenlactone compounds, has excellent inhibitory activity against Pityrosporum ovale, causative of dandruff in humans and has superior curative effects on dandruff and scalp itching and preventive effect on depilation, and effectively inhibits the activity of Malassezia pachydermatis, causative of dandruff in dogs, and shows good deodorizing effects in animals, without side effects such as skin erythema, has excellent at antibacterial, pesticidal and insect repellent effects.

[17] Disclosure of Invention Technical Solution [18] It is an object of the present invention to provide a composition useful in the treatment of dandruff, scalp itching, and depilation.

[19] It is another object of the present invention to provide a shampoo composition for use in animals.

[20] It is a further object of the present invention to provide a composition as an active ingredient for antifungal, pesticides and insect repellents.

[21] In accordance with the present invention, the above objects could be accomplished by a provision of a composition comprising ginkgolide A, ginkgolide B and bilobalide which are the terpenlactone compounds, as an active ingredients.

[22] Brief Description of the Drawings [23] FIG. 1 shows a process flow of extracting, separating and purifying ginkgolide A, ginkgolide B and bilobalide from Ginkgo biloba leaves.

[24] Best Mode for Carrying Out the Invention [25] In order to achieve the above object, the present invention provides a composition comprising ginkgolide A, ginkgolide B and bilobalide which are the terpenlactone compounds, as an active ingredients.

[26] The composition of the present include shampoos composition for treating dandruff and preventing depilation, shampoos composition for animals, and antifungal agents, pesticides and insect repellents.

[27] Hereinafter, the present invention is described in detail.

[28] The composition of the present comprises 50-80 wt% of ginkgolide A, 10-25 wt% of ginkgolide B, and 10-20 wt% of bilobalide.

[29] In the composition of the present invention, these terpenlactone compounds may be in the form of pharmaceutically acceptable salts, hydrates or solvates.

[30] The terpenlactone compounds may be obtained from Ginkgo biloba leaves by ordinary processes such as extraction, isolation and purification or from commercially available drugs.

[31] For example, extraction, isolation and purification from Ginkgo biloba leaves may be executed as follows.

[32] Ginkgo biloba leaves are washed, dried and cut into a size of 1-2 mm. The leaf scraps are put in a round-bottom flask containing distilled water and heated for 2-4 hours at 70-90°C in a water bath. Filtration is conducted by use of GF/C filter paper (pore size 1. 2for). A mixture of the filtrate and ethyl acetate is allowed to stand for 2 hours in a separatory funnel to remove the organic layer. The ethyl acetate thus obtained is combined again with distilled water and allowed to stand for 2 hours, after which the organic layer is removed. Following filtration, the organic layer is con- centrated under reduced pressure.

[33] Next, ginkgolide A, ginkgolide B and bilobalide are separated from the above ethyl acetate concentrate using high performance liquid chromatography (H2O:THF:MeOH = 68.5:10.5:21).

[34] Upon extraction, hot water may destroy ginkgolide A, ginkgolide B and bilobalide.

In the present invention, the water used for the extraction is warmed to 70-90°C and preferably 80°C to effectively obtain the terpenlactone compounds without destruction thereof.

[35] For mass production, the extraction solvent may be water, alcohol, ethyl acetate or a mixture of water and alcohol, with preference for a mixture of alcohol and water in terms of sterilization and extraction efficiency. The aqueous alcohol solution has an alcohol content of 20-50 % and preferably 40 %. More preferable is a 40 % aqueous ethanol solution.

[36] As for a shampoo composition of the present invention, it contain, as effective in- gredients, the terpenlactone compounds, that is, ginkgolide A, ginkgolide B and bilobalide in the amount of 0. 001-10 wt%, based on the total weight of the shampoo composition. For example, if less than 0.001 wt% of the terpenlactone compounds are used, desired effects cannot be obtained. On the other hand, greater than 10 wt% of the terpenlactone compounds may give excellent preventive effects on dandruff and scalp itching, but may also irritate the skin, and increase the production cost.

[37] A combination of 50-80 wt% of ginkgolide A, 10-25 wt% of ginkgolide B and 10-20 wt% of bilobalide in shampoo composition of the present invention gives synergistic and additive effects so that it can effectively inhibit the activity of Pityrosporum ovale, reduce the population of the dandruff-causative fungus, increase dandruff reduction rate and prevent the progress of depilation, as compared with individual terepenlactone compounds.

[38] Additionally, the shampoo composition of the present invention can be used for animals as it is found to have inhibitory activity against Malassezia pachydermatis, a dandruff-causative bacteria in dogs, and have deodorizing activity without occurrence of side effects such as erythema.

[39] The shampoo composition of the present invention can be in addition to above effective ingredients formulated with necessary constituents into various commercial formulas, including rinses, hair tonics, pomades, hair treatments, hair lotions, as well as shampoos.

[40] Constituents necessary to make a shampoo composition are exemplified by a synthetic surfactant, a pearl essence aid, a preservative, a thickener, a viscosity controlling agent, a pH-adjusting agent, flavor, dye, a hair conditioning agent, and water. The synthetic surfactant is used in an amount of 10-70 wt% and preferably in an amount of 10-30 wt% based on the total weight of the shampoo composition. Any of anionic, amphoteric, and non-ionic surfactants may be used in the present invention.

Examples of the synthetic anionic surfactant include alkylsulfate and alkylethersulfate such as sodium laurylsulfate, ammonium laurylsulfate, triethanolamine laurylsulfate, polyoxyethylene sodium laurylsulfate, polyoxyethylene ammonium laurylsulfate, etc.

Alkyl betaine and alkylamidopropyl betaine are representative for synthetic amphoteric surfactants the concrete examples of which include oooodimethyl car- boxymethyl betaine, lauryldimethyl carboxymethyl betaine, lauryldimethyl alpha- carboxyethyl betaine, cetyldimethylcarboxymethyl betaine, and oocamidopropyl betaine. Alkanolamide and alkylamine oxide are used as non-ionic surfactants. Lau- ryldiethylamine oxide, coconut alkyldimethylamine oxide, laurylic add di- ethanolamide, coconut fatty aid diethanolamide, and coconut fatty acid monode- thanolamide fall into a non-ionic surfactant category. The surfactants mentioned above may be used individually or in combination in the shampoo composition of the present invention.

[41] Acting as a medium, water is used in an amount of 20-90 wt% and preferably in an amount of 40-85 wt% based on the total weight of the shampoo composition.

[42] As mentioned above, other constituents include pearl essence aids such as ethylene glycol monostearate and ethylene glycol distearate; preservatives such as methyl parabenzoate, methylchloroisothiazolinone and methylisothiozolinone; thickeners or viscosity controlling agents such as sodium chloride, ammonium chloride, and propylene glycole ; pH-adjusting agents such as citric aid, phosphoric acid, sodium hydroxide, and potassium hydroxide; hair conditioning agents such as polyquaternium 10, polyquaternium-7, methylpolysiloxane, dimethylmncopolyol, and hydrolyzed animal proteins; dyes such as water-soluble tars; and flavors.

[43] Ginkgolide A, ginkgolide B and bilobalide in the composition of the present invention can be also used as natural active ingredients in antibacterial agents, pesticides and insect repellents. In these compositions, the amounts of the active in- gredients are not specified, but used as is suitable for the purposes.

[44] When used for killing bacteria, fungus and insects, the natural active ingredients, ginkgolide A, ginkgolide B and bilobalide, may be diluted with water or combined with other natural antibacterial, antifungal agents or pesticides. Also, the terpenlactone compounds may be used in combination with fertilizers, cleaning agents, or other additives such as dye, surfactants or carriers. For use in practice, they may be formulated into powders, pellets, granules, tablets or other forms.

[45] Aqueous solutions or dilutions of the formulations may be sprayed to apply the active ingredients to targets. Amounts of the formulations may vary depending on purpose, target, application types, and other parameters.

[46] Mode for the Invention [47] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[48] [49] EXAMPLE : Extraction, Separation and Purification of Ter- penlactone compounds from Ginkgo biloba Leaves [50] After being washed and dried, Ginkgo biloba leaves were cut into a size of 1-2 mm.

100 g of the leaf scraps was put into a round-bottom flask containing 1 liter of water (or 40% ethanol solution) and heated at 80°C for 2 hours in a water bath. Thereafter, the solution was filtered through GF/C filter paper (pore 1. 2for). The filtrate thus obtained was combined with 50 ml of ethyl acetate and the mixture was allowed to stand for 2 hours in a separatory funnel to remove the ethyl acetate layer. This layer was combined with 50 ml of distilled water and allowed to stand for 2 hours to remove the ethyl acetate layer separated. Filtration of the organic layer was followed by con- centration in vacua.

[51] The ethylacetate concentrate thus obtained was subjected to HPLC (water: THF: MeOH=68. 5: 10.5 : 21) to separate ginkgolide A, ginkgolide B and bilobalide therefrom.

[52] [53] <Analysis aDnditions> [54] Instrument Model: Shimadzu LC-1OAD [55] Column: C18 preparative column [56] Injection: with syringe [57] Volume injected: 50 ml [58] Detection: 219 nm [59] Flow rate: 10 ml/min [60] Mobile phase: water: THF: MeOH=68. 5: 10.5 : 21 [61] Running Time: 30 min [62] [63] <Chemical Formula of Ginkgolides> [64] o HO pi \/'\ ! P2 R3 Ginkaoiid _.. __ C) H i A O R2 lWo X) N Otq H OH t J , -p/'"OH OH OH C ! JR-i j CfCH, OH OH 1-i i3 [65] [66] <Chemical Formula of Bilobalide> [67] 0 0 tu ; ho OH 0 0 0 Bilobalide [68] [69] PREPARATION EXAMPLE : Preparation of Shampoo Composition [70] A mixture of a surfactant and ethyleneglyool distearate in filtered water was heated to 55°C to uniform dissolution. The solution was slowly cooled down to 40°C with stirring. Following the addition of ginkgolide A, ginkgolide B, bilobalide and other suitable ingredients shown in Table 1, below, the solution was cooled to room temperature with stirring, so as to afford a shampoo composition.

[71] Table 1 Composition shampoo composition(wt%) Surfactants Ammonium laurylsulfate 10. 0 Polyoxyethylene sodium 5.0 laurylsulfate Cccoamidopropylmetaine 5.0 Lauroylmirystyldiethanola 3.0 mide ActiveIngredients Ginkgolide A (65 0. 001-10 wt%) +Ginkgolide B (20 wt%) +Bilobalide (15 wt%) Otheringredients Ethyleneglycol distearate 1.0 Polyquaternium-7 1.0 Blue dye #1 0.0001 Yellow dye #4 0.0004 Methyl parabenzoate 0.1 Aroma oil 0.5 Citric acid 2.0 Water Remainder to 100 [72] [73] EXPERIMENTAL EXAMPLE 1 : Antifungal Effect on Dandruff- Causing Fungus [74] To examine whether the composition of the present invention has inhibitory activity against Pityrosporum ovale, the following experiment was conducted using a colony counting method.

[75] Pityrosporum ovale was controlled to a population of 2x10 cells and cultured under optimal growth condition for 1-2 days in a PDA medium (potato dextrose broth 24g + Ox-Gall powder 5g + agar 15 g + distilled water 1.0 liter) over which ginkgolide A, ginkgolide B and bilobalide were spread. Colonies formed on the medium were counted. As a positive mntrol, ketoconazole was used. The results are given in Table 2, below.

[76] Table 2 Samples ColonyNo. InhibitionRate (%) Non-treated group 270 0 Ketooonazole (lOOmg/f) 205 24.1 Ginkgolide A (lOOmg/f) 115 57.4 Ginkgolide B (100mg/l) 97 64.1 Bilobalide (100mg/l) 56 79.3 Ginkgolide A (65 mg/l)+ 5 98.2 Ginkgolide B (20 mg/f) + Bilobalide (15 mg/f) [77] [78] As shown in Table 2, a combination of ginkgolide A 65 wt%, ginkgolide B 20 wt% and bilobalide 15 wt% removed 98.2 % of the fungus and is superior in antifungal activity to individual ones, that is, ginkgolide A with a removal rate of 57.4%, ginkgolide B with a removal rate of 64. 1 %, and bilobalide with a removal rate of 79.3%. Consequently, a combination of ginkgolide A, ginkgolide B and bilobalide shows synergistic and additive effects and thus more effectively inhibits the growth of Pitysporum ovale than individual ingredients.

[79] [80] EXPERIMENTAL EXAMPLE 2 : Inhibitory Activity Against Dandruff-Causing Fungus [81] To examine whether the composition of the present invention kills Pitysporum ovale, the following experiment was conducted.

[82] 104-105 cells of Pitysproum ovale were cultured on a medium which was sup- plemented with the shampoo composition prepared in the Preparation Example in an amount of 5 wt% based on the total weight of the medium. After culturing for 12 hours, the cell number of the dandruff-causing fungus was counted. For a control, a commercially available shampoo containing no terpenlactone compounds was employed. The results are given in Table 3, below.

[83] Table 3 Non-treated After treatment with Shampoo Control composition of the present invention cell number of the 14,000 930 10,500 dandruff-causing fungus (cells/g) [84] [85] As shown in Table 3, the treatment with the shampoo composition of the present invention reduced the cell number of the dandruff-causing fungus by 95% compared with non-treatment.

[86] [87] EXPERIMENTAL EXAMPLE 3 : Dandruff-Reducing Effect [88] To examine whether the composition of the present invention reduces dandruff, the following experiment was conducted.

[89] 48 men aged 19-35 who suffered from severe dandruff were divided into 7 groups of 6 persons each and tested for one month.

[90] They washed their hair with a conventional shampoo every three days for one month and dandruff formed at the end of three days was collected. Likewise, the shampoo composition prepared in Preparation Example was used to wash their hair every three days and dandruff formed at the end of three days was collected. Dandruff was collected directly from the scalp by use of an aspirator and measured for weight.

Dandruff reduction rates were calculated according to Equation 1.

[91] For comparison, a commercially available shampoo composition containing no ter- penlactone compounds was employed.

[92] <Equation 1> [93] Dandruff Reduction Rate (%) = Wt. of Dandruff formed Before Experiment (mg) - Wt. of Dandruff formed After Experiment (mg) x100 Wt. of Dandruff Before Experiment (mg) [94] [95] The results are given in Table 4, below.

[96] Table 4 Shampoo Composition Inventive Conventional Dandruff Reduction 65.3 4.2 Rate (%) [97] [98] As shown in Table 4, with a superior anti-dandruff effect to conventional shampoo, the shampoo composition of the present invention reduces dandruff by 65.3%.

[99] [100] EXPERIMENTAL EXAMPLE 4 : Curing Effect on Scalp Itching [101] 30 men and 30 women from their teens to their sixties, who were all troubled with scalp itching, were made to wash their hair once every three days for two weeks with the shampoo composition prepared in Preparation Example. Measurement was made of the itching-curative effects of the shampoo composition of the present invention according to the standards given below. For comparison, a commercially available shampoo containing no terpenlactone compounds was used.

[102] The results are given in Table 5, below.

[103] Table 5 Shampoo Composition Inventive Conventional Curing for scalp itching 4.6 2. 1 [104] x Excellent (5 points), Good (4 points), Moderates points), [105] Poor (2 points), Very Poor (l point).

[106] [107] As shown in Table 5, the shampoo composition of the present invention has far better curative effects on scalp itching than does the conventional one.

[108] [109] EXPERIMENTAL EXAMPLE 5 : Depilation-Preventive Effect [110] 30 men and 30 women from their teens to their sixties who were all troubled with severe or mild depilation used the shampoo composition to wash their hair for one month and then were examined for depilation reduction.

[111] After being washed with the shampoo composition and completely rinsed, their hair was completely dried. During washing and drying, the scalp was lightly massaged with the fingers only, using neither brushes nor driers.

[112] For comparison, a commercially available shampoo containing no terpenlactone compounds was used.

[113] The results are given in Table 6, below.

[114] Table 6 Shampoo Composition Inventive Conventional Depilation Redaction 77.4 2.2 rate (%) [115] [116] As seen in Table 6, the shampoo composition of the present invention has good depilation-retardant activity.

[117] [118] EXPERIMENTAL EXAMPLE 6 : Inhibitory Activity Against Dandruff-Causing Fungus in Animals [119] To examine whether the composition of the present invention has antifungal activity against fungus that causes dandruff in dogs, the following experiments were conduced.

[120] [121] 1. Test for antifungal activity [122] Used for this test was Malassezia pachydermatis, which was isolated from the skin of a patient dog with atopy dermatitis.

[123] Under an optimal growth condition, Malassezia pachydermatis was cultured for 3-4 days in an SDA medium (Saboraud's Dextrose Broth 30g + agar 15g), and the culture was diluted with a phosphate buffer to adjust the cell number to a population of 2x104 cells/ml. The cells were cultured for 3-4 days on an SDA plate supplemented with ginkgolide A, ginkgolide B and bilobalide, prepared as in the Preparation Example, followed by counting the number of colonies formed. Ketooonazole was used as a positive control.

[124] The results are given in Table 7, below.

[125] Table 7 Samples ColonyNo. RemovalRate (%) Non-treated group 270 0 Ketooonazole (lOOmg/f) 205 24.1 Ginkgolide A (100mg/f) 115 57.4 Ginkgolide B (lOOmg/f) 97 64.1 Bilobalide (100mg/l) 56 79.3 Ginkgolide A (65 mg/f) + 15 94.5 Ginkgolide B (20 mg/f) + Bilobalide (15 mg/f) [126] [127] As shown in Table 7, a combination of ginkgolide A 65 wt%, ginkgolide B 20 wt% and bilobalide 15 wt% removed 94.5 % of the fungus and is superior in antifungal activity to individual compounds, that is, ginkgolide A with a removal rate of 57.4%, ginkgolide B with a removal rate of 64. 1 %, and bilobalide with a removal rate of 79.3%. Consequently, a combination of ginkgolide A, ginkgolide B and bilobalide shows synergistic and additive effects and thus inhibits the growth of Malassezia pachydermatis, a pathogen to animals such as dogs and cats, more effectively than individual ingredients.

[128] [129] 2. Test for deodorization and skin irritation [130] 12 beagles (4 males and 4 females) eight weeks old, were bathed once with the composition of the present invention and since then, deodorization and skin irritation were measured for the next 12 days.

[131] The results are given in Table 8, below.

[132] Table 8 Days Dogs After #1 #2 #3 #4 #5* #6 #7 #8 #9 #10 #11 #12 bath 1 O O O O O O O O O O O O 2 O O O O O O O O O O O O 3 O O O O O O O O O O O O 4 + + + + + + + + + + + + 5 + + + + + + + + + + + + 6 + + + + + + + + + + + + 7 + + + + + + + + + + + + 8 + + + + + + + + + + + + 9 x + + x + x x x x + + + 10 x x x x x x x x x x x x 11 x x x x x x x x x x x x 12 x x x x x x x x x x x x [133] i O : free of odor [134] + : some bad odor and shampoo flavor coexisting.

[135] x : offensive odor with no shampoo flavor remaining.

[136] dog (*) #5 : greasy scales formed after shampooing [137] [138] No bad odor was generated, as shown in Table 8, until three days after bathing with the shampoo composition of the present invention. From four to eight days after bathing, the dogs started to develop a bad odor while losing the shampoo smell.

Generally, the dogs regained offensive odors nine days after bathing. Therefore, it is preferred to bathe indoor dogs once a week and outdoor dogs twice a week. Over- sensitive responses such as skin erythema were not observed.

[139] The shampoo composition of the present invention can be useful for animals as it is found to inhibit the activity of Malassezia pachydermatis and shows good deodorizing effects without the occurrence of side effects such as skin erythema.

[140] [141] EXPERIMENTAL EXAMPLE 7 : Test for Antibacterial Activity [142] To examine the antibacterial activity of the composition of the present invention, the following experiment was carried out.

[143] Escherichia coli (AB1157) as bacteria; Aspergillus oryzae var. oryzaeKCTC 6095) as fungi; Pseudomonas corrugataKACC10141), Pseudomonas syringae (KACC10396) and Ralstonia solanacearumKACC10475), all causative of tomato and cucumber pith necrosis; Glomerella cingulata, causative of anthrax; and Phytophthora drechsleri, pathogenic to lettuce, were used for the test.

[144] E. coli (AB1157) was cultured in an LB medium (Bacto-trypton lOg/1 + Bacto- Yeast extract 5g/1 + NaCl lOg/1 + Bacto-Agar 15g/1 + distilled water l. Og/1, pH 7.0) at 37°C for 24 hours with agitation. Cell concentration was determined using a serial dilution method with 50 mM potassium phosphate buffer (pH 7.0). Biomass which survived active ingredients was suitably diluted and 1000 of the dilution was spread over a nutrient agar medium and cultured for 24 hours, followed by cell counting.

[145] A. oryzae was inoculated in a slant medium prepared from potato dextrose (Eifoo) plus agar (1.5%, Junsei) and then incubated for 4 days at 30°C. Conidia formed from the strain were removed by vortexing with 3 ml of Tween 80 (0.02%, Junsei) added.

Using a hemacytometer, oonidia were determined to have a concentration of 10 conidia/ml before entrance into a test. After sterilization, cell fractions were diluted in series and 1000 of the dilution was spread over a PDA plate supplemented with 0.1% ox-gall, followed by determining the number of the fungus surviving.

[146] All of the other cells were cultured in King's B media (proteose peptone # 3 20. 0g + K HPO 1. 5g + MgSO 7H O 1. 5g + glycerol 15.0 ml + agar 15. 0g + distilled water 1 24 42 liter, pH 7.2) at 27°C for 24 hours with agitation.

[147] Under the above-mentioned conditions, the composition of the present invention (ginkgolide A 65 wt%, ginkgolide B 20 wt%, bilobalide 15 wt%) was tested for inhibitory activity against the microorganisms, with ampiållin used as a control.

[148] The results are given in Table 9, below.

[149] Table 9 Strains Minimal Inhibitory Conc. (MIC, mg/1) Composition of the present Ampiållin invention E. coli 2.5 0.02 Aspergillus oryzae 5.25 0.08 Pseudomonas corrugata 1.45 0.01 Pseudomonas syringe 2.2 0.01 Ralstonia solanacearum 1.85 0.01 Glomerella cingulata 1.85 0.02 Phytophthora drechsleri 1.65 0.02 [150] [151] As shown in Table 9, demonstrating superior inhibitory activity against various pathogens, higher MIC values were obtained in the composition of the present invention over all the strains than in the control.

[152] [153] EXPERIMENTAL EXAMPLE 8 : Test for Pesticidal Activity [154] To examine the pesticidal activity of the composition of the present invention, Tetranychus urticae and Myzus persicae were tested as follows.

[155] Toxicity to Tetranychus urticae was determined by a leaf disk method. For a test for acaricadal activity against female imagoes, kidney bean leaves were cut into a size of 2x2 cm and put on a piece of water-soaked sanitary cotton in a Petri dish after which 30 female imagoes were transferred to the leaf scraps by use of a fine brush and allowed to stand for 30 min. At a distance of 25 cm from the Petri dish, the composition prepared in the Preparation Example (ginkgolide A 65 wt%, ginkgolide B 20 wt%, bilobalide 15 wt%) was hand-sprayed for 5 sec in such an amount as to suf- ficiently wet the leaf scraps which were than dried in a shady place. After storing the Petri dish at room temperature for 24 and 72 hours, measurement was made of the acricidal rate. If they were observed to be unable to move a distance as long as their body length under an anatomy microscope when being contacted with a brush, the insects were considered dead.

[156] A bioassay with seedlings was conducted for Myzus persicae. In this experiment, the insects were transferred to pepper seedlings and the number of insects settling on the seedlings was counted. Thereafter, the composition prepared in Example 1 was hand-sprayed three times over the insects on the pepper seedlings and insecticidal activity was examined within 24 and 72 hours after the spraying.

[157] The results are given in Table 10, below.

[158] Table 10 No. ofSpraying Insecticidal Rate (%) Tetranychus urticae Myzus persicae <24 hrs <72 hrs <24 hrs <72 hrs The 1st 96. 7 100.0 84.3 95.8 composition 2na g6, 2 96. 6 78. 0 87.2 of the present 3rd 93.1 96.6 86.2 99.6 invention(Ex Avg. 92.0 97.7 82.8 94.2 ample) [159] [160] As shown in Table 10, the composition of the present invention (ginkgolide A 65 wt%, ginkgolide B 20 wt%, bilobalide 15 wt%) was found to kill 92.0 % of Tetranychus urticae within 24 hours and 97.7% within 72 hours. As for Myzus persicae, it was killed at a rate of 82.8% within 24 hours and 94.2% within 72 hours.

[161] These data demonstrate that the composition of the present invention has high in- sectiddal activity.

[162] [163] EXPERIMENTAL EXAMPLE 9 : Test for Insect-repellency [164] To examine whether the composition of the present invention repels insects, ex- periments with Tetranychus urticae, Spodoptera litura, Plutella xylostella and Myzus persicae were carried out as follows.

[165] For Tetranychus urticae, 30 female adults were transferred onto kidney bean leaf scraps with a size of 2x2cm over which the composition of the present invention (ginkgolide A 65 wt%, ginkgolide B 20 wt%, bilobalide 15 wt%) was sprayed. And at a distance of 5cm from the Petri dish, it isolated the composition of the present invention at room temperature. After 24 and 72 hours, the repellency rate was examined.

[166] In the case of Spodoptera litura, 10 larvae 2-3 days old were introduced to a pot in which Chinese cabbage was planted, after which the composition of the present invention was hand-sprayed over the Chinese cabbage. After 24 and 72 hours, the larvae residing on the Chinese cabbage were counted. This experiment was repeated three times.

[167] A Bioassay with seedlings was conduced for Myzus persicae. In this experiment, the insects were transferred to pepper seedlings and the number of the insects settling on the seedlings was counted. Thereafter, the composition prepared in Example was hand-sprayed three times over the insects on the pepper seedlings and was examined for repellency within 24 and 72 hours after the spraying.

[168] In all of the experiments, no insects were found to remain on the food leaves.

Therefore, the composition of the present invention has superior repellent activity against insects.

[169] Industrial Applicability [170] Compared with keteoonazole, used as a dandruff treatment agent, the composition comprising ginkgolide A, ginkgolide B and bilobalide according to the present invention has excellent inhibitory activity against Pityrosporum ovale, causative of dandruff in humans and has superior curative effects on dandruff and scalp itching and preventive effect on depilation. Also, the composition of the present invention ef- fectively inhibits the activity of Malassezia pachydermatis, causative of dandruff in dogs, and shows good deodorizing effects in animals, without side effects such as skin erythema. Thus, the composition of the present invention is suitable for use in shampoo for dandruff treatment and depilation prevention as well as in animal shampoo. Furthermore, the composition comprising ginkgolide A, ginkgolide B and bilobalide according to the present invention can be used in antibacterial and antifungal agents, pesticides and insect repellents thanks to its high activity against bacteria, fungi, pests and insects.