(12) United States Patent (10) Patent No.: US 6,355,627 B1 Ishida Et Al

Home , Hinokitiol

USOO6355627B1 (12) United States Patent (10) Patent No.: US 6,355,627 B1 Ishida et al. (45) Date of Patent: Mar. 12, 2002

(54) BRANCHED CYCLODEXTRIN CLATHRATE 5,730,969 A * 3/1998 Hora et al...... 424/85.1 COMPOUND OF HINOKITOLS AND 5,811,114 A * 9/1998 Knight et al...... 536/54 COMPOSITION CONTAINING THE SAME 5,955,093 A * 9/1999 Woo et al...... 424/401 (75) Inventors: Kenya Ishida; Kazutoshi Sakurai, FOREIGN PATENT DOCUMENTS both of Kanagawa (JP) EP O 822 447 A1 12/1998 (73) ASSignee: ItsInternation Corporation, * cited by examiner (*) Notice: Subject to any disclaimer, the term of this Primary Examiner James O. Wilson patent is extended or adjusted under 35 (74) Attorney, Agent, or Firm Sughrue Mion, PLLC U.S.C. 154(b) by 0 days. (57) ABSTRACT (21) Appl. No.: 09/244,106 The invention has an object of providing a hinokitiol which 1-1. is not damaged in the antibacterial activity and improved in (22) Filed: Feb. 4, 1999 the water solubility, heat stability (anti-sublimation (51) Int. Cl." ...... A61K 31/715 characteristics) and corrosion to metals and also providing a (52) U.S. Cl...... 514/58; 536/46; 536/103; composition comprising a hinokitiol. The invention resides 512/2; 512/3; 512/4 in a clathrate compound comprising clathration of hinokiti (58) Field of Search ...... 512/2, 3, 4, 536/16, ols by using a branched cyclodextrin Such as a branched 536/103; 514/58 B-cyclodextrin. The clathrate compound can be used in various applications requiring an antibacterial activity, Such (56) References Cited as hair cosmetics, bath agents, skin cosmetics and oral compositions. U.S. PATENT DOCUMENTS 5,024,998 A 6/1991 Bodor ...... 514/58 14 Claims, 5 Drawing Sheets U.S. Patent Mar. 12, 2002. Sheet 1 of 5 US 6,355,627 B1

C S Sd

YN SQ s

Cld

a. S S U.S. Patent Mar. 12, 2002. Sheet 2 of 5 US 6,355,627 B1

S Sd

6S.s s s

a. S. U.S. Patent Mar. 12, 2002. Sheet 3 of 5 US 6,355,627 B1

Sld

6S.g5 s

a S s U.S. Patent Mar. 12, 2002. Sheet 4 of 5 US 6,355,627 B1

S S>

6S. s

2. 3 s U.S. Patent Mar. 12, 2002. Sheet 5 of 5 US 6,355,627 B1

OOOO- S OOOO - S

O8969 -N. N is 9,869 – 1860s 911. 981 \ N 600E \ 19960)- y O6881C.19.1 - 196=|s 91781 - K si\ ao)= 8,671 - 1960RE Ef1899.1 1. 1666 OR Se e 66891 e e

S. S. US 6,355,627 B1 1 2 BRANCHED CYCLODEXTRIN CLATHRATE BRIEF DESCRIPTION OF THE DRAWINGS COMPOUND OF HINOKITOLS AND COMPOSITION CONTAINING THE SAME In the accompanying drawings, there are shown illustra tive embodiments of the invention from which these and BACKGROUND OF THE INVENTION other of its objectives, novel features, and advantages will be 1. Field of the Invention readily apparent. The present invention relates to a clathrate compound of In the drawings: hinokitiol or a metal Salt of hinokitiol and to a composition containing the clathrate compound. FIG. 1 is an analysis graph showing the result of a 2. Prior Art differential thermal analysis of a hinokitiol; Hinokitiols have a high antibacterial activity against Vari 1O FIG. 2 is an analysis graph showing the result of a ous Gram-positive bacteria and Gram-negative bacteria and differential thermal analysis of a G-B-CD; have been known to exhibit an antibacterial activity against FIG. 3 is an analysis graph showing the result of a mesitylene-resistant Staphylococcus aureus (MRSA) in differential thermal analysis of a mixture of a G-B-CD and recent years. By using the characteristics of a hinokitiol a hinokitiol which are blended at a ratio by weight of 1:9; based on its antibacterial activity, it is utilized in various 15 FIG. 4 is an analysis graph showing the result of a applications Such as medicament, fragrant cosmetic and differential thermal analysis of a compound prepared in food fields. Example 1; and However, hinokitiols have the drawbacks of high FIG. 5(A) shows the H-NMR spectrum of a hinokitiol at Sublimation, deficient thermal Stability and Strong corrosive the aromatic ring region and FIG. 5(B) shows the H-NMR neSS to metals, e.g. stainless, as well as poor Solubility in Spectrum of a compound prepared in Example 1. Water. For this, trials for water-solubilization of hinokitiols have DETAILED DESCRIPTION OF THE been made and clathrate compounds of disbranched cyclo INVENTION dextrins (hereinafter described as “disbranched CD” as the case may be) have been proposed (Japanese Patent Appli 25 The present invention will be explained in detail. cation Publication (JP-B) No. 1-53,856). However, the solu Examples of hinokitiols include, other than a hinokitiol, bilities of clathrates of disbranched CD in water at 15 C. are its metal Salts. Such as Sodium, potassium, magnesium, 13 g/100 ml in the case of C-CD clathrates containing 38 calcium, barium, aluminum, Zinc, copper, and Silver Salts. mol% of a hinokitiol, 0.3 g/100 ml in the case of B-CD The branched CD in the present invention are those clathrates containing 92 mol% of a hinokitiol and 0.9 g/100 having the Structure in which one or two molecules of an ml in the case of Y-CD clathrates containing 60 mol% of a oligosaccharide Such as glucose, maltose and maltotriose are hinokitiol. The solubility of each clathrate including the bound with a CD such as an OL-CD, B-CD, or Y-CD at a-1,6 B-CD clathrate is insufficient. Moreover, hinokitiols have a positions. Those having the Structures in which one and two solubility insufficient to be formulated into fragrant 35 glucose molecules are bound are called "glucosyl CD cosmetics, health and Sanitary materials or medicaments. (hereinafter abbreviated as “G-CD”) and “diglucosyl CD” SUMMARY OF THE INVENTION (hereinafter abbreviated as “G-G-CD”) respectively. Likewise, those having the Structures in which one and two Present inventors have conducted various studies to solve maltose molecules are bound are called “maltosyl CD' the above problems and as a result found that the solubilities (hereinafter abbreviated as “G-CD”) and “dimaltosyl CD” of hinokitiols in water, fragrant cosmetics, health and Sani 40 (hereinafter designated as "G-G-CD) respectively. In the tary materials and medicaments can be improved in contrast present invention, preferred examples of the branched CD with clathrates of disbranched CD without damaging the include branched B-CDs such as a G-B-CD and G-B-CD. antibacterial activity of a hinokitiol by clathration of hino These branched CDs may be used either singly or in kitiols by using branched cyclodextrin (hereinafter combinations of two or more. described as “branched CD” as the case may be). The 45 present inventors have also found that the Sublimation, the The clathrate compound of the present invention can be corrosive characteristics and the like of a hinokitiol can be produced by bringing the branched CD into contact with at improved and the invention was thus completed. least one compound Selected from a hinokitiol and its metal According to a first aspect of the present invention, there Salts in the presence of water to produce a clathrate com 50 pound of the hinokitiols and branched CD and, as required, is provided a clathrate compound comprising clathration of by drying and powdering the resulting clathrate compound. hinokitiols by using a branched cyclodextrin. The above contact operation is generally performed by In preferred embodiments, the hinokitiols are at least one dissolving the branched CD in water, adding the hinokitiols compound Selected from the group consisting of a hinokitiol to the Solution and Stirring or shaking the mixture vigorously and a metal Salt of hinokitiol; 55 for Several Seconds to Several hours by using a stirrer, the branched cyclodextrin is a branched B-cyclodextrin; homogenizer or the like. Further possible clathration meth and ods include a method of Shaking the above components in a the clathrate compound is a water-Soluble powder. closed container and a method of treating the above com According to another aspect of the present invention, ponents ultraSonically. The hinokitiols may be added either there is provided a composition comprising the clathrate 60 in the form of a crystal as it is or in the form of a solution compound. produced by dissolving in an organic Solvent. In preferred embodiments, the composition comprising Given as examples of the organic Solvent are acetone, the clathrate compound has an antibacterial activity; and ethanol and methanol, in which acetone is most preferable. the composition comprising the clathrate compound is a There are no limitations to the amount of the organic Solvent composition Selected from the group consisting of a coS 65 as long as it can dissolve hinokitiols. The amount of the metic composition, a bath agent, a detergent and an oral hinokitiols is generally 0.1 to 1 equivalent mol and prefer composition. ably 0.5 to 1.0 equivalent mol based on the branched CD. US 6,355,627 B1 3 4 The contact reaction temperature is generally 0 to 60° C. and 12 ml of water was added a Solution produced by dissolving preferably 5 to 35° C. 0.4 g (2.44 mmol, the ratio to the CD: 11.1% by weight) of The resulting clathrate compound is dried and powdered a hinokitiol (manufactured by Takasago International as it is thereby producing a clathrate compound powder Corporation) in 8 ml of acetone. The mixture was stirred at 35 C. for about one hour and acetone was then distilled containing the hinokitiols. under reduced pressure to obtain a transparent Solution. The The fact that the clathrate compound of the present resulting Solution was treated for one minute in an ultrasonic invention is not a simple mixture but forms a clathrate can homogenizer (manufactured by Kaijo Electric Corporation; be confirmed by a differential thermal analysis using a 0.8A; frequency: 38 KHZ) and was then allowed to stand in differential Scanning calorimeter (DSC) and by a measuring a refrigerator at 10 C. all night. The solution made cloudy method using a nuclear magnetic resonance instrument was freeze-dried and then powdered in a mortar to obtain (NMR) 3.90 g of the object branched CD clathrate compound of The branched CD clathrate compound of hinokitiol pre hinokitiol as a white powder. pared in the present invention may be used in various forms, The resulting clathrate compound was Subjected to the for example, in the form of a powder or Solution as it is. 15 measurements of the proton nuclear magnetic resonance The branched CD clathrate compound of hinokitiol pre spectrum (H-NMR)(solvent: deuterated dimethylsulfoxide pared in the present invention must be varied in its concen (CD) SO and UV-ray absorption spectrum (241 nm) with tration to be adaptable to the proper range according to the respect to its aqueous Solution. As a result, it was confirmed types and object of products to which an antibacterial that the mol ratio of G-B-CD:hinokitiol of this compound activity is to be imparted. The clathrate compound of the was about 1:0.93 (a hinokitiol was contained in an amount present invention is used in a concentration of generally 0.01 of 105 mg/1000 mg). to 10% by weight and preferably 0.1 to 5% by weight based In order to confirm that the resulting compound was not on the total amount of all components of the product. a simple mixture but formed a clathrate, a mixture of a In the object product compositions to which the clathrate G-B-CD and a hinokitiol which were mixed in a ratio by compound of the present invention is applied, optional 25 weight of 1:9 was formed. Four Samples consisting of the components corresponding to its object in use are appropri above mixture, a hinokitiol, a G-B-CD and the above ately compounded thereby providing these compositions as resulting compound were Subjected to a differential thermal fragrant cosmetics, health and Sanitary materials or medi analysis. FIGS. 1 to 4 show the results of the analysis when caments. Specifically, the clathrate compound of the present the temperature was raised at a rate of 10 C. per minute. invention can be compounded in, for example, Skin FIGS. 1 to 4 are the graphs of the differential thermal cosmetics, shampoos, rinses, fragrances, colognes, hair analysis (DSC:+4 mcal/sec) of the hinokitiol, G-B-CD, tonics, hair cosmetics including hair creams, bath agents, mixture of G-f-CD and a hinokitiol blended in a ratio by Soaps, detergents, Softeners, room fragrances, furniture weight of 1:9 and the compound prepared in this example cares, disinfectants, insecticides, bleaching agents, dental respectively. creams, oral detergents, or ointments. It is preferably used, 35 Comparing FIGS. 1 to 4, the endothermic peak of the particularly, for preparations, Such as lotions for all body, hinokitiol is observed in FIG. 3, but is not observed at all in FIG. 4. Therefore, it is confirmed that, in the compound of oral detergents and Shampoos, which use water as a major this example, the G-B-CD and the hinokitiol form a clath component and preparations, Such as bath agents, which is rate. used by dissolving in water, these preparations being shown FIG. 5 shows the "H-NMR spectrums (heavy water in the examples described later. 40 solution) of (A) the hinokitiol and of (B) the compound of DESCRIPTION OF THE PREFERRED this example, namely, the clathrate compound of the G-B- EXAMPLES CD and hinokitiol at the aromatic ring region of a hinokitiol by using tetramethylsilane (TMS) as the external standard. The present invention will be explained in detail by way The difference between the spectrums of (A) and (B) is due of examples and comparative examples which are not 45 to the clathration by the G-f-CD. The measurement of intended to be limiting of the present invention. The clath "H-NMR of a substance containing a clathrate material rate compounds (branched CD) used in the following extracted by an organic Solvent from a Solution of the above examples are as follows: clathrate compound showed that the substance exhibited the (i) G-f-CD: glucosyl-B-cyclodextrin Same spectrum pattern as a hinokitiol. From this result, it (ii) G-B-CD: maltosyl-B-cyclodextrin 50 was confirmed that a hinokitiol formed a clathrate, for (iii) Isoeleat P(trademark): manufactured by Ensuiko Sugar instance, without being resolved. Refining Co., Ltd., the amount of a branched G-CD: 50% or more, the ratio of C-CD: B-CD:Y-CD=6:3:1. EXAMPLE 2 The following instruments were used in the following (Preparation of a G-?-CD Clathrate Compound of measurementS. 55 Hinokitiol) Nuclear magnetic resonance spectrum: 'H-NMR:AM-400 3.6 g (2.47 mmol) of a G-f-CD (manufactured by (400 MHz) (manufactured by Brucker Co., Ltd.) Ensuiko Sugar Refining Co., Ltd.) was dissolved with stir Differential scanning calorimeter: DSC-50 (manufactured ring in 12 ml of water. To the mixed Solution was added a by Shimadzu Corporation) solution produced by dissolving 0.4 g (2.44 mmol, 0.99 UV-ray absorption spectrum (UV): UV-260 (manufactured 60 molar equivalents, the ratio to the CD: 11.1% by weight) of by Shimadzu Corporation) a hinokitiol in 8 ml of acetone. The mixture was stirred at 35 C. for about one hour and acetone was then distilled EXAMPLE 1. under reduced pressure to obtain a transparent Solution. The (Preparation of a G-?-CD Clathrate Compound of resulting Solution was treated in the same manner as in Hinokitiol) 65 Example 1 to obtain 3.95 g of a dry clathrate compound of To 3.6 g (2.78 mmol) of a G-B-CD (manufactured by a white color. The amount of a hinokitiol in the clathrate Ensuiko Sugar Refining Co., Ltd.) which was dissolved in compound was measured by means of an absorption Spec US 6,355,627 B1 S 6 trum in a UV range (241 nm) using an aqueous Solution hinokitiol. The resulting clathrate compound was Subjected containing the clathrate compound. The measurement to the measurements of the proton nuclear magnetic reso showed that a clathrate compound containing a hinokitiol in nance spectrum (H-NMR)(solvent: deuterated dimethyl a ratio by mol of 1:0.71 (a hinokitiol is contained in an sulfoxide (CD) SO and UV-ray absorption spectrum (245 amount of 74 mg/1000 mg) was produced. 5 nm) with respect to its aqueous Solution. As a result, it was confirmed that the mol ratio of B-CD:hinokitiol of this EXAMPLE 3 compound was about 1:0.90 (a hinokitiol was contained in (Preparation of a G-f-CD Clathrate Compound of a an amount of 115 mg/1000 mg). Sodium Salt of Hinokitiol) The Solubilities of the above various branched CD clath 3.6 g (2.47 mmol) of a G-B-CD was dissolved with rate compounds prepared in the above examples and refer stirring in 12 ml of water. To the mixed solution was added ence example in water at 15 C. are shown in Table 1. a solution produced by dissolving 0.4 g (2.15 mmol, 0.77 molar equivalents, the ratio to the CD: 11.1% by weight) of TABLE 1. a sodium salt of hinokitiol. The mixture was stirred at 35 C. Hinokitiols % for about one hour and acetone was then distilled under 15 reduced pressure to obtain a transparent Solution. The result Hinokitiol <0.05 ing Solution was Successively treated in the same manner as B-CD clathrate compound of hinokitiol O.3 in Example 1 to obtain 3.86 g of a dry clathrate compound G-B-CD clathrate compound of hinokitiol 15.O prepared in Example 1 of a slightly yellowish white color. The amount of a sodium G-B-CD clathrate compound of hinokitiol 47.5 Salt of hinokitiol in the clathrate compound was measured by prepared in Example 2 means of an absorption spectrum in a UV range (244 nm) Isoeleat P clathrate compound of hinokitiol 24.7 using an aqueous Solution containing the clathrate com prepared in Example 4 pound. The measurement showed that a clathrate compound containing a Sodium Salt of hinokitiol in a ratio by mol of 1:0.62 (a Sodium Salt of hinokitiol is contained in an amount Test Example 1 of 80 mg/1000 mg) was produced. 25 (Evaluation of the Corrosion Resistance of a Hinokitiol) Test pieces made of StainleSS were placed in each aqueous EXAMPLE 4 Solution of a hinokitiol, a Sodium Salt of hinokitiol and the (Preparation of an Isoeleat P Clathrate Compound of G-B-CD clathrate compound of a hinokitiol obtained in Hinokitiol) Example 1. These aqueous Solutions were Stirred in the same 3.6 g of an Isoeleat P (manufactured by Ensuiko Sugar condition at room temperature (25 C.) and 90° C. to inspect Refining Co., Ltd., the amount of a branched CD: 50% or the corrosion of stainless. The results are shown in Table 2. more, the ratio of C-CD: B-CD:Y-CD=6:3:1) was dissolved Test panel with stirring in 12 ml of water. To the mixed solution was Sus-316L (manufactured by Nippon Test Panel Industries added a Solution produced by dissolving 0.4 g (2.44 mmol, Co., Ltd.); and the ratio to the CD: 11.1% by weight) of ahinokitiol in 8 ml 35 Sus-304 (manufactured by Nippon Test Panel Industries of acetone. The mixture was stirred at 35 C. for about one Co., Ltd.). hour and acetone was then distilled under reduced preSSure Consequently, as shown in Table 2, in the condition of to obtain a transparent Solution. The resulting Solution was room temperature, Sus-316L and Sus-304 (stainless test Successively treated in the same manner as in Example 1 to piece) were reduced in thickness by 0.57 mm/year and 0.67 obtain 3.72 g of a dry clathrate compound of a white color. 40 mm/year respectively in an aqueous 0.1% Solution of the The amount of a hinokitiol in the clathrate compound was hinokitiol whereas Sus-316L and Sus-304 (stainless test measured by means of an absorption Spectrum in a UV range piece) were reduced in thickness by 0.05 mm/year and 0.10 (244 nm) using an aqueous Solution containing the clathrate mm/year respectively in an aqueous 0.1% Solution of the compound. The measurement showed that a clathrate com 45 Sodium Salt of hinokitiol. By using a Sodium Salt of pound containing a hinokitiol in a ratio by weight of 1:0.03 hinokitiol, the corrosion could be reduced to about one-tenth (a hinokitiol is contained in an amount of 30 mg/1000 mg) that of a hinokitiol. Moreover, in the case of using the was produced. clathrate compound of the present invention, SuS-316L and Sus-304 (stainless test piece) were reduced in thickness by Reference Example 1 0.01 mm/year and 0.03 mm/year respectively in an aqueous (Preparation of a f-CD Clathrate Compound of Hinokitiol) 50 To 4.56 g (4.0 mmol) of a B-CD (manufactured by Nippon 1.0% Solution of the clathrate compound at room tempera Food Industrial Co., Ltd.) which was dissolved in 70 ml of ture. water at 30° C. was added a solution produced by dissolving 0.66 g (4.0 mmol) of a hinokitiol in 16 ml of acetone. The TABLE 2 mixture was heated to about 50 C. and was stirred for about 55 Material one hour to obtain a transparent Solution. The resulting Solution was treated for one minute in a homogenizer Corrosion rate at 25 C. (mm/year) SuS 316L SuS 304 (manufactured by Kaijo Electric Corporation; 0.8 A; fre 0.1% aqueous solution of hinokitiol 0.57 mm O.67 mm quency: 38 KHZ) and was then allowed to stand in a 0.1% aqueous solution of sodium salt of 0.05 mm 0.10 mm refrigerator at 10 C. all night. The solution made cloudy 60 hinokitiiol was added to 300 ml of acetone whose temperature was -35 1.0% aqueous solution of clathrate of 0.01 mm O.O3 mm. C. and the resulting Solution was mixed with Stirring. Next, hinokitiol prepared in Example 1 Suction filtration was carried out immediately to Separate and collect a white crystal, which was then dried under Next, as shown in Table 3, in the condition of a tempera reduced pressure for 8 hours in a vacuum desiccator. The 65 ture of 90° C., Sus-316L and Sus-304 (stainless test piece) resulting clathrate compound was powdered in a mortar to were reduced in thickness by 1.96 mm/year and 3.27 obtain 4.69 g of the object B-CD clathrate compound of mm/year respectively in an aqueous 0.1% Solution of the US 6,355,627 B1 7 8 hinokitiol whereas Sus-316L and Sus-304 (stainless test Aerobic Bacteria piece) were reduced in thickness by 0.15 mm/year and 0.30 Sa: Staphylococcus aureus IAM 1011 mm/year respectively in an aqueous 0.1% Solution of the Se: Staphylococcus epidermidis JCM 2414 Sodium Salt of hinokitiol. Moreover, in the case of using the Cm: Corynebacterium minutissiumum ATCC 23348 clathrate compound of the present invention, Sus-316L and Ec: Escherichia coli IFO 3972 Sus-304 (stainless test pieces) were reduced in thickness by Sm: Serratia marcessenes IFO 3736 0.05 mm/year and 0.08 mm/year respectively in an aqueous Pv: Proteus vulgaris IFO 3167 1.0% solution of the clathrate compound. By using the Mf: Malassezia furfur IFO O656 G-B-CD of hinokitiol, no production of rust was observed Kp: Klebsiella pneumoniae IFO 13277 and it was thus clarified that a corrosion to Stainless could be CX: Corinebacterium Xerosis JCM 1324 1O greatly reduced. Anaerobic Bacteria Pa: Propionibacterium acnes ATCC 12818 TABLE 3 Fn: Fusobacterium nucleatum JCM 6328

Material The Samples used in the antibacterial test are as follows in 15 which butylparabene was used as a control compound. The Corrosion rate at 90° C. (mm/year) SuS 316L SuS 304 concentrations of clathrate compounds were all measured by converting into the concentrations of a hinokitiol. Samples 0.1% aqueous solution of hinokitiol 1.96 mm 3.27 mm 0.1% aqueous solution of sodium salt of 0.15 mm. O.30 mm of antibacterial agents hinokitiiol 1. Hinokitiol (Takasago International Corporation) 1.0% aqueous solution of clathrate of 0.05 mm. O.08 mm 2. G-B-CD clathrate compound of hinokitiol (the com hinokitiol prepared in Example 1 pound of the present invention, described as “G-CD clathrate” in the tables) 3. Butylparabene (manufactured by Tokyo Chemical Indus Test Example 2 try Co., Ltd.) (Evaluation of the Heat Stability (Sublimation) of a Method: Serial Double Dilution Method Hinokitiol) 25 1000 mg of each of a hinokitiol, a sodium salt of AS for the culture medium, an aqueous Solution contain hinokitiol, the G-B-CD clathrate of hinokitiol prepared in ing 38 g/l of a Muller-Hilton agar medium (manufactured by Example 1, the G-B-CD clathrate of hinokitiol prepared in DEFCO) was used for aerobic bacteria and an aqueous Example 2 and the Isoeleat P clathrate of hinokitiol prepared Solution containing 59 g/l of a GAM agar medium in Example 4 were allowed to stand at 90° C. for 20 hours (manufactured by Nissui Pharmaceutical Co., Ltd.) for to measure the residual weight. The results of the residual anaerobic bacteria. Each Subject compound was added to weight, sublimation rate (%) and sublimation rate (%) each corresponding medium in a manner that the concen converted into a hinokitiol were shown in Table 4. tration of a hinokitiol was 400 ug/ml and diluted according As shown in Table 4, it was confirmed that the hinokitiol to the Serial double dilution method using the Same medium. was Sublimated in an amount of 99.2% and even the Sodium 35 10 ml of each resulting medium was poured into a plastic Salt of hinokitiol was Sublimated in an amount of 23.6%. On Schale with an inside diameter of 90 mm and was solidified. the other hand, the Sublimation of each branched CD clath The Solidified medium in the Schale was divided. 0.1 ml of rate was significantly restrained. Particularly, the Sublima a distilled water Suspension containing the Subject microor tion rate of the G-B-CD clathrate was 0.4% and when it was ganisms (the number of microorganisms: 10-10"/ml) was converted into the Sublimation rate of a hinokitiol, it was inoculated into each compartment and incubated at 37 C. confirmed that the Sublimation rate of a hinokitiol was 40 for 24 hours. The groSS growth of each bacteria was reduced to 4.3%. Specifically, it was confirmed that the observed to determine the minimal inhibitory concentration Sublimation of a hinokitiol, if it was made into a branched (MIC, ug/ml) which inhibited the growth of microorgan CD clathrate, can be prevented and a reduction in the isms. The results are shown in Tables 5 and 6. Incidentally amount of a hinokitiol due to Sublimation can be restrained the incubation of anaerobic bacteria was performed using a more Significantly than in the case of a hinokitiol or alkaline 45 metal Salts. gas pack jar for incubating anaerobic bacteria. TABLE 5 TABLE 4 Se Cm Cx Mf Sa Ec Sm Pw Sublimation rate 50 Residual (%) Hinokitiol 50 50 50 50 50 50 200 50 weight (Converted into G1-CD clathrate 50 25 50 25 50 25 200 25 Hinokitiols (mg) hinokitiol) Butylparabene 1OO 100 1 OO 100 100 4OO >4OO 100 Hinokitiol 8 99.2 Nasalt of hinokitiol 764 23.6 G-B-CD clathrate of hinokitiol 996 0.4(4.3) 55 prepared in Example 1 TABLE 6 G-B-CD clathrate of hinokitiol 991 0.9(12.6) prepared in Example 2 Kp Pa Fn Isoeleat P clathrate of hinokitiol 996 0.4(13.0) prepared in Example 4 Hinokitiol 50 1OO 1OO 60 G-CD clathrate 50 1OO 1OO Butylparabene 1OO 2OO 1OO Test Example 3 (Test for Antibacterial Activity) As is clear from Tables 5 and 6, the clathrate compound The clathrate compound of the present invention and of hinokitiol of the present invention possesses the same or non-clathrate compounds were each Subjected to an anti 65 higher antibacterial activity than a hinokitiol, showing that bacterial test. Aerobic and anaerobic bacteria used in the test the clathration does not cause a reduction in the antibacterial are as follows: activity. US 6,355,627 B1 9 EXAMPLE 5 (Antibacterial Agent) -continued 5 parts by weight of the G-B-CD clathrate compound of hinokitiol prepared in Example 1 was dissolved in 95 parts Proportion by weight of distilled water to obtain a liquid antibacterial Component (% by weight) agent. This product as it is can be advantageously used as the Perfume O.05% antibacterial agent for antibacterial composition Such as Purified water Balance cosmetics and daily needs. EXAMPLE 6 (Bath Agent) EXAMPLE 9 Using the G-f-CD clathrate compound of hinokitiol prepared in Example 1, the following components were (Lotion) mixed according to a usual method to produce 100 g of a Using the G-f-CD clathrate compound of hinokitiol bath agent. 15 prepared in Example 1, the following components were mixed according to a usual method to produce 100 g of a lotion. Proportion Component (% by weight) G-?-CD clathrate compound of 0.5% Proportion hinokitiol prepared in Example 1 Component (% by weight) Sodium chloride 10.0% Silicic acid anhydride 0.5% G-B-CD clathrate compound of 2.0% Pigment O.OO1% hinokitiol prepared in Example 1 Perfume O.1% 25 Loofah extract liquid 2.0% Neutral sodium sulfate anhydride Balance 1,3-butylene glycol 5.0% Sodium citrate O.1% Ethanol 15.0% Pigment O.OOO5% Perfume O.05% EXAMPLE 7 Purified water Balance (Hair Lotion) Using the G-B-CD clatrate compound of hinokitiol pre pared in Example 1, the following components were mixed according to a usual method to produce 100 g of a hair EXAMPLE 10 lotion. 35 (Shampoo) Using the G-f-CD clathrate compound of hinokitiol Proportion prepared in Example 1, the following components were Component (% by weight) mixed according to a usual method to produce 100 g of a G-B-CD clathrate compound of 2.0% 40 Shampoo composition. hinokitiol prepared in Example 1 f-glycyrrhhetic acid O.2% 1-menthol O.1% Polyoxyethylene hydrogenated castor oil 5.0% Proportion BHT O.O.3% Component (% by weight) Perfume O.05% 45 Purified water Balance GB-CD clathrate compound of 1.0% hinokitiol prepared in Example 1 Decanoic acid O.05% Triethanolamine lauryl sulfate 18.5% EXAMPLE 8 Hydroxypropylmethyl cellulose 15.0% 50 Ammonium lauryl sulfate 8.0% (Oral Detergent) Cocamide 4.0% Using the G-B-CD clathrate compound of hinokitiol Palmitic acid O.3% prepared in Example 1, the following components were 1,3-dimethylol-5,5-dimethylhydantoin O.15% mixed according to a usual method to produce 100 g of an Disodium ethylenediamine tetraacetate O.05% oral detergent. Citric acid Trace 55 Common salt Trace Perfume 0.07% Purified water Balance Proportion Component (% by weight) G-C-CD clathrate compound of 1.0% 60 EXAMPLE 11 hinokitiol prepared in Example 1 Ethanol 12.5% (Detergent) Sodium lauryl sulfate 1.25% Glycerol 10.0% 1-menthol O.1% Using the G-f-CD clathrate compound of hinokitiol Saccharin O.OO1% 65 prepared in Example 1, the following components were Pigment O.OO3% mixed according to a usual method to produce 100 g of a detergent. US 6,355,627 B1 11 12 were mixed in the same manner as in Example 9 to produce a lotion. Proportion Component (% by weight) GB-CD clathrate compound of 2.0% Proportion hinokitiol prepared in Example 1 Component (% by weight) Senecioic acid O.1% White vaseline 6.0% B-CD clathrate compound of 2.0% Alkylallyl polyether sulfonate 50.0% hinokitiol prepared in Reference Cholesterol 2.0% Example 1 Perfume O.1% Loofah extract liquid 2.0% Purified water Balance 1,3-butylene glycol 5.0% Sodium citrate O.1% Ethanol 15.0% Pigment O.OO5% Comparative Example 1 Perfume O.05% (Hair Lotion) 15 Purified water Balance Using the f-CD clathrate compound of hinokitiol pre pared in Reference Example 1, the following components This product was a white Suspension and produced a were mixed in the same manner as in Example 7 to produce white precipitate when it was allowed to Stand at room a hair lotion. temperature with the result that it was unsuitable for the use as the lotion.

Proportion Comparative Example 4 Component (% by weight) (Shampoo) B-CD clathrate compound of 2.0% 25 Using the B-CD clathrate compound of hinokitiol pre hinokitiol prepared in Reference pared in Reference Example 1, the following components Example 1 were mixed in the same manner as in Example 10 to produce f-glycyrrhhetic acid O.2% a shampoo composition. 1-menthol O.1% Polyoxyethylene hydrogenated castor oil 5.0% BHT O.O.3% Perfume O.05% Purified water Balance Proportion Component (% by weight) This product was a white Suspension and produced a B-CD clathrate compound of 1.0% hinokitiol prepared in Reference white precipitate when it was allowed to Stand at room 35 Example 1 temperature with the result that it was unsuitable for the use Decanoic acid O.05% as the hair lotion. Triethanolamine lauryl sulfate 18.5% Hydroxypropylmethyl cellulose 15.0% Comparative Example 2 Ammonium lauryl sulfate 8.0% (Oral Detergent) Cocamide 4.0% Using the f-CD clathrate compound of hinokitiol pre 40 Palmitic acid O.3% pared in Reference Example 1, the following components 1,3-dimethylol-5,5-dimethylhydantoin O.15% Disodium ethylenediamine tetraacetate O.05% were mixed in the same manner as in Example 8 to produce Citric acid Trace an oral detergent. Common salt Trace Perfume 0.07% 45 Purified water Balance Proportion Component (% by weight) This product was a white Suspension and produced a white precipitate when it was allowed to Stand at room B-CD clathrate compound of 1.0% temperature with the result that it was unsuitable for the use hinokitiol prepared in Reference 50 Example 1 as the Shampoo. Ethanol 12.5% Sodium lauryl sulfate 1.25% Comparative Example 5 Glycerol 10.0% 1-menthol O.1% (Detergent) Saccharin O.OO1% 55 Using the B-CD clathrate compound of hinokitiol pre Pigment O.OO3% pared in Reference Example 1, the following components Perfume O.05% were mixed in the same manner as in Example 11 to produce Purified water Balance a detergent. This product was a white Suspension and produced a white precipitate when it was allowed to Stand at room 60 temperature with the result that it was unsuitable for the use Proportion as the oral detergent. Component (% by weight) B-CD clathrate compound of 2.0% Comparative Example 3 hinokitiol prepared in Reference (Lotion) 65 Example 1 Using the f-CD clathrate compound of hinokitiol pre Senecioic acid O.1% pared in Reference Example 1, the following components US 6,355,627 B1 14 formulated, the following fact is clarified from Table 7. -continued Specifically, all of the products of Comparative Examples 1 to 5 in which the disbranched CD was formulated had a Proportion minimal inhibitory concentration exceeding 400 ppm, Show Component (% by weight) ing that the antibacterial activity of a hinokitiol was reduced. White vaseline 6.0% On the other hand, all of the products of Examples 7 to 11 Alkylallyl polyether sulfonate 50.0% in which the branched CD clathrate was formulated had a Cholesterol 2.0% minimal inhibitory concentration as low as 100 ppm, Show Perfume O.1% ing that they maintained high antibacterial activities. Purified water Balance AS is evident from the above descriptions, according to the present invention in which a hinokitiol is formed into a This product was a white Suspension and produced a branched cyclodextrin clathrate, the antibacterial activity of white precipitate when it was allowed to Stand at room the hinokitiol is not damaged, its heat Stability is made high, temperature with the result that it was unsuitable for the use the corrosion to metals is inhibited and the solubility in as the detergent. water can be Significantly improved. Therefore, the clathrate 15 compound of the present invention, as an antibacterial agent Test Example 4 having high water-Solubility, is Suitable for the purpose (Comparative Test for Antibacterial Activity) intended to impart an antibacterial activity to many products Solutions of the hair lotions prepared in Example 7 and Such as hair cosmetics, bath agents, skin cosmetics, deter Comparative Example 1 respectively, Solutions of the oral gents and oral compositions. Particularly when a branched detergents prepared in Example 8 and Comparative Example B-cyclodextrin is used as the cyclodextrin, its effect is great. 2 respectively, Solutions of the lotions prepared in Example What is claimed is: 9 and Comparative Example 3 respectively, solutions of the 1. A clathrate compound comprising a clathrate of a Shampoos prepared in Example 10 and Comparative hinokitiol and a branched cyclodextrin wherein Said com Example 4 respectively and Solutions of the detergents pound has bactericidal activity of at least about twice the prepared in Example 11 and Comparative Example 5 respec 25 magnitude of a clathrated hinokitiol formed with tively were Subjected to a test for antibacterial activity unbranched cyclodextrins. against Staphylococcus aureus (Sa). 2. A clathrate compound according to claim 1, wherein the Sa: Staphylococcus aureus IAM 1011 hinokitiol is at least one compound Selected from the group Method: Like Test Example 3, as for the culture medium, an consisting of hinokitiol and a metal Salt of hinokitiol. aqueous Solution containing 38 g/l of a Muller-Hilton agar 3. A clathrate compound according to claim 1 or 2, medium (manufactured by DEFCO) was used for aerobic where in the branched cyclodextrin is a branched bacteria. Each Subject compound was added to each B-cyclodextrin. corresponding medium in a manner that the theoretical 4. A clathrate compound according to claim 1 or 2, amount of a hinokitiol was 400 ug/ml and diluted accord wherein the clathrate compound is a water-Soluble powder. ing to the Serial double dilution method using the same 35 5. A clathrate compound according to claim3, wherein the medium. 10 ml of each resulting medium was poured into clathrate compound is a water-Soluble powder. a plastic Schale with an inside diameter of 90 mm and was 6. A composition comprising the clathrate compound Solidified. The Solidified medium in the Schale was according to claim 1 or 2 and a carrier or diluent. divided. 0.1 ml of a distilled water Suspension containing 7. A composition comprising the clathrate compound the Subject microorganisms (the number of microorgan 40 according to claim 3 and a carrier or diluent. isms: 10°-10/ml) was inoculated into each compartment 8. A composition comprising the clathrate compound and incubated at 37 C. for 24 hours. The gross growth of according to claim 4 and a carrier or diluent. each bacteria was observed to determine the minimal 9. A composition according to claim 6, wherein the inhibitory concentration (MIC, ppm) which inhibited the composition comprising the clathrate compound has anti growth of microorganisms. The results are shown in 45 bacterial activity, Tables 7. wherein the composition is a composition Selected from the group consisting of a cosmetic composition, a bath TABLE 7 agent, a detergent and an oral composition. 10. A clathrate compound according to claim 3, wherein Hair lotion (G-f-CD clathrate) prepared in Example 7 1OO Hair lotion (B-CD clathrate) prepared in Comparative >400 50 the branched cyclodextrin is a branched B-cyclodextrin Example 1 Selected from the group consisting of a glucosyl Oral detergent (G1-?3-CD clathrate) prepared in Example 1OO B-cyclodextrin, a diglucosyl 3-cyclodextrin, a maltosyl 8 B-cyclodextrin, and a dimaltosyl B-cyclodextrin. Oral detergent (3-CD clathrate) prepared in Comparative >400 Example 2 11. A clathrate compound according to claim 10, wherein Lotion (G-B-CD clathrate) prepared in Example 9 1OO 55 the clathrate compound is a water-Soluble powder. Lotion (B-CD clathrate) prepared in Comparative Example >400 12. A composition comprising the clathrate compound 3 according to claim 10 and carrier or diluent. Shampoo (G-f-CD clathrate) prepared in Example 10 1OO Shampoo (B-CD clathrate) prepared in Comparative Example >400 13. A composition comprising the clathrate compound 4 according to claim 11 and carrier or diluent. Detergent (G-f-CD clathrate) prepared in Example 11 1OO 60 14. A composition according to claim 12, wherein the Detergent (3-CD clathrate) prepared in Comparative >400 composition comprising the clathrate compound has anti Example 5 bacterial activity, wherein the composition is a composition Selected from Making a comparison between the antibacterial activities the group consisting of a cosmetic composition, a bath of Examples 7 to 11 in which the branched CD clathrate was 65 agent, a detergent, and an oral composition. formulated and the antibacterial activities of Comparative Examples 1 to 5 in which the disbranched CD was k k k k k