Description

METHOD OF PRODUCING EXTRACT OF HORSE CHESTNUT

LEAF

Technical Field

[1] The present invention relates to a method of producing extract of Horse chestnut leaf with enhanced matrix metalloproteinases (MMPs) inhibitory activities and a composition comprising the extract as an active ingredient for treating or preventing MMPs -dependent diseases including periodontal disease. Background Art

[2] Matrix metalloproteinases (MMPs) are endopeptidase, which degrade or proteolyze the components of the extracellular matrix such as collagen, proteoglycan, and gelatin, and are classified into four groups: collagenase, gelatinase, stromelysin, and membrane-type MMP. They are secreted as proenzyme and their activation by degradation is a prerequisite for function (Bond, J. S., et al., Int. J. Biochem., 75, pp565-574 (1985); Chen, J. M., Chen, W. T., Cell, 48, ppl93~203 (1987).

[3] Increased expression or activation of MMPs is observed in many pathological states, such as atherosclerosis, restenosis, MMP-dependent-osteopathy, inflammation of the central nervous system, Alzheimer's disease, sMn aging, rheumatoid arthritis, osteoarthritis, septic arthritis, corneal ulcer, corneal synechia, iris synechia, osteoporosis, proteinuria, abdominal aortic aneurysm, regressive cartilage loss, myelinated nerve loss, multiple sclerosis, liver fibrosis, nephroglomerular disease (nephritic syndrome), Premature rupture of fetal membrane, inflammatory bowel disease, periodontal disease, periodontitis, gingivitis, senile macular degeneration, diabetic retinopathy, proliferate vitreous body retinopathy, immature retinopathy, eye inflammation, conical cornea, Sjogren's syndrome, myopia, eyes tumors, rejection of cornea implantation, an- giogenesis, cancer infiltration and metastasis, etc. (Woessner Jr., Ann. NY. Acad. ScL , 732, ppl l-21, 1994; Warner et al., Am. J. Pathol., 1^ pp2139-44, 2001; Stetler- Stevenson, Surg. Oncol. Clin. N. Am. , K), pp383-92, 2001).

[4] For example, strαnelysins are known to be the major enzyme for disruption of cartilage (Murphy, G. et al., Biochem. J., 248, pp265-268, 1987). Collagenases, gelatinases and stromelysins are responsible for the degradation of the extracellular matrix in many retinopathies (Brans, F.R. et al., Invest. Opthalmol. and Visual ScL, 32, ppl569-1575, 1989). Collagenases and stromelysins are identified in fibroblast from gingiva in inflammation, and the activity of the enzyme is dependent on the degree of

inflammation (Overall, CM. et aU J Periodontal Res, 22, pp81-88, 1987). MMP activity is highly enhanced in response to the bacterial infection and inflammation in gingival crevicular fluid taken from patients with periodontal disease. Destruction of collagen, a major structural component of the periodontal matrix, by MMP leads to gingival recession, pocket formation and tooth loss(Goulb, LM., Ryan ME. Williams RC, Dent Today, H, pp 102- 109, 1998).

[5] Chemically modified non- antimicrobial tetracyclines have been shown to suppress periodontal destruction and tooth loss by inhibiting pathologically elevated collagenase and other MMP activity ( Llavaneras A et aU J Periodontal, 72, pp 1069-77, 2001).

[6] There are several patents issued and disclosed using horse chestnut for ameliorating the periodontal diseases, but the effects were not clear.

[7] For example, US 4,486,404 had disclosed that horse chestnut seed extract was added in toothpaste as a blowing agent to ameliorate gingivitis, however it had no effect on gingival bleeding when using horse chestnut seed extract only. KR 138,248 had disclosed that horse chestnut extract has bactericidal effect on the cariogenic bacteria to prevent plaque formation and ameliorate the gingival inflammation, but it had written only the amelioration of the early gingivitis rather than treatment of periodontitis by inhibition of MMP activity. KR 533,777 and WO 03/035092 had described anti-angiogenic and MMP-inhibitory effect of 80% methanol extract of horse chestnut leaves or seeds, but the MMP-inhibitory effect of the extract was not high.

[8] Therefore the efficient method of producing extract of horse chestnut leaf with enhanced MMPs inhibitory activities is required for treating or preventing MMPs- dependent diseases including periodontal disease. Disclosure of Invention Technical Problem

[9] It is an objection of the present invention to provide a method of producing extract of

Horse chestnut leaf with enhanced matrix metalloproteinases (MMPs) inhibitory activities and a composition comprising the extract as an active ingredient for treating or preventing MMPs-dependent diseases including periodontal disease. Technical Solution

[10] Accordingly, the present invention provides a method of producing extract of Horse chestnut leaf for maximizing its MMPs inhibitory activities.

[11] Below, the method of producing extract of Horse chestnut leaf are described in

detail.

[12] The method of producing extract of Horse chestnut leaf comprises the following steps:

[13] 1) drying and crushing horse chestnut leaves;

[14] 2) subjecting crushed horse chestnut leaves to extraction with a 25% aqueous alcohol with heating;

[15] 3) filtering the aqueous extract from step 2) ; and

[16] 4) concentrating the filtrate from step 3) to recover the extract.

[17]

[18] In the method of the present invention, the alcohols used in step 2) may be C i~C ₄ lower alcohol, preferably C ₁ alcohol (methanol), C ₂ alcohol (ethanol) or the mixture of Ci and C ₂ alcohol.

[19] Between step 1) and 2), the process of supercritical fluid (SCF) extraction can be further included. SCF treatment helps removing the non-polar materials in plant cell and modifying plant cell structure. The object of pre-treating SCF is to improve the solvent extraction efficiency of biological active substances. In the conditions of SCF, fluidal gas, temperature and average pressure are CO ₂ , 25-45°C, 200-300bars, respectively. The extraction time is 1-6 hours, preferably 3 hours.

[20]

[21] The method of the present invention can include additional solvent partition step to divide polar and non-polar fractions of aqueous alcohol extract from step 4). The solvent partition step helps to increase the contents of biological active substances. In the solvent partition step, it is preferable to use the solvents in following order: n- hexane, ethyl acetate, butanol and H ₂ O.

[22] Also, the method of the present invention can also include colunn chromatography step to increase the contents of MMPs inhibiting substances.

[23]

[24] In step 2), the range of heating temperature can be from 50 to 65 ⁰ C, preferably 6O ⁰ C.

The methods of extraction in step 2) can include ultrasonic waves, refluxing or conventional aqueous or solvent extraction. The extraction time can be from 0.5-6 hours, preferably from 1 to 5 hours.

[25]

[26] For the present invention, Horse chestnut of the present invention comprise all

Aescidus species, preferably Japanese Horse chestnut (Aescidus turbinata Blume), Chinese Horse chestnut (Aescidus chinensis Bunge and Aescidus wilsonii Rehder),

Indian Horse chestnut(Aesculus indica Colebr.), North American Horse chestnut ( Aescidus calif ornica Nutt, Aescidus flava Ait, Aescidus glabra Willdenow, and Aescidus pavia Linnaeus) and European Horse chestnut (Aescidus hippocastanum Linnaeus). The extract of Horse chestnut leaf can be extracted using the leaves harvested from spring to autumn and dried naturally or artificially(hot wind), the leaves on the market.

[27]

[28] The present invention provides a composition for treating or preventing MMPs- dependent diseases, comprising the extract produced by above method as an active ingredient.

[29] Also, the present invention provides a use of the said composition for the manufacture of a medicament for treating or preventing MMPs-dependent diseases, and a method of treating or preventing MMPs-dependent diseases comprising administrating to a subject a therapeutically effective amount of the said composition.

[30] In the above, the MMPs-dependent diseases can be atherosclerosis, restenosis,

MMPs-dependent-osteopathy, inflammation of the central nervous system, Alzheimer's disease, sMn aging, rheumatoid arthritis, osteoarthritis, septic arthritis, corneal ulcer, corneal synechia, iris synechia, osteoporosis, proteinuria, abdominal aortic aneurysm, regressive cartilage loss, myelinated nerve loss, multiple sclerosis, liver fibrosis, nephroglomerular disease(nephritic syndrome), Premature rupture of fetal membrane, inflammatory bowel disease, periodontal disease, periodontitis, gingivitis, senile macular degeneration, diabetic retinopathy, proliferate vitreous body retinopathy, immature retinopathy, eye inflammation, conical cornea, Sjogren's syndrome, myopia, eyes turors, rejection of cornea implantation, angiogenesis, cancer infiltration and metastasis, preferably, periodontal disease.

[31] The pharmaceutical composition can comprise the extract of Horse chestnut leaf as an active ingredient, in combination with pharmaceutically acceptable excipients, carriers or diluents.

[32] Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, mannitol, starches, gun acacia, alginates, gelatin, calciun phosphate, calciun silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesiun stearate and mineral oil. Appropriate with pharmaceutically acceptable excipients, carriers or diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

[33] The pharmaceutical composition can additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the present invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[34] A formulation may be prepared by using the composition in accordance with any of the conventional procedures. In preparing the formulation, the active ingredient is preferably admixed or diluted with a carrier or enclosed within a carrier, which may be in the form of a capsule, sachet or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient.

[35] The pharmaceutical composition can be prepared in any form, such as oral dosage form (tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

[36] The unit dosage of the formulation prepared above should contain 5 mg to 2000 mg, or preferably 10 to 1000 mg of Horse chestnut extract in oral and injectable dosage forms. In general, 0.05 to 200 mg/kg of Horse chestnut extract can be administrated in a single dose or 1-3 divided doses per day. For a topical application, QOl to 20 % of horse chestnut leaf extract should be included in the preparations such as emulsion, ointment, spray and toothpaste.

[37] The pharmaceutical composition of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, intra- arterial, rectal, nasal, ocular, and topical introduction.

[38] The pharmaceutical composition may be applied differently according to the purpose of dosing and diseases. It should be understood that the amount of active ingredient has to be determined with various factors. These factors include the severity of the patient's symptoms, other co- administrated drugs (e.g., chemotherapeutic agents), age, sex, body weight of the individual patient, food, dosing time, the chosen route of administration, and the ratio of the composition.

[39]

[40] In accordance with another aspect of the present invention, there is also provided a toothpaste composition comprising Horse chestnut produce by the said method as an active ingredient for prevention and treatment of MMPs-dependent diseases such as

periodontal disease, for example, gingivitis and periodontitis. Also, the present invention provides a use of a composition comprising the extract produced by the said method as an active ingredient for the manufacture of a toothpaste for treating or preventing periodontal disease, periodontitis or gingivitis.

[41] The toothpaste composition contains an abrasive cleaning agent, a humectant, a binder and a flavoring agent and Horse chestnut extract.

[42] It is preferable that the present toothpaste composition contains QO 1-20% by the weight of the Horse chestnut extract based on the total weight of the composition. The other components may be a mixture of the gradients of a conventional toothpaste composition.

[43]

[44] In accordance with another aspect of the present invention, there is also provided a cosmetic composition comprising horse chestnut extract produced by the said method as an active ingredient for treating or preventing sMn aging.

[45] It is preferable that the present cosmetic composition contains QO 1-20% by the weight of the horse chestnut extract based on the total weight of the composition. The other components may be a mixture of the gradients of a conventional cosmetic composition known in the art.

Advantageous Effects

[46] The method of the present invention provides advanced extraction condition to obtain extract of Horse chestnut leaf with enhanced MMPs inhibitory activities, thus the extract according to the present invention may advantageously be used for treating or preventing MMPs-dependent diseases including periodontal disease. Mode for the Invention

[47] The following examples are intended to further illustrate the present invention.

However examples and experimental examples are shown only for better understanding the present invention without limiting its scope.

[48]

[49] Example 1 : Production of aqueous alcohol extract from horse chestnut leaves

[50] Horse chestnut leaves harvested in Daejeon City, Korea, were dried and crushed. 1Og of Dried Horse chestnut leaves were extracted with 500ml of 0, 25, 50, and 75% aqueous methanol, respectively. After extraction at 6O ⁰ C for 2hours and filtration, the filtrates were concentrated under vacuun and 1.39, 1.73, 1.83 and 21 Ig of M-O, M- 25, M-50 and M-75 were obtained, respectively.

[51]

[52] Example 2 : Production of aqueous alcohol extract from supercritical fluid pre- treated horse chestnut leaves

[53] Horse chestnut leaves harvested in Daejeon City, Korea, were dried and crushed. 50g of crushed leaves were treated by supercritical fluid extraction (CO ₂ , 3O ⁰ C, 300 bars, 3 hours) to remove non-polar substances like wax. The residue (1Og) was extracted with 500ml of 0, 25, 50, and 75% aqueous methanol, respectively. After extraction at 6O ⁰ C for 2 hours and filtration, the filtrates were concentrated under vacuun and 1.43, 1.93, 1.53 and 1.82g of SF-O, SF-25, SF-50 and SF-75 were obtained, respectively.

[54]

[55] Example 3 : Production of solvent partition of alcohol extract from horse chestnut leaves

[56] Horse chestnut leaves harvested in Daejeon City, Korea, were dried and crushed. 2Og of crushed leaves were extracted with 1,000ml of cone, ethanol. After extraction at 6O ⁰ C for two hours and filtration, the filtrate was concentrated under vacuun to yield 3.4g of crude extract, E-10Q E-100(20g) was dissolved with ethanol, then chroma- tographed on a silica gel colunn(70-230mesh, 10Og, 3.4 x 20cm) at a flow rate of 7ml/min, eluting with a gradient of hexane/ethyl acetate[l:l (0.5L of eluent volune); group I-H, 1:3 (1.0L) ;group H-V, cone, ethanol (0.5L); group VI] to give 40 fractions (about 50ml per each fracton). The group was divided by R _f values compared with standard compounds (kaempferol and quercetin), respectively. Group ^fractions 1-6, 624mg), group Infraction 7-13, 84mg), group Infraction 14-16, 21mg), group IV(fraction 17-20, l lmg), group V(fraction 21-30, 9mg) and group V^fraction 31-40, 683mg, cone, ethanol) were obtained.

[57]

[58] <Confirmation of increased MMPs inhibitory activities of horse chestnut leaf extract obtained by the method of the present invention>

[59]

[60] ( 1 ) Preparation of MMPs

[61] Hunan MMP- 1 ,MMP-2, MMP-9 and MMP- 13 were cloned and prepared from insect cells (Sf21 insect cell) by using a Baculovirus system.

[62] MMP- 1 cDNA (GENEBANK No. XM_040735), MMP-2 cDNA (GENBANK No.

XM_048244), MMP-9 cDNA(GENEBANK No. XM_009491), and MMP-13 cDNA(GENEBANK NO. NM_002427) were cloned respectively to a pBlueBac4.5 transfer vector (Invitrogen, Cat No. V1995-20) and then transfected to Sf21 cells re-

spectively with a Bac-N-Blue linear Transfection Kit (Invitrogen, Cat No. K855-01). The transfected Sf21 cells were incubated with a TNM-FH (Sigma Co, Cat. No. T 1032) media containing 10% fetal bovine seran at 27 ⁰ C. The media containing a virus stock was harvested and suspended with Sf 21 cells. The cell suspension was incubated with a virus stock containing the cloned gene for 1 hr at room temperature. Infected Sf21 cells were grown at 27 ⁰ C for 72 hrs and the mediun was recovered, and each MMP was purified.

[63] MMP-2 and MMP-9 were purified by a gelatin-sepharose affinity colunn chromatography (Sigma, Cat. No. G-5384) from the recovered media with 5% DMSO as an eluent.

[64] MMP-I and MMP- 13 were purified by SP-sepharose colunn chromatography

(Pharmacia, Cat. No. 17-0729-01) from the recovered media with 2OmM Tris-Cl, pH7.0, 5mM CaCl ₂ as an eluent. Each purified MMP was activated with ImM APMA(p-aminophenyl mercuric acetate) before enzymatic assay.

[65]

[66] (2) MMP assay with spectrofluorcmeter

[67] In order to investigate MMPs inhibitory activities by Horse chestnut leafextract of the present invention, MMPs enzyme activities were assayed by using a spectrofluorcmeter (PerMn-Elmer LS50B).

[68] The substrate for MMP-2 and MMP-9 was MCA- Arg-Pro-Lys-Pro-Tyr- Ala- Nva-

Trp-Met-Lys(Dnp)-NH ₂ (Bachem, Cat. No. M-2105) [MCA=methyl coumaryl amide; Nva=L-Norvaline; Dnp : 2,4-dinitrophenyl].

[69] The substrate for MMP-I and MMP- 13 was MCA-

Pro-Cha-Gly-Nva-His-Ala-Dpa-NH ₂ (Calbiochem, Cat. No. 444235) [MCA=methyl counaryl amide; Cha=L-Cyclohexylalanine; Dpa=3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl; Nva=L-Norvaline].

[70] As a control, 10 nM of each MMP and ImM of corresponding MMP substrate were mixed in 2 mH of reaction buffer (50 mM Tricine (pH 7.5), 10 mM CaCl ₂ , 200 mM NaCl) in a cuvette. Fluorescence intensity was measured for 5-10 min at room temperature with a spectrofluorometer under an excitation wavelength of 325 nm and an emission wavelength of 393 nm.

[71] MMPs inhibitory activities of Horse chestnut leaf extract of the present invention were assayed at the final concentration of 25μg/m#, and fluorescence intensity was measured in the same manner.

[72]

[73] (3) MMPs inhibitory activities of aqueous alcohol extracts of Horse chestnut leaves according to Example 1. [74] When Horse chestnut leaves were extracted with different concentrations of aqueous methanol of 25% (M-25), 50% (M-50) and 75% (M-75) respectively, especially 25% aqueous methanol extract (M-25) showed excellent MMPs inhibitory activities (refer table 1).

[75] In detail, M-25 showed much better MMP-2 inhibition by 88.50% than 18.51% inhibition of commercial seed extract (Finzelberg) and 49.98% inhibition of commercial leaf extract for injection (Schwabe). M-25 also showed excellent MMP-9 inhibition by 91.84% comparing with 27.48% and 55.73% inhibition of commercial seed extract and commercial leaf extract for injection, respectively. Comparing with Doxycycline, M- 25 showed similar inhibitory activities against MMP-2 and MMP-9.

[76] [77] Table 1 [Table 1] [Table ] MMPs Inhibitory activities of aqueous alcohol extracts (%)

[78] * In the above table, n.d. is abbreviation of "not determined" and means "it was not measured."

[79] [80] (4) MMPs inhibitory activities of aqueous alcohol extracts of Horse chestnut leaves pretreated by supercritical fluid extraction according to Example 2

[81] When Horse chestnut leaves were pretreated by supercritical fluid extraction to increase the extraction yield and the residue was extracted with different concentrations of aqueous methanol of 25% (SF-25), 50% (SF-50) and 75% (SF-75) respectively , especially 25% aqueous methanol extract pretreated by supercritical fluid extraction (SF-25) showed excellent MMPs inhibitory activities (refer table X).

[82] In detail, SF25 showed much better MMP-2 inhibition by 90.53% than 18.51% inhibition of commercial seed extract (Finzelberg) and 49.98% inhibition of commercial leaf extract for injection (Schwabe). SF-25 also showed excellent MMP-9 inhibition by 91.90% comparing with 27.48% and 55.73% inhibition of commercial seed extract and commercial leaf extract for injection, respectively. Comparing with Doxycycline, SF- 25 showed similar inhibitory activities against MMP-2, MMP-9 and MMP- 13.

[83] [84] Table 2 [Table 2] [Table ]

MMPs Inhibitory activities of aqueous alcohol extracts pretreated by supercritical fluid extraction (%)

[85] * In the above table, n.d. is abbreviation of "not determined" and means "it was not measured."

[86] [87] (5) MMPs inhibitory activities of solvent partitions of alcohol extract of Horse chestnut leaves according to Example 3

[88] When alcohol extract of Horse chestnut leaves were partitioned by polarity, Group Vl, the polar fraction, showed excellent MMPs inhibitory activities (refer table 3). [89] In detail, Group VI showed much better MMP-2 inhibition by 94.82% than 18.51% inhibition of commercial seed extract (Finzelberg) and 49.98% inhibition of commercial leaf extract for injection (Schwabe). Group VI also showed excellent MMP-9 inhibition by 95.40% comparing with 27.48% and 55.73% inhibition of commercial seed extract and commercial leaf extract for injection, respectively. Comparing with Doxycycline, Group VI showed similar inhibitory activities against MMP-2, MMP-2, MMP-9 and MMP- 13.

[90] [91] Table 3 [Table 3] [Table ] MMPs Inhibitory activities of partitions of alcohol extract by polarity (%)

[92] * In the above table, n.d. is abbreviation of "not determined" and means "it was not measured."