WO2004043170A1 - Use of lower alcohol-insoluble extract of hovenia dulcis thunb and polysaccharide isolated therefrom for relieving fatigue or enhancing exercising ability - Google Patents

Use of lower alcohol-insoluble extract of hovenia dulcis thunb and polysaccharide isolated therefrom for relieving fatigue or enhancing exercising ability Download PDF

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WO2004043170A1
WO2004043170A1 PCT/KR2003/002418 KR0302418W WO2004043170A1 WO 2004043170 A1 WO2004043170 A1 WO 2004043170A1 KR 0302418 W KR0302418 W KR 0302418W WO 2004043170 A1 WO2004043170 A1 WO 2004043170A1
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Prior art keywords
lower alcohol
hovenia dulcis
insoluble fraction
dulcis thunb
isolated therefrom
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PCT/KR2003/002418
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French (fr)
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Chun-Soo Na
Nam-Chul Jung
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Lifetree Biotech Co., Ltd.
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Priority to AU2003282400A priority Critical patent/AU2003282400A1/en
Publication of WO2004043170A1 publication Critical patent/WO2004043170A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/31—Foods, ingredients or supplements having a functional effect on health having an effect on comfort perception and well-being

Definitions

  • the present invention relates to a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb and a polysaccharide isolated therefrom for relieving fatigue and enhancing an exercising ability.
  • Lactate dehydrogenase plays the role of catalyzing the reversible conversion between pyruvate and lactate, and the rise in the metabolic rate caused by muscular exercise or stress induces increases in the amount of lactate and the activity of LDH in the tissues (Yoshida T. et al., Eur. J. Appl. Physiol. 56:7(1987)). It has been reported that elevated serum levels of LDH, aspartate aminotransaminase and alanine ammotransaminase are signs of the fatigue caused by overwork or stress (Cho T. S. et al, YakhakHoeji 39: 548(1995); Gay R. J. et al., Clin. Chem.
  • a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for relieving fatigue in a mammal.
  • a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for enhancing the exercising ability of a mammal.
  • Fig. 1 a schematic procedure for preparing a methanol-insoluble fraction of an extract of Hovenia dulcis Thunb and a polysaccharide isolated therefrom;
  • Fig. 2 a DSC scan of the methanol-insoluble fraction of the fruit-peduncle of Hovenia dulcis Thunb
  • Fig. 3 a MALLS spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
  • Fig. 4 an IR spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
  • Fig. 5 a UV spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
  • Fig. 6 GC spectrums of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
  • Fig. 7 MALLS spectrums of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb
  • Fig. 8 an IR spectrum of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb;
  • Fig. 9 an NMR spectrum of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb;
  • Fig. 10 sustained aerobic exercise time
  • Fig. 11 the maximum oxygen consumption of an aerobic exerciser
  • Fig. 12 the mean power of an anaerobic exerciser
  • Fig. 13 LDH release inhibitory activity of the methanol-insoluble fraction and the polysaccharide.
  • the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb of the present invention may be prepared in two steps. First, a hot- water extract of dried Hovenia dulcis Thunb is obtained using a high-pressure extraction procedure and then an insoluble fraction is obtained by treating the hot- water extract thus obtained with a lower alcohol. In the first step, the fruit peduncle, wood, branch, leaf, bark of the tree or root of Hovenia dulcis Thunb is sliced and dried in the shade.
  • An appropriate amount of water is added to the dried slices of the Hovenia dulcis Thunb and the resulting mixture is kept at a temperature ranging from 110 to 150 ° C, preferably 120 to 125 ° C, under a pressure ranging from 1 to 3 arm., preferably 1.5 arm., for a period ranging from 15 minutes to 48 hours, preferably 30 minutes to 12 hours. Then the mixture is cooled to room temperature and filtered, and the filtrate is lyophilized pursuant to a conventional lyophilizing method, to obtain a hot-water extract.
  • the hot- water extract is further dried at room temperature under a reduced pressure e.g., -1.5 arm, and extracted with a lower alcohol, preferably methanol, ethanol or butanol, most preferably 100% methanol, to remove lower alcohol- soluble components therefrom, to obtain the intended lower alcohol-insoluble fraction.
  • a lower alcohol preferably methanol, ethanol or butanol, most preferably 100% methanol
  • Hovenia dulcis Thunb is selected from the group comprising Hovenia dulcis Thunb. var. Koreana NAKAI, Hovenia dulcis Thunb. var tomentella Makino and Hovenia dulcis Thunb. var. latifolia NAKAI.
  • the lower alcohol-insoluble fraction thus obtained has the following characteristics: gel permeation chromatography (GPC) peaks at mean M.W. of 1,330,000 dalton (Da), 142,800 Da, 70,540 Da and 102,400 Da; IR (KBr, nm) absorption bands at 1000-1300 nm (ether, phenol, sulfoxide, vinyl peak);, and a UV absorption at 200-300 nm (cyclic ring peak).
  • GPC gel permeation chromatography
  • a polysaccharide having high fatigue-relieving and exercising ability- enhancing activities can be isolated from the lower alcohol-insoluble fraction by the following procedure.
  • the lower alcohol-insoluble fraction is dissolved in distilled water, an ion exchange column is charged with the resulting solution, polysaccharide fractions are eluted stepwise using solutions having increasing NaCl concentrations from 0 to 5M, dialyzed, concentrated, and lyophilized (see Fig. 1).
  • either a cation exchange resin or an anion exchange resin may be used.
  • exchange resins which can be used for this purpose are: strong acidic cation exchange resins such as AG 50W- x8, Amberlite IR-120 and Dowex 50W-x8; weak acidic cation exchange resins such as Amberlite IRC-50, Bio-Rex 70 and Duolite-436; weak basic cation exchange resins such as Amberlite IRA-67 and Dowex 3-x4A; strong basic cation exchange resins such as AG 2x8, Amberlite IRA-400 and Dowex 2-x8; modified cellulose cation exchange resins such as CM-Celluose and SE-Cellulose; a modified cellulose anion exchange resins such as DEAE Celluose; cationic sephadex-type resins such as G-25 and G-50 bead type cross-linked dextran resins; and modified bead-type ion exchange resins made from agarose such as Cepha
  • polysaccharide fractions are obtained according to the above isolation process and a polysaccharide that is eluted with 0.2 M NaCl solution shows the significant activity in relieving the fatigue and in enhancing the exercising ability.
  • the lower alcohol-insoluble fraction and the polysaccharide isolated therefrom can be administered to a mammal in the form of a composition containing, e.g., a pharmaceutical composition, a food composition or a beverage composition.
  • the pharmaceutical composition of the present invention may additionally include pharmaceutically acceptable excipients, carriers or diluents for the purpose of enhancing the intended effect.
  • the pharmaceutical composition of the present invention can effectively relieve fatigue caused by overwork or stress, by way of reducing LDH level, and also can enhance the exercising ability by restoring the serum and body fluid lost by an exercise.
  • the pharmaceutical composition containing the lower alcohol-insoluble fraction and the polysaccharide isolated therefrom shows little toxicity or mitogenicity in test using mice and exert no adverse effects.
  • a pharmaceutical formulation may be prepared in accordance with any of the conventional procedures.
  • 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.
  • 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.
  • the formulation may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.
  • Suitable carriers, excipients, or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • the formulation may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsif ⁇ ers, preservatives and the like.
  • the composition of the invention may be formulated so as to provide a quick, sustained or delayed release of the active ingredient after it is administrated to a patient, by employing any one of the procedures well known in the art.
  • the pharmaceutical formulation of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction.
  • a typical daily dose of the above-mentioned extraction or polysaccharide isolated therefrom may range from about 0.01 to 10 g/kg body weight, preferably 1 to 5 g/kg body weight, and can be administered in a single dose or in divided doses.
  • the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
  • the above-mentioned lower alcohol-insoluble fraction and the polysaccharide isolated therefrom can be added to food or beverage for relieving fatigue and enhancing the exercising ability.
  • the amount of said fraction and/or polysaccharide that may be added to food or beverage for the purpose of relieving fatigue or enhancing the exercising ability may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % based on the total weight of food, and 0.1 to 15 g, preferably 1 to 10 g based on 100 m& of beverage.
  • the health care beverage composition of the present invention may contain other components, e.g., deodorants and natural carbohydrates as in conventional beverages.
  • natural carbohydrates are monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; conventional sugars such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol.
  • a natural deodorant such as taumatin, levaudioside A, and glycyrrhizin, or a synthetic deodorant such as saccharin and aspartam may be used.
  • the amount of the above-described natural carbohydrate is generally in the range of about 1 to 20 g, preferably 5 to 12 g based on 100 m# of beverage.
  • compositions that may be added to the inventive food or beverage composition are various nutrients, vitamins, minerals, synthetic flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal adhesives, pH controlling agents, stabilizers, preservatives, glycerin, alcohol, carbonizing agents used in carbonated beverage.
  • the amount of the above-described additives is generally in the range of about 0 to 20 weight portions based on 100 weight portions of the composition.
  • the foods containing the lower alcohol-insoluble fraction or the polysaccharide isolated therefrom to develop health supplementary food may include various foods, various beverages, various gums, vitamin complexes.
  • the percentage with respect to the solid/solid mixture, liquid/liquid, and solid/liquid is each considered at weight/weight, volume/volume,, and weight/volume, respectively and unless it is specifically instructed, all experiments are carried out at room temperature.
  • the molecular weight measurement was carried out using a GPC apparatus, equipped with a pump (spectra system, p2000 model), a guard column (TSK PWH, Tosoh Company), an Rl-detector (Shodex SE71 model), SEC (size exclusion cliromatography) columns (TSK gel 3000pw, 4000pw, 5000pw (7.8x300 mm, Tosoh Company)), and a MALLS (multi angle laser light dispersion, Dawn DSP-F, Wyatt Technology Co.) detector, using a 0.02% sodium azide as developing solvent containing 0.15 M NaNO ⁇ at a flow rate of 0.5 m- /min.
  • Example 1 Preparation a lower alcohol-insoluble fraction of Hovenia dulcis Thunb
  • the extract was centrifuged at 4,000 rpm for 10-20 minutes to obtain a methanol- insoluble fraction of Hovenia dulcis Thunb (dry weight 65.71 g, yield: 4.3% w/w) and a methanol-soluble fraction (dry weight 152.29 g, yield: 10.27% w/w).
  • Example 2 Analysis of the methanol-insoluble fraction of Hovenia dulcis Thunb
  • Example 1 The properties of the methanol-insoluble fractions obtained in Example 1 were analyzed as follows.
  • the melting temperature and the melting entalphy were determined by DSC (Differential Scanning Calorimeter, Seiko Instruments Inc. DSC 6100). Samples of the methanol-insoluble fraction of Hovenia dulcis Thunb was placed in an aluminum pan, sealed, and then heated from 20 ° C to 200 ° C at a rate of 10 ° C/min to obtain a melting heat absorption curve and the melting temperature, and the crystallinity of the sample was determined based thereon. The DSC scans of the methanol-insoluble fractions showed a main peak starting at 164.9 ° C and reaching a maximum melting temperature 185.3 ° C. The methanol-insoluble fraction was formed of several carbohydrates (mp: 60 ⁇ 100 ° C) and a minor amount of proteins (mp: 60 ⁇ 100 ° C) (Fig. 2).
  • a 500 ⁇ g sample was methylated, and then, the methylated product was collected using an ethanol-soaked g 8x10 cartridge column (Sep-Pak). Acidic sugar moieties of the methylated product were reduced using LiB(C 2 H 5 ) 3 D (Super-Deupride, 1 i, Aldrich Company) in THF and the reduction product was recovered using a Cis 8x10 cartridge column(Sep-Pak). Subsequently, the treated sample was subjected sequentially to: hydrolysis at 121 ° C for 2 hours in 1.0 M TFA; reduction by NaBD ; and acetylation. The resulting partially methylated alditol acetate was analyzed by GLC and GC-EIMS, and the peak areas were measured with an FID (flame ionization detector).
  • FID flame ionization detector
  • the sample was analyzed by IR spectroscopy (Vector 22 model, Bruker Analytician Messtechnik GMBH): resolution, 4.0; source, sphere; velocity, 6, 10 KHz; capture mode, dual wall/forward-backward condition).
  • IR spectroscopy Vector 22 model, Bruker Analytician Messtechnik GMBH: resolution, 4.0; source, sphere; velocity, 6, 10 KHz; capture mode, dual wall/forward-backward condition).
  • IR (KBr, cm “1 ): ether, phenol, sulphoxide and vinyl peak (1000-1300nm; main 1039nm), aromatic ring peak (665, 939, 1313, 1663), hydroxyl peak (3435nm).
  • the sample was analyzed by UV-Vis spectroscopy (HP 8453 model, Hewlett Packard Company) and the result showed the maximum absorbance at 200-300 nm, suggesting the existence of a cyclic ring.
  • Example 3 Isolation of polysaccharides having fatigue-relieving and exercising ability-enhancing activities
  • Fractions 1 to 5 0.3 and 3 M NaCl were designated as Fractions 1 to 5, respectively.
  • the contents of total sugar and polyphenol components were determined by the phenol-s ⁇ lfuric acid method (Dubois M. et al.; Anal. Chem. 28, 350- 356(1956)), and the result thus obtained is shown in Table 2.
  • GC analyses were conducted (Varian CP-3800 model, set-up condition; detector : FID, column: SP-2380 (30 m x 0.25 mm x 0.2 ⁇ m), temperature of column: 230 °C, temperature of injector: 250 °C, temperature of detector: 250 °C, mobile phase: N 2 gas(1.0ml/min)) to identify the sugar components of the above fractions and the relative amounts of mannose, glucose, galactose, ramanose, arabinose and xylose present in each fraction were determined. The result is shown in Table 3 and Fig. 6.
  • the lower alcohol-insoluble fraction- administered experimental group has a much longer sustained aerobic exercise time than the control group.
  • Each member of the two groups was asked to run at heart rate of 140 to 170 for 30 minutes and the maximum oxygen consumption of the aerobic exerciser was measured using JAEGER Tradmill (Quinton, U.S.A.) while increasing the running rate by 20m/min until the exerciser was exhausted.
  • the difference of the average values between the two groups was determined according to the independent-sample t-test method. A statistically significant difference level was set as 0.05(P ⁇ 0.05).
  • Each member of the two groups was asked to exercise intermittently using a bike cycle ergo meter (Monag, Sweden) until the exerciser was exhausted.
  • the exerciser was allowed to exercise at an individual load (an individual body weight x 0.075 kp) and a constant rate of 90 rpm for 20 min, followed by resting for 20 min. The above procedure was repeated.
  • the mean power per the body weight was determined by the following formula.
  • Mean power total exercise amount (kpm)/exercise time (sec) x 9.8066/the body weight
  • Test Example 2 Activity for relieving fatigue of the lower alcohol-insoluble fraction of Hovenia dulcis Thunb
  • Sprague-Dawley rats were evenly divided into three dietary groups by a randomized block design.
  • the rats of two groups (blank-test group and control group) were allowed to ingest food and water.
  • the methanol-insoluble fraction was orally administered to the rats of the remaining group (experimental group) in an amount of 100 mg per 1 kg of mouse body weight everyday for 3 days.
  • the rats of the control group and experimental group were forced to swim for 20 minutes, and then, anesthetized with diethyl ether.
  • a blood sample was taken from the heart of each rat, allowed to stand for 30 minutes and centrifuged at 3,000 x g for 15 minutes and the supernatant serum was separated.
  • the administration of the methanol- insoluble fraction reduces the serum LDH level by 20%, as compared with the control group. Accordingly, it has been confirmed that the lower alcohol- insoluble fraction of the present invention has the fatigue-relieving activity, as it effectively prevents the release of LDH which is closely related to general fatigue caused by stress.
  • the lower alcohol-insoluble fraction of the present invention and the polysaccharide isolated therefrom can be used in preparing a pharmaceutically effective powder, tablet, capsule, injection or liquid composition according to any one of the known conventional methods, as exemplified below.
  • Example 1 100 mg of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tabletted to obtain a tablet preparation.
  • An exemplary health care food was prepared by the following method.
  • a scorched dried meal mixture of brown rice, barley, glutinous rice and Job's tear was pulverized and sieved to obtain grain particles of 60 mesh or less.
  • a mixture of black bean, black sesame and wild sesame was steamed, dried, scorched, pulverized and sieved to obtain seed particles of 60 mesh or less.
  • the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom was pulverized and sieved to obtain particles of 60 mesh or less, which were mixed with the grain particles and seed particles in the following proportions to prepare a granule type health food.
  • Grains brown rice 30 w%, Job's tear 15 w%, barley 20 w%, Seeds : wild sesame 7 w%, black bean 8 w%, black sesame 7 w%, Dried powder of Hovenia dulcis Thunb : 3 w%, Shiitake mushroom 0.5 w%, rehmania root 0.5 w%>
  • Exemplary health care beverages were prepared by the following method.
  • a health care beverage was prepared by carrying out the above procedure except for using 0.4 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of soft drink.
  • a health care beverage was prepared by carrying out the above procedure except for using 0.25 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of sports drink.
  • a health care beverage was prepared by carrying out the above procedure except for using 0.15 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of vegetable or fruit juice.
  • a health care beverage was prepared by carrying out the above procedure except for using 0.5 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of drink for relieving fatigue.
  • a health care beverage was prepared by carrying out the above procedure except for using 0.5 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of alcoholic drink.

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Abstract

Disclosed in this invention is a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for relieving fatigue or for enhancing the exercising ability.

Description

USE OF LOWER ALCOHOL-INSOLUBLE EXTRACT
OF HOVENIA DULCIS THUNB AND POLYSACCHARIDE
ISOLATED THEREFROM FOR RELIEVING FATIGUE
OR ENHANCING EXERCISING ABILITY
Field of the Invention
The present invention relates to a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb and a polysaccharide isolated therefrom for relieving fatigue and enhancing an exercising ability.
Background of the Invention
There have been many attempts to develop a functional food or beverage for health care using a new natural material. However, most of the commercially functional foods and beverages are aimed at enhancing moisture absorption moisture or to furnish electrolyte materials to the body, and no effective food or beverage for directly relieving fatigue and enhancing the exercising ability are presently available. Thus, there is a need to develop a functional food or beverage which can supply oxygen and energy by restoring the serum and body fluid lost by an exercise.
Lactate dehydrogenase (LDH) plays the role of catalyzing the reversible conversion between pyruvate and lactate, and the rise in the metabolic rate caused by muscular exercise or stress induces increases in the amount of lactate and the activity of LDH in the tissues (Yoshida T. et al., Eur. J. Appl. Physiol. 56:7(1987)). It has been reported that elevated serum levels of LDH, aspartate aminotransaminase and alanine ammotransaminase are signs of the fatigue caused by overwork or stress (Cho T. S. et al, YakhakHoeji 39: 548(1995); Gay R. J. et al., Clin. Chem. 14: 740 (1968)). The present inventors have endeavored to develop an effective drug of natural origin for relieving fatigue and enhancing the exercising ability, and, as a result, have discovered that a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom is effective in relieving fatigue by reducing the LDH level and enhancing the exercising ability by restoring the serum and body fluid lost during exercise. Summary of the Invention
Accordingly, it is an object of the present invention to provide a pharmacologically active substance for relieving fatigue. It is another object of the present invention to provide a pharmacologically active substance for enhancing the exercising ability.
In accordance with one aspect of the present invention, there is provided a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for relieving fatigue in a mammal. In accordance with another aspect of the present invention, there is provided a use of a lower alcohol-insoluble extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for enhancing the exercising ability of a mammal.
Brief Description of the Drawings
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:
Fig. 1 : a schematic procedure for preparing a methanol-insoluble fraction of an extract of Hovenia dulcis Thunb and a polysaccharide isolated therefrom;
Fig. 2: a DSC scan of the methanol-insoluble fraction of the fruit-peduncle of Hovenia dulcis Thunb; Fig. 3: a MALLS spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
Fig. 4: an IR spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
Fig. 5: a UV spectrum of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
Fig. 6: GC spectrums of the methanol-insoluble fraction of the fruit- peduncle of Hovenia dulcis Thunb;
Fig. 7: MALLS spectrums of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb; Fig. 8: an IR spectrum of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb;
Fig. 9: an NMR spectrum of the methanol-insoluble polysaccharide of the fruit-peduncle of Hovenia dulcis Thunb;
Fig. 10: sustained aerobic exercise time;
Fig. 11: the maximum oxygen consumption of an aerobic exerciser;
Fig. 12: the mean power of an anaerobic exerciser; and Fig. 13: LDH release inhibitory activity of the methanol-insoluble fraction and the polysaccharide.
Detailed Description of the Invention
The lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb of the present invention may be prepared in two steps. First, a hot- water extract of dried Hovenia dulcis Thunb is obtained using a high-pressure extraction procedure and then an insoluble fraction is obtained by treating the hot- water extract thus obtained with a lower alcohol. In the first step, the fruit peduncle, wood, branch, leaf, bark of the tree or root of Hovenia dulcis Thunb is sliced and dried in the shade. An appropriate amount of water is added to the dried slices of the Hovenia dulcis Thunb and the resulting mixture is kept at a temperature ranging from 110 to 150 °C, preferably 120 to 125 °C, under a pressure ranging from 1 to 3 arm., preferably 1.5 arm., for a period ranging from 15 minutes to 48 hours, preferably 30 minutes to 12 hours. Then the mixture is cooled to room temperature and filtered, and the filtrate is lyophilized pursuant to a conventional lyophilizing method, to obtain a hot-water extract.
The hot- water extract is further dried at room temperature under a reduced pressure e.g., -1.5 arm, and extracted with a lower alcohol, preferably methanol, ethanol or butanol, most preferably 100% methanol, to remove lower alcohol- soluble components therefrom, to obtain the intended lower alcohol-insoluble fraction.
The above-mentioned Hovenia dulcis Thunb is selected from the group comprising Hovenia dulcis Thunb. var. Koreana NAKAI, Hovenia dulcis Thunb. var tomentella Makino and Hovenia dulcis Thunb. var. latifolia NAKAI.
The lower alcohol-insoluble fraction thus obtained has the following characteristics: gel permeation chromatography (GPC) peaks at mean M.W. of 1,330,000 dalton (Da), 142,800 Da, 70,540 Da and 102,400 Da; IR (KBr, nm) absorption bands at 1000-1300 nm (ether, phenol, sulfoxide, vinyl peak);, and a UV absorption at 200-300 nm (cyclic ring peak).
A polysaccharide having high fatigue-relieving and exercising ability- enhancing activities can be isolated from the lower alcohol-insoluble fraction by the following procedure.
The lower alcohol-insoluble fraction is dissolved in distilled water, an ion exchange column is charged with the resulting solution, polysaccharide fractions are eluted stepwise using solutions having increasing NaCl concentrations from 0 to 5M, dialyzed, concentrated, and lyophilized (see Fig. 1).
In carrying out the ion exchange, either a cation exchange resin or an anion exchange resin may be used. Examples of exchange resins which can be used for this purpose are: strong acidic cation exchange resins such as AG 50W- x8, Amberlite IR-120 and Dowex 50W-x8; weak acidic cation exchange resins such as Amberlite IRC-50, Bio-Rex 70 and Duolite-436; weak basic cation exchange resins such as Amberlite IRA-67 and Dowex 3-x4A; strong basic cation exchange resins such as AG 2x8, Amberlite IRA-400 and Dowex 2-x8; modified cellulose cation exchange resins such as CM-Celluose and SE-Cellulose; a modified cellulose anion exchange resins such as DEAE Celluose; cationic sephadex-type resins such as G-25 and G-50 bead type cross-linked dextran resins; and modified bead-type ion exchange resins made from agarose such as Cepharose CL, Biogel A Cepharose resin, Fractogels and Toyopearl. The preferred are Toyopearl DEAE type exchange resins and the most preferred are Toyoprearl DEAE-650C type exchange resins.
Several polysaccharide fractions are obtained according to the above isolation process and a polysaccharide that is eluted with 0.2 M NaCl solution shows the significant activity in relieving the fatigue and in enhancing the exercising ability. The polysaccharide obtained from Hovenia dulcis Thunb according to the above procedure shows the following characteristics: It is composed of mannose, glucose, galactose, rhamanose and arabinose in the ratio of 1: 2.51: 12.53: 187: 13.43; absolute molecular weight is 114,500 Da; and IR (KBr, nm) shows peaks at 3550-3450 Da(broad, OH), 1660-1600 Da(C=C), and 1290- 1420 Da(=CH-OH); and 1H-NMR (600MHz, D20) exhibits a peak at 4.4-4.8 ppm (sugar peak).
Various experiments clearly show that the lower alcohol-insoluble fraction and the polysaccharides isolated therefrom are effective for relieving fatigue and enhancing the exercising ability.
In order to relieve fatigue, or to enhance the exercising ability, the lower alcohol-insoluble fraction and the polysaccharide isolated therefrom can be administered to a mammal in the form of a composition containing, e.g., a pharmaceutical composition, a food composition or a beverage composition. The pharmaceutical composition of the present invention may additionally include pharmaceutically acceptable excipients, carriers or diluents for the purpose of enhancing the intended effect.
The pharmaceutical composition of the present invention can effectively relieve fatigue caused by overwork or stress, by way of reducing LDH level, and also can enhance the exercising ability by restoring the serum and body fluid lost by an exercise.
Moreover, in spite of its potent efficacies, the pharmaceutical composition containing the lower alcohol-insoluble fraction and the polysaccharide isolated therefrom shows little toxicity or mitogenicity in test using mice and exert no adverse effects.
A pharmaceutical formulation may be prepared 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. Thus, the formulation may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.
Examples of suitable carriers, excipients, or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulation may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifϊers, preservatives and the like. The composition of the invention may be formulated so as to provide a quick, sustained or delayed release of the active ingredient after it is administrated to a patient, by employing any one of the procedures well known in the art.
The pharmaceutical formulation of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction. For treating a human patient, a typical daily dose of the above-mentioned extraction or polysaccharide isolated therefrom may range from about 0.01 to 10 g/kg body weight, preferably 1 to 5 g/kg body weight, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
The above-mentioned lower alcohol-insoluble fraction and the polysaccharide isolated therefrom can be added to food or beverage for relieving fatigue and enhancing the exercising ability. The amount of said fraction and/or polysaccharide that may be added to food or beverage for the purpose of relieving fatigue or enhancing the exercising ability may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % based on the total weight of food, and 0.1 to 15 g, preferably 1 to 10 g based on 100 m& of beverage.
The health care beverage composition of the present invention may contain other components, e.g., deodorants and natural carbohydrates as in conventional beverages. Examples of such natural carbohydrates are monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; conventional sugars such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. As the deodorant, a natural deodorant such as taumatin, levaudioside A, and glycyrrhizin, or a synthetic deodorant such as saccharin and aspartam may be used. The amount of the above-described natural carbohydrate is generally in the range of about 1 to 20 g, preferably 5 to 12 g based on 100 m# of beverage.
Other components that may be added to the inventive food or beverage composition are various nutrients, vitamins, minerals, synthetic flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, organic acids, protective colloidal adhesives, pH controlling agents, stabilizers, preservatives, glycerin, alcohol, carbonizing agents used in carbonated beverage. The amount of the above-described additives is generally in the range of about 0 to 20 weight portions based on 100 weight portions of the composition. Moreover, the foods containing the lower alcohol-insoluble fraction or the polysaccharide isolated therefrom to develop health supplementary food, may include various foods, various beverages, various gums, vitamin complexes.
The following Reference Examples, Examples, Test Examples and Formulation Examples are intended to further illustrate the present invention without limiting its scope.
Also, in the examples below, the percentage with respect to the solid/solid mixture, liquid/liquid, and solid/liquid is each considered at weight/weight, volume/volume,, and weight/volume, respectively and unless it is specifically instructed, all experiments are carried out at room temperature.
Reference Example 1: Determination of absolute molecular weight by Gel Permeation Chromatography
The molecular weight measurement was carried out using a GPC apparatus, equipped with a pump (spectra system, p2000 model), a guard column (TSK PWH, Tosoh Company), an Rl-detector (Shodex SE71 model), SEC (size exclusion cliromatography) columns (TSK gel 3000pw, 4000pw, 5000pw (7.8x300 mm, Tosoh Company)), and a MALLS (multi angle laser light dispersion, Dawn DSP-F, Wyatt Technology Co.) detector, using a 0.02% sodium azide as developing solvent containing 0.15 M NaNOβ at a flow rate of 0.5 m- /min.
Example 1: Preparation a lower alcohol-insoluble fraction of Hovenia dulcis Thunb
Dried slices (1.5 kg) of fruit-peduncles of Hovenia dulcis Thunb. Collected at the Korean National Arboretum site located at Jikdong-ri, Sohol- myun, Pochun-kun Kyunggi-do, were extracted with 10 I of hot-water at 120 °C for 3 hours under a high pressure (1.5 arm) to obtain a hot water extract. The resulting extract was filtered through Wattman paper and the filtrates (218.5 g) was lyophilized, and then, the resulting powder was subjected to 3 cycles of reflux-extraction, each with 3 -8 of HPLC-grade pure methanol for 1 hour. The extract was centrifuged at 4,000 rpm for 10-20 minutes to obtain a methanol- insoluble fraction of Hovenia dulcis Thunb (dry weight 65.71 g, yield: 4.3% w/w) and a methanol-soluble fraction (dry weight 152.29 g, yield: 10.27% w/w).
Example 2: Analysis of the methanol-insoluble fraction of Hovenia dulcis Thunb
The properties of the methanol-insoluble fractions obtained in Example 1 were analyzed as follows.
(1) Determination of the melting temperature and the melting entalphy
The melting temperature and the melting entalphy were determined by DSC (Differential Scanning Calorimeter, Seiko Instruments Inc. DSC 6100). Samples of the methanol-insoluble fraction of Hovenia dulcis Thunb was placed in an aluminum pan, sealed, and then heated from 20 °C to 200 °C at a rate of 10 °C/min to obtain a melting heat absorption curve and the melting temperature, and the crystallinity of the sample was determined based thereon. The DSC scans of the methanol-insoluble fractions showed a main peak starting at 164.9 °C and reaching a maximum melting temperature 185.3 °C. The methanol-insoluble fraction was formed of several carbohydrates (mp: 60 ~ 100 °C) and a minor amount of proteins (mp: 60 ~ 100 °C) (Fig. 2).
(2) Analysis for sugar chains
To examine whether the above main-peak components contain sugar chains, a methylation analysis was conducted according to the method described by Hakomori et al. (J. Biochem. Tokyo, 55, 205-209(1964)) and Waeghe T. J. et al. (Carbohydrate Research, 123, 281-304(1983)).
A 500 μg sample was methylated, and then, the methylated product was collected using an ethanol-soaked g 8x10 cartridge column (Sep-Pak). Acidic sugar moieties of the methylated product were reduced using LiB(C2H5)3D (Super-Deupride, 1 i, Aldrich Company) in THF and the reduction product was recovered using a Cis 8x10 cartridge column(Sep-Pak). Subsequently, the treated sample was subjected sequentially to: hydrolysis at 121 °C for 2 hours in 1.0 M TFA; reduction by NaBD ; and acetylation. The resulting partially methylated alditol acetate was analyzed by GLC and GC-EIMS, and the peak areas were measured with an FID (flame ionization detector).
(3) Determination of molecular weight by GPC
The result of GPC conducted as in Reference Example 1 showed that the methanol-insoluble fraction was composed of 4 peak components as shown in Fig. 3 and in Table 1.
Table 1
Figure imgf000009_0001
(4) IR spectrum (Fig. 4)
The sample was analyzed by IR spectroscopy (Vector 22 model, Bruker Analytische Messtechnik GMBH): resolution, 4.0; source, sphere; velocity, 6, 10 KHz; capture mode, dual wall/forward-backward condition).
IR (KBr, cm"1): ether, phenol, sulphoxide and vinyl peak (1000-1300nm; main 1039nm), aromatic ring peak (665, 939, 1313, 1663), hydroxyl peak (3435nm).
(5) UV spectrum (Fig. 5)
The sample was analyzed by UV-Vis spectroscopy (HP 8453 model, Hewlett Packard Company) and the result showed the maximum absorbance at 200-300 nm, suggesting the existence of a cyclic ring.
Example 3: Isolation of polysaccharides having fatigue-relieving and exercising ability-enhancing activities
To isolate active compounds having fatigue-relieving and exercising ability-enhancing activities from the methanol-insoluble fraction obtained in Example 1, 200 mg of the methanol-insoluble fraction was dissolved in distilled water, charged to a Toyopearl® DEAE-650C column (4.0x 30 cm), and eluted successively with 0, 0.1, 0.2, 0.3 and 3M NaCl solutions. The eluted fractions were dialyzed using a dialysis membrane permeable at a M.W. of 1000 or below, concentrated, and then lyophilized to obtain purified fractions weighing 38 mg, 64 mg, 73 mg, 5 m . and 4 mg, respectively.
Example 4: Characterization of the isolated fractions of Hovenia dulcis Thunb
The isolated fractions obtained in Example 3 by eluting with 0, 0.1, 0.2,
0.3 and 3 M NaCl were designated as Fractions 1 to 5, respectively. For each fraction, the contents of total sugar and polyphenol components were determined by the phenol-sύlfuric acid method (Dubois M. et al.; Anal. Chem. 28, 350- 356(1956)), and the result thus obtained is shown in Table 2.
Figure imgf000011_0001
GC analyses were conducted (Varian CP-3800 model, set-up condition; detector : FID, column: SP-2380 (30 m x 0.25 mm x 0.2 μm), temperature of column: 230 °C, temperature of injector: 250 °C, temperature of detector: 250 °C, mobile phase: N2 gas(1.0ml/min)) to identify the sugar components of the above fractions and the relative amounts of mannose, glucose, galactose, ramanose, arabinose and xylose present in each fraction were determined. The result is shown in Table 3 and Fig. 6.
Table 3
Figure imgf000011_0002
Fraction 3 among the above fractions was further characterized as below.
Characterization of Fraction 3
The absolute molecular weight of Fraction 3 determined by the procedure of Reference Example 1 was 114,500 Da (Fig. 7). An IR analysis (KBr) showed peaks (cm'1) at 3550-3450 (broad, OH), 1660-1600 (C=C) and 1290-1420 (=CH- OH) (Fig. 8). 1H-NMR (600MHz, D20) showed a peak at 4.4-4.8ρpm (sugar peak) (Fig. 9).
Test Example
To examine the exercising ability-enhancing activity of Hovenia dulcis Thunb extract, the aerobic exercising abilities in terms of sustained time, maximum oxygen consumption and mean power of the exerciser were measured as follows.
(1) Experimental subject group
26 high school male hockey players having the characteristics shown in Table 4 divided into 2 groups, the experimental group and the control group.
Table 4
Figure imgf000012_0001
(2) Administration of methanol-insoluble fraction to the experimental group
Each member of the experimental group was asked to take the methanol- insoluble fraction of Hovenia dulcis Thunb extract for 12 weeks in an amount of 3.6 g/day, while those of the control group were allowed to ingest just water. Both groups took normal meals and excises.
(3) Measurement of sustained aerobic exercise time
Each member of both groups was asked to run at 70 to 75% of the maximum oxygen consumption rate (140 to 170 of heart rate) for 30 minutes, and the sustainable aerobic exercise time was measured using JAEGER Tradmill (Quinton, U.S.A.) while increasing the running rate by 20m/min until the exerciser was exhausted. The difference of the average values between the two groups was determined according to the independent-sample t-test method. The result is shown in Table 5 and Fig. 10. Table 5
Figure imgf000013_0001
Figure imgf000013_0003
As shown in Table 5 and Fig. 10, the lower alcohol-insoluble fraction- administered experimental group has a much longer sustained aerobic exercise time than the control group.
(4) Measurement of maximum oxygen consumption of aerobic exercisers
Figure imgf000013_0002
Each member of the two groups was asked to run at heart rate of 140 to 170 for 30 minutes and the maximum oxygen consumption of the aerobic exerciser was measured using JAEGER Tradmill (Quinton, U.S.A.) while increasing the running rate by 20m/min until the exerciser was exhausted. The difference of the average values between the two groups was determined according to the independent-sample t-test method. A statistically significant difference level was set as 0.05(P< 0.05).
The result showed in Table 6 and Fig. 11.
Table 6
Figure imgf000013_0004
As shown in Table 6 and Fig. 11, after 7 weeks of administering, a significant difference between the experimental group and control group in terms of the maximum oxygen consumption was observed. This result shows that the lower alcohol-insoluble fraction of Hovenia dulcis Thunb is effective in enhancing the aerobic exercising ability.
(5) Measurement of mean power of anaerobic exercisers
Each member of the two groups was asked to exercise intermittently using a bike cycle ergo meter (Monag, Sweden) until the exerciser was exhausted. In order to induce artificial muscle fatigue, the exerciser was allowed to exercise at an individual load (an individual body weight x 0.075 kp) and a constant rate of 90 rpm for 20 min, followed by resting for 20 min. The above procedure was repeated. The mean power per the body weight was determined by the following formula.
Formula
Mean power = total exercise amount (kpm)/exercise time (sec) x 9.8066/the body weight
The difference of the average values between the two groups was determined according to the independent-sample t-test method. A statistically significant difference level was set as 0.05(P<0.05). The result showed in Table 7 and Fig. 12.
Table 7
Figure imgf000014_0001
As shown in Table 7 and Fig. 12, the lower alcohol-insoluble fraction induced increased anaerobic exercises performance ability, thereby demonstrating their superior exercises-enhancing activity.
Test Example 2: Activity for relieving fatigue of the lower alcohol-insoluble fraction of Hovenia dulcis Thunb
(1) Preparation of experimental animals
Two-week old Sprague-Dawley rats were allowed to freely ingest food
(Purina Korea) and water while being kept in a cage under the condition of temperature 20±22°C, moisture 50±10% and photoperiod 12L/12D for 4 weeks. Six-week old Sprague-Dawley rats each weighing about 200 to 225 g 90 to 110 g were used for the following experiments.
(2) Administration of methanol-insoluble fraction and exercise
15 Sprague-Dawley rats were evenly divided into three dietary groups by a randomized block design. The rats of two groups (blank-test group and control group) were allowed to ingest food and water. The methanol-insoluble fraction was orally administered to the rats of the remaining group (experimental group) in an amount of 100 mg per 1 kg of mouse body weight everyday for 3 days. Then, the rats of the control group and experimental group were forced to swim for 20 minutes, and then, anesthetized with diethyl ether. A blood sample was taken from the heart of each rat, allowed to stand for 30 minutes and centrifuged at 3,000 x g for 15 minutes and the supernatant serum was separated.
(3) Measurement of the amount of LDH released in the serum
To determine the fatigue-relieving activity of the methanol-insoluble fraction, the amount of LDH (lactate dehydrogenase) released in the serum was determined with an LDH meter (Express plus; Chiron, USA). The result is shown in Table 8 and Fig. 13. Table 8
Figure imgf000016_0001
As can be seen in Table 8 and Fig. 13, the administration of the methanol- insoluble fraction reduces the serum LDH level by 20%, as compared with the control group. Accordingly, it has been confirmed that the lower alcohol- insoluble fraction of the present invention has the fatigue-relieving activity, as it effectively prevents the release of LDH which is closely related to general fatigue caused by stress.
The lower alcohol-insoluble fraction of the present invention and the polysaccharide isolated therefrom can be used in preparing a pharmaceutically effective powder, tablet, capsule, injection or liquid composition according to any one of the known conventional methods, as exemplified below.
[Formulation Example 1]
2 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom was mixed with 1 g of lactose to obtain a powder preparation, which was filled and sealed in a sealed package.
[Formulation Example 2]
100 mg of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tabletted to obtain a tablet preparation.
[Formulation Example 3]
100 mg of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom, 100 mg of the corn starch, 100 mg of lactose, 2 mg of magnesium stearate were mixed and filled in a gelatin capsule to obtain a capsule preparation. [Formulation Example 4]
100 mg of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom, distilled water, and an appropriate amount of a pH controller were dissolved to obtain an injection formulation, which was filled in a 2 £ ample and sterilized according to a conventional injection preparation method, to obtain a injection preparation.
An exemplary health care food was prepared by the following method.
[Preparation of health care food]
A scorched dried meal mixture of brown rice, barley, glutinous rice and Job's tear was pulverized and sieved to obtain grain particles of 60 mesh or less. Also, a mixture of black bean, black sesame and wild sesame was steamed, dried, scorched, pulverized and sieved to obtain seed particles of 60 mesh or less. The dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom was pulverized and sieved to obtain particles of 60 mesh or less, which were mixed with the grain particles and seed particles in the following proportions to prepare a granule type health food.
Grains : brown rice 30 w%, Job's tear 15 w%, barley 20 w%, Seeds : wild sesame 7 w%, black bean 8 w%, black sesame 7 w%, Dried powder of Hovenia dulcis Thunb : 3 w%, Shiitake mushroom 0.5 w%, rehmania root 0.5 w%>
Exemplary health care beverages were prepared by the following method.
[Preparation of health care beverage 1]
3.6 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb obtained in Example 1 or the polysaccharide isolated therefrom, 1000 mg of citric acid, 2g of plum extract, lOOg of oligo-saccharide and lg of taurin were added to 1000 ml of distilled water and mixed at 85 °C for 60 minutes while stirring to make a syrup. The syrup thus obtained was filtered and sterilized to obtain a health care beverage having pH 3.2 and 9% of sweetness.
[Preparation of health care beverage 2]
A health care beverage was prepared by carrying out the above procedure except for using 0.4 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of soft drink.
[Preparation of health care beverage 3] A health care beverage was prepared by carrying out the above procedure except for using 0.25 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of sports drink.
[Preparation of health care beverage 4] A health care beverage was prepared by carrying out the above procedure except for using 0.15 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of vegetable or fruit juice.
[Preparation of health care beverage 5]
A health care beverage was prepared by carrying out the above procedure except for using 0.5 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of drink for relieving fatigue.
[Preparation of health care beverage 6]
A health care beverage was prepared by carrying out the above procedure except for using 0.5 g of the dried methanol-insoluble fraction of Hovenia dulcis Thunb or the polysaccharide isolated therefrom and 100 ml of alcoholic drink.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

What is claimed is :
1. A use of a lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for relieving fatigue in a mammal.
2. The use of claim 1, wherein the mammal is human.
3. The use of claim 1, wherein the lower alcohol is selected from the group consisting of methanol, ethanol, butanol and a mixture thereof.
4. The use of claim 1, wherein the polysaccharide is prepared by subjecting the lower alcohol-insoluble fraction to ion exchange column cliromatography and obtaining a fraction containing the polysaccharide.
5. The use of claim 1, wherein the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom is administered to the mammal in the form of a composition containing same, said composition being selected from the group consisting of: a pharmaceutical composition, a food composition and a beverage composition.
6. The use of claim 5, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers or diluents.
7. The use of claim 5, wherein the food composition comprises the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom in an amount ranging from 0.1 to 15 w/w %.
8. The use of claim 5, wherein the beverage composition comprises the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom in an amount ranging from 0.1 to 15 g based on 100 ml of beverage.
9. A use of a lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or a polysaccharide isolated therefrom for enhancing the exercising ability of a mammal.
10. The use of claim 9, wherein the mammal is human.
11. The use of claim 9, wherein the lower alcohol is selected from the group consisting of methanol, ethanol, butanol and a mixture thereof.
12. The use of claim 9, wherein the polysaccharide is prepared by subjecting the lower alcohol-insoluble fraction to ion exchange column cliromatography and obtaining a fraction containing the polysaccharide.
13. The use of claim 9, wherein the lower alcohol-insoluble fraction of a hot- water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom is administered to the mammal in the form of a composition containing same, said composition being selected from the group consisting of: a pharmaceutical composition, a food composition and a beverage composition.
14. The use of claim 13, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers or diluents.
15. The use of claim 13, wherein the food composition comprises the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom in an amount ranging from 0.1 to 15 w/w %.
16. The use of claim 13, wherein the beverage composition comprises the lower alcohol-insoluble fraction of a hot-water extract of Hovenia dulcis Thunb or polysaccharide isolated therefrom in an amount ranging from 0.1 to 15 g based on 100 mi of beverage.
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JPH04282318A (en) * 1991-03-08 1992-10-07 Suntory Ltd Food and drink for ameliorating drunken sickness
KR970073403A (en) * 1996-05-20 1997-12-10 구형우 Beverage composition containing Hovenia dulcis extract and method for its production
KR19990037871A (en) * 1999-02-08 1999-05-25 장철수 Beverage for Curing Hangover and Manufacturing Method thereof
KR20010069022A (en) * 2000-01-11 2001-07-23 문혜연 Functional beverage containing water extract of Hovenia dulcis and process for preparation thereof
KR20020064151A (en) * 2001-01-31 2002-08-07 (주)생명의나무 Lower alcohol insoluble extract and a polysaccharide therein isolated from hovenia dulcis var. koreana nakai having antihepatotoxic and anti-hangover activity and composition containing same
KR20020076980A (en) * 2001-03-29 2002-10-11 박근형 Antiaging Agent Using Water Extracts from the Leaves and Stems of Hovenia dulcis Thunb Containing Natural Antioxidants

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