KR100828068B1 - Composition for preventing or treating hyperlipemia comprising fucoxanthin or marine plant extract containing same - Google Patents

Abstract

A composition comprising fucoxanthin or marine plant extracts containing the same is provided to inhibit accumulation of cholesterol and neutral fat in blood, and reduce cholesterol absorption from food, so that the composition is useful for preventing or treating hyperlipidemia. A composition for preventing or treating hyperlipidemia comprises fucoxanthin represented by the formula(1) or marine plant extracts containing the fucoxanthin including brown seaweed extract, sea tangle extract, gulfweed extract and laver extract, which are obtained by heating marine plants in water, alcohol, hexane, ethyl acetate or isopropylalcohol at 10-50 deg. C for 1-48 hours, and filtering and vacuum concentrating the marine plant extracts, wherein the fucoxanthin is obtained by adding hexane into the marine plant extract to remove oil-soluble materials, solubilizing the marine plant extract in acetone, passing the solution through the silica gel column, eluting the adsorbed components, and vacuum concentrating, filtering and drying the eluted solution. Further, the composition for preventing or treating hyperlipidemia is manufactured by mixing the fucoxanthin or the brown seaweed extract with pharmaceutically acceptable carriers.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating hyperlipemia, which comprises fucoxanthin or a seaweed extract containing the same. TECHNICAL FIELD The present invention relates to a composition for preventing or treating hyperlipidemia,

The present invention relates to a composition for preventing or treating hyperlipemia, which contains fucoxanthin as an active ingredient or a seaweed extract containing the same.

Hyperlipidemia refers to any one of four types of lipid components, such as cholesterol, triglycerides, phospholipids, and free fatty acids, that play important roles in cell function maintenance and energy metabolism, resulting in abnormally high levels of lipid metabolism It is a symptom that causes various diseases by being present in large quantities.

Hyperlipidemia is generally classified into hypercholesterolemia with high total cholesterol level in the blood, hypertriglyceridemia with high triglyceride level, and all of the above two levels, and such hyperlipemia induces atherosclerosis, In severe cases, angina and myocardial infarction may occur.

Causes of hyperlipidemia include high fat and high calorie diets, drinking, genetic, liver dysfunction, diabetes, contraceptive use, hypothyroidism, etc. In particular, in modern people, hyperlipidemia As the number of patients is soaring, it is very necessary to develop materials that can effectively prevent and treat them.

On the other hand, fucosanthin having a structure represented by the following general formula (1) is a major carotenoid which is brownish in the seaweeds such as seaweed, mackerel, kelp, and red sea bream. literature.. [.... Das , SK et al, Biochim Biophys Acta, 2005, 1726 (3): 328-335]; [Kotake-Nara, E. et al, Cancer Lett 2005, 220 (1): 75 -84]; [Kotake-Nara, E. et al, Biosci Biotechnol Biochem, 2005, 69 (1):.... 224-227]; [Hosokawa M. et al, Biochim Biophys Acta, 2004,... [Okuzumi, J. et al., Cancer (2001), 131 (12): 2921-2927]; Kotake-Nara, E. et al., J. Nutr . Lett, 1993, 68 (2-3) :. 159-168]; and [Okuzumi, J. et al, Cancer Lett, 1990, 55 (1):.. 75-78]), an anti-inflammatory effect (as described in Shiratori, K. et al., Exp Eye Res 2005, 81 (4): 422-428), angiogenesis inhibitory effects (Sugawara, T. et al., J. Agric. (26): 9805-9810), anti-obesity effect (Naeda, H. et al., Biochem. Biophy s. Res. Commun. , 2005, 332 (2): 392-397), which has been proved effective:

 However, no study has yet been reported that fucosanthin is effective in the prevention and treatment of hyperlipidemia.

[Document 1] Das, SK et al., Biochim. Biophys. Acta ., 2005, 1726 (3), pp. 328-335

[Literature 2] Kotake-Nara, E. et al., Cancer Lett. 2005, 220 (1), pp. 75-84

[Literature 3] Kotake-Nara, E. et al., Biosci. Biotechnol. Biochem. , 2005, 69 (1), 224-227

[Document 4] Hosokawa M. et al., Biochim. Biophys. Acta , 2004, 1675 (1-3), 113-119

[Literature 5] Kotake-Nara, E. et al., J. Nutr ., 2001, 131 (12), 2921-2927

[Document 6] Okuzumi, J. et al., Cancer Lett ., 1993, 68 (2-3), pp. 159-168

[Document 7] Okuzumi, J. et al., Cancer Lett. , 1990, 55 (1), pp. 75-78

[Literature 8] Shiratori, K. et al., Exp Eye Res. 2005, 81 (4), 422-428

[Literature 9] Sugawara, T. et al., J. Agric. Food Chem . 2006, 54 (26), 9805-9810

[Literature 10] Naeda, H. et al., Biochem. Biophys. Res. Commun. , 2005, 332 (2), 392-397

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating hyperlipemia, which comprises fucoxanthin or an algae extract containing the same as an active ingredient.

Yet another object of the present invention is to provide a food composition containing the above-mentioned components as an active ingredient for the purpose of preventing or improving hyperlipidemia.

According to an aspect of the present invention,

A pharmaceutical composition for preventing or treating hyperlipemia, which comprises fucoxanthin of the following formula (1) or an algae extract containing the same as an active ingredient, together with a pharmaceutically acceptable carrier.

The present invention also provides a food composition for preventing or ameliorating hyperlipidemia comprising fucosanthin of the following formula 1 or a seaweed extract containing the same:

[Chemical Formula 1]

 The composition of the present invention comprising fucosanthin or a seaweed extract containing the same exhibits an excellent effect in preventing and treating hyperlipidemia by inhibiting the accumulation of cholesterol and triglycerides in the blood and reducing the absorption of cholesterol from the diet, It can be usefully used as a food composition.

The fucoxanthin used in the present invention is a carotenoid compound present in an amount of about 0.05 to 1.0% (w / w) in seaweed, seaweed, seaweed, mackerel and so on of seaweed.

Examples of seaweed extracts used in the present invention include seaweed extract, sea tangle extract, mung bean extract, and top extract, but any natural product containing fucoxanthin can be used and is not limited to the above extract.

The extract of seaweed containing fucoxanthin used in the present invention is obtained by extracting the dried seaweeds at least once at 10 to 50 ° C for 1 to 48 hours in water, alcohol, isopropanol or mixtures thereof, , And the obtained extract is filtered and concentrated under reduced pressure.

Specifically, in a preferred embodiment of the present invention, the seaweed powder is extracted once in a mixture of water and alcohol at 25 ° C for 6 hours, and then the obtained extract is filtered and concentrated under reduced pressure to obtain a fucoxanthin-containing seaweed extract.

The fucoxanthin used in the present invention can be obtained by separating and purifying from seaweed extracts obtained through the above-mentioned procedures by a conventional method.

Specifically, in a preferred embodiment of the present invention, hexane is added to the seaweed extract and filtered to remove the high-solubility substance from the extract, which is then dissolved in acetone and passed through a silica gel column to remove unadsorbed impurities. Eluted, concentrated in vacuo, filtered and dried to obtain high purity fucosanthin.

According to the present invention, fucosanthin inhibits the accumulation of blood cholesterol or triglyceride in rats induced by hyperlipidemia by supplying a high fat diet, and has an effect of reducing the absorption of cholesterol from the diet, and thus has an excellent effect of preventing and treating hyperlipemia.

The composition for prevention and treatment of hyperlipidemia comprising fucoxanthin of the present invention can be prepared by using fucoxanthin purified at high purity as a raw material or by using an extract containing fucozanthin as a raw material.

According to the present invention, the fucoxanthin or an algae extract containing the fucoxanthin can be mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition for the prevention or treatment of hyperlipidemia. Examples of suitable carriers are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, mannitol, starch, acacia rubber, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain an excipient, a filler, an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, an antiseptic, and the like. The pharmaceutical compositions of the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. Formulations may be in the form of tablets, capsules, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical composition of the present invention may be administered parenterally or orally, and may be administered orally or parenterally. In the case of parenteral administration, 0.01 to 50 mg, preferably 0.1 to 30 mg per kg of body weight per day And the oral administration may be administered in one to several divided doses so as to be administered in an amount of 0.01 to 100 mg, preferably 0.1 to 50 mg per kg of body weight per day on the basis of the fucoxanthin content. It should be understood that the dosage for a particular patient should be determined in light of various relevant factors such as the patient's body weight, age, sex, health condition, diet, time of administration, method of administration, severity of disease, And are not intended to limit the scope of the invention in any way.

In addition, the present invention provides a health functional food composition for preventing or ameliorating hyperlipidemia, which comprises the fucosanthin or a seaweed extract containing the same. The health functional food composition of the present invention is characterized by inhibiting the accumulation of cholesterol or triglycerides in the blood and reducing the absorption of cholesterol from the diet. In order to exhibit the above effects, the seaweed extract of the present invention or the fucoxanthin isolated therefrom can be prepared into capsules, tablets, granules, powders, beverages, and rings of health functional foods. The composition comprising the fucoxanthin or the fucosanthin-containing extract in an amount of 0.01 to 100 mg, preferably 0.1 to 50 mg per kg of body weight per day based on the fucoxanthin content .

In addition, food can be produced by adding fucoxanthin or fucosanthin-containing extract to various foods, meat, beverage, chocolate, snack, confectionery, pizza, ramen, other noodles, gum, ice cream, In this case, the content of fucozanthin in the finally produced food may be in the range of 0.01 to 50% by weight.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1  : Preparation of Seaweed Extracts Containing Fucoxanthin

240 L of water and 40 L of water were added to 45 kg of dried seaweed powder and extracted for 6 hours in a 1 ton concentration tank (JEIL KOGO CO., LTD., Model name: J003) at 25 ° C. The dried seaweed residue was removed by pressing with a filter press having 12 pads each having a size of 300 mm x 300 mm. The extract solution recovered from the filter press was concentrated under reduced pressure to 20 L in a concentration tank at a temperature of 25 캜 and a pressure of 740 mmHg for 3 hours and then concentrated in a vacuum concentrator at a temperature of 60 캜 and a pressure of 50 mmHg for 8 hours. The final concentrate was lyophilized for 48 hours in a lyophilizer maintained at a temperature of -40 DEG C to prepare a sample of the desired seaweed extract.

The calorie content and general composition of the seaweed extract are shown in Table 1 below.

The HPLC method was used to determine the concentration of fucoxanthin in the seaweed extract prepared through the above process. The analytical column was a column of Symmetry C18 (4.6x250mm, Waters, Ireland) and fucosanthin was detected at 450nm wavelength using UV (Ultraviolet). The mobile phase of the column was a 1: 9 (v / v) mixture of hexane and acetone, and eluted at a flow rate of 0.5 mL per minute for about 15 minutes. At this time, the content of the sample was measured in terms of an area ratio based on 94% fucoxanthin from CaroteNature (Switzwrland) as a standard substance. The fucosanthin concentration of the wakame extract was 3.5% by weight.

Example 2 : Preparation of high purity fucosanthin

In order to prepare high purity fuco xanthine, the seaweed extract obtained in Example 1 was placed on No. 2 watt filter paper, and hexane was poured into the volume of about 2 times of the extract, and the process of filtering by vibration was repeated twice Highly soluble materials were removed. Through this procedure, 7.5 g of a sample containing a fucosanthin having a purity of about 50% was obtained.

The fucoxanthin-containing sample was dissolved in about 50 mL of acetone, and the solution was added to a 2,000 mL silica gel column (resin: Merck Kieselgel 66; 70-230 mesh, inner diameter 7.5 cm x length 60 cm) So that the fuco xanthine component was adsorbed onto the silica gel. After the adsorption of the sample was completed, about 2,000 mL of a solvent having a ratio of hexane and acetone of 8: 2 (v / v) was injected into the column to remove undesired impurities. Then, hexane and acetone were added to the column in a ratio of 6: 4 / v) was passed through the eluent to elute the components adsorbed on the silica gel. Thereafter, about 2,000 mL of the eluate was concentrated in vacuo until the volume of the concentrate reached about 10 mL by using a vacuum concentrator at an internal temperature of 40 DEG C or less in the evaporation tube, and about 10 mL of tertiary distilled water was added to the concentrate And allowed to stand at -20 DEG C for about 4 hours to obtain a red precipitate. The precipitate was collected by filtration using No. 2 Wattmann filter paper, and then dried in a vacuum dryer at 40 캜 for about 12 hours to obtain 3.6 g of a dried product. The purity of the obtained dried product fucosanthin was 97.5%.

Experimental Example 1 : Effect of Fucoxanthin-Containing Seaweed Extract and High-Purity Fucozanthin on Hyperlipidemia

1-1. Experimental animal care and empirical formula

Seventy weeks old male C57BL / 6N / CriBgi mice weighing 14 g and purchased from Orient Co., Ltd. were purchased from the Orient Co., Ltd., and incubated in an animal breeding room maintained at a temperature of 24 ° C and a relative humidity of 55% And kept in individual cages. Five week old mice, weighing between 18.5 and 18.7 g, which were fed pelleted diets for one week, were divided into seven groups in a randomized block design to provide different diets, respectively.

The experimental diet consisted of a semisynthetic diet of Teklad (Madison, WI, USA), a "normal diet" group, and a normal diet containing 10% corn oil and lard, respectively, Quot; group ", and those obtained by adding the sea mustard extract of Example 1 such that the fucoxanthin content was 0.05% in the above-mentioned high fat-fat-derived diets and that the fucosanthin content was 0.2% in the high fat- And the high-purity fucoxanthin powder of Example 2 was added such that the fucoxanthin content was 0.05% in the high-fat-content diets as "Experiment Group III ", and the high fat- The addition of the high-purity fucoxanthin powder of Example 2 so that the fucoxanthin content was 0.2% was classified as "Experimental Group Ⅳ", and the six dietary groups (normal diet group, high fat dietary group, experimental groups I, II, III and IV ) Were injected into experimental animals for 6 weeks It was fed. Dietary intake was recorded daily and body weight was measured weekly.

The composition of each experimental diet is shown in Table 2 below.

1-2. The empirical formula shows the weight change during the pay period

In this experiment, we tried to confirm that the weight gain was inhibited in the experimental group fed with the fucoxanthin-containing waxy extract and the high-purity fucoxanthin-treated experimental groups, compared with the control group fed with the high fat diet. The mean weight of each group was measured by the one-way ANOVA method, and the test was performed using the Duncan's multiple range test ), And the results were expressed as mean ± standard deviation.

As a result, all of the experimental diets of Experiment Groups I to IV in which the fucoxanthin-containing waxy extract and the high-purity fucoxanthin were added showed a lower body weight gain than the high fat dietary group. As a result, the waxy extract containing fucoxanthin and the high purity It has been confirmed that fucosanthin can effectively inhibit the weight gain resulting from the high fat diet. The weekly weight changes of the experimental groups are shown in Table 3 below.

1-3. Test for the inhibition of hyperlipemia of fucosanthin

After 6 weeks of experimental diets, the animals were fasted for 12 hours, first anesthetized through ether inhalation, and secondly anesthetized by intramuscular injection with ketamine-HCl (Yuhan) anesthetic. After that, blood was collected from the abdominal subclavian vein, collected directly into a heparinized test tube, and centrifuged at 3,000 rpm for 15 minutes to separate the plasma. Separated plasma was kept at -70 ° C until analysis. Total cholesterol and triglyceride in plasma and cholesterol in feces were determined by the following method.

① Quantification of plasma cholesterol

The amount of plasma cholesterol was determined by applying the principle of color development method using a measuring solution (ASAN kit, Korea) using the enzyme method. Since plasma cholesterol exists in two forms of cholesteryl ester (CE) and free cholesterol, cholesterol esterase is used to quantify all of them, and cholesteryl ester is converted to fatty acid and free cholesterol . ≪ / RTI > In this manner process the conversion of free cholesterol is cholesterol, oxy-zero Δ ₄ - cholesterol was converted to the non-(Δ ₄ -cholestenon), wherein the hydrogen peroxide oxy occurred buffer oxidase (peroxidase), phenol (phenol) and 4-amino-anti-soft (4-amino-antiptrine), and the absorbance at 500 nm was measured and compared with the cholesterol standard solution (300 mg / dL).

② Quantification of plasma triglyceride

The amount of plasma triglyceride was determined by applying the principle of the coloring method using a measuring solution (ASAN kit, Korea) using the enzyme method. Plasma neutral lipids were treated with lipase, digested with glycerin and fatty acids, and then converted to L-α-phosphoglycerol by the addition of ATP and glycerol kinase (GK). Hydrogen peroxide is generated by adding oxygen (O ₂ ) and glycerophosphooxidase to the converted L-α-phosphoglycerol and reacting the generated hydrogen peroxide with peroxidase and 4-amino-antiputrin And the absorbance was measured at 550 nm, and quantified in comparison with glycerol standard solution (300 mg / dL).

③ Cholesterol determination in feces

The mixture was homogenized with 4 ml of a chloroform: methanol (2: 1) solution and allowed to stand at 4 ° C for 24 hours. The mixture was centrifuged at 3,000 rpm for 10 minutes, Lt; / RTI > 1 ml of a chloroform: methanol (2: 1) solution was further added thereto, and 200 μl of the solution was transferred to two test tubes and dried with nitrogen gas at 50 ° C. Then, 5 ml of ethanol was added to each test tube, and the cholesterol content in the feces was quantified by the same method as the above-mentioned determination of cholesterol in plasma.

The results of measurement of triglyceride, cholesterol, and cholesterol content in plasma by the above method are shown in Tables 4 and 5.

As shown in Table 4, in the animal group fed with the high fat diet, the triglyceride and cholesterol contents in the plasma were significantly higher than those in the normal diet group, but the fucoxanthin or the fucoxanthin-containing seaweed extract was added to the high fat diet In the case of one dietary group (experimental group Ⅰ, Ⅱ, Ⅲ and Ⅳ), triglyceride and cholesterol contents in plasma were significantly decreased to the level of normal dietary group. These results show that fucosanthin is very effective for the prevention and treatment of hyperlipidemia induced hyperlipidemia.

As shown in Table 5, in the experimental groups I to IV, which are groups of the diet containing the fucoxanthin or fucoxanthin-containing seaweed extract, the fecal cholesterol content was significantly increased compared with the high fat dietary group, Which is effective for the prevention of hyperlipemia.

≪ Preparation Example 1 >

The following components were mixed and filled in an airtight container according to a conventional acid preparation method to prepare a powder:

50 mg of fucoxanthin isolated and purified from Example 2

Crystalline cellulose 2 g

≪ Preparation Example 2 >

After mixing the following ingredients, tablets were prepared by tableting according to a conventional tablet preparation method:

50 mg of fucoxanthin isolated and purified from Example 2

Crystalline cellulose 400 mg

5 mg of magnesium stearate

≪ Preparation Example 3 >

After mixing the following ingredients, tablets were prepared by tableting according to a conventional tablet preparation method:

400 mg of wakame extract containing 3.5% of fucosanthin of Example 1

100 mg of crystalline cellulose

5 mg of magnesium stearate

≪ Preparation Example 4 >

A mixture of 55% by weight of Spirulina, 10% by weight of guar gum enzyme hydrolyzate, 0.01% by weight of vitamin B1 hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, And 10% by weight of the fucoxanthin-containing marrow extract of Example 1 were combined and tableted by a conventional method.

≪ Preparation Example 5 >

The following ingredients were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules:

30 mg of fucosanthin, isolated and purified from Example 2

100 mg whey protein

Crystalline cellulose 400 mg

6 mg of magnesium stearate

≪ Preparation Example 6 >

The following ingredients were mixed and filled in gelatin capsules according to the conventional preparation method of capsules to prepare capsules:

300 mg of wakame extract containing 3.5% fucosanthin of Example 1

Corn starch 100 mg

100 mg of crystalline cellulose

5 mg of magnesium stearate

≪ Preparation Example 7 >

The active ingredient was dissolved in distilled water for injection and the pH was adjusted to about 7.5. Then, the whole of the following ingredients were filled in distilled water for injection and filled in 2 ml of ampoule and sterilized to prepare an injection:

100 mg of fucoxanthin isolated and purified from Example 2

Distilled water for injection

pH adjuster

≪ Production Example 8 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then powdered into a powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame, and perilla were steamed and dried by a known method, and then powdered into 60 mesh powder by a grinder.

The grains, seeds and fucoxanthin-containing waxy extracts prepared above were formulated in the following proportions.

Cereals: 30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley, 9% by weight of glutinous rice,

Seeds: 7% by weight of perilla, 8% by weight of black beans, 7% by weight of black sesame,

3% by weight of sea mustard extract, 0.5% by weight of manure, 0.5%

≪ Production Example 9 >

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of a flavor and 2% by weight of water and 0.1% by weight of a fucoxanthin-containing seaweed extract of Example 1.

≪ Production Example 10 >

Candy was prepared by combining 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of flavor and 0.1% by weight of fucoxanthin-containing marrow extract of Example 1.

≪ Production Example 11 >

A mixture of 0.75% by weight of sodium chloride, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonium, 0.17% by weight of sodium bicarbonate and 0.16% by weight of sodium bisulfite, 25.59% by weight of Grade I, 22.22% 1.45 wt% of rice flour, 0.0001 wt% of vitamin B1, 0.0001 wt% of vitamin B2, 0.04 wt% of milk fractions, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% 0.29% by weight of spray salt and 7.27% by weight of spray oil and 1% by weight of fucoxanthin-containing seaweed extract of Example 1 were mixed to prepare biscuits by a conventional method.

≪ Production Example 12 >

0.0001 wt% of nicotinic acid amide, 0.0001 wt% of sodium riboflavin hydrochloride, 0.0001 wt% of pyridoxine hydrochloride, 0.001 wt% of inositol, 0.002 wt% of orthoacetic acid and 98.7362 wt% of water were mixed with 0.26 wt% of honey, 0.0002 wt% 1 < / RTI > of fucoxanthin-containing marrow extract of the present invention was added to prepare a health drink.

Claims (6)

A pharmaceutical composition for preventing or treating hyperlipidemia comprising fucoxanthin of the formula (1) or an algae extract containing the same as an active ingredient, together with a pharmaceutically acceptable carrier: [Chemical Formula 1]

The method according to claim 1, Wherein said composition inhibits the accumulation of cholesterol or triglycerides in the blood and reduces the absorption of cholesterol from the diet. A food composition for preventing or improving hyperlipidemia comprising fucosanthin of the following formula (1) or an algae extract containing the same as an active ingredient: [Chemical Formula 1]

The method of claim 3, Wherein the composition inhibits the accumulation of cholesterol or triglycerides in the blood and reduces the absorption of cholesterol from the diet. The method according to claim 1 or 3, Wherein the seaweed extract is a seaweed extract, a seaweed extract, a mung bean extract, a top extract or a mixture thereof. The method according to claim 1 or 3, Wherein the algae extract is obtained by extracting seaweed from water, alcohol, hexane, ethyl acetate, isopropyl alcohol or a mixture thereof at 10 ° C to 50 ° C for 1 to 48 hours.