KR101592251B1 - Purification method of Phlorotannins from brown algae - Google Patents

Abstract

The present invention relates to a method for producing purified Florotannin, which comprises extracting brown algae with an aqueous ethanol solution, selectively adsorbing phlorotannin by using a hydrophobic adsorbent resin, thereby reducing the arsenic content while increasing the content of phlorotannin. The purified Florotannin produced according to the present invention can improve the content of fluorotannine having affinity to GABA _A -benzodiazepine receptor which can effectively work to relieve anxiety, improve seizure, sedation, and induce and improve sleep It has excellent sedation and sleep inducing effect, but it also improves safety by effectively lowering arsenic content. It can induce sedation and induce sleeping effect even with small amount. When used as tablets, tablets or injections, This is easy.

Description

Purification method of Phlorotannin from brown algae [

The present invention relates to a method for separating and purifying phlorotannin from brown algae, which comprises purifying a brown algae extract to increase the content of phlorotannin, and producing purified purified phlorotannin from brown algae with reduced arsenic content .

Many people in the modern world suffer from sleep disturbance because of stress, anxiety, and agitation. According to the statistics, about 15% of adults are known to need medication due to insomnia due to anxiety symptoms. Insomnia is caused by stress, tension, and fear. Therapeutic drugs include benzodiazepines and serotonin Drugs, etc., are used. However, these drugs have a serious side effect of being formed with tolerance and dependency when they are used for a long period of time.

Therefore, the need for natural supplements is increasing as a means of replacing sleeping pills in the case of long-term sleepers and restrictors, and the demand for functional foods for sleep enhancement is increasing.

GABA _A Gamma-aminobutyric acid (GABA) acts on calming, sleeping, anxiety, muscle tension, convulsions, memory loss and the like, which is a pterameric protein that forms a membrane ion channel. Many drugs, including benzodiazepines, as anxiolytics, sedatives or sleep inducers, bind to this receptor and perform pharmacological actions. When a drug or adjuvant is bound to the benzodiazepine site, GABA _A Enhances receptor affinity and increases intracellular influx of chlorine ions, thereby relieving anxiety, improving seizure, sedation, and inducing and improving sleep. However, it still shows drug dependence and can cause serious problems such as muscle relaxation and forgetfulness.

On the other hand, phlorotannin, a phenolic compound specifically present in a seaweed, a kind of brown algae, has an affinity for GABA _A - benzodiazepine receptor, and a composition containing it has an anxiolytic effect, an improvement in seizure, sedation, And it is known that it is very useful as a food composition such as a pharmaceutical composition and a health functional food because it is effective for improvement and uses a substance obtained from a natural substance and does not cause side effects and can secure safety.

Seaweed contains trace amounts of coloring matter and arsenic in addition to the above-mentioned active ingredients such as phlorotannin. Arsenic is a common element in soils, materials, and the natural world that is widely distributed in the air. Generally, 0.01 to 1.0 mg / kg is also contained in vegetables, grains and meat. On the other hand, the average content of arsenic contained in seaweed is 10 to 60 mg / kg, and it contains a larger amount of arsenic, which is different depending on the area where seaweed is collected and the type of seaweed, but it is included in seaweed collected from the East Coast The content of arsenic is about 30.650 mg / kg in the case of kelp, 41.003 mg / kg in the sea tangle, about 42.594 mg / kg in the seaweed and 167.390 mg / kg in the case of the sea bream [Kim, 2011 Kanto National University]. Arsenic contained in seaweeds is safe because it exists in the form of organic arsenic, which is almost non-toxic. However, due to recent causes of various environmental pollution, there is a problem of increase in inorganic arsenic content in aquatic products, It is preferable that the food be consumed in a reduced amount because it may cause a health hazard when accumulated over a long period of time.

Korean Patent No. 1260696 discloses a composition for activating GABA _A -benzodiazepine receptor comprising an extract of brown algae as an active ingredient. The extract is prepared by using at least one solvent selected from methanol, ethanol and water, Patent No. 1260697 discloses a composition for activating GABA _A - benzodiazepine receptor, which comprises phloroglucinol or phlorotannin as an active ingredient. The brown algae having excellent GABA _A - benzodiazepine receptor binding activity are selected and dissolved in methanol, And water to prepare an extract. The above-mentioned inventions are prior art patents of the present inventors. In order to utilize the above-mentioned brown algae extract industrially, when the content of phlorotannin contained in brown algae extract is increased to be used as tablets, tablets or injections, And should be able to solve the part of the increase of the heavy metal content which is often occurred when concentrating natural products. Accordingly, the present inventors found that the content of heavy metals, especially arsenic, which is accumulated in the brown algae extract is increased while the content of fluorotannine is being increased, and it is safe when applied to large-scale processes without using dangerous organic chemicals, The inventors of the present invention have completed the present invention with an effort to provide a process for producing the easily purified fluorotannin purified product.

The first problem to be solved by the present invention is to provide a method for producing purified Florotannin having reduced arsenic content while improving the content of Florotannine in brown algae extract.

According to an aspect of the present invention,

1) preparing a brown algae extract; And

And 2) adsorbing phlorotannin from the brown algae extract using a hydrophobic adsorbent resin.

According to one embodiment of the present invention, the brown algae extract may be a brown algae ethanol extract obtained by extracting dried brown algae with an aqueous solution of ethanol at 30-95 vol%

The brown algae may be selected from the group consisting of Ecklonia cava, Dictyopteris prolifera Okamura, Dictyota dichotoma Lamouroux, Sargassum horneri C. Agardh, Sargassum patens C. Agardh, And Ishige okamurae Yendo, and may be at least one selected from the group consisting of:

The extraction can be carried out by refluxing at 50 to 90 ° C for 5 to 48 hours. The extraction can be performed by adding 1 to 50 parts by weight of an aqueous ethanol solution of 30 to 95% by volume to 1 part by weight of dry brown algae.

According to another embodiment of the present invention, the hydrophobic adsorbent resin may include any one selected from styrene-divinylbenzene and aliphatic ester polymers, wherein the porosity is 0.9 to 2.0 mL / g, and the surface area Of from 400 to 800 m ² / g, a pore radius of from 50 to 300 Å, and a particle size of from 0.2 to 0.85 mm.

According to another embodiment of the present invention, the hydrophobic adsorption resin is at least one resin selected from the group consisting of Diaion HP-20, Amberlite XAD-4, Amberlite XAD-7HP, Amberlite XAD-1180 and Amberlite XAD-2000 Lt; / RTI >

More specifically, it may be selected from Diaion HP-20 and Amberlite XAD-7HP.

According to another embodiment of the present invention, the sugar content of the brown algae extract may be 15-25 Brix.

According to another embodiment of the present invention, the step 2) is performed by performing at least one method selected from static adsorption using a hydrophobic adsorption resin and dynamic adsorption using a hydrophobic adsorption resin to obtain a brown algae extract Lt; RTI ID = 0.0 > fluorotannine < / RTI >

The fixed adsorption method comprises: i) a hydrophobic adsorption resin; Brown algae extract; And distilled water are mixed and placed in a container and stirred at 50 to 500 rpm for 1 to 24 hours to adsorb fluorotannin to the hydrophobic adsorbent resin; Ii) after the adsorption is completed, separating the fluorotannine into the adsorbent filtrate with the adsorbed hydrophobic adsorbent resin; Iii) adding a 50 to 97 vol% ethanol aqueous solution to the hydrophobic adsorbent resin on which the fluorotannine has been adsorbed and stirring to prepare a desorbent liquid; And iv) concentrating and drying the desorption liquid,

The dynamic adsorption method comprises the steps of: a) filling a column tube with a hydrophobic adsorption resin; b) loading the brown algae extract on the top of the packed hydrophobic adsorbent resin and flowing distilled water at a flow rate of 2 to 7 BV / hr to adsorb phlorotannin to the hydrophobic adsorbent resin; And c) flowing 20 to 60% by volume of an aqueous ethanol solution at a flow rate of 0.5 to 7 BV / hr to desorb the fluorotannine adsorbed on the hydrophobic adsorption resin,

It is preferable that the hydrophobic adsorbent resin is pretreated by washing it with at least one selected from an aqueous solution of 50-97 vol% of ethanol and distilled water before use in a fixed adsorption method or a dynamic adsorption method, more preferably 50-97 vol% It can be used after first washing with aqueous solution and then second washing with distilled water.

Also, it may be carried out by mixing 1 to 10 parts by weight of the hydrophobic adsorbent resin with 1 part by weight of the brown algae extract,

The purified floatotannin purified by the above method can have a fluorotannine content of 700 mg / g or more and an arsenic content of 70 mg / kg or less based on the solid content.

The purified fluorotannine can exhibit an anxiolytic, an anticonvulsant, sedative, and induction and improvement of sleep with an improved dose of fluorotannine having an affinity for GABA _A - benzodiazepine receptor It can be very useful as a pharmaceutical composition, a health functional food and a food composition.

The method for producing the fluorotannine purified product according to the present invention is characterized in that the content of fluorotannine having an affinity to the GABA _A -benzodiazepine receptor which can effectively work to relieve anxiety, improve seizure, sedation, and induce and improve sleep, As the content of arsenic, which is a heavy metal, is lowered effectively to improve the stability of the human body, it can induce sedation and induce sleeping with a small amount, and when it is used as tablets, tablets or injections, it can be easily taken by lowering the dose once. In addition, since the content of phlorotannin is low and it is difficult to commercialize brown algae, it is possible to selectively extract and purify phlorotannin by using the production method according to the present invention, Does not use organic solvents harmful to human body and has a simple process and is easy to use for mass production and is excellent in industrial application.

FIG. 1 is a two-dimensional reaction surface graph of a condition for extracting fluorotannine from a brown algae set by a central synthesis program under optimum conditions using a reaction surface analysis method according to an embodiment of the present invention. FIG.
FIG. 2 is a graph showing adsorption amounts and desorbed amounts of phlorotannin contained in refined floatotannin purified according to a fixed adsorption method according to an embodiment of the present invention.
3 is a graph showing adsorption and desorption amounts of Example 1.1 prepared according to the present invention over time.
4 is a graph showing the affinity between the adsorbent resin and the concentration and reaction temperature of Example 1.1 prepared according to the present invention.
FIG. 5 is a graph showing desorption characteristics of the solvent concentration of Example 2.1 prepared according to the present invention. FIG.
FIG. 6 is a graph illustrating the desorption characteristics of Example 2.1 prepared according to the present invention with respect to the flow velocity. FIG.
FIG. 7 shows the HPLC (high performance liquid chromatography) analysis results of the brown algae extract and fluorotannin purified according to the present invention (a brown algae extract (menthol), b-fluorotannin purified product)
8 is a diagram illustrating a sleep induction experiment according to the present invention.
FIG. 9 is a graph showing the results of hypnotic pentobarbital induced sleep test of the preparation example according to the present invention and the example 2.1.

GABA _A receptors are associated with anxiety relief, seizure improvement, sedation, and sleep induction and improvement. Drugs acting on GABA _A receptors include benzodiazepines, diazepam (DZP), sol Zolpidem, and the antagonist is flumazenil (FLU). As mentioned above, the drugs acting on the GABA _A receptor have drawbacks that cause dependency and side effects.

Unlike green algae or red algae, brown algae extracts contain phlorotannin that acts on GABA _A- benzodiazepine (benzodiazepine) receptors and has a sedative, sleep-inducing effect. However, marine organisms and seaweeds contain different amounts of heavy metals, but most of them contain trace amounts of heavy metals such as arsenic, and there is a large difference in content depending on the location and season of harvest. When raw materials are used as food, the intake is not so high. Therefore, heavy metals such as arsenic, which are ingested together, are rarely consumed in excess of the human tolerable standard. However, when the raw materials are processed and concentrated, heavy metals are also concentrated together with the active ingredient , It is difficult to apply it to food additives, pharmaceutical compositions and health functional foods without solving the heavy metal concentration problem.

It is known that arsenic contained in seaweed is mostly present in the form of organic arsenic, and it is known that there is almost no toxicity. However, since arsenic is also concentrated when the brown algae extract is concentrated, problems such as increase in heavy metal content, It can be used as a composition.

In addition, in order to facilitate industrial use, it is necessary to exhibit a high effect in a small amount when used as tablets, tablets or injections, and to reduce the use of organic solvents which are harmful to human body during the manufacturing process.

According to Food Standards and Food Standards and Standards (published in 2013) issued by the Food and Drug Administration, capsules are formed from food additives such as gelatin, glycerin, and foods as raw materials. The standard of arsenic is limited to 1.5 mg / kg or less. Accordingly, the present inventors have found that, in order to facilitate the use of food additives, health functional foods, and pharmaceutical compositions, a simple and environmentally friendly method of purifying fluorotannine, which can reduce the arsenic content to a level below the standard value while increasing the content of fluorotannine, As a result of effort, the present inventors have completed the present invention by developing a method for producing purified flood tannin which has a high content of fluorotannine and a significantly reduced arsenic content by a simple method using an adsorbent resin and a reaction surface analysis method.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a method for producing brown algae comprising the steps of: 1) preparing a brown algae extract; And 2) adsorbing phlorotannin from the brown algae extract using a hydrophobic adsorbent resin.

According to the present invention, the brown algae are selected from the group consisting of Ecklonia cava, Dictyopteris prolifera Okamura, Dictyota dichotoma Lamouroux, Sargassum horneri C. Agardh, patens C.Agardh) and paddle (Ishige okamurae Yendo).

The brown algae extract can be obtained by extracting dried brown algae with an aqueous solution of 30-95% by volume of ethanol, wherein 5-30 parts by weight of 70-95% by volume ethanol aqueous solution is added to extract brown algae Can be prepared.

In addition, the extract can be extracted by refluxing at 50 to 90 ° C for 5 to 48 hours. If the temperature is lower than the above range, it is difficult to extract the phlorotannin of brown algae sufficiently in the aqueous ethanol solution. It is possible to produce a brown algae extract having a high content of phlorotannin as an active ingredient having a water-surface inducing effect within the above-mentioned range, It is possible to produce a brown algae extract having a higher content of phlorotannin by refluxing at 80 ° C for 16 to 24 hours.

The extract may be prepared by adding 1 to 50 parts by weight of an aqueous ethanol solution of 30 to 95% by volume as a solvent to 1 part by weight of dried brown algae, but 5-30 parts by weight of 70-95% Is added to extract the brown algae having a high content of phlorotannin, which is preferable.

 When the amount of the solvent is less than the above range, the amount of the solvent is so small that the dry brown algae are not sufficiently dissolved or suspended in the solvent, so that it is difficult to sufficiently extract the fluorotannin component. When the amount of the solvent exceeds the above range, There is a problem that it is time-consuming and economical to concentrate the extracted solvent of the fluorotannine.

According to the present invention, the hydrophobic adsorbent resin used for purifying the brown algae extract may be any one selected from styrene-divinylbenzene and aliphatic ester polymers,

The hydrophobic adsorbent resin may have a porosity of 0.9 to 2.0 mL / g, a surface area of 400 to 800 m ² / g, a pore radius of 50 to 300 Å, a particle size of 0.2 to 0.85 mm The above condition is that the adsorbed phlorotannin contained in the brown algae extract is adsorbed with excellent efficiency, but the additive components such as arsenic, dye and polymeric water-soluble substance are not adsorbed, so that arsenic is reduced even when the content of fluorotannine is high Whereby a purified tannin product can be obtained.

According to the present invention, the hydrophobic adsorption resin may be at least one resin selected from the group consisting of Diaion HP-20, Amberlite XAD-4, Amberlite XAD-7HP, Amberlite XAD-1180 and Amberlite XAD-2000, Preferably, it may be a resin selected from Diaion HP-20 and Amberlite XAD-7HP. Since this resin absorbs fluorotannine contained in the brown algae extract with excellent efficiency, it does not adsorb additional components such as arsenic and coloring matter , It is preferable to enhance the phlorotannin component and purify the brown algae extract so that the arsenic content can be reduced.

The sugar content of the brown algae extract can be adjusted to 15-25 Brix, but the sugar content is not limited to the purifying efficacy of the brown algae extract.

According to the present invention, the step 2) separates the phlorotannin from the brown algae extract by performing at least one method selected from static adsorption using a hydrophobic adsorbent resin and dynamic adsorption using a hydrophobic adsorbent resin. However,

The hydrophobic adsorbent resin, the brown algae extract and water are mixed to allow the hydrophobic adsorbent resin to adsorb the phlorotannin component contained in the brown algae extract, and the hydrophobic adsorbent resin adsorbed on the phlorotannin component is washed with an aqueous ethanol solution to obtain phlorotannin By allowing the components to be desorbed, the phlorotannin contained in the brown algae extract can be selectively extracted.

The water may be a general water, but it is preferable to use distilled water because it can reduce the influx of unnecessary components and ions, and the aqueous solution of ethanol in which fluorotannine has been desorbed can be concentrated and dried to produce purified fluorotannin have. The step 2) will be described in more detail as follows.

The fixed adsorption method comprises: i) a hydrophobic adsorption resin; Brown algae extract; And distilled water are mixed and placed in a container and stirred at 50 to 500 rpm for 1 to 24 hours to adsorb fluorotannin to the hydrophobic adsorbent resin; Ii) after the adsorption is completed, separating the fluorotannine into the adsorbent filtrate with the adsorbed hydrophobic adsorbent resin; Iii) adding a 50 to 97 vol% ethanol aqueous solution to the hydrophobic adsorbent resin on which the fluorotannine has been adsorbed and stirring to prepare a desorbent liquid; And iv) concentrating and drying the desorption liquid,

According to the present invention, the reaction can be carried out at a temperature of 10 to 60 ° C. If the stirring temperature is lower than the above range, the amount adsorbed on the hydrophobic adsorption resin (hereinafter referred to as adsorption resin) The temperature is too high, and the pigment, the high-molecular water-soluble substance and other components contained in the brown algae ethanol extract are converted into a size suitable for adsorption to the adsorbent resin and adsorbed on the adsorbent resin, so that the content of the fluorotannin adsorbed on the adsorbent resin Which is undesirable.

If the stirring speed is less than the above range, it is too slow to adsorb uniformly on the adsorbent resin, which is not preferable because the amount of adsorbed fluorotannine on the adsorbent resin is low. If the stirring speed exceeds the above range, The fluorotannine adsorbed by the adsorbed or hardly adsorbed fluorotannine can be eluted again, which is not preferable. If the agitation time is less than 0.5 hour, the amount of fluorotannine to be adsorbed is so small that the adsorption time is too short, which is not preferable. Even if the stirring time exceeds 12 hours, the increase of the adsorption of fluorotannine on the adsorption resin is insignificant It is not.

According to the present invention, the dynamic adsorption method comprises the steps of: a) filling a column tube with a hydrophobic adsorption resin; b) loading the brown algae extract on the top of the packed hydrophobic adsorbent resin and flowing distilled water at a flow rate of 2 to 7 BV / hr to adsorb phlorotannin to the hydrophobic adsorbent resin; And c) 20 to 60% by volume aqueous ethanol solution at a flow rate of 0.5 to 7 BV / hr to desorb the fluorotannine adsorbed on the hydrophobic adsorption resin.

According to the present invention, it is preferable that the hydrophobic adsorbent resin is pretreated by washing with at least one selected from an aqueous solution of 50-97 vol% of ethanol and distilled water before use in a fixed adsorption method or a dynamic adsorption method, more preferably 50- 97% by volume of ethanol aqueous solution, and then washed with distilled water for secondary washing. Since the above procedure can remove impurities introduced during the production of the hydrophobic adsorption resin or during the distribution and storage, It is possible to produce a purified fluorotannin product having a high content.

According to the present invention, it is preferable that the flow rate of the distilled water flowing through the column tube is 2 to 7 BV / hr. If the flow velocity is slower than the above rate, it takes a long time to adsorb the fluorotannine, which is not economical. As long as fluorotannine can not be adsorbed on the hydrophobic adsorbent resin at the earliest, most of it can be discharged, which is not preferable.

The flow rate of the aqueous ethanol solution of 20 to 60% by volume used for the desorption may be 0.5 to 7 BV / hr, preferably 3 to 5 BV / hr. When the flow rate of the aqueous solution of 20 to 60% by volume of ethanol is less than the above range, the flow rate is too slow and the amount of desorbed is large, but it takes a long time and is not economical. When the flow rate exceeds the above rate, Is not sufficiently desorbed.

The fluorotannine purified product according to the present invention may have a fluorotannine content of 700 mg / g or more and an arsenic content of 70 mg / kg or less based on the solids content of brown algae extract, May be 800 mg / g or more, and the content of arsenic may be 50 mg / kg or less.

Flow tannin purified water GABA _A in accordance with the present invention acts in the benzodiazepine receptor binding by GABA _A - is characterized by having an affinity to the benzodiazepine receptor. As the brown algae extracts extracted by the extraction conditions set by the central synthetic design method using the reaction surface analysis method according to the present invention, for example, a menthol which is known to have a high content of fluorotannin among brown algae was selected, the IC ₅₀ values of the Ecklonia cava extract extracted according to 1,260,697 number), while the 0.456 mg / mL, IC ₅₀ values of the Ecklonia cava extract extracted by the extraction conditions set by the central composite design by using the response surface methodology according to the invention 0.120 mg / mL. The IC ₅₀ value of the chelated fluorotannin purified by the purification method according to the present invention was 0.012 mg / mL. The affinity of the purified brown algae according to the present invention for the GABA _A -benzodiazepine receptor Which means that it can exhibit a high sleep inducing effect even in a small amount.

The purified brown algae according to the present invention exhibits high anxiolytic, sedative, sleep inducing or sleeping enhancing effect even at a low dose by increasing the content of fluorotannine as described above, and is easy to use as tablets, tablets and injections, It can be applied as an additive, a health promotion food and a pharmaceutical composition, and has a stability against the accumulation of heavy metals by effectively lowering the content of arsenic.

The sleep inducing effect may be an effect of decreasing sleep latency and increasing sleep duration, but the scope of the present invention is not limited thereto,

The surface time of the brown algae extract extracted according to the extraction conditions set by the central synthesis program using the reaction surface analysis method according to the present invention is 65 to 85 minutes, The sleeping time of the purified water was increased from 120 to 140 minutes at the same concentration.

When the purified fluorotannine according to the present invention is used as an active ingredient additive for general food, the purified fluorotannin according to the present invention can be used as it is or can be used in combination with other food or food ingredients, Can be used.

Generally, the purified product of the floatotinin according to the present invention can be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range. Further, since the present invention uses an extract from a natural product, Or more. However, taking into consideration that the daily allowance of arsenic is 1.5 mg / kg per 1 kg of body weight, it is desirable to take it so that it does not exceed the daily allowance of arsenic.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

The beverage food in the food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient like ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the functional food according to the present invention.

When the tablets of the present invention are used as a health functional food for relieving anxiety, sedation, inducing sleep or promoting sleep, there is no particular limitation on the kind thereof, and a powder containing the purified product of the floatotannin as an active ingredient , Granules, tablets, capsules or beverages, but the present invention is not limited thereto. The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use such as prevention, health or treatment.

When the purified product of the fluorotannine according to the present invention is used as a pharmaceutical composition for relieving anxiety, sedation, inducing sleep or promoting sleep, it may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols The composition may be formulated in the form of an oral preparation, a external preparation, a suppository, and a sterile injection solution. The amount of the active ingredient to be mixed may be appropriately determined according to each use purpose such as prevention, health or treatment.

When the tablets of the present invention are formulated into powders, granules, tablets or capsules, they may further contain appropriate carriers, excipients and diluents conventionally used in the manufacture of foods. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid preparations for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of liquid formulations for oral use include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of a composition for use in the induction, amelioration and sedation of sleep according to the present invention depends on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. In addition, the scope of the present invention is not limited by the dosage, and the dosage may be increased or decreased according to necessity. If there is no arsenic ingested through another route or less than 1.35 mg / kg, the fluorotannine tablet according to the present invention may be increased to include less than the arsenic dose per day (1.5 mg / kg) The dosage may be increased under the conditions in which it is taken.

According to the daily intake limit of arsenic announced by the Food and Drug Administration, the allowable amount of arsenic per kg of body weight is 1.5 mg. For example, in order to exhibit a desired effect, arginine, which is ingested at a daily dose of 2,000 mg, is administered to 1 kg of the solid of the fluorotannine purified product according to the present invention. Was 0.14 mg or less and the content of fluorotannine was 1400 mg or more, which was stable because it was below the allowable daily intake of arsenic. On the other hand, referring to Table 11 below, the following Comparative Example 3 according to the conventional method has a total content of fluorotannine of 118 mg / g and an arsenic content of 188.1 mg / kg, (Total fluro tannin content: 1400 mg), it was difficult to take the fluro tannin because 11,864 mg of Comparative Example 3 had to be consumed. At this time, the content of arsenic was 2.231 mg, Arsenic is contained in an amount of 16 times as much as that of the purified fluorotannine purified product.

The fluorotannine tablet according to the present invention solves the above problems and exhibits an excellent effect even with a small amount of intake. When used as tablets, tablets or injections, it is easy to take and has a high stability by lowering arsenic content.

Compositions for use in the induction, amelioration and sedation of sleep according to the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The health functional food or pharmaceutical composition containing the purified fluorotannine according to the present invention as an active ingredient may be used as a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a pectic acid and its salt, an alginic acid and its salt, A stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated beverage, and these components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the total composition of the present invention.

On the other hand, the active ingredient of the purified product of the floatotin may be at least one selected from the group consisting of phloroglucinol, ecol, exolol, triphloretol-A and diacyl, and the structure of the active ingredient is as follows.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

Example

In order to carry out the purification method according to the present invention, as the brown algae, the menthol showing the highest content of phlorotannin in the following Test Example 1 was selected and the experiment was conducted. The senses used in the present invention were collected directly from Jeju coastal area and used as a sample. The methanol used in the analysis was JT baker (Pilipsburg, NJ, USA) and the distilled water was samchun pure chemical co., Ltd (Pyeongtaek, Korea). The ethanol used in the experiment was purchased from Daejeong Chemical Were purchased and used.

Amberlite XAD-7, Amberlite XAD-7HP, Amberlite XAD-16, Amberlite XAD-4, Amberlite XAD-7, and Amberlite XAD-7 were purchased from Mitsubishi Chemical (Japan) 1180, and Amberlite XAD-2010 were purchased from Sigma-Aldrich (USA). Folin &Ciocalteu's phenol reagent used in the total polyphenol content experiment was purchased from Sigma-Aldrich and Na ₂ CO ₃ was purchased from Junsei chmical co., Ltd (Japan).

Physical properties of the hydrophobic adsorbent resin according to the present invention are shown in Table 1 below.

Resin name
Structural Series Porosity (mL / g) Surface area
(m ² / g) Pore radius
(A) Particle size
(mm) Diaion HP-20 Polystyrene type polymer 1.3 600 260 250-850 Amerlite XAD-2 Acrylic ester 0.65 330 90 250-840 Amberlite XAD-4 Polystyrene type polymer 0.98 750 100 560-710 Amberlite XAD-7 Acrylic ester 0.5 380 300-400 560-710 Amberlite XAD-7HP Polystyrene type polymer 1.14 450 90 250-840 Amberlite XAD-16 Polystyrene type polymer 0.55 800 200 560-710 Amberlite XAD-1180 Polystyrene type polymer 1.68 600 300 560-710 Amberlite XAD-2010 Polystyrene type polymer 1.8 660 280 560-710 sepabead SP850 Polystyrene type polymer 1.2 1000 35 300-800

The column used for dynamic adsorption was a glass chromatographic column (25 mm.times.400 mm, DURAN, Germany). The column used for static adsorption was SI-600R (Jeio Tech Inc., Seoul, Korea) ) Was used. The total polyphenol content was measured using a V-530 UV / VIS spectrophotometer (Jasco Inc., Japan) and a 10-mm disposable cuvette (Ratiolab, Germany). For HPLC analysis, Agilent TC-C18 (4.6 x 250 mm, 5 μm) was used for the analysis of the contents of purified and untreated phlorotannin tablets using Shimadzu LC-20 liquid chromatography (Shimazu Co., Ltd., Kyoto, Japan) Were used.

Production example . Response Surface Methodology (Response Surface Methodology , RSM ) Menthol ethanol extract

end. Experimental Design

In order to optimize extraction conditions to increase the content of phlorotannin, 17 experimental conditions were derived using Response Surface Methodology (RSM), and the content of fluorotannine was analyzed Conditions.

Experimental design was established by Central Composite Design (CCD). The independent variable, which is a parameter affecting the reaction, was set as the ethanol concentration (X ₁ ), the extraction temperature (X ₂ ) and the extraction time (X ₂ ) of the aqueous ethanol solution. The center values and ranges of the independent variables were set through preliminary experiments and were statistically analyzed by coding them at five levels (-1.682, -1, 0, +1, +1.682) as shown in Table 2 below. The total fluorotannine content (Y ₁ ) and yield (Y ₂ ) were set as dependent variables. Experimental groups were randomly selected to reduce unexpected errors in the experiment. All statistical analyzes were performed using MINITAB (Version 15, Minitab Inc., USA) and a 3-D graph was used with Maple (Version 7, Waterloo Maple Inc., Canada).

Independent variable Mark Range and levels -1.682 -One 0 +1 +1.682 Ethanol concentration (vol%) X _One 55 63 75 87 95 Temperature (℃) X ₂ 40 48 60 72 80 Time (h) X ₃ 6 9.6 15 20.4 24

RSM is a statistical technique that is very effective in optimizing the manufacturing conditions and monitoring the influence on the parameters, and is widely used in food processing in recent years. The 17 experimental groups determined according to the central synthetic program were run randomly and the values of the total fluorotannine content (Y ₁ ) and yield (Y ₂ ), which are dependent variables thereof, are shown in Table 3 below . Dependent variables were set as the content of phlorotannin which is an active ingredient for sleep enhancement and the yields important for industrialization.

Experiment number Uncoded independent variables Response X _One X ₂ X ₃ Y _One Y ₂ Factorial
portion One -One -One -One 379.6 16.2 2 One -One -One 443.2 13.3 3 -One One -One 315.4 15.7 4 One One -One 464.2 14.8 5 -One -One One 344.6 15.4 6 One -One One 416.8 15.4 7 -One One One 444.3 14.4 8 One One One 498.1 14.5 Star
portion 9 -1.682 0 0 381.1 15.4 10 1.682 0 0 518.2 11.9 11 0 -1.682 0 402.3 13.9 12 0 1.682 0 412.2 16.5 13 0 0 -1.682 398.2 16.2 14 0 0 1.682 381.1 16.2 Center
portion 15 0 0 0 389.8 16.2 16 0 0 0 416.7 16.0 17 0 0 0 459.6 15.8

X ₁ , aqueous ethanol concentration (volume%); X ₂ , temperature (캜); X ₃ , time (h).

Y ₁ , total fluro tannin content (mg PGE / g); Y ₂ , Yield (%).

I. Independent and Dependent Variables Influence

The most basic principle of the RSM is to determine the response surface model equation that represents the interrelationship between the independent variables that affect and the dependent variables that respond accordingly. The response surface model equation is statistically significant with a coefficient of determination (R ² ) and a significance level (P-value) of 0.85 or more and 0.05 or less, respectively, and is shown in Table 4 below.

Quadratic polynomial model equations R ² P- value Y ₁ = 422.152 + 41.662 X ₁ + 11.309 X ₂ + 5.319 X ₃ + 9.354 X ₁ ² - 5.637 X ₂ ² - 11.859 X ₃ ² + 8.350 X ₁ X ₂ - 10.800 X ₁ X ₃ + 28.025 X ₂ X ₃ 0.871 0.020 Y ₂ = 15.997 - 0.820 X ₁ + 0.373 X ₂ - 0.052 X ₃ - 0.848 X ₁ ² - 0.284 X ₂ ² + 0.075 X ₃ ² + 0.306 X ₁ X ₂ + 0.274 X ₁ X ₃ - 0.129 X ₂ X ₃ 0.900 0.009

X ₁ , aqueous ethanol concentration (volume%); X ₂ , temperature (캜); X ₃ , time (h).

Y ₁ , total fluro tannin content (mg PGE / g); Y ₂ , Yield (%).

It is easy to see the influence of ethanol concentration (X ₁ ), extraction temperature (X ₂ ) and extraction time (X ₃ ) which are dependent variables on the total fluro tannin content (Y ₁ ) and yield (Y ₂ ) A three-dimensional reaction surface graph for each raw material is shown in FIG. The higher the aqueous ethanol concentration (X ₁ ) and the extraction temperature (X ₂ ), the higher the total fluorotannine content (Y ₁ ) and the lower the yield (Y ₂ ). As the extraction time (X ₃ ) was shortened, the total fluorotannine content (Y ₁ ) increased and the yield (Y ₂ ) tended to decrease. However, compared with the aqueous solution concentration (X ₁ ) and extraction temperature (X ₂ ) The effect on the total fluorotannine content (Y ₁ ) and yield (Y ₂ ) was low.

All. Optimum condition setting

As a result of the optimal extraction conditions, the concentration of aqueous ethanol solution (X ₁ ) was 70-95%, the extraction temperature (X ₂ ) was 70-80 ° C, The extraction time (X ₃ ) was 16-24 hours. In the optimum condition, the total fluorotannine content (Y ₁ ) was 420-560 mg PGE / g and the yield (Y ₂ ) was 13-16%.

la. Extraction by Optimum Condition

400 Kg of the dryness was washed with water to remove the salts, sand and the like, and then 4000 L of 70 vol% ethanol aqueous solution was added and the mixture was extracted at 70 ° C for 16 hours. After the extraction was completed, the mixture was filtered using a filter, and then the filtrate was concentrated under reduced pressure to prepare a 20 brix chewing ethanol extract.

hemp. Determination of Total Florotannine Content and Yield of Ethanol Extract

Actual measured values of CPT ethanol extracts were 435-620 mg PGE / g and 14-18%, respectively, indicating the total fluorotannine content (Y ₁ ) and yield (Y ₂ ) And the results are shown in Table 5 below.

Multiple response optimization Dependent variables Y ₁ Y ₂ Optimal conditions X ₁ = 70-95.0 (volume%), X ₂ = 70-80 (° C), X ₃ = 16-24 (h) Predicted value 400 - 600 13 - 16 Experimental value 435 - 620 14 - 18

X ₁ , aqueous ethanol concentration (volume%); X ₂ , temperature (캜); X ₃ , time (h).

Y ₁ , total fluro tannin content (mg PGE / g); Y ₂ , Yield (%).

Example  1. Purification by fixed adsorption

Example 1.1. Diaion HP-20 (hereinafter HP 20)

The cutaneous ethanol extract prepared in Preparation Example was purified by a fixed adsorption method. 10 g of Diaion HP-20 (hereinafter referred to as HP 20) was added to the flask, 200 mL of distilled water was added, and the mixture was pre-treated by stirring at 180 rpm and 25 ° C for 12 hours. After the pretreatment was completed, the distilled water contained in the resin was removed, and then 7.55 g of the extract of the menthol ethanol (20 brix) prepared in Production Example and 200 mL of fresh distilled water were added thereto. The mixture was stirred at 180 rpm and 25 ° C for 12 hours, Adsorption filtrate was collected.

The adsorbent resin adsorbed with the ethanol extract of Ganoderma lucidum was eluted (desorbed) with 95 vol.% Ethanol aqueous solution.

Example 1.2. Amberlite XAD-4 (hereinafter referred to as XAD-4)

The chewing ethanol extract was purified by the method of Example 1.1, except that Amberlite XAD-4 was used instead of Diaion HP-20 as a hydrophobic resin.

Example 1.3. Amberlite XAD-7HP (hereinafter referred to as XAD-7HP)

The ethidium ethanol extract was purified by the method of Example 1.1, except that Amberlite XAD-7HP was used instead of Diaion HP-20 as a hydrophobic resin.

Example 1.4. Amberlite XAD-1180 (hereinafter referred to as XAD-1180)

The ethidium ethanol extract was purified by the method of Example 1.1, except that Amberlite XAD-1180 was used instead of Diaion HP-20 as a hydrophobic resin.

Example 1.5. Amberlite XAD-2010 (hereafter referred to as XAD-2010)

The chewing ethanol extract was purified by the method of Example 1.1, except that Amberlite XAD-2010 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 1.1. Example 1.2. Sepabead SP-850 (hereinafter referred to as SP-850)

The cephem ethanol extract was purified by the method of Example 1.1, except that Sepabead SP-850 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 1.2. Amerlite XAD-2 (hereinafter XAD-2)

The ethidium ethanol extract was purified by the method of Example 1.1, except that Amerlite XAD-2 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 1.3. Amerlite XAD-7 (hereinafter referred to as XAD-7)

The ethidium ethanol extract was purified by the method of Example 1.1, except that Amerlite XAD-7 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 1.4. Amerlite XAD-16 (hereinafter referred to as XAD-16)

The ethidium ethanol extract was purified by the method of Example 1.1, except that Amerlite XAD-16 was used instead of Diaion HP-20 as the hydrophobic resin.

Example  2. In the dynamic adsorption method  Refining by

Example 2.1. Diaion HP-20 (hereinafter HP 20)

The ethanol extracts from the preparations prepared in Preparation Example were purified by dynamic adsorption. A glass column tube of 25 mm x 400 mm was filled with 30 g of a hydrophobic adsorption resin Diaion HP-20 (hereinafter referred to as HP-20), washed with a 95% by volume aqueous ethanol solution and then washed with distilled water for 2 hours to stabilize the resin . 22.65 g of the cutaneous ethanol extract (20 brix) prepared in Production Example was loaded and 1360 mL of distilled water was flowed at a flow rate of 4 BV / hr to obtain an adsorption filtrate.

Thereafter, a component of the filtered ethanol extract adsorbed on the adsorbent resin was eluted (desorbed) using a 40 vol% ethanol aqueous solution at a flow rate of 4 BV / hr.

Example 2.2. Amberlite XAD-4 (hereinafter referred to as XAD-4)

The cephem ethanol extract was purified by the method of Example 2.1 except that Amberlite XAD-4 was used instead of Diaion HP-20 as the hydrophobic resin.

Example 2.3. Amberlite XAD-7HP (hereinafter referred to as XAD-7HP)

The chewing ethanol extract was purified by the method of Example 2.1 except that Amberlite XAD-7HP was used instead of Diaion HP-20 as a hydrophobic resin.

Example 2.4. Amberlite XAD-1180 (hereinafter referred to as XAD-1180)

The ethidium ethanol extract was purified by the method of Example 2.1 except that Amberlite XAD-1180 was used instead of Diaion HP-20 as a hydrophobic resin.

Example 2.5. Amberlite XAD-2010 (hereafter referred to as XAD-2010)

The ethidium ethanol extract was purified by the method of Example 2.1 except that Amberlite XAD-2010 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 2.1. Example 1.2. Sepabead SP-850 (hereinafter referred to as SP-850)

The cephem ethanol extract was purified by the method of Example 2.1 except that Sepabead SP-850 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 2.2. Amerlite XAD-2 (hereinafter XAD-2)

The ethidium ethanol extract was purified by the method of Example 2.1 except that Amerlite XAD-2 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 2.3. Amerlite XAD-7 (hereinafter referred to as XAD-7)

The ethidium ethanol extract was purified by the method of Example 2.1 except that Amerlite XAD-7 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 2.4. Amerlite XAD-16 (hereinafter referred to as XAD-16)

The ethidium ethanol extract was purified by the method of Example 2.1 except that Amerlite XAD-16 was used instead of Diaion HP-20 as a hydrophobic resin.

Comparative Example 3 Ethanol extraction according to the conventional method (Korean Patent No. 1260697)

4000 kg of dryness was washed with water to remove the remaining salts and sand, and then 4000 L of 70 vol% ethanol aqueous solution was added and the mixture was extracted at 50 캜 for 3 hours.

Test Example  1. In the brown algae extract GABA _A - Benzodiazepine  Detection of receptor binding activity

Seven species of brown algae methanol extracts were purchased from Jeju Technopark 's Jeju Biological Resources Extraction Bank to investigate seaweeds with anxiety relief, seizure improvement, sedation, and sleep induction and improvement effects. The GABA _A - benzodiazepine receptor binding Were measured. A voucher specimen was deposited with the Jeju Biological Resources Extraction Bank.

GABA _A - benzodiazepine receptor binding activity was measured by homogenizing the cerebral cortex in 20 mL of 30 mM Tris - HCl buffer (pH 7.4, keep at 4) and treating with ultrasonication for 10 seconds. After centrifugation at 27,000 × g and 4 ° C. for 15 minutes, the supernatant was discarded, and 20 mL of the buffer was centrifuged. This procedure was repeated three times. To remove GABA in the brain tissue, the cells were incubated in a water bath at 37 ° C for 30 minutes, centrifuged, and then the pellets were collected and stored frozen at -80 ° C. After thawing the frozen membranes, centrifuge at 27,000 × g and 4 ° C for 10 minutes, discard the supernatant, add 20 mL of 50 mM Tris-citrate buffer (pH 7.1, keep at 4 ° C) Respectively. This procedure was repeated three times and the membrane was suspended in binding buffer at a concentration of 500 mL buffer / original tissue g and used for the binding force test. In a 96-well plate, add 180 μl of membrane suspension, 10 μl of each extract, and 10 μl of [3 H] flumazenil (1 nM, final concentration) (Ro 15-1788; PerkinElmer Life and Analytical Sciences, Waltham, Mass., USA) and incubated in ice-bath for 40 min. And then collected using a glass fiber filter (GF / C, Whatman). The radioactivity of the samples was measured using Tri-Carb Liquid Scintillation Analyzers (Perkin-Elmer, Shelton, CT, USA). The nonspecific binding (NSB) was determined using clonazepam (1 uM, final concentration) and the binding displacement value (%) was calculated according to the following equation (1).

Equation (1)

Binding displacement (%) = [1 - ((DPM - NSB DPM) / (TB DPM - NSB DPM)

(DPM: disintegrations per minute, TB: total binding, NSB: nonspecific binding)

The results of GABA _A - benzodiazepine receptor binding activity of the methanol extract of brown algae are shown in Table 6, with different concentrations of extracts of 0.1 mg / mL, 1 mg / mL and 10 mg / mL, respectively.

Family Species Displacement (%) of ^[3 H] -flumazenil binding to the GABA _A receptor -BZD 0.1 mg / mL 1 mg / mL 10 mg / mL Alariaceae Ecklonia cava Kjellman 5.2 ± 6.4 43.1 ± 6.2 91.3 ± 2.3 Dictyotaceae Dictyopteris prolifera Okamura 18.5 ± 1.0 31.5 ± 4.9 63.8 ± 4.9 Sargassaceae
Dictyota coriacea 16.2 + 1.7 16.2 ± 5.6 42.1 ± 2.0 Dictyota dichotoma Lamouroux 26.3 ± 14.7 25.7 ± 9.3 72.6 ± 1.6 Sargassacea
Sargassum hornier C.Agardh 4.4 ± 3.6 18.8 ± 1.3 70.4 ± 2.4 Sargassum patens C.Agardh 3.9 ± 2.8 15.9 ± 4.7 55.7 ± 1.5 Shigeaceae Ishige okamurae Yendo -8.3 ± 4.1 20.0 ± 2.3 73.8 ± 1.8 Alariaceae Undaria pinnatifida Harvey Suringar 26.7 ± 3.2 38.6 ± 7.7 70.6 ± 1.1

True leather nets on the end of brown algae (Dictyota coriacea ) and twin moths ( Sargassum patens C. Agardh), the binding activity of GABA _A - benzodiazepine receptor was more than 55% at 10 mg / mL concentration. Especially, Ecklonia cava Kjellman) showed more than 90% GABA _A - benzodiazepine receptor binding activity.

Test Example  2. According to the fixed adsorption method Florotannine  The adsorption amount and Desorption amount  Measure

The total fluorotannin content was measured by measuring the total polyphenol content from the adsorbed and desorbed solution prepared in Example 1, and the adsorption / desorption rate was calculated using the following equation, Respectively.

Adsorption evaluation

Where V _e is the adsorption solvent (mL), W is the weight of the resin (mg / mL), q _e is the adsorption capacity (mg / g dry resin), C ₀ is the initial concentration, C _e is the equilibrium concentration (g).

Desorption evaluation

Wherein, q _d is the adsorption capacity (mg / g dry resin) and, C _d represents the elution concentration (mg / mL) of the desorption solution, V _d is the desorption solvent (mL), W is the weight (g) of the resin .

Total fluorotannine content was measured using the Folin-Ciocalteu method. Add 0.5 mL of Folin-Ciocalteu reagent to 0.5 mL of sample, add 6.5 mL of distilled water, and allow to stand at room temperature for 3 minutes. Then, 1 mL of 20% Na ₂ CO ₃ reagent and 1.5 mL of distilled water were added, and the final volume was adjusted to 10 mL. Then, the reaction was carried out in a dark room for 1 hour, and then, using a V-530 UV / VIS spectrophotometer (Jasco Inc., Japan) And the absorbance at 765 nm was measured.

division Adsorption amount (mg) Desorption amount (mg) Removal rate (%) Example 1.1 57.86 56.71 98.04 Example 1.2 53.15 51.22 94.23 Example 1.3 54.67 52.18 95.46 Example 1.4 51.35 49.27 91.71 Example 1.5 51.81 49.21 93.93 Comparative Example 1.1 38.69 35.95 93.00 Comparative Example 1.2 21.87 16.69 86.88 Comparative Example 1.3 24.21 20.14 84.64 Comparative Example 1.4 20.16 17.23 82.88

The content analysis of Example 1.1 showing the highest adsorption amount and desorption amount was performed. The content was determined by using a column of Agilent TC-C18 (4.6 x 250 mm, 5 μm) at a column oven temperature of 24 ° C. and a flow rate of 0.8 mL / min. Then, the mobile phase solvent 90% H ₂ O (A) B) for 60 minutes, and the separated chromatograms were obtained using a PDA detector at UV 230 nm.

As shown in FIG. 3, the influence of adsorption and desorption amount on the HP-20 resin of Example 1.1 was examined. As a result, in the case of adsorption, equilibrium was observed after 6 hours of stirring, and 30 Min and the elution was completed within 10 minutes.

Test Example  3. Adsorption affinity measurement by temperature and concentration

In order to confirm the affinity between the concentration of the solute and the adsorbent resin depending on the temperature, the HP-20 resin of Example 1.1, which showed the best result in Test Example 2, was used. 3.02 g, 1.51 g, 0.755 g, 0.377 g, 0.188 g and 0.094 g of the sentinel extract prepared in Preparation Example (20 brix) were placed in a container, and 20 mL of distilled water was added thereto. While stirring at 180 rpm, (25, 35, and 45 ° C, respectively). Adsorption capacity values were shown in the following Table 8 and FIG. 4A. The adsorption capacity values were used to calculate the adsorption capacity values according to Langmuir and Freundlich equations 4B and 4C, respectively.

Langmuir isotherm equation

Where q _e is the concentration of the solute adsorbed per weight of adsorbent resin (mg / g), C _e is the equilibrium concentration (mg / mL), K is the Langmuir constant and q _m is the experimental it means.

Freundlich model equation

q _e is the same as defined in langmuir, K is the Freundlich constant, C _e is the equilibrium concentration (mg / mL), and 1 / n is the constant related to the adsorption power.

Experiment number One 2 3 4 5 6 Safflower ethanol extract (g) 3.02 1.51 0.75 0.37 0.18 0.09 By temperature
Adsorption capacity
(mg / g) 25 63.02 51.49 38.86 21.66 9.93 8.23 35 73.40 53.18 40.26 22.99 12.51 7.55 45 100.88 68.745 46.78 24.71 13.745 8.36 By temperature
Equilibrium concentration (mg / mL) 25 10.22 4.12 1.53 0.76 0.44 0.22 35 10.64 4.28 1.67 0.80 0.55 0.26 45 9.32 3.47 1.53 0.76 0.39 0.23

The isothermal adsorption experiments were carried out for 6 hours at 25 ° C, 35 ° C and 45 ° C using HP-20 resin. As the temperature increased, the adsorption capacity increased as the Langmuir and Freundlich isotherm In the Langmuir model, the binding force between the adsorbent and the adsorbent is higher than that of the Freundlich model as the R ² value increases as the temperature increases. The results are shown in Table 9 below.

Temperature
(° C) Langmuir equation R ² Freundlich equation R ² 25 q _e = (72.70Ce) / (1.695 + Ce) 0.981 q _e = 25.24Ce ^{0 .4136} 0.947 35 q _e = (89.07 Ce) / (2.317 + Ce) 0.987 q _e = 26.11Ce ^{0 .4581} 0.898 45 q _e = (129.70Ce) / (2.848 + Ce) 0.994 q _e = 32.96Ce ^{0 .5128} 0.898

Test Example  4. Dynamic adsorption  Desorption flow rate test

In order to effectively desorb the fluorotannine in the dynamic adsorption method using the column, desorption experiments were carried out on the fluorotannine adsorbed by the dynamic adsorption method based on the results of the fixed adsorption method. It progresses at a flow rate of BV / hr and is shown in Fig. As can be seen from FIG. 5, the highest desorption rate of phlorotannin in the 40% aqueous ethanol solution was confirmed.

Flow velocities ranging from 1 to 10 BV / hr were tested to find a flow rate suitable for desorption of fluorotannine. As a result of elution test at a flow rate of 4 BV / hr, all the adsorbed fluorotannine was eluted from the 20 minutes zone and selected at a suitable rate, which is shown in FIG.

Test Example  5. In the dynamic adsorption method  by Florotannine  The adsorption amount and Desorption amount  Measure

The total polyphenol content was measured from the adsorbed and desorbed solution prepared according to Example 2 and the total fluorotannin content was measured. The adsorption / desorption amount was calculated using the following equation, which is shown in Table 10 below.

division Adsorption amount (mg) Desorption amount (mg) Desorption rate Example 2.1 47.51 32.15 92.63 Example 2.2 42.14 26.02 89.25 Example 2.3 42.35 27.78 90.41 Example 2.4 41.02 27.97 86.42 Example 2.5 39.12 27.15 82.63 Comparative Example 2.1 26.12 11.08 80.06 Comparative Example 2.2 22.06 7.05 73.76 Comparative Example 2.3 20.06 4.14 70.24 Comparative Example 2.4 18.45 2.07 64.52

As shown in Table 10, even the purification by the dynamic adsorption method showed the best effect on adsorption and desorption in the HP-20 resin.

Test Example  6. Content analysis

High - performance liquid chromatography (HPLC) analysis was used to analyze the contents of pre - and post - purification ethanol extracts. As shown in FIG. 7, since the fluorotannine component detected from 230 nm exhibits a higher content after purification than that before purification, the synthetic adsorbent HP-20 not only selectively purifies the fluorotannin but also is industrially useful It can be utilized.

Test Example  7. Analysis of heavy metal content

The content of heavy metals contained in the chewable ethanol extract and chewing gum phlorotannin purified according to the preparation examples of the present invention and Example 2 was analyzed and shown in Table 11 below.

division Salinity
(%) Total Florotannine
(mg / g) lead
(mg / kg) arsenic
(mg / kg) Mercury
(mg / kg) cadmium
(mg / kg) Raw material 14.0 128.4 Non-detection 109.5 0.001 Non-detection Manufacturing example 2.0 457 Non-detection 243.9 0.01 Non-detection Example 2.1 0.0 855 Non-detection 49.2 Non-detection Non-detection Example 2.2 0.0 749 Non-detection 53.8 Non-detection Non-detection Example 2.3 0.0 735 Non-detection 56.6 Non-detection Non-detection Example 2.4 0.0 746 Non-detection 59.6 Non-detection Non-detection Example 2.5 0.0 717 Non-detection 58.7 Non-detection Non-detection Comparative Example 2.1 0.0 551 Non-detection 95.5 0.001 Non-detection Comparative Example 2.2 0.0 558 Non-detection 108.9 Non-detection Non-detection Comparative Example 2.3 0.0 524 Non-detection 118.8 Non-detection Non-detection Comparative Example 2.4 0.0 519 Non-detection 109.4 0.001 Non-detection Comparative Example 3 2.0 118 Non-detection 188.1 0.01 Non-detection

Test Example  8. Moth  Extract GABA _A - Benzodiazepine  Receptor binding activity

GABA _A - benzodiazepine ligand [ ³ H] flammazenil was tested for binding activity of GABA _A - benzodiazepine receptor by the binding substitution of radiolabeled GABA _A - benzodiazepine ligand, as shown in Table 12 below.

division IC ₅₀ (mg / mL) Manufacturing example 0.120 Example 2.1 0.012 Example 2.2 0.109 Example 2.3 0.078 Example 2.4 0.101 Example 2.5 0.258 Comparative Example 2.1 0.155 Comparative Example 2.2 0.189 Comparative Example 2.3 0.193 Comparative Example 2.4 0.256 Comparative Example 3 0.456

Test Example  9. Sleep induction test

Animal experiments were conducted to measure the sleep inducing effect.

ICR mouse (18-22 g, male) was purchased from Koatech Co., Ltd., and was adapted for one week in an experimental animal breeding box. Animals were fed at a temperature of 23 ± 1 ° C, a humidity of 55 ± 5%, a light-dark cycle (light on from 09:00 to 21:00) and an illumination of 300 Lux. Feed and water were free. All animals were maintained by the KFRI-IACUC (Laboratory of Animal Care and Use Committee) guidelines

The Pentobarbital sleep induction experiment was conducted within a certain period of time from 1 pm to 5 pm. Ten mice (n = 10) were used per group and mice were fasted for 24 hours before the experiment. All samples were prepared using 0.5% CMC-saline solution and administered orally (p.o.) 45 minutes before pentobarbital administration. In general control group, 0.5% CMC-saline solution was treated at a concentration of 10 mg / kg, and diazepam (DZP) was used as a positive control to compare the effect of water on the water extract . Pentovagalite was used by intraperitoneal injection (i.p.) at a concentration of 45 mg / kg (hypnotic dosage) according to the experimental design. After the pentobarbital treatment, each individual was moved to a separate space and the sleep latency (duration) and duration (sleep time) were measured. Sleep latency was defined as the time elapsed from the time of righting reflex to lapse of more than 1 min after peritoneal injection of pentobarbital, and the sleep duration was set as the time until the specular recovery was restored. Respectively. A mouse that did not exhibit sleeping behavior after 10 minutes of pentobarbital administration was excluded from the study. In the sub-hypnotic dosage of pentobarbital-treated experiment, the sleep initiation calculation method is shown in the following equation (2).

Equation (2)

Sleep onset (%) = (No. falling asleep) / (Total No.) x 100

Results of all animal studies were expressed as mean and standard error (mean ± SEM). Statistical comparisons between each group and control were made using Dunnett's one-way analysis of variance (ANOVA). Significant levels of significance were marked with asterisks at P <0.05 (*) and P <0.01 (**) levels. Significant differences between the two groups were also assessed by t-test and were expressed as P <0.05 (#), P <0.01 (##).

Test Example  10. Sub - hypnotic  Concentration On the pen  Induced Sleep Induced Effect in Rats

Example 2.1, Preparation Example and Comparative Example 3 were repeated at 100, 250, 500, and 1,000 mg / kg, respectively, in order to evaluate the effect of Gamboa extract on the GABA _A - benzodiazepine receptor binding activity of Example 2.1, (30 mg / kg), ip) at a concentration lower than the induction threshold for sleep induction after oral administration (po) at a concentration of 1 mg / kg / day.

* Means p <0.05 compared with control, *** means Dunnet's test at p <0.001 compared with control, # means p <0.05, ## when p <0.01 Meaning there is a significant difference between co-administered and single administered groups.

The positive control group, DZP, showed a significant decrease in sleep latency (p <0.01) and an increase in sleep duration (p <0.01), as expected by the sub- hypnotic pentobarbital-induced sleep test p < 0.01). (P <0.01), which is similar to that of the positive control group (DZP), in the case of Example 2.1, and a significant decrease in sleep latency And increased sleep duration (p <0.01).

The results of the water-induced induction by the treatment of 250 mg / kg of the preparation example were 18.1 ± 2.4 minutes, compared with the results of the water-induced induction (15.1 ± 4.4 minutes) of 250 mg / kg of the comparative example (19.7 +/- 3.4 minutes), which was obtained by treating the comparative example at a concentration of 500 mg / kg, and it was found that the total amount of the total extract from the brown algae Florotannine demonstrates that the content is extracted at a significant level.

In addition, the water-surface induction result obtained in Example 2.1 at a concentration of 62.5 mg / kg was 19.5 ± 5.2, which was similar to that of the water-surface induction effect at the concentration of 250 mg / kg of the preparation example and 500 mg / kg of the comparative example As a result, purification of the hydrophobic adsorbent resin according to the present invention revealed that the purified product of the fluorotannin having a higher level of purity was produced.

group Dose (mg / kg) Sleep state / Total (Wed) Sleep initiation (%) Sleep time (minutes) CON - 2/12 17 7.3 ± 1.1 DZP 2 12/12 100 55.4 ± 4.8 ***
Manufacturing example
100 7/12 58 15.7 ± 2.9 250 7/12 58 18.1 ± 2.4 500 10/12 83 25.7 ± 2.1 1000 11/12 92 40.1 ± 5.1 *
Example 2.1

62.5 8/12 67 19.5 ± 5.2 125 10/12 83 24.8 ± 2.8 250 11/12 92 40.4 ± 4.5 500 12/12 100 58.8 ± 5.8 **
Comparative Example 3 100 7/12 58 12.7 ± 2.1 250 8/12 67 15.1 ± 4.4 500 10/12 83 19.7 ± 3.4 1000 11/12 92 38.7 ± 6.3 *

Test Example  11. Hypnotic  Concentration On the pen  Induced Sleep Induced Effect in Rats

According to the results of the sleep induction by the sub-hypnotic pentobarbital-induced sleep test according to Test Example 9, the preparation and the Example 2.1 were orally administered (po) at 125, 250 and 500 mg / (45 mg / kg, ip) was used to induce sleep induction.

In the case of the preparation example, the sleep latency was 3.28 ± 0.05 minutes at a concentration of 500 mg / kg, and the sleep duration was 103.82 ± 2.89 in the concentration of 500 mg / kg, latency was 2.58 ± 0.04 minutes, and sleep duration was 142.50 ± 3.78 minutes, which is shown in FIG. In addition, as in the case of the sub-hypnotic pentobarbital-induced sleep test, a significant sleep enhancement effect was observed at a concentration range of 125 mg / kg of Example 2.1, which is similar to that of the 500 mg / kg of the preparation Example. Showed excellent effect even in a small amount and proved to be improved for easy industrial use.

Test Example  12. Statistical Analysis

The 50% inhibitory concentration (IC ₅₀ ) values were calculated by applying the one-site competition binding model using the Prism 5.0 program. The results of all experiments were expressed as mean and standard error (means ± SEM). Statistical comparisons of each group and control were evaluated by Dunnett's one-way analysis of variance (ANOVA). Significance levels were marked with asterisks in the p <0.05 (*), P <0.01 (**), p <0.001 (***) levels. Data comparisons between the two groups were analyzed by Unpaired Student's t-test and statistically significant differences between the two groups at p <0.05 (#), p <0.01 (##), p <0.001 . The statistical analysis program was performed using Prism 5.0 software.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

20 mg of the fluorotannine purified water powder obtained in Example 2.1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

10 mg of the fluorotannine purified water powder obtained in Example 2.1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

10 mg of the fluorotannine purified water powder obtained in Example 2.1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

10 mg of the fluorotannine purified water powder obtained in Example 2.1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

20 mg of the fluorotannine purified water powder obtained in Example 2.1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water and adjusted to 100 as a whole. To prepare a liquid agent.

Formulation example  6. Manufacture of health functional foods

1,000 mg of the flourotannin purified water powder obtained in Example 2.1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation example  7. Manufacture of functional beverages

1,000 mg of the flourotannin purified water powder obtained in Example 2.1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain a total of 900 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (18)

A method for producing a floatotannin purified product from brown algae,
1) A brown algae extract having a sugar content of 15-25 Brix which is obtained by adding 1 to 50 parts by weight of 70 to 95% by volume aqueous ethanol solution to 1 part by weight of dried brown algae and refluxing the mixture at 70 to 90 ° C for 16 to 24 hours Lt; / RTI &gt; And
2) Amberlite XAD-1180 and Amberlite XAD-2010 having a porosity of 0.9 to 2.0 mL / g, a surface area of 400 to 800 m ² / g, a pore radius of 50 to 300 Å and a particle size of 0.2 to 0.85 mm And adsorbing phlorotannin from the brown algae extract by a dynamic adsorption method using a hydrophobic adsorbent resin selected from the group consisting of ethanol,
The dynamic adsorption method comprises the steps of: a) filling a column tube with a hydrophobic adsorption resin;
b) loading the brown algae extract on the top of the packed hydrophobic adsorbent resin and flowing distilled water at a flow rate of 2 to 7 BV / hr to adsorb phlorotannin to the hydrophobic adsorbent resin; And
c) flowing 20 to 60% by volume of an aqueous ethanol solution at a flow rate of 3 to 5 BV / hr to desorb the fluorotannine adsorbed on the hydrophobic adsorption resin,
Wherein the refined product has a fluorotannine content of not less than 700 mg / g and an arsenic content of not more than 70 mg / kg based on the solid content.
The method according to claim 1, wherein the brown algae is selected from the group consisting of Ecklonia cava ), spikelet net horses ( Dictyopteris prolifera Okamura), true netted horse ( Dictyota dichotoma Lamouroux, Sargassum horneri C.Agardh), twins with Sargassum patens C.Agardh and Ishige okamurae Yendo). &lt; / RTI &gt; delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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