KR101731065B1 - Method for preparing novel extract of salicornia herbacea having improved anti-diabetic efficacy and toxicity, and composition comprising the extract of salicornia herbacea - Google Patents

Abstract

The present invention relates to a novel method for producing a green tea extract having decreased toxicity and an increased antidiabetic effect and a novel green tea extract obtained by the method. The novel green tea extract of the present invention has an increased antidiabetic effect and reduced toxicity compared to the green tea extract obtained by the conventional extraction method, and therefore is useful as a pharmaceutical composition for preventing or treating diabetes, a food composition for preventing diabetes or improving diabetic symptoms And can be more advantageously used for health functional foods for the prevention of diabetes or the improvement of diabetic symptoms.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing a novel green tea extract having improved toxicity and antidiabetic effect and a composition containing the green tea extract and a composition containing the green tea extract.

The present invention relates to a method for producing a novel green tea extract having reduced toxicity and an increased antidiabetic effect, and the use of the green tea extract.

Green tea ( Salicornia , Scientific name: Salicornia herbacea L., Korea Name: Salicornia (Hamcho)) is the nation's leading halophytes that inhabit the surrounding waters and close grouped tidal or salt as an annual herb belonging to chenopodiaceae (Chenopodiaceae) (a window, a plant native to Korea, hyangmunsa, p.990, 1985).

Recently, a variety of in vitro experiments have been carried out to investigate the antioxidant activity of antimicrobial antioxidants and antioxidants in green tea (Korean J. Food Sci. Ani. Cholesterol, Antibacterial, Anti-hyperlipidemic, and Anti-aging (Korean J. Herbology 17 (2): 2004, 61-69, 2002) have been reported. Especially, it was predicted that green tea has antioxidative effect twice as much as that of sun saliva and bamboo salt, and antioxidant ingredient which has acne suppression and ultraviolet shielding effect. Recently, tungtungmadic acid and isorhamnetin-3-O-β-D-glucopyranoside compounds (Biol. Pharm. Bull. 28 (5): 916-918, 2005) have been identified as major antioxidant and antidiabetic components.

In addition, the study of physiological activity of green tea powder and extracts showed that the consumption of green onion in the STZ-induced diabetic rats caused the hypoglycemic effect of STZ-induced diabetic rats (J Korean Soc. Food Sci. Nutr. 31: 840-846, (Arch Pharm. Res. 29: 256-264, 2006) reported that the intake of Hamcho was inhibited by hyperglycemia and hyperlipidemia induced by high-fat diets (Korean J. Phys. Anthropol. 19 (2): 117-124, 2006) reported that mice were immunized with macrophages, Journal of Life Science 16 (1): 95-100, 2006) reported that a water extract of Hamchoa has an inhibitory effect on the hepatotoxicity induced by CCl ₄ administration. As described above, various physiological activities such as anti-diabetes, antioxidant, lipid metabolism improvement effect and immunity activity promoting effect of green tea have been revealed and various processed foods and health supplement foods using them have recently been developed.

It is an object of the present invention to provide a method for producing a green tea extract having decreased toxicity and an increased antidiabetic effect and a novel green tea extract prepared by the method.

It is another object of the present invention to provide a composition for preventing and treating diabetes mellitus containing a novel green tea extract having reduced toxicity and increased antidiabetic effect as an active ingredient.

As a result of intensive studies to solve the above problems, the inventors of the present invention found that the novel green tea extract obtained by the extraction method of the present invention is significantly superior to the green tea extract obtained by the conventional extraction method, , And the toxicity was significantly lower in the toxicity test results, thereby completing the present invention.

Accordingly, the present invention provides a method for producing a novel green tea extract having decreased toxicity and increased antidiabetic effect.

The present invention provides a composition for preventing or treating diabetes mellitus containing a novel green tea extract obtained by the above production method as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes mellitus containing a novel green tea extract obtained by the above production method as an active ingredient.

The present invention provides a food composition for preventing or treating diabetes mellitus containing a novel green tea extract obtained by the above production method as an active ingredient.

The composition of the present invention can be suitably applied to the prevention or treatment of diabetes by various causes.

More specifically, the present invention provides a process for preparing a novel Leuco Acid extract having reduced toxicity and improved anti-diabetic effect, comprising the steps of:

(a) a primary extraction step in which a first organic solvent is added to a green tea plant and heated;

(b) a second extraction step of further adding and heating the first organic solvent to the first extracted sample;

(c) filtering and concentrating the secondly extracted sample, followed by lyophilization;

(d) an ultrasonic treatment process in which the second organic solvent is added to the lyophilized sample and subjected to ultrasonic treatment;

(e) recovering and filtering the supernatant of the ultrasonic treated sample;

(f) adding a third organic solvent to the filtered sample and centrifuging to recover the supernatant; And

(g) concentrating the supernatant.

The present invention is a method for producing the green tea extract, wherein the first organic solvent, the second organic solvent and the third organic solvent are selected from a lower alcohol having 1 to 6 carbon atoms, water, acetone, ethyl acetate or a mixture thereof to provide.

The present invention provides a method for producing the green tea extract, wherein the first organic solvent is 70% ethanol, and the second organic solvent and the third organic solvent are 100% ethanol.

The present invention provides a method wherein the amount of the second organic solvent added in the step (d) ranges from 700 parts by weight to 800 parts by weight based on 100 parts by weight of the lyophilized sample.

The present invention provides a pharmaceutical composition for preventing or treating diabetes mellitus containing the green tea extract obtained by the above-described production method as an active ingredient.

The present invention provides a health functional food containing the green tea extract obtained by the above-described production method as an active ingredient and having reduced toxicity and improved antidiabetic efficacy.

The present invention provides a food composition containing a green tea extract obtained by the above-described production method as an active ingredient with reduced toxicity and improved antidiabetic effect.

According to the composition of the present invention, hyperglycemia due to type 1 or type 2 diabetes can be reduced, and symptoms due to diabetes can be improved. In addition, the composition according to the present invention is less toxic and can reduce adverse effects on the patient. Therefore, the composition according to the present invention is useful for the prevention or treatment of diabetes, and it is also possible to orally administer the composition, thus the patient's compliance with the medication is excellent.

1 shows the results of a comparison of the antidiabetic effects of the novel green tea extract (experimental group) of the present invention and the green tea extract (comparative group) obtained by the conventional extraction method in a diabetic zebrafish model induced by alloxan (NOR: Normal group, CON: untreated control group, PPT green tea group: test group, green tea group: comparison group).
2 shows the result of evaluating the survival rate after 60 hours of exposure to a fresh green tea extract (experimental group) of the present invention and a green tea extract (comparative group) obtained by a conventional extraction method in a zebrafish model (PPT: green tea: Comparison group).
FIG. 3 shows the result of observing the morphological changes of the heart after 60 hours of exposure to a fresh green tea extract (experimental group) of the present invention and a green tea extract (comparative group) obtained by a conventional extraction method in a zebrafish model (Normal: normal Group, PPT: green pepper: experimental group, green pepper: comparative group).
FIG. 4 shows the result of confirming the anti-diabetic effect according to the concentration of the organic solvent in the ultrasonic treatment of the novel Leekyota extract of the present invention in a diabetic zebrafish model induced by alloxan.

A novel green tea extract having reduced toxicity and improved antidiabetic effect of the present invention is obtained by a manufacturing method comprising the following steps:

(a) a primary extraction step in which a first organic solvent is added to a green tea plant and heated;

(b) a second extraction step of further adding and heating the first organic solvent to the first extracted sample;

(c) filtering and concentrating the secondly extracted sample, followed by lyophilization;

(d) an ultrasonic treatment process in which the second organic solvent is added to the lyophilized sample and subjected to ultrasonic treatment;

(e) recovering and filtering the supernatant of the ultrasonic treated sample;

(f) adding a third organic solvent to the filtered sample and centrifuging to recover the supernatant; And

(g) concentrating the supernatant.

The heating temperature in the primary extraction step and the secondary extraction step is 20 占 폚 to 180 占 폚, preferably 70 占 폚 to 150 占 폚, and more preferably 80 占 폚 to 120 占 폚. Further, the extraction time can be appropriately set according to the extraction temperature, and the primary extraction process can be performed in the range of about 30 minutes to one week, and the secondary extraction process can be performed in the range of about 10 minutes to several hours have. Preferably, the primary extraction process may be performed for 60 minutes to 3 hours, and the secondary extraction process may be performed for 30 minutes to 1 hour.

The first organic solvent used in the first extraction step and the second extraction step may be a lower alcohol having 1 to 6 carbon atoms, water, acetone, ethyl acetate, or a mixture thereof, preferably ethanol, 70% ethanol (aqueous solution) can be used.

In the first extraction step, the first organic solvent is added in an amount of 100 parts by weight to 3000 parts by weight, preferably 500 parts by weight to 2000 parts by weight based on 100 parts by weight of the green tea plant. In the second extraction step, Is added in an amount of 30 to 200 parts by weight, preferably 50 to 100 parts by weight per 100 parts by weight of the tea extract.

The filtration performed in the above production method may be carried out by a conventional filtration method including atmospheric pressure filtration, reduced pressure filtration, pressure filtration and the like, and filtration methods using a filter paper such as a nonwoven fabric, gauze or cotton can be used. In addition, the concentration performed in the above production method can be carried out using a conventional concentration method including evaporation under reduced pressure, evaporation in boiling, drying in a hot bath, and the like.

In the step (c), the conditions for freeze-drying may be a range generally set for drying the extract, preferably freeze-drying at a temperature of -30 ° C or lower, more preferably -50 ° C or lower After freeze-drying, store in the freezer.

The second organic solvent used in the ultrasonic treatment may be a lower alcohol having 1 to 6 carbon atoms, water, acetone, ethyl acetate or a mixture thereof, preferably ethanol, more preferably 100% Ethanol is used. When 100% ethanol is used as the second organic solvent, it is most preferable since more improved toxicity and antidiabetic effect can be obtained.

In the ultrasonic treatment, the second organic solvent is added in an amount of 500 parts by weight to 1500 parts by weight, preferably 700 parts by weight to 800 parts by weight, based on 100 parts by weight of the lyophilized sample obtained in the previous step. Particularly preferably, when the second organic solvent is 100% ethanol, it is added in an amount of 700 to 800 parts by weight based on 100 parts by weight of the lyophilized sample. (Ethanol density: 0.789 g / cm3) in terms of the volume (ml) of 100% ethanol relative to the weight (g) of the lyophilized sample, 100% When added at a rate of about 10 ml, improved antidiabetic efficacy and toxicity can be obtained. In the ultrasonic treatment step, the sample mixed with the second organic solvent is treated with ultrasonic waves of 10 to 1000 kHz, preferably 20 to 80 kHz, more preferably 40 kHz as in the embodiment.

As the third organic solvent used in the step (f), a lower alcohol having 1 to 6 carbon atoms, water, acetone, ethyl acetate or a mixture thereof may be used. Preferably, ethanol is used, 100% ethanol is used. The third organic solvent is added in an amount of 50 parts by weight to 2000 parts by weight, preferably 100 parts by weight to 1000 parts by weight, based on 100 parts by weight of the filtered sample obtained in the previous step.

In the step (f), centrifugation after the addition of the third organic solvent is carried out at 100 to 30,000 rpm, preferably 5,000 to 15,000 rpm, more preferably 12,000 rpm, for several seconds to several minutes, preferably one minute To 5 minutes, more preferably for 3 minutes.

In addition, in the above production method, in the case where an organic solvent is added to the sample, a step of further dispersing the sample in the organic solvent and mixing the sample and the organic solvent may be further included so as to facilitate contact between the solvent and the sample .

In the above production method, the extract obtained by carrying out the first extraction step or the second extraction step may be obtained in the form of a liquid in which impurities are removed by filtration according to a conventional method, or the obtained liquid form extract Followed by an intermediate step of obtaining a powdery form by concentration under reduced pressure and / or drying.

The green tea extract obtained by the above production method is remarkably toxic as compared with the green tea extract obtained through the conventional extraction method, for example, (a) the first extraction step, (b) the second extraction step and (c) And an antidiabetic effect exhibits an increased action effect. Therefore, the novel green tea extract obtained by the production method of the present invention can be more advantageously used for the prevention or treatment of diabetes.

The present invention also relates to a pharmaceutical composition for preventing or treating diabetes mellitus containing a novel green tea extract having reduced toxicity and increased antidiabetic effect obtained by the above production method as an active ingredient.

The present invention also relates to a food composition for prevention or improvement of diabetes mellitus containing a novel green tea extract having reduced toxicity and increased antidiabetic effect obtained by the above production method as an active ingredient.

The composition of the present invention can be suitably applied to the prevention or treatment of diabetes by various causes. As used herein, the term "diabetes" includes type 1 diabetes and type 2 diabetes.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. The pharmaceutical composition of the present invention can be administered orally, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, Formulations, external preparations, suppositories, and sterile injectable solutions.

Such pharmaceutically acceptable carriers may be those conventionally used in the art such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, But are not limited to, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention includes diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants, and other pharmaceutically acceptable additives.

When the pharmaceutical composition of the present invention is formulated into an oral solid preparation, it includes tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, Tosse, gelatin, and the like, including, but not limited to, lubricants such as magnesium stearate, talc, and the like.

When the pharmaceutical composition of the present invention is formulated orally for oral use, it includes suspensions, solutions, emulsions, syrups and the like, and includes, but is not limited to, diluents such as water and liquid paraffin, wetting agents, sweeteners, fragrances and preservatives.

When the pharmaceutical composition of the present invention is formulated for parenteral use, it may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository. Examples of the non-aqueous solvent include propylene glycol, polyethylene glycol, But are not limited to, injectable esters such as oil, ethyl oleate, and the like. Examples of suppository bases include, but are not limited to, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The dose of the green tea extract contained in the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the age, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For example, the PPT leach extract may be administered at a dose of 1 to 2000 mg / kg per day, preferably 10 to 2000 mg / kg per day, and the administration may be administered once a day or divided into several times.

The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like by various routes, for example, oral, intraperitoneal or intravenous, muscular, subcutaneous, intrauterine, .

The present invention also includes a food composition for preventing or ameliorating diabetes, comprising a green tea extract as an effective substance.

The food composition of the present invention can be used as a health functional food. The term "functional food" as used herein means a food prepared and processed using a raw material or ingredient having a useful function in the human body pursuant to Law No. 6727 on Health Functional Foods, and the term "functional" And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action.

The food composition of the present invention may contain conventional food additives. Unless otherwise specified, the suitability of the food additives for the above-mentioned "food additives" Of the present invention.

The food composition of the present invention may contain 0.01 to 95% by weight, preferably 1 to 80% by weight, of the green tea extract against the total weight of the composition for the purpose of preventing and / or improving diabetes. The green tea extract contained in the food composition of the present invention can be obtained in the same manner as the extraction method mentioned in the production of the above pharmaceutical composition.

In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings which can be usually prepared for the purpose of preventing and / or improving diabetes.

The definitions of the above excipients, diluents, binders, disintegrators, etc. are described in documents known in the art and include the same or similar functions (refer to Korean Pharmacopoeia, Moon Sung, Korean Pharmacological Association, , p. 33-48, 1989).

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples and test examples.

Example

Example  One. PPT green tea  Preparation of extract

500 g of domestic green tea was firstly extracted with 8 L of 70% ethanol at 90 캜 for 120 minutes. 4 liters of 70% ethanol was further added and the mixture was subjected to secondary extraction at the same temperature for 40 minutes. The extract was filtered and concentrated to 70-80 rpm at 40 ° C in a water bath. The concentrate was frozen at -50 ° C and lyophilized for 7 days. After freeze-drying, it was stored in a -50 ° C freezer. The total amount of 500g was extracted to obtain 185.332g of Hamcho extract, and the yield was 17.1%.

Then, 9 g of 100% ethanol was added to 1 g of the green tea extract, sonicated at 40 kHz for 30 minutes, and then overnight at 4 캜. The supernatant was filtered, 10 ml of 100% ethanol was added, and centrifuged at 12,000 rpm for 3 minutes. The obtained supernatant was concentrated to obtain a green tea extract (hereinafter referred to as "PPT green tea"). The yield was 12.03%.

Comparative Example  One. Green tea  Preparation of extract

The green tea extract (hereinafter, referred to as "green tea") of Comparative Example 1 was used as the green tea extract (the green tea extract before the ultrasonic treatment) obtained after lyophilization in Example 1.

Test Example

Test Example  One. Zebra fish Pancreatic islet ( Zebrafish  pancreatic islet (PI))

Five days post-fertilization zebrafish larvae were placed in 96 wells and stained with 25 μM 2-NBDG for 12 hours. After exposure to 100 μM alooxane for 15 min, it was replaced with 0.03% sea salt solution and exposed for up to 5 h. Fluorescence microscopy (Olympus 1 × 70, Olympus, Japan) was used for direct pancreatic islet observation, and 25 μM 2-NBDG was re-stained for 1 h before fluorescence microscopy. After the first microscope photographing, the samples were exposed to the PPT green tea prepared in Example 1 and 100 μg / ml of the green tea prepared in Comparative Example 1 for 1 hour, respectively. Secondary microscopic photographs were taken to observe changes in the pancreatic islets caused by the leachate of PPT in Example 1 and Comparative Example 1, and 25 μM 2-NBDG was observed for 1 hour before fluorescence microscopy Lt; / RTI > Images obtained through fluorescence microscopy were analyzed for the intensity of the pancreatic islets of the zebrafish and the intensity of the fluorescence intensity of the pancreatic islets using the Focus Lite and Image J software.

As a result, PI size increase of PPT green tea was significantly larger than increase of PI size of green tea (Fig. 1). As a result, it was confirmed that the anti-diabetic effect of PPT green tea was better.

Test Example  2. Zebra fish  Comparison of toxicity through embryo survival rate

Zebrafish embryo was placed in 6 wells and exposed to the green tea extract of Example 1 (PPT green tea extract) and the green tea extract of Comparative Example 1 (green tea) by concentration. Survival rates were compared after 60 hours of exposure.

Evaluation result, the LC ₅₀ of Salicornia appeared 18.57 ㎍ / ㎖ the other hand, the LC ₅₀ of the PPT Salicornia 0.7208 ㎍ / ㎖ (Fig. 2). As a result, it was confirmed that the toxicity of PPT green tea was remarkably low.

Test Example  3. Zebra fish  Comparison of toxicity through morphological changes of embryo

Zebrafish embryo was placed in 6 wells and exposed to 100 쨉 g / ml of each of the extracts of Example 1 (PPT anchovy) and the green tea extract of Comparative Example 1 (green tea). After 60 hours of exposure, the morphology of the zebrafish was compared using a microscope.

As a result of evaluation, in case of Hamcho, edema of pericardium was observed, and deformity due to yolk sac edema was shown. However, PPT green tea was not morphologically different from normal (Fig. 3).

Test Example  4. ultrasonic wave Before processing  Depending on the concentration of added organic solvent Anti-diabetic  Check the effect

Except that 70% ethanol, 95% ethanol, and 100% ethanol were used as the organic solvents added before the ultrasonic treatment. The same procedure as in Test Example 1 was repeated to prepare PPT Changes in the size of pancreatic islets induced by green tea extract were observed.

As a result of the analysis, it was found that the PI size greatly increased in the experimental group treated with 100% ethanol as the organic solvent during the ultrasonic treatment (FIG. 4). Thus, when 100% ethanol was used in the ultrasonic treatment, the antidiabetic effect was significant.

Formulation example

The following formulation was prepared using the PPT rapeseed extract prepared in Example 1 above. However, the following formulation examples are illustrative of the present invention, and the contents of the present invention are not limited thereto.

Formulation example  1. Preparation of tablets

PPT Green tea extract 200 mg

Lactose 100 mg

100 mg of starch

Magnesium stearate qs

The above components were mixed and tableted according to a conventional tablet preparation method to prepare tablets.

Formulation example  2. Liquid  Produce

PPT Green tea extract 1000 mg

20 g of CMC-Na

20 g per isomer

Lemon incense quantity

Purified water was added to adjust the total volume to 1000 ml. The above components were mixed according to a conventional method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.

Formulation example  3. Preparation of capsules

PPT Leaf extract 300 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

0.2 mg of magnesium stearate

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

Formulation example  4. Preparation of injections

PPT Leaf extract 300 mg

180 mg mannitol

2974 mg of sterile distilled water for injection

Na ₂ HPO _{4 12} H ₂ O 26 mg

(2 ml) per ampoule according to the usual injection preparation method.

Claims (8)

(a) a primary extraction step in which a first organic solvent is added to a green tea plant and heated;
(b) a second extraction step of further adding and heating the first organic solvent to the first extracted sample;
(c) filtering and concentrating the secondly extracted sample, followed by lyophilization;
(d) an ultrasonic treatment process in which a second organic solvent is added to the lyophilized sample, followed by ultrasonication and overnight at 4 ° C;
(e) recovering and filtering the supernatant of the ultrasonic treated sample;
(f) adding a third organic solvent to the filtered sample and centrifuging to recover the supernatant; And
(g) concentrating the supernatant,
Wherein the first organic solvent and the third organic solvent are selected from lower alcohols having 1 to 6 carbon atoms, water, acetone or a mixture thereof,
And the second organic solvent is 100% ethanol.
A method for preparing a green tea extract having reduced toxicity. delete The method according to claim 1,
Wherein the first organic solvent is 70% ethanol. delete The method according to claim 1,
Wherein the amount of the second organic solvent added in the step (d) is 700 parts by weight to 800 parts by weight with respect to 100 parts by weight of the lyophilized sample. A pharmaceutical composition for preventing or treating diabetes mellitus containing the green tea extract obtained by the production method according to any one of claims 1, 3, and 5 as an active ingredient. A food composition for lowering blood glucose levels, comprising the green tea extract obtained by the production method according to any one of claims 1, 3, and 5 as an active ingredient. A health functional food for lowering blood glucose, comprising the green tea extract obtained by the production method according to any one of claims 1, 3, and 5 as an active ingredient.

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