KR101322721B1 - Anti-obesity composition containing the Platycodon grandiflorum fraction from the Platycodon grandiflorum roots with high purity platycosides - Google Patents

Abstract

The present invention relates to a composition for anti-obesity comprising a gilyeong fraction containing praticoside as an active ingredient, and more particularly, to remove the precipitate formed by dissolving the gilyeong ethanol crude extract in ethanol and adding acetone, Obtained by dissolving concentrated ethanol-acetone fraction in ethyl acetate and sequentially using water and saturated butanol, and separating and purified by reverse phase (C18) cartridge chromatography using ethanol-containing aqueous solution as an elution solvent. The present invention relates to an anti-obesity composition containing as an active ingredient a long-length fraction containing one praticoside in high purity.
According to the present invention, by separating and purifying the Praticoside with high purity by a different method than the conventional method, containing a large amount of gilyeong Praticoside and Praticodine D in the fraction in high purity, diet by the oral administration of the gilyeong extract and Gilyeong fraction It can interfere with the digestion and digestion of triglycerides and lipids accumulated in cells and tissues, promote the consumption and excretion of cholesterol, and induce weight loss, which can be expected to be used as a potential material with anti-obesity activity. have.

Description

Anti-obesity composition containing the Platycodon grandiflorum fraction from the Platycodon grandiflorum roots with high purity platycosides}

The present invention relates to a composition for anti-obesity comprising, as an active ingredient, a gilyeong fraction containing high purity platycosides, which are saponins contained in gilyeong.

Obesity is a disease caused by metabolic disorders in which excess energy is converted into fat and accumulated in fat tissue excessively, resulting in abnormally high body fat. The causes of obesity are largely genetic and environmental (eating meat-based diet, reduced exercise, etc.). Causes other than these include neuroendocrine causes and drug causes.

Obesity, already defined as a disease in 1996 by the World Health Organization (WHO), is one of the most proliferating chronic diseases in the world in recent years and is a major risk factor for the development of “sexual diseases” such as diabetes, hyperlipidemia, arteriosclerosis, and cardiovascular disease It is a factor. The global obesity and adult disease-related market is rapidly growing due to the increase in the direct and indirect expenditures due to the increase of the obese population, and the amount of social losses is also increasing rapidly.

As a target for treating such obesity, there are appetite control, fat metabolism control, adipocyte differentiation control, fat absorption control and energy metabolism control, and active research is being conducted mainly on them. There are currently two anti-obesity drugs approved by the U.S. FDA for long-term use: sibutramine (REDUCTIL®) with a mechanism of action that inhibits reuptake of norepinephrine and serotonin. There is an orlistat (XENICAL, trade name) which has an effect by inhibiting lipase secreted from the machine.

Sibutramine was initially developed as an antidepressant, but it is currently used as an anti-obesity agent because of its excellent anti-obesity effect. Recent studies have shown that sibutramine, when used for at least one year, loses more than 4.3 kg or 4.5% of body weight compared to placebo. However, when using sibutramine, there are side effects such as blood pressure rise, insomnia, dry mouth, dizziness, etc., there is a disadvantage that can not be used in patients with cardiovascular diseases such as hypertension.

Orlistat reduces the absorption of fat in foods by 30% by inhibiting the action of lipase secreted by the pancreas and digestive system. The results reported to date indicate that olistat, when administered for one year, lost 2.7 kg or 2.9% of body weight compared to placebo. However, when using olistat, there are side effects such as diarrhea, fatty stool, and loss money, and the effect of the drug is not obvious when the fat intake is less than that of Westerners such as Koreans. Due to the limitations of drugs developed so far for the treatment of obesity, there is an urgent need for the development of drugs with high anti-obesity effect and new side effect mechanism.

Regarding the improvement of obesity, Korean Patent Publication No. 2010-061212 discloses obesity or lipid-related metabolism, which contains two or more herbal extract mixtures selected from the group consisting of cypress leaf extract, white porcelain extract, juniper extract, and sesame extract. A pharmaceutical composition for preventing or treating a disease is disclosed, and Korean Patent Laid-Open Publication No. 2009-0113554 discloses Eupolyphaga sinensis ) discloses a composition for preventing and treating obesity containing an extract or fractions thereof as an active ingredient, and Korean Laid-Open Patent No. 2005-0027506 includes sujijihwang and yiyiin as the main components, and antiseptic, licorice, neck, herb, talc Disclosed is a bodily tanguitang composition for treating obesity, characterized by using an active ingredient extracted from a herbal mixture consisting of, moth, baekryepi, donkey, goji berry, cornus, cheonggung, sulphides, joiner and sirloin as an auxiliary ingredient, Korea Patent No. 0947278 discloses an oral preparation having a therapeutic activity and inhibitory activity against obesity-related diseases consisting of about 10 kinds of natural extracts of alcohols of which the lower lobe is the main ingredient, and the oyster and the seaweed as sub-components, and a method for preparing the same and including the composition An anti-obesity agent is disclosed, and Korean Patent No. 0806133 discloses a low back muscle A composition for inhibiting and treating obesity containing an extract or aylanton is disclosed, and Korean Unexamined Patent Publication No. 2010-0067299 discloses a mixed herbal extract and ephedra of ephedra, gypsum and cinnabar. ) An anti-obesity composition containing a mixture of extracts as an active ingredient is disclosed, Korean Patent No. 0990260 discloses a composition for treating or preventing obesity or hyperlipidemia comprising a fermented black garlic extract as an active ingredient, Korea Patent Publication No. 2010-0137852 discloses a pharmaceutical composition for weight control and obesity prevention and treatment comprising the black onion extract as an active ingredient.

On the other hand, the bellflower (Kilyeong, 桔梗) [ Platycodon grandiflorum A. DC) is a perennial herb belonging to the Campanulaceae, whose roots are mainly used as traditional herbs and foods. At this time, when used as a herbal medicine is called Gilgyeong, when used as food is often called bellflower. Various health benefits of the gilyeong extract have been reported, especially for four years, it is known to be effective in bronchitis, asthma, pulmonary tuberculosis, diabetes.

Recently, the immunological effects of Gil-Gil extract were studied, and several active substances such as triterpene saponin were isolated and identified. Known avenues include glycosides of platyodigenin, platinumcodin A, C, D, D2 and two monoacetate praticodine D3, deepio-praticodine D3. Glycosides of polygalacic acid include polygalacin D and two monoacetates, polygalacin D2 and monoacetate, and a glycoside of platicogenic acid A Ten kinds of triterpene saponins, such as praticonate A and methyl-2-methoxy praticonate A, have been reported. Other aglycones have reported the presence of praticogenic acids B and C.

On the other hand, it has been reported to protect the high cholesterol and hyperlipidemia, and liver damage induced by CCl ₄ of the Bellflower extract, obesity and insulin-independent diabetes, dyslipidemia, hypertension, cardiovascular disease Increasingly, research into active substances that can reduce the concentration of blood lipids such as cholesterol from natural products including saponins is underway. New drugs and functional foods are being developed to improve or prevent the treatment of such diseases.

The present invention is a diet of the Gilyeong fraction obtained by the characteristic method of the present invention along with a high-fat diet to the experimental animal is reduced intake of diet and body weight of the experimental animal, inhibit the digestive absorption of cholesterol in the blood and liver, The present invention was completed by confirming that the amount of cholesterol and lipid ingested is excreted in feces.

Therefore, the present invention has no concern about side effects and prevents the accumulation of cholesterol in the blood and liver and facilitates lipid metabolism, and thus the object of the present invention is to provide a new use of Gilkyung as a natural product that safely induces weight loss in the human body.

As an example for achieving the above object, the present invention, after dissolving the crude ethanol crude extract in ethanol and removing the precipitate formed by the addition of acetone, the filtrate was concentrated in ethyl acetate to dissolve the gilyeong ethanol-acetone fraction in ethyl acetate The long-length butanol fraction obtained by sequentially using saturated butanol was partitioned and purified by reverse phase (C18) cartridge chromatography using an ethanol-containing aqueous solution as an eluting solvent. An anti-obesity composition is characterized.

As another example for achieving the above object, the present invention includes a food and a medicament containing the anti-obesity composition.

Hereinafter, the present invention will be described in detail.

In the present invention, the method for obtaining the long-length fraction is as follows.

Crude ethanol crude extract is dissolved in ethanol, the acetone is added to remove the precipitate formed, and the filtrate is concentrated to obtain the citric ethanol-acetone fraction.

The long-length ethanol crude extract is obtained by extracting the long-length dry powder with an ethanol-containing aqueous solution. As the ethanol-containing aqueous solution, it is suitable to use 40 to 90 volume% aqueous solution of ethanol. For extraction, various well-known extraction methods may be adopted, and the extraction efficiency may be increased when performing ultrasonic extraction. After extraction, the mixture is left to stand, and the solid is filtered. The process of extracting, leaving and filtering by adding ethanol-containing aqueous solution to the filter residue is repeated several times. The repetition is preferably repeated three or more times three to five times in terms of extraction efficiency. The extracts were collected and filtered again to remove the solids. The filtrate was concentrated under reduced pressure at 50 to 60 ° C. to remove ethanol. The long ethanol crude extract, which has been blown off by ethanol concentration under reduced pressure, is not solidified by the sugar component contained therein, is concentrated to water, frozen at -50 ° C or lower, and lyophilized to be processed into a powder. The component contained in the crude ethanol crude extract can be confirmed by HPLC chromatogram performed by dissolving it in methanol [FIG. 1].

The crude length ethanol crude extract was dissolved in ethanol, and the precipitate formed by adding acetone was removed and the filtrate was concentrated to obtain the length ethanol-acetone fraction.

Specifically, in order to dissolve the crude ethanol crude extract, it is used at a rate of about 1 L of ethanol relative to 10 g of the crude ethanol crude extract. At this time, a precipitate is generated, and the ethanol solution in which the crude ethanol crude extract is dissolved is carefully transferred to another container, and continuously dissolved in an undissolved residue by adding a certain amount of ethanol. Acetone, which is three times the amount of ethanol used, is added to the solution in which the crude ethanol crude extract is completely dissolved.

In general, saponins and praticodine D, such as Gilgon, are extracted with a solvent having high polarity, and then dissolved in water, and then fractionated in the order of hexane, ethyl acetate, butanol and water, and the butanol fraction is concentrated and HP- A conventional separation method such as passing through a column such as 20 is used. In this case, at least four open column chromatography should be performed in order to obtain saponin from the cirrhosis extract.In each step, the substances fractionated by TLC and the like are identified. There was a lot of uneconomical and unproductive problems, such as taking at least 7 to 9 months for separation.

Thus, this step of the present invention, unlike the conventional use of acetone characteristically. When acetone is added to the ethanol solution in which the crude ethanol crude extract is dissolved, a white precipitate is generated, which corresponds to some water-soluble polysaccharides and proteins, which are filtered off. At this time, the amount of acetone used is preferably 2 to 5 times the volume of ethanol used to dissolve the crude ethanol crude extract, preferably 3 to 4 times the volume in terms of removal efficiency of the water-soluble polysaccharide and protein.

The filtrate, from which the white precipitate was removed, was concentrated completely to obtain a long ethanol-acetone fraction.

The long ethanol-acetone fraction is dissolved in water and partitioned with ethyl acetate to give a water fraction by concentrating the water layer.

The lengthwise ethanol-acetone fraction is completely dissolved in distilled water, and the same amount of ethyl acetate (EtoAC) and distilled water used therein are added and shaken vigorously to allow water and EtoAC to be mixed and then left to recover an upper layer of EtoAC. The same amount of EtoAC is added to the lower water layer and shaken to separate the layers several times to separate the water layer and the EtoAC layer. The layer separation is effective to repeat three or more times, preferably three to five times, it is possible to remove some of the sugar components and non-saponin-based mesopolar material contained in the EtoAC layer through this process. The EtoAC layer was concentrated and then dissolved in methanol, and the result of HPLC was shown in FIG. 2, through which the EtoAC layer contained a sugar component and a substance having a medium polarity. The lower water layer is concentrated using a vacuum concentrator at 40 to 50 ° C. to remove all the EtoAC contained in the water layer.

Saturated butanol is mixed with the water layer of the above step, the layers are separated into a water layer and a butanol layer, and the butanol layer is concentrated to obtain a butanol fraction.

In the above step, the same amount of saturated butanol is added to the water layer from which the EtoAC is removed, shaken, mixed, and left to stand to separate the water layer and the butanol layer. Adding the saturated butanol to the separated water layer again, shaking, mixing and standing still is effective to perform the layer separation several times, preferably 3 to 5 times to separate the water layer and the butanol layer again. All butanol layers separated into upper layers were recovered and concentrated under reduced pressure at 50 to 60 ° C. to obtain a butanol fraction. The butanol fraction can be confirmed by the HPLC chromatogram of FIG. On the other hand, it can be confirmed through the HPLC chromatogram of FIG. 4 that the highly polar compound and the sugar component which were not removed by the EtoAC layer separation were dissolved in the water layer of the lower layer separated. There is an effect that they are removed by the distribution used.

The saturated butanol is the same amount of butanol and ultrapure water mixed and violently shaken and left to completely separate the layer after discarding the water layer of the lower layer, to recover the upper layer to be prepared in advance to prepare.

The butanol fraction was dissolved in distilled water and purified by reverse phase (C18) cartridge chromatography using an ethanol-containing aqueous solution as an eluting solvent.

Looking at the HPLC chromatogram of the butanol fraction shown in Figure 3 it can be seen that the relatively high polar component is still included in the butanol fraction compared to the saponin component, solvent distribution using several times EtoAC and saturated butanol It can also be seen that they are not completely removed.

Therefore, in the present invention, the butanol fraction was separated and purified by reverse phase (C18) cartridge chromatography. At this time, the reverse phase (C18) cartridge can use SEP-PAK vac 6cc, the mobile phase may use an ethanol-containing aqueous solution. The butanol fraction completely dissolved in distilled water was loaded in a reverse phase (C18) cartridge and the butanol fraction was eluted while changing the ethanol concentration of the ethanol-containing aqueous solution corresponding to the mobile phase in the range of 20 to 90% by volume. C18) It is possible to elute praticosides and praticodine D, which are indicators of long-lived saponins, which are indicative of bellflower adsorbed on cartridges.

In other words, it can be seen that the long-length fraction obtained by the above method from the crude long-ethanol crude extract contains high amounts of high purity Praticodine D and Praticoside [Table 1 and FIG. 11].

In the present invention, after the oral administration of high fat, high cholesterol diet and the ethanol extract and the gilyeong fractions for 4 weeks to investigate the effect of gilyeong fractions containing a large amount of high purity Praticodine D and praticoside on fat metabolism , Dietary intake, weight change, blood cholesterol content, blood lipid content, cholesterol and lipid content in feces, and enzyme activity and pancreatic lipase inhibitory activity of blood ALT and AST were measured.

As a result, it was confirmed that the experimental animals ingesting the gilyeong extract and gilyeong fraction decreased the dietary intake, and increased the content of lipids and cholesterol in the feces.

In other words, when oral administration of the gilyeong extract and gilyeong fractions, dietary intake is reduced, it can be expected to be effective in obese patients who are difficult to control the amount of food. And the activity of lipoprotein-forming enzymes are reduced, it can be predicted that the cholesterol and lipids ingested by the high cholesterol diet or high fat diet are all digested and absorbed by the body and excreted without being consumed, thereby suppressing the absorption of excessive calories.

In addition, the gilyeong saponin, which is contained in large quantities in high quantities in the gilyeong extract and gilyeong fraction, praticoside can be easily combined with cholesterol and lipids to confirm the phenomenon of discharge into the feces.

According to the present invention described above, the Giltyeong fraction has an anti-obesity activity, suggesting the possibility that the composition comprising the same as a composition for the prevention, improvement and treatment of obesity as a variety of functional food, pharmaceutical material.

On the other hand, the composition for anti-obesity containing the cirrhosis fraction as an active ingredient can be administered orally or parenterally, for example, intravenous, subcutaneous, intraperitoneal or topical at the time of clinical administration, in the form of a general health food Can be used.

The compositions may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Is formulated. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection injection method. To formulate into a parenteral formulation, the composition may be mixed in water with stabilizers or buffers to prepare a solution and formulated in unit dosage forms of ampoules or vials.

The above-mentioned lengthwise fraction is contained in the composition of 1 μg / mL or more, preferably in the range of 1 to 100 μg / mL, more preferably in the range of 1 to 10 μg / mL, which is good for the expression of anti-obesity activity.

The effective dosage of the composition of the present invention described above may vary according to the age, physical condition, weight, etc. of the patient, but is generally 1 to 50 mg / day per 1 kg of adult patient weight, preferably 5 to 20 mg / Day, and may be divided into several times a day, preferably two to three times a day at regular time intervals according to the judgment of the doctor or pharmacist.

In addition, since the composition of the present invention is derived from edible bellflower, which is safe for human body and suitable for use in food, it can be developed into various foods that exhibit functionality for the prevention, improvement and treatment of obesity.

When the anti-obesity composition of the present invention is included in food, it may be used in the range of 0.01 to 50% (w / w), preferably 1 to 30% (w / w) of the total weight.

That is, various foods can be prepared by a conventionally known method by adding them in conventional palates such as noodles, tofu, cereals, breads, chewing gum, candy, confectionary, such as ramen noodles, raw noodles, tablets, granules, It may be formulated into a general formulation such as pills, hard capsules, soft capsules, or liquid formulations, and may be prepared in a juice, pouch, beverage, or a variety of ingredients. It can select and mix.

According to the present invention described above, the gilyeong fraction containing a large amount of gilyeong extract and gilyeong praticoside of high purity is applied when the intake can not be controlled autonomously by causing a decrease in dietary intake by oral administration to exhibit an excellent anti-obesity effect It is expected to be.

In addition, according to the present invention, the cirrhosis fraction decreases the accumulation of cholesterol or lipids in the body and the activity of lipoprotein-forming enzymes, so that both cholesterol and lipids ingested by the high cholesterol diet or the high fat diet are not digested and absorbed by the body, so that the amount of excess calories is increased. The effect which can suppress absorption can be expected.

In addition, according to the present invention, the gilyeong fraction containing the high purity of the salicinin praticoside, the high content of high purity praticosides saponin contained in high purity was confirmed to be a phenomenon that is discharged to the feces combined with cholesterol and lipids excessively intake Since cholesterol and lipids are excreted without being used in the body, an effect that can suppress the absorption of excessive calories can be expected.

In addition, according to the present invention, by inhibiting the digestion and absorption of excess cholesterol and lipids in the body and excreted in the feces, it can be expected that the effect of inducing weight loss due to suppression of calorie absorption.

Taken together, it can be predicted that the Giltyeong fraction can be utilized as a potential material with anti-obesity activity.

1 is a reversed phase HPLC chromatogram analyzing the components of the crude ethanol crude extract.
FIG. 2 is an HPLC chromatogram analyzing the components removed from the ethanol crude extract of long length by EtoAC layer separation.
3 is an HPLC chromatogram analyzing the components recovered in the butanol fraction.
FIG. 4 is an HPLC chromatogram analyzing the ethanol crude extract [Crude extract] and the component [water fraction after BuOH partition] removed by the butanol fraction.
FIG. 5 is an HPLC chromatogram analyzing the butanol fraction [BuOH fraction] obtained by saturated butanol partitioning and [Loading effluent of Sep-pak] components which were removed by reverse phase cartridge chromatography and then eluted.
6 is a HPLC chromatogram of the butanol fraction [BuOH fraction] obtained by saturated butanol partitioning and the component [Sep-pak with 20% EtOH] recovered by dropping it in reverse phase cartridge chromatography and eluting with a 20% ethanol aqueous solution. It is a comparison.
FIG. 7 shows an HPLC chromatogram of a butanol fraction obtained by saturated butanol partitioning and a component [Sep-pak with 40% EtOH] recovered by eluting it with 40% ethanol aqueous solution in the reverse phase cartridge chromatography. It is a comparison.
8 is a HPLC chromatogram of the butanol fraction [BuOH fraction] obtained by saturated butanol partitioning and the component [Sep-pak with 60% EtOH] which is recovered when the mixture is eluted with a 60% aqueous ethanol solution in the reverse phase cartridge chromatography. It is a comparison.
9 is a HPLC chromatogram of the butanol fraction [BuOH fraction] obtained by saturated butanol partitioning and the components [Sep-pak with 80% EtOH] recovered when the mixture is eluted with an aqueous 80% ethanol solution in the reverse phase cartridge chromatography. It is a comparison.
FIG. 10 shows an example of a result of performing LC / MS mass spectrometry on a component having a retention time of 21.13 among components eluted in FIG. 8, and FIG. 11 shows a result of identifying components eluted in FIG. 8.
Figure 12 shows the changes in body weight and intake after 4 weeks of oral administration of high cholesterol and high fat diet and Gilt ethanol extract and Gilt fraction for experimental animals.

Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited to the following Examples and the like.

Example  One.

One) Gakyung  ethanol Crude extract  Produce

Washed and dried 6-year-old bellflower [Gilyeong] grown by a dog farmer in Samcheok, Gangwon-do, and prepared it in powder form, and then added 80 times of aqueous solution containing 80% by volume of ethanol to the weight of powder for 30 minutes at 40 ℃. After ultrasonic extraction, the mixture was left at room temperature for 30 minutes, and Whatman No. It was filtered using two filter papers. After the filtration, the same amount of the aqueous solution containing 80% by volume of ethanol was added again to the residue, and the ultrasonic extraction, standing and filtration were repeated three times. Collect all the extraction filtrate obtained in the above process, Whatman No. The filter was filtered again using two filter papers, ethanol was blown off using a vacuum concentrator at 50 to 60 ° C., and concentrated to water. The concentrate was frozen and lyophilized at a low temperature of −50 ° C. or lower and then processed into a powder state (longitudinal ethanol crude extract, hereinafter “longitudinal extract” has the same meaning).

Some of the crude ethanol crude extract was dissolved in methanol and analyzed by HPLC under reversed phase conditions to confirm the composition of the components. The results are shown in the chromatogram of FIG. 1. According to FIG. 1, the aspect which dozens of constituent components coexist is confirmed.

2) Gakyung Butanol  Preparation of Fractions

10 g of the crude ethanol crude extract is dissolved in about 1 L of ethanol, and a precipitate is generated. The ethanol solution is carefully transferred to a beaker, and a certain amount of ethanol is added to the residue to dissolve, and ethanol is continuously added to the remaining precipitate. Dissolved.

200 mL of the ethanol solution in which the crude ethanol crude extract was dissolved was transferred to a 1000 mL beaker and mixed with 600 mL of acetone to induce white precipitate formation. Filtration with two filter papers removed some poorly soluble polysaccharides and proteins. The ethanol and acetone solution from which the white precipitate was filtered was completely concentrated using a reduced pressure concentrator at 50 ° C., and about 400 mL of distilled water was added to the resulting concentrate to dissolve it. The same amount of EtoAC and distilled water in which the concentrate was dissolved were added thereto, and layer separation was performed three times in succession. This process is intended to remove some sugars and non-saponin-based mesopolar substances in the extract.

The EtoAC layer, the upper layer obtained by the layer separation result, was collected, concentrated, dissolved in methanol, and the components removed from the extract by EtoAC layer separation were confirmed by HPLC analysis, and the results are shown in FIG. 2. According to FIG. 2, the EtoAC layer separated from the crude ethanol crude extract was found to have many components corresponding to the retention time of 8 to 17 minutes during HPLC analysis. The above results can be seen to mean that the material having a polarity corresponding to this part is removed when the EtoAC layer separation is induced from the long-length ethanol crude extract.

The water layer, which is the separated layer, is partially concentrated in a reduced pressure concentrator at 40 ° C. to completely remove the EtoAC, recover the saponin-based material from the water layer from which the EtoAC is removed, and a highly polar compound and a polarity that were not removed from the EtoAC fraction. In order to remove the sugar component, the same amount of saturated butanol was added thereto, and the layer separation was performed four times in the same manner. After the separation, the upper butanol layer was recovered.

The saturated butanol is shaken vigorously by putting the same amount of butanol and ultrapure water and left to completely separate the layers, and then used to recover the upper layer.

The butanol layer separated by the upper layer was collected and concentrated under reduced pressure using a vacuum concentrator at 60 ° C. to prepare a butanol fraction, and the components removed by fractionation were removed into a water-based layer and a gilyeong saponin-based material recovered by the butanol fraction. Butanol and water fractions were analyzed by HPLC to confirm, and the results are shown in FIGS. 3 and 4.

According to FIG. 3, the butanol fraction has been removed from the crude ethanol crude extract by EtoAC fraction, but the remaining components are eluted. In particular, the retention time during the HPLC analysis corresponds to 21 to 25 minutes, it can be seen that the tendency that a large number of components of the various speculative compounds that are supposed to be a long saponin compound. In FIG. 4, the water layer removed from the crude ethanol crude extract by the butanol fraction treatment contains a large amount of fully polar compounds, so that the removal effect of the completely polar compounds corresponding to the retention time of 3 to 15 minutes during HPLC analysis can be confirmed. have.

3) Gakyung Butanol  Separation and Purification of Fractions

The butanol fraction obtained by concentrating the butanol layer contains a large amount of Praticodine D and Praticoside, which are supposed to be saponin components corresponding to the indicators of Gil-Gil, but as shown in the results of FIG. In spite of the solvent fraction of, it can be seen that there are still many polar compounds with extremely high polarity compared to the saponin components, which have a retention time of 3 to 10 minutes.

Since these components can no longer be removed by solvent fractionation, different purification and purification procedures were applied.

That is, 2 g of butanol fraction obtained by concentrating the butanol layer was completely dissolved in 100 mL of distilled water, and was loaded on reverse phase (C18) cartridge chromatography (Sep-pak vac 6cc), followed by 20% ethanol concentration of an aqueous solution containing 0 to 80% by volume of ethanol. After elution with 10 mL volume, the elution component of each ethanol concentration was confirmed by HPLC, and the removal pattern of the polar compound of each eluting ethanol concentration and the recovery pattern of the saponin component corresponding to the indicator component of Gil-Long were shown. It was confirmed at 9.

FIG. 5 shows a butanol fraction, which is presumed to contain a large amount of gilgon saponin obtained by solvent precipitation and two solvent distributions of gilyeong extract, and the eluate which is passed through reversed phase cartridge after being loaded onto reverse phase cartridge chromatography. As a result of the chromatogram analysis, the result shows that a completely polar compound having a retention time of about 3 minutes among the components contained in the butanol fraction is not adsorbed to the reverse phase cartridge and is removed.

FIG. 6 is a result of eluting the adsorbed components by using 10 mL of 20% ethanol solution in the reverse phase cartridge of FIG. 5, wherein the polar compounds were stored for 3 to 6 minutes by retention of 20% ethanol solution. Further removal could be confirmed, and no elution of the relative mesopolar compound after 10 minutes of retention time was found.

FIG. 7 is a result of eluting the adsorbed components using 10 mL of 40% ethanol solution continuously in the reversed phase cartridge of FIG. 6, and the retention time removed by treatment of 20% ethanol solution is between 3 minutes and 6 minutes. The additional removal of some of the remaining components of the polar compound and the relatively large elution of the relative mesopolar compound between the retention time of 10 to 15 minutes can be confirmed, and the elution of the components that are presumed to be the main saponin components of Gilkyung after 15 minutes of retention time Not confirmed at all.

FIG. 8 is a result of eluting the adsorbed components using 10 mL of 60% ethanol solution in the reverse phase cartridge of FIG. 7. Most of polar compounds having a retention time of less than 10 minutes by treatment with ethanol solution up to 40% This almost entirely removed pattern was confirmed, and the large amount of components eluted between the retention time of 13-15 minutes and 17-18 minutes, which is relatively mesopolar, and the retention time of 21-24 minutes, which is estimated to be the main saponin component, You can check it.

FIG. 9 is a result of eluting the adsorbed components by using 10 mL of 80% ethanol solution in the reverse phase cartridge of FIG. 8, which is separated by almost all butanol fractions by treatment with ethanol solution up to 60%. It can be seen that the component is eluted and no residue is present.

4) Gakyung Butanol  In fractions Inverse phase Cartridge  Identification of Tablet Ingredients

About 10 kinds of saponin components contained in Gil-gil have been reported in the literature, including the indices praticodine D and various praticosides worldwide.

Therefore, the chemical structure of the eluted component of the reverse phase cartridge using 60% ethanol finally purified by the sequential process was identified by molecular weight measurement using LC / MS.

FIG. 10 is an LC / MS representative of the components of the retention time 21.13 components, which is one of the main components between 13 to 24 minutes of retention time estimated as the main saponin component of Gilyeong among the components eluted by reverse phase cartridge separation using 60% ethanol. Mass spectrometry results are shown. As a result, the molecular weight of 1223.4 is identified and identified as Praticodine D, which is known as an indicator component of cirrhosis activity. In addition, by the same method, in addition to praticodine D of the main components eluted by reverse phase cartridge separation using 60% ethanol shown in Fig. By using the method, 9 kinds of Praticosides including Praticodine D, which is known as an indicator component of Gilkyung, were extracted from 10 kinds of Praticosides contained in Gilgil to date. High purity purification was possible at a relative purity of 70% or more relative to the total area of the ultraviolet light absorbing component by HPLC analysis.

Peak No. Retention time
(min.) Platycoside Molecular formula [MH] ^- One 13.14 Deapi-Platycoside E C ₆₄ H ₁₀₄ O ₃₄ 1415.4 2 13.68 Platycoside E C ₆₉ H ₁₁₂ O ₃₈ 1547.2 3 17.06 Platycodin D ₃ C ₆₃ H ₁₀₂ O ₃₃ 1385.4 4 20.65 Deapi-Platycodin D C ₅₂ H ₈₄ O ₂₄ 1091.4 5 21.13 Platycodin d C ₅₇ H ₉₂ O ₂₈ 1223.4 6 21.57 Polygalacin D C ₅₇ H ₉₂ O ₂₇ 1207.4 7 22.02 3 " -O- acetyl polygalacin D C ₅₉ H ₉₄ O ₂₈ 1249.4 8 22.67 Platycodin a C ₅₉ H ₉₄ O ₂₉ 1265.4 9 23.20 2 " -O- acetyl polygalacin D C ₅₉ H ₉₄ O ₂₈ 1249.4

Example  2. High Fat Diet  Ingested In rats Gakyung  Extract and Gakyung Fraction Anti-obesity  effect

1) Dietary Animals

Male ICR mice were purchased and used at 5 weeks of age (25 ± 3g, Central Lab Animal Inc.) and independently (20-23 ° C, 12 h periods of light and dark) under environmental control of a stainless steel cage. Breeding.

The high-cholesterol diet used Dyets # 101556 [40% (by weight ratio) Beef Tallow, Central Lab Animal Inc. in the basic diet, 200g casein per kg, 3g DL- Methionine, 150 g corn starch, 500 g sucrose, 50 g cellulose, 50 g corn oil, 35 g salt mix (# 200000), 10 g vitamin mixture (# 300050), 2 g choline bitartrate It consisted of. The nutritional composition of the high-fat diet consisted of 17.7% protein, 40.0% fat, 5.0% fiber, 4.0% ash, 3.3% moisture, and 31.4% sugar.

There was no restriction on the feed and water supplied to the experimental animals until the experiment. One week after the start of the high fat diet, blood was collected and the blood cholesterol content was measured. Only the individuals whose blood cholesterol content was 200 mg / ㎗ or more were selected for the experiment. Used. The 24 selected individuals were randomly divided into 4 groups, and the general diet group was the control group (C), and the diets containing the high fat diet were the negative control group (HCC), and the high fat diet was selected as a Gilkyung extract (PDE). ) And the Gilt fractions were divided into dietary groups (PDS) [n = 6].

For the application of Gil-Gil extract and Gil-Gil fractions, oral administration of Gil-Gil extract and Gil-Gil fractions was 15 mg / kg, respectively, for 4 weeks, and 0.9% physiological saline was orally administered to the control group containing only the high fat diet. Weight change and intake were measured every two days for four weeks after the experiment. After 4 weeks, blood was collected from eyeballs of experimental animals after 14 hours of oral administration of gilyeong extract and gilyeong fractions. The collected blood was stored at room temperature for 2 hours and then centrifuged (3500 × g, 10 Min) plasma was obtained and stored at −80 ° C. until analysis. After the blood collection, the experimental animals were sacrificed by cervical dislocation and the livers were extracted, and the livers were weighed and stored at -80 ° C until analysis. Feces of ICR mice were collected about 40 ~ 70 mg for each individual before the end of the experiment and weighed after lyophilization.

2) Measurement of body weight and intake of experimental animals

The high cholesterol and high fat diet, and the gilyeong extract and gilyeong fractions were orally administered to the experimental animals and body weight and intake were examined 4 weeks later (Fig. 12, p = 0.05).

The weight change of the group administered orally with the gilyeong extract and the gilyeong fraction for the weight change of the group treated with the high cholesterol diet alone showed that the gilyeong extract did not show any significant weight change, Case weight loss was about 52.6% compared to the group treated with high cholesterol diet alone. In the high cholesterol alone diet group, there was no significant difference in weight gain compared to the general diet group, which means that the diet of cholesterol alone does not affect the weight gain.

In the case of oral administration of the Gil-Gil extract and the Gil-Gil fraction, the weight gain rate was lower than the high-cholesterol diet group or the general diet group for 4 weeks. In the weekly intake during the experimental period, when the diet treated with high cholesterol diet was 100, the general diet group showed a slightly lower intake, and the oral administration of Gil-Gil ethanol extract and Gil-Gil fraction showed a clear decrease in intake. It was. In particular, the group administered orally to the Gilt fraction showed a clear reduction of intake of about 35% compared to the group treated with high cholesterol diet alone until 2 weeks of dietary experiments [Table 2, n = 6].

A decrease in intake is associated with a decrease in the cholesterol content in the blood, and it can be said that praticosides contained in the gilded ethanol extract and the gilded fraction can affect the homeostasis of cholesterol and can be effectively used to limit the intake. .

Week One 2 3 4 C ^d (HC-free) 89.2 ± 6.6 ^a 85.2 ± 2.1 ^a 91.1 ± 5.8 ^a 82.5 ± 7.4 ^a HCC 100.2 ± 1.1 100.6 ± 5.4 99.6 ± 8.3 100.1 ± 3.2 PDE ^d 84.7 ± 5.8 ^a 82.4 ± 7.3 ^a 89.5 ± 3.1 ^b 78.8 ± 9.2 ^b PDS ^d 65.4 ± 9.3 ^a 64.6 ± 8.3 ^b 85.4 ± 2.9 ^c 76.4 ± 5.3 ^b The data are mean ± SD and significantly different at ^a P <0.05, ^b P <0.01 and ^c P <0.001 by the unpaired Student's t-test.
^d The results are expressed as% of the high cholesterol control.

3) plasma, liver tissue and Feces  Cholesterol Metabolism Analysis

Extraction of lipids from plasma, liver tissue and feces was performed by the method by Folch et al. (1951). Tissue samples of each experimental group were subjected to sonication for 5 minutes by adding 1 mL per 1 mg of tissue sample mixed with chloroform and methanol (2: 1), followed by centrifugation (3000 × g, 10 minutes). The insoluble material was removed, water was added to the supernatant, mixed, and then centrifuged again (3000 × g, 10 minutes) to collect the layers below and dried under nitrogen charging. The dried extract was dried in 100 μl. Resuspended in chloroform and separated using an aminoprophyl-bonded solid phase extraction column (500 mg, Alltech, Deerfield, IL). The aminopropyl-fixed column was washed twice with 4 mL of hexane and passed through the resuspended lipid extract and again eluted with 6 mL of chloroform and 2-propanol (15: 1, v / v) and dried. The weight was then quantified. The concentration of high density lipoprotein-cholesterol (HDL) in the blood was measured by an automated analyzer (Hitachi-747 auto-analyzer, Hitachi Co., Tokyo, Japan) using a high density lipoprotein-cholesterol diagnostic reagent (Kyowamedex Co. Ltd, Japan). Low density lipoprotein-cholesterol (LDL) was measured using a Roche 2nd generation low density lipoprotein-cholesterol plus reagent and an automatic analyzer (Hitachi-7170 auto-analyzer, Hitachi Co., Ltd.). Tokyo, Japan). In addition, plasma triglyceride and cholesterol content were measured using a colormetric kit (Eiken Chemicla C. Ltd., Tokyo), and the absorbance of the final product was measured by a microplat reader (Molecurlar device, Sunnyvale, CA). It was measured at 490 ~ 520 nm using. The content of triglycerides and cholesterol in liver tissue and feces was measured in the same way as in plasma.

Group C (HC-free) HCC PDE ^d PDS ^d TG (mg / 100mL) 112.6 ± 16.5 140.9 ± 17.6 ^c 118.8 ± 13.3 ^b 126.2 ± 12.1 ^c TC (mg / 100mL) 116.8 ± 12.6 ^b 152.3 ± 6.4 ^c 98.2 ± 17.1 112.1 ± 13.9 ^b HDL-C (mg / 100mL) 75.7 ± 23.9 ^a 69.2 ± 29.3 ^a 74.5 ± 22.9 ^a 71.5 ± 16.2 ^b LDL-C (mg / 100mL) 60.4 ± 24.2 ^b 71.2 ± 27.2 ^b 47.2 ± 13.3 48.1 ± 27.9 ^a HDL-C / LDL-C ^d 1.5 ± 0.2 1.0 ± 0.2 ^c 1.6 ± 0.6 ^a 1.4 ± 0.4 ^c ALT (KU / L) ^e 20.8 ± 2.7 25.4 ± 3.8 22.3 ± 2.7 19.8 ± 4.3 AST (KU / L) ^e 53.4 ± 4.6 70.2 ± 3.7 42.2 ± 6.6 45.9 ± 2.8 The data are mean ± SD and significantly different at ^a P <0.05, ^b P <0.01 and ^c P <0.001 by the unpaired Student's t-test. ^d The discrete parameters of each measurement were used in statistical analysis after being counted and averaged. ^e KU (Karmen unit).
PDE, ethanol extract from root of Platycodon grandiflorum ., PDS, saponin fraction from root of Platycodon grandiflorum , HCC, high cholesterol., TG, triglyceride., TC, total cholesterol, HDL-C, high density lipoprotein cholesterol, LDL-C, low density lipoprotein cholesterol.

As shown in Table 3, the total cholesterol, total triglycerides, LDL (low demsity lipoprotein), HDL (high density lipoprotein) in the blood after 4 weeks of oral administration of high cholesterol diet, gilyeong extract and gilyeong fractions for the experimental animals As a result of assaying the enzyme activity of ALT and AST, total cholesterol, total triglyceride and LDL in blood were increased and HDL content was decreased when high cholesterol was fed alone. This demonstrates that it is suitable to induce hypercholesterolemia from ICR mice through the diet of cholesterol added feed.

In the group administered orally with the gilyeong extract and the gilyeong fraction, the triglyceride contents decreased to 84.3% (PDE) and 89.6% (PDS) levels compared to the high cholesterol group alone, and the total cholesterol content was lower than that of the cholesterol alone diet group. The levels of 64.5% (PDE) and 73.6% (PDS) were also found in the LDL content. PDS) showed a tendency to decrease. On the other hand, HDL did not show any change in the group fed with cholesterol alone, the general diet group, or the group administered orally to Gilgil extract and Gilgil fraction. The oral administration of the pathogenic fractions resulted in a sharp decrease in LDL compared to other types of cholesterol, and the results showed that the change in HDL was not significant. Some synthetic saponins, such as tiqueside and parnaqueside, reduced cholesterol in plasma. The effect of lowering cholesterol content by interfering with the absorption was similar, but it was similar to the report that it did not affect HDL. It is believed that the metabolism of cholesterol is regulated by the various praticoside saponins present in the pathogenic fraction.

As shown in Table 3 above, the enzyme activity of ALT and AST in plasma was slightly increased in AST activity compared to the general diet group in the cholesterol-only group, but no statistically significant change was observed. The degree of activity of each enzyme showed a tendency to decrease even in the group administered orally, Gil-Gil extract and Gil-Gil fraction.

Group C (HC-free) HCC PDE ^d PDS ^d Liver mass (g) 1.83 ± 0.14 1.92 ± 0.13 1.54 ± 0.21 1.67 ± 0.14 Total lipid extract 24.5 ± 3.2 ^a 64.8 ± 2.3 ^c 38.6 ± 2.5 ^b 42.6 ± 3.3 ^a TG (mg / g liver mass) 15.2 ± 1.8 ^b 19.9 ± 1.6 12.8 ± 0.8 ^a 14.2 ± 0.7 ^a TC (mg / g liver mass) 16.5 ± 1.8 ^b 34.3 ± 2.2 ^c 20.2 ± 1.1 ^b 20.1 ± 3.9 ^b The data are mean ± SD and significantly different at ^a P <0.05, ^b P <0.01 and ^c P <0.001 by the unpaired Student's t-test. ^d The discrete parameters of each measurement were used in statistical analysis after being counted and averaged.

As shown in Table 4, the liver weight, fat content, total cholesterol and total triglyceride content in the liver extracted 4 weeks after the oral administration of high cholesterol diet and the gilyeong extract and gilyeong fractions for the experimental animals .

Oral administration of gilyeong extract and gilyeong fraction decreased to 59.6 ~ 65.7% compared to the fat content of the group fed cholesterol alone, triglyceride content decreased to 64.3 ~ 71.3%, especially total cholesterol content was 58.6 Considering that the cholesterol content in the liver is mainly in the form of cholesterol esters, the ciliary extract and the ciliary fraction prevent the accumulation of cholesterol esters in the arteries and impede the formation of lipoproteins in the liver to prevent vascular walls. It is thought that it can be utilized as a powerful anti-arteriosclerosis agent by preventing the proliferation of foam cells in the inner body.

Liver weight was 1.92 ± 0.13 g in the cholesterol-only group, but slightly decreased to 1.54 ± 0.21 g and 1.67 ± 0.14 g in the oral administration of the Gilt extract and the Gilt fraction, respectively. Based on the results of Table 3 and Table 4, it can be seen that the reduction of ALT and AST in plasma has the inhibitory effect on hepatic damage stress associated with high cholesterol and on the formation of fatty liver through the reduction of ALT and AST. there was.

Considering studies showing that saponin forms a water-insoluble complex by physical binding with cholesterol to prevent the absorption of cholesterol into the tissue, Gilpyeong praticoside saponin is a competitive bond with cholesterol in the tissue May be able to prevent the accumulation of cholesterol. On the other hand, saponins with lower polarity of saponin and cholesterol have higher polarity and higher binding capacity, and thus they can be combined with cholesterol. I think it should be done.

Group C (HC-free) HCC PDE PDS Total lipid extract ^b 6.8 ± 1.6 ^a 10.2 ± 2.8 16.2 ± 0.5 ^a 21.6 ± 2.4 ^a TG 3.2 ± 0.9 4.5 ± 1.0 4.8 ± 0.9 5.2 ± 0.7 TC ^c 1.5 ± 0.3 2.1 ± 0.4 4.2 ± 0.4 ^a 4.8 ± 0.9 ^a The data are mean ± SD and significantly different at P <0.05 by the unpaired Student's t-test.
^b Total lipid extract was obtained by Soxhlet lipid extraction.
^c Cholesterol was determined by commercial kits and includes cholesterol ester, free cholesterol and some cholesterol metabolites which remained as an unoxidized 3-hydroxyl residues.

Table 5 shows the results of assaying the contents of fat content, total cholesterol, and total triglycerides in ICR mouse feces, which are in contrast to the lipid metabolism index contents in the blood and liver of Tables 3 and 4 The group administered orally with the gilyeong extract and the gilyeong fraction showed higher values than the group fed with cholesterol alone. The amount of excreted fat increased about 1.5 times in the group administered orally with Gil-Gil extract, and 2.1 times in the group administered orally with Gil-Gil fraction, and the content of triglyceride was not significantly changed. Compared to one group, the group administered orally with Gil-Gil extract showed an increase of about 2 times, and the group administered orally with Gil-Gil fraction showed a 2.3-fold increase. In the fecal group, the oral administration of Gil-Gil extract and Gil-Gil fraction was higher in lipid-related molecular sieve than in blood and liver. It is thought to be the result of excretion without affecting.

Unlike saponin and cholesterol complex formation, saponins and triglycerides did not form complexes, but the contents of cholesterol and triglycerides were increased in the feces of the group administered orally with the Gilt extract and the Gilt fraction.

4) pancreatic lipase ( Pancreatic lipase ) Active and Low performance  Measure

According to the oral administration of the Gilt Extract and the Gilt Fractions to the experimental animals, Praticoside Saponin contained in the Gilt Extract and the Gilt Fractions was excreted without formation of cholesterol complexes and showed high cholesterol content in feces. Feces also showed high triglyceride contents, which are generally known to not complex with saponin compounds. Therefore, it is judged that Praticoside saponin contained in Gil-Gil may have an effect on triglyceride metabolism, and thus it is a key enzyme (key enzyme, Bitou) that decomposes triglyceride into 2-monoacylglycerol and fatty acid from plasma of each treatment group. et al., 1999). The inhibitory activity of pancreatic lipase enzymes was assayed.

The activity and inhibitory ability of pancreatic lipase was modified by the method of Bitou et al. (1999), and the lipase-induced plasma was reacted with lipase-induced plasma by separating plasma from the experimental animals to which the gill extract and the gill fraction were administered. It was analyzed by measuring oleic acid isolated from (triolein). The absorbance of lipase activity increased by high cholesterol diet was decreased by administration of Gil-Gil extract and Gil-Gil fraction, and the relative enzyme was obtained when the lipase activity was 100% in plasma of experimental animals fed the normal diet. The degree of inhibitory activity was assayed.

Substrate is 90 μmol in 9 mL of 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pH 7.0) with 0.1 M NaCl added. Triolein, 45 mg Arabian gum and 9.45 μmol taurocholic acid were added and prepared by ultrasonic treatment for 5 minutes. The reaction solution for measuring lipase activity was prepared by mixing 20 μl of protein quantified plasma and 180 μl of substrate solution to make 200 μl of the final reaction solution, and reacting for 30 minutes at pH 7.0 and reaction temperature of 37 ° C. Quantification of oleic acid produced after enzymatic reaction was determined according to the method of Zapf et al. (1981). After enzymatic reaction, 3 mL of chloroform / heptane (1: 1) solution containing 2% (v / v) methanol was added to 0.2 mL of the reaction solution, stirred for 10 minutes, and then centrifuged for 10 minutes (2,000 × g). The aqueous layer was removed. 1 mL of copper reaction solution was added thereto and stirred for another 10 minutes, followed by centrifugation (2,000 × g) for 10 minutes, and 1 mL of an organic solvent layer containing oleic acid and copper salt extracted was 0.05%. (w / v) 1 mL of a 0.1% (w / v) bathocuprione solution containing 3-tert-butyl-4-hydroxyanisole was added. Then, the enzyme activity was measured by quantifying oleic acid by measuring the absorbance at 480 nm.

Enzymatic source
(serum) Lipase activity C (HC-free) HCC PDE PDS Inhibition rate (%) 100.4 ± 1.2 168.2 ± 8.5 69.5 ± 2.6 48.1 ± 5.6

As a result of assaying pancreatic lipase activity using the plasma of each treatment group as an enzyme solution to investigate the cause of the increase in triglyceride content in the feces of the group administered orally with the high cholesterol diet, the table 6 above. As shown in the figure, when the enzyme activity of the pancreatic lipase of the general diet group was 100%, the group with the high cholesterol diet increased to 168%. The activity was 69.5% and 48.1%, respectively, and it is thought that the praticoside saponin contained in the pathogen binds to the pancreatic lipase enzyme, thereby inhibiting the enzyme activity, so that triglycerides are not digested and excreted more in feces.

A representative pancreatic lipase inhibitor inhibits about 30% of the fat consumed as tetrahydrolipstatin (Orlistat, Ro 18-0647), a derivative of lipstatin derived from Streptomyces toxitricini. Although it is known to have excellent efficacy, tetrahydrorifstatin is known to have side effects such as gastrointestinal disorders, hypersensitivity, bile secretion disorders, and fat-soluble vitamin absorption inhibition. Therefore, recently, research for the development of pancreatic lipase inhibitors from foods and natural products having no side effects has been conducted. The results described above indicate that the Gilt Extract and Gilt Fraction can be used as pancreatic lipase inhibitors.

In conclusion, praticosides contained in the gilyeong extract and the gilyeong fraction decrease the dietary intake, inhibit the digestion and absorption of lipids and cholesterol in the body, and promote the excretion of cholesterol and lipids in anti-obesity activity It can be used as a potential material.

Formulation example .

1) Preparation of Syrup

A syrup containing the Giltyeong fraction or Giltyeong extract including the Giltyeong fraction according to the present invention as an active ingredient was prepared by the following method.

After dissolving the Gilyeong fraction (200 ㎍), saccharin, sugar in 80 g of warm water, and cooled, a solution consisting of glycerin, sweetener, flavoring agent, ethanol, sorbic acid and distilled water was prepared and mixed, and water was added thereto. 100 mL was made.

2) Preparation of Tablets

Tablets containing the gilyeong fraction according to the present invention as an active ingredient were prepared by the following method [active ingredient 10 ㎍ / mg].

250 g of the lengthwise fraction was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, to which 10% gelatin solution was added, followed by grinding to pass through a 14 mesh sieve. It was dried and tablets were prepared from a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate.

3) manufacture of beverages

After dissolving 500 μg of the gilyeong fraction in an appropriate amount of water, an appropriate amount of vitamin C, citric acid, sodium citrate, and oligosaccharides was added, and an appropriate amount of sodium benzoate was added as a preservative to make 100 mL by adding water to prepare a beverage composition.

4) Manufacture of health food

Health food containing the Gilyeong fraction according to the present invention as an active ingredient was prepared by the following method.

100 μg of Gilt fraction, appropriate amount of vitamin mixture, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, vitamin C 10 mg, biotin 10 μg, nicotinic acid Amide 1.7mg, Folic acid 50㎍, Calcium pantothenate 0.5mg, Inorganic mixture appropriate amount, Ferrous sulfate 1.75mg, Zinc oxide 0.82mg, Magnesium carbonate 25.3mg, Potassium phosphate monobasic 15mg, Dibasic calcium phosphate 55mg, Potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg were prepared by mixing according to a conventional health food preparation method.

The present invention described above is not limited to the above-described embodiment and the accompanying drawings, and various substitutions, modifications, and changes are possible within the scope without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

1 is evacuated in Figure 11 - A plastic Tycho side E (Deapi-Platycoside E), 2 is a plastic tigo and side E (Platycoside E), 3 is plastic Tycho Dean D ₃ (Platycodin D _3), and 4 is to evacuate the plastic Deapi-Platycodin D, 5 is Platycodin D, 6 is Polygalacin D, and 7 is 3 “ -O -acetyl polygalactin D (3”). - and O -acetyl polygalacin D), 8 is a plastic Tycho Dean a (Platycodin a), 9 is 2 "- O - acetyl-poly go raksin D (2" - a O -acetyl polygalacin D).

Claims (6)

Gil-gil ethanol fraction obtained by dissolving the crude ethanol crude extract in ethanol, removing the precipitate formed by adding acetone, and dispersing the filtrate in ethyl acetate, and using water and saturated butanol sequentially. Has anti-obesity activity, characterized in that it contains high purity praticoside having a relative purity of 70% or more relative to the total area of the ultraviolet light absorbing component separated and purified by reverse phase (C18) cartridge chromatography using an ethanol-containing aqueous solution as an elution solvent. Separation and Purification Method of Cultivated Fractions.
delete The method according to claim 1,
The separation purification method,
1) dissolving the crude ethanol crude extract in ethanol and removing the precipitate formed by adding acetone and concentrating the filtrate to obtain the gilyeong ethanol-acetone fraction;
2) dissolving the lengthwise ethanol-acetone fraction in water and partitioning with ethyl acetate to obtain a water fraction by concentrating the water layer;
3) mixing the saturated butanol in the water layer of step 2), separating the water layer and the butanol layer, and concentrating the butanol layer to obtain a butanol fraction; And
4) Gil-length fraction containing long-length Praticoside with high purity of 70% or more relative to the total area of UV absorbing component by reverse phase (C18) cartridge chromatography in which the butanol fraction is dissolved in distilled water and an ethanol-containing aqueous solution is used as an eluting solvent. Separating and purifying it;
Separation and purification method of Gilyeong fraction having anti-obesity activity, characterized in that it comprises a.
The method according to claim 3,
Ethanol and acetone in the step 1) is the separation and purification method of the gilyeong fraction having anti-obesity activity, characterized in that consisting of 1: 3 to 5 by volume. delete delete

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