KR101322503B1 - Composition for Prevention and Treatment of Obesity Comprising Liriope Seed Extracts - Google Patents

Abstract

The present invention relates to an anti-obesity composition comprising an extract of Macmundong seed, more specifically, to confirm that the enzyme extract activity of alpha-glucosidase, alpha-amylase, and lipase from the extract of the Macmundong seed and the red pigment separated therefrom will be.
According to the present invention can provide a composition for the treatment, improvement and prevention of obesity, including the extract of the mammary sinus seed.

Description

Composition for prevention and treatment of Obesity Comprising Liriope Seed Extracts}

The present invention relates to an anti-obesity composition comprising the extract of Macmundong seed.

Overweight and obesity have long been known to be closely linked to various chronic diseases. Obesity is well known as a risk factor for cardiovascular disease (heart disease, stroke), diabetes, cancer, liver disease, hypertension, etc. and increases the risk of surgery or pregnancy (Levinson, 1977). In particular, the World Health Organization (WHO) assessed obesity as a major factor in adult diseases such as diabetes and defined it as the greatest risk factor that threatens human health and well-being in the 21st century (Taubes, 1998).

What is currently being marketed or considered as a weight-controlling drug or product usually works by mechanisms that inhibit the digestion and absorption of energy nutrients or inhibit appetite. For example, pancreatic lipase inhibitors such as orlistat and carbohydrate degrading inhibitor Acarbose have been developed as drugs that limit the absorption of energy sources by inhibiting carbohydrates or lipoproteinases.Acarbose effectively inhibits obesity. (Vedula et) al . , 1991).

On the other hand, alpha-glucosidase inhibitory activity was detected from various food materials, and its efficacy has been reported in human experiments (Fujita et. al . , 2001), among the herbal medicines, Eucommia ulmoides ), coriander ( Communelina communis ), golden ( Scutellaria alpha-glucosidase inhibitors have been isolated from baicalensis , et al . (Kim et al . , 1999; Watanabe et al . , 1997; Nishioka et al . , 1998; Yoshikawa et al . , 1998).

In addition, alpha-amylase inhibitors are considered to be very potent diabetes treatment or prevention material by effectively suppressing post-prandial blood sugar rise, and when the alpha-amylase of the pancreas is strongly inhibited, the digestion and absorption of the small intestine of the starch component is restricted. It is reported that there is a high possibility of preventing obesity by inactivating enzymes in the duodenum (Layer et. al . , 1985).

Due to the recent westernized diet, nutritional dependence on fat is increasing, and research on the lipase inhibitory activity is very active because it is effective in controlling weight in people who consume a lot of energy from fat. Recent studies have reported that most of the materials with lipase inhibitory activity are effective in preventing and improving obesity, fatty liver and hyperlipidemia (Yamamoto et. al . , 2000). For example, it is well known that saponins or polyphenol components of bellflower or green tea have anti-obesity effects through inhibition of lipase enzyme activity (Han et. al . , 2001).

Plants, including herbal medicines, are known to have abundant distribution of carbohydrates and fat-inhibiting enzymes. Therefore, it is expected that they can effectively control obesity by delaying postprandial blood sugar elevation and fat digestion. Lankisch et al . , 1998; Matsuo et al . , 1992; Goese et al . , 2000; Yamamoto et al . , 2000). Moreover, weight control through digestion of digestive enzymes is considered to be a relatively low side effect and high compliance method for obese patients.

On the other hand, Liriope platyphylla Wang et Tang) is a perennial herbaceous plant belonging to a lily family that grows well in wet areas and has purple flowers in July and has a black fruit in October. It is ovary and the fruit is round and curd. Peels off early to expose purple-black seeds. Root plant of Ophiopogon japonicus ), Liriope spicata ).

In Chinese medicine, McMun-Dong is a bohemian medicine, which acts as a positive effect of Yang Yin Yun, Xin Sansheng, and Cheong Shim Jeon (거 心 除煩). ), It is used for nourishment, tonic, diuresis, and jigal, and it is used to control mammundong such as Ongyeong-tang, Jagamcho-tang, Cheongsimyeon-ja-myeon, Jeonjeonjeon, Saengmaek-san. have. It has been reported that the effects of pulmonary edema are hypoglycemic action, anti-inflammatory action, IgM antibody production inhibitory action, antiarrhythmic effect, anticancer action of butanol fraction, and leukopenia antagonist.

As described above, the various pharmacological effects of the known mammundong means bulge bulge of the mammundong, it was peeled and dried casserole has been generally used as the mammundong.

That is, until now, research on other parts of the pulmonary tract except for the pulmonary muscle of the pulmonary tract has not been made, and the research on the fruit of the pulmonary tract is almost never conducted. There is no mention of the fruit of McMoon-dong in the old literature, and its composition has not been studied, nor is it known as a medicinal or edible product. In addition, since most of the farms are grown in the Munmundong farming method, the fruit is not currently used.

Recently, however, the physiological activity of the extracts of the seedlings of Macmundong was studied, so that acute toxicity was not induced by the single dose of Macmundong hot water extract, and it has a protective effect against liver damage in rats induced by carbon tetrachloride (CCl ₄ ). It has been reported that it is toxic. While searching for a substance exhibiting such physiological activity, he became interested in various types of anthocyanins contained in the pulmonary sinus seeds, resulting in delphinidin 3-glucoside and delphinidin 3-rutinoside. Four anthocyanins were reported, Petunidin 3-rutinoside, and Malvidin 3-rutinoside. It was first isolated from the pulmonary seed, but it is a known substance.

Anthocyanin is a phenolic compound belonging to the flavonoid family, which is a water-soluble pigment widely found in plants, and is widely found in fruits and vegetables. In particular, most of the black pigment contained in the seed of plant seeds is due to the high concentration of anthocyanins. Anthocyanins, the main constituents of plant pigments such as red, purple, and blue, which are expressed in flowers, fruits, and many other plant tissues, have been applied as a natural coloring agent in the food industry. , Antioxidant, anti-cancer effect by induction of apoptosis (apoptosis) has been reported.

The inventors of the present invention, while verifying the activity of the extract of the seedlings Mengmundong Seed extract has an inhibitory activity against the carbohydrate-related enzymes alpha-glucosidase and alpha-amylase and lipolytic enzyme pancreatic lipase, The present invention was completed by knowing that the extract can be used as an anti-obesity material for weight control.

Therefore, an object of the present invention is to provide a medicinal seed extract extract as a new anti-obesity material.

As an example for achieving the above object, the present invention is characterized by an anti-obesity composition comprising the extract as the active ingredient.

As another example for achieving the above object, the present invention is characterized by an anti-obesity composition, which is separated from the pulse extract seeds, containing an anthocyanin-based pigment represented by the following formula (1) as an active ingredient.

In Formula 1, R ₁ is OH or OCH ₃ , R ₂ is H, OH or OCH ₃ , R ₃ is O-beta-glucoside or O-beta-lutinoside (O -β-rutinoside) and R ₄ is OH.

As another example for achieving the above object, the present invention is characterized by anti-obesity health foods using the extract as the active ingredient.

Hereinafter, the present invention will be described in detail.

Obtaining the pulse extract of the present invention, the method of extracting and separating the red pigment from it is as follows.

First, the pulmonary dong seeds are dried at room temperature (below 30 ° C.) and pulverized as necessary to form a dry powder, which is immersed in water, an alcohol or an aqueous alcohol solution, extracted at room temperature, and filtered to obtain pulmonary seed extract obtained by removing insoluble matters. At this time, the extract is made by adding the dried powder of dried munmun dong seeds or ganmun dong seeds to water, alcohol or an aqueous solution of alcohol at room temperature to repeat the immersion for 24 to 72 hours 2 to 4 times Immersion temperature is to be made at room temperature specifically below 30 ℃ as room temperature. When the immersion temperature is higher than 30 ℃, the yield of the ragmundong seed extract is high, but the stability of the anthocyanin compound which is the active ingredient contained in the ganmundong seed extract cannot be guaranteed.

The alcohol may be a lower alcohol having 1 to 4 carbon atoms, the alcohol concentration of the aqueous alcohol solution used for immersion is preferably in the range of 20 to 80%. Preferably, an acid is added thereto.

As the acid, hydrochloric acid or organic acid may be used, and the addition amount may be adjusted according to the acidity thereof, and the pH of the acid-containing alcohol or the aqueous alcohol solution used is suitable for extraction of the anthocyanin compound. For this purpose, the concentration is preferably 0.1 to 1% when using hydrochloric acid, 1 to 10% when using an organic acid. As the organic acid, one or more selected from formic acid, acetic acid, nitric acid, succinic acid and citric acid may be used.

If the immersion time is short, it is difficult to completely extract the anthocyanin-based compound in the makmundong seed extract, and when the immersion time is long or the number of repetitions is more than four times, the efficiency tends to be lowered.

The extract of Macmundong seed was removed by concentrating the solvent, followed by column chromatography to obtain an anthocyanin-based pigment fraction, each fraction was concentrated to dryness and redissolved, and then purified by preparative HPLC.

The structure of the obtained anthocyanin compound was identified by various spectroscopic methods such as NMR, MS, etc. As a result, an anthocyanin-based red pigment having the structure shown in Chemical Formula 1 was obtained.

As described above, the pulmonary tract seed extract was administered to the mouse at a dose of 10 times the normal dose by a single dose toxicity test, but it did not appear to cause toxicity. In addition, the test results using alpha-glucosidase extracted from rat small intestine powder showed inhibitory activity, and the test results using alpha-amylase obtained from pig pancreas showed inhibitory activity against this. As a result of the test using the lipase obtained in, it was confirmed that there is also inhibitory activity, it can be used as an anti-obesity composition containing it as an active ingredient.

On the other hand, the anti-obesity composition comprising the pulse extract seed extract or the anthocyanin-based red pigment of Formula 1 as an active ingredient can be administered orally or parenterally, for example intravenous, subcutaneous, intraperitoneal or topical during clinical administration. .

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. It is formulated as. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into 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 a stabilizer or buffer to prepare a solution and formulated in unit dosage form of ampoules or vials.

The effective dose of the extract of the present invention or the compound of formula 1 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, 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, depending on the judgment of the doctor or pharmacist.

In addition, since the extract of Macmundong seed of the present invention was confirmed that there is no toxicity by a single dose toxicity test, it is safe for human body, so it is suitable for use in food, and has an anti-obesity health food having an improvement and prevention effect of obesity. Can be developed.

0.01 to 50% (w / w) of the total weight, preferably 1 to 30% (w / w) of the total weight when the ganmundong seed extract of the present invention or the anthocyanin-based red pigment represented by Formula 1 is included in the health food It can be used as

That is, various foods can be prepared by conventionally known methods by adding them in conventional palates such as noodles, tofu, cereals, breads, chewing gum, candy, confectionary, such as ramen noodles, raw noodles, etc. It may also be formulated into a general formulation such as tablets, granules, pills, hard capsules, soft capsules or liquid formulations, and may be prepared in fresh juice, pouches, beverages, or teas. Depending on the formulation, those skilled in the art can appropriately select and combine.

In the present invention, it is confirmed that the extract of Macmundong seed, that is, the extract containing an anthocyanin-based red pigment represented by Chemical Formula 1 as an active ingredient exhibits an inhibitory effect on alpha-glucosidase, alpha-amylase and lipase, which is a natural product of Macmundong seed extract The effect which can be used as an antiobesity composition can be anticipated.

In addition, according to the present invention, by using the seed of the ragmundong that has not been utilized as a raw material can be expected to expand the range of use of medicinal medicinal medicinal herbs.

In addition, according to the present invention, it can be expected to find a new activity of anthocyanin isolated from the pulmonary sinus seeds and to use it as an anti-obesity composition for the treatment, improvement and prevention of obesity.

1 is a graph showing the alpha-glucosidase inhibitory activity of the extracts of the makmundong seeds.
Figures 2 and 3 are graphs showing the alpha-amylase inhibitory activity of Macmundong seed extract.
Figure 4 is a graph showing the lipase inhibitory activity of Macmundong seed extract.
5 is an HPLC chromatogram of Macmundong seed extract.

Hereinafter, the present invention will be described concretely with examples and the like, but the present invention is not limited by the following examples and the like.

Example  1. Preparation of Macmundong Seed Extract

(1) in vivo And in Preparation of Macmundong Seed Extract for In Vitro Test

1 kg of dried dried Mengmundong seeds were extracted by immersion for 48 hours at 4 ° C using 1% HCl-20% ethanol 3L. The extract was filtered through an Advantec Toyo No. 2 filter paper, the residue was extracted three times, and the extracts were combined and concentrated in a 30 ° C. rotary evaporator. The concentrated Macmundong seed extract was frozen at −70 ° C. and then lyophilized to be used as a sample.

Example  2. of Macmundong Seed Extract Single dose  Toxicity Test

(1) Experimental animals

Four to five week old ICR male mice were used. Ten mice were used in each group, and the breeding conditions were maintained at 23 ± 2 ° C., 60 ± 5% humidity, and 12 hours of indoor lighting (specifications 08:00 to 20:00).

(2) Administration of sample solution and determination of acute toxicity

The lyophilized Macmundong seed extract was diluted in distilled water. It was prepared to have a concentration of 500 mg / kg, 5,000 mg / kg, respectively, and injected into the abdominal cavity of mice at a dose of 10 mL / kg.

After intraperitoneal injection of the pulmonary sinus extract, the survival rate of the mice was observed and body weights were measured at daily intervals for 7 days. The survival rates are shown in Table 1 and weight changes in Table 2, respectively.

Experimentalal
group Days after administration Death
rate One 2 3 4 5 6 7 A 0 0 0 0 0 0 0 0 B 0 0 0 0 0 0 0 0 A: 500 mg / kg from the extract of Liriopis seeds.
B: 5,000 mg / kg from the extract of Liriopis seeds.

Experimentalal
group  body weight (g / mouse) Mean ± SD 1 day 4 days 7 days A 28.00 ± 0.97 29.94 ± 0.98 31.94 ± 2.99 B 28.41 ± 1.91 30.36 ± 2.25 30.82 ± 2.41 A: 500 mg / kg from the extract of Liriopis seeds.
B: 5,000 mg / kg from the extract of Liriopis seeds.

As shown in Tables 1 and 2, the acute toxicity test of the pulmonary sinus sinus was administered by intraperitoneal injection of 500 mg / kg, which is a normal normal dose, and 5,000 mg / kg, which is 10 times the normal dose. Survival of the mice for 7 days after sample administration showed 100% survival rate in both 500 mg / kg and 5,000 mg / kg groups. In addition, the body weight of the 500 mg / kg administration group and 5,000 mg / kg administration group was also slightly increased at 1, 4, 7 days after administration respectively.

From the above results, it was shown that the pulmonary sinus seeds were not toxic because the dose of 10 times the normal dose was administered but did not cause toxicity.

Example  3. Biochemical Characterization of Macmundong Seed Extract

(1) alpha-glucosidase inhibitory activity

Alpha-glucosidase enzyme solution derived from rat small intestine was added to 0.1 U sodium phosphate buffer (pH 7.0) containing 0.2% bovine serum albumin (BSA) and 0.02% NaN ₃ . Dissolve to mL. The alpha-glucosidase enzyme solution was added 100 mg of rat small intestine powder to 3 mL of 0.9% NaCl solution, sonicated 12 times for 30 seconds in an ice bath to extract the enzyme, and centrifuged at 10,000 g for 30 minutes. The supernatant was used to measure the enzyme inhibitory activity. The substrate was used by dissolving para-nitrophenyl-alpha-di-glucopyranoside ( p- nitrophenyl-α-D-glucopyranoside, Sigma N 1377) at a concentration of 5 mM in 0.1 M phosphate buffer (pH 7.0). 50 μl of enzyme solution and 10 μl of sample extract (control for extraction) were placed in a 96-well plate and absorbance was measured at 405 nm (A at time = 0). After reacting for 5 minutes at room temperature, the absorbance (A at time = 5 min) was again measured at 405 nm. The degree of inhibition of enzyme activity was calculated by the following equation.

Lower enzyme activity (%) = (ΔAo-ΔA) / ΔAo × 100

ΔAo: Absorption change of control group without sample

ΔA: Change in absorbance after sample addition

Soybean seed extract was dissolved in dimethylsulfoxide (dimethylsulfoxide, DMSO) solvent at different concentrations (50, 200, 500 ㎍ / mL), and then the inhibitory activity against alpha-glucosidase enzyme extracted from the small intestine of the rat was It measured, and the result is shown in FIG.

As shown in FIG. 2, the alpha-glucosidase inhibitory activity of the extract of Macmundong seed showed a inhibition rate of 7.8% in the positive control group Acarbose at a low concentration of 50 μg / mL, whereas the extract of Macmundong seed showed 2.3% inhibition. In the case of 200μg / mL treatment, Acarbose showed 28.5% inhibition and Macmundong seed extract showed higher inhibition rate of 34.2%. However, when treated with high concentration of 500㎍ / mL, Acarbose of positive control group showed inhibition rate of 71.4%, which increased concentration-dependent inhibitory activity, whereas similar activity was obtained with Megmundong seed extract when treated with 200㎍ / mL. At higher concentrations, the inhibitory activity did not change significantly.

In general, most herbal extracts with high inhibitory activity of alpha-glucosidase enzymes show a concentration-dependent increase and toxicity to related cells according to their concentrations. Due to the difficulty of standardization, it is difficult to develop a new herbal extract.

In the case of Acarbose, carbohydrates, which are not dose-dependently absorbed at high concentrations, are fermented by the intestinal bacteria in the colon to produce gas, which leads to gastrointestinal spasms and bloating, and impairs mineral absorption and metabolism. It is reported that it causes dizziness, hypoglycemia, dizziness, and skin diseases such as pruritus and rash due to insufficient metabolic control.

However, the extract of Macmundong seeds exhibited the best and best alpha-glucosidase enzyme inhibitory activity at 200 µg / mL, thereby avoiding the side effects caused by standardization and dose-dependent pharmacological activity of the herbal extracts. It is thought that various products such as functional foods can be developed.

(2) Determination of alpha-amylase inhibitory activity

The alpha-amylase inhibitory activity of the extracts of the pulsed seed was used as pancreatic alpha-amylase (α-amylase, Sigma A 3176; 100U) in pigs, and 0.1M containing 2g / L BSA and 0.2g NaN ₃ / L. nitrophenyl-alpha-D-phosphate buffer (pH 7.0) 5mM para as, the substrate was used to dissolve so that 0.7U / mL in the malto hexanoate side (p -nitrophenyl-α-D- maltohexanoside, Sigma 73681) 0.1 It was dissolved in M phosphate buffer (pH 7.0) and used. Enzyme reaction time was adjusted to 20 minutes and enzyme activity was measured in the same manner as the alpha-glucosidase activity measurement method.

After extracting the ginmundong seed extract in dimethylsulfoxide (DMSO) solvent at different concentrations (50, 200, 500 ㎍ / mL), the inhibitory activity of the alpha-amylase enzyme of the pig pancreas was measured by the above method. The results are shown in FIGS. 2 and 3, respectively.

According to FIGS. 2 and 3, the inhibitory activity of the alpha-amylase enzyme of the extract of Macmundong seed was measured to be 14.9% when treated at a level of 200 μg / mL, and 50 μg for the acarbose positive control group. Inhibitory rate of 100% at / mL was 8% at the same concentration in extracts of Megmundong seeds, while the rate of inhibition was 11% and 15% as the concentration was increased to 100, 500㎍ / mL. It was found that the inhibition rate was increased in a concentration dependent manner.

According to the results of the recent search for a plant material that has an inhibitory activity of the alpha-amylase enzyme related to carbohydrate digestion from a plant natural resource including 300 kinds of agricultural products, only 24 kinds of extracts by site of all 300 plant natural resources are alpha-amylase. Enzyme inhibitory activity was shown, and the same method and extraction concentration (200㎍ / mL) as the method used in the present invention showed that -56%, Lichen -37%, and Cinnamon bean belonged to the same family Liliaceae, such as Macmundong. -30%, licorice seeds -22%, snow horse riding 13%, pearweed fragrance 14%, hyangbu seeds 11%, etc. In addition, oak tree, clear bamboo mugwort, moth, etc. showed less than 10% inhibition rate Reported.

The alpha-amylase enzyme inhibitory activity of the extracts of the pulsed seed was reported to indicate that the 60% ethanol extract of Rhododendron, which is being developed as a weight control food, exhibited 39.5% alpha-amylase enzyme inhibitory activity at the level of 200 µg / mL. Compared to other plant natural resources, alpha-amylase enzyme inhibitory activity was found to be relatively low or low compared to the low level. It is thought that it can be actively used as an anti-obesity material.

In addition, comparing the alpha-amylase enzyme inhibitory activity of the extracts of the organolpi fruit extract and makmundong seed extracts containing various polyphenols such as anthocyanins and flavonoids, the alpha-amylase enzyme inhibitory activity of the extracts of the ganmundong seed extract was higher than that of the organolpi fruit extract at all concentrations. It showed at least twofold activity. Kim et al. (2000) found that some flavonoids, such as luteolin and their glycosides, amentoflavones, daidzein and genistein, are alpha-glucosidase and alpha-amylase. Phenolic compounds and flavonoids containing many anthocyanins were included in the pulsed seeds, as reported by high inhibitory activity against anthocyanin and ancylate added acylate. It is thought that alpha-glucosidase inhibitory activity is exhibited through these.

(3) Lipase inhibitory activity measurement

Lipase inhibitory activity was assessed using porcine pancreatic enzyme and methylmethylbelliferyl oleate (4-MU oleate) as substrate. In other words, 100 μl (0.1mM) 4-MU oleate, 60 μl McIvavaline buffer (0.1 M citrate-Na ₂ HPO ₄ , pH 7.4) and 10 μl sample solution were mixed in a total of 200 μl of reaction solution, and pancreatic lipase 30 The reaction was initiated by adding [mu] l (0.03 U). After reacting at 37 ° C. for 15 minutes, the reaction was terminated by adding 1 mL of 0.1N HCl solution and 2 mL 0.1M sodium citrate. 4-methylumbelliferone liberated by lipase was measured on a fluorophotometer (Ex 320 / Em450nm). Lipase inhibitory activity was calculated by the following formula.

% Inhibition = (A-B) / A × 100

A: Fluorescence intensity when no sample is added

B: Fluorescence intensity when sample is added

The results of assaying the inhibitory activity of the lipase enzymes involved in fat metabolism of the extracts of Macmundong seeds are shown in FIG. 4. As shown in FIG. 4, orlistat, a positive control group, showed high activity of 68.4% at a low concentration of 1 μg / mL, but showed a low activity of 4% in the case of Macmundong seed extract, orlistat at a level of 500 μg / mL. Was 97.8%, and the extract of Macmundong seeds was 51.8%, and the lipase enzyme inhibitory activity was rapidly increased as the concentration was increased. The above results can be predicted that the extract of Macmundong seed may have an effect on triglyceride metabolism, and the major enzyme that extracts triglyceride into 2-monoacylglycerol and fatty acid (key enzyme) (Bitou et al . , 1999), by inhibiting the enzyme of pancreatic lipase, it can be predicted to reduce the fat digestion and have an anti-obesity effect.

Example  4. Isolation and Structure Identification of Seed Containing Ingredients

(1) Extraction and Separation Conditions of Pigments

100 g of wormwood seeds were immersed in 3 L of a solution containing 1% HCl-20% ethanol, and extracted by standing at 4 ° C for 48 hours. The extract was filtered through an Advantec Toyo No. 2 filter paper, the residue was extracted three times, and the extracts were combined and concentrated in a 30 ° C. rotary evaporator. The concentrated Macmundong seed extract was frozen at −70 ° C. and then lyophilized to be used as a sample. Concentrated Macmundong seed extract was 250 × 9.4 mm i.d. Fractionation was carried out using semipreparative HPLC equipped with a Zorbax SB-C18 column (Agilent Technologies, Wilmington, DE), with the detector having a wavelength of 520 nm and a column temperature of 30 ° C. Was carried out.

The fractionation condition of semi-preparative HPLC was controlled by gradient elution of two solvents. First, solvent A was made of ultrapure distilled water containing 5% formic acid, and solvent B contained 5% formic acid. It was made into one acetonitrile. The flow rate of the fractionation solvent was adjusted to 3mL per minute, and fractionation was carried out under the following solvent conditions.

Solvent conditions are 10-18% B solvents for 0-10 minutes, 18-28% B solvents for 10-18 minutes, 28-40% B solvents for 18-19 minutes, 40% B solvents for 19-21 minutes, 21 40-10% B solvent by -23 minutes and 10% B solvent by 23-25 minutes were used.

Sample injection volume was adjusted to 2mL, and a total of four major anthocyanins were isolated from the extracts of Macmundong seeds. The relative purity of the four major anthocyanins isolated was 150 × 4.6 mm i.d. Purity was determined using RP-HPLC using a TSK gel ODS-120T column (Supelco Inc, Bellefonte, PA), and all were found to have a purity of 98% or higher.

Analytical conditions of RP-HPLC were adjusted to a flow rate of 0.7 mL / min and analyzed under the same gradient illumination conditions as the solvent of semi-preparative HPLC described above. The detector was adjusted to a wavelength of 200 to 700nm by the photodiode array detection method, the sample injection amount was adjusted to 20μl. During injection, the analytical sample was filtered using a 0.45 μm membrane filter, and a Nova-Pak C ₁₈ guard insert column (Waters, Milford, MA) was used to protect the analytical column. Analysis was performed.

(2) analyzer and condition

¹ H-NMR (500 MHz) and ¹³ C NMR (125 MHz) spectra were measured using Varian Unity Plus 500 NMR, and 0.5% DCl-CD ₃ OD containing TMS as an internal standard was measured with a solvent. FAB-MS was measured using NBA (m-NO ₂ -Benzyl-OH) as a matrix, and molecular weight was measured using a Jeol JIMS 700 High Resolution Mass Spectrometer. LC / ES-MS analysis was performed by combining a Thermo Finnigan AQA single-quadrupole mass spectrometer with a Spectra system P-4000 HPLC system to measure molecular weight on-line.

Anthocyanin pigments in pulsed seeds were collected using a semipreparative HPLC system consisting of a Spectra system P-4000 pump, a Spectra system UV-1000 UV-Vis variable wavelength detector, and a Hitachi D-2500 integrator. Rheodyne 7725i injector with sample loop was used.

Purity assays and analytical HPLC systems were performed using an Agilent 1100 HPLC system equipped with a G1315B Agilent diode array detector, and HPLC instrumentation and data analysis were performed using the G2180AA Agilent ChemStation for LC 3D Spectral SW Module (version A.08.01).

(3) Analysis result

A total of seven anthocyanins were separated as a result of separating anthocyanin pigments from Macmundong seeds using semipreparative HPLC [FIG. 5]. A total of seven pigment peaks were identified in the extract of Macmundong seed through HPLC, and the UV-Vis and MS data and chemical structures of the seven pigment components separated are shown in Table 3, among which compounds 1 and 2 were high in content. , 5 and 7 were identified.

Compound HPLC
retention (min) UV-Vis. MS [M ⁺ ] Chemical
formular One  17.8 278, 526  465, 303 C ₂₁ H ₂₁ O ₁₂ ⁺ 2  18.3 278, 530  611, 303, 465 C ₂₇ H ₃₁ O ₁₆ ⁺ 3  20.2 280, 527  449, 287 C ₂₁ H ₂₁ O ₁₁ ⁺ 4  20.4 278, 527  479, 317 C ₂₂ H ₂₃ O ₁₂ ⁺ 5  20.8 278, 529  625, 317, 479 C ₂₈ H ₃₃ O ₁₆ ⁺ 6  22.7 278, 531  493, 331 C ₂₃ H ₂₅ O ₁₂ ⁺ 7  23.6 282, 533  639, 331, 493 C ₂₉ H ₃₅ O ₁₆ ⁺

1) Identification of the Structure of Compound 1

Compound (compound) 1 was determined as NMR, LC / ES-MS, FAB-MS, UV / Vis spectral data analysis to Delphinidin-3-glucoside.

1 of Compound 1 showing 1H singlet at δ 8.95 (H-4), 2H singlet at δ 7.76 (H-2 ′, 6 ′), 2H AX system at δ 6.65 (H-6) and 6.87 (H-8) The aromatic region in the dimension ¹ H-NMR spectrum coincided with the delphinidin nucleus.

^{In the 13} C spin echo Fourier transform NMR spectrum, the spectral region between δ 60 and 80 has five resonances with an anmeric carbon that matches one hexose.

¹ H- ¹ H pairing of sugar ring protons (7-10 Hz) revealed by ¹ H and ¹³ C shift values in one bond heteronuclear shift correlation NMR and iterative spin simulation (PANIC) Coupling constants correspond to beta-di-glucopyranoside. The binding site of sugar was identified as position 3 of aglycone through the long range correlation peak between anmeric proton and aglycone C-3 in HMBC. In addition, the UV-Vis spectrum of Compound 1 obtained by ON-LINE HPLC shows an absorption maximum at 526 nm, which is consistent with 3-glycosides with delphinidin nuclei.

The LC / ES-MS and FAB-MS spectra of Compound 1 obtained [M ⁺ ] consistent with delphinidin hexose at m / z 465, and a base peak at m / z 303 indicating the presence of delphinidin aglycone.

The NMR data of Compound 1 is consistent with the literature values of delphinidin-3-glucoside.

2) Identification of Structure of Compound 2

Compound 2 showed λmax at 278 and 530 nm as a result of UV-Vis measurement in 0.5% HCl-MeOH solution, and obtained [M ⁺ ] at m / z 611 in FAB-MS. This shows the molecular formula of C ₂₇ H ₃₁ O ₁₆ ⁺ with a fragment consistent with delphinidin-3-glucoside (m / z 465), delphinidin (m / z 303).

The proton and carbon signal of delphinidin, an aglycone of Compound 2, was expressed using information on pairing constants and chemical shifts. Hexose was identified by one-dimensional ¹ H NMR spectrum and ¹ H- ¹ H COSY.

All pairing constants adjacent to sugars appeared at 7.8-9.5 Hz with anomer protons at δ 5.32 (d, J = 7.8 Hz). Thus the sugar is beta-di-glucopyranoside. Irradiation of glucosyl (δ 5.32) H-1 "observed strong negative NOE in the nucleus H-4 (δ 8.87), which linked the sugar moiety to 3-OH of delphinidin. Also, a weak negative NOE between H-6 of glucosyl (δ 4.06; 3.59) and H-1 ″ of rhamnosyl (δ 4.65) indicates a 1 → 6 linkage. . The spectral data of Compound 2 is consistent with the data shown in the literature of delphinidin-3-rutinosides.

3) Identification of the structure of compound 5

The UV-Vis spectrum of Compound 5 obtained by on-line HPLC showed an absorption maximum wavelength at 529 nm. The NMR spectrum of Compound 5 was shown to be much similar to that of Compound 2. Proton resonance analysis showed that Compound 5 was consistent with petunidin as an asymmetric anthocyanidin B-ring with one methoxy group (δ 3.92) and two hydroxyl groups. .

Cross peak at 5.34 / 145.1 (H-1 ″ / C-3) of the HMBC spectrum confirmed binding to 3-OH of glucoserk aglycone. ¹³ C of Compound 5 A downfield shift of C-6 "(67.7 ppm) in the spectrum indicates that 3-glucose and rhamnose are combined at 6" -OH. 4.70 / 67.7 (H-1 '"/ C of the HMBC spectrum) -6 "), cross peaks at 4.10 / 102.2 (H-6" a / C-1 '"), and 3.59 / 102.2 (H-6" b / C-1'"). (inner glucose) can be seen that the bond is made in 6 "-OH.

Thus compound 5 was identified as petunidine-3-rutinoside.

4) Structure Identification of Compound 7

[M ⁺ ] of compound 7 was 14 mass units larger than compound 5. ¹ H-NMR spectra show that the B-ring has two equivalent aromatic protons (δ 7.88) and two methoxy groups (δ 3.98), thus the aglycone of compound 7 Vidin. ¹ H and ¹³ C-NMR spectrum of the sugar residue can be made substantially equal to the compound 2, 5, was confirmed by ^{1 H- 1 H COSY, HMBC,} NOE spectrum.

UV-Vis spectra showed λ max at 282 nm and 533 nm in 0.5% HCl-MeOH solution and [M ⁺ ] at m / z 639 in FAB-MS. The molecular formula of C ₂₉ H ₃₅ O ₁₆ ⁺ was obtained with fragments consistent with malvidin-3-glucoside (m / z 493) and malvidin (m / z 331).

Thus, Compound 7 was confirmed to be malvidin-3-rutinoside.

To summarize the above results, Compound 1 is isolated from delphinidin 3-glucoside, Compound 2 is delphinidin 3-rutinoside, Compound 5 is petunidine 3 Petunidin 3-rutinoside, chemical 7 was identified as malvidin 3-rutinoside. The structurally identified anthocyanins are all known substances [Formula 1 and Table 4].

[Formula 1]

No. compound R ₁ R ₂ R ₃ R ₄ One  Delphinidin 3-glucoside OH OH  O-β-glucoside OH 2  Delphinidin 3-rutinoside OH OH  O-β-rutinoside OH 3  Cyanidin 3-glucoside OH H  O-β-glucoside OH 4  Petunidin 3-glucoside OCH ₃ OH  O-β-glucoside OH 5  Petunidin 3-rutinoside OCH ₃ OH  O-β-rutinoside OH 6  Malvidin 3-glucoside OCH ₃ OCH ₃  O-β-glucoside OH 7  Malvidin 3-rutinoside OCH ₃ OCH ₃  O-β-rutinoside OH

Formulation example .

1) Preparation of Syrup

Syrup containing the ganmundong seed extract according to the present invention as an active ingredient was prepared by the following method.

The mammundong seed extract (2% by weight), saccharin and sugar were dissolved in 80 g of warm water and cooled, and a solution consisting of glycerin, sweetener, flavoring agent, ethanol, sorbic acid and distilled water was prepared and mixed, and water was added thereto. Add to 100 mL.

2) Preparation of Tablets

Tablets containing the ganmundong seed extract according to the present invention as an active ingredient were prepared by the following method [active ingredient 15mg].

250 g of the pulsed seed extract 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 mg of the pulse extract seeds in an appropriate amount of water, a suitable amount of vitamin C, citric acid, sodium citrate, oligosaccharides were added, and an appropriate amount of sodium benzoate was added as a preservative to add 100 ml of water to prepare a beverage composition.

4) Manufacture of health food

Health foods containing the extract of Mengmundong seed according to the invention as an active ingredient was prepared by the following method.

1,000 mg of ginmundong seed extract, suitable amount of vitamin mixture, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg, biotin 10 μg, Nicotinic acid amide 1.7 mg, folic acid 50 ㎍, calcium pantothenate 0.5 mg, mineral mixture appropriate amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, dicalcium phosphate 55 mg, citric acid 90 mg of potassium, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride 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.

In Figure 5, 1 is delphinidin 3-glucoside, 2 is delphinidin 3-rutinoside, 5 is petunidin 3-rutinoside, 7 is Malvidin 3-rutinoside.

Claims (4)

Delphinidine 3-glucoside, delphinidin 3-lutinoside, cyanidin 3-glucoside, petunidine 3-glucoside, petunidine 3-lutinoside, malvidin 3-glucoside represented by Formula 1 And an anthocyanin-based pigment of malvidin 3-lutinoside as an active ingredient:
[Chemical Formula 1]

In Formula 1, R ₁ is OH or OCH ₃ , R ₂ is H, OH or OCH ₃ , R ₃ is O-beta-glucoside or O-beta-lutinoside (O -β-rutinoside) and R ₄ is OH.
delete The method according to claim 1,
The pulsed seed extract is characterized in that the anti-obesity composition characterized in that extracted by a solvent selected from water, lower alcohol having 1 to 4 carbon atoms or mixtures thereof.
Delphinidine 3-glucoside, delphinidin 3-lutinoside, cyanidin 3-glucoside, petunidine 3-glucoside, petunidine 3-lutinoside, malvidin 3-glucoside represented by Formula 1 And an anti-obesity health food comprising an anthocyanin pigment of malvidin 3-rutinoside as an active ingredient:
[Chemical Formula 1]

In Formula 1, R ₁ is OH or OCH ₃ , R ₂ is H, OH or OCH ₃ , R ₃ is O-beta-glucoside or O-beta-lutinoside (O -β-rutinoside) and R ₄ is OH.

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