KR20080109243A - Ishigeaceae extracts having anti-diabetes activity - Google Patents

Abstract

The extracts of the Ishigeaceae plant showing the antidiabetic activity is provided. A pharmaceutical composition comprising the extracts of the Ishigeaceae plant is provided to improve anti-diabetic and anti-oxidative activities. The Ishigeaceae plant extract for prevention and improvement of diabetes is obtained from the Ishigeaceae plant, and is prepared by washing Ishige sinicola; dipping the washed Ishige sinicola in 70% ethanol and agitating it at room temperature for 2-3 days; filtering the leached product of Ishige sinicola to prepare the crude extract; and sequentially fractioning the crude extract with n-hexane, ethylacetate, and butanol to obtain fractions.

Description

Extract of the brachial plant showing antidiabetic activity {ISHIGEACEAE EXTRACTS HAVING ANTI-DIABETES ACTIVITY}

Figure 1 is a process for producing a single plant separated from the defoliant extract of the present invention

Figure 2 is a Prep-HPLC analysis chromatogram of the ethyl acetate layer of shell extract

Figure 3 HPLC chromatogram of crude extract of the paddle

Figure 4 HPLC chromatogram of the ethyl acetate layer of shell extract

Figure 5 HPLC chromatogram of Peak 1 isolated from shell extract

Figure 6 HPLC chromatogram of Peak 2 isolated from shell extract

Figure 7 LC-MS-MS spectrum of Peak 1 isolated from shell extract

8 is a ¹ H-NMR spectrum of Peak 1 isolated from the shell extract

FIG. 9 shows ¹³ C-NMR spectra of Peak 1 isolated from the extracts of the perennial plants.

10 is a molecular structure of Peak 1 isolated from the shell extract

11 is a graph showing the results of alpha-glucosidase inhibitory effect on the extracts

A: broad leaf extract, B: shell extract

12 is a graph showing the results of the alpha-glucosidase inhibitory effect on the shell extract and Peak 1, Peak 2 separated therefrom

Figure 13 is a graph showing the results of DPPH radical scavenging activity for the extract

A: fraction of broad-leaf extract, B: fraction of shell extract

14 is a graph showing the results of DPPH radical scavenging activity against the shell extract and Peak 1, Peak 2 separated therefrom

15 is a graph showing the NO production inhibitory activity results for the extracts

A: fraction of broad-leaf extract, B: fraction of shell extract

16 is a graph showing the NO production inhibitory activity results for the shell extract and Peak 1, Peak 2 separated therefrom

17 is a graph showing the results of xanthine oxidase inhibitory activity against the extracts of the plants

A: fraction of broad-leaf extract, B: fraction of shell extract

18 is a graph showing the results of xanthine oxidase inhibitory activity against the shell extract and Peak 1, Peak 2 separated therefrom

19 is a graph showing the results of peroxide scavenging activity for the extract of the Perennial plant

A: fraction of broad-leaf extract, B: fraction of shell extract

20 is a graph showing the results of peroxide scavenging activity for the shell extract and Peak 1, Peak 2 separated therefrom

The present invention relates to extracts of the defoliated plants showing antidiabetic activity.

Paegwa (Ishigeaceae) plant is a plant belonging to galjo galjo plant door (phacophyta), Num of malmok (Chordariales), the representative species is L (Ishige okamurae Yendo and Ishige sinicola (Setchell et Gardner) Chihara).

L ( Ishige okamurae Yendo) It is distributed throughout the southern coast, Jeju Island (each coast), and Japan, and it looks like a hard twig in morphological features, dark brown, and black when dried, and the inner structure of the filamentous tangled filamentous cells. The outer layer is composed of small cells with heat and perpendicular to the surface of the body.

Widespread ( Ishige sinicola (Setchell et Gardner) Chihara) is distributed all over the southern coast, Jeju Island (each region), Japan and China. Its morphological features are foliate, blackish brown, and bubble-like areas are yellowish brown and black when dried. They live in colonies on the middle rocks of the intertidal zone where the waves are quiet and are used for food.

In Korean Patent Application Publication No. 2003-0015536 (Whitening Cosmetic Containing Flask Extract), an extraction solvent is added to the dried and pulverized slabs, and the extract is obtained by distillation, and whitening containing the broadleaf extract obtained by concentrating through distillation under reduced pressure. The present invention relates to cosmetics, and to extracts that have an inhibitory effect on tyrosinase activity and are effective for whitening.

In addition, Korean Patent Publication No. 10-0386417 (environmentally friendly antifouling agent using an extract of seaweed), using the extract of the broad shell shows a strong antifouling activity against marine adherent organisms such as spiny seaweed and mussels, causing problems such as destruction of the ecosystem. It is disclosed that the broad leaf extract that can replace the tributyl tin having a.

However, no research has been conducted on more diverse physiological activities other than the degree of research on neurotoxicity inhibition, antifouling activity, and whitening effect of the prior art.

Diabetes mellitus is a series of metabolic disorders characterized by hyperglycemia, a chronic degenerative disease caused by a lack of insulin, a hormone secreted from the pancreas, or by insulin resistance.

Insulin is a hormone secreted by β-cells of the pancreas when blood sugar is increased, which induces glucose in the blood into cells of the human body.The cells use glucose to produce the necessary energy or to convert glucose into other substances (glycogen). Promotes the reaction to store, keeping the blood sugar level normal.

If something goes wrong with insulin, cells can't produce the energy they need, and blood sugar goes up.

Currently, oral hypoglycemic agents among the therapeutic agents of diabetes have side effects, so recent studies on the treatment of diabetes have been trying to use hypoglycemic substances using natural products.

There is a high possibility of finding new active ingredients from traditional medicine, natural plants, or ingredients that have been used for edible use, and has been used for a long time, so there is little concern about toxicity due to developed drugs.

These natural plants have not been found to have a distinctly active ingredient, but because various components act in combination to strongly enhance the immune or therapeutic functions for the survival of the human body, thereby enabling the treatment or prevention of diseases. It has been taken as a herbal medicine since ancient times, and researches for using it in various ways, such as functional foods and food additives, are being actively conducted.

Seaweeds have been studied because they contain high-molecular sugars, low-molecular sugars, pigments, and various bioactive substances of carbohydrates having various structures.

Extracts of seaweeds have been developed and utilized to prevent crystal formation and emulsion stabilization. They are also used as tissue-improving agents for hardening jelly, preventing food drying, and fish paste.

In recent years, attempts have been made to commercialize the seaweed, seaweed, maize, ecstasy, and fern, which are distributed on the coast of Jeju.

In particular, fucoidan isolated from brown algae such as seaweed and kelp is a sulfated polysaccharide combined with sugar, uronic acid, and H ₂ SO ₂ , and is known to have anti-cancer effects, lower blood cholesterol, and boost immunity. It is widely regarded as a very valuable material such as functional drinks, health foods and medicines.

Recently, the interest in brown algae has been increased, so research and development on brown algae has been actively conducted.

Meanwhile, Korean Patent Publication No. 10-0527094 (a composition for reducing neurotoxicity caused by beta-amyloid) includes a beta-amyloid containing as an active ingredient at least one seaweed extract selected from the group consisting of Ecklonia spp. Disclosed is a composition for inhibiting neurotoxicity.

However, research on the physiological activity of the defoliated plants is lacking, so more active research is needed.

In order to solve the above problems, an object of the present invention is to provide an antidiabetic extract having excellent antidiabetic effect and a substance separated therefrom.

In addition, an object of the present invention is to provide an antidiabetic composition comprising the extract of the perennial plant as an active ingredient, and to provide an antioxidant composition.

In addition, an object of the present invention is to provide a beverage composition and food additive to which the anti-diabetic and anti-diabetic extract of the present invention is added.

The present invention relates to extracts of the defoliated plants showing antidiabetic activity.

The extract of the present invention, prepared by selecting one of the plaques and broad leafs, washed and then immersed in 70% ethanol and leached by stirring at room temperature for 2 to 3 days, then filtered through a filter to prepare a crude extract In addition, the crude extracts were composed of fractions using a different polar solvent, n-hexane, ethyl acetate and butanol, sequentially to prepare a fraction.

In addition, the present invention consists of preparing an antidiabetic composition and an antioxidant composition, the above-mentioned patio plant extract as an active ingredient.

In addition, the present invention consists of preparing an anti-diabetic composition and an antioxidant composition containing Diphlorethohydroxycarmalol (Diphlorethohydroxycarmalol) isolated from the extract of the defoliant as an active ingredient.

The inventors of the present invention have been found to have excellent anti-diabetic activity in shellfish algae, such as shellfish and broadleaf shells, belonging to brown algae, while conducting research on seaweeds having excellent antidiabetic effect.

Alpha-glucosidase is an enzyme that breaks down disaccharides into monosaccharides and has been reported to increase post-prandial blood sugar in the small intestine mucosa.

Therefore, anti-diabetic experiments were performed on the extracts of the present invention through the alpha-glucosidase inhibitory effect test.

As a result, α-glucosiase inhibitory effect on the broad-leaf extract of Example 1 of the present invention was crude extract (IC ₅₀ ; 0.1103 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.0815 ㎍ / ㎖), butanol fraction (IC ₅₀ 0.0836 μg / ml) and hexane fractions (IC ₅₀ ; 0.5182 μg / ml) showed very high inhibitory effects and water fractions (IC ₅₀ ; 3.7915 μg / ml) (Table 5, FIG. 11A).

In addition, α-glucosiase inhibitory effect on the shell extract of Example 2 of the present invention is crude extract (IC ₅₀ ; 0.8117 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.0480 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 0.0683 μg / ml) and hexane fractions (IC ₅₀ ; 0.2921 μg / ml) showed very high inhibitory effects and water fractions (IC ₅₀ ; 8.5007 μg / ml) (Table 5, FIG. 11B).

On the other hand, the inhibitory effect of the already commercialized acarbose, the IC ₅₀ value of 242.08 ㎍ / ㎖, it was found that the extract of the present invention showed a very excellent effect compared to the α-glucosiase inhibitory effect of acarbose.

Therefore, it was found that the anti-diabetic effect of the extract of the present invention is significantly superior antidiabetic effect.

In addition, as a result of the α-glucosidase inhibitory effect on the diphloorthohydroxycarmalol and Peak 2 of the extract of the present invention and the single substance Peak 1 separated therefrom, P1 is IC ₅₀ ; 0.63 μg / ml, P2 is IC ₅₀ ; 0.093 μg / ml, shell extract was IC ₅₀ ; 0.812 μg / mL, the ethyl acetate fractions were IC ₅₀ ; 0.048 μg / ml, the control acarbose is IC ₅₀ ; It was found to be 242.08 μg / ml (FIG. 12).

Therefore, it was found that the single substance isolated from the extract of the perennial plant of the present invention also has an excellent antidiabetic effect.

In addition, the antioxidant effect was tested for the extract of the present invention.

First, as a result of DPPH radical scavenging activity experiment, commercialized ascorbic acid (IC ₅₀ ; 8.23 ㎍) showed a high active radical inhibitory effect.

Crude extract, water fraction, and hexane fraction of the broad leaf of the present invention showed a low effect (IC ₅₀ ;> 400 μg / ml), but ethyl acetate fraction (IC ₅₀ ; 111.09 μg / ml), butanol fraction (IC ₅₀ ; 178.17). ㎍ / ㎖) layer showed more than twice the active oxygen inhibitory effect than the crude extract (Fig. 13A).

In addition, crude extract of the present invention had a low active oxygen inhibitory effect, ethyl acetate fraction (IC ₅₀ ; 40.44 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 74.21 ㎍ / ㎖) is crude extract ((IC ₅₀ ; > 400 [mu] g / ml) showed higher free radical inhibition effect (FIG. 13B).

In addition, the active oxygen inhibitory effect of the peak 1 and peak 2 fractions as a single substance was Peak 1 (IC ₅₀ ; 100 ㎍ / mL) and Peak 2 (IC ₅₀ ; 200 ㎍ / mL), with Peak 1 being somewhat higher (Fig. 14).

In addition, as a result of the NO production inhibitory activity test for the extract of the present invention, crude crude extract and water fraction (IC ₅₀ ; 591.14) of the broad leaf as shown in Table 5, the inhibition rate was very low, but ethyl acetate fraction ( IC ₅₀ ; 82.59 μg / ml) and butanol fraction (IC ₅₀ ; 147.08 μg / ml) showed high inhibitory effect (FIG. 15A).

In addition, the shell extract of the present invention showed a low inhibitory effect in all samples except hexane fraction (IC ₅₀ ;> 192.58 ㎍ / ㎖) as shown in Figure 15B (IC ₅₀ ;> 300 ㎍ / ㎖).

In addition, the NO scavenging effect of the peak 1, Peak 2 fraction as a single substance is IC ₅₀ ; > 400 μg / ml), the NO scavenging power appeared somewhat lower (FIG. 16).

In addition, as a result of the xanthine oxidase inhibitory activity test on the extract of the present invention, xanthine oxidase inhibitory activity against the broad leaf extract of the present invention is crude extract (IC ₅₀ ; 20.63 ㎍ / ㎖), ethyl acetate fraction (IC _{50; <10.42 ㎍ / ㎖)} , butanol fraction _{(IC 50; 7.21 ㎍ / ㎖} ), the hexane fraction _{(IC 50; 58.77 ㎍ / ㎖} ), the water fraction (IC _50:> as 314.97 ㎍ / ㎖), ethyl acetate layer It showed high activity in the butanol layer (FIG. 17A).

Xanthine oxidase inhibitory activity against the shell extract of the present invention is crude extract (IC ₅₀ ; 187.47 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.71 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 22.2 ㎍ / ㎖), Xanthine oxidase inhibitory activity as hexane fraction (IC ₅₀ ; 26.56 ㎍ / ml) and water fraction (IC ₅₀ :> 400 ㎍ / ml) showed high activity in ethyl acetate fraction and butanol fraction (FIG. 17B).

In addition, P1. A single substance isolated from the shell extract of the present invention. The inhibitory activity of x2 was shown to be slightly higher as P1 (IC ₅₀ ; 10 μg / ml) and P2 (IC ₅₀ ; 30 μg / ml) (FIG. 18).

In addition, the peroxide scavenging activity of the broad-leaf extract of the present invention is crude extract (IC ₅₀ 37.7 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 19.95 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 37.91 ㎍ / ㎖), hexane Fractions (IC50; 78.07 μg / ml) and water fractions (IC50; 210.82 μg / ml) showed high effects in the ethyl acetate fractions (FIG. 19A).

Peroxide scavenging activity of the shell extract of the present invention was crude extract (IC ₅₀ ; 81.5 ㎍ / mL), ethyl acetate fraction (IC ₅₀ ; 11.24 ㎍ / mL), butanol fraction (IC ₅₀ ; 17.65 ㎍ / mL), hexane fraction (IC50; 70.80 μg / ml) and water fraction (IC50; 114.95 μg / ml) showed high effect in the ethyl acetate fraction (FIG. 19B).

In addition, P1. A single substance isolated from the shell extract of the present invention. The peroxide scavenging activity of P2 was P1 (IC ₅₀ ; 8 μg / ml) and P2 (IC ₅₀ ; 35 μg / ml), which was somewhat higher than P1 (FIG. 20).

Therefore, it was found that the extract of the perennial plant of the present invention is excellent in antioxidant activity.

On the other hand, the peak 1 separated from the extract of the present invention was subjected to mass measurement using LC-MS-MS, and the result of analysis confirmed that the molecular weight was 512 (Fig. 7).

In addition, as a result of ¹ H-NMR and ¹³ C-NMR analysis through nuclear magnetic resonance (NMR) analysis, Peak 1 of the extract-derived single substance of the present invention was obtained as follows.

The molecular name is Diphlorethohydroxycarmalol, molecular weight is 512.383, molecular formula is C ₂₄ H ₁₆ O ₁₃ , and the composition ratio is C 56.26%; H 3.15%; It was confirmed that the material is O 40.59%.

Difluoroethoxycarmarol is known as a substance having HIV-1 reverse transcriptase inhibitory activity.

On the other hand, the defoliant extract of the present invention and the substances separated therefrom can be used in various ways as food additives, beverage compositions and the like.

In addition, the present invention can prepare an antidiabetic composition and an antioxidant composition comprising as an active ingredient the defoliant extract and the substances separated therefrom exhibiting the physiological activity as described above.

At this time, the composition of the present invention may be prepared in various forms such as pills, capsules, granules, suspensions.

On the other hand, when preparing a composition containing the extract of the present invention and a single substance separated therefrom as an active ingredient, the amount of the addition can be variously adjusted according to the use and form of the composition.

Hereinafter, the brachial extract showing the anti-diabetic effect of the present invention will be described in more detail through Examples and Experimental Examples, but these are not intended to limit the scope of the present invention.

Example 1 Preparation of Broadleaf Extract

The strip was collected from the intertidal zone on the eastern coast of Jeju Island, and then dried and prepared.

5 g of 70% ethanol was added to 500 g of the prepared plaque and leached at room temperature for 2 to 3 days.

The leached solution was concentrated under reduced pressure after filtration.

After freezing the concentrate in DEEP FREEZER using a freeze dryer to remove the residual solvent to obtain a crude extract.

Then, 50 g of the crude extract was suspended in distilled water, and fractionated sequentially using a nonpolar solvent with hexane, ethyl acetate, and butanol in a separatory funnel.

The fractions were filtered, concentrated under reduced pressure, and freeze-dried to obtain 29.7 g of water fraction, 7.1 g of butanol fraction, 9.7 g of ethyl acetate fraction, and 3.5 g of hexane fraction.

Example 2 Preparation of Shell Extract

To prepare an extract in the same manner as in Example 1 of the present invention, a paddle extract was prepared using the paddle.

50 g of the crude extract was suspended in distilled water, and fractionated sequentially using a nonpolar solvent with hexane, ethyl acetate, and butanol in a separatory funnel.

The fractions were filtered, concentrated under reduced pressure, and lyophilized to obtain 14.5 g of water fraction, 15.5 g of butanol fraction, 14 g of ethyl acetate fraction, and 7.5 g of hexane fraction.

Example 3 Separation of Shell Extract-derived Single Material

The ethyl acetate fraction of the pad prepared in Example 2 of the present invention was prepared.

The prepared fractions were dissolved in methanol for HPLC and then passed through a 0.45 μm sample filter to isolate using stabilized HPLC.

The column was a Waters Co. Model 2695 alliance equipped with Nova-Pak silica 8 μm (7.8 × 300 mm), and a mobile phase solvent degassed by mixing chloroform and methanol in a ratio of 95: 5. The flow was analyzed at a flow rate of 2 ml / min.

The UV absorption pattern of the material was observed with a Waters 2996 UV detector (UV Detecter) and the wavelength was measured at 254 nm.

The HPLC instrument conditions are shown in Table 1, and the gradient elution conditions of the mobile phase are shown in Table 2.

As a result, it was confirmed that Peak 1 appeared in 23.962 squad and Peak 2 in 38.579 squad (FIG. 2), and these materials were separated.

Experimental Example 1 Analysis of a Single Substance Isolated from the Extract of the Plants

1) HPLC assay

The crude extract prepared in Example 2 and each fraction were prepared.

In addition, the single material Peak 1 and Peak 2 separated in Example 3 were prepared.

The prepared sample was used by HPLC (Alliance2695) of Water, and the eluent was analyzed by gradient elution using water (5% acetic acid) and acetonitrile (5% acetic acid) to confirm purity.

At this time, HPLC instrument conditions are shown in Table 3, and the gradient elution conditions of the mobile phase are shown in Table 4.

<Table 3> HPLC Apparatus Conditions for Extraction Analysis of Shelled Plants

HPLC Waters Alliance 2695 system Column Sunfire ^TM C18 5μm (4.6 × 250mm) Detecter Photodiode Array Detector Flow rate 1.0 ml / min Injection Volume 10 μl Column temperature 25 ℃ Sample Temperature 10 ℃

TABLE 4 Mobile phase conditions for HPLC gradient elution

Program order Time Flow H2O Acetonitrile Curve One 0.00 1.00 95.0 5.0 2 5.00 1.00 95.0 5.0 6 3 10.00 1.00 90.0 10.0 6 4 30.00 1.00 80.0 20.0 6 5 40.00 1.00 80.0 20.0 6 6 50.00 1.00 70.0 30.0 6 7 60.00 1.00 70.0 30.0 6 8 70.00 1.00 50.0 50.0 6 9 80.00 1.00 50.0 50.0 6 10 81.00 1.00 0.0 100.0 6 11 90.00 1.00 0.0 100.0 6 12 91.00 1.00 95.0 5.0 6 13 100.00 1.00 95.0 5.0 6

2) Structural analysis experiment on a single substance

To check the molecular weight, purely isolated Peak 1 It was dissolved in methanol for HPLC.

Peak 1 dissolved in methanol was passed through a 0.2 μm sample filter, followed by mass measurement using LC-MS-MS stabilized at a concentration of 1 mg / mL.

As a result of negative analysis, 512 molecular weights were obtained, and the results are shown in FIG. 7 (ESI-MS, m / z 511 [M-H]).

In addition, nuclear magnetic resonance (NMR) analysis was completely dried 6 mg pure purified compound dissolved in DMSO (0.6mL) for NMR and injected into a 5mm NMR tube using FT-NMR as a Giol model model (JMX-700) ¹ H-NMR and ¹³ C-NMR analysis were performed to show the results in FIGS. 8 and 9, respectively.

Peak 1 of the single plant-derived extract of the present invention obtained from the above results is as follows.

Molecular name: Diphlorethohydroxycarmalol

Molecular formula: 7- (3,5-Dihydroxyphenoxy) -3- (2,4,6-trihydroxyphenoxy) dibenzo [b, e] [1,4] dioxin-1,2,6,8-tetrol

9CI Chapman & Hall Number: CJY33-G

CAS Registry Number: 138529-04-1

Type of Compound Code (s): VG1000

Molecular Formula: C ₂₄ H ₁₆ O ₁₃

Molecular Weight: 512.383

Accelate Mass: 512.059095

Composition ratio: C 56.26%; H 3.15%; O 40.59%

In addition, the molecular structure of Peak 1, which is a single substance isolated from the extract of the perennial plant of the present invention, is shown in FIG.

Example 4 Manufacture of Rings Using Broadleaf Extract

The broad pad extract prepared by the method of Example 1 of the present invention was lyophilized to prepare a powder.

200 g of water and 100 g of honey were put in 1 kg of the prepared powder, kneaded, put into a pill manufacturing apparatus to make a ring shape, and then put into a drier and dried at 30 ° C. for 12 hours to prepare a ring.

Example 4 Manufacture of Capsules Using Materials Derived from Shell Extracts

Peak 1 (difluoroetohydroxycarmarol) isolated from the shell extract was prepared by the method of Example 3 of the present invention.

Capsules were prepared in a conventional manner using the prepared material.

Example 5 Preparation of a Beverage Composition Containing Shell Extract

The shell extract prepared by the method of Example 2 of the present invention was prepared.

20 g of extract prepared per 1 liter of purified water, 4 g of vitamin B2, 6 g of vitamin B6, 10 g of nicotinic acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herb flavor were added to the extract of the present invention. A beverage composition was prepared.

<Example 6> Preparation of the beverage composition containing the substance derived from the broad-leaf extract

Using the broad extract of Example 1 of the present invention was prepared Peak 1 (difluoroethoxy hydroxycarmarol) separated by the method of Example 3.

10 g of prepared substance per 1 liter of purified water, 4 g of vitamin B2, 6 g of vitamin B6, 10 g of nicotinic acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herb flavor are added to the substance derived from the broad-leaf extract of the present invention. Prepared beverage composition.

Example 7 Preparation of Food Additives Containing Broadleaf Extract

The broad-leaf extract prepared by the method of Example 1 of the present invention was prepared as a powder after lyophilization.

2 wt% of the above powder was used to prepare a conventional food.

Example 8 Preparation of a Food Additive Containing a Substance-Derived Substance

Peak 1 (difluoroetohydroxycarmarol) isolated from the shell extract was prepared by the method of Example 3 of the present invention.

2 wt% of the above-mentioned substance was used to prepare a conventional food.

Experimental Example 2 Antidiabetic Effect of the Extract of the Perennial Plant of the Present Invention

Alpha-glucosidase is an enzyme that breaks down disaccharides into monosaccharides and has been reported to increase post-prandial blood sugar in the small intestine mucosa.

The inhibitory effect was added to 5 mM PNP (p-nitrophenyl-α-D-glucopyranoside) solution in 0.1 M phosphate buffer (pH7.0) and α-glucosidase (0.7U / ml) and sample for 10 minutes at room temperature. Stop using stop solution (1M glycine-NaOH, pH9.0).

Absorbance was expressed as the concentration of the sample (IC ₅₀ ) that appears when the absorbance decreases by more than 50% after measuring at 405 nm.

As a result, α-glucosiase inhibitory effect on the broad-leaf extract of Example 1 of the present invention was crude extract (IC ₅₀ ; 0.1103 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.0815 ㎍ / ㎖), butanol fraction (IC ₅₀ 0.0836 μg / ml) and hexane fractions (IC ₅₀ ; 0.5182 μg / ml) showed very high inhibitory effects and water fractions (IC ₅₀ ; 3.7915 μg / ml) (Table 5, FIG. 11A).

In addition, α-glucosiase inhibitory effect on the shell extract of Example 2 of the present invention is crude extract (IC ₅₀ ; 0.8117 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.0480 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 0.0683 μg / ml) and hexane fractions (IC ₅₀ ; 0.2921 μg / ml) showed very high inhibitory effects, and water fractions (IC ₅₀ ; 8.5007 μg / ml) (Table 5, FIG. 11B).

On the other hand, the inhibitory effect of the already commercialized acarbose, the IC ₅₀ value of 242.08 ㎍ / ㎖, it was found that the extract of the present invention showed a very excellent effect compared to the α-glucosiase inhibitory effect of acarbose.

Therefore, it was found that the anti-diabetic effect of the extract of the present invention is significantly superior antidiabetic effect.

<Table 5> Antidiabetic Experimental Results for the Fruit Extracts

division α-glucosidase inhibitory effect (IC ₅₀ )   Shell Extract Crude extract 0.8117 Hexane fraction 0.2921 Ethyl acetate fraction 0.0480 Butanol fraction 0.0683 Water fraction 8.5007   Broadleaf extract Crude extract 0.1103 Hexane fraction 0.5182 Ethyl acetate fraction 0.0815 Butanol fraction 0.0836 Water fraction 3.7915 Control (Akabos) 242.08

Experimental Example 3 Antidiabetic Effect Test on Substances Isolated from Shell Extracts

Peak 1 (DifluoroEthoxycarmarol) and Peak 2, which were separated from the ethyl acetate fraction of the pad prepared in Example 2 of the present invention, were prepared.

Acabose, which is already commercialized, was prepared as a control.

Antidiabetic experiments were performed on the prepared samples and are shown in Table 6 below.

<Table 6> Antidiabetic activity test results for shell extract and single substance isolated

division IC ₅₀ (μg / ml) Pea Extract 0.812 Ethyl acetate fraction 0.048 Peak 1 0.063 Peak 2 0.934 Akabos 242.08

As shown in Table 6 above, Peak 1 is IC ₅₀ ; 0.63 μg / mL, Peak 2 is IC ₅₀ ; 0.093 μg / ml, shell extract was IC ₅₀ ; 0.812 μg / mL, the ethyl acetate fractions were IC ₅₀ ; 0.048 μg / ml, control acarbose was IC ₅₀ ; It was found to be 242.08 μg / ml (FIG. 12).

Therefore, it was found that the single substance isolated from the extract of the perennial plant of the present invention also has an excellent antidiabetic activity.

Experimental Example 4 Antioxidant Experiments on the Perishable Plant Extracts of the Present Invention

1. DPPH radical scavenging activity experiment

Crude extracts and fractions of the extracts of the perennial plants prepared by the methods of Examples 1 and 2 of the present invention were prepared.

In addition, a single material Peak 1 and Peak 2 were prepared from the ethyl acetate fraction of the pad prepared in Example 2.

Electron donating ability was measured by DPPH free radical scavenging method by Blosis method.

First, 100 μl of various concentrations of samples dissolved in methanol were dispensed into 96 well plates, and 0.4 mM DPPH solution was added in the same amount to react for 10 minutes at room temperature, and then measured at 517 nm.

At this time, ascorbic acid and trolox were used as a control.

DPPH radical scavenging activity was calculated from the following equation, and the absorbance of DPPH was expressed as the concentration of the 50% sample (IC ₅₀ ).

DPPH radical scavenging activity (%) = (A _control -A _sample ) / A _control × 100

A _sample = absorbance of the reaction solution to which the sample was added

A _control = absorbance of reaction solution with methanol instead of sample

As a result, the commercialized ascorbic acid (IC ₅₀ ; 8.23 μg) showed a high active radical inhibitory effect.

Crude extracts, water fractions, and nucleic acid fractions of the broad leaf of the present invention showed low effects (IC ₅₀ ;> 400 μg / ml), but ethyl acetate fraction (IC ₅₀ ; 111.09 μg / ml), butanol fraction (IC ₅₀ ; 178.17). ㎍ / ㎖) layer showed more than twice the active oxygen inhibitory effect than the crude extract (Fig. 13A).

In addition, crude extract of the present invention had a low active oxygen inhibitory effect, ethyl acetate fraction (IC ₅₀ ; 40.44 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 74.21 ㎍ / ㎖) is crude extract ((IC ₅₀ ; > 400 [mu] g / ml) showed higher free radical inhibition effect (FIG. 13B).

In addition, the active oxygen inhibitory effect of the peak 1 and peak 2 fractions as a single substance was Peak 1 (IC ₅₀ ; 100 ㎍ / mL) and Peak 2 (IC ₅₀ ; 200 ㎍ / mL), with Peak 1 being somewhat higher (Fig. 14).

2. Nitrogen Compound Scavenging Activity Test

Sodium nitroprusside (SNP), a material that naturally produces nitric oxide, was used to analyze the NOx removal activity of the sample.

Samples were added to 200 μl of 10 mM SNP solution by concentration, and reacted at 25 ° C. for 3 hours. Then, the same amount of Griess reagent (2.5% phophoric acid, 1% sulfanilamide, 0.1% naphylethylendiamine) was added for 10 minutes at room temperature. Absorbance at 540 nm was measured.

The NO scavenging activity was expressed by the concentration of the sample (IC ₅₀ ) that appears when the absorbance was reduced by 50%.

As a result, as shown in Table 7, the crude extract and the water fraction (IC ₅₀ ; 591.14) of the broad-leaf was very low, but the ethyl acetate fraction (IC ₅₀ ; 82.59 ㎍ / ml) and the butanol fraction (IC ₅₀ ; 147.08 ㎍) / Ml) showed a high inhibitory effect (Fig. 15A).

In addition, the shell extract of the present invention showed a low inhibitory effect in all samples except hexane fraction (IC ₅₀ ;> 192.58 ㎍ / ㎖) as shown in Figure 15B (IC ₅₀ ;> 300 ㎍ / ㎖).

In addition, the NO scavenging effect of the peak 1, Peak 2 fraction as a single substance is IC ₅₀ ; > 400 μg / ml), the NO scavenging power appeared somewhat lower (FIG. 16).

(3) Xanthine oxidase inhibition and peroxide scavenging activity assay

Uric acid production by xantihine / xanthine oxidase was measured by increased absorbance at 290 nm.

The amount of superoxide was measured by nitroblue tetrazolium (NBT) reduction method.

Uric acid production was induced by adding 50 mU / ml xanthine oxidase to the reaction solution (0.5 mM xanthine, 200 mM phosphate buffer (pH7.5) 1 mM EDTA).

Superoxide scavenging activity was reacted by adding 0.5 mM NBT to the reaction solution.

Xanthine oxidase inhibition and peroxide scavenging activity were expressed by the concentration of the sample (IC ₅₀ ) which appeared when the absorbance of the produced uric acid and peroxide decreased by 50% or more, respectively.

As a result, xanthine oxidase inhibitory activity against the broad-leaf extract of the present invention was crude extract (IC ₅₀ ; 20.63 ㎍ / mL), ethyl acetate fraction (IC ₅₀ ; <10.42 ㎍ / mL), butanol fraction (IC ₅₀ ; 7.21 ㎍) / ML), hexane fractions (IC ₅₀ ; 58.77 ㎍ / mL), and water fractions (IC ₅₀ :> 314.97 ㎍ / mL) showed high activity in the ethyl acetate layer and the butanol layer (FIG. 17A).

Xanthine oxidase inhibitory activity against the shell extract of the present invention is crude extract (IC ₅₀ ; 187.47 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 0.71 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 22.2 ㎍ / ㎖), The hexane fraction (IC ₅₀ ; 26.56 μg / ml) and the water fraction (IC ₅₀ :> 400 μg / ml) showed high activity in the ethyl acetate fraction and butanol fraction (FIG. 17B).

In addition, P1. A single substance isolated from the shell extract of the present invention. The inhibitory activity of x2 was shown to be slightly higher as P1 (IC ₅₀ ; 10 μg / ml) and P2 (IC ₅₀ ; 30 μg / ml) (FIG. 18).

In addition, the peroxide scavenging activity of the broad-leaf extract of the present invention is crude extract (IC ₅₀ 37.7 ㎍ / ㎖), ethyl acetate fraction (IC ₅₀ ; 19.95 ㎍ / ㎖), butanol fraction (IC ₅₀ ; 37.91 ㎍ / ㎖), hexane Fractions (IC50; 78.07 μg / ml) and water fractions (IC50; 210.82 μg / ml) showed high effects in the ethyl acetate layer (FIG. 19A).

Peroxide scavenging activity of the shell extract of the present invention was crude extract (IC ₅₀ ; 81.5 ㎍ / mL), ethyl acetate fraction (IC ₅₀ ; 11.24 ㎍ / mL), butanol fraction (IC ₅₀ ; 17.65 ㎍ / mL), hexane fraction (IC 50; 70.80 μg / ml) and the water fraction (IC50; 114.95 μg / ml) showed high effects in the ethyl acetate layer (FIG. 19B).

In addition, P1. A single substance isolated from the shell extract of the present invention. The peroxide scavenging activity of P2 was P1 (IC ₅₀ ; 8 μg / ml) and P2 (IC ₅₀ ; 35 μg / ml), which was slightly higher than P1 (FIG. 20).

<Table 7> Antioxidant Test Results for the Extracts

division DPPH (IC ₅₀ ) NO (IC ₅₀ ) Xanthine oxidase (IC ₅₀ ) Peroxide (IC ₅₀ )   Shell Extract Crude extract - 924.58 187.47 81.5 Hexane fraction 217.74 192.58 26.56 70.8 Ethyl acetate fraction 40.44 343.03 0.71 11.24 Butanol fraction 74.21 - 22.2 17.65 Water fraction - - 531.45 114.95   Broadleaf extract Crude extract - 591.14 20.63 37.7 Hexane fraction - 240.09 58.77 78.07 Ethyl acetate fraction 111.09 82.59 10.42 19.95 Butanol fraction 178.17 147.08 7.21 37.91 Water fraction - - 314.97 210.82 Control Asco 8.23 Qucertin 380.56 Allo 0.24 Allo 0.028

According to the present invention, there is provided a perennial extract excellent in antidiabetic activity and a substance separated therefrom.

In addition, there is provided an antidiabetic and antioxidant composition comprising the extract of the present invention as an active ingredient.

In addition, there is provided a beverage composition and food additive to which the anti-diabetic and anti-diabetic extract of the present invention is added.

Claims (6)

Extract from Ishigeaceae plants, characterized by having an anti-diabetic effect, A deciduous plant extract for the prevention and improvement of diabetes. The method of claim 1, Peel plant extract is prepared by washing one selected from the plaque and broad leaf, and then immersed in 70% ethanol and leached by stirring at room temperature for 2 to 3 days, then filtered through a filter to prepare a crude extract, Characterized in that the extract is sequentially fractionated using solvents of different polarity n-hexane, ethyl acetate, butanol, A deciduous plant extract for the prevention and improvement of diabetes. Antidiabetic composition comprising the extract of the deciduous plant as an active ingredient. Antioxidant composition comprising a deciduous plant extract as an active ingredient. An antidiabetic composition comprising Diphloretohydroxycarmalol (Diphlor-Ethohydroxycarmalol) isolated from the extract of the perennial plant as an active ingredient. Anti-oxidant composition comprising Diphloretohydroxycarmalol (Diphlor- ethohydroxycarmalol) isolated from the extract of the deciduous plant.

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