KR20140148226A - A composition having activity of blood glucose regulation and aldose reductase inhibition comprising the extract of magnolia grandiflora l. as an active ingredient for preventing and treating diabetic complications - Google Patents

Abstract

The present invention relates to a composition containing an extract of Mgnolia grandifloral L. as an active component for preventing and treating diabetic complications. More specifically, the present invention relates to a composition containing an extract of Mgnolia grandifloral L. as an active component for preventing and treating diabetic complications, which contains an extract of Mgnolia grandifloral L. having activity of blood glucose regulation and aldose reductase inhibition to prevent or treat diabetic complications as an active component.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating diabetic complications comprising, as an active ingredient, an extract of a perennial plant of Magnolia grandiflora L. having an effect of inhibiting blood glucose level and aldose reductase. As an active ingredient for preventing and treating diabetic complications,

The present invention relates to a composition for preventing and treating diabetic complications containing an extract of Magnoliaceae as an active ingredient. More particularly, the present invention relates to a composition for prevention and treatment of diabetic complications, which comprises an extract of black granular extract capable of preventing or treating diabetic complications due to its excellent blood glucose control and aldose reductase inhibitory activity.

Diabetes mellitus (DM) is a progressive disease often associated with obesity characterized by insulin deficiency and insulin resistance, or both. An increase in fasting and postprandial blood glucose may result in the patient being exposed to acute and chronic complications (micro- and macro-vascular) that cause blindness, kidney failure, heart disease, stroke and amputation do. Improved blood glucose control has been shown to reduce the risk of such complications.

Because of the progressive nature of the disease, an evolving therapeutic strategy is needed to maintain blood glucose control. There are two types of diabetes: type 1, or childhood diabetes or insulin dependent diabetes mellitus (IDDM), and type 2, or adult type diabetes or non-insulin dependent diabetes mellitus (NIDDM). Patients with type 1 diabetes are absolutely insulin deficient due to immunological destruction of pancreatic beta cells that synthesize and secrete insulin. Type 2 diabetes is characterized by a more complex and relative insulin deficiency, reduced insulin action, and insulin resistance. Early-onset NIDDM or adult-onset diabetes mellitus (MODY) share many of the most common forms of NIDDM in the middle-aged (Rotter et al., 1990). A clear mode of inheritance (autosomal dominant) was observed for MODY. At least three distinct mutations have been identified in the MODY family (Bell et al., 1996).

It is known that inhibition of aldose reductase in the polyol pathway, which is one of the main causes of diabetic complications, plays an important role in the treatment and prevention of diabetic complications. It has been reported that the aldose reductase inhibitors zopolrestat, ponalrestat, sorbinil, tolrestat, fidarestat, ranireatat and epalrestat, which have been developed so far, prevent and delay diabetic complications in various animal experiments.

However, in clinical practice, zopolrestat and ponalrestat were ineffective and stopped during development due to side effects such as sorbinil hypersensitivity and tolrestat liver dysfunction. Clinical trials of ranireatat and fidarestat are underway in Japan and the United States. Epalrestat has not been approved by the US Food and Drug Administration (FDA), but has been approved and marketed only in Japan in 1992.

In Korean Patent Registration No. 10-0204500, a variety of plant extracts have been studied in order to develop a therapeutic agent capable of treating sepsis. As a result, the extracts of Magnolia grandiflora L. have been induced by lipopolysaccharide from leaves and stems of Magnolia grandiflora L. The activity of inhibiting the synthesis of Nitric oxide, tumor necrosis factor-alpha and prostaglandin E2 was identified, and the active substance was isolated and purified. The chemical structure of the active substance was identified, and it was confirmed that the sesquiterpene lactone compound, , The present invention provides a novel use of the extract of the black granular extract and the phytorenolide compound as a therapeutic agent for sepsis, but does not disclose any effect of the black granular extract on the blood glucose lowering effect.

Korean Patent Registration No. 10-0321312 discloses a sesquiterpene lactone compound extracted from the leaves of Magnolia grandiflora L. and has a structure of Formula 1 which can be used as a therapeutic agent for inflammatory diseases and immune diseases. Disclose the costunolide and describe that these compounds can be used as effective treatments for inflammatory diseases and immune diseases caused by overproduction of nitric oxide or tumor necrosis factor-alpha or activation of NF-κB However, no attention is paid to the blood glucose lowering effect or the inhibitory effect of aldose reductase.

The authors of the present invention found that the extracts of the genus Magnolius exhibited a blood glucose lowering effect during the study of natural extracts that could effectively prevent and treat diabetes and diabetic complications without side effects and found that the excellent single dose reductase inhibition As the efficacy has been confirmed, this study has been conducted to obtain a composition that can be used for the treatment of diabetes and diabetic complications from extracts of Magnoliaceae.

The present invention relates to a composition for preventing and treating diabetic complications containing an extract of Magnoliaceae as an active ingredient.

It is another object of the present invention to provide a novel process for obtaining an effective extract from Magnolia grandiflora L. Specifically, the roots, leaves or stems of the magnolia are extracted using an organic solvent such as lower alcohols, ether, methylene chloride, ethyl acetate, a mixed solvent thereof, etc., followed by performing silica gel column chromatography and recrystallization, have.

The present invention relates to a composition for preventing and treating diabetic complications containing an extract of Magnoliaceae as an active ingredient. More particularly, the present invention relates to a composition for prevention and treatment of diabetic complications, which comprises an extract of black granular extract capable of preventing or treating diabetic complications due to its excellent blood glucose control and aldose reductase inhibitory activity.

Magnolia grandiflora is a tree of evergreen, with large, leafy leaves. It is planted mainly in North America and South Korea in South Korea. It is an evergreen large tree planted ornamental plant, also called Olan. It is an evergreen tree leaf and is said to be used for Christmas decoration in the United States. It is about 20m in height and the bark is grayish brown with many small peel eyes. It is hairy in branches and winter eyes. Leaves are alternate, long elliptical, with flat edges and dull ends. Leaves are leather, the surface is dark green, shiny, with brown hairs on the back and plain edges. Petiole is 2 ~ 3cm in length. Flowers bloom in May ~ June, white, 15 ~ 20cm in diameter, one branch runs on top of the branch. It has strong aroma, 3 calyxes, 9-12 petals. There are many pistils and stamens, and the operating table is purple. The fruit is oolgwon, ripened in September, oval, short hairs, and two red seeds hanging in red thread.

According to one preferred embodiment of the present invention, the extracts and fractions of the genus Penicillium can be obtained for each of the roots, stems, and leaves, and all of them except for the difference of some components exhibit effective plasma lowering effect.

It is another object of the present invention to provide a novel process for obtaining an effective extract from Magnolia grandiflora L.

The extract of Magnoliaceae may be one prepared by a conventional method for producing plant extracts. For example, the extract may be prepared by adding an extracting solvent to leaves, branches, roots, fruits or shells or crushed products of Magnolia, or extracting with an extraction solvent The crude extract may be prepared by fractionating a fraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as an alcohol having 1 to 5 carbon atoms, a diluted alcohol such as ethyl acetate or acetone, a nonpolar solvent such as ether, chloroform, benzene, hexane or dichloromethane, or a mixture thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, or isopropanol, but is not limited thereto. In addition, the alcohol dilution water may be an alcohol diluted with water at 50 to 99.9% (v / v).

The extraction solvent used to obtain the extract of the present invention may preferably be at least one selected from the group consisting of water, an alcohol having 1 to 5 carbon atoms, an alcohol-diluted water, and a mixture thereof, more preferably water, To 4% alcohol, and mixtures thereof, and most preferably from 30% to 99% ethanol aqueous solution or alcohol. The extraction process may be performed, for example, at 4 ° C to 120 ° C, or 50 ° C to 90 ° C, or 70 ° C to 85 ° C, but is not limited thereto. The extraction time is not particularly limited, but may be 10 minutes to 12 hours, preferably 1 hour to 6 hours.

The extract of the present invention may be one prepared by a conventional method for producing a plant extract. Specifically, it may be a cold extraction method, a warm extraction method, a heat extraction method, an ultrasonic extraction method, or the like, and may be a conventional extraction apparatus, .

According to a preferred embodiment of the present invention, roots, leaves or stems of the magnolius can be extracted with lower alcohols in order to obtain the magnolius extract. The alcohol used here is preferably a lower alcohol having from 1 to 4 carbon atoms.

Further, the extract extracted with the solvent may be further fractionated with any one solvent selected from the group consisting of hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. Two or more kinds of solvents may be used for the fractionation, and the solvent extracts may be sequentially used or mixed according to the polarity of the solvent. In addition, the fractionation process may be performed, for example, at 4 ° C to 120 ° C, or 50 ° C to 90 ° C, or 70 ° C to 85 ° C, but is not limited thereto.

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. The drying can be performed by, for example, freeze drying.

The effect of the extract of Magnoliaceae on the activity of aldose reductase, a catalytic enzyme of the polyol pathway, is known to play an important role in the development of diabetic complications. An aldose reductase inhibitor is an enzyme inhibitor that inhibits aldose reductase that converts glucose to sorbitol and aims to lower the production of sorbitol. In many studies, polyol metabolism abnormalities are known to cause diabetic complications, so that aldose reductase inhibitors may be useful as a therapeutic agent for diabetic complications.

Thus, in order to demonstrate the therapeutic effect of diabetic complications, the present example tested the ability of the extracts and fractions of Magnolia of Magnolia to inhibit the aldose reductase activity.

The composition containing the black granular extract of the present invention as an active ingredient exhibits an excellent blood glucose lowering effect and is effective for prevention and treatment of diabetic complications.

The present invention also provides a novel production method for obtaining an effective extract from magnolia.

Figure 1 illustrates an exemplary method for obtaining a blackcurrant extract and fractions.
FIGS. 2A to 2C show the results of TLC determination of the solvent-dependent fractions.
3A to 3C show the results of analysis of the fractional material distribution by solvent using HPLC.
FIG. 4 is a graph showing the effect of oral administration of root and stem extracts of D-glucose to D-glucose rats on blood glucose.
FIG. 5 is a graph showing the effect of roots and stem extracts of D-rosacea on D-glucose rats on blood glucose after administration of spinal cord.
Figure 6 is a schematic of an on-line HPLC-ABTS ⁺ system.
Figure 7 is a graph showing the antioxidant activity of the composition of the present invention (root EtOAc fr.) Using the on-line HPLC-ABTS ⁺ system.
Figure 8 is a graph showing the antioxidant activity of the composition of the present invention (Leaf EtOAc fr.) Using the on-line HPLC-ABTS ⁺ system.
Figure 9 is a schematic of an off-line HPLC-microfractionation system for measuring Rat lens aldose reductase inhibitory activity.
10 is a graph showing the inhibitory activity of HPLC-microfractionated (B) of the ethyl acetate fraction of the root of the root of the magnolia in a 96-well plate.
FIG. 11 is a graph showing the inhibitory activity of HPLC chromatogram (A) and HPLC-microfractionated (B) of the ethyl acetate fraction of Magnoliaceae leaf in a 96-well plate.
12 is a ¹ H-NMR spectrum of Compound 1 (Protocatechuic acid).
13 is a ¹³ C-NMR spectrum of Compound 1 (Protocatechuic acid).
14 is a structural formula of Compound 2. Fig.
15 is a structural formula of compound 3.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples illustrate the present invention in detail and are not intended to limit the scope of the present invention.

Experimental Example  1. Preparation of sample

Magnolia was divided into root, stem and leaf. The extracts and the fractions were extracted with 80% ethanol of the dried magnolia roots 280g, 183g of the leaves, 553g of the stem and 10 times of 70% ethanol in the aqueous solution at 80 ° C for 2 hours and then filtered to obtain an extract. The extraction process was repeated three times. The resulting extract is concentrated under reduced pressure at 40 ° C or below and lyophilized to obtain a powdery extract.

As can be seen from FIG. 1, 2 L of distilled water was added to the extracts of roots, roots, leaves, and the mixture was suspended in 2 L of hexane, followed by separation into hexane-soluble fraction and water-soluble fraction. The same procedure was repeated three times to collect only the hexane soluble fraction, followed by drying under reduced pressure to obtain a hexane soluble extract powder. Similarly, methylene chloride, ethyl acetate, butanol soluble fraction and water soluble fraction were collected, And dried to obtain a powder.

Experimental Example  2. Thin layer chromatogram ( Thin Layer Chromatography , TLC ) Component Analysis Test

In order to confirm the distribution of the substance, the sample was dissolved in methanol at a concentration of 10 mg / ml in a sample (hyphae root, stem, leaf) by TLC (pre-coated with silicagel 60 (Merk Co. USA) F254) And developed in a solvent (chloroform: methanol: water = 65: 35: 5).

After development, UV (254, 366 nm) was observed, and 10% sulfuric acid was sprayed onto the expanded surface and dried at 100 ° C to confirm the band. The organic component of the sample was developed to show all of the organic components. The results obtained by testing the obtained extracts and fractions using this method are shown in FIG. As can be seen from FIG. 2, the reason for developing sulfuric acid is that the material is carbonized and dehydration reaction occurs, and when heat is applied in this state, carbon carbonizes and changes to black.

Experimental Example  3. HPLC Analysis of Fractional Substance Distribution by Solvent

The analytical column was eluted with 0: A: B = 100: 0 20 min A: B using a mobile phase water (A) and methanol (B) at a flow rate of 0.7 mL / min using Agilent Eclips XDB-C18 B: 100: 23: A: B = 0: 100 27 minutes A: B = 100: 0 30 minutes A: B = 100: 0 The peak was detected by UV detection at 254 nm, Respectively.

Experimental Example  4. D- Glucose  In the rat model Magnolia tree  Effect of Root and Stem Extract on Changes in Blood Glucose

After 30 or 60 minutes of oral administration, 2 g / kg of D-glucose was orally administered to rats at a concentration of 50 mg / kg, and 30, 60 and 120 minutes after the administration of blood, .

In addition, roots or stem extracts of the roots were treated with 50 ug / 5 цl of the rat spinal cord, and after 10 minutes, 2 g / kg of D-glucose was administered. After 30, 60 and 120 minutes, A small amount of blood was extracted from the vein.

The glucose in the blood was measured using an Accu-check blood glucose meter, which had already been established as an accurate blood glucose meter by the extracted blood. At this time, PEC (PEG400 + EtOH + CMC) solvent was used as a control group in place of roots or stem extracts of Magnolia root. As a result, it was confirmed that the root roots or stem extract of the present invention showed remarkable blood glucose lowering effect in a rat model fed with D-glucose.

4, 50 mg / kg of root extract or stem extract was orally administered and 30 minutes later, in the group fed with D-glucose, 50 mg / dl in 30 minutes and 60 minutes It can be confirmed that the blood glucose lowering effect is present.

5, 50 μg / 5 μl of the root of the root or stem extract was administered to the spinal cord, and in the group fed with D-glucose at 10 minutes, 25 mg / dl was found in the magnoliophyte extract, Root extract showed a blood glucose lowering effect of 50 mg / dl.

Experimental Example  5. ABTS Free radical  Scavenging activity test

ABTS (3-ethylbenzothiazoline-6-sulfonic acid-Diammonium salt, purchased from Fluka) was mixed with 3.5 mM potassium persulfate at a concentration of 2 mM and dissolved in 10 ml of tertiary distilled water . To the obtained solution, 290 ml of tertiary distilled water was added, diluted 30 times, and then allowed to stand for overnight to induce ABTS radical formation to prepare an ABTS radical solution. 10 μl of the prepared extracts of roots, stem, leaves and 290 μl of ABTS radical solution were reacted at room temperature for 10 minutes and absorbance was measured at 750 nm.

The radical scavenging activity of the root extracts (roots, stems, leaves) was calculated by IC ₅₀ values by calculating the concentration required to eliminate 50% of the ABTS radicals, and the results are shown in Table 1 below.

Calculation formula 1 = (ABTS radical scavenging rate (%)) = {1- (Sample / Control)} * 100

(control: without sample, ABTS and solvent (DMSO) only)

Trolox (CALBIOCHEM purchased, catalog number-648471, also known as ABTS radical scavenger, was used as a positive control.

Concentration
(/ ml) ABTS radical
% Inhibition IC ₅₀ (/ ml) Magnolia root extract 16.67 68.26 ± 1.24 11.68 ± 0.26 3.33 20.83 ± 0.53 1.67 11.67 + - 0.35 Hexane fr. 33.33 37.14 + 0.18 - CHCl ₃ fr. 16.67 74.65 ± 0.35 10.10 ± 0.13 3.33 26.60 ± 1.6 1.67 16.06 + -0.53 EtOAc fr. 16.67 88.71 + - 0.01 3.87 ± 0.01 3.33 45.67 ± 0.89 1.67 28.11 + - 0.89 n-BuOH fr. 16.67 72.52 + 0.18 10.94 + 0.09 3.33 21.46 ± 0.71 1.67 11.79 ± 0.53 Water fr. 33.33 37.01 + - 0.35 - Magnolia stems extract 33.33 83.59 + - 0.35 11.86 + 0.04 16.67 55.54 + 0.17 3.33 13.90 ± 0.17 Hexane fr. 33.33 22.11 + - 0.35 - CHCl ₃ fr. 16.67 71.46 ± 1.56 6.1 ± 0.25 3.33 37.42 ± 0.17 1.67 21.98 ± 0.52 EtOAc fr. 16.67 89.10 ± 0.17 3.72 + 0.24 3.33 47.09 + - 3.46 1.67 29.21 + - 1.04 n-BuOH fr. 33.33 88.73 + - 0.69 9.31 + 0.07 16.67 69.14 + - 0.01 3.33 17.82 + 0.17 Water fr. 33.33 43.29 + - 0.17 - Magnolia leaf extract 16.67 62.19 + - 0.18 13.11 + 0.03 3.33 17.19 ± 0.18 1.67 9.94 + - 0.01 Hexane fr. 33.33 24.26 ± 0.18 - CHCl ₃ fr. 16.67 60.77 + - 0.01 13.37 ± 0.01 3.33 18.71 + - 0.01 1.67 9.03 + - 0.18 EtOAc fr. 16.67 89.03 + - 0.18 7.36 ± 0.09 3.33 36.26 ± 0.73 1.67 22.97 + 0.18 n-BuOH fr. 16.67 79.74 ± 1.28 9.82 + 0.26 3.33 22.45 + - 0.55 1.67 13.94 + - 2.74 Water fr. 33.33 38.71 + - 0.18 - Trolox 3.33 86.75 ± 2.3 1.86 ± 0.01 1.67 49.42 ± 0.1 0.33 5.77 ± 0.2

The results of ABTS free radical scavenging activity were shown by IC ₅₀ values using the extracts of Magnoliaceae and fractions. The IC ₅₀ value of Trolox used as a positive control group was 1.86 / ml. In comparison, it was found that the fractions had excellent free radical scavenging ability at 3.87 and 3.72 7.36 / ml in roots, stem and leaf EtOAc fr.

Experimental Example  6. On-line HPLC - ABTS ^{· +} System test

ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) -diammonium salt (Fluka) was mixed with 3.5 mM potassium persulfate at a concentration of 2 mM and dissolved in 10 ml of tertiary distilled water The ABTS radical solution was prepared by adding 290 ml of tertiary distilled water to the obtained solution, diluting it 30 times, and allowing it to stand overnight to induce ABTS radical formation. The analytical column was Agilent Eclips XDB-C18 (5 [mu] m, 4.6 * 250 mm id) flow rate 0.7 mL / min, mobile phase 0.1% TFA (trifluoroacetic acid) / water (A) and methanol (B) 0 min A: B = 85: 15 25 min A: B = 50 35 minutes A: B = 50:50 45 minutes A: B = 35:65 55 minutes A: B = 30:70 60 minutes A: B = 0: 100 62 minutes A: B = 85: 15 75 minutes A: B = 85: 15 and detected at UV 254 nm to confirm the peak.

Experimental Example  7. Aldos  Reductase inhibition test

A lens of Sprague-Dawly (SD) rats (male, 12 weeks old, 250 to 300 g, Central laboratory animal center) weighing 250-300 g or more was taken out, After centrifugation (centrifuge UNION 55 R Hanil, Korea) at 4 ° C and 10,000 rpm for 20 minutes, the supernatant was taken and aldose reductase test As an enzyme source.

The inhibitory activity of Aldose reductase activity was modified by Haymanh and Kinoshita (Hayman S, Kinoshita JH 1965. Isolation and properties of lens aldose reductase. J. Biol. Chem. ): 877-882). To the 530 μL of 0.05 M potassium phosphate buffer (pH 7.0), add 10 μL of the prepared enzyme source, 160 μL of the prepared enzyme source, 100 μL of 1.6 mM NADPH (Applichem, Germany) and the magnoliophyte root, stem and leaf extract obtained in the sample (Example 1) And finally 100 μL of 0.1 M DL-glyceraldehyde (DL-Glyceraldehyde, Sigma, USA) was added thereto. The reaction was allowed to proceed for 4 minutes in the cuvette to measure the decrease rate of NADPH absorbance at 340 nm. The reaction was carried out for 4 minutes in the cuvette to calculate the absorbance of the sample at the extinction values of 0 s and 240 s.

Calculation formula 2 = absorbance value at {0 s} - absorbance value at -240 s} / reaction time}

Using the absorbance value obtained in the above equation 2, the inhibition rate was calculated by the following equation.

3 = {(control-sample) / (control-blank)} * 100

(control: enzyme and substrate in the absence of drug,

blank: with all the rest of the substrate removed)

In addition, the decrease rate of the absorbance was measured using quercetin (Quercetin, Sigma Chemical Co., St. Louis, MO, USA) as a control substance known to have an aldose reductase inhibitory effect, Were compared with the results of roots, stem and leaf extracts of the magnoliops.

The concentration of each sample (IC ₅₀ ) indicating the decrease of the absorbance by 50% was indicated and the samples were repeated three times and averaged. The results are shown in Table 2 below.

Concentration (/ ml) Aldose reductase inhibition (%) IC ₅₀ (/ ml) Magnolia root extract 10 44.75 - Hexane fr. 10 28.84 - CHCl ₃ fr. 10 53.75 - EtOAc fr. 10 83.25 1.24 5 71.21 One 46.86 n-BuOH fr. 10 45.33 - Water fr. 10 30.55 - Magnolia stems extract 10 78.30 3.40 5 58.11 One 38.86 Hexane fr. 10 57.97 - CHCl ₃ fr. 10 37.97 - EtOAc fr. 10 97.32 0.99 5 72.81 One 49.24 n-BuOH fr. 10 80.36 3.84 5 55.75 One 35.98 Water fr. 10 41.55 - Magnolia leaf extract 10 62.19 6 5 48.29 One 33.1 Hexane fr. 10 56.22 - CHCl ₃ fr. 10 51.65 - EtOAc fr. 10 88.55 2.99 5 62.35 One 38.27 n-BuOH fr. 10 70.92 3.31 5 59.4 One 25.98 Water fr. 10 41.19 - Quercetin 5 82.25 1.02 One 51.34 0.5 34.11

As can be seen from Table 2, by measuring the rat lens aldose reductase (RLAR) activity using a magnolia extract and the fractions are shown as IC ₅₀ values. It had a IC ₅₀ value of the quercetin in RLAR inhibitory activity as a positive control showed up to 1.02 / ml, In comparison Magnolia root, stem, leaf extract is respectively 1.24, 0.99, 2.99 / ml of the IC ₅₀ in the fraction EtOAc fr. Show the value.

Therefore, the present invention of the present invention indicates that the root extract, stem and leaf extracts exhibit equivalent IC ₅₀ values and inhibition rates at the respective concentrations, as compared with quercetin, which is known as the conventional aldose reductase inhibitor, in EtOAc fr. , Showing excellent aldose reductase inhibitory activity.

Experimental Example  8. off -line HPLC - micro - fractionation - aldose reductase inhibitor  Securing analytical conditions for activity

HPLC-microfractionation was performed using a Thermo Electron Spectra HPLC system and a Foxy 200 fraction collector (ISCO, Lincoln, NE). The analytical column was eluted with a gradient of 0: 1 using an Agilent Eclips XDB-C18 (5 μm, 4.6 * 250 mm id) flow rate 0.7 mL / min, mobile phase 0.1% trifluoroacetic acid / water (A) B = 85:15 25 minutes A: B = 50:50 35 minutes A: B = 50:50 45 minutes A: B = 35:65 55 minutes A: B = 30:70 60 minutes A: B = 0: 100 62 minutes A: B = 0: 100 65 minutes A: B = 85: 15 75 minutes A: B = 85:15 and detected at UV 254 nm to obtain chromatograms separated.

The eluate separated from the column was directly loaded into a 96-well plate tube at a rate of 1 min / well and used as an analytical sample by vacuum concentration (EZ-2 plus Evaporator, Genevac Ltd., Ipswich, UK) ).

A sample obtained through off-line HPLC-microfractionation was collected at a rate of 1 min / well to perform an experiment for inhibiting Rat lens aldose reductase (RLAR) for each substance. The results are shown in FIGS. 10 and 11 .

Experimental Example  9. Magnolia tree  Root, From leaves  Identification of active ingredients and structure

3 g of the EtOAc fractions of roots, stems, and leaves of each magnolia were loaded on a column (36 × 460 mm, glass column) packed with RP-18, and then the methanol ratio (5% Water → 100% methanol) were successively subjected to MPLC (medium pressure liquid chromatography).

The eluate was separated and recovered in a volume of 70 mL. The classification standard of the separated product was classified according to HPLC chromatogram using HPLC. The active component was separated using MPLC, and the precise structural analysis was further performed using the separated active compound using NMR and ESI-MS.

1) HPLC-ESI-MS ⁿ

The LC-MS analysis was carried out using an LCQ Advantage Max system (Thermo Quest, San Jose, Calif.) At a flow rate of 0.7 mL / min using Agilent Eclips XDB-Phenyl (3.5 μm, 4.6 × 150 mm id) A: B = 50: 50 50 min A: B = 0: 100 60 min A: B = 95: 5 40 min A: B = 50: 50 using 0.1% TFA (trifluoroacetic acid) and Acetonitrile : B = 0: 100 65 minutes A: B = 95: 5 72 minutes A: B = 95: 5. Detection at UV 254 nm gives chromatograms separated. Mass spectrometry was performed using the positive and negative modes of the electrospray ionization source (ESI). The capillary temperature was 250 ° C, the spray voltage was 4.8 kV, and the capillary voltage was 5.0 V. MS / MS Respectively.

2) NMR

Instrumental analysis such as ¹ H-nuclear magnetic resonance ( ¹ H-NMR), ¹³ C-nuclear magnetic resonance ( ¹³ C-NMR) and 2-D NMR was performed using Fourier Transform (FT) -NMR (Bruker DPX 400 MHz, Bruker Avance 600 MHz ), And MeOH-d ₄ and DMSO-d ₆ were used as analytical solvents. The structure of the compound was identified by comparing ¹ H-NMR and ¹³ C-NMR analysis. The related data is shown in Fig. 12 and Fig.

Compound ^{1: 1 H-NMR (CD3OD} , 400 MHz) δ 6.79 (1 H, d, J = 8.0 Hz), δ 7.42 (1 H, dd, J = 8.0 Hz and J = 2.0 Hz), δ 7.43 (1H , < / RTI > d, J = 2.0 Hz); ^{13 C-NMR (CD3OD, 100} MHz) δ 168.15 (C-7), 115.99 (C-2), 117.38 (C-5), 122.47 (C-6), 122.47 (C-1), 145.73 (C- 3), 150.85 (C-4);

ESI-MS (m / z) 155 [M + H] +, MS-MS (m / z) 109 [M-COOH] +; UV (MeCN, lambda max nm) 259sH, 294.

Compound 2: ESI-MS (m / z) 353 (MH), 191 [quinic acid-H], 179 [caffeic acid-H] -; UV (MeCN, lambda max nm) 298, 346 (max).

Compound ^{3: 1 H-NMR (DMSO-} d 6, 600 MHz) δ 0.92 (3H, d, J = 6.3 Hz, H-6 "'), 4.32 (1H, s, H-1"'), 5.28 ( (1H, s, H-8), 6.78 (1H, d, J = 8.2 Hz, H- 5 '), 7.47 (1H, s, H-2'), 7.48 (1H, s, H-6 ').

_{^{13 C-NMR (DMSO- d 6}} , 150 MHz,) δ 17.68 (C-6 "'), 66.95 (C-6"), 68.19 (C-5 "'), 69.97 (C-3"'), C-3 "), 70.33 (C-2"), 70.53 (C-4 "), 71.81 (C-6), 100.70 (C-1 "), 101.16 (C-1"), 103.92 (C-10), 115.18 2 '), 121.14 (C-1'), 121.54 (C-6 '), 133.28 (C-3), 144.71 156.56 (C-9), 161.18 (C-5), 164.06 (C-7), 177.33 (C-4).

Protocatechuic acid, chlorogenic acid, and protocatechuic acid, chloroacetic acid, and rutin were detected in the ethyl acetate fraction of the perennials. As a result of identifying the constituent patterns, the constituent patterns of the leaves and stems were similar.

Experimental Example  10. From active ingredients Aldos  Reductase inhibition test

Table 3 shows the IC ₅₀ values of the Rat Lens Aldose Reductase (RLAR) inhibitory activity of the three single substances isolated from the ethyl acetate fraction of the peroxidase. The IC ₅₀ value of Compound 2 was 3.16 uM, and the IC ₅₀ value of Compound 3 was 2.99 uM. On the other hand, the IC ₅₀ value of the positive control group, Quercetin, was 3.38 μM.

Number Compound IC ₅₀ ([mu] M) ^a) One Protocatechuic acid - 2 Chlorogenic acid 3.16 3 Routine 2.99 Quercetin ^b) 3.38

^{a) The IC50 value was defined as the concentration at  50% inhibition .}

^{b) Quercetin , positive control .}

Experimental Example  11. From active ingredients ABTS Radical  Used gun Antioxidant  Measure

ABTS radical inhibition test was carried out with the derived compounds. The mixture was mixed with 7.4 mM ABTS and 2.6 mM potassium persulfate, and incubated overnight (16-24 h) to form ABTS ⁺ . Absorbance was measured at 750 nm after adding 10 μL of each sample to 290 μL of the prepared ABTS solution. Ten minutes later, the absorbance was measured at 750 nm. As a positive control for the comparison of the elimination effect, trolox was used. Respectively.

Number Compound IC ₅₀ ([mu] M) ^a) One Protocatechuic acid 11.79 2 Chlorogenic acid 20.40 3 Routine 10.56 Trolox ^b) 14.24

^{a) The IC50 value was defined as the concentration at  50% inhibition .}

^{b) Trolox , positive control .}

Referring to Table 4, it was found superior or equal to the IC ₅₀ values of the isolated compounds 1 to 3 from the extract of the present invention is compared to the IC ₅₀ value of the positive control trolox.

Claims (8)

A composition for preventing and treating diabetic complications containing an extract of Magnoliaceae as an active ingredient. The composition for preventing and treating diabetic complications according to claim 1, wherein the extract is an extract from roots, leaves or stems of the magnolia. A process for preparing a composition according to claim 1,
Adding a lower alcohol to the magnolia tree; And
Concentrating the extract and concentrating under reduced pressure. 4. The process according to claim 3, wherein the lower alcohol is ethanol. The process according to claim 3 or 4, wherein the ethanol is 70% ethanol. 4. The process according to claim 3, wherein said extracting is carried out at 80 < 0 > C. 4. The method of claim 3, wherein the extracting step is performed two or more times. The method according to claim 3, further comprising the step of fractionating the concentrate after the step of concentration under reduced pressure and / or freeze-drying.

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