JP5610179B2 - Anti-obesity agent and diabetes-improving agent - Google Patents

Description

  The present invention relates to an anti-obesity agent and an anti-diabetic agent, and more particularly to an anti-obesity agent and an anti-diabetes agent containing an extract of a legume plant, in particular, a flower part of Nemnoki (Nejuhana).

  In recent years, obesity has increased due to excessive intake of high-calorie foods. Obesity is attracting attention as a cause of lifestyle-related diseases and diabetes. In particular, metabolic syndrome, which is a pathological condition in which diabetes, hypertension, hyperlipidemia and the like are accumulated, is focused on visceral obesity.

According to the 2007 National Health and Nutrition Survey by the Ministry of Health, Labor and Welfare in Japan, about 8.9 million people are strongly suspected of having diabetes. It is estimated that there are about 13.2 million people who cannot deny the possibility of diabetes, and about 22.1 million people in total. At age 40-74, one in two men and one in five women are considered highly suspected or a reserve group of metabolic syndrome.

In obese people, fat cells are enlarged, and neutral fat (triglyceride: TG) accumulates in the enlarged fat cells, and tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) It is said that the increase in secretion of inflammatory adipocytokines represented by the above and the production of anti-inflammatory adipocytokines such as adiponectin are decreased and insulin resistance is induced.

In obesity, macrophages infiltrate adipose tissue, which causes nitric oxide (NO), tumor necrosis factor α (TNF-α), monocyte chemotactic protein-1 (MCP-1), etc. It has been reported that cytokines and chemokines are generated and a chronic bioinflammatory reaction occurs, leading to the onset and exacerbation of insulin resistance.

Therefore, as a measure for suppressing obesity, diabetes, and hypertension, it is necessary to suppress adipocyte differentiation and suppress an increase in fat cells, and to suppress accumulation of triglyceride more than necessary. Furthermore, it is also necessary to suppress the secretion of inflammatory cytokines such as TNF-α by suppressing the activation of macrophages.

It has been reported that compounds such as resveratrol, quercetin, vitisin A, xanthohumol, isoxanthohumol, capsaicin derived from natural products suppress adipocyte differentiation and accumulation of triglycerides. In addition, it has been reported that compounds such as phloretin, retrofractaminde A, narigenin, hesperetin, catechin, 6-gingerol, curcumin and capsaicin increase the secretion of adiponectin from adipocytes. On the other hand, compounds such as curcumin and resveratrol are known to decrease TNF-α secretion from adipocytes.

Japanese Patent Application Laid-Open No. 2002-138044 contains at least one plant extract selected from Akane, Asunaro, Achacha, Greater Crested Beetle, Gaiyou, Hakukayumatou, Lotus, Kakikoshi and Houkigi, and induces differentiation of preadipocytes. Disinfecting drugs, cosmetic compositions or food and beverage products are disclosed.

  Furthermore, JP 2007-269757 A discloses an adipocyte differentiation inhibitor having an excellent inhibitory effect on induction of preadipocyte differentiation without side effects.

JP 2002-138044 A JP 2007-269757 A

Nemnoki (Albizzia julibrissin Durazz.) Is a plant of the leguminous family (Leguminosae), which is distributed from Iran, India, Southeast Asia, China, to the northeastern region of Japan. Nemunoki is a deciduous tree that grows naturally in the mountains, thick forests and river fields. The height reaches more than 10 m, and the leaves are even-numbered bilobed twice, and are 9-23 cm long. The flowers are pale red inflorescences and the flowering period is from June to August. A flower that has been dried in the early stages of flowering is known as a joy flower under the name of herbal medicine.

In the private sector, infused with insomnia, antidepressant, analgesia, sedation, diuresis. In addition, it is listed as a moderate item in the name of Neon in Shenong Hongsheng, the oldest drug book in China.
Although it contains Flavonoids such as Quercetin and Quercitrin as components of the joyous flowers, it is not known to have a differentiation action from preadipocytes to adipocytes.
The inventor of the present invention aims to obtain a novel ingredient having anti-obesity action and diabetes refining action, paying attention to Nemunoki.

  In order to achieve the above-mentioned object, the present invention is characterized by being an anti-obesity agent and an anti-diabetic agent containing an extract of a flower of anemone tree, in particular, a pleasant flower.

An extract of the flower part of anemone, its fractions and isolated compounds suppress the differentiation of preadipocytes into adipocytes, suppress the accumulation of triglycerides (TG), and improve obesity . It also improves diabetes with insulin resistance by eliminating obesity.

ADVANTAGE OF THE INVENTION According to this invention, the novel antiobesity agent and diabetes improvement agent which consist of a natural component of the flower part of a Nemnoki can be provided.

It is the list figure which showed the structural formula of the compound (plurality) which belongs to the extract of a joy flower. It is a table | surface which shows the effect | action which suppresses the differentiation of 3T3-L1 preadipocyte and the accumulation of triglyceride of each fraction of the extract of a joy flower. 3 is a table showing 1H-NNR spectrum data of Compound 1-4. 6 is a table showing 13C-NNR spectrum data of Compound 1-4. FIG. 5 is a view showing the properties of compound 1-4. 2 is a structural formula showing a partial structure of Compound 2. . It is a table | surface which shows the result of the TG accumulation | storage suppression test and the GPDH activity test about each compound isolated from the extraction part of the joy flower.

The present inventor extracted the joyous flowers, isolated the components and determined the structure, 10 kinds of Flavonol and its glycoside (compound 1-10), 5 kinds of Flavone (compound 11-15), 1 kind Flavanonol (Compound 16), 1 Calcone (Compound 17), 2 terpenoids (Compound 18-19), 3,3'-Dithiodipropanoic acid (Compound 20), indole derivative (Compound 21), Alloxazine derivative (Compound) 22) and phenylpropanoid (Compound 23) were obtained. Of these, compound 1-4 is a novel compound, and compound 20 is the first compound isolated from nature. FIG. 1 shows the structural formulas of Compounds 1 to 23.

Compound 1 is 3 ''-(E) -p-Coumaroylquercitrin,
Compound 2 is 3 ″-(E)-(3 ′ ″-Methoxycaffeoyl) quercitrin,
Compound 3 is 2 ''-(E) -Cinnamoylquercitrin
Compound 4 is 3 ''-(E) -Cinnamoylquercitrin
Compound 5 is Quercetin,
Compound 6 is Isorhamnetin,
Compound 7 is Rhamnetin,
Compound 8 is Quercitrin
Compound 9 is Kaempferol,
Compound 10 is Afzelein,
Compound 11 is Chrysoeriol,
Compound 12 is Apigenin,
Compound 13 is Tricin,
Compound 14 is Luteolin,
Compound 15 is Amentoflavone,
Compound 16 is Taxifolin,
Compound 17 is Isoliquiritigenin,
Compound 18 is α-Spinasterol-3-O-β-D-glucopyranoside,
Compound 19 is (6S) -Menthiafolic acid,
Compound 20 is 3,3'-Dithiodipropanoic acid,
Compound 21 is Indole-3-carboxylic acid,
Compound 22 is 7,8-Dimethylalloxazine and
Compound 23 is (E) -p-Coumaric acid.

Furthermore, the inhibitory effect of these compounds on 3T3-L1 preadipocyte differentiation and neutral fat accumulation was examined.

Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples.

A 15 kg flower portion of the tree (Albizzia julibrissin Durazz.) Was extracted with 80% ethanol, and the extract was concentrated and dried to obtain an extract (3.5 kg). This extract was suspended in water and partitioned and extracted sequentially with n-hexane, chloroform, ethyl acetate, and n-butanol. Each fraction was subjected to a TG production suppression test, and at a sample concentration of 30 μg / ml, the hexane elution fraction (55.5%), chloroform elution fraction (58.6%), ethyl acetate elution fraction (70.9%), n-butanol Suppression of TG accumulation was observed in the fraction (82.1%, Cytotoxicity) and the water fraction (91.3%). Furthermore, the GPDH activity test was performed about each fraction. The test results are shown in FIG. Methods for testing TG accumulation inhibition and GPDH activity will be described later.

1) Separation of ethyl acetate elution fraction The ethyl acetate elution fraction (165.5 g) was applied to a Silica gel column (12.5 φ × 21 cm) and eluted with CHCl3: MeOH = 100: 0 to 0: 100. Fractions were divided into A to B. At this time, the precipitated precipitate was collected by filtration and recrystallized to obtain Compound 18 (423.8 mg) (Fig. 2).

  AJE-B was applied to a Silica gel column (4.5 φ × 16 cm) and eluted with Hexane: EtOAc = 100: 0 to 0: 100, and fractionated into AJE-B1 to 11. Crystals precipitated from AJE-B2, and were collected by filtration and recrystallized to obtain Compound 20 (230 mg) (Fig. 3).

  Since the spots were similar by TLC, AJE-B4 and B5 were combined, applied to an ODS column (2.7φ × 18 cm), and eluted with H2O: MeOH = 100: 0 to 0: 100. AJE-B4 + Fractions into 5a ~ i (Fig.3). At this time, a precipitate was deposited on B4 + 5e. This was collected by filtration and recrystallized to obtain Compound 22 (10.5 mg) (Fig. 6).

  AJE-B4 + 5c was applied to a Sephadex LH-20 column, eluted with CHCl3: MeOH = 50: 50, and fractionated into B4 + 5c-1 to 6 (Fig. 6). Further, B4 + 5c-3 was subjected to HPLC (COSMOSIL Cholester 10φ × 250 mm, 0.1% TFA: MeOH = 50: 50) to obtain B4 + 5c-3a to c. Further, B4 + 5c-3b was subjected to HPLC (Shodex GF310 HQ 4.6φ × 250 mm, CHCl 3: MeOH = 95: 5) to obtain Compound 23 (1.8 mg, retention time 22 m00s) (FIG. 7).

  AJE-B4 + 5c-5 was subjected to HPLC (COSMOSIL πNAP 10φ × 250 mm, 0.1% TFA: MeOH = 50: 50) to obtain compound 21 (4.4 mg, retention time 16 m48s) (FIG. 6).

  AJE-B4 + 5h was applied to a Sephadex LH-20 column, eluted with H2O: MeOH = 40: 60, and fractionated into AJE-B4 + 5h-1 to 6, of which B9 + 5h-6 Obtained (Fig.4).

  AJE-B4 + 5h-3 was subjected to HPLC (COSMOSIL Cholester 10φ × 250 mm, 0.1% TFA: MeOH = 63: 37), compound 11 (2.6 mg, retention time 30 m00s), 12 (1.4 mg, retention time 32 m12s) 13 (3.2 mg, retention time 34 m06 s) was obtained.

  AJE-C was applied to a Silica gel column (8φ × 16 cm) and eluted with Hexane: EtOAc = 100: 0 to 0: 100 to obtain C1 to 5 (Fig. 8).

  AJE-C2 was subjected to HPLC (COSMOSIL 5C18-MS-II 10φ × 250 mm, H2O: CH3CN = 70: 30) and fractionated into C2a and C2b. Of these, C2b was Compound 5 (64 mg, retention time 13m48s) (Fig.8). C2a was purified by HPLC (COSMOSIL 5C18-MS-II 10φ × 250 mm, H 2 O: CH 3 CN = 82: 18) to obtain Compound 16 (2.7 mg, retention time 19 m 12 s) (FIG. 8).

  AJE-C3 was applied to an ODS column (2.7φ × 15 cm), eluted with H 2 O: MeOH = 100: 0 to 0: 100, and fractionated into AJE-C3a to h. Among these, C3d was subjected to HPLC (COSMOSIL πNAP 10φ × 250 mm, 0.1% TFA: MeOH = 50: 50) to obtain compounds 1 (18.6 mg, retention time 13 m12s) and 2 (19.5 mg, retention time 16 m10s) (Fig. .9)

  AJE-C3e was subjected to HPLC (COSMOSIL Cholester 10φ × 250 mm, 0.1% TFA: CH3CN = 60: 40) and fractionated into C3e-1 to 5. Further, C3e-4 was fractionated into C3e-4a to 4c by HPLC (COSMOSIL πNAP 10φ × 250 mm, 0.1% TFA: MeOH = 32: 68), 4c of which was obtained as compound 15 (3.3 mg, retention time 22m48s). (Fig.10).

  AJE-C3e-5 was subjected to HPLC (COSMOSIL πNAP 10φ × 250 mm, 0.1% TFA: MeOH = 40: 60) to obtain compounds 3 (4.5 mg, retention time 13 m50s) and 4 (3.3 mg, retention time 17 m24s). (Fig.11)

AJE-D (250 mg / 162.9 g) was subjected to HPLC (COSMOSIL πNAP 10φ × 250 mm, 0.1% TFA: MeOH = 40: 60), and compound 8 (129 mg, retention time 9m00s) and 10 (6.6 mg, retention time) 13m48s) was obtained (Fig.11).

2) Separation of the fraction eluted with chloroform
AJC was applied to a Silica gel column (12φ × 20 cm), eluted with Hexane: EtOAc = 100: 0 to 0: 100, and fractionated into AJC-A to O. Among them, AJC-G was applied to a Silica gel column (3.5φ × 20 cm) and eluted with CHCl 3: MeOH = 100: 0 to 0: 100 and fractionated into AJC-G 1 to 5 (FIG. 12). Further, AJC-G3 was subjected to HPLC (COSMOSIL 5C18-MS-II 10φ × 250 mm, H 2 O: MeOH = 57: 43) to obtain Compound 19 (342 mg, retention time 13 m00s) (FIG. 13).

Next, the results of the structural analysis of compound 1-4 will be described.
Compound 1 (3 ''-(E) -p-Coumaroylquercitrin)
Compound 1 is obtained as a yellow powder and exhibits [α] 25D -146 °. From Negative FAB-MS, m / z 593 [MH]-and HR-FAB-MS are simulated at m / z 593.1301 (calcd. 593.1294). The molecular formula C30H26O13 was estimated from the molecular ion peak. In IR, strong absorption based on a hydroxyl group was observed at 3378 cm-1, a carbonyl group at 1654 cm-1, and an absorption based on a conjugated double bond at 1603 cm-1. In the UV spectrum, maximum absorption was observed at 267 nm and 315 nm, and a flavonoid derivative was estimated (FIG. 5).

  In 1H-NMR spectrum, proton signal to metacouple [δH 6.23 (d, J = 2.0 Hz), 6.42 (d, J = 2.0 Hz)], proton signal to split into ABX type [δH6.89 (d, J = 8.3 Hz), 7.31 (d, J = 2.1 Hz) and 7.33 (dd, J = 8.3, 2.1 Hz)], A2B2 type proton signal [δH6.82 (d, J = 8.6 Hz), 7.50 (d, J = 8.6 Hz)] and olefinic protons [δH6.42 (d, J = 15.8 Hz), 7.62 (d, J = 15.8 Hz)] having a trans double bond were observed. Furthermore, a signal that appears to be a chelate hydroxyl group at δH12.6 and a sugar anomeric proton at δH5.23 was observed (FIG. 3).

  In the 13C-NMR spectrum and DEPT spectrum, 11 quaternary carbons (δC: 104.6, 121.2, 125.7, 134.9, 145.8, 149.0, 156.9, 157.8, 160.2, 161.7, 164.7), 16 methines (δC: 68.2, 68.7, 71.3, 74.2, 94.2, 99.2, 102.5, 115.2, 115.6, 116.3 × 3, 121.8, 130.7 × 2, 145.0,), 1 methyl (δC: 17.6), 2 carbonyl groups (δC: 166.9, 178.2 ) (Fig. 4).

  Furthermore, the partial structure of Compound 1 was estimated by analyzing the HMQC spectrum. Quercetin and Rhamnose were detected by acid hydrolysis of compound 1. The sugar was estimated to be α-Rhamnose by 1H-1H COZY and HMQC spectra.

  From the HMBC spectrum, it was estimated that (E) -p-Coumaric acid was bound to the 3 ″ position of α-Rhamnose. Thus, Compound 1 was determined to be 3 ″-(E) -p-Coumaroylquercitrin).

Compound 2 (3 ''-(E)-(3 '''-Methoxycaffeoyl) quercitrin)
Compound 2 is obtained as a yellow powder, shows [α] 25D -158 °, and is simulated by m / z 623 [MH]-from Negative FAB-MS and m / z 623.1398 (calcd. 623.1399) from HR-FAB-MS. The molecular formula C31H27O14 was estimated from the molecular ion peak. In IR, strong absorption based on hydroxyl groups was observed at 3422 cm-1, absorption based on conjugated double bonds at 1653 cm-1 carbonyl groups, and 1603 cm-1. In the UV spectrum, maximum absorption was observed at 267 nm and 333 nm, and a flavonoid derivative was estimated (FIG. 5).

  In 1H-NMR spectrum, proton signal metacoupling [δH6.24 (d, J = 2.0 Hz), 6.45 (br)], proton signal splitting into ABX type [δH6.90 (d, J = 8.8 Hz) , 7.32 (br) and 7.33 (br), 6.83 (d, J = 8.0 Hz), 7.13 (dd, J = 8.0, 2.0 Hz) and 7.32 (br)] and olefinic protons with trans double bonds [ δH6.50 (d, J = 16.0 Hz), 7.61 (d, J = 16.0 Hz)] was observed. Furthermore, a proton signal based on the methoxyl group was observed at δH3.83 (s), a chelate hydroxyl group at δH12.4, and a signal that was considered to be an anomeric proton of the sugar at δH5.22 (d, J = 1.5 Hz) (Fig. 3).

  From 13C-NMR spectrum and DEPT spectrum, 12 quaternary carbons (δC: 104.5, 121.2, 126.2, 135.0, 145.8, 148.5, 149.1, 149.8, 156.9, 157.8, 161.7, 164.8), 15 methines (δC: 68.2, 68.8, 71.3, 74.2, 94.2, 99.3, 102.6, 111.5, 115.6, 116.0 × 2, 116.1, 121.8, 123.6, 145.4), 1 methyl (δC: 17.9), 1 methoxyl group (δC: 56.2 ) And two carbonyl groups (δC: 166.9, 178.2) were observed (FIG. 4).

    Further, the partial structure of Compound 2 was estimated by analyzing the HMQC spectrum. In addition, since the spectrum data of 1H-NMR and 13C-NMR were very similar when compared with compound 1, it was estimated that compound 2 was the same Quercitrin derivative as 1. Therefore, it is presumed that the partial structure is derived from Quercetin.

  From 1H-1H COZY and HMQC spectra, the continuity was traced from the anomeric proton signal δH5.23 (d, J = 1.5), and the proton and carbon signals of the sugar moiety were assigned as shown in Fig. 26. In addition, since 1JCH shows 176.2 Hz, the hydrogen at the 1-position was determined to be the α-position. From this, the constituent sugar was estimated to be α-Rhamnose.

  From the HMBC spectrum, long range correlations were observed from δH7.61 (H-7 '' ') to δC111.5 (C-2' '') and 123.6 (C-6 '' '). Long range correlations were observed from δH3.83 (OMe) and δH6.83 (H-5 '' ') and 7.32 (H-2' '') to δC148.5 (C-3 '' '). Furthermore, long range correlations were observed from olefin protons at δH7.61 (H-7 '' ') and 6.50 (H-8' '') at δC166.9 (C-9 '' '). Long range correlations were observed from hydrogen at δH6.50 (H-8 '' ') and 6.83 (H-5' '') to carbon at δC126.2 (C-1 '' '). Based on the above, the partial structure as shown in FIG.

As a result of analyzing the HMBC spectrum to determine the binding position of α-Rhamonose, a long range correlation was observed from δH5.22 (H-1 ') to δC135.0 (C-3). It was revealed that it was linked to the third position of Quercetin. Furthermore, as a result of analyzing the HMBC spectrum in order to determine the bonding position of the structure of FIG. 6, long range correlation was observed in δH4.95 (H-3 ''') and δC166.9 (C-9'''). As a result, it became clear that the structure of Fig. 6 was bonded to the 3 "position of α-Rhamnose.
Based on the above results, Compound 2 was determined to be 3 ″-(E)-(3 ′ ″-Methoxycaffeoyl) quercitrin.

Compound 3 (2 ''-(E) -Cinnamoylquercitrin)
Compound 3 is obtained as a yellow powder and exhibits [α] 25D -143 °. From Negative FAB-MS, m / z 577 [MH]-and HR-FAB-MS are simulated at m / z 577.1350 (calcd. 577.1345). The molecular formula C30H26O12 was estimated from the molecular ion peak. In IR, a strong absorption based on a hydroxyl group was observed at 3418 cm-1, a carbonyl group at 1652 cm-1, and an absorption based on a conjugated double bond at 1606 cm-1. In the UV spectrum, maximum absorption was observed at 267 nm and 333 nm, and a flavonoid derivative was estimated (see FIG. 5).

  In 1H-NMR spectrum, proton signal to metacouple [δH 6.22 (d, J = 2.0 Hz), 6.42 (d, J = 2.0 Hz)], proton signal to split into ABX type [δH6.90 (d, J = 8.3 Hz), 7.30 (dd, J = 8.3, 2.3 Hz) and 7.35 (d, J = 2.3 Hz)] and the presence of mono-substituted benzene due to the presence of 5H proton signals at δH7.43 and 7.74 Was estimated. In addition, olefinic protons with trans double bonds [δH6.64 (d, J = 16.0 Hz), 7.67 (d, J = 16.0 Hz)] and δH5.51 (d, J = 1.5 Hz) and sugar anomeric A signal that seems to be a proton was observed (see FIG. 3)).

In the 13C-NMR spectrum and DEPT spectrum, 10 quaternary carbons (δC: 104.2, 120.7, 133.5, 134.1, 145.5, 148.8, 156.6, 157.5, 161.4, 164.5), 17 methines (δC: 68.7, 70.9, 71.6, 72.0, 93.9, 98.5, 98.9, 116.0, 116.1, 118.1 121.8, 128.6 × 2, 129.1 × 2, 130.7, 145.3), 1 methyl (δC: 17.6), 2 carbonyl groups (δC: 165.6, 178.2) was observed (see Fig. 4).
Further, the structure of Compound 3 was analyzed using the results of 13C-NMR spectrum, DEPT spectrum, HMQC spectrum, and HMBC spectrum.

Compound 4 (3 ''-(E) -Cinnamoylquercitrin)
Compound 4 is obtained as a yellow powder and exhibits [α] 25D -125 °, from Positive FAB-MS m / z 579 [M + H] +, HR-FAB-MS m / z 579.1496 (calcd. 579.1501) The molecular formula C30H26O12 was estimated from quasi-molecular ion peak. In IR, strong absorption based on hydroxyl groups was observed at 3422 cm-1, carbonyl groups at 1653 cm-1, and absorption based on conjugated double bonds at 1607 cm-1. In the UV spectrum, maximum absorption was observed at 270 nm and 340 nm, and the flavonoid derivative was estimated (FIG. 5).

  In 1H-NMR spectrum, proton signal to metacouple [δH 6.22 (d, J = 2.0 Hz), 6.41 (d, J = 2.0 Hz)], proton signal to split into ABX type [δH6.90 (d, J = 8.3 Hz), 7.30 (dd, J = 8.3, 2.0 Hz) and 7.32 (d, J = 2.0 Hz)] and δH7.44 7.74, because there is a proton signal for 5H, the presence of mono-substituted benzene Was estimated. In addition, olefinic protons with trans double bonds [δH6.67 (d, J = 16.0 Hz), 7.72 (d, J = 16.0 Hz)] and δH5.24 (d, J = 1.5 Hz) and sugar anomeric A signal that seems to be a proton was observed (Fig. 3).

In the 13C-NMR spectrum and DEPT spectrum, 10 quaternary carbons (δC: 104.3, 120.8, 134.3, 134.6, 145.5, 148.7, 156.7, 157.5, 161.5, 164.4), 17 methines (δC: 68.4, 67.8, 70.9, 74.2, 93.8, 98.9, 102.1, 115.6 × 2, 118.8 121.5, 128.5 × 2, 129.2 × 2, 130.6, 144.5), 1 methyl (δC: 17.6), 2 carbonyl groups (δC: 166.2, 177.8) was observed (Fig. 4)
Furthermore, the structure of Compound 4 was analyzed using the results of 13C-NMR spectrum, DEPT spectrum, HMQC spectrum, and HMBC spectrum.

Triglyceride (TG) accumulation inhibition test and glycerol triphosphate dehydrogenase (GPDH) activity test
1) Differentiation induction and promotion test from 3T3-L1 preadipocytes to adipocytes
3T3-L1 preadipocytes were cultured at 37 ° C. with 5% CO 2. For the adipocyte differentiation inhibition test, cells in the logarithmic growth phase were prepared at 1.0 × 10 5 cells / mL and used. After culturing in basic medium (DMEM medium containing 10% FCS) for 2 days (Day 0), 3-isobutyl-1-methylxanthine (IBMX, 500 μM), dexamethasone (DEX, 1 μM) and insulin (10 μg / mL) ) -Containing differentiation induction medium (DMEM medium containing 10% FBS) and cultured for 3 days (Day 3). Furthermore, the test drug was added to a differentiation maintenance medium containing insulin (10 μg / mL), and the culture medium was replaced with the same medium every two days and cultured for 8 days (Day 8). The supernatant of the differentiated cells was collected in a tube, and the adipocytes were washed twice with phosphate buffered saline (PBS (−)). 500 μl of 25 mM Tri buffer (pH 7.5) containing Tris-HCl buffer containing 1 mM EDTA was added, the cells were detached, and collected in a 1.5 ml plastic tube. While cooling with ice water, the cell membrane was disrupted by sonication, and then centrifuged at 4 ° C., 15,000 rpm for 2 minutes.

2) Measurement of triglyceride (TG) content A portion of the cell homogenate was taken, and triglyceride (TG) accumulated in the cells was quantified using a lab assay triglyceride (Wako Pure Chemical Industries).
Triglyceride coloring reagent was added to the cell homogenate of each sample, mixed well, and heated at 37 ° C. for 5 minutes. Using the blank as a control, the absorbance of the sample and the reference solution at a wavelength of 595 nm was measured. The inhibition rate was calculated by the following formula.

Suppression rate (%) = {(Y-X) / Y} x 100
X: The amount of TG per well when the sample is added divided by the amount of DNA per well
Y: The amount of TG per well when DMSO is added divided by the amount of DNA per well.
3) A portion of glycerol 3-phosphate dehydrogenase (GPDH) active cell homogenate was taken and GPDH was used with a GPDH activity measurement kit (Primary Cell Co., Ltd.).

The reaction substrate solution was placed in a spectrophotometer cell, a sample was added to the cell, and the decrease in absorbance at a wavelength of 340 nm was measured over time to determine the amount of change in absorbance per minute (ΔO.D.).

Calculation of activity values:
The GPDH per 1 ml of the sample consumed 1 μM NADH per minute as 1 U, and the GPDH activity was calculated by the following formula (when the optical path length was 1 cm).

GPDH activity (U / ml) = ΔO.D. × 0.482
ΔO.D .: Change in absorbance at a wavelength of 340 nm per minute
Suppression rate (%) = {(Y-X) / Y} x 100
X: GPDH activity per well when the sample is added divided by the amount of DNA per well
Y: GPDH activity per well when DMSO is added divided by the amount of DNA per well.

4) Quantification of DNA A portion of the cell homogenate was taken, and the amount of intracellular DNA was quantified using a DNA activity measurement kit (Primary Cell Co. Ltd.). As a result, when a TG accumulation suppression test and a GPDH activity test were performed on the extract of Heiseihana and each fraction, a strong effect was recognized on the water fraction and the ethyl acetate fraction as shown in FIG. . The results are shown by the ± SD (n = 3) method. The sample concentration is 30 μg / ml.

Furthermore, a TG accumulation suppression test and a GPDH activity test were performed on the compounds obtained from Heiseihana. The result is shown in FIG. The sample concentration is 30 μM. Compared with quercetin (5), which is known to have an adipocyte differentiation and TG accumulation inhibitory effect used as a positive control, all flavonoids showed similar or strong inhibition of TG accumulation and GPDH activity as compound 5. It was. In particular, strong activity was observed for Compound 2 and Compound 7.

  As explained above, the fraction and active ingredient of the extract of the flower part of Nemnoki suppress the differentiation of the adipocytes into adipocytes by the TG accumulation inhibitory action and the GPDH activity inhibitory action, and the fat By inhibiting the accumulation of TG in cells, it achieves anti-obesity action and diabetes improvement effect.

In order to apply the active ingredient of the present invention to humans or animals, all known dosage forms such as internal use, injection, and external use can be used. The active ingredient of the present invention can be used as a drug, health food, and external preparation.

Claims (2)

An anti-obesity agent or a diabetes-improving agent characterized by containing 3 ″-(E)-(3 ′ ″-Methoxycaffeoyl) quercitrin or Rhamnetin as an active ingredient , which is an extract of Nemunoki. The anti-obesity agent or diabetes improving agent according to claim 1, wherein the extract belongs to a fraction of ethyl acetate or water in the flower part .