JP2013177355A - Natural ingredient and extract derived from organic body for preventing and treating metabolic syndrome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a herbal medicine ingredient and an extract that are derived from an organic body for preventing and treating a metabolic syndrome by paying attention to white peach blossom.SOLUTION: The herbal medicine ingredient is aromadendrin as a natural ingredient included in the extract of a methyl acetate fraction derived from a white peach blossom. Further, the extract is an extract of the methyl acetate fraction or a water fraction. The herbal medicine ingredient and the extract can promote the differentiation of a precursor adipose cell into an adipose cell to prevent and treat a metabolic syndrome. The herbal medicine ingredient and the extract can be made into active ingredients in optional drugs, quasi drugs, cosmetics or food and drink such as health supplements.

Description

  The present invention relates to natural ingredients and extracts derived from organisms for preventing and treating metabolic syndrome, and foods and drinks such as pharmaceuticals and health supplements using the natural ingredients and extracts, cosmetics, and quasi drugs About.

  Metabolic syndrome (metabolic syndrome) is a pathological condition that combines two or more of diabetes, hyperlipidemia, and hypertension, and has recently attracted attention as one of the risk factors for cardiovascular diseases such as myocardial infarction and cerebral infarction. ing. Metabolic syndrome is roughly classified into two types, that is, metabolic syndrome caused by obesity and metabolic syndrome caused by fat atrophy (FIG. 1).

  Metabolic syndrome due to obesity is caused by the accumulation of visceral fat due to the enlargement of healthy small fat cells. The enlargement of small fat cells is caused by lifestyle habits such as intake of a high fat diet and lack of exercise. In hypertrophic adipocytes, secretion of inflammatory adipocytokines (eg, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6)) increases and anti-inflammatory adipocytokines (eg, insulin sensitivity). Production of the cytokine adiponectin) is reduced and insulin resistance is induced.

  Therefore, one method of suppressing metabolic syndrome due to obesity is to suppress differentiation from small adipocytes to hypertrophic adipocytes and reduce secretion of inflammatory adipocytokines. Patent Document 1 or 2 discloses a natural component derived from an organism and an extract for suppressing differentiation from preadipocytes to small adipocytes.

  On the other hand, in metabolic syndrome due to obesity, it is known that blood adiponectin level can be increased and insulin resistance can be improved by promoting differentiation from preadipocytes to small adipocytes (Non-patent Document 1). .

  Moreover, in the metabolic syndrome due to fat atrophy, the differentiation of preadipocytes is impaired, resulting in a decrease in blood adiponectin level and thus insulin resistance. Therefore, in order to suppress metabolic syndrome due to fat atrophy, it is necessary to promote differentiation from preadipocytes to small adipocytes.

  Furthermore, the decrease in the amount of adiponectin secretion promotes inflammatory changes in adipocytes and macrophages, which are secreted from macrophages activated by enlarged fat cells and free fatty acids secreted from fat cells. It is known that it is caused by

  From the above, in order to prevent and improve metabolic syndrome, it is in the art to identify a natural component that can promote differentiation from preadipocytes to small adipocytes, or a natural component that can suppress macrophage activation. It is an important issue.

JP 2010-202558 A JP 2010-202602 A Japanese Patent Application Laid-Open No. 2010-083838 JP 2010-202536 A JP 2011-153093 A

Yamauchi T et al .: Nat. Med. , 2001; 7: 941. Jeon, S.J. H. Chun, W .; Choi,. Y. J. et al. Kwon, Y .; S. Cytotoxic Constituents from the Bark of Salix ulteni. Arch. Pharm. Res. , 32 (8), 978-982 (2008) Takahashi, H .; Li, S .; Harigaya, Y .; Onda, M .; Heterocycles. XXII. Stereoselective Synthesis of (+)-Aromadendrin Trimethyl Ether and It's Enantiomer, and Their Reduction. Chem. Pharm. Bull. , 36 (5), 1877-1881 (1988) Kwak, J .; H. Kang, M .; W. Roh, J .; H. Choi, S .; U. Zee, O .; P. Cytotoxic Phenolic Compounds from Chiananthus retusus. Arch. Pharm. Res. , 32 (12), 1681-1687 (2009) Zhang, Y. et al. Que, S .; Yang, X .; Wang, B .; Qiao, L .; Zhao, Y .; Isolation and identification of metabolites from dihydromyricin. Magn. Reson. Chem. 45, 909-916 (2007) Li Y. L. Li J .; Wang N .; L. Yao X .; S. Moleculars, 13, 1931-1941 (2008). Gao, D .; F. Xu, M .; Yang, C .; R. Xu, M .; Zhang, Y .; Phenolic Antioxidants from the leaves of Camellia pachyandra Hu. J. et al. J. et al. Agric. Food Chem. , 58, 8820-8824 (2010) Jeon, S.J. H. Chun, W .; Choi, Y .; J. et al. Kwon, Y .; S. Cytotoxic Constituents from the Bark of Salix ulteni. Arch. Pharm. Res. , 31 (8), 978-982 (2008) Nessa, F .; Ismail, Z .; Mohamed, N .; Harris, M .; R. H. K. Free radical-scavenging activity of organic extracts and of pure flavonoids of Blume balsamifera DC leaves. Food Chem. , 88, 243-252 (2004) Rey, P.M. Rossi, J .; C. Taylores, J .; Gros, G .; Nore, O .; Hydrology of Nitsles using an Immobilized Nitrase: Applications to the Synthesis of Methineline Hydrology Analogue Derivatives. J. et al. Agric. Food Chem. , 52, 8155-8162 (2004) Karl C.I. Muller G .; Pedersen P .; , Phytochemistry, 15, 1084-1085 (1976).

  Differentiation of preadipocytes is controlled by transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and peroxisome proliferator-activated receptor γ (C / EBP) family. Pioglitazone, a therapeutic agent for diabetes, is a ligand of PPARγ and increases insulin resistance by increasing small adipocytes through activation of PPAR and thus increasing blood adiponectin levels.

  Patent Documents 3 to 5 disclose organic-derived natural components and extracts that can promote differentiation from preadipocytes to small adipocytes, and are known to have the same effects as pioglitazone. . In addition, it is known that natural ingredients and extracts derived from a large number of organisms (for example, vanillin, sakuranelin, nobiletin) have the same effect.

  Natural components and extracts derived from organisms (ie, natural herbal medicines) are superior to non-natural component pharmaceuticals in terms of safety, so natural components derived from organisms for the prevention and improvement of metabolic syndrome And the use of extracts is particularly sought in the art.

  An object of the present invention is to provide a novel herbicidal ingredient derived from an organism for preventing and treating metabolic syndrome, focusing on white peach blossoms.

  In one aspect of the present invention, the herbal medicine component of the present invention is an extract derived from an organism for promoting differentiation of preadipocytes, and is an extract derived from an organism containing aromadendrin.

  In one embodiment of the present invention, the organism is a white peach flower.

  In another embodiment of the present invention, the organism-derived extract of the present invention is an extract of an ethyl acetate fraction.

  In another aspect of the present invention, the herbal medicine component of the present invention is an extract derived from white peach blossom for promoting the differentiation of preadipocytes.

  In one embodiment of the present invention, the white peach flower-derived extract of the present invention is an extract of an ethyl acetate fraction or an extract of a water fraction.

  In an alternative embodiment of the present invention, an extract derived from an organism such as white peach blossom in the present invention may be included in any pharmaceutical product, food or drink such as health supplement, cosmetics or quasi drugs.

  Another aspect of the present invention is a food / beverage product, a cosmetic product or a quasi-drug such as a pharmaceutical product and a health supplement for promoting differentiation of preadipocytes, which contains aromadendrin as an active ingredient.

  Since the aromadendrin derived from an organism has an effect of promoting differentiation of preadipocytes and an effect of promoting adiponectin secretion in adipocytes, an effect of preventing and improving metabolic syndrome is achieved.

  Since the kaempferol derived from the organism has an inhibitory effect on macrophage activation, the prevention and improvement effect of metabolic syndrome is achieved.

  Prevention and improvement of metabolic syndrome because the extract of ethyl acetate or water fraction derived from white peach blossoms has the effect of promoting differentiation of preadipocytes, the effect of promoting adiponectin secretion in adipocytes, and the effect of suppressing macrophage activation The effect is achieved.

  Since the above-mentioned natural ingredients and extracts derived from organisms are superior to non-natural ingredients from the viewpoint of safety, they are widely used as foods and drinks such as pharmaceuticals and health supplements, cosmetics or quasi-drugs. It is suitable for various uses.

FIG. 1 is a schematic diagram showing the role of adipocytes in the development of metabolic syndrome. FIG. 2 is a graph showing the increase rate of glycerol 3-phosphate dehydrogenase (GPDH) activity relative to control (100%) in the sample of the present invention as an index for promoting differentiation of preadipocytes. FIG. 3 is a graph showing the rate of increase in the amount of triglyceride (TG) accumulated in adipocytes relative to control (100%) in the sample of the present invention. FIG. 4 is a graph showing the results of Oil Red O staining of small adipocytes in an 80% ethanol extract, and the lower graph is a graph showing the increase rate of TG accumulation relative to the control (100%). FIG. 5 is a graph showing the increase rate of adiponectin secretion with respect to the control (100%) in the sample of the present invention. FIG. 6 is a graph showing the inhibition rate of nitric oxide (NO) production relative to the control (100%) in the sample of the present invention as an index of suppression of macrophage activation.

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

In the present invention, focusing on white peach blossoms of the rose family peach (Prunus persica) as an organism, the natural components of white peach blossoms were separated and purified for component analysis. Furthermore, the effect of treatment and prevention of metabolic syndrome in the extract derived from white peach blossoms and the natural components analyzed for ingredients was examined.

[material]

  In the present invention, white peach flowers of the family Rosaceae are used as the organic material.

  The peach is a deciduous tree of the Rosaceae family and is native to the Upper Yellow River in northwestern China. The peach grows over 5 meters in height. The peach leaves are alternating, skin-shaped and hairy, and the peach blossoms that bloom in early spring are white to peach-red petal flowers. “Momojin” is a herbal medicine name for dried peach seeds. "White peach blossom" is the name of herbal medicines for dried flowers and buds.

  White peach blossoms are known to have diuretic, laxative and blood circulation improving effects and are used in the private sector to treat edema and constipation. According to recent studies, the blood circulation improvement effect is due to the production-suppressing action of plasminogen activator inhibitor 1 (PAI-1) and that prunasinic acid and neoprnasinic acid are one of the active ingredients. It has become clear.

Furthermore, flavonoids such as kaempferol and naringenin, flavonoid glycosides such as triphorin, and coumarins such as coumarin have been clarified as natural components of white peach flowers.

[Component Separation Equipment and Component Analysis Equipment]

<Component Separation Equipment>
In an embodiment of the present invention, as a column chromatography packing material:
Silica gel 60N (Kanto Chemical);
YMC-GEL ODS-AS-150 (YMC);
Sephadex® LH-20 (GE Healthcare Japan);
Activated Charcoal (or Wako Pure Chemical Industries); or
MCI GEL CHP20P (Mitsubishi Chemical);
Was used.

In an embodiment of the present invention, in thin layer chromatography (TLC):
Silica gel 60 F ₂₅₄ (MERCK); or RP-18F ₂₅₄ (MERCK);
Was used.

<Component analysis equipment>
In high performance liquid chromatography (HPLC), as a detector:
JASCO UV-2075 Plus;
JASCO UV-1575; or
SSC-5410;
Use as a pump:
JASCO PU-2089 Plus;
JASCO PU-1580; or
JASCO PU-986;
As a column:
COSMOSIL πNAP (Φ10 × 250 mm);
COSMOSIL 5C ₁₈ -MS-II (Φ10 × 250 mm); or
Shodex GF310 HQ (Φ4.6 × 250 mm);
Was used.

  The nuclear magnetic resonance (NMR) apparatus used was: JEOL Delta 600; or JEOL Delta 500;

  JEOL GCmate was used as the mass spectrometer (MS) apparatus.

As the polarimeter, JASCO P-1020 Polarimeter was used.

[Separation and purification of natural components of white peach blossom]

  A commercially available white peach flower (1.5 kg) was extracted with 15 L of 80% ethanol, and the extract was concentrated and dried to obtain 300 g of an ethanol extract. Thereafter, the ethanol extract was dissolved in 2 L of water and extracted sequentially step by step using an equal volume of n-hexane, ethyl acetate, and n-butanol. The weight of the n-hexane extract fraction was 35.0 g, the weight of the ethyl acetate extract fraction was 54.4 g, and the weight of the n-butanol extract fraction was 64.1 g.

  Among these extract fractions, the ethyl acetate extract fraction exhibited a strong preadipocyte differentiation promoting effect (Example 1), and therefore the ethyl acetate extract fraction (54.4 g). Is subjected to normal phase silica gel column chromatography (Silica gel 60N) and eluted in an n-hexane-ethyl acetate system with the ratio of n-hexane: ethyl acetate changed from 100: 0 to 0: 100, then 100 Fractionation into 7 fractions (PrPE-A to PrPE-G) eluting with% acetone and 100% methanol.

  In order to obtain a natural component of white peach blossom, 3 fractions out of 7 fractions (PrPE-C (6.94 g), PrPE-D (7.28 g), and PrPE-F (21.3 g)) Further purification of was performed.

<Separation of fraction PrPE-C>
Fraction PrPE-C (6.94 g) was subjected to normal phase silica gel column chromatography (Silica gel 60N), and the ratio of chloroform: methanol was changed from 100: 0 to 0: 100 in a chloroform-methanol system. Elution and fractionation into 10 fractions (PrPE-CA to PrPE-C-J).

  Of these 10 fractions, 4 fractions (PrPE-C-D (2.02 g), PrPE-CE (121 mg), PrPE-C-F (153 mg), and PrPE-C-H ( Further separation and purification of 50 mg)) was carried out.

<Separation of fraction PrPE-CD and purification of compound>
Fraction PrPE-C-D (2.02 g) was subjected to reverse phase column chromatography (YMC-GEL ODS-AS-150), water-methanol system, water: methanol ratio from 100: 0. Elution was carried out until 0: 100, and fractionated into 10 fractions (PrPE-C-D-1 to PrPE-C-D-10).

  Of these 10 fractions, the compound (477 mg) was purified from the fraction PrPE-CD-4 (477 mg) (“compound 7” in the present specification).

<Separation of fraction PrPE-CE and purification of compound>
Fraction PrPE-CE (121 mg) was subjected to high performance liquid chromatography (COSMOSIL Cholester Φ10 × 250 mm, 0.1% trifluoroacetic acid: acetonitrile (60:40), flow rate 4.0 mL / min, wavelength 210 nm). And fractionated into 4 fractions (PrPE-CE-1 to PrPE-CE-4).

  Of these four fractions, the compound (20.2 mg) was purified from the fraction PrPE-CE-2 (20.2 mg) (“Compound 1” in the present specification).

  Furthermore, the compound (31.6 mg) was purified from the fraction PrPE-CE-4 (31.6 mg) (“compound 4” in the present specification).

<Separation of fraction PrPE-CF and purification of compound>
Fraction PrPE-C-F (153 mg) was subjected to high performance liquid chromatography (COSMOSIL Cholester Φ10 × 250 mm, 0.1% trifluoroacetic acid: acetonitrile (60:40), flow rate 4.0 mL / min, wavelength 210 nm). And fractionated into three fractions (PrPE-C-F-1 to PrPE-C-F-3).

  Of these three fractions, the compound (18.2 mg) was purified from the fraction PrPE-C-F-2 (18.2 mg) (“compound 3” in the present specification).

<Separation of fraction PrPE-C-H and purification of compound>
Fraction PrPE-C-H (50 mg) was subjected to high performance liquid chromatography (COSMOSIL Cholester Φ10 × 250 mm, 0.1% trifluoroacetic acid: acetonitrile (60:40), flow rate 4.0 mL / min, wavelength 210 nm). And fractionated into three fractions (PrPE-C-H-1 to PrPE-C-H-3).

  Of these three fractions, fraction PrPE-C-H-2 (18 mg) was further subjected to high performance liquid chromatography (COSMOSIL πNAP φ10 × 250 mm, 0.1% trifluoroacetic acid: acetonitrile (60:40), flow rate. 4.0 mL / min, wavelength 210 nm), and fractionated into three fractions (PrPE-C-H-2-1 to PrPE-C-H-2-3).

  Of these three fractions, the compound (7 mg) was purified from the fraction PrPE-C-H-2-1 (7 mg) (“Compound 9” in the present specification).

<< Separation of fraction PrPE-D >>
Fraction PrPE-D (7.28 g) was subjected to activated charcoal column chromatography and then eluted with 100% methanol, 1: 1 ratio of chloroform: methanol (chloroform-methanol system).

  The eluted fraction PrPE-D (3.61 g) was subjected to normal phase silica gel column chromatography (Silica gel 60N), and the chloroform: methanol ratio was changed from 100: 0 to 0: 100 in a chloroform-methanol system. And fractionated into 7 fractions (PrPE-D-1 to PrPE-D-7). Of these 7 fractions, the fraction PrPE-D-4 (1.25 g) was further separated.

  Fraction PrPE-D-4 (1.25 g) was subjected to reverse phase column chromatography (YMC-GEL ODS-AS-150), water-methanol system, water: methanol ratio from 100: 0. The elution was carried out until 0: 100, and fractionated into 14 fractions (PrPE-D-4-1 to PrPE-D-4-14).

  Of these 14 fractions, 2 fractions (PrPE-D-4-5 (68.9 mg), PrPE-D-4-7 (89.9 mg)) were further separated and purified.

<Separation of fraction PrPE-D-4-5 and purification of compound>
Fraction PrPE-D-4-5 (68.9 mg) was subjected to high performance liquid chromatography (COSMOSIL Cholester Φ10 × 250 mm, 0.1% trifluoroacetic acid: methanol (60:40), flow rate 4.0 mL / min, wavelength 254 nm. ) And fractionated into 5 fractions (PrPE-D-4-5-1 to PrPE-D-4-5-5).

  Of these 5 fractions, PrPE-D-4-5-4 (23.5 mg) was further purified by high performance liquid chromatography (COSMOSIL πNAP φ10 × 250 mm, 0.1% trifluoroacetic acid: methanol (55:45) At a flow rate of 4.0 mL / min, wavelength 210 nm) to yield compound (14.5 mg) (“Compound 10” herein).

  Further, among these 5 fractions, PrPE-D-4-5-5 (20.6 mg) was subjected to high performance liquid chromatography (COSMOSIL πNAP φ10 × 250 mm, 0.1% trifluoroacetic acid: methanol (55:45). ), A flow rate of 4.0 mL / min, a wavelength of 254 nm), and fractionated into two fractions (PrPE-D-4-5-5-1, PrPE-D-4-5-5-2). .

  Of these two fractions, the compound (14.2 mg) was purified from the fraction PrPE-D-4-5-5-2 (14.2 mg) (“compound 2” in the present specification).

<Separation of fraction PrPE-D-4-7 and purification of compound>
Fraction PrPE-D-4-7 (89.9 mg) was subjected to high performance liquid chromatography (COSMOSIL πNAP φ10 × 250 mm, 0.1% trifluoroacetic acid: methanol (70:30), flow rate 4.0 mL / min, wavelength 210 nm. ) And fractionated into three fractions (PrPE-D-4-7-1 to PrPE-D-4-7-3).

  Of these three fractions, the compound (62.0 mg) was purified from the fraction PrPE-D-4-7-2 (62.0 mg) (“Compound 8” in the present specification).

<< Separation of fraction PrPE-F >>
Fraction PrPE-F (21.3 g) was subjected to normal phase silica gel column chromatography (Silica gel 60N), and the ratio of chloroform: methanol was changed from 100: 0 to 0: 100 in a chloroform-methanol system. Elution and fractionation into 7 fractions (PrPE-F-1 to PrPE-F-7). Of these 7 fractions, the fraction PrPE-F-6 (8.70 g) was further separated.

  Fraction PrPE-F-6 (8.70 g) was subjected to reverse phase column chromatography (YMC-GEL ODS-AS-150), water-methanol system, water: methanol ratio from 90:10 Elution was carried out until 0: 100, and fractionated into 10 fractions (PrPE-F-6-1 to PrPE-F-6-10). Of these 10 fractions, the fraction PrPE-F-6-4 (1.14 g) was further separated.

  Fraction PrPE-F-6-4 (1.14 g) was subjected to normal phase column chromatography (MCI GEL CHP20P), water-methanol system, water: methanol ratio from 90:10 to 0: 100. Elution was carried out by changing, and fractionated into 6 fractions (PrPE-F-6-4-1 to PrPE-F-6-4-6). Of these 6 fractions, the fraction PrPE-F-6-4-2 (747 mg) was further separated.

  Fraction PrPE-F-6-4-2 (747 mg) was subjected to normal phase column chromatography (Sephadex® LH-20) eluting with 70% methanol, 100% methanol to yield 12 Fractions were fractionated (PrPE-F-6-4-2-1 through PrPE-F-6-4-4-2-12).

Of these 12 fractions, further separation and purification of 2 fractions (PrPE-F-6-4-2-5 (191 mg), PrPE-F-6-4-2-7 (168 mg)) Went.

<Separation of PrPE-F-6-4-2-5 and purification of the compound>
Fraction PrPE-F-6-4-2-5 (191 mg) was subjected to normal phase column chromatography (Sephadex® LH-20) followed by a 1: 1 ratio of chloroform: methanol (chloroform-methanol). System), eluting with 100% methanol and fractionating into 9 fractions (PrPE-F-6-4-4-2-5-1 to PrPE-F-6-4-4-2-5-9).

  Of these 9 fractions, the compound (96.4 mg) was purified from the fraction PrPE-F-6-4-2-5-4 (96.4 mg) (“compound 5” in the present specification).

<Separation of PrPE-F-6-4-2-7 and purification of the compound>
Fraction PrPE-F-6-4-2-7 (168 mg) was subjected to high performance liquid chromatography (COSMOSIL Cholester Φ10 × 250 mm, 0.1% trifluoroacetic acid: methanol (55:45), flow rate 4.0 mL / min, And was fractionated into 5 fractions (PrPE-F-6-4-4-2-7 to PrPE-F-6-4-2-2-7-5). Of these 5 fractions, the fraction PrPE-F-6-4-2-7-4 (87.4 mg) was further separated.

  Fraction PrPE-F-6-4-2-7-4 (87.4 mg) was subjected to high performance liquid chromatography (Shodex GF-310HQ φ4.6 × 250 mm, 0.1% trifluoroacetic acid: methanol (10:90). And a fraction of 5 (PrPE-F-6-4-2-7-4-1 to PrPE-F-6-4-2-7-7-4) at a flow rate of 4.0 mL / min and a wavelength of 210 nm. It fractionated to -5).

Of these 5 fractions, the compound (38.5 mg) was purified from the fraction PrPE-F-6-4-2-7-4-2 (38.5 mg) (“compound 6” in the present specification). ).

[Component identification of compounds purified from white peach flowers]

<< Identification of Compound 1 >>
Compound 1 was a white powder, and exhibited [α] ²⁵ _D -47.8 ° (c = 0.1, methanol), and m / z = 288 [M] ⁺ by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 5.86 (1H, d, J = 2.1 Hz), 5.91 (1H, d, J = 2.1 Hz));
A ₂ B ₂ type proton signal (δ _H 6.78 (2H, d, J = 8.7 Hz), 7.30 (2H, d, J = 8.7 Hz));
Signals from 1,2-diaxial protons (δ _H 4.55 (1H, dd, J = 11.3, 5.9 Hz), 5.04 (1H, d, J = 11.3 Hz));
A signal from a chelating hydroxyl group (δ _H 11.87 (1H, s));
Was detected.

^{For 13} C-NMR and DEPT spectra:
Signals by 15 sp ² carbons (δ _C 94.9, 96.0, 100.4, 114.8 × 2, 127.5, 129.3 × 2, 157.7, 162.5, 1,163 .2, 166.7, 197.7);
Signals due to two sp ³ carbons (δ _C 71.4, 82.8);
Was detected.

By estimating the molecular structure from the above optical rotation and m / z value, and comparing the spectrum data of ¹ H-NMR and ¹³ C-NMR with the data described in Non-Patent Documents 2 and 3, ) -Identified as aromadendrin.

  (−)-Aromadendrine is a compound that is rarely found from organic substances as compared to its enantiomer (+)-aromadendrin. Furthermore, (−)-aromadendrin has been isolated and purified from white peach flowers for the first time.

<< Identification of Compound 2 >>
Compound 2 was a yellow powder, and m / z = 304 [M] ⁺ was obtained by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 5.84 (1H, d, J = 2.1 Hz), 5.89 (1H, d, J = 2.1 Hz));
Signals from 1,2-diaxial protons (δ _H 4.46 (1H, dd, J = 11.3, 6.0 Hz), 4.95 (1H, d, J = 11.3 Hz));
A signal by a chelating hydroxyl group (δ _H 11.88 (1H, s));
Was detected.

In addition, since proton signals (δ _H 6.72 (2H, d, J = 1.0 Hz), 6.86 (1H, d, J = 1.0 Hz)) were detected, 1,3,5-3 The presence of substituted benzene was estimated.

^{For 13} C-NMR and DEPT spectra:
Signals by 15 sp ² carbons (δ _C 95.4, 96.4, 100.9, 115.5, 115.7, 119.8, 128.4, 145.3, 146.2, 163.0 , 163.7, 167.2, 198.2);
Signals from two sp ³ carbons (δ _C 72.0, 83.5);
Was detected.

By estimating the molecular weight from the above m / z value and comparing the spectrum data of ¹ H-NMR and ¹³ C-NMR with the data described in Non-Patent Documents 4 and 5, Compound 2 is 5, 7, 3 ′. , 5'-tetrahydroxyflavanonol.

  This is the first time that 5,7,3 ', 5'-tetrahydroxyflavanonol has been isolated, purified and identified from white peach flowers.

<< Identification of Compound 3 >>
Compound 3 was a yellow powder, and m / z = 302 [M] ⁺ was obtained by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 6.18 (1H, d, J = 2.1 Hz), 6.40 (1H, d, J = 2.1 Hz));
ABX type proton signal (δ _H 6.87 (1H, d, J = 8.5 Hz), 7.53 (1H, dd, J = 8.5, 2.3 Hz), 7.67 (1H, d, J = 2.3 Hz));
A signal from a chelating hydroxyl group (δ _H 12.49 (1H, s));
Was detected.

^{For the 13} C-NMR spectrum:
Signals by 15 sp ² carbons (δ _C 94.1, 98.9, 103.7, 115.7, 116.2, 120.6, 122.6, 136.3, 145.6, 147.3 , 148.2, 156.6, 161.2, 164.5, 176.2);
Was detected.

The molecular weight was estimated from the above m / z value, and ¹ H-NMR and ¹³ C-NMR spectrum data was compared with the data described in Non-Patent Document 6, whereby Compound 3 was identified as quercetin.

<< Identification of Compound 4 >>
Compound 4 was a yellow powder, and m / z = 286 [M] ⁺ was obtained by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 6.19 (1H, d, J = 2.3 Hz), 6.44 (1H, d, J = 2.3 Hz));
A ₂ B ₂ type proton signal (δ _H 6.92 (2H, d, J = 8.8 Hz), 8.03 (2H, d, J = 8.8 Hz));
A signal from a chelating hydroxyl group (δ _H 12.45 (1H, s));
Was detected.

^{For 13} C-NMR and DEPT spectra:
Signals by 15 sp ² carbons (δ _C 93.4, 98.1, 102.9, 115.3 × 2, 121.6, 129.4 × 2, 135.5, 146.8, 156.1 159.1, 160.6, 163.8, 175.8);
Was detected.

The molecular weight was estimated from the above m / z value, and ¹ H-NMR and ¹³ C-NMR spectral data were compared with the data described in Non-Patent Document 6, whereby Compound 4 was identified as kaempferol.

<< Identification of Compound 5 >>
Compound 5 was a yellow powder, and a pseudo ion peak was detected at m / z = 449 [M + H] ⁺ in FAB-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 6.20 (1H, d, J = 2.1 Hz), 6.38 (1H, d, J = 2.1 Hz));
ABX type proton signals (δ _H 6.85 (1H, d, J = 8.4), 7.23 (1H, dd, J = 8.4, 2.3 Hz) and 7.29 (1H, d, J = 2.3 Hz));
A signal from a chelating hydroxyl group (δ _H 12.64 (1H, s));
Detects and further:
A signal presumed to be an anomeric proton of the sugar (δ _H 5.24 (1H, d, J = 1.6 Hz));
Proton signal of the methyl group (δ _H 0.80 (3H, d, J = 6.2 Hz));
Was detected.

^{For 13} C-NMR and DEPT spectra:
Signals by 15 sp ² carbons (δ _C 93.7, 98.7, 104.1, 115.5, 115.7, 120.8, 121.1, 134.2, 145.2, 148.5 156.5, 157.3, 161.3, 164.2, 177.8);
Was detected.

In addition, since six carbon signals (δ _C 17.5, 70.1, 70.4, 70.6, 71.2, 101.8) were detected in the aliphatic region, deoxy sugars such as rhamnose Presumed existence.

In the HMBC spectrum, a cross peak was detected from δ _H 5.24 (Rha-1) to δ _C 135.5 (C-3).

The molecular weight was estimated from the above-mentioned m / z value, and ¹ H-NMR and ¹³ C-NMR spectral data were compared with the data described in Non-Patent Document 6, whereby Compound 5 was identified as quercitrin.

<< Identification of Compound 6 >>
Compound 6 was a yellow powder, and a pseudo ion peak was detected at m / z = 465 [M + H] ⁺ in FAB-MS.

^{In the 1} H-NMR spectrum:
Proton signal to metacouple (δ _H 6.18 (1H, d, J = 2.4 Hz), 6.36 (1H, d, J = 2.4 Hz));
ABX type proton signal (δ _H 6.85 (1H, d, J = 10.0 Hz), 7.23 (1 H, dd, J = 2.4, 10.0 Hz), 7.71 (1 H, d, J = 2.4 Hz));
A signal from a chelating hydroxyl group (δ _H 12.60 (1H, s));
Detects and further:
Signal presumed to be an anomeric proton of sugar (δ _H 5.23 (1H, d, J = 7.5 Hz));
Was detected.

^{For 13} C-NMR and DEPT spectra:
Signals by 15 sp ² carbons (δ _C 94.8, 99.9, 105.6, 116.0, 117.6, 123.0, 123.2, 135.6, 145.8, 149.8 , 158.4, 159.0, 162.9, 166.1, 179.4);
Was detected.

In addition, the presence of sugar was estimated from the detection of six carbon signals (δ _C 62.5, 71.2, 75.7, 78.1, 78.3, 104.4) in the aliphatic region.

In the HMBC spectrum, a cross peak was detected from δ _H 5.23 (Glc-1) to δ _C 135.6 (C-3).

The molecular weight was estimated from the above m / z value, and ¹ H-NMR and ¹³ C-NMR spectrum data was compared with the data described in Non-Patent Document 7, whereby Compound 6 was identified as isoquercitrin.

  This is the first time that isoquercitrin has been isolated, purified and identified from white peach blossoms.

<< Identification of Compound 7 >>
Compound 7 was a yellow powder, and exhibited [α] ²⁵ _D −80.3 ° (c = 0.1, methanol), which was m / z = 272 [M] ⁺ by EI-MS.

^{In the 1} H-NMR spectrum:
A ₂ B ₂ type proton signal (δ _H 6.79 (1H, d, J = 8.7 Hz), 7.31 (1H, d, J = 8.7 Hz));
From the presence of 1,4-disubstituted benzene, and further:
Proton signal (δ _H 12.16 (1H, s));
From this, the presence of chelate hydroxyl group was estimated.

^{For 13} C-NMR and DEPT spectra:
Signals by 13 sp ² carbons (δ _C 95.0, 95.9, 101.8, 115.2 × 2, 128.4 × 2, 128.9, 157.8, 163.0, 163.5 , 166.7, 196.5);
With signals from two sp ³ carbons (δ _C 42.0, 78.5);
Was detected.

By estimating the molecular structure from the above optical rotation and m / z value and comparing the spectrum data of ¹ H-NMR and ¹³ C-NMR with the data described in Non-Patent Document 8, the compound 7 is (-)- Identified as naringenin.

<< Identification of Compound 8 >>
Compound 8 was obtained as a yellow powder and showed [α] ²⁵ _D −48.4 ° (c = 0.1, methanol), which was m / z = 288 [M] ⁺ by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal (δ _H 5.87 (d, J = 1.9 Hz)) for 2H meta-coupling;
And the presence of 1,2,3,5-tetrasubstituted benzene is estimated:
Proton signal (δ _H 6.74 (2H, s), δ _H 6.87 (1H, s);
And the presence of 1,3,5-trisubstituted benzene is estimated, and further:
Signal due to chelate hydroxyl group (δ _H 12.15 (1H, s));
Was detected.

^{For 13} C-NMR and DEPT spectra:
Signals from 13 sp ² carbons (δ _C 95.5, 96.3, 102.3, 114.9, 115.9, 118.5, 130.0, 145.7, 146.2, 163.4 , 164.0, 167.3, 196.9) and;
With signals from two sp ³ carbons (δ _C 42.1, 79.0);
Was detected.

By estimating the molecular structure from the above optical rotation and m / z value, and comparing the spectrum data of ¹ H-NMR and ¹³ C-NMR with the data described in Non-Patent Document 9, the compound 8 is (-)- It was identified as 5,7,3 ′, 5′-tetrahydroxyflavanone.

  This is the first time that (-)-5,7,3 ', 5'-tetrahydroxyflavanone has been isolated, purified and identified from white peach blossoms.

<< Identification of Compound 9 >>
Compound 9 was a white powder, and a molecular ion peak was detected at m / z = 152 [M] ⁺ by EI-MS.

^{In the 1} H-NMR spectrum:
Proton signal of aromatic region (δ _H 7.20 (1H, t, J = 7.3 Hz), 7.23 (2H, t, J = 7.3 Hz), 7.35 (2H, d, J = 7 .3 Hz));
And the presence of monosubstituted benzene was estimated.

^{For 13} C-NMR and DEPT spectra:
Signals from seven sp ² carbons (δ _C , 127.4 × 2, 128.7, 128.9 × 2, 140.3, 175.7);
A signal from one sp ³ carbon (δ _C 73.6);
And the presence of a carbonyl group (δ _C 175.7) was estimated from the chemical shift.

The molecular weight was estimated from the above m / z value, and ¹ H-NMR and ¹³ C-NMR spectral data were compared with the data described in Non-Patent Document 10, whereby Compound 9 was identified as mandelic acid.

  This is the first time that mandelic acid has been isolated, purified and identified from white peach flowers.

<< Identification of Compound 10 >>
Compound 10 was a white powder, and a molecular ion peak was detected at m / z = 164 [M] ⁺ by EI-MS.

^{In 1} H-NMR, in the aromatic region:
Proton signal split into A ₂ B ₂ types (δ _H 6.79 (2H, d, J = 8.7 Hz), 7.51 (2H, d, J = 8.7 Hz));
, And even:
Peak based on transcoupling olefinic proton signal (δ _H 6.28 (1H, d, J = 15.5 Hz), 7.49 (1H, d, J = 15.5 Hz));
Also detected.

^{For 13} C-NMR and DEPT spectra:
Signal from 6 sp ² carbons (δ _C 115.3, 115.7 × 2, 125.3, 130.1 × 2, 144.1, 159.6, 168.0);
And the presence of a carbonyl group (δ _C 167.9) was estimated from the chemical shift.

The molecular weight was estimated from the above-mentioned m / z value, and ¹ H-NMR and ¹³ C-NMR spectral data were compared with the data described in Non-Patent Document 11, whereby Compound 10 was identified as p-coumaric acid.

[Bioactivity of extracts and compounds derived from white peach flowers]

Samples separated or isolated and purified from white peach blossoms:
n-hexane fraction extract (PrPH);
Extract of ethyl acetate fraction (PrPE);
n-butanol fraction extract (PrPB);
Extract of water fraction (PrPW);
(−)-Aromadendrin (Compound 1);
Kaempferol (compound 4);
(−)-Naringenin (compound 7); and (−)-5,7,3 ′, 5′-tetrahydroxyflavanone (compound 8);
about,
Effects on differentiation of preadipocytes;
Effect on adipose triglyceride (TG) accumulation;
Effects on suppression of macrophage activation in adipocytes;
Was examined.

<< Effect of Sample of the Present Invention on Preadipocyte Differentiation >>
<the purpose>
It is known that an increase in the activity of glycerol 3-phosphate dehydrogenase (GPDH) is an indicator that preadipocytes have differentiated into adipocytes. Therefore, in order to examine the effects of the extract and compound of the present invention on GPDH activity, in vitro experiments were performed using 3T3-L1 preadipocytes of mouse fibroblasts.

<Control sample in the control test>
Dimethyl sulfoxide (DMSO) was used as a control sample in this example. In addition, pioglitazone (SIGMA), a compound that promotes differentiation into small adipocytes, is used as a positive control sample, and GW9662 (a Japanese compound that suppresses differentiation into small adipocytes as a negative control sample). Optical pure drug industry) and quercetin (Tokyo Kasei Kogyo) were used. Further, a white peach flower extract (80% EtOH ext.) With 80% ethanol was used as a sample for comparison.

<Method>
1. Medium preparation

  Three types of media used in this example were prepared as follows.

(1) Preparation of cell growth medium To 500 mL Dulbecco's modified Eagle medium (DMEM: high glucose, GIBCO 11965):
a) Fetal calf serum (FCS) prepared at a final concentration of 10%, immobilized at 60 ° C. for 30 minutes;
b) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%:
Was added to prepare a cell growth medium.

(2) Preparation of cell differentiation induction medium To 500 mL Dulbecco's modified Eagle medium (GIBCO11965):
a) fetal bovine serum (FBS) prepared to a final concentration of 10%;
b) IBMX (50 mM 3-isobutyl-1-methylxanthine) prepared to a final concentration of 500 μM;
c) DEX (dexamethasone) prepared to a final concentration of 1 μM;
d) a 10 mg / mL insulin solution prepared to a final concentration of 10 μg / mL;
e) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%;
Was added to prepare a cell differentiation induction medium.

(3) Preparation of cell differentiation promoting medium To 500 mL Dulbecco's modified Eagle medium (GIBCO11965):
a) fetal bovine serum (FBS) prepared to a final concentration of 10%;
b) a 10 mg / mL insulin solution prepared to a final concentration of 5 μg / mL;
c) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%;
Was added to prepare a cell differentiation promoting medium.

2. Preparation of preadipocytes

3T3-L1 preadipocytes were cultured until confluent, and then the dish medium was removed, the cell surface was washed with phosphate buffered saline (PBS (−)), and then PBS was aspirated. Next, a 0.25% trypsin-EDTA solution (SIGMA T4049) was added to the cells and cultured in a CO ₂ incubator.

After confirming that the cells had detached from the bottom of the dish using a microscope, 10 mL of cell growth medium was added and injected into a 50 mL Falcon tube. The cells were centrifuged at 1000 rpm for 3 minutes at 4 ° C. and the supernatant was removed. 50 mL of fresh medium was added and suspended, and 1 mL each was dispensed into two 24-well plates (Sumitomo Bakelite), and 50-well cells were cultured in a CO ₂ incubator for 4 days.

After 4 days, the medium in all wells was removed and 1 mL / well of cell differentiation induction medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 3 days.

After 3 days, all wells were removed and 1 mL / well of cell differentiation promoting medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 3 days.

After 3 days, all wells were removed and 1 mL / well of cell differentiation promoting medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 2 days.

Two days later, a cell lysate was prepared as follows.

3. Preparation of cell debris

The medium was collected from each well into a tube (Ring Look Microtube BM-15). After removing the medium, the cell surface was washed twice with PBS (−). 500 μL of 25 mM Tris buffer containing 1 mM EDTA (12.5 mL of 1M Tris-HCl (Tris-HCl) at pH 7.5 and 0.1 M EDTA was added to ultrapure water to prepare 500 mL) was added. . Cells were detached using a micropipette and collected in a tube. The collected cells were crushed with ultrasonic waves for 1 minute under ice-cooling, and then the cells were centrifuged at 1500 rpm for 2 minutes at 4 ° C.

4). Quantification of DNA in cell disruption The amount of intracellular DNA was measured using a DNA quantification kit (Primary Cell). Cell debris, buffer and color former were added and mixed thoroughly. The mixed solution was subjected to fluorescence measurement using a 356 nm excitation filter and a 458 nm fluorescence filter. In order to construct a standard curve, a standard solution (100 μg / mL of DNA) was diluted with purified water and prepared to be 100, 50, 25, 12.5, and 0 μg / mL.

5. Measurement of GPDH activity

  Intracellular GPDH activity was measured using a GPDH activity measurement kit from Primary Cell Corporation.

  4.2 mL of purified water was added and dissolved in one vial of the reaction substrate (freeze-dried) in the kit to prepare a reaction substrate solution. The prepared reaction substrate solution was added to a spectrophotometer cell (quartz microcell). Next, the cell lysate was added to a spectrophotometer cell (quartz microcell) and sufficiently stirred.

  The amount of change in absorbance of each sample at a wavelength of 340 nm was measured every 30 seconds for 4 minutes, and the amount of change in absorbance per minute (ΔOD) was calculated.

If GPDH per mL of cell lysate consumes 1 μM nicotinamide adenine dinucleotide (NADH) per minute is 1 U, GPDH activity is represented by the following formula:
GPDH activity (U / mL) = ΔO. D. × 0.482
(When the optical path length is 1 cm).

The rate of increase in GPDH activity relative to control (100%) is:
GPDH activity (% of Control) = (X / Y) × 100;
Calculated from Here, X is a value obtained by dividing GPDH activity per well when each sample is added by the amount of DNA per well, and Y is GPDH per well when a control sample (DMSO) is added. It is a numerical value obtained by dividing the activity by the amount of DNA per well.

<result>

FIG. 2 shows the rate of increase in GPDH activity relative to the control (100%) in the sample of the present invention. In the extract, an increase in GPDH activity was observed in the ethyl acetate fraction (PrPE) and the water fraction (PrPW), and in the isolated compound, a significant increase in GPDH activity was observed in (−)-aromadendrin (symbol 1). It was.

《Effects of white peach blossom samples on triglyceride accumulation in adipocytes》
<the purpose>
It is known that excessive TG accumulation in adipocytes causes fat cell hypertrophy and thus causes obesity and thus metabolic syndrome. Therefore, in order to examine the influence of the extract and compound derived from white peach flower of the present invention on triglyceride (TG) accumulation in adipocytes, in vitro experiments were performed using 3T3-L1 preadipocytes of mouse fibroblasts. .

<Method>

  The same method as in Example 1 was used for setting the control sample, the method for preparing the medium, the method for preparing the preadipocytes, the method for preparing the cell disruption, and the method for quantifying the DNA in the control test. The method for measuring the TG content is described below.

  The TG content accumulated in the cell lysate of each sample was quantified using LabAssay (trademark) Triglyceride (Wako Pure Chemical Industries) (GPO / DAOS method). A triglyceride coloring reagent was added to the cell disruption of each sample, mixed well, and heated at 37 ° C. for 5 minutes. Using DMSO as a control sample, the absorbance of each sample at a wavelength of 595 nm was measured. In order to prepare a standard curve, a standard solution was prepared and added so that final concentrations were 100, 200, 300, 596, and 882 mg / dL.

The ratio (% of Control) of the amount of TG accumulated to the control (100%) is:
TG accumulation amount (% of Control) = (X / Y) × 100;
Calculated from Here, X is a value obtained by dividing the TG amount per well when each sample is added by the DNA amount per well, and Y is the TG per well when the control sample (DMSO) is added. It is a numerical value obtained by dividing the amount by the amount of DNA per well.

<result>

FIG. 3 shows the increase rate of the TG accumulation amount with respect to the control (100%) in the sample of the present invention. In the extract, an increase in the TG accumulation amount was observed in the water fraction (PrPW), and in the isolated compound, a significant increase in the TG accumulation amount was observed in the (−)-aromadendrin (symbol 1).

<< Effects of white peach flower extract with 80% ethanol on differentiation of small adipocytes and increase of TG accumulation >>
<the purpose>
In this example, when only insulin is added without adding DEX and IBMX at the induction of differentiation of preadipocytes, an extract of white peach blossoms with 80% ethanol (80% EtOH ext.) In order to examine the effects on differentiation and TG accumulation, in vitro experiments were performed using 3T3-L1 preadipocytes of mouse fibroblasts.

Differentiation of small adipocytes in this experiment was confirmed visually by using Oil Red O staining. Moreover, the comparison of the amount of TG accumulated in this experiment was performed in a control experiment with a control sample (DMSO).

<Method>
1. Medium preparation

  Three types of media used in this example were prepared as follows.

(1) Preparation of cell growth medium To 500 mL Dulbecco's modified Eagle medium (DMEM: high glucose, GIBCO 11965):
a) Fetal calf serum (FCS) prepared at a final concentration of 10%, immobilized at 60 ° C. for 30 minutes;
b) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%:
Was added to prepare a cell growth medium.

(2) Preparation of cell differentiation induction medium To 500 mL Dulbecco's modified Eagle medium (GIBCO11965):
a) fetal bovine serum (FBS) prepared to a final concentration of 10%;
b) a 10 mg / mL insulin solution prepared to a final concentration of 10 μg / mL;
c) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%;
Was added to prepare a cell differentiation induction medium.

(3) Preparation of cell differentiation promoting medium To 500 mL Dulbecco's modified Eagle medium (GIBCO11965):
a) fetal bovine serum (FBS) prepared to a final concentration of 10%;
b) a 10 mg / mL insulin solution prepared to a final concentration of 5 μg / mL;
c) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%;
Was added to prepare a cell differentiation promoting medium.

2. Reagent preparation

  Three types of reagents used in this example were prepared as follows.

(1) 10% (v / v) formaldehyde solution 1.5 mL of formaldehyde solution was diluted with water for injection to make a total volume of 15 mL.

(2) 60% isopropanol 15 mL of isopropanol was diluted with water for injection to make a total volume of 25 mL.

(3) Oil Red O solution 150 mg of Oil Red O was dissolved in 50 mL of isopropanol and mixed for 10 minutes to purify the stock solution of Oil Red O. Next, a stock solution of Oil Red O and water for injection were mixed at a ratio of 6: 4, allowed to stand at room temperature for 10 minutes, and filtered through a filter having a pore diameter of 0.5 μm. The prepared Oil Red O solution was used within 1 to 2 hours after preparation.

3. Cell preparation

3T3-L1 preadipocytes were cultured until they became confluent, and then the dish medium was removed, the cell surface was washed with PBS (−), and then PBS was aspirated. Next, a 0.25% trypsin-EDTA solution (SIGMA T4049) was added to the cells and cultured in a CO ₂ incubator.

After confirming that the cells had detached from the bottom of the dish using a microscope, 10 mL of cell growth medium was added and injected into a 50 mL Falcon tube. The cells were centrifuged at 1000 rpm for 3 minutes at 4 ° C. and the supernatant was removed. 50 mL of fresh medium was added to suspend, 10 mL each was dispensed into a φ60 mm dish, and cultured in a CO ₂ incubator for 4 days.

After 4 days, the medium in all wells was removed, and 10 mL of cell differentiation induction medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 3 days.

Three days later, the medium in all wells was removed, and 10 mL of cell differentiation promotion medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 3 days.

Three days later, the medium in all wells was removed, and 10 mL of cell differentiation promotion medium was added. Further, the sample was added and cultured in a CO ₂ incubator for 2 days.

Two days later, Oil Red O staining was performed as follows.

4). Oil Red O staining

  The dish medium was removed and washed twice with 10 mL of PBS (−). 10 mL of 10% formaldehyde solution was added and fixed at room temperature for 10 minutes. After fixation, the plate was washed twice with 10 mL of PBS (−) and replaced with 60% isopropanol. 60% isopropanol was removed, 10 mL Oil Red O solution was added, and the mixture was allowed to stand at room temperature for 15 minutes.

After 15 minutes, the Oil Red O solution was removed and washed with 10 mL of 60% isopropanol. The plate was washed once with 10 mL of PBS (−), and microscopic observation was performed in PBS (−).

5. Measurement of TG content

  After observation of Oil Red O staining, PBS (-) was removed, 10 mL of isopropanol was added to elute the dye, and the absorbance at a wavelength of 520 nm was measured.

The ratio (% of Control) of the amount of TG accumulated to the control (100%) is:
TG accumulation (% of Control) = (Ab.sam./Ab.con.);
Calculated from Here, X is the absorbance when each sample is added, and Y is the absorbance when a control sample (DMSO) is added.

<result>

  FIG. 4 shows the results of Oil Red O staining of small adipocytes in an 80% ethanol extract and the increase rate of TG accumulation relative to control (100%).

  In Oil Red O staining, the addition of 80% ethanol extract increased the number of small adipocytes compared to the control.

In addition, when 80% ethanol extract was added, the amount of TG accumulation increased compared to the control.

《Effects of white peach flower samples on adiponectin secretion in adipocytes》
<the purpose>
It is known that an increase in adiponectin secretion in adipocytes can increase blood adiponectin levels and thus improve insulin resistance. Therefore, in order to examine the effect of white peach flower-derived samples on adiponectin secretion of adipocytes, in vitro experiments were performed using 3T3-L1 preadipocytes of mouse fibroblasts.

This experiment was performed in a control experiment with a control sample (DMSO), and a sample derived from white peach flowers:
White peach blossom extract with 80% ethanol (80% EtOH ext.);
An extract of ethyl acetate fraction (PrPE);
(−)-Aromadendrin (Compound 1);
The effects of adipocytes on adiponectin secretion were examined.

<Method>
1. Reagent preparation

  In the measurement of this example, a mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.) was used, and five types of reagents in this kit were prepared as follows.

(1) Washing solution 960 mL of purified water was mixed with a total amount of 40 mL of cleaning stock solution. When crystals were precipitated in the stock solution, it was prepared after heating to dissolve. The prepared washing solution was stored at 2 to 8 ° C.

(2) Sample dilution solution 200 mL of purified water was mixed with a total volume of 50 mL of sample dilution stock solution. The prepared specimen diluent was stored at 2 to 8 ° C.

(3) Standard solution A standard product of 8.0 ng / mL is diluted 2-fold with a sample diluent, and 4.0, 2.0, 1.0, 0.5, and 0.25 ng / mL standard solutions are prepared. Prepared. A standard product of 8.0 ng / mL was used as a standard solution of 8.0 ng / mL, and a specimen diluent was used as a standard solution of 0 ng / mL.

(4) Enzyme-labeled streptavidin solution Streptavidin stock solution was mixed at a ratio of 60 μL to 12 mL of the streptavidin diluted solution, and the required amount was prepared before use.

(5) Substrate solution The substrate solution A was mixed with 6 mL of the substrate solution B at a ratio of 6 mL, and the required amount was prepared before use.

2. Dilution of culture supernatant

10 μL of the medium (culture supernatant) collected in the tube (Ring Look Microtube BM-15) in Example 1 was added to 1.0 mL of the sample diluent and mixed (becomes a culture supernatant diluted 101 times). Of this, 50 μL was transferred to a new container and mixed with 1.0 mL of the sample diluent (resulting in a culture supernatant diluted 2,121 times).

3. Preparation of culture supernatant for measurement

  About 350 μL of the washing solution was added to each well of the antibody plate, and the solution in the well was completely removed. Next, the antibody plate was turned upside down and tapped on a paper towel to remove the washing solution remaining in the well. 100 μL of each concentration standard solution and diluted culture supernatant were added to each well, the antibody plate was covered with a plate seal, and allowed to stand at 22 to 28 ° C. for 60 minutes.

  Then, the plate seal was removed from the antibody plate, and the liquid in the well was completely removed. About 350 μL of the washing solution was added to each well of the antibody plate, and the solution in the well was completely removed. This operation was repeated a total of 3 times, and the antibody plate was turned upside down and tapped against a paper towel to remove the washing solution remaining in the well. 100 μL of biotin standard antibody solution was added to each well of the antibody plate, the antibody plate was covered with a plate seal, and allowed to stand at 22 to 28 ° C. for 60 minutes.

Then, the plate seal was removed from the antibody plate, and the liquid in the well was completely removed. About 350 μL of the washing solution was added to each well of the antibody plate, and the solution in the well was completely removed. This operation was repeated a total of 3 times, and the antibody plate was turned upside down and tapped against a paper towel to remove the washing solution remaining in the well. 100 μL of enzyme-labeled streptavidin solution was added to each well of the antibody plate. The antibody plate was covered with a plate seal and washed as above. 100 μL of the substrate solution was added to each well of the antibody plate and allowed to stand at 22 to 28 ° C. for 15 minutes to prepare a culture supernatant for measurement.

4). Measurement of adiponectin secretion

  100 μL of the reaction stop solution was added to each well of the antibody plate, the absorbance at 450 nm was measured using the absorbance at 650 nm as a control, and the adiponectin concentration in each well was calculated.

The adiponectin concentration in the culture supernatant was calculated by multiplying the calculated adiponectin concentration by the dilution rate of the culture supernatant (2,121 times). The ratio (% of Control) of adiponectin secretion to control (100%) is:
Adiponectin secretion (% of Control) = (X / Y) × 100;
Calculated from Here, X is the amount of adiponectin per well when the sample is added, and Y is the amount of adiponectin per well when the control sample (DMSO) is added.

<result>

FIG. 5 shows the increase rate of adiponectin secretion with respect to the control (100%) in the sample of the present invention. All adiponectin secretion levels of white peach flower extract (80% EtOH ext.), Ethyl acetate fraction extract (PrPE), and (−)-aromadendrin (symbol 1) with 80% ethanol Increased.

《Effects of white peach-derived samples on suppression of macrophage activation in adipocytes》
<the purpose>
Decreased secretion of adiponectin, which contributes to metabolic syndrome, is caused by inflammation of adipocytes and macrophages due to TNF-α secreted from macrophages activated by enlarged fat cells and free fatty acids secreted from fat cells. It is known to occur by promoting sexual changes. Furthermore, it is known that the production of nitric oxide (NO) in macrophages is an indicator of macrophage activation.

  Therefore, in order to examine the influence of the extract and compound derived from white peach flower of the present invention on the production of NO in adipocytes, an in vitro experiment was performed using RAW264.7 cells, which are mouse macrophage-like cells.

As a control sample of this example, quercetin (IC ₅₀ 26.8) was used, and 100% was added to which no test drug was added. In this example, a white peach blossom extract (80% EtOH ext.) With 80% ethanol was used as a sample for a comparative example.

<Method>
1. Medium preparation

To 500 mL of F-12 HAM medium (SIGMA N4888):
a) L-glutamine (SIGMA) prepared to a final concentration of 1.8 mM;
b) Fetal bovine serum (FBS) prepared at a final concentration of 10%, immobilized at 60 ° C. for 30 minutes;
c) Penicillin Streptomycin (SIGMA) prepared to a final concentration of 1%;
Was prepared.

2. Reagent preparation

  Four types of reagents used in this example were prepared as follows.

(1) Preparation of Recombinant Mouse IFN-γ (Genzyme / Techne) 0.3 mg / mL INF-γ stock solution was diluted 100-fold with the above-prepared medium to a concentration of 3 μg / mL at −35 ° C. saved.

(2) Preparation of LPS (SIGMA O55: B5) LPS was diluted with a medium to a concentration of 5 mg / mL and stored at -35 ° C (LPS stock solution). The LPS stock solution was diluted 5-fold with the medium and stored at -35 ° C.

(3) Preparation of 0.1% naphthylethylenediamine solution Before use, 5 mg of naphthylethylenediamine dihydrochloride (Wako) was dissolved in 5 mL of water for injection. The prepared 0.1% naphthylethylenediamine solution was stored in the dark.

(4) Preparation of sulfanilamide solution Before use, 50 mg of sulfanilamide (Wako) was dissolved in 250 μL of phosphoric acid (Wako), and water for injection was added to prepare 5 mL. The prepared sulfanilamide solution was stored in the dark.

3. Cell preparation

Macrophage-like cells RAW264.7 cells were cultured in F-12 HAM medium containing 10% fetal calf serum until confluent. Thereafter, the cultured RAW264.7 cells were injected into a 50 mL Falcon tube. The cells were centrifuged at 1000 rpm for 3 minutes at 4 ° C. and the supernatant was removed. 10 mL of fresh medium was added and suspended to a concentration of 1.2 × 10 ⁶ cells / mL. The prepared cells were dispensed at 200 μL each into a 96-well plate (Sumitomo Bakelite 8096R), and cultured for 60 to 60 minutes in a CO ₂ incubator for 1 to 2 hours to adhere the cells.

Subsequently, 2 μL of 10 μg / mL LPS (SIGMA O55: B5) was added, 2 μL of 30 ng / mL mouse IFN-γ (Genzyme) was added, and 0.4 μL of sample was added. Thereafter, the cells were cultured for 16 hours in a CO ₂ incubator. The final concentration is 0.33 ng / mL for IFN-γ and 100 ng / mL for LPS. Moreover, the sample was prepared so that it might melt | dissolve in DMSO and the content with respect to a culture medium might be 0.2%.

4). Evaluation of NO production by the grease method

  100 μL of the culture supernatant was collected, 50 μL of 0.1% naphthylethylenediamine solution was added, 50 μL of sulfanilamide solution was added, and the mixture was allowed to stand at room temperature for 10 minutes in a light-shielded state. Thereafter, the change in absorbance at 520 nm (OD) was measured using the absorbance at 655 nm as a control.

The NO production inhibition rate (% of Control) is expressed by the following formula:
Inhibition rate (%) = {1- (X−Y) / (Z−Y)} × 100;
Calculated from Here, X is the absorbance induced by IFN-γ and LPS in the presence of the sample, Y is the absorbance control sample induced in the absence of the sample, IFN-γ and LPS, and Z is the IFN− Absorbance induced by γ and LPS.

5. Cytotoxicity measurement

  Cytotoxicity was measured by microscopic observation and normal MTT method. The outline of the method for measuring cytotoxicity in the MTT method is described below.

RAW264.7 cells were cultured in F-12 HAM medium containing 10% fetal calf serum until confluent. Thereafter, the cultured RAW264.7 cells were injected into a 50 mL Falcon tube. The cells were centrifuged at 1000 rpm for 3 minutes at 4 ° C. and the supernatant was removed. 10 mL of fresh medium was added and suspended to a concentration of 1.2 × 10 ⁶ cells / mL. The prepared cells were dispensed at 200 μL each into a 96-well plate (Sumitomo Bakelite 8096R), and cultured for 60 to 60 minutes in a CO ₂ incubator for 1 to 2 hours to adhere the cells.

Subsequently, 2 μL of 10 μg / mL LPS (SIGMA O55: B5) was added, 2 μL of 30 ng / mL mouse IFN-γ (Genzyme) was added, and 0.4 μL of sample was added. Then, CO ₂ and 16 hours incubator culturing, the culture supernatant was 100μL harvested and 1 mg / mL of MTT were incubated for 4 hours by adding 25μL to 100μL of NO assay cell. Thereafter, the medium was removed, and formazan produced by adding 150 μL of DMSO to the cells was completely dissolved and allowed to stand for 5 minutes, and then the absorbance at 570 nm was measured using the absorbance at 655 nm as a control.

<result>

  In FIG. 6, the NO production suppression rate in the sample of this invention is shown. In the extract, a strong NO production inhibitory activity is observed in the n-hexane fraction (PrPH), and the ethyl acetate fraction (PrPE), the n-butanol fraction (PrPB), and the water fraction (PrPW) are weak in this order. Production inhibitory activity was observed. In the isolated compound, NO production inhibitory activity was observed in kaempferol (symbol 4).

No cytotoxicity was observed in all samples tested.

[Discussion]

  According to the examples, the extract of the ethyl acetate fraction derived from white peach blossoms showed the effect of promoting differentiation of preadipocytes, the effect of promoting adiponectin secretion in adipocytes, and the effect of suppressing nitric oxide production. In the extract of the ethyl acetate fraction, since the amount of triglyceride accumulated in adipocytes does not increase, insulin resistance is improved without hypertrophy of adipocytes. Therefore, by applying the extract of the ethyl acetate fraction derived from white peach blossom as a crude drug component, the prevention and improvement effect of metabolic syndrome can be achieved.

  According to Examples, the extract of the water fraction derived from white peach blossoms showed the effect of promoting differentiation of preadipocytes, the effect of promoting adiponectin secretion in adipocytes, and the effect of suppressing nitric oxide production. In the extract of the water fraction, although the adiponectin secretion is remarkably promoted although the triglyceride accumulation amount of the adipocytes is increased, the insulin resistance is improved without the fat cells being enlarged. This suggests that the effect of preventing and improving metabolic syndrome can be achieved by applying an extract of a water fraction derived from white peach blossom as a herbal component. In addition, the fact that a strong preadipocyte differentiation promoting effect was observed in the water fraction suggests that there is a high possibility that such an effect is observed in an unknown herbal medicine component contained in the water fraction.

  The effect of preventing and improving metabolic syndrome in the extract derived from white peach flowers as described above is a novel effect discovered in the present invention.

  According to Examples, (−)-aromadendrin derived from white peach blossoms was found to have a preadipocyte differentiation promoting effect and an adiponectin secretion promoting effect in adipocytes. (-)-Aromadendrin increases the amount of triglyceride accumulated in adipocytes, but adiponectin secretion is significantly promoted, so that insulin resistance is improved without hypertrophy of adipocytes. Therefore, the prevention and improvement effect of metabolic syndrome is achieved by applying (−)-aromadendrin as a crude drug component.

  An antifungal action is known as a medicinal effect of aromadendrin. However, the preventive and ameliorating effects of metabolic syndrome in aromadendrin are effects newly discovered in the present invention.

  According to the examples, the extract of all the fractions derived from white peach flowers showed an inhibitory effect on nitric oxide production in RAW264.7 cells. This suggests that macrophage activation is suppressed, production of inflammatory adipokines is suppressed, and insulin resistance is improved.

  According to the Example, kaempferol derived from white peach flower showed an inhibitory effect on nitric oxide production in RAW264.7 cells. Kaempferol has no effect of promoting the differentiation of preadipocytes, but can suppress the production of nitric oxide. This suggests that since the activation of macrophages is suppressed, the production of inflammatory adipokines is suppressed, and as a result, insulin resistance is improved. Furthermore, kaempferol does not increase the amount of triglyceride accumulated in adipocytes, so that adipocytes do not enlarge. Therefore, by applying kaempferol as a crude drug component, prevention and improvement effects of metabolic syndrome can be achieved.

  According to the present invention, it has been suggested that the herbal medicine component derived from white peach blossom can exert the effect of preventing and improving metabolic syndrome for humans and animals. Accordingly, the herbal medicine component of the present invention can be applied to humans or animals as a pharmaceutical using a known dosage form. Moreover, the active ingredient of this invention can be utilized as food / beverage products, such as a pharmaceutical and a health supplement, cosmetics, or a quasi-drug.

  The dosage form of pharmaceuticals is not limited, but injections, suspensions, suppositories, capsules, powders, granules, solids, solutions, gels, foams, emulsions, ointments, creams, gels, patches An agent, a sheet, and an inhalant may be used.

  Either an oral administration or a parenteral administration can be adopted as a method for administering a pharmaceutical product. The herbal medicine component of the present invention may be mixed with a solid or liquid pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and administered in the form of a known pharmaceutical preparation. The preparation is not limited, but it may be a solid preparation such as a tablet, granule, powder, capsule, etc., a liquid preparation such as a solution, suspension, emulsion, or a lyophilized preparation that can be prepared by known preparation preparation means. The pharmaceutical carrier is not limited, but glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, It may be physiological saline. The pharmaceutical carrier may further contain additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent.

  The herbal medicine component of the present invention is not limited, but can be used as a health food or health food, supplement, food material. Further, it may be formed into granules, granules, tablets, capsules, pastes, and the like using known means so as to be suitable for food and drink. Further, the herbal ingredients of the present invention are not limited, but processed fishery products such as kamaboko and chikuwa, livestock products such as sausages and ham, Western confectionery, Japanese confectionery, raw noodles, Chinese noodles, boiled noodles, buckwheat noodles, sauces, Condiments such as soy sauce, sauce, sugar, honey, powdered candy, syrup, etc., spices such as curry powder, mustard powder, pepper powder, jam, marmalade, chocolate spread, pickles, vegetables, sprinkles, or various vegetables and fruits Canned and bottled processed vegetables and fruits, cheese, butter, yogurt and other dairy products, miso soup, soup, fruit juice, vegetable juice, whey beverage, soft drinks, alcoholic beverages, and liquid food Good. Moreover, you may use for animal and plant feeds, such as pet food.

The herbal medicine component of the present invention is not limited, but lotion, milky lotion, cream, ointment, lotion, oil, pack, facial cleanser and skin cleanser, massage agent, cleansing agent, hair remover, depilatory agent, shaving cream, After-shave lotion, pre-show lotion, shaving cream, foundation, lipstick, blusher, eye shadow, eyeliner, mascara, perfume, nail polish, nail enamel, nail enamel remover, poultice, plaster, tape, Sheet agent, patch, aerosol, shampoo agent, rinse agent, hair treatment agent, pre-hair treatment agent, permanent liquid, hair dye, hair styling agent, hair nicking agent, hair growth / hair restorer, poultice, plaster agent, tape agent, sheet Agent, patch, aerosol, bath agent, odor control agent and Agents, deodorants, antiperspirants, hygiene, sanitary cotton acids, wet tissue, tooth acids, mouthwashes, gargles, may be used in cosmetics or quasi Hinto such dishwashing detergent.

Claims (14)

  An extract derived from an organism for promoting differentiation of preadipocytes, wherein the extract derived from the organism contains aromadendrin.   2. The extract according to claim 1, wherein the organism is a white peach flower.   An extract derived from white peach blossoms for promoting differentiation of preadipocytes.   4. The extract according to claim 1, wherein the extract is an extract of an ethyl acetate fraction.   The extract according to claim 3, which is an extract of a water fraction.   A pharmaceutical comprising the extract according to any one of claims 1 to 5.   A food or drink comprising the extract according to any one of claims 1 to 5.   Cosmetics comprising the extract according to any one of claims 1 to 5.   A quasi-drug comprising the extract according to any one of claims 1 to 5.   A pharmaceutical product containing aromadendrin as an active ingredient for promoting differentiation of preadipocytes.   A food or drink containing aromadendrin as an active ingredient for promoting differentiation of preadipocytes.   The food or drink according to claim 7 or 11, which is a nutritional supplement.   Cosmetics for promoting differentiation of preadipocytes, comprising aromadendrin as an active ingredient. A quasi-drug for promoting the differentiation of preadipocytes, comprising aromadendrin as an active ingredient.