JP5255862B2 - Antidiabetic - Google Patents

Description

  The present invention relates to an antidiabetic agent. More specifically, the present invention relates to 6-hydroxy kaempferol-3-O-glycoside (1), which is a flavonoid glycoside having excellent antidiabetic activity.

  In recent years, excessive calorie intake has been a cause of lifestyle-related diseases, and therefore, restriction of calorie intake has been demanded. Among typical lifestyle-related diseases, diabetes is one of the most common lifestyle-related diseases in Japan. A characteristic of diabetes is that when a sugar derived from a carbohydrate taken by a meal is absorbed from the digestive tract and enters the blood, the blood sugar level is too high or a hyperglycemic state persists. Therefore, suppressing the digestion of carbohydrates and preventing it from being converted into energy in the living body is considered to be effective for the prevention and treatment of lifestyle-related diseases such as diabetes. It is thought to be a preventive / therapeutic agent for

  Here, 6-hydroxy kaempferol-3-O-glycoside (1) which is a flavonoid glycoside will be described below as a matter related to the present invention.

6-hydroxykaempferol-3-O-glycoside is a flavonoid glycoside of the chemical formula C ₂₁ H ₂₀ O ₁₂ , kaempferol (3,4 ′, 5,7 -tetrahydroxyflavonol) is a compound having a hydroxyl group at the 6-position, that is, a glycoside in which glucose is bonded to the 3-position of 6-hydroxykaempferol.

  Regarding 6-hydroxy kaempferol-3-O-glycoside (1), for example, there are the following documents.

  Non-Patent Document 1 discloses the isolation of 6-hydroxy kaempferol-3-O-glycoside (1) from an Egyptian plant (Ononis serrata Forssk (Leguminosae)). Reference 3 discloses isolation from safflower petals. Non-patent document 4 and Non-patent document 5 report biosynthesis of 6-hydroxykaempferol-3-O-glycoside (1) in safflower petals, and non-patent document 6 describes safflower. A method for identifying 6-hydroxykaempferol-3-O-glycoside (1) in petals using thin layer chromatography is described in Non-Patent Document 7 using high-performance liquid chromatography (HPLC). A method of performing the measurement is described.

Furthermore, Patent Document 2 exemplifies 6-hydroxy kaempferol-3-O-glycoside (1) as a kaempferol derivative for a prophylactic / therapeutic agent for cerebrovascular disorder. In Non-Patent Document 8, there is a report on the influence of a methanol extract of safflower petals on cultured bacteria. However, no antidiabetic activity has been reported so far for 6-hydroxykaempferol-3-O-glycoside (1).
Indo Patent Application Publication No. 183773 Specification Chinese Patent Application No. 1403080 Alexandria Journal of Pharmaceutical Sciences (2001), 15 (2), 99-102 Bioscience, Biotechnology, and Biochemistry (2000), 64 (8), 1588-1599 Phytochemistry (1992), 31 (11), 4001-4004 Foods & Food Ingred. J. Jpn (2000), No.189, 5-14 Yaoxue Xuebao (1998), 33 (8), 626-628 Zhongguo Zhongyao Zazhi (2003), 28 (11), 1086-1087 Zhongcaoyao (2001), 32 (8), 707-708 Shoyakugaku Zasshi (1992), 46 (3), 210-16

  An object of the present invention is to provide an antidiabetic agent that suppresses the hyperglycemic state after meal and exhibits an excellent effect in the prevention and treatment of diabetes.

  In order to obtain the antidiabetic agent as described above from a natural plant with few side effects, the present inventors searched for various unknown plants and conducted various evaluations. As a result, the asteraceae plant safflower (scientific name “Carthamus tinctorius L. It was found that the petal crude extract had an anti-diabetic action, and the petal crude extract was purified by contacting with an adsorbent to obtain an anti-diabetic composition (see WO 2005/094858). ).

  This anti-diabetic composition extracts safflower petals using, for example, water, an alcohol having 1 to 8 carbon atoms, a mixed solvent of the alcohol and water, an extraction solvent of ethyl acetate or acetone, Next, the safflower petal crude extract is brought into contact with an adsorbent, and then chromatographed using one or more of an organic solvent, water, a mixed solution of an organic solvent and water, an aqueous solution of an acid or an alkali. Elution and recovery of the fraction can be obtained, but the active ingredient was unknown.

  Therefore, the present inventor conducted intensive studies on a method for extracting safflower petals and a method for purifying petal crude extracts in order to isolate and purify active ingredients from various components contained in the antidiabetic composition. As a result, 6-hydroxy kaempferol-3-O-glycoside (1), which is a flavonoid glycoside, was separated as an active ingredient.

  That is, the present invention relates to 6-hydroxy kaempferol-3-O-glycoside, which is a flavonoid glycoside as an antidiabetic agent that suppresses post-meal hyperglycemia and exerts excellent effects in the prevention and treatment of diabetes (1) is provided.

  Although 6-hydroxy kaempferol-3-O-glycoside (1) has been reported so far as a prophylactic / therapeutic agent for cerebrovascular disorders, it has not been known to date regarding the postprandial blood glucose increase inhibitory effect. It was.

  6-hydroxy kaempferol-3-O-glycoside (1) has a structure represented by the following “Chemical Formula 2” and exhibits the inhibitory activity and / or suppressive activity of α-glucosidase which is a carbohydrate digestive enzyme. is there. Accordingly, a prophylactic and / or therapeutic agent for diabetes and a food composition containing an antidiabetic agent containing 6-hydroxykaempferol-3-O-glycoside (1) as an active ingredient suppresses hyperglycemia after eating. By doing so, it can contribute to the prevention and treatment of diabetes.

  The antidiabetic agent according to the present invention is effective for the prevention and treatment of diabetes.

  Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  The application amount of the antidiabetic agent according to the present invention varies depending on age, symptoms, etc., but for example, about 6-hydroxy kaempferol-3-O-glycoside (1) 1 to 500 mg, preferably 5 to 300 mg per adult. Degree.

  When the antidiabetic agent according to the present invention is used as a prophylactic and / or therapeutic agent for diabetes, in addition to the antidiabetic agent according to the present invention, other known compounds commonly used in the pharmaceutical field, and A compound necessary for molding into a form suitable for oral administration, for example, lactose, starch, hydroxypropylcellulose, kaolin, talc, calcium carbonate and the like may be contained. In addition, the drug may contain the antidiabetic agent in the form of a pharmaceutically acceptable salt, and may contain an excipient, a preservative, a coloring agent, a corrigent and the like as appropriate. In addition, it may be used in various forms such as powders, liquids, granules, tablets, capsules and the like by a known method in the field of pharmaceutical or food production.

  The antidiabetic agent according to the present invention can be applied to health foods. Health food means a food composition that is more active than ordinary food and drink, and is intended for health, health maintenance and promotion, etc. Is included. Specifically, for example, liquid, semi-solid or solid products such as cookies, rice crackers, jelly, yokan, yogurt, manju and other confectionery, soft drinks, nutritional drinks, soups and the like can be mentioned. In addition, it may be taken in hot water or water as it is. In the production process of these foods and drinks, or to the final product, an antidiabetic agent can be added by mixing, coating, spraying, or the like to obtain health food. These anti-diabetic food compositions indicate that they are used for the prevention and / or improvement of diabetes as necessary.

  Examples showing the effects of the present invention as antidiabetic agents will be described in detail below.

<Preparation of antidiabetic agent according to the present invention>
A hexane soluble part and an ethyl acetate soluble part were removed from 20 g of 85% ethanol extract of dried safflower petals to obtain a water soluble part. This was sequentially fractionated into a water elution part, methanol / ethanol elution part, and 1% ammonia / methanol elution part on a polyamide column, water elution part 13.5 g, methanol / ethanol elution part 1.10 g, and 1% ammonia / methanol elution part. 2.10 g was obtained.

  Elution of the methanol / ethanol elution part 2.3g obtained by repeating the above-mentioned operation on a Sephadex LH-20 column with 60% methanol, 80% methanol, and 100% methanol in sequence while changing the developing solvent. The eluted product (1) having an HPLC retention time of 10 min was confirmed by HPLC and concentrated. Furthermore, this sample was recrystallized with water-containing methanol containing water: methanol = 70: 30 to 80:20 to obtain 146 mg of the intended 6-hydroxy kaempferol-3-O-glucoside (1).

  The results of HPLC measurement and UV measurement are shown in FIGS. 1 and 2, respectively. The measurement conditions are as follows.

(1) HPLC measurement conditions
Column: Inertsil ODS-3 4.6mmφx250mm
Eluent: MeOH / H ₂ O / AcOH = 40/60 / 0.5
Detecter: HITACHI Diode Array L-7455
Detect: 350nm
Sample inject Volume: 10μl
(2) UV measurement condition model: HITACHI 150-20 double beam spectrophotometer
Mode: Abs
Limts: 0.0 to 1.50
WL scan speed: 200nm / min
WL Limts: 200.0 ton 600.0nm
Back round: no
No. of Repeat: 1
Cycle Time: 0
Response: Medium
Lamp Chane: Auto
Recording: no
WL Scale: nm / cm
Line: Solid
Cell: Quartz glass cell 10mm

Next, the structure of the isolated and purified 6-hydroxykaempferol-3-O-glycoside (1) was determined by nuclear magnetic resonance spectroscopy (NMR). The 1H-nuclear magnetic resonance spectrum and 13C-nuclear magnetic resonance spectrum data are as follows. Further, the isolated and purified 6-hydroxy kaempferol-3-O-glycoside (1) was a colorless crystal and had a melting point of 257 to 261 ° C. The molecular formula was represented by C ₂₁ H ₂₀ O ₁₂ , the molecular weight was 464.41 in the molecular formula, and it was 463 [MH] ^{− as} analyzed by Fast Atom Bombardment Mass Spectrometry (FAS-MAS).

(1) 1H-nuclear magnetic resonance spectrum
1H-NMR (500 MHz, DMSO-d6) δ:
6.53 (1H, s) 8-H
8.04 (2H, dd, J = 8.9Hz, 2.1Hz) 2'-H, 6'-H
6.89 (2H, dd, J = 8.9Hz, 2.1Hz) 3'-H, 5'-H
5.48 (1H, d, J = 7.7Hz) 3-O-Glc, 1-H
3.23 to 3.16 (1H, m) 3-O-Glc, 2-H
3.10 ~ 3.09 (1H, m) 3-O-Glc, 3-H
3.23 to 3.16 (1H, m) 3-O-Glc, 4-H
3.10 ~ 3.09 (1H, m) 3-O-Glc, 5-H
3.72 (1H, d, J = 10.3) 3-O-Glc, 6a-H
3.34 (1H, d, J = 6.8Hz) 3-O-Glc, 6b-H
(2) 13C-nuclear magnetic resonance spectrum
13C-NMR (125 MHz, DMSO-d6) δ:
156.0 (C-2)
132.7 (C-3)
177.4 (C-4)
146.3 (C-5)
128.9 (C-6)
153.5 (C-7)
93.4 (C-8)
148.8 (C-9)
104.2 (C-10)
121.1 (C-1 ')
130.7 (C-2 ', C-6')
114.9 (C-3 ', C-5')
159.7 (C-4 ')
100.8 (3-O-Glc, C-1)
74.1 (3-O-Glc, C-2)
76.3 (3-O-Glc, C-3)
69.7 (3-O-Glc, C-4)
77.4 (3-O-Glc, C-5)
60.7 (3-O-Glc, C-6)

<Measurement of α-glucosidase inhibitory activity>
Using the 6-hydroxykaempferol-3-O-glycoside (1) prepared in Example 1, an α-glucosidase activity inhibition test was conducted.

  A 9-fold amount of 0.05 M maleic acid buffer (pH 6.0) was added to rat acetone powder (manufactured by SIGMA), subjected to ultrasonic treatment, centrifuged at 3000 rpm for 10 minutes, and the supernatant was used as an enzyme solution. Add 50 μL of 2% sucrose dissolved in maleate buffer (pH 6.0) to 50 μL of test substance dissolved in maleate buffer (pH 6.0) at the specified concentration, and pre-incubate for 5 minutes at 37 ° C. Then, 50 μL of an enzyme solution diluted twice with maleic acid buffer solution (pH 6.0) was added and reacted at 37 ° C. for 60 minutes. After completion of the reaction, the enzyme was inactivated at 95 ° C. for 10 minutes, centrifuged at 15000 rpm for 10 minutes, and the amount of glucose in the supernatant was measured with SYNCHRON CX (Beckman).

  As a control, a sample solution using only a maleic acid buffer (pH 6.0) (negative control) was used. Further, as the sample solution, an acarbose solution that is an α-glucosidase inhibitor and is used as a postprandial hyperglycemia improving agent was used as a positive control.

  The results are shown in “Table 1”. The numerical values in the table are values (%) indicating the enzyme inhibitory activity, and the glucose amount when each sample is used is divided by the glucose amount when the maleate buffer is used (negative control). Calculated.

  As shown in “Table 1”, when 0.005% of 6-hydroxykaempferol-3-O-glycoside (1) was added, α-glucosidase activity was suppressed to 80%. Furthermore, when 0.05% of 6-hydroxykaempferol-3-O-glycoside (1) was added, α-glucosidase activity was markedly suppressed to 34%. From this, it is clear that 6-hydroxy kaempferol-3-O-glycoside (1) has an excellent α-glucosidase inhibitory activity.

<Examination of blood glucose level rise suppression effect>
Regarding 6-hydroxykaempferol-3-O-glycoside (1), the effect of suppressing the increase in blood glucose level was examined.

  Rats (SD, male, 5 weeks old) were purchased from Oriental yeast and acclimated for 1 week. After fasting for 17 hours from the day before the test, body weight was measured. Further, blood was collected from the tail vein, and the blood glucose level was measured with Antsense II (manufactured by Bayer Medical). The groups were divided so that the body weight and blood glucose level were uniform in each group, and 3 animals per group. Test substance 15 mg / kg and sucrose (Wako Pure Chemical Industries) 2 g / kg were orally administered by gavage using a sonde. Since 6-hydroxy kaempferol-3-O-glycoside (1) has low solubility, the administration solvent was 40% DMSO and 60% distilled water. Blood was collected from the tail vein 30 minutes, 60 minutes and 120 minutes after administration, and the blood glucose level was measured with Anthsen II.

  For the control, instead of the administration solution, a solution containing only 40% DMSO and 60% distilled water and 2 g / kg were administered in the same manner.

  The amount of increase in blood glucose level after administration (blood glucose increase value (mg / dL)) is shown in “Table 2”. In addition, the blood glucose increase value was calculated | required by the following formula. "Elevated blood glucose level = administration group blood glucose level-administration group blood glucose level before administration start".

  In addition, the blood glucose level increase inhibition rate (%) was determined by the following formula. “Inhibition rate of increase in blood glucose level = [1 − {(blood glucose level in administration group−administration group blood glucose level before administration start) / (blood glucose level in control group−control blood glucose level before administration start)}] × 100” The inhibition rate (%) of blood glucose level is shown in “Table 3”. In the table, “AUC” represents an area under the curve (Area Under Curve) and represents an increase in blood glucose level over time.

  As shown in “Table 2”, in rats administered with 6-hydroxykaempferol-3-O-glycoside (1), increase in blood glucose level was suppressed at all times of 30, 60 and 120 minutes after administration. It was. Thereby, it was shown that 6-hydroxy kaempferol-3-O-glycoside (1) has a remarkable blood glucose rise inhibitory effect (refer to "Table 3").

  Since the anti-diabetic agent according to the present invention is a natural product derived from plants, there is less concern about side effects and the like, and it can be effectively used as a preventive and / or therapeutic agent for diabetes. Moreover, the food composition which mix | blended the antidiabetic agent can be taken easily, and possibility that it can continue taking continuously for a long period of time is high.

It is a figure which shows the HPLC chart of 6-hydroxy kaempferol-3-O-glycoside (1). It is a figure which shows the UV chart of 6-hydroxy kaempferol-3-O-glycoside (1).

Claims (2)

An α-glucosidase inhibitory and / or inhibitory active agent comprising 6-hydroxykaempferol-3-O-glycoside (1), which is a flavonoid glycoside having a structure represented by the following “chemical formula 1”.
Claim 1, wherein the α- glucosidase inhibiting and / or suppressing active agent and pharmaceutically containing a acceptable additives α- glucosidase inhibiting and / or suppressing activator.