JP2000239164A - Glycosidase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a highly safe glycosidase inhibitor derived from natural matter and having potent α-glucosidase activity inhibitory effect and α-amylase activity inhibitory effect by including a specific hibiscus acid compound. SOLUTION: This glycosidase inhibitor contains e.g. 0.01-30, pref. 0.1-20 w/w% of a hibiscus acid compound of the formula [R is H or a (substituted) alkyl] (specifically, hibiscus acid, hibiscus acid methyl ester, hibiscus acid ethyl ester, or the like), wherein the hibiscus acid compound is pref. an extract obtained by subjecting a plant belonging to the genus Hibiscus, Malvaceae (e.g. Hibiscus sabdariffa L., Hibiscus rosa-sinensis L.) to extraction with a hydrophilic solvent (e.g. water, methanol, a mixture thereof). The daily dose of this inhibitor is, for example, normally 1-5,000, pref, 10-1,000 mg/adult or so, in terms of the hibiscus acid compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a glycosidase inhibitor, and more particularly to a glycosidase inhibitor and a therapeutic agent for diabetes derived from natural products and having high safety.

[0002]

2. Description of the Related Art Usually, human carbohydrates represent about
80-90% is said to be starch and sucrose. When starch is ingested, it is decomposed into dextrin by α-amylase in saliva, and further decomposed by pancreatic α-amylase to maltose and isomaltose. On the other hand, sucrose reaches the small intestine without being decomposed in the gastrointestinal tract. These disaccharides reaching the small intestine are absorbed after being decomposed into monosaccharides by α-glucosidase localized on the small intestinal brush border membrane. α-glucosidase is a general term for an exo-type glucosidase that cleaves a monosaccharide from the reducing end of a compound having a glucoside bond,
Contains maltase, sucrase, isomaltase and the like. Ingestion of starch and sucrose raises blood sugar levels, which in turn secretes insulin and regulates blood sugar levels. However, it is known that, when the state of high blood sugar level continues, the function of the pancreas decreases, the required insulin is not secreted, and this causes diabetes.
It is also known that obesity reduces insulin sensitivity and causes pancreatic function to decline.
For this reason, it is effective for prevention and treatment of diabetes and obesity to suppress a rapid increase in blood sugar level after eating.

In recent years, it has been found that administration of a glycosidase inhibitor such as α-glucosidase or α-amylase suppresses a rapid increase in blood glucose and insulin after a meal (for example, Japanese Patent Application Laid-Open No.
-122342, Diabetic Medicine, 1993, Vol. 10,
See pages 688-693). Acarbose, an α-glucosidase inhibitor, has been clinically applied as a therapeutic agent for non-insulin-dependent diabetes. However, administration of acarbose not only requires a strict prescription by a physician, but also reports side effects such as abdominal distention, increased flatus, loose stools, and diarrhea, making it difficult to use as a food or food material. is there.

[0004]

DISCLOSURE OF THE INVENTION The present invention does not have the drawbacks of the conventional glycosidase inhibitors as described above,
Not only α-glucosidase but also α-amylase activity is strongly suppressed, and it is contained in a large amount in plants and the like, and a substance which is easy to extract and process and which is highly safe is required, and the substance is contained as an active ingredient. Another object of the present invention is to provide a glycosidase inhibitor and an antidiabetic agent which can be expected to have an effect of suppressing postprandial hyperglycemia.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, roselle (Hisel) which is a plant of the genus Hydrangea and is drunk as herbal tea.
biscus sabdariffa L.) and bushes (Hibiscus ros)
a-sinensis L.) It was revealed for the first time that the extract from petals showed a high glycosidase activity inhibitory effect, and that the active substance was hibiscus acids, and if this hibiscus acid was included as an active ingredient, Remarkable glycosidase inhibitory action prevents diabetes and obesity
The inventors have found that the present invention is effective for improvement and the like, and have completed the present invention based on these findings. That is, the present invention
Lower formula

[0006]

Embedded image (Where R in the formula represents a hydrogen atom or a substituted or unsubstituted alkyl group) is a glycosidase inhibitor containing a hibiscus acids as an active ingredient, A glycosidase inhibitor containing, as an active ingredient, an extract obtained by extraction with a neutral solvent.

[0007]

Embedded image (Where R in the formula represents a hydrogen atom or a substituted or unsubstituted alkyl group). Hereinafter, the present invention will be described in detail.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hibiscus acids referred to in the present invention are represented by the above formula.
Any such material is acceptable. A hibiscus acid wherein R in the formula is a hydrogen atom,
Or an alkyl ester thereof, for example, those wherein R in the formula is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, for example, a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, an isobutyl group, and a hexyl group. It is particularly preferable because it has high water solubility and is easy to handle. Specific examples of preferable hibiscus acids in the present invention include, for example, hibiscus acid, methyl hibiscate, and ethyl hibisate. And their production means is not particularly limited, and may be a method by chemical synthesis, or a method of extracting from a plant containing hibiscus acids using a conventional technique, etc., but the latter is cheaper and easier. It is preferably used because it can be obtained. In the present invention, any of the extract such as the extract thus obtained, its intermediate purified product, and its purified product can be used.

In order to extract the hibiscus acids from the plant or the like, for example, the plant itself containing hibiscus acid or a crushed product thereof (whether raw or dried) can be extracted with water or an organic solvent, Alternatively, extraction may be performed by a usual method using a mixture of water and an organic solvent. Plants containing hibiscus acids include plants belonging to the genus Hydrangea, such as the annual rosel (Hibiscus s.
abdariffa L.), a bush bud (Hibiscus r.)
osa-sinensis L.), Chinese Rosewood (Hibiscus syri) native to China
acus L.), Kenaf (Hibiscus cannabinu) native to Africa
s L.), a frog mallow (Hibiscus coccineus) native to North America
L.), Hamabo (Hibiscus hamabo) native to Japan, and Japanese Fir (Hibiscus mutabilis L.) native to China. And to extract hibiscus acids,
Although any part of the plant may be used, petals of roselle or bushuge are particularly preferably used in view of the content.

The solvent used for extracting hibiscus acids from the hibiscus acids-containing material is preferably water, an organic solvent, or a mixture thereof. Examples of the organic solvent include hydrophilic solvents such as methanol, ethanol, isopropanol, DMF, and acetone, and ethyl acetate, chloroform, dichloromethane, pentane, hexane, heptane, and the like. Among these solvents, water or a hydrophilic organic solvent such as methanol, or a mixture of water and a hydrophilic organic solvent is preferable, and water is particularly preferable in terms of extraction rate and safety.

In order to obtain an extract using the above-mentioned solvent,
A known method may be used. For example, the petals of the above-described plants are used as raw materials, and after appropriately crushing the treated petals, the crushed products are treated with the above-described solvent using a known method. Specifically, 1 to 100 times (weight ratio) of the raw material, preferably 3 to 2 times
0 times (weight ratio) solvent, temperature 0 ° C or higher, preferably 1
The extraction treatment is carried out for 1 minute to 8 weeks, preferably 10 minutes to 1 week, with stirring, at a temperature of 0 ° C. to the boiling point of the solvent or lower. A method in which a cooling tube is attached to the upper portion and extraction is performed while refluxing may be used.

The extracted substance obtained as described above may be used as it is in the present invention. Preferably, the organic solvent used for the extraction is removed by a usual method, for example, using a rotary evaporator under reduced pressure. Is good. If necessary, the extract from which the organic solvent has been removed is freeze-dried,
Conventional drying treatment such as heat drying may be used.

In order to purify the hibiscus acids from the above-mentioned extract, a known method used for separating and purifying natural organic compounds may be employed. For example, adsorption and desorption using activated carbon, silica gel, chemically modified silica gel, a polymer carrier, or chromatography, liquid-liquid extraction,
The target substance can be purified by a technique such as fractional precipitation. Specifically, for example, the extract is subjected to ODS-column chromatography, and eluted with 5% methanol,
The obtained eluted fraction was further subjected to ODS-HPLC,
The mixture is eluted and fractionated with a water-methanol mixed solvent containing 0.1% trifluoroacetic acid. The target fractions separated by chromatography are collected, concentrated and dried to obtain higher purity hibiscus acids.

As described in Examples, the above-mentioned extract and its purified product, ie, hibiscus acids, have a strong activity inhibiting activity against rat small intestine-derived α-glucosidase (sucrase) and porcine pancreas-derived α-amylase. It was confirmed that. It was confirmed that these hibiscus acids also had extremely high safety.

Therefore, by containing any of the above-mentioned extracts, purified products or intermediate purified products containing the above hibiscus acids, the glycosidase inhibitor or the glycosidase activity inhibitory activity of the present invention, which is expected to exhibit the above-mentioned effects. Can be used as a therapeutic agent for diabetes. If necessary, the above-mentioned hibiscus acids may be added with a suitable excipient usually used in medicines, for example, lactose, starch, fats and the like, to give the glycosidase inhibitor of the present invention or the therapeutic agent for diabetes. . In the present invention, one or two or more hibiscus acids may be used in combination, if necessary.

The glycosidase inhibitor or the therapeutic agent for diabetes according to the present invention comprises hibiscus acids, for example, from 0.01 to 30.
% (W / w), preferably 0.1 to 20% (w / w)
It is used by containing. Further, an emulsifier, a stabilizer, a bulking agent, and the like may be mixed with the hibiscus acids alone or as a mixture with another drug. Further, the glycosidase inhibitor or the therapeutic agent for diabetes of the present invention can be administered orally. Powders as oral preparations,
Any dosage form such as tablets, granules, capsules and the like can be employed.

The dosage of the glycosidase inhibitor or antidiabetic agent of the present invention varies depending on the condition to be treated, age, body weight and the like. For example, usually, 1 to 5000 mg of hibiscus acids per adult per day is preferable, It is about 10 to 1000 mg. Also, health food,
Normal intake when eating as foods and drinks, food additives, etc., for example, per adult day, as hibiscus acids,
About 1 mg to 3000 mg, preferably 5 mg to 200 mg
mg.

[0018]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Experimental Examples, and Examples, but the present invention is not limited to these examples. Reference Example 1 (Preparation of Hibiscus Acid from Roselle) 100 ml of water was added to 10 g of dried rose petals, and the mixture was extracted at room temperature for 1 day with stirring. The residue was removed by suction filtration to obtain an extract. The extract was dried under reduced pressure using a rotary evaporator to obtain an extract. After dissolving this in water to remove insolubles, H using Inertsil PREP-ODS (manufactured by GL Sciences) with an inner diameter of 20 mm × 25 cm was used.
20% containing 0.1% trifluoroacetic acid by PLC
It was eluted with a methanol-water mixture. When this fraction was dried under reduced pressure by a rotary evaporator, 60 mg
Of hibiscus acid was obtained. In addition, the molecular weight of the obtained substance by mass spectrum was 190. The proton NMR spectrum was as follows. Proton NMR spectrum (500 MHz) (acetone-d
6) δ (ppm): 5.34 (1H, S), 3.24 (1H, d, J = 17.2)
Hz), 2.77 (1H, d, J = 17.2Hz). From these values, the substance was identified as Boll et al. (Acta Chemica Sca
ndinavica, 1969, Vol. 23, pages 286-293). That is, this purified product is a hibiscus acid in which R in the above formula is a hydrogen atom.

Reference Example 2 (Preparation of hibiscus acid from bushuge) Hibiscus acid was extracted and purified in exactly the same manner as in Reference Example 1 except that 10 g of dried flower petals of Boussouget was used instead of 10 g of dried petals of roselle. Acid 59
mg was obtained.

Reference Example 3 (Preparation of 6-methyl hibiscus acid) 100 ml of 50% methanol was added to 10 g of dried rosel petals and left at room temperature for one day. The residue was removed by suction filtration to obtain an extract. The extract was dried under reduced pressure using a rotary evaporator to obtain an extract. This was dissolved in water to remove insolubles, and then subjected to column chromatography of Cosmosil 75C18-OPN (manufactured by Nacalai Tesque) having an inner diameter of 4 cm × 18 cm. Fractions eluted with 5% methanol were collected. This fraction was concentrated under reduced pressure by a rotary evaporator, and further subjected to HPLC using an InertsilPREP-ODS (manufactured by GL Sciences) using an inner diameter of 20 mm × 25 cm, and containing 20% containing 0.1% trifluoroacetic acid.
The mixture was eluted with a methanol / water mixed solvent. This fraction was evaporated to dryness under reduced pressure using a rotary evaporator.
mg of hibiscus acid 6-methyl ester were obtained.
The molecular weight of the obtained substance by mass spectrum was 2
04. The proton NMR spectrum and the carbon-13 NMR spectrum were as shown below. Proton NMR spectrum (500 MHz) (D2O) δ
(Ppm): 5.43 (1H, S), 3.89 (3H, S), 3.44 (1H, S)
d, J = 17.8 Hz), 2.87 (1 H, d, J = 17.8 Hz). Carbon-13 NMR spectrum (125 MHz) (D2O)
δ (ppm): 178.3, 174.5, 172.3, 86.6, 81.1, 56.7, 44.
9. From these values, the substance was identified as Boll et al. (Acta Chemica Sca
ndinavica, 1969, Vol. 23, pp. 286-293). In addition, HMBC between the methyl proton and the carbon at position 6
Since a correlation was observed in the spectrum, the position of methylation was determined to be position 6. That is, this purified product
The hibiscus acid 6-methyl ester in which R in the above formula was methyl was determined.

Experimental Example 1 [Measurement of α-glucosidase (sucrase) activity inhibitory effect] Hibiscus acid sample obtained in the same manner as in Reference Example 1 and hibiscus acid 6-methyl ester sample obtained in the same manner as Reference Example 3 Using the product, the α-glucosidase (sucrase) activity inhibitory effect was measured as follows. As a substrate solution, a solution prepared by dissolving sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) in a 10 mM phosphate buffer (pH 7.0) to a concentration of 28 mM was used. The enzyme solution was prepared from rat small intestine acetone powder (manufactured by Sigma). The partially purified enzyme dissolved in a 10 mM phosphate buffer (pH 7.0) was used. The amount of glucose generated by the sucrase reaction was quantified using a glucose test kit, Wako (manufactured by Wako Pure Chemical Industries, Ltd.). In this system, a test substance dissolved in 50% dimethyl sulfoxide (eg, hibiscus acid or hibiscus acid 6) was used.
-Methyl ester) at various concentrations (1.58, 2.1
0, 2.63, 3.16, 6.31 mM). The rate of decrease in glucose production at this time was defined as the sucrase activity inhibitory activity.

The results are shown in FIG. 1. Hibiscus acid and hibiscus acid 6-methyl ester inhibited sucrase activity to the same extent. The concentration that inhibits the sucrase reaction by 50% is about 2.3 in each case.
mM.

Experimental Example 2 (Measurement of α-Amylase Activity Inhibiting Effect) A sample of hibiscus acid obtained in the same manner as in Reference Example 1 and a sample of 6-methyl hibiscus acid obtained in the same manner as in Reference Example 3 were used. Then, the α-amylase activity inhibitory effect was measured as follows. As a substrate, 10 mg of Starchiazur (manufactured by Sigma) was used, and as an enzyme solution, porcine pancreatic α-amylase (manufactured by Sigma) was 2.36 units /
50m containing 10mM calcium chloride to make ml
What was dissolved in M Tris-HCl buffer (pH 6.9) was used. The blue dye produced by the α-amylase reaction was quantified by measuring the absorbance at 595 nm. In this system, a test substance (hibiscus acid or hibiscus acid 6-methyl ester) dissolved in 50% dimethyl sulfoxide was added at various concentrations (1.14, 1.43, 1.72,
(2.29, 3.43 mM). The α-amylase activity inhibitory activity was defined as the rate of decrease in the amount of blue dye produced at this time.

The results are shown in FIG. 5. Hibiscus acid and 6-methyl hibiscus acid suppressed α-amylase activity to the same extent. And
The concentration at which the α-amylase reaction was inhibited by 50% was about 1.6 mM in all cases.

From these facts, it can be seen that hibiscus acids have a strong sucrase activity inhibitory effect and an α-amylase activity inhibitory effect, and if they are contained as active ingredients, the prevention and improvement of diabetes and obesity can be achieved. It turns out that it is effective for treatment.

Experimental Example 3 (single dose toxicity test) ICR / crj male mice weighing 29 to 32 g (5 weeks old)
(5 animals per group), using the guidelines for toxicity test methods for pharmaceuticals (February 15, 1984, Pharmaceutical Affairs Commission No. 118, Notification of the second section of the Ministry of Health and Welfare Pharmaceutical Affairs Bureau's Examination Section 2 to the Prefectural Health Administration Bureau) A single dose toxicity test was performed according to the criteria. That is, a physiological saline test substance (hibiscus acid or hibiscus acid 6-methyl ester) was dissolved and orally administered to mice at a rate of 2 g per kg of body weight, and observed for 14 days. As a result, there were no dead animals and no side effects were observed in all cases, and no microscopic abnormalities of tissues and organs were observed at necropsy on day 14, so that the hibiscus acids used in the present invention were It can be seen that the toxicity is extremely low.

Example 1 (Glycosidase inhibitor (powder)
Production of Hibiscus acid sample obtained in the same manner as described in Example 1, lactose, and crystalline cellulose were mixed in the following composition, kneaded with purified water, and then powdered or finely powdered by a conventional method. 1 kg of granulated powder was prepared. This powder is 1 packet 1
g is a glycosidase inhibitor (powder) containing 100 mg of hibiscus acid per dose. <Composition of powder> Hibiscus acid 100 g Lactose 700 g Crystalline cellulose 200 g

Example 2 (Glycosidase inhibitor (powder)
Production of Hibiscus acid 6 obtained in the same manner as described in Example 3.
-Methyl ester sample, lactose, and crystalline cellulose were mixed in the following composition, kneaded with purified water, and then 1 kg of powder or fine powder was prepared by a conventional method. This powder is a glycosidase inhibitor (powder) containing 200 mg of hibiscus acid 6-methyl ester in one packet of 1 g per dose. <Composition of powder> Hibiscus acid 6-methyl ester 200 g Lactose 600 g Crystalline cellulose 200 g

Example 3 (Preparation of a health food (powder form) containing an extract) A rosel extract (containing about 5% hibiscus acid) obtained in the same manner as described in Reference Example 1 was added. 1 kg of a health food (in powder form) having the following composition (unit: g) and having a glycosidase inhibitor action was prepared. As the soybean separated protein in the composition, "New Fuji Pro 1200" manufactured by Fuji Purina Protein Co., Ltd. was used. <Composition of health food (per kg)> Roselle extract 90.0 Soybean isolated protein 864.446 Hydrous crystalline glucose 30.3 Dextrin 3.0 Vitamin B ₁ 0.05 Vitamin B ₂ 0.06 Vitamin B ₆ 0.15 Vitamin B ₁₂ 0.00018 Folic acid 0.024 Vitamin A 0.22 Ascorbic acid 2.0 α-dl-tocopherol 0.75 Calcium lactate 9.0 Eating 30 g of this health food a day gives 135 mg of hibiscus acids can do.

Example 4 (Preparation of Healthy Food (Soft Capsule) Containing Extract) A roselle extract (containing about 5% of hibiscus acid) obtained in the same manner as in Reference Example 1 was added to obtain the following composition ( Unit: m
g) A health food (soft capsule) having a glycosidase inhibitory action was prepared. <Composition of health food (per capsule)> Roselle extract 100.0 β-carotene 20.0 α-dl-tocopherol 0.75 DHA 30.0 olive oil 849.25 Eating 30 can take 5-150 mg of hibiscus acids.

[0031]

EFFECT OF THE INVENTION The glycosidase inhibitor and antidiabetic agent of the present invention containing hibiscus acids as active ingredients have a remarkable inhibitory effect on α-glucosidase activity and α-amylase activity. Hibiscus acids are highly safe in use. Therefore, the glycosidase inhibitor of the present invention containing this substance as an active ingredient is extremely useful for preventing and improving diabetes and obesity. Also,
Since the food material containing the hibiscus acids has an α-glucosidase activity inhibitory effect and an α-amylase activity inhibitory effect, it can be used as a health food material, and the present invention is extremely useful in industry.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the concentration (mM) of hibiscus acid and hibiscus acid 6-methyl ester and sucrase inhibitory activity (%) in Experimental Example 1.

FIG. 2 is a graph showing the relationship between the concentration (mM) of hibiscus acid and hibiscus acid 6-methyl ester and α-amylase inhibitory activity (%) in Experimental Example 2.

 ────────────────────────────────────────────────── ─── Continued on front page F term (reference) 4C086 AA01 AA02 BA03 GA17 MA01 MA04 NA14 ZA70 ZC20 ZC35 4C088 AB13 AC01 AC03 BA08 BA09 BA10 BA11 BA31 MA01 NA14 ZA70 ZC20 ZC35

Claims (6)

[Claims] 1. The following formula: (Where R in the formula represents a hydrogen atom or a substituted or unsubstituted alkyl group). A glycosidase inhibitor comprising a hibiscus acid as an active ingredient. 2. A glycosidase inhibitor comprising, as an active ingredient, an extract obtained by extracting a Malvaceae plant of the genus Malvaceae with a hydrophilic solvent. 3. The Malvaceae plant of the family Malvaceae is Roselle.
The glycosidase inhibitor according to claim 2. 4. The glycosidase inhibitor according to claim 2, wherein the Malvaceae plant of the genus Malvaceae is bushweed. 5. The glycosidase inhibitor according to claim 2, wherein the hydrophilic solvent is water or methanol or a mixture of water and methanol. 6. The following formula: (Where R in the formula represents a hydrogen atom or a substituted or unsubstituted alkyl group). A therapeutic agent for diabetes comprising a hibiscus acid as an active ingredient.