JP2002275191A - New insulin-like active substance and composition comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition capable of exhibiting insulin-like actions by oral administration and having effects on prophylaxis or amelioration of diabetes in the field of medicines or foods. SOLUTION: This saponin compound is specifically represented by the following formula (wherein, R<1> denotes a sugar chain; R<2> denotes a sugar or the like substituted with angeloyl group or tigloyl group; and R<3> and R<4> denote each hydrogen atom or acetyl group) and has actions with promoting effects on sugar uptake by cells similar to those of insulin. The composition comprises the compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel substance having an effect on preventing or improving diabetes. More specifically, a mechanism of action similar to insulin, that is,
The present invention relates to a novel plant-derived substance that is effective in preventing or ameliorating diabetes by suppressing an increase in postprandial blood glucose level by promoting glucose uptake into cells.

[0002]

2. Description of the Related Art Diabetes is a general term for a group of metabolic diseases characterized by sustained hyperglycemic state due to insufficient action of insulin, and is not a single disease. Reduced insulin sensitivity and reduced insulin secretion develop in any combination, resulting in abnormalities in sugar, lipid, and protein metabolism.
Recently, it is said to be one of lifestyle-related diseases and is expected to be improved by appropriate measures.However, when it becomes severe, complications such as neuropathy, retinopathy or nephropathy occur,
It is a disease that significantly reduces daily activities.

At present, there are about 7 million people in Japan who are strongly suspected of diabetes, so-called diabetic patients, and about 7 people who cannot deny the possibility of diabetes, so-called diabetic reserves.
It is estimated to be one million, and it is estimated to reach 14 million if both are combined.
The number has increased, and it can be seen that it has increased dramatically. Globally, the number of patients from 100 million in 1994 was 20
The number is expected to reach 220 million in 2010, and the surge is particularly acute in the Asian region.

It is needless to say that this is mainly due to environmental factors such as the westernization of lifestyles.
Analysis of NPs (Single nucleotide polymorphisms: subtle differences in DNA sequence between individuals) reveals that Japanese (Asian) have a genetic predisposition to be more susceptible to diabetes than Westerners It is thought that such facts are also accelerating the increase in the number of patients worldwide. Therefore, it can be said that measures are a global issue.

[0005] Diabetes is insulin dependent (IDDM)
And insulin-independent type (NIDDM). The insulin-dependent type is a type in which insulin cannot be synthesized and secreted by pancreatic β-cells necrotic or dysfunctional due to an autoimmune mechanism caused by a virus or the like. is there. Insulin-independent type is a type showing insulin deficiency caused by unclear and various factors such as aging and stress, and hyperglycemia due to insulin resistance, and about 90% of diabetics have this type. include. Diet therapy and exercise therapy are mainly used for treatment of mild or moderate patients of this type, and blood glucose levels are stabilized by calorie restriction of diet and promotion of glucose metabolism by exercise.

[0006] However, if the postprandial increase in blood glucose level and its continuation (postprandial hyperglycemia) continue for many years, it eventually leads to impaired glucose tolerance, which promotes vascular disorders as the diabetes worsens, and increases neuropathy, There is a risk of developing complications such as nephropathy, retinopathy, and even myocardial infarction and stroke. Suppression of postprandial hyperglycemia is said to be effective in the development of insulin-independent diabetes, and α-glucosidase inhibitors are used as medicaments for suppressing glucose absorption, and sulfonylurea agents are used to promote insulin secretion. From the viewpoint of prevention or prevention of deterioration, further enhancement of diet therapy is desired.

[0007] In addition, there is stress as a factor that causes a hyperglycemic state other than diabetes. In today's stressful society, many people, from children to adults, feel stress. In addition, there are not a few people who drink or eat under this stress. It is said that stress excites the sympathetic nerve and promotes the secretion of adrenaline and noradrenaline, thereby increasing the blood sugar level. Therefore, the risk of hyperglycemia is increased when stress, daily drinking and overeating continue in daily life, leading to complications similar to diabetes. Therefore, it is always necessary to control the blood glucose level by suppressing postprandial hyperglycemia on a daily basis.

[0008] As described above, abnormalities in glucose metabolism are associated with many diseases of lifestyle-related diseases. Therefore, drugs and foods for preventing or preventing exacerbation of lifestyle-related diseases are desired.

Insulin is the only hormone in vivo that promotes glucose metabolism in the liver and lowers blood sugar levels by causing increased glucose uptake in peripheral tissues such as muscle and adipose tissue. One of the mechanisms of action in peripheral tissues is to recruit sugar transporter type 4 (GLUT4) present in the intracellular pool onto the cell membrane. The signal transduction mechanism involved in the promotion of glucose uptake in muscle and fat cells by insulin has been considered to date as follows.

That is, insulin binds to an insulin receptor (insulin receptor: IR), activates an intrinsic tyrosine kinase, and reacts with an insulin receptor substrate (insulin receptor substrate: IRS).
s) Tyrosine phosphorylate the family. Tyrosine phosphorylated IRSs are phosphatidylinositol-3
Activating kinase (PI3K), followed by some signal transduction, resulting in potential GLUT4
Is believed to be translocated onto the cell membrane (Shepherd PR, Kahn BB. Et al.,: N. Engl. J. Med.,
1999 Jul 22; 341 (4): 248-57. Review .: Glucose tran
sporters and insulin action--implications for insu
lin resistance and diabetes mellitus.).

[0011] Insulin is used as a pharmaceutical, but is a peptide having a molecular weight of 5,000 or more.
It cannot be absorbed orally and can only be used effectively by administration by injection. Therefore, in the field of medicines and foods, there is a demand for an orally active substance that exhibits an insulin-like action, that is, an action of promoting glucose uptake into cells, thereby suppressing an increase in blood glucose level. Under these circumstances, as a recent research result, the discovery of a low-molecular-weight insulin-like substance derived from microorganisms (Zhang B. et al.,: Science 199
9 May 7; 284 (5416): 974-7: Discovery of a small molec
ule insulin mimetic with antidiabetic activity in
mice.) has been reported. However, none has yet been put to practical use.

[0012]

In view of the current situation,
The present inventors have found a non-peptide compound exhibiting an insulin-like action in vivo, and in the field of medicine and food, develop a composition which exhibits an insulin-like action by oral administration and is effective in preventing or improving diabetes. This is an object of the present invention.

[0013]

Means for Solving the Problems The present inventors have used the blood glucose elevation suppressing effect (in the present specification, the blood glucose elevation suppressing effect and the blood glucose lowering effect are used as synonyms unless otherwise specified. ) And did not show any harmful effects on the human body. As a result of extensive research on natural products, we found that some of the saponins derived from Aesculaceae plants contained sugars, which are similar to insulin, to cells. The present inventors have found that there is a novel compound group having an uptake promoting effect, and have completed the present invention.

Saponin is a group of glycosides distributed in the plant kingdom and is a generic term for compounds containing polycyclic compounds as aglycones, which form aqueous colloids which foam remarkably like soap. ) Is found in plants in more than 60 families. They are classified into triterpenoid saponins and steroid saponins according to the difference in the structure of aglycone called sapogenin (Physical and Chemical Dictionary (Iwanami Shoten), Chemical Dictionary (Kyoritsu Shuppan) and others).

In general, saponin is used as an emulsifier or a detergent because it produces an aqueous solution such as soap which remarkably foams as described above and has an effect of emulsifying oils.
Saponin has hemolytic activity, irritates mucous membranes and has an toxic effect on fish, but is generally harmless when taken orally by humans (Rikken Dictionary (Iwanami Shoten) , Chemical Dictionary (Kyoritsu Shuppan) and others.

Also, the horse chestnut ( Aesculus)
Hippocastanum L. , Hipocast
anaceae) Saponin mixture (e) contained in seeds
Scin) is known in Europe as the most important saponin that is used clinically as an anti-inflammatory drug and as a therapeutic agent for swelling after surgery and trauma, and also widely used in cosmetics and the like. In Japan as well, it is used as a drug or raw material for treating inflammation or swelling. Although escin has been reported to have anti-inflammatory activity and anti-tumor activity, all previous studies have examined using saponin mixtures, and detailed studies such as the correlation between saponin structure and activity have been performed. (Masayuki Yoshikawa et al.,
Pharmaceutical Journal, 119 (1), 81-87 (1999)).

Thus, Yoshikawa et al. Conducted research on biologically active saponins contained in Japanese and Chinese herbs and medicinal plants. As a part of these studies, a mixture of saponins obtained from western horse chestnut seeds and commercially available β-escin were added to blood glucose in glucose-loaded rats. It has been found that there is a remarkable inhibitory activity on the increase in the value and an inhibitory activity on alcohol absorption. That is, 12 kinds of saponins were isolated from a saponin mixture, their chemical structures were clarified, and four of the compounds had an activity of suppressing blood sugar level increase and an activity of suppressing alcohol absorption, and their activities were remarkable. And found that there were significant differences. further,
A comparative study of the activities of related compounds revealed that the presence of an acyl group is essential for the expression of the activity, and that the type of the acyl group and the oligosaccharide chain structure are closely related to the activity intensity (Yoshikawa M. et al, Chem. Pharm. Bull., 42
(6), 1357-1359 (1994)). And, it was clarified that the mechanism of action of the escins for inhibiting the increase in blood glucose level is due to the activity of inhibiting gastric emptying and glucose absorption in the small intestine (Matsuda H. et al, Bioorg. Med. Chem., 6, 1019-10
23 (1998)).

As described above, since it is known that saponin has a compound exhibiting a blood glucose level-inhibiting activity, the present inventors have the insulin-like action aimed by the present inventors. As a result of vigorous search for the active substance targeting saponins derived from plants using the inhibition rate of fatty acid release using rat epididymal adipocytes as an index, Aesculus, a horse chestnut plant growing in Yunnan region of China,
assamica Griff. The root extract was found to have a strong activity, and the active component was isolated and purified to confirm that it was a saponin having three new chemical structures. The present invention was completed by confirming that all of these compounds have a strong glucose uptake promoting activity, which is an insulin-like action.

These compounds are known from the aforesaid escin, which is already known in the structure of its aglycone, sapogenin.
It had the same structure as that of the glycosides, but it was found that there was a characteristic difference in the structure of the sugar chain portion of the glycoside.
That is, in the sugar chain that binds to the hydroxyl group at the 3-position of the aglycone moiety, glucose is usually bonded to the hydroxyl group at the 4-position of glucuronic acid in escins. Has a unique structure in which rhamnose binds to the hydroxyl group of.

Further, the substituent bonded to the hydroxyl group at position 21 of the aglycone moiety also has structural features,
In cins, an angeloyl group or a tigloyl group is usually bonded, whereas in two of the compounds of the present invention, deoxyglucose is bonded as a glycoside,
It had a structure in which two angeloyl groups were bonded to a hydroxyl group on this deoxyglucose. The present inventors have also clarified that this unique structure plays an important role in its activity.

The mechanism of action of the compound of the present invention is clearly different from that of the above-mentioned conventionally known saponins in the action of suppressing an increase in blood glucose level. That is, the compound of the present invention suppresses an increase in blood sugar level by promoting the sugar once taken up into the body from the digestive tract into the tissue in peripheral tissue cells such as fat cells. . On the other hand, the mechanism of action of the above-mentioned escins for suppressing the increase in blood glucose level is the activity of inhibiting the gastric emptying ability and the activity of inhibiting glucose absorption in the small intestine, ie, inhibiting the absorption of sugar from the digestive tract into the body. This is clearly different from the insulin-like action.

From these results, the novel substance of the present invention is:
It has been clarified that as an active substance having a blood glucose level-inhibiting action similar to insulin, its efficacy is widely expected in the prevention and treatment of diabetes.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION

The novel compound used in the present invention is, for example, Aesculus assami, a horse chestnut plant that grows widely from Yunnan, China to Assam, India.
ca Giff. Can be obtained by extracting and purifying from plants containing the compound, such as roots, bark and nuts. Further, in the present invention, not only high-purity products obtained by isolating and purifying each of these compounds, but also plant extracts containing one or more of these compounds in the form of a mixture as they are Can also be used.

As the solvent used for extracting the compound of the present invention from these plants, water, alcohols such as methanol or ethanol, and ketones such as acetone are used. These solvents may be used alone or as a mixed solvent in which two or more kinds of solvents are arbitrarily mixed, considering that the extract is finally incorporated into foods and the like. , In terms of safety, water,
It is preferable to use ethanol or a mixed solvent thereof.

The ratio of the plant to the solvent at the time of extraction is not particularly limited.
000 weight times, especially 5 to 100 in terms of extraction operation and efficiency
Weight times are desirable. The extraction temperature is conveniently in the range of the boiling point of the solvent at room temperature to normal pressure, and the extraction time varies depending on the extraction temperature, but is preferably in the range of several hours to two days.

Also, a shredded plant itself containing the compound of the present invention may be used in a form to be extracted at the time of final use, and this form is also included in the present invention.

The plant extract thus obtained is further subjected to a purification method such as an adsorbent treatment method, a membrane separation method, a solvent fractionation method, and a column chromatography operation using a commonly used resin. As a result, a highly pure compound of the present invention can be obtained. For example, a highly pure compound of the present invention can be obtained by adsorbing a plant extract or a solvent fraction thereof or subjecting it to chromatography in another separation mode and eluting with one or more solvents.

In order to fractionate the plant extract or a solvent fraction thereof by chromatography using a resin generally used in a separation mode such as adsorption or distribution, gel filtration, etc. Is dissolved in a small amount of water, a solvent such as methanol or ethanol or a mixed solvent thereof, and Diaion HP20 (manufactured by Mitsubishi Kasei Kogyo), Se
p-Pak (Waters), Develosil
After being adsorbed on an adsorbent column such as ODS (manufactured by Nomura Chemical) or Sephadex LH-20 (manufactured by Pharmacia), the mixture is sufficiently washed with water, and then mixed with a hydrophilic solvent such as methanol, ethanol, acetone or a mixed solvent thereof. It may be eluted. Further, by combining two or more adsorption or column chromatography in another separation mode, the compound of the present invention with higher purity can be obtained.

For example, Aesculus assami
ca Giff. The dried roots were cut into 10 times (in terms of weight) 50% (v / v) ethanol at 40 ° C.
For 24 hours, and after filtration, after distilling off the solvent by vacuum concentration, adsorbed on Sep-Pak (Waters), a resin for solid phase extraction, washed with water, and washed with 20, 40, 60, 80, 1
The fraction eluted stepwise with a 00% by volume methanol aqueous solution and then fractionated with a 80% by volume methanol was fractionated by high performance liquid chromatography using a reversed phase column to finally obtain a single fraction. It can be purified.
At this time, it is particularly effective to perform fractionation under a plurality of solvent conditions in which the pH has been changed. For example, CAPCELL PAK C18 exhibiting good durability even under alkaline conditions for the first time.
(4.6 mmΦ × 250 mm, manufactured by Shiseido)
0 mM ammonium acetate-acetonitrile system (pH 8.
Gradient fractionation using 9), the second time, fractionation under the same column using 0.1% trifluoroacetic acid (TFA) -acetonitrile system allows effective isolation and purification. Finally, by repeating high-performance liquid chromatography using these reversed-phase columns, it becomes possible to purify each of the target active substances to a pure product.

In addition to the method for obtaining the compound of the present invention by isolation and purification from the natural products described above, the compound of the present invention can also be obtained by total synthesis or semi-synthesis by ordinary organic chemical techniques. .

The compound of the present invention thus obtained is represented by the general formula (I):

[0033]

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Wherein R ¹ is a group represented by the formula (II):

[0035]

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Or formula (III):

[0037]

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R ² represents an angeloyl group, tigloyl (tig)
loyl) group or formula (IV):

[0039]

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And a substituent represented by R ^{3 or} R ⁴
Represents a hydrogen atom or an acetyl group].

More specifically, the following compounds can be mentioned. That is, the following equation (V):

[0042]

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A compound represented by the following formula (hereinafter referred to as "compound 1")
And the following formula (Vb):

[0044]

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A compound represented by the following formula (hereinafter referred to as “Compound 1
b "); and the following formula (VI):

[0046]

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(Hereinafter referred to as "compound 2")
And the following formula (VIb):

[0048]

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The compound represented by the following formula (hereinafter referred to as "Compound 2
b)); and the following formula (VII):

[0050]

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A compound represented by the following formula (hereinafter referred to as "compound 3")
And the following formula (VIIb):

[0052]

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The compound represented by the following formula (hereinafter referred to as “Compound 3
b ”).

The structures of these compounds can be analyzed by ordinary instrumental analysis.
Can be easily determined by the method using. Ingredient
Physically, mass spectrum (MS), nuclear magnetic resonance spec
Torr (NMR), ultraviolet absorption spectrum (UV) and
Obtained by infrared absorption spectrum (IR), etc.
Information on the molecular weight, molecular formula and partial structure of the compound
Thus, the overall structure of the compound can be determined. For example,
Compound 1 was analyzed by FAB-MS at m / z 1115 (M +
H)⁺, Molecular formula C₅₅H₈₆O₂₃And the compound
2 and 3 are m / z 1343 (M + H)⁺, Molecular formula C
₆₆H₁₀₂O ₂₈It was decided. And this is NM
By taking into account the structural data from R
The structures of 1, 2, and 3 are represented by Formula (V), Formula (VI),
It has been clarified that the compound is represented by the formula (VII).

The compound of the present invention obtained as described above can be used as it is for preventing and improving diabetes by lowering the blood sugar level of the present invention as it is. In addition, a formulation prepared by appropriately mixing an optional auxiliary agent, excipient, water for use as a solution, or an organic solvent or the like can be used.

When the compound having a hypoglycemic effect of the present invention is used as a medicament such as an antihyperglycemic agent or an antidiabetic agent, the compound may be administered in the form of an oral preparation such as a powder, granule, tablet, capsule, or pill. Agents, internal solutions, suspensions, emulsions and the like can be mentioned, and they can be used alone or in combination depending on their symptoms and the like.
These preparations are prepared according to a conventional method, according to the purpose, into the compound of the present invention, excipients, binders, preservatives, oxidation stabilizers, disintegrants,
It can be formulated using known auxiliaries usually used in the technical field of pharmaceutical preparations such as lubricants and flavoring agents.

As the carrier to be used, those usually used in accordance with the dosage form can be used without particular limitation. Examples of preferred carriers include starch, lactose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts. And solid carriers such as distilled water, physiological saline, aqueous glucose solutions, alcohols such as ethanol, liquid carriers such as propylene glycol and polyethylene glycol, and various animal and vegetable oils, and oil carriers such as white petrolatum, paraffin and wax.

When the substance having a blood glucose lowering effect of the present invention is used as an oral preparation such as a blood glucose elevation inhibitor, an antidiabetic agent, etc., the dose may be appropriately changed depending on the age and symptoms of the patient. In general, the compound of the present invention may be used in a range of 0.5 to 5,000 mg / day in terms of a pure product.

The food or drink of the present invention is prepared by mixing the compound of the present invention as it is or with various components conventionally used in foods. For the production of these foods and drinks, various components can be used depending on the type, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, Malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erysorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester Used as normal food ingredients such as gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamins B, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, flavors, preservatives, etc. What it is can be produced by appropriately blending.

The form of the food to be produced can be any food form such as solid food, creamy or jam-like semi-liquid food, gel food, beverage and the like. For example, soft drinks, juices, coffees, teas, liqueurs, milk, whey drinks, lactic acid bacteria drinks, candies (candy) using capsules, granules, tablets, drinks, etc., and any commonly used base materials. , Chewing gum, chocolate, gummy, yogurt, ice cream, pudding, water cans and the like.

The substance exhibiting a blood glucose lowering effect of the present invention can be used as an additive for food. This food additive can be prepared by mixing the substance having a blood sugar lowering effect of the present invention as it is or in combination with a carrier used for ordinary production, in the form of powder, granules, paste, capsule, syrup, solid, gel, liquid, suspension, etc. It can be used in the form of a suspension, an emulsion or the like. The food additive can be added to any food at the time of manufacturing the food or for the purpose of imparting an anti-diabetic effect to the increase in blood sugar level.

[0062]

Insulin promotes glucose uptake into muscle and fat by translocating GLUT4 onto the cell membrane via signal transduction from the insulin receptor, and free fatty acids by epinephrine for fat. Is known to have the effect of suppressing the recruitment of such materials.

It was confirmed that all of the compounds 1 to 3 of the present invention exhibited the same fatty acid release inhibitory action as insulin and also had the activity of promoting the uptake of sugar into the preadipocyte 3T3L1 differentiated similarly to insulin. Therefore, the compound of the present invention is a novel substance that is expected to be useful in the prevention and treatment of diabetes as a compound having a blood sugar level-suppressing action similar to insulin.

[0064]

EXAMPLES Next, the present invention will be described in more detail with reference to Production Examples of Compounds 1, 2, and 3, and various biological test examples and formulation examples. Needless to say, the present invention is not limited to these embodiments and the like.

Production Example 1: Aesculus assam
ica Griff. Of compound 1,2,3 containing composition (composition A) from Aesculus assa growing in Yunnan area of China
mica Griff. After chopping 1 kg of the dried root, the mixture was put into an extraction tank, 10 L of 50% (v / v) ethanol was added, and the mixture was extracted with stirring at 40 ° C. for 24 hours. This was filtered, the obtained filtrate was concentrated by evaporating the solvent by vacuum concentration, and then lyophilized to obtain 225 g of an extract.
This was designated as composition A (hereinafter, referred to as "composition A").

Production Example 2: Purification method of compounds 1, 2, and 3 1.13 g of composition A was dissolved in water, and Sep-Pak was prepared.
The solution was loaded on C18 (manufactured by Waters) (10 g), washed with water, and then eluted stepwise with 20, 40, 60, 80, or 100% by volume aqueous methanol solution. The fraction (598 mg (composition B)) eluted with 80% by volume methanol was subsequently subjected to CA
PCELL PAK C18 (4.6 mm x 250 m
m, manufactured by Shiseido Co., Ltd.) at a flow rate of 1 ml / min under alkaline conditions by high-performance liquid chromatography with stepwise gradient fractionation. The gradient conditions are as follows: solution A; 10 mM ammonium acetate (pH
8.9), solvent B; using acetonitrile,
Conditions increasing from 0% to 80% in 35 minutes were used.

The retention time obtained above was about 15,1
The peaks around 9, 23 minutes were collected, and fractions D-1-2 (113.2 mg) and D-1-4 (2
3.2 mg) and D-1-6 (19.8 mg).

These active fractions were further separated on the same column under acidic and isocratic conditions. Solution A: 0.1% TFA, solution B: acetonitrile, solution B-D-1-2 (11
3.2 mg), and an active peak eluted at a retention time of about 31 minutes was isolated.
(D-2-3) 7.8 mg was obtained.

Next, D-1-4 (23.2 mg) was fractionated under the condition of 44% of the solvent B, and the retention time was about 26%.
The activity peak eluted in minutes was isolated to obtain 4.6 mg of the purified compound 2 (D-4-3).

Further, D-1-6 (19.8 mg) was fractionated under the conditions of a solvent B of 46%, and a retention time of about 3% was obtained.
The activity peak eluted at 4 minutes was isolated to obtain 2.4 mg of purified compound 3 (D-6-3).

These isolation and purification schemes are shown in FIG. Further, the elution pattern of the composition A, which was performed in Production Example 2, by high performance liquid chromatography is shown in FIG.

Structural analysis The physicochemical properties of the isolated active substance are summarized in Table 1.

[0073]

[Table 1]

The NMR was measured in heavy pyridine. These ¹ H-NMR analysis diagrams are shown in FIGS. 3 to 5. From these various properties and analytical data, the obtained active compound 1,
It was confirmed that the structures of 2, 3 were represented by the formulas (V), (VI) and (VII), respectively, as described above.

Biological Test Example 1: Effect on fatty acid release (lipolysis) of adipocytes Method:

Male Wistar rats (weight: about 20
0g) was killed by exsanguination under ether anesthesia, and the epididymal fat was excised. This was added to a KRB buffer containing 2% BSA (120 mM NaCl, 4.75 mM KCl, 1.
27 mM CaCl ₂ , 1.2 mM MgSO ₄ ,
_{2mM KH 2 PO 4, 24.0mM NaHCO} 3:
2mg after removing large blood vessels etc. in pH 7.4)
The mixture was allowed to react at 37 ° C. for 1 hour in a KRB buffer containing 2% BSA containing 1% / ml collagenase, and fat cells were separated. 25 isolated fat cells
After passing through a 0 μm mesh, the cells were washed three times with a buffer solution, and prepared so that the number of cells became about 2 × 10 ⁶ cells / ml.

The cell suspension was transferred to a polypropylene (PP) test tube (1.0 × 7.0)
cm), water was added as a test compound or a control, and preincubation was performed at 37 ° C for 30 minutes. Next, epinephrine, a lipolysis-inducing substance, was added to a final concentration of 10 μM, and the mixture was further incubated at 37 ° C. for 3 hours. The reaction was stopped by ice-cooling, and free fatty acids (FFA) released from the cells into the buffer were measured using a free fatty acid measurement kit (NEFA).
C-Test Wako: manufactured by Wako Pure Chemical Industries, Ltd.).

Results: It is known that the FFA release from adipocytes by the addition of epinephrine is suppressed by insulin, but Aesculus assamic
a Griff. Therefore, all of the novel substances isolated and purified from the above-mentioned Production Examples and structurally analyzed showed a strong FFA release inhibitory action at a concentration of 50 μg / ml. From these results, it was shown that the novel compounds 1, 2, and 3 suppress lipolysis by epinephrine similarly to insulin. These results are shown in FIG.

Biological test example 2: mouse preadipocyte 3T3
Evaluation of activity using L1 (sugar incorporation into cells) Method: 3T3L1 mouse fat precursor cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine serum (CS) under 5% CO ₂ under type 1 collagen-coated Petri dishes. (Made by Iwaki). According to a conventional method, cells recovered by trypsin treatment were similarly spread on a type 1 collagen-coated 96-well plate at 4000 cells / well, and cultured for 3 days to reach confluence. After removing the medium, 0.5 mM isobutylmethylxanthine (MIX), 1 μM dexamethasone (DEX), 10 μM
g / ml insulin, 10% fetal bovine serum (FBS) -added DMEM with 200 µl / well
The cells were cultured for 8 hours and differentiated into adipocytes. Thereafter, 10% FB excluding the medium, MIX, DEX, and insulin
200 μl of S-added DMEM was added to each well, and 1
What was cultured for about 5 days while changing the medium every day was used for the assay.

100 μl KRH buffer (136 mM)
NaCl, 4.7 mM KCl, 1.25 mM CaC
_{l 2, 1.25mM MgSO 4 · 7H} 2 O, 20mM
Hepes, 2 mg / ml BSA: pH 7.4) was carefully washed so as not to detach the cells, conditioned for 30 minutes, replaced with 90 μl of sample solution, and pre-incubated for 10 minutes. To this were added tritium-labeled 2-deoxyglucose (2-DG) and cold 2-deoxyglucose (final concentration 1 mM) and incubated for 30 minutes. 100 μl of 20
The uptake was stopped by the addition of μM cytochalasin B. After washing four times, the cell membrane was dissolved by treating with 50 μl of 1% Nonidet P40 solution, and after leaving for 30 minutes or longer, 40 μl was added.
1 sampled, dried in a luma plate (manufactured by Packard), and tritium label 2 taken up into cells.
-The amount of DG was quantified by a top count microplate scintillation-luminescence counter (manufactured by Packard) to measure the sugar uptake promoting activity.

Results: The addition of insulin (final concentration 100 nM) was compared with the sugar uptake without sample addition.
Uptake of up to about 13 to 14 times was observed. Under these conditions, compounds 1, 2, and 3 were 40, 5, 30 μm, respectively.
At a concentration of M, the activity of promoting uptake was about three-fold.
FIG. 7 shows the results.

The following are specific examples of pharmaceutical preparations containing the compounds provided by the present invention alone or as a mixture, and examples of foods and drinks. The above ingredients are kneaded, granulated, dried and tableted in a conventional manner,
A tablet weighing 200 mg containing 80 mg of compound 2 as an active ingredient in one tablet was obtained.

Food and Drink Example 1 (Tablet) Composition: Composition A 0.75 g Sorbitol 2.13 g Sucrose fatty acid ester 0.12 g The above ingredients were kneaded, granulated, dried and compressed into tablets by a conventional method.
A tablet weighing 200 mg containing 50 mg of composition A as an active ingredient in one tablet was obtained.

[0084] Using the composition having the above blending percentage, candy was obtained according to a conventional method.

[0085]

The novel saponin compound of the present invention is
The present invention has for the first time been found to have various activities similar to insulin. This leads to the provision of a pharmaceutical composition and a food or drink or an additive for food and drink that exhibit an insulin-like blood glucose increase inhibitory action by oral administration, and has remarkable efficacy and usefulness in the prevention or treatment of diabetes. .

[Brief description of the drawings]

FIG. 1 is a diagram showing a scheme for isolating and purifying an active substance, performed in Production Examples 1 and 2 of the present invention.

FIG. 2 is a view showing an elution pattern of composition A performed in Production Example 2 of the present invention by high performance liquid chromatography.

FIG. 3 is a ¹ H-NMR chart of Compound 1 provided by the present invention.

FIG. 4 is a ¹ H-NMR chart of Compound 2 provided by the present invention.

5 is a chart of ^{1 H-NMR} of Compound 3 provided by the present invention.

FIG. 6 is a diagram showing the results of Biological Test Example 1 of the present invention.

FIG. 7 is a diagram showing the results of Biological Test Example 2 of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 35/78 A61K 35/78 C (72) Inventor Keiichi Abe 1 Wakayamadai, Shimamotocho, Mishima-gun, Osaka Chome 1-1 F-term in Suntory Limited Health Science Laboratory (reference) 4B014 GB06 GB07 GB08 GE06 GG18 GL03 4B018 LB01 MD27 MD43 ME03 4C057 BB04 DD01 JJ52 4C086 AA01 AA02 AA03 EA10 MA01 MA04 MA52 NA14 ZC35 4C088 AB12 BA08 BA08 BA08 NA14 ZC35

Claims (15)

[Claims] 1. A compound of the general formula (I): Wherein R ¹ is a group represented by the formula (II): Or formula (III): Wherein R ² is angeloyl (a
ngeloyl) group, tigloyl group or formula (IV): And R ³ and R ⁴ each represent a hydrogen atom or an acetyl group.] 2. The following formula (V): The compound according to claim 1, which is represented by the formula: 3. The following formula (VI): The compound according to claim 1, which is represented by the formula: 4. The following formula (VII): The compound according to claim 1, which is represented by the formula: 5. A pharmaceutical composition comprising as an active ingredient one or a mixture of two or more of the compounds according to any one of claims 1 to 4. 6. The pharmaceutical composition according to claim 5, which is for preventing or treating diabetes or an increase in blood sugar level based on an insulin-like action. 7. The pharmaceutical composition according to claim 6, wherein the insulin-like action is an activity of promoting glucose uptake in peripheral tissues. 8. A method for producing a pharmaceutical composition for preventing or treating diabetes, comprising isolating and purifying a mixture comprising one or more of the compounds according to any one of claims 1 to 4. Or a mixture thereof, or in the form of a plant extract containing a mixture thereof. 9. A food or drink or an additive for food or drink comprising a mixture comprising one or more of the compounds according to any one of claims 1 to 4. 10. The food or drink or the additive for food and drink according to claim 9, for preventing or treating diabetes or an increase in blood sugar level due to an insulin-like action. 11. The food or drink or the food or drink additive according to claim 10, wherein the insulin-like action is an activity of promoting glucose uptake in peripheral tissues. 12. When it is in the form of a solid or powder, it contains a total of at least 0.01% by weight of a mixture comprising one or more of the compounds according to any one of claims 1 to 4. The food or drink or the additive for food or drink according to any one of claims 9 to 11, wherein 13. A compound comprising one or more of the compounds according to any one of claims 1 to 4 when the form is a liquid, containing a total of 0.0001% by weight or more. Item 12. A food or drink or an additive for food or drink according to any one of Items 9 to 11. 14. A method for producing a food or drink or an additive for a food or drink for preventing or improving diabetes.
A method comprising adding a mixture consisting of one or more of the compounds described in any one of (1) to (4) after isolation and purification, or adding the mixture in the form of a plant extract containing these mixtures. 15. A food or drink or a raw material thereof which does not contain the compound according to any one of claims 1 to 4, and the food or drink raw material comprising one or more of the compounds according to any one of claims 1 to 4. The food or drink or food additive according to claim 14, wherein the mixture is added after isolating and purifying the respective compounds or in the form of a plant extract containing these compounds. Manufacturing method.