JP2005089419A - Garlic extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a garlic extract containing allicin which is a component of garlic and spirostanol which is a kind of saponin at the same time. <P>SOLUTION: The garlic extract containing the alliin and the spirostanol is produced by separately extracting the alliin and the spirostanol and using the components in combination. The garlic extract is effective for suppressing the increase in blood sugar level and increasing glucose tolerance. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a garlic extract containing a combination of alliin and spirostanol, and a diabetes preventive / therapeutic agent comprising such a garlic extract as an active ingredient.

  Diabetes is a disease group mainly having chronic hyperglycemia due to insufficient insulin action and accompanied by various characteristic metabolic abnormalities. Both genetic and environmental factors are involved in its development. Diabetes is roughly classified into insulin-dependent diabetes (type 1 diabetes) and non-insulin-dependent diabetes (type 2 diabetes), and is known to be a risk factor for stroke and heart disease. Deterioration of quality of life due to complications such as infectious diseases and kidney problems is also a serious problem. Furthermore, the number of patients with diabetes has shown a rapid increase in recent years, and it is said that there are 6.9 million people who are strongly suspected of having diabetes, and 13.7 million people who can not deny the possibility of diabetes. This is one of the most important health problems of the people as a lifestyle-related disease.

  A typical drug used for the treatment of diabetes is insulin, which is generally used by intravenous injection. In addition, as oral antidiabetic drugs, 1) sulfonylureas that have the effect of stimulating the pancreas to promote insulin secretion, 2) suppressing the absorption of glucose from the intestine, and the liver adding glucose to the blood A biguanide agent that has the effect of suppressing the delivery; 3) an α-glucosidase inhibitor that suppresses the action of the saccharide-degrading enzyme α-glucosidase and delays the digestion of carbohydrates; 4) an effect that facilitates glucose uptake into cells Insulin resistance improving drugs having the above are known. These drugs are appropriately selected depending on the type of diabetes and are used alone or in combination.

  However, diabetes is an intractable disease, which is also called a modern disease associated with an environment rich in food, and it has been necessary to continue to take the above-mentioned improving drugs mainly composed of insulin and various oral diabetes drugs for a long time. Therefore, there was also a problem that undesirable side effects occurred. Therefore, attempts have been made to use natural products having no side effects or degradation products thereof, such as extracts of flower-turned flowers, carrots or persimmons (see Patent Document 1), Agaricustake (see Patent Document 2), pomegranate, taranoki, red fox, The effectiveness is known to many plants such as a red celestial genus plant, a Salacia genus plant (see Patent Document 3), and a bunashimeji fruit body (see Patent Document 4).

  However, it is the actual situation that no anti-diabetic agent / therapeutic agent so far satisfies all of stability, safety and efficacy.

  The garlic used in the present invention is widely used as a food having a nourishing tonic effect, and many pharmacological activities thereof have been studied. For example, alliin contained in garlic has a cancer prevention effect (see Patent Document 5), antibacterial action (see Patent Documents 6 and 7), immunostimulatory action (see Patent Document 8), and the like. Known saponins include antifungal activity (see Patent Document 9), dementia prevention / treatment agent (see Patent Document 10), and the like.

JP 2001-39882 A JP 2001-213773 A JP 2002-20308 A JP 2002-187851 A JP 2001-302531 A Japanese translation of PCT publication No. 2002-520343 JP-A-10-28546 JP 7-247220 A JP-A-1-224396 JP 2001-518477 A

  However, since alliin is decomposed into allicin by an enzyme or the like, it cannot be extracted simultaneously with saponin. Therefore, the first object of the present invention is to provide a garlic extract containing allicin contained in garlic and spirostanol, which is a kind of saponin.

  Extracting alliin and spirostanol separately and using them in combination yields a garlic extract containing both alliin and spirostanol, and this garlic extract has an effect of suppressing blood sugar level elevation and improving glucose tolerance As a result, the present invention has been completed.

  The composition for preventing / ameliorating diabetes according to the present invention is derived from garlic, which is a natural product, has high safety, and has an effect of suppressing an increase in blood glucose level and an effect of improving glucose tolerance. Ingesting foods can be expected to prevent diabetes and treat diabetes.

Garlic used in the present invention, Allium (Allium) belonging to the genus garlic (Allium s ativum L.), and their analogous plants, can be used varieties and cultivation varieties. As a site for obtaining the garlic extract, a bulb portion is preferable.

  To obtain alliin from garlic, first, enzyme inactivation treatment of garlic is performed. The enzyme deactivation treatment can be performed by treating garlic with heat, microwave, high pressure treatment, enzyme, acid or alcohol, and among these, heat treatment is simple and preferable. The temperature for the heat treatment is preferably 50 to 200 ° C. and 1 to 60 minutes, particularly preferably 90 to 121 ° C. and 10 to 30 minutes.

  Next, the enzyme-inactivated raw garlic is crushed and water is added to extract alliin to obtain a crude garlic extract. For crushing this garlic, a high performance mixer, a homogenizer or the like is preferably used. In addition, the ratio of the raw garlic in the crude extract is preferably in the range of 1 to 20% by weight of the raw garlic, particularly about 5% by weight. The crushed garlic contains insoluble fiber and the like, and water is added to the crushed product of enzyme-inactivated garlic, and the stirred solution is filtered or centrifuged to remove insoluble components. The garlic crude extract is put in a bottle, filled with an inert gas such as nitrogen gas, carbon dioxide gas, helium gas or argon gas, and stored in a cool dark place at 25 ° C. or lower, preferably 4 ° C. or lower. Such a crude extract may be used as it is, or may be used after the purification step shown below.

  The crude garlic extract from which insoluble components have been removed is separated by liquid chromatography using a reverse-phase column, and alliin is separated and purified. As the column used here, for example, a reverse phase column ZORBAX TMS manufactured by DuPont is preferably used. When chromatography of a garlic crude extract using this kind of reversed-phase column is performed, the crude garlic extract is supplied to the column to adsorb alliin, and then pure water is separated as an eluent, and UV is extracted. By using a detector and monitoring the UV absorption rate of the eluent with a specific absorption wavelength of about 210 nm of alliin as a measurement wavelength, it is possible to easily obtain the alliin-containing fraction.

  When separating the garlic crude extract by liquid chromatography using a reversed-phase column, details will be given in the experimental examples described later, but there are almost other components other than alliin in the alliin effluent fraction. Without purification, it is possible to fractionate purified alliin only by liquid chromatography using a reverse phase column.

  In addition, when carrying out this liquid chromatography, when carrying out continuous large-scale fractionation, the garlic crude extract is unstable, and when exposed to air, insoluble components tend to precipitate and become cloudy. Therefore, the garlic crude extract is put in a bottle and filled with an inert gas such as nitrogen gas, carbon dioxide gas, helium gas, or argon gas. This prevents white turbidity of the garlic crude extract and enables continuous fractionation.

  In order to obtain spirostanol from garlic, for example, it can be obtained by the method described in JP-A-1-22439. Specifically, it can be extracted after hydrolyzing raw garlic with an enzyme or acid to purify spirostanol, or after extracting the saponin fraction after deactivating the enzyme activity of garlic and then acid. Alternatively, it can be obtained by hydrolysis with an enzyme. In addition, spirostanol can also be obtained using the garlic crude extract extracted when obtaining the above-mentioned alliin.

  In the present invention, alliin and spirostanol are used in combination. The blending ratio is not particularly limited, and can be appropriately determined within a range of about 1: 100 to 100: 1.

Extraction Example 1 Crude Alliin Extract 4 kg of garlic bulb peeled, weighed 4 kg, boiled in boiling water for 15 minutes, heat-treated, then pulverized and homogenized using a disper, and filtered using filter paper. Was made up to 100 mL with pure water to obtain an alliin crude extract.

Extraction Example 2 Purified Alliin Using the crude alliin extract shown in Extraction Example 1, an alliin fraction was collected by liquid chromatography using a reverse phase column, and separated and purified.
Liquid chromatography preparative conditions and temperature: 25 ° C
・ Flow rate: 0.5ml / min.
・ Eluent: Pure water ・ Column: ZORBAX TMS (9.4 mm × 25 cm)
・ Detector: UV detector (measurement wavelength: 210 nm)

Extraction Example 3 Spirostanol Garlic bulb 2 kg was crushed in 3 L of water and extracted overnight at room temperature. This extract was chromatographed by MCI GEL in the same manner as in Experimental Example 1 to obtain crude saponin fraction 5.0 g was obtained. This mixture was subjected to silica gel chromatography [eluent: chloroform-methanol-water (7: 3: 0.5)], and further subjected to reverse phase chromatography (eluent: 90% methanol) using MCI GEL CHP 20P, and spirostanol ( 26 mg) was obtained.

Extraction Example 4 Spirostanol 2
After 4 kg of garlic bulbs were frozen and crushed in 6 L of methanol, the mixture was heated and extracted at a bath temperature of about 70 ° C. After filtration, the residue was further heated and extracted with 2 L of methanol. After the extracts were combined, methanol was distilled off, and water was added to make the total amount about 3. This extract was subjected to column chromatography using MCI GEL CHP 20P (Mitsubishi Kasei), and eluted sequentially with water, 20% methanol and methanol to obtain 8.6 g of a crude saponin fraction from the fraction eluted with methanol. This mixture was subjected to silica gel chromatography [eluent: chloroform-methanol-water (6: 4: 1)] to obtain a crude fraction containing saponin (0.6 g). Furthermore, this fraction was subjected to reverse phase column chromatography using MCI GEL CHP 20P (eluent: 80% methanol), and the resulting fraction (0.49 g) was made into an acetone: water (2: 5) solution. After heating at 95 ° C. for 4 hours, the solvent was distilled off to obtain 0.45 g of purified saponin. After reacting 0.25 g of this purified saponin in 50 ml of 0.1 M acetic acid buffer (pH 4.3) of β-glucosidase (0.25 g, derived from almonds) at 37 ° C. for about 2 hours, 50 ml of water was added to the reaction solution. GEL CHP 20P was added and the solution was passed through the column and eluted with water, 50% methanol and methanol sequentially. The fraction eluted with methanol was distilled off under reduced pressure to obtain spirostanol (0.17 g).

  The extracts shown in Extraction Example 1 to Extraction Example 4 were mixed at a weight ratio shown in Table 1 to obtain a garlic extract according to an example of the present invention.

Effect on Type 1 Diabetes Using a streptozotocin (STZ) diabetes model rat, which is a type 1 diabetes model animal, the diabetes prevention / amelioration effect of the garlic extract according to the present invention was examined as follows. About 4 weeks old male SD rats were given 65 mg / kg streptozotocin intraperitoneally to induce diabetes. One week later, rats that showed hyperglycemia, urine sugar, multiple meals, and multiple drinks were subjected to the following experiment. A group consisting of 10 STZ diabetic model rats prepared, and a basic diet group administered with a solid feed for rats (CRF-1 manufactured by Charles River Japan) as a basic diet, and the above implementation so that the basic diet was 5% by weight It divided into the Example 1-4 mixed food group which mixed the garlic extract of Examples 1-4. For comparison, the extraction examples 2 and 3 mixed food group in which extraction example 2 or extraction example 3 was mixed so as to be 5% by weight in the basic food were also tested at the same time. These were bred for 4 weeks in an environment where food and water could be freely ingested at a room temperature of 23 ° C. and a 12 hour light / dark cycle. Blood was collected from the rat tail vein after fasting for 20 hours every week from the start of the breeding, and the blood glucose level was measured. Furthermore, a glucose tolerance test was performed at 0, 2, and 4 weeks. That is, after fasting for 20 hours, 2 g / kg of glucose was orally administered using a sonde. Blood samples were collected from the tail vein before glucose administration (0 hour), 0.5 hours, 1 hour, 1.5 hours, and 2 hours after administration, and blood glucose levels were measured to calculate average values. Changes in fasting blood glucose levels during the test period are shown in Table 2, and the results of the glucose tolerance test are shown in Tables 3 to 5, respectively.

  As can be seen from Table 2, it was shown that the increase in fasting blood glucose level tended to be suppressed in the example mixed diet group as compared with the basic diet group. In the sampled mixed diet group, it was shown that the increase in fasting blood glucose level tended to be suppressed compared to the basic diet group, but the effect was not sufficient. In addition, as shown in Table 3, the results of the glucose tolerance test showed no difference between the basic diet group, the example mixed diet group, and the extracted example mixed diet group at the start of the test, as shown in Table 4 and Table 5. In the second and fourth week of breeding, the increase in blood glucose level immediately after glucose administration was suppressed, and the recovery of blood glucose level was accelerated. Therefore, compared with the basic food group, the glucose tolerance was improved in the Example mixed food group. Moreover, although the glucose tolerance of the extraction example mixed food group tended to be superior to that of the basic food group, the improvement effect was not as good as that of the mixed food group of Example. From the above, it has been shown that garlic extract containing alliin and spirostanol has an effect of preventing and improving type 1 diabetes.

Effect on Type 2 Diabetes The effect of garlic extract was examined as follows using type KKA ^Y diabetes model mouse, which is one of type 2 diabetes model animals, in the same manner as type 1 diabetes. About 4 weeks old KKA ^Y / Ta Jcl mice were preliminarily raised for 1 week, and 10 animals were grouped into a basic diet group, an example mixed diet group, and an extracted example mixed diet group, and the test was started. Mice were individually housed in cages and bred for 5 weeks in an environment where food and water could be freely fed in a 12 hour light / dark cycle at room temperature of 23 ° C. Every week from the start of breeding, blood was collected from the fundus of the mouse after fasting for 5 hours using a capillary blood collection tube, and the blood glucose level was measured. Furthermore, a glucose tolerance test was performed 36 days after the start of the breeding. That is, after fasting for 20 hours, 2 g / kg of glucose was orally administered using a sonde. Before glucose administration (0 hour), 0.5 hours, 1 hour, 1.5 hours, and 2 hours after administration, blood was collected from the fundus oculi, blood glucose level was measured, and an average value was calculated. Table 6 shows changes in blood glucose level during the test period, and Table 7 shows the results of the glucose tolerance test.

  As shown in Tables 6 and 7, it was confirmed that in the example mixed diet group, the increase in blood glucose level immediately after glucose administration was suppressed and the recovery of blood glucose level was quicker than that in the basic diet group. In addition, in the extraction example mixed meal group, such an effect could not be confirmed. From the above, it was shown that the garlic extract of the present invention containing alliin and spirostanol in combination has an effect of preventing / ameliorating type 2 diabetes.

Claims (3)

Garlic extract containing alliin and spirostanol saponin. The garlic extract according to claim 1, comprising alliin obtained by inactivating the enzymatic activity of raw garlic and then extracting with a solvent. The diabetes preventive / improving agent which uses the garlic extract of Claim 1 or Claim 2 as an active ingredient.