JP2005132837A - Hyperglycemia inhibitor and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new hyperglycemia inhibitor having high α-glucosidase inhibitory action, capable of producing a treating effect more excellent that those of already known treating agents for diabetes, and derived from a plant. <P>SOLUTION: The hyperglycemia inhibitor contains a metal conjugate of a pyrrolizidine alkaloid glycoside expressed by the formula as an active ingredient. The hyperglycemia inhibitor is produced by concentrating a methyl alcohol decoction of bark of Syzygium malaccense belonging to the family Myrtaceae under reduced pressure, subjecting the obtained concentrated residue to partition treatment between ethyl acetate and water, and further subjecting the obtained water layer to partition treatment between itself and n-butyl alcohol, so as to recover a solid product from the water phase. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel antihyperglycemic agent for suppressing an increase in blood glucose level after a meal and a method for producing the same. More specifically, the present invention relates to a novel antihyperglycemic agent utilizing an α-glucosidase inhibitory action obtained from a plant native to a tropical region such as the South Pacific Islands, and a method for producing the same.

  Recently, many antidiabetic drugs have been known to inhibit the increase in blood glucose levels by inhibiting the action of disaccharide-degrading enzyme, α-glucosidase, present in the gastrointestinal mucosa and suppressing the production of glucose from starch and oligosaccharides. However, most of them are derived from natural plants.

  By the way, diabetes is divided into insulin dependence (type I) and non-dependence (type II), and the latter accounts for about 90% of all diabetes, and its pathological condition is delayed insulin secretion, liver and muscle, etc. Insulin resistance in other tissues is characterized by hyperglycemia after meal intake.

  Therefore, when hyperglycemia, especially postprandial hyperglycemia, is observed even after diet and exercise therapy, insulin secretion from pancreatic β cells can be reduced by delaying the digestion and absorption of starch and sucrose and suppressing the increase in blood glucose level. It is possible to control blood sugar without promoting or injecting insulin.

On the other hand, α-glucosidase inhibitors widely used in the treatment of mildly ill diabetes have a pharmacological effect that inhibits postprandial hyperglycemia by delaying sugar absorption by inhibiting α-glucosidase in the small intestine, Improvement in fasting blood glucose level and HbA _{1c level} (adult hemoglobin, which is an index of blood glucose management in diabetes) has been observed with long-term use, but this α-glucosidase inhibitor is conspicuous in postprandial hyperglycemia, but fasting It is said that it is best applied to symptoms with relatively low blood glucose, that is, relatively mild type II diabetic patients.

The raw materials for these anti-diabetic drugs are mainly prepared from plants as raw materials. Examples of plants used as the raw materials include Ephedra Herb (see Patent Document 1), Heracleum lanatum, and Heracleum lanatum. Populus (see Patent Document 2), Salacia prinoides (see Patent Document 3), perilla (see Patent Document 4), Rhodiola plant (see Patent Document 5) , Plants of the genus Fuaceae (see Patent Document 6), Pelvetia wrighti Okama (see Patent Document 7), Trichocaulon or Hoodia genus plants (see Patent Document 8), Hanaba leaves (La) erstroemia Speciosa, Linn or Pers.) (see Patent Document 9), and the like.
On the other hand, it is known that pyrrolidide alkaloids (see Patent Documents 10 and 11) and glycosides thereof (see Non-Patent Documents 1 and 2) exhibit weak glucosidase inhibitory activity.

JP-A-9-2963 (Claims and others) JP 10-508880 A (Claims and others) JP-A-11-29472 (Claims and others) JP 2000-102383 (Claims and others) JP 2000-128793 A (Claims and others) JP 2000-239164 A (Claims and others) JP 2000-342224 A (Claims and others) JP 2002-68994 A (Claims and others) JP 2001-39880 A (Claims and others) Japanese Patent Laid-Open No. 3-106883 (Claims and others) Japanese Patent Laid-Open No. 3-141280 (Claims and others) "Tetrahedron Letters", Vol. 35, No. 42, 1994, p. 7849-7852 “Carbohydrate Letters”, Vol. 2, No. 3, 1996, p. 167-174

  The present invention has been made for the purpose of providing a novel plant-derived antihyperglycemic agent that has a high α-glucosidase inhibitory action and exhibits a therapeutic effect superior to that of known therapeutic agents for diabetes. It is.

Recently, in the South Pacific countries including Fiji, due to changes in the dietary environment, there has been a rapid increase in the number of diabetic patients that had not existed until 1960, which has become a major social problem.
By the way, in order to treat this diabetes, several plants are used, and among them, the male yellow peach (Syzygium malaccense (L.) Merrill & Perry) belonging to Myrtaceae, Banjiro guava [ Psidium guajava (L.)], Decasperum fruticosum Forst. And the like, but as a result of the inventor's research, Malay peach called Kabika in Fiji It has been found that a substance obtained from bark exhibits a strong α-glucosidase inhibitory action, and the present invention has been made based on this finding.

で表わされるピロリジデインアルカロイド配糖体の金属結合体を有効成分としてなる血糖上昇抑制剤、及びフトモモ科（Ｍｙｒｔａｃｅａｅ）、マレーフトモモ［Ｓｙｚｙｇｉｕｍ ｍａｌａｃｃｅｎｓｅ（Ｌ．）Ｍｅｒｒｉｌｌ ＆ Ｐｅｒｒｙ］植物の樹皮のメチルアルコール浸出物を有効成分としてなる血糖上昇抑制剤、及びフトモモ科（Ｍｙｒｔａｃｅａｅ）、マレーフトモモ［Ｓｙｚｙｇｉｕｍ ｍａｌａｃｃｅｎｓｅ（Ｌ．）Ｍｅｒｒｉｌｌ ＆ Ｐｅｒｒｙ］植物の樹皮のメチルアルコール浸出液を減圧下に濃縮し、得られた濃縮残渣を酢酸エチルエステルと水との間で分配処理し、得られた水層についてさらにｎ‐ブチルアルコールとの間の分配処理を行ったのち、水層から固形分を回収することを特徴とする上記の血糖上昇抑制剤の製造方法を提供するものである。 That is, the present invention

A blood glucose elevation inhibitor comprising a metal conjugate of a pyrrolidideine alkaloid glycoside represented by the formula, and methyl alcohol in the bark of a plant of Myrtaceae, Syzygium malaccense (L.) Merrill & Perry Antihyperglycemic agent comprising leachables as an active ingredient, and methyl alcohol exudate from bark of Myrtaceae and Syzygium malaccense (L.) Merrill & Perry plants under reduced pressure, and the resulting concentration The residue is partitioned between acetic acid ethyl ester and water, and the resulting aqueous layer is further partitioned between n-butyl alcohol and then the solid content is recovered from the aqueous layer. Said blood sugar elevation inhibitor There is provided a manufacturing method.

The blood sugar elevation inhibitor of the present invention can be produced, for example, using fresh bark of male peach peach as a raw material.
That is, first, the bark of male peach is preferably pulverized into fine granules. The finer the chip size, the greater the extraction efficiency. However, if the chip size is too small, it becomes difficult to handle, so it is usually ground to 0.1 to 3 mm, preferably 0.3 to 1.5 mm. .

  Next, the granular material thus obtained is leached with an extraction solvent such as methyl alcohol. The amount of methyl alcohol used is selected in the range of 2 to 10 times, preferably 3 to 6 times, in terms of the volume ratio of the granular material. The leaching is carried out by standing at room temperature, but can be heated or agitated to increase the leaching rate if desired.

  It can also be carried out while refluxing methyl alcohol. As the extraction solvent at this time, a solvent in which ethyl alcohol, dimethyl sulfoxide, dimethylformamide, or the like is added to methyl alcohol to enhance the dissolving power can also be used. The leaching time depends on the leaching conditions, but is usually in the range of 50 to 180 hours, and in many cases 70 to 150 hours is sufficient.

Next, the solvent is distilled off from the leachate thus obtained to obtain a solid residue. At this time, it is preferable to promote distillation by reducing pressure or heating.
The resulting residue is then partitioned with a water-insoluble organic solvent such as ethyl acetate and water, the aqueous layer is further partitioned with n-butyl alcohol, and the aqueous layer is separated and lyophilized. By doing so, a metal conjugate of the compound of the general formula (I) can be obtained.
This can be purified by, for example, dissolving it in water, adding barium hydroxide powder, removing the formed precipitate, and lyophilizing.

The purified product thus obtained showed 99.5% α-glucosidase inhibitory activity. On the other hand, acarbose obtained from a culture solution of Actinoplanes genus which is one of the actinomycetes of commercially available α-glucosidase inhibitors, and amino acid variol obtained from the culture solution of actinomycetes The α-glucosidase inhibitory effect of voglibose obtained by dehydrating glycerol with glycerol was 90.6% and 98.7%, respectively.
From this, it can be seen that the blood sugar elevation inhibitor of the present invention exhibits a pharmacological effect comparable to or superior to conventional ones.

As a result of measuring and analyzing nuclear magnetic resonance spectra ¹ H-NMR and ¹³ C-NMR for the purified product thus obtained, this main component is a pyrrolidideine alkaloid having a chemical structure of the general formula (I). It was identified as a glycoside (Casuarine-6-O-α-D-glucoside).
As a result of fluorescent X-ray elemental analysis, it was found that the purified product contained metals such as magnesium, potassium, calcium, iron, copper, and zinc.
And, although the enzyme inhibitory activity of the purified mold (H) fraction is as high as 2.4 μg / ml, the enzyme inhibitory activity of the isolated and purified pyrrolididein alkaloid glycoside is as weak as 43 μg / ml, When magnesium salt or zinc salt is added to this, the increase in blood glucose level is increased to 18.5 μg / ml or 5.7 μg / ml. It can be seen that it is based on a conjugate of a didein alkaloid glycoside and a metal.

  The blood glucose level elevation inhibitor of the present invention exhibits a suppression effect of about 3 times or more compared to conventional blood sugar level elevation inhibitors extracted from bunjirou guava. In addition, since it exhibits the same or better inhibitory effect as compared with commercially available products such as acarbose or voglibose, it is suitable as a blood glucose level increase inhibitor by an α-glucosidase inhibitory action.

Next, the best mode for carrying out the present invention will be described in more detail by way of examples, but the present invention is not limited to these at all.
In addition, the α-glucosidase inhibitory action in this example was measured by the following method.

(1) Preparation of Enzyme / Substrate Solution The upper mucosa of the small intestine of Wistar rats (6 weeks old, male) was washed out and scraped out, and 100 mM potassium phosphate buffer (containing 5 mM EDTA per 1 g mucosa) 1 ml of pH 7.0) was added and homogenized at 4 ° C. The treated product thus obtained was centrifuged at 21,000 × g for 60 minutes, and then 100 mM potassium phosphate buffer (pH 7.0) containing 3% Triton X-100 in the precipitate was centrifuged. The same amount as the supernatant was added and solubilized by stirring at 4 ° C. for 60 minutes. The obtained solution was centrifuged at 110,000 × g for 90 minutes, and the obtained supernatant was dialyzed for 24 hours using 10 mM potassium phosphate buffer as an external solution to prepare an enzyme solution. Further, sucrose was used as the substrate solution, and the final concentration was 80 mM.

(2) Enzyme activity measurement In a test tube, add 100 μl of the sample solution to 400 μl of the substrate solution to a final concentration of 1 mg / ml, add 100 μl of the enzyme and react at 37 ° C. for 30 minutes, and then boil the test tube The reaction was stopped by immersing in a water bath for 2 minutes.
Next, 3 ml of a coloring reagent (trade name “Glucose B-Test Wako”, manufactured by Wako Pure Chemical Industries, Ltd.) was dispensed, and a part of the reaction solution (20 μl) was added thereto, followed by reaction at 37 ° C. for 20 minutes. The glucose concentration was determined by measuring the absorbance (A ₅₀₅ ). In consideration of the influence of the specimen on GOD activity, the substrate was reacted at a final concentration of 240 mg / dl glucose for 20 minutes at 37 ° C., and the numerical value was corrected based on the result.

(3) Protein quantification It was performed by the Lowry method [see “J. Biol. Chem.”, Vol. 193, page 265 (1951)].

4.0 kg of fresh male peach bark (average particle size: 1 mm) was immersed in 3 volumes of methyl alcohol and extracted for 5 days. The solvent was distilled off from the extract under reduced pressure to obtain a crude extraction residue (A).
Next, this crude extraction residue (A) was partitioned between the same volume of ethyl acetate and water, and 2.7 g of ethyl acetate soluble matter (B) was obtained from the ethyl acetate layer. On the other hand, the water layer (C) is further divided between n-butyl alcohol and water of the same volume, and is divided into an aqueous layer and an n-butyl alcohol layer. (D) 9.5 g was recovered, and the aqueous layer was freeze-dried to obtain 108 g of a solid (E) [hereinafter referred to as unpurified mold (E)].
Subsequently, this unpurified mold (E) is dissolved in water, and a barium hydroxide powder is added to form a precipitate. After removing this precipitate, the liquid phase portion is freeze-dried to obtain purified mold (F) about Obtained in a yield of 60% by weight. A process diagram of the above method is shown in FIG.

The α-glucosidase inhibition rate of the fractions thus obtained was as follows: crude extraction residue (A) 98.0%, ethyl acetate soluble fraction (B) 9.0%, n-butyl alcohol soluble fraction ( D) 79.0%, unpurified mildew (E) 99.1%, and purified mildew (F) 99.5%.
Further, the substrate specificity (IC ₅₀ ) of the inhibitory activity was measured for unpurified mold (E) and purified mold (F), and the results are shown in Table 1 together with those of commercially available voglibose and acarbose.

  As can be seen from this table, unpurified mold (E) and purified mold (F) have a strong activity equivalent to that of commercially available products and have an inhibitory action on a wide range of enzyme substrates.

The following experiment was conducted in order to identify a compound exhibiting the suppression of blood glucose elevation.
That is, 10 g of unpurified mildew (E) was subjected to column chromatography using Shephadex LH-20, and 3 g of purified mildew (G) was obtained from the methanol-solubility part of the first flowing out portion. In purified mold (G), the enzyme inhibitory activity when sucrose was used as the enzyme substrate showed an extremely high IC ₅₀ value of 4.4 μg / ml. Further, 3 g of purified mold (G) was subjected to high performance liquid chromatography using a column (Waters Carbohydrate Analysis Column), and was eluted with solvent acetonitrile: water = 4: 1 to obtain 1 g of purified mold (H). The yield was about 0.25% by weight based on fresh barberry peach bark and about 1% by weight based on unrefined mildew (E). When the enzyme inhibitory activity of this purified mold (H) was examined, it was found that the activity was further increased to IC ₅₀ 2.4 μg / ml. A process diagram of the above method is shown in FIG.

で示されるカズアリン‐６‐Ｏ‐α‐Ｄ‐グルコシドと同定された。
また、蛍光Ｘ線元素分析装置［堀場製作所製、製品名「メサ（ＭＥＳＡ）−５００Ｗ」を用いて分析した結果、炭素、水素、酸素、窒素以外に表２に示す元素が含まれていることが分った。 After 2 g of purified mold (G) was adsorbed using an ion exchange resin (Amberlite CG120, manufactured by Organo Corporation), it was discharged with 0.1 M aqueous ammonia to obtain 50 mg of a pure substance on high performance liquid chromatography. FIG. 3 shows the nuclear magnetic resonance spectrum and ¹ H-NMR of this substance, and FIG. 4 shows the ¹³ C-NMR. These spectra are known data [Tetrahedron Letters], Volume 35 (No. 42), 1994, pages 7849-7852 and Carbohydrate Letters], Volume 2. (No. 3), 1996, as a result of comparison with pages 167 to 174,

It was identified as kazualin-6-O-α-D-glucoside.
Moreover, as a result of analysis using a fluorescent X-ray elemental analyzer [manufactured by Horiba, product name “MESA-500W”, the elements shown in Table 2 are included in addition to carbon, hydrogen, oxygen, and nitrogen. I found out.

The α-glucosidase inhibitory action of the pyrrolidine alkaloid glycoside obtained in Example 3 was tested using sucrase. The IC _{50 was} 43 μg / ml, and the inhibitory activity IC ₅₀ of purified mold (G) was 44 μg / ml. In comparison, the activity was found to be remarkably weak at about 1/10.

Separately, add magnesium acetate (49 wt% in terms of Mg) or zinc acetate (2.3 wt% in terms of Zn) separately to the aqueous solution of isolated and purified kazualin-6-O-α-D-glucoside. The inhibitory action of α-glucosidase was investigated.
As a result, it was found that the activity increased with the addition of magnesium acetate, IC ₅₀ 18.5 μg / ml, and the addition of zinc acetate with IC ₅₀ 5.7 μg / ml.

(Inhibition effect on blood sugar level increase)
Normal Wistar rats, streptozotocin diabetic rats (hereinafter referred to as STZ) and spontaneously diabetic rats (hereinafter referred to as GK rats) prepared by dissolving unpurified mold (E) in purified water at a concentration of 7.5 mg / kg A single oral dose was given. In addition, 7-week-old normal Wistar rats were used as STZ rats, and STZ 30 mg / kg was administered twice daily from the tail vein, and the glucose level during glucose loading reached 300 mg / dl-350 mg / dl. . The same amount of purified water was administered to the control group. 15 minutes after sample administration, sucrose was orally administered at 3.75 mg / kg, blood was collected from the tail vein at 30, 60, and 120 minutes after administration, and the plasma glucose was measured by the GOD method. The results are shown in FIG. 5 as a graph showing the amount of glucose over time. In the figure, ■ indicates the results for the test group, and ◆ indicates the results for the control group.

  As can be seen from this figure, when the unpurified mildew (E) administration group was compared with a single administration, the increase in blood glucose after administration of sucrose was suppressed in both normal rats and diabetic rats. In particular, significant suppression was observed 30 minutes after administration of sucrose. That is, in normal rats, the blood glucose level, which was 198.4 mg / dl in the non-administration group 30 minutes after sucrose loading, was significantly suppressed to 160.5 mg / dl (risk rate of 1% or less), and in 60 minutes 179.9 mg / dl was reduced to 158.3 mg / dl and 165.7 mg / dl was reduced to 152.7 mg / dl in 129 minutes, respectively (risk rate 5% or less). Similarly, similar effects were confirmed in rats that were caused by diabetes by streptozotocin and rats that were spontaneously affected.

(Long-term continuous administration effect)
Two groups of 7 rats each were administered unpurified mildew (E) at a dose of 7.5 mg / kg or 37.5 mg / kg for 28 days. In addition, purified water was administered to the control group. On days 0, 7, 21 and 28, blood was collected from the tail vein and the plasma glucose was measured. The results are shown as a graph in FIG. In the figure, the control group is indicated by ♦, 7.5 mg / kg administration is indicated by ■, and 37.5 mg / kg administration is indicated by ▲. Further, plasma glucose at 30 minutes after glucose loading was measured, and the results are shown as a graph in FIG. Each numerical value is an average value of 7 pieces.

  As can be seen from these figures, even in the long-term administration, the group administered with unpurified mold (E) tends to suppress or improve the increase in blood glucose level as compared with the control group. That is, after 2 to 3 weeks of administration, the fasting blood glucose of rats caused diabetes with streptozotocin was changed from about 450 mg / dl to 350 to 400 mg / dl (FIG. 6), and the blood sugar 30 minutes after glucose load was 500 mg. / Dl was reduced from about 400 to 450 mg / dl (FIG. 7).

In order to examine the state of β cells in pancreatic islets of Langerhans, the amount of insulin in β cells was immunostained using an insulin antibody. Using a staining kit (VACTASTAIN Elite ABC Rabbit IgG kit (Vector Laboratories, CA, USA), Anti-porin Insulin antibody (CAPPEL RESERCH REAGENTS) as a primary antibody, and secondary antibody as a secondary antibody kit Rabbit IgG antibody was used.
The amount of fat in the liver was observed by oil red O (Oil Red O) method, staining lipid droplets and neutral fat.
Further, in order to examine the state of the glomeruli of the kidney, PAM staining was performed, the kidney glomerular basement membrane was stained, and nuclear staining / post-staining was performed with HE (hematoxylin-eosin).

  As a result, it was found that the islets of Langerhans in the long-term administration group of unpurified mold (E) had a higher insulin content than that in the control group. In terms of morphology, the islets of Langerhans in the sample administration group were often close to normal. The liver had less fat compared to the control group. Furthermore, the glomerular basement membrane (mesangial region) was nearly normal in the administration group, whereas that in the control group was thickened, and the glomeruli also showed a tendency to atrophy.

(Toxicity test)
(1) Toxicity after a single oral administration Unpurified mildew (E), 75, 750 and 1000 mg / kg, were each administered once to two normal rats in each group, and the toxicity was observed for 14 days after administration.
As a result, it was found that the blood sugar elevation inhibitor of the present invention has no acute toxicity.

(2) Toxicity caused by large-scale oral administration No death was observed in the group that received large-scale administration of unpurified mildew (E). There was no suppression of weight gain. GOT and GPT values showed a slight increase in GOP value 1 day after administration in the 750 mg / kg administration group, but no abnormality was observed after 7 and 14 days. In addition, changes in appearance and histopathological abnormalities were examined, but no abnormalities were observed in any group.

  The present invention is useful as a blood glucose increase inhibitor for suppressing an increase in blood glucose level after a meal.

FIG. 3 is a process diagram illustrating the operation of the first embodiment. Process drawing which shows operation of Example 2. FIG. ¹ H-NMR spectrum diagram of the substance obtained in Example 3. FIG. ¹³ C-NMR spectrum diagram of the substance obtained in Example 3. FIG. The graph which shows the effect of the unpurified moldy (E) component with respect to suppression of a blood glucose level raise of the normal rat which administered sucrose. The graph which shows the effect of an unpurified moldy (E) ingredient with respect to a diabetic rat. The graph which shows the effect of an unpurified moldy (E) ingredient 30 minutes after sugar load of a diabetic rat.

Claims (3)

A blood glucose elevation inhibitor comprising a metal conjugate of a pyrrolidideine alkaloid glycoside represented by the formula:   The blood sugar elevation inhibitor according to claim 1, wherein the metal is at least one selected from magnesium, calcium and zinc. The methyl alcohol leachate of bark of Myrtaceae and Syzygium malaccense (L. Merrill & Perry) plants was concentrated under reduced pressure, and the resulting concentrated residue was partitioned between ethyl acetate and water. And the obtained aqueous layer is further subjected to a partitioning treatment with n-butyl alcohol, and then the solid content is recovered from the aqueous layer. Method.

Images