JPH062762B2 - New Polysuka Ride - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention provides a novel branched heteropolysaccharide having an excellent antiemetic action, and can be used in the field of antiemetic agents.

[Prior art] It is mixed with Kampo medicines for the purpose of antiemetic, antitussive, Meniere's syndrome, early pregnancy suppressant, sedation, expectorant, etc. Especially, it is used as an antiemetic agent which is excellent for preventing emesis of pregnant women. There is. In the half-summer, the rhizomes of the pearl shrimp ( Pinellia ternata Breitenbach) belonging to the family Araceae are dried and removed. The crow bishaku is widely distributed from the Chinese continent to the Korean Peninsula, and even in Japan, it grows naturally in each region. There are various reports on the anti-emetic effect in the half-summer, and the anti-emetic effect in the half-summer petroleum ether extract [Tohoku J. Exp.Med. Vol. 17, p. 528 (1931)] and in the mid-summer methionine [ J. Taiwan Pharm. Asso. 27, 41
(1975)], in 50% methanol extract [Biopharmaceutical magazine 3
7:73 (1983); 37: 367 (1983 name); 38: 102 (1984)
Year)], and the antiemetic action was observed in the water-soluble fraction in the summer [Gifu Medical University Bulletin, Vol. 6, p. 243 (195).
8 years)]], but the view is different, and the effective substance showing the antiemetic action has not been clarified yet.

The difference in the reports on the effective substances for antiemetic action in the summer is due to the fact that until now, no appropriate animal antiemetic bioassay method has been established.

[Problems to be Solved by the Invention] In order to find an antiemetic substance that has an extremely small antitoxicity and side effects and has an excellent antiemetic effect, it is necessary to clarify an effective antiemetic substance in the summer. It is desired to do. For that purpose, it is necessary to first establish an antiemetic bioassay method, and it is necessary to clarify the effective antiemetic substance by separating and refining the components contained in the half summer using this assay method. .

[Means for Solving Problems] The present inventors have conducted various studies to find a novel anti-emetic substance having less toxicity and side effects. An antiemetic bioassay is required to find an antiemetic substance, but it was judged based on how many times the number of vomiting caused by copper sulfate was suppressed by using the Japanese toad frog. However, the frog's tongue is attached to the inside of the mouth from the tip of the mouth unlike the way the tongue of warm-blooded animals is attached, so it is not necessary to vomit an object out of the mouth during the vomiting test unless it is too much. It was possible. Therefore, in such a conventional method, the judgment is often erroneous. Therefore, whether or not the stomach contents digested substance is vomited into the oral cavity in order to make a more accurate judgment without compensating for this defect We established a method of observing with an open mouth, and used this method to search for antiemetic active ingredients during the summer months. As a result, while succeeding in isolating a novel branched heteropolysaccharide having the following physicochemical properties, the polysaccharide has 100% antiemetic action on animals such as frogs and cats. The present invention has been completed based on these findings. The active ingredient has the following properties:

(a) UV measurement shows no absorption in the ultraviolet.

(b) Elemental analysis does not contain nitrogen as a constituent atom.

(c) Average molecular weight: When measured by high performance liquid chromatography using TSKG4000SW (manufactured by Toyo Soda Co., Ltd.) as a column and pullulan Shodex P-82) as a standard product, the average molecular weight is 750,000 to 950,000.

(d) Approximately 10 mg / ml aqueous solution: shows mucus with a slight sweetness. The solubility in water is 5 to 10 mg / ml.

(e) Optical rotation dispersion of 1 wt% aqueous solution: levorotatory, ▲ [α] ²⁵
_D ▼ -6.0 ° (c = 1%, H ₂ O) (f) Content of neutral sugar (% by weight): L-arabinose about 5
0, L-fucose about 7, D-glucose about 5, L-rhamnose about 4, D-ribose about 2, D-galactose about 2.

(g) Content of uronic acid: Approximately 1/6 weight of the total amount in terms of D-galacturonic acid.

(h) Nuclear magnetic resonance spectrum (in D ₂ O): 102.8,79.1,7
Major signals are shown at 7.7, 72.8, 71.4, 63.2, 22.9 ppm.

(i) It has an acetyl group at a ratio of 1 in about 15 sugar residues.

The branched heteropolysaccharide of the present invention is obtained by cold-extracting half-summer with hydrous alcohol, drying the extraction residue and then cold-extracting with water, centrifuging the aqueous extract, and dialyzing the resulting supernatant. The compound with a molecular weight of less than 10,000 is then purified by dialysis with an internal solution by column chromatography, the fraction with an average molecular weight of about 1,000,000 is further purified by column chromatography, and the fraction with an average molecular weight of about 850,000 is deproteinized by heating. Can be obtained by

As the summer (Pinelliae Tuber), tubers excluding the cork layer of Calaspishak are used as they are or after being appropriately crushed. As the hydrous alcohol, for example, 5
0 to 90% by weight of methanol, ethanol, butanol or the like is used, and preferably 80% by weight of methanol is used. Cold-soaking extraction using hydrous alcohol can be performed according to a conventional method, for example, at 15 to 25 ° C.
Or it may be extracted several times. The extraction residue is dried. The drying method is preferably natural drying. As a method for cold-immersion extraction of the dried residue with water, a known cold-immersion extraction method similar to the cold-immersion extraction with the above-mentioned hydrous alcohol is used. Centrifugation of the cold-soaked extract of water can be performed according to a conventional method. The centrifugation is usually performed at 2500 to 12000 rpm, preferably 3000 to 4000 rpm. The supernatant obtained by this centrifugation is dialyzed to remove substances having a molecular weight of less than 10,000.

The dialysis method is not particularly limited as long as the purpose is achieved.
For example, it can be carried out by using a dialysis membrane that has a molecular weight of less than 10,000 and which does not pass through while flowing water. For the dialysis, the above-mentioned supernatant may be used as it is, but lyophilized one may also be used. Next, the dialysis inner solution obtained by this dialysis is dissolved in water as it is or after freeze-drying, and loaded on column chromatography. The column is not particularly limited as long as the target fraction is obtained,
Preferably, for example, Sephacryl S-300 (manufactured by Pharmacia) is used.

Elution may be performed with water or the like. Outflow rate is typically 0.5
~ 5 ml / min, preferably 1 ml / min. For example, a differential refractometer or the like can be used to detect the target fraction. By this column chromatography, it is usually divided into four fractions having an average molecular weight of about 3 million, about 1 million, about 10,000 and about 1,000. Of these four fractions, the fraction having an average molecular weight of about 100,000 is directly or after lyophilization, dissolved in water and further purified by column chromatography. As the column for this purification, for example, TSKG4000SW (manufactured by Toyo Soda Co., Ltd.) or the like is used, and other than that, it can be purified in the same manner as the column chromatography of the above dialysis inner solution. In this purification column chromatography, the average molecular weight is usually about 1.08 million, about 8
It is divided into four fractions of 50,000, about 820,000, and about 770,000.
When the protein is removed from this fraction having an average molecular weight of about 850,000, the desired branched heteropolysaccharide having an antiemetic rate of 100% is obtained by the following antiemetic bioassay method. To remove protein,
The fraction can be heated by heating, for example, 60 to 90 ° C. to coagulate the protein and filtration. This heating deproteinization step may be performed in the final step as described above, but can be performed in any step of the step after dialysis.

The physicochemical properties of the desired branched heteropolysaccharide thus obtained are as follows.

(a) UV measurement shows no characteristic absorption in the ultraviolet.

(b) In the elemental analysis, since the nitrogen content is 0%, nitrogen is not contained as a constituent atom.

(c) As for the average molecular weight, TSKG4000SW (manufactured by Nippon Soda) is used as a column, and pullulan “pullula” is used as a standard product.
n "(Sodex" Shodex "P-82) was measured by high performance liquid chromatography (HPLC) using a differential refractometer (RI) as a detector to find that it was 750,000-950,000, which was approximately 800,000 (± 10-10). 15%).

(d) Approximately 10 mg / ml aqueous solution is mucous and slightly sweet. The solubility in water is 5-10 mg / ml.
Is.

(e) The optical rotation dispersion of a 1 wt% aqueous solution is ▲ [α] ²⁵ _D ▼-
It is 6.0 ° (solvent = water, c = 1%) and exhibits levorotatory properties.

(f) Neutral sugar is analyzed according to the method described in Journal of the American Chemical Society (JACS), 73, 5849 (1951), etc. Specifically, 2.5 mg of the branched heteropolysaccharide of the target substance is added to 5 ml of 2N-sulfuric acid.
Was added, dissolved, hydrolyzed on a boiling water bath for 8 hours, cooled to room temperature, neutralized with IRA-45 (OH type), filtered through a glass filter, and the filtrate was concentrated under reduced pressure to obtain borate buffer pH 7.0. After adding a fixed amount and coloring with orcinol-sulfuric acid, use a sugar analyzer [sugar analyzer: JEOL JLC-GAH JEOL, autorotation analyzer: JASCO J-20] to prepare and measure a 1% aqueous solution of the sample. To do. ] To measure 440 nm. Furthermore, a part of the sample was trimethylsilylated (after TMS conversion 9 and then gas chromatography (GC)
Search for constituent sugars with [column: Shimadzu GC-RIA, FID, column packing: 3% 0V-17 (Chromosorb AW), 80-120 mesh,
2m, column temperature: 140 ° C, inlet temperature: 180 ° C, flow rate:
60 ml / min]. As a result, as the content (% by weight) of the neutral sugar, about 50 L-arabinose, about 7 L-fucose and D-
About 5 glucose, about 4 L-rhamnose, about 2 D-ribose, and about 2 D-galactose are detected, respectively.

(g) A type of sugar oxidation product, which is represented by the formula CHO | (CH.OH) _n | COOH [wherein n represents a positive integer] of a saccharide derivative having one carboxyl group together with an aldehyde group. The content of uronic acid (naturally believed to be glucuronic acid, mannuronic acid, and galacturonic acid) is D
-Approximately 1/6 weight of the total amount converted to galacturonic acid.

Specifically, Ministry of Health and Welfare Food Chemistry Report No. 39, 172
It is measured according to the carbazole method described on page (1985). That is, 0.05 N-hydrochloric acid was added to 5 mg of the branched heteropolysaccharide of the present invention, and the mixture was heated in a boiling water bath for 1 hour. After cooling, transfer to a 50 ml volumetric flask and add water to make exactly 50 ml. Accurately measure 5 ml of sulfuric acid test solution, put it in a 25 ml stoppered test tube, and cool with ice. 1 ml sample solution
Accurately weigh and add it carefully to form a layer on sulfuric acid,
Shake. The solution is heated in a boiling water bath for 10 minutes and cooled in ice water. To this solution, 0.2 ml of carbazole reagent solution was added and mixed, and then heated in a boiling water bath for 15 minutes and cooled to room temperature. About this solution 1 ml of water instead of the sample solution
As a control, the solution prepared by performing the same operation using
The absorbance at 525 nm was measured. When a calibration curve was prepared using D-galacturonic acid as a standard product and the concentration in the sample was calculated, the content of uronic acid was 16.5% by weight. This content corresponds to about 1/6 of the total weight.

(h) The target branched heteropolysaccharide was dissolved in D ₂ O using a 5 mm tube, and ¹³ C NMR was measured at 60 ° C. with sodium trimethylsilylprobansulfonate (TPS-Na) as an internal standard [manufactured by JEOL Ltd. JEO
L FX-270 type (270 MHz) is used],
102.8 (± 1), 79.1 (± 1), 77.7 (± 1), 72.8 (± 1), 71.4 (±
The main signals are obtained at 1), 63.2 (± 1) and 22.9 (± 1).

(i) Chemical and Pharmaceutical Bulletin (Chem, Pharm.Bull.), 26, 2768 (1978)
According to the method for quantifying acetyl groups described above, 0.5 mg of N-hydrochloric acid and 0.5 ml of propionic acid were added to 4 mg of the branched heteropolysaccharide of the target substance and hydrolyzed in a 100 ° C water bath for 4 hours.
Directly measure by GC [column: Shimadzu Gc-RIA, FID, column packing: 5% -Therm on 1000 (Chromosorb AW) 80-120 mesh, 2m, column temperature: 115 ° C, injection temperature: 150 ° C, Nitrogen flow rate: 40 ml / min]. When acetyl groups were quantified using acetic acid as a standard product, it was about 2.3% by weight. This quantitative value shows that the target product has an acetyl group in a ratio of about 1 to about 15 sugar residues.

The branched heteropolysaccharide of the present invention having such physicochemical properties is capable of forming a salt with an alkali metal such as sodium and potassium, an alkaline earth metal such as calcium and magnesium, and the like. Salts are also within the scope of this invention.

The thus-obtained branched heteropolysaccharide of the present invention and salts thereof have an excellent antiemetic action, and thus can be used as a safe antiemetic agent against vomiting of humans and livestock.

The branched heteropolysaccharide of the present invention or a salt thereof can be used alone or in combination with other active ingredients containing a herbal medicine in any of various pharmaceutical preparations including a herbal medicine, for example, a capsule, It can be used as a tablet, powder, solution, suspension or elixir.
They can usually be administered orally, if necessary intravenously or intramuscularly.

Capsules, powders and tablets used for oral administration include common excipients such as binders such as syrup, arabic gum, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc., fillers such as lactose, sugars, corn starch, calcium phosphate, sorbitol, glycine. Etc., such as lubricants such as magnesium stearate, talc, polyethylene glycol, silica and the like, demulsifiers such as potato starch and the like or available lubricants such as sodium lauryl sulphate. The tablets may be coated by methods well known in the art. Oral liquid preparations can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and the like, and are dry products which are dissolved in water or other suitable solvent before use. Good. Such liquid preparations include suspensions such as sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils such as almond oil, fractionated coconut oil, oily esters. Such as propylene glycol or ethyl alcohol, preservatives such as methyl or propyl p-hydroxybenzoate, sorbic acid and the like. The suppository can use a common suppository base such as cocoa butter or other glycerides.

Injectable compositions may be presented in unit dose form in ampoules or in containers containing a preservative. The composition may be in the form of a suspension, solution or emulsion in an oily or aqueous solvent, optionally containing auxiliary agents such as suspending agents, stabilizers and / or dispersants. You may have. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

It may also be formulated in a suitable form for absorption by the mucous membranes of the nose and throat or bronchial tissue, for example powders, liquid sprays or inhalants, rosein, throat paints and the like.

In addition to the carrier, it may also contain other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, thickening agents or flavoring agents. In addition, the composition may include other active ingredients, including herbal medicines, to provide broader pharmaceutical use.

For livestock, it may be formulated as an intramammary formulation in a long-acting or fast-release matrix.

The branched heteropolysaccharide of the present invention or a salt thereof can be used as an antiemetic agent, for example, for the treatment of vomiting symptoms in mammals, especially emesis and obesity in obstetrics and gynecology.
The daily dose depends on the condition and weight of the patient to be treated, the administration method (parenteral method or oral method) and frequency, and the like. In general, the oral daily dose will consist of about 1 to 50 mg, preferably 4 to 12 mg of active ingredient per kg of body weight of the patient in one or more applications per day. A suitable daily dose for an adult human is about 6 to 1 as the active ingredient per kg of body weight.
0 mg, divided into 2 to 4 times daily, about 1.5 to 5 mg
It is suitable to administer orally in an amount of / kg.

The composition containing the object of the present invention or a salt thereof can be administered in several unit-use modes such as a solid or liquid ingestible orally. The liquid or solid composition per unit use contains 0.5 to 99% of the object of the present invention. The preferred range is about 10-60%.

[Action] The branched heteropolysaccharide of the present invention has an excellent antiemetic action. In particular, it shows an antiemetic effect with a 100% antiemetic rate in the following test methods for frogs and cats.

(1) Anti-emetic test for frogs Rana nigromaculata and Rana frog (R
ana japonica) is bred for 3 days, and a bioassay is carried out using a group of 5 normal frogs having a body weight of 10-15 g. For the frog used, the earthworm is forcibly administered orally the day before the test. For bioassay, 100 μg of target substance in 0.2 ml of aqueous solution
Immediately after the preparation, the test solution is orally administered in a desposable manner such that the target substance is 100 μg per 10 g body weight. The frog was allowed to stand for 30 minutes as much as possible without receiving irritation from external fitting, and then orally administered apomorphine hydrochloride 1,000 μ / 0.2 ml and copper sulfate 1,000 μg / 0.2 ml as emetics. Immediately after that, 0 minute is set, and 5 minutes at 60 minute intervals for a total of 5 times. It is observed whether or not the gastric contents digestive substance is vomited into the oral cavity of the frog. The result of 15 minutes after the administration of the emetic was used as a criterion, and the antiemetic rate was 100% when the antiemetic rate of the object of the present invention was expressed as a percentage (%) divided by the total number of animals used for the experiment. is there.

(2) Antiemetic test for cats Whether or not the antiemetic effect of this target can be seen using large warm-blooded animals, a bioassay for apomorphine using 3 cats (hybrid, male, weight 0.4-0.5 kg) To do. Apomorphine (25 mg / kg) administered orally alone, repeated vomiting intensely for about 3-5 minutes, and abnormal behavior continued for about 20 minutes. On the other hand, a cat orally administered the target substance (26 mg / kg body weight) Even after administration of apomorphine (25 mg / kg) 30 minutes later, no vomiting behavior was observed.

[Example] Half summer was crushed and homogenized, and 80% methanol 10% was added to 1 kg thereof.
After adding 00 ml and stirring at room temperature for 1 hour, the mixture is allowed to stand for 1 day and night, cooled, extracted and filtered. The extraction residue obtained by repeating this operation twice was naturally dried, water 1 was added, and cold immersion extraction was carried out 3 times by adding water 1 and stirring at room temperature for 1 hour and then leaving it for 1 day. The extracts were combined, centrifuged at 3,500 rpm, and the supernatant was dialyzed for 2 days while flowing a dialysis membrane for removing 10,000 molecular weight. 2.2 g of freeze-dried dialysis solution was obtained.

This freeze-dried product showed an antiemetic rate of 67% against apomorphine in the above antiemetic bioassay using frog, and contained 13.9% nitrogen by elemental analysis. Dissolve 2 g of this freeze-dried product in about 40 ml of water and add Sephacryl "Sep
hacryl "S-300 (Pharmacia) (51D x 10
0 cm) and eluted with water using a differential refractometer as a detector (speed 1 ml / min), and the average molecular weights shown in Fig. 1 are 3 million, 1 million, 11,000 and 1000 A, B, respectively. 4 fractions of C and D were obtained.

When each fraction was freeze-dried and subjected to an antiemetic bioassay using a frog, the B fraction having an average molecular weight of 1.1 million showed an antiemetic rate of 75% with respect to apomorphine. This B fraction (freeze-dried product) was dissolved in about 10 ml of water and passed through a TSKG-4000SW column (manufactured by Toyo Soda) (4.6 x 60 cm) to elute with about 30 to 50 ml of water using a differential refractometer as a detector (speed. 1 ml / min) and the average molecular weights shown in Fig. 2 are 1.08 million and 85, respectively.
40,000, 820,000 and 770,000 F ₁ , F ₂ , F ₃ , and F ₄ fractions were obtained. Each of the obtained fractions was freeze-dried to give F ₁ 2.2 m
g, F ₂ 28.9 mg, F ₃ 28.4 mg, F ₄ 108.3 mg were obtained, and when an antiemetic biological study was performed using frogs, the F ₂ fraction (containing 5.9% nitrogen by elemental analysis) was 86% antiemetic. Showed the rate.
Dissolve this F ₂ fraction (freeze-dried product) in about 10 ml of water and
After heating to 0 to 70 ° C. for about 30 minutes, the supernatant obtained was freeze-dried to obtain about 20 mg of the target white cotton. This white cotton-like material had a nitrogen content of 0% by elemental analysis, and a frog,
An antiemetic bioassay using cats showed an antiemetic rate of 100% for apomorphine. This target product is dissolved in D ₂ O to dissolve TPS.
¹³ C NMR was measured at —60 ° C. using —Na as an internal standard, and the spectrum of FIG. 3 was obtained. In this spectrum,
Major signals can be seen at 102.8,79.1,77.7,72.8,71.4,63.2,22.9. JACS, 73, 5849 (1951)
By the described sugar analysis method, gas chromatography, and mass spectrometry, D-Gal (β1-3) LA was formed in the molecule of the target substance.
ra, D-Gal (β1-4) D-Glc, D-Glc (β1-
3) D-Glc, D-Glc (α1-4) D-Glc, LF
The presence of oligosaccharides of uc (α-2) L-Fuc (Gal is galactose, Ara is arabinol, Glc is glycol, and Fuc is fucose) was confirmed. In addition, in the X-ray fluorescence analysis, the calcium content (% by weight) of the target substance was 1.9% [Device: Kevex 0600 (Ultra trace, Rigaku) Fluorescence X]
Line device, data processing system 7000 tube: germanium,
It was measured under the conditions of 30 kv, 30 mA, and 100 seconds of irradiation under normal pressure. Quantitative analysis: An atomic absorption spectrophotometer equipped with Varian AA-975 type and GTA-95 was used. Calcium analysis line: 4226.7A, acetylene 1.5 kg / cm, air 13.0 kg / cm. ].

[Effect of the Invention] Since the branched heteropolysaccharide of the present invention is a novel compound having an excellent antiemetic action, a preparation containing this object can be used as an effective antiemetic agent.

[Brief description of drawings]

FIG. 1 shows the column chromatography elution pattern for the freeze-dried product of the dialysate obtained in the Example, FIG. 2 shows the column chromatography elution pattern for the B fraction (freeze-dried product), and FIG. Target ¹³ C NM
The R spectrum is shown.

Claims (1)

[Claims] 1. A branched heteropolysaccharide having the following physicochemical properties, and a salt thereof. (a) No absorption in the ultraviolet region. (b) Does not contain nitrogen as a constituent atom. (c) Average molecular weight: 750 to 950,000 (e) About 10 mg / ml aqueous solution: Shows mucus with a slight sweetness. (d) Optical rotation dispersion of 1% by weight aqueous solution: levorotatory, ▲ [α] ²⁵
_D ▼ -6.0 ° (c = 1%, H ₂ O) (f) Content of neutral sugar (% by weight): L-arabinose about 5
0, L-fucose about 7, D-glucose about 5, L-rhamnose about 4, D-ribose about 2, D-galactose about 2. (g) Content of uronic acid: Approximately 1/6 weight of the total amount in terms of D-galacturonic acid. (h) Nuclear magnetic resonance spectrum (in D ₂ O): 102.8,79.1,7
Major signals are shown at 7.7, 72.8, 71.4, 63.2, 22.9 ppm. (i) It has an acetyl group at a ratio of 1 in about 15 sugar residues.