JPS6216957B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel berberine compound, a method for producing the same, and an antihypertensive agent. More particularly, the present invention provides a product precipitated by mixing berberine or a salt thereof with an aqueous extract of rhubarb in water, a process for making the same, and an antihypertensive agent containing the product. Traditional herbal medicine Ouren (Coptis)
Japonica Makino root), Oubaku (Kiwada;
The alkaloid berberine C ₂₀ H ₁₉ O ₅ N and its salts (e.g., acid acid, sulfate, etc.) are known as antidiarrheal and bactericidal agents. , and the herbal medicine rhubarb has been used medicinally as a laxative since ancient times. The inventors of this invention have discovered a new fact that a yellow colored precipitate is formed by mixing berberine or its salt with an aqueous extract of rhubarb in water, and that this substance exhibits a strong hypotensive effect when administered orally. We discovered this and arrived at this invention. As the berberine or its salt in this invention, substantially pure berberine is used. For example, berberine chloride is listed in the Japanese Pharmacopoeia (9th revision). However, it may also be a herbal medicine containing berberine, such as a salt of substantially berberine obtained from Orensis, Orientalis or the like, or one containing berberine. That is, for example, Aurenia or Aureoleum is extracted with water, alcohol (preferably methanol), or hydrous alcohol in accordance with a conventional method, and if alcohol is used, it is concentrated and added to water. The water immersion liquid obtained in this way is further treated to remove the resin, and when a mineral acid is added to the resulting aqueous solution, berberine salt is precipitated. Further, by neutralizing this in the form of a hot aqueous solution, an aqueous solution of berberine can be obtained. The berberine salt or berberine produced in this manner substantially contains these and a small amount of other berberine-based alkaloids, and such salts may be used as raw materials for the present invention. As the salt of berberine in this invention, mineral acid salts such as sulfate and hydrochloride are usually used, but water-soluble and non-toxic salts such as citrate and benzoate may also be used. In this invention, an aqueous rhubarb extract is obtained as follows. First, rhubarb (Rheum)
palmatum Linne′, Rheum tangusticum
Maximowice, Rhum careanum Nakai, Rueum
officinale Baillon or their interspecific hybrids) is used as raw material. Water or water-containing lower aliphatic alcohol is used as the extraction solvent.
Do this when the extraction is hot. The amount of extraction solvent and extraction time are selected so that the active ingredients can be extracted efficiently. For example, when using water, the amount is about 10 times that of rhubarb, and the extraction time is about 1 hour when hot, and about 1 hour when cold. About 24 hours is convenient. After extraction, insoluble matter is separated. When water is used as the extraction solvent, the obtained extract may be mixed as is with an aqueous solution of berberine or its salt, or this extract may be mixed with ether, benzene, hexane, etc. as necessary. This may be used as a raw material extract by adding water and a resinous organic solvent that is difficult to dissolve in each other and shaking well to transfer and remove contaminating oils, fats, and free anthraquinones. On the other hand, when using a hydrous lower aliphatic alcohol as an extraction solvent, the residue obtained by evaporation to dryness is redissolved in water (if there is any insoluble portion, it is removed by filtration), and this is used for raw material extraction. It is used as a liquid or treated with a fat-soluble organic solvent as described above to remove impurities in the same manner, and used as a raw material extract. Evaporation to dryness is preferably carried out under reduced pressure at a temperature not exceeding 60°C. The aqueous rhubarb extract and berberine or its salt are mixed in water, but it is usually preferably carried out under heat. For example, an aqueous rhubarb extract is gradually added to a hot aqueous solution of berberine or its salts while stirring well (or vice versa). A yellow colored precipitate is precipitated by mixing the two. This operation is carried out until an excess of one of the raw materials is added until no precipitate is observed (usually 200 to 300 g of rhubarb aqueous extract is required for 4 g of berberine). Thus, even if one of the raw materials is used in large excess, a precipitate of a constant nature is always produced. After the addition (mixing) is completed and the mixture is left to stand (usually left overnight), the supernatant liquid is removed, for example, by a decanting method, and the precipitate is separated and collected. The precipitate thus obtained is usually thoroughly washed with water. Washing is continued until the washing solution no longer turns red even when aqueous ammonia is added to it. The desired compound is then obtained after drying, preferably at a temperature below 60°C. The compound thus obtained exhibits the following properties. (i) Tasteless, odorless, yellow and colored powder, hardly soluble in water, alcohol, chloroform, ether,
Insoluble in benzene, 50% aqueous acetone solution,
Dissolve in 50% acetic acid aqueous solution. Furthermore, it does not dissolve in artificial gastric fluid, artificial intestinal fluid, or artificial digestive fluid containing pepsin even when shaken at 37°C for 30 minutes. (ii) Dissolve 150 mg of this product in 5 ml of N·N-dimethylformamide (PH 7.0) and add 10 ml of distilled water, and the pH of the solution is 5.75. (iii) Add 2 mg of this product to N-hydrochloric acid: methanol: water (2:
The maximum ultraviolet absorption wavelength of this product when dissolved in 200 ml of a mixture of 100:98) and the E ¹ % ₁ cm at that wavelength are λmax: 210nm (501), 268nm (313), 345n
m (157), 420 nm (61). (iv) The infrared absorption spectrum (KBr) of this product is νmax: 3300 (broad), 1600, 1500,
1440, 1275, 1360, 1095, 1025 ^{cm -1} . Figure 2 shows the infrared absorption spectrum (KBr) of this product. (v) Dissolve 0.1 g of this product in 20 ml of 50% acetic acid aqueous solution and use it as a sample solution. Looking at its color reaction to the precipitation reagent, a. Add 0.2 ml of hydrochloric acid to 5 ml of the sample solution, shake for 1 minute, and add potassium iodide test solution. When added, a yellow precipitate is formed (berberine coloration). b When 0.5 ml of ferric chloride test solution is added to 5 ml of the sample solution, a dark blue color appears, and when left for 5 minutes, a black and colored precipitate forms (presence of tannin). c 0.5ml of Dragendorff test solution to 5ml of sample solution
When adding , a reddish-orange precipitate is formed (presence of berberine alkaloid). TLC (thin film chromatography) also showed a single spot. The fact that TLC shows only a single spot and that the precipitation formation by adding the aqueous extract of rhubarb to a certain amount of berberine or its salt is completed at a certain amount is that this precipitated product is berberine or its salt. This suggests that the salt and the active ingredient extracted from rhubarb are bound in some way at a certain ratio. This substance has the following physical properties. (vi) Melting point 191-193℃ (decomposition) (vii) Elemental analysis Empirical formula C ₄₀ H ₃₇ N ₁ O ₁₈ 2H ₂ O C H N Calculated value (%) 56.1 4.8 1.6 Actual value (%) 56.2 4.7 1.7 ( viii) Molecular weight: 855.76 This substance is a very low toxicity substance that does not show any abnormality even when administered orally once to rats at 1 g/kg, and is a hypotensive agent for essential hypertension, arteriosclerosis, autonomic imbalance, etc. It has a wide range of uses. The dosage of this product is approximately 300 mg to 500 mg per time, and is administered, for example, three times a day. The dosage form usually includes oral dosage forms such as powders, tablets, emulsions, capsules, granules, and tea preparations. Preferred excipients used in these formulations include, for example, magnesium stearate, starch, lactose, glucose, sucrose, rice flour, talc and white sugar. Next, the results of pharmacological tests and clinical tests regarding the antihypertensive effect of the compound according to the present invention will be described. Pharmacological test test method: Wistar rats (body weight 120-150 g) were used as test animals, and each group consisted of 5 animals.
This product was administered orally at a rate of 400 mg per dose to a human weighing 60 kg, which was 10 times the amount calculated from the rat body weight. That is, in the case of a 120 g rat, 8 mg was well suspended in 1 ml of distilled water, forcefully administered using a gastric tube, and blood pressure values before administration and 1 hour, 3 hours, 5 hours, 7 hours, and 24 hours after administration were determined. Blood pressure values were measured after hours and their fluctuations were investigated. The test was carried out using 1 ml of distilled water as a blank. Test results:

[Table] As mentioned above, oral administration of this product causes a blood pressure drop of approximately 20% in rats after 1 hour, which continues for 6 hours, and returns to normal after 24 hours.
In this way, administration of this product brings about a significant and specific blood pressure lowering effect. Clinical study (1) A 39-year-old woman diagnosed with essential hypertension, a 51-year-old man diagnosed with arteriosclerosis, and a 42-year-old woman diagnosed with autonomic nervous system imbalance were tested in the supine position for 15 minutes.
The subjects were allowed to rest for a minute, and the systolic and diastolic blood pressures in the left brachial artery were measured. Then, 400 mg of this product was orally administered, and the fluctuations in the systolic and diastolic blood pressures over the next 1 hour were measured. The results are as follows (measured values are the average values of three measurements).

[Table] (2) For a 48-year-old woman diagnosed with essential hypertension, administer 400 ml of this product 1 hour and 30 minutes before bedtime using the same method as in (1).
mg orally, 10 days after administration with sleep time in between.
Changes in systolic and diastolic blood pressure values over time were measured. The results are as follows.

[Table] As shown above, oral administration of 400 mg of this product lowered the maximum and minimum blood pressure values in all patients and showed improvement in their medical conditions. The compounds used in the above pharmacological tests and clinical tests were produced in the same manner as in Example 1. Next, production examples of compounds according to the present invention will be described. Example 1 200 g of Japanese Pharmacopoeia rhubarb powder was added with boiling water from Step 2, and heated and extracted using a reflux condenser for 1 hour, cooled and filtered. Separately, Japanese Pharmacopoeia berberine chloride was dissolved in boiling water, and the rhubarb water extract was poured into the solution while stirring well. At this time, a yellow colored precipitate was deposited. After all the injections were completed, a small amount of the rhubarb soaked liquid prepared in the same manner was added to the supernatant liquid, and after confirming that no precipitate was formed, it was allowed to stand overnight, and the supernatant liquid was removed by tilting. , and the precipitate was filtered under reduced pressure. The precipitated material obtained was washed with water until it no longer turned pink even when an ammonia test solution was added to the washing solution. The obtained yellow colored precipitate was dried at below 60°C to obtain 13 g of product. The characteristics of this product are as described above. Example 2 200g of Japanese Pharmacopoeia rhubarb powder was added with boiling water from Step 2, heated and extracted using a reflux condenser for 1 hour, cooled and filtered. Versol (1) was added to this liquid, and the mixture was shaken well and left to stand, and the separated aqueous layer was collected. Separately, 4 g of berberine was dissolved in 300 ml of water, and a defatted water extract of rhubarb was gradually added thereto. A large amount of yellow precipitate was produced as the mixture was added. After all the additions were completed, a small amount of the rhubarb immersion solution prepared in the same manner was added to the supernatant liquid, and after confirming that no precipitate was formed, it was left overnight, and the supernatant liquid was poured onto the slant. Then, the precipitate was filtered under reduced pressure. The obtained precipitate was washed with water until it no longer turned pink even when an ammonia test solution was added to the washing solution, and the precipitate was dried at 60° C. or lower to obtain 13.4 g of a product. It was confirmed that this product had exactly the same properties and properties as the product obtained in Example 1. Example 3 A 50% aqueous methanol solution 2 was added to 200 g of Japanese Rhubarb powder, heated and extracted using a reflux condenser for 1 hour, cooled, and filtered. The liquid was concentrated to dryness under reduced pressure at a temperature below 60°C. Two portions of boiling water was added to the colored residue, stirred well for 10 minutes, cooled, and filtered. Ether (1) was added to the solution, and the mixture was shaken and allowed to stand, and the aqueous layer was separated. Separately, berberine sulfate was dissolved in 500 ml of hot water, and the fractionated rhubarb soluble solution was gradually added to this solution while stirring. At this time, a yellow colored precipitate was deposited. After all the addition was completed, the mixture was left to stand overnight, the supernatant liquid was removed by decanting, and the precipitate was filtered under reduced pressure. The resulting precipitate was washed with water until it no longer turned pink even when an ammonia test solution was added to the washing solution, and the precipitate was dried at below 60°C to give 12.8 g.
products were obtained. Example 4 100 g of Japanese Pharmacopoeia Oren powder was extracted twice with 1 part of water for 30 minutes each time, and the liquids were combined. Separately, add boiling water from step 2 to 200 g of Japanese Rhubarb powder, heat and extract with a reflux condenser for 1 hour, and filter after cooling.
Hexane (1) was added to the liquid, and the mixture was shaken and allowed to stand, and the aqueous layer was separated. The hot rhubarb degreased water solution was gradually poured into the water-soaked solution of Japanese orchid while stirring well. At this time, a large amount of yellow colored precipitate was produced. After all the injections were completed, a small amount of rhubarb immersion solution prepared in another similar manner was added to the supernatant, and after confirming that no precipitate had formed, the supernatant was allowed to stand overnight, and the supernatant was decanted. The precipitate was filtered under reduced pressure. The obtained precipitate was washed with water until no light color appeared even when an ammonia test solution was added to the washing solution, and the precipitate was dried at 60° C. or lower to obtain 12.7 g of a product.

[Brief explanation of the drawing]

Figure 1 is a graph showing changes in blood pressure values when Wistar rat rats were administered the compound according to the present invention (solid line) and when distilled water was administered (blank: dotted line). It is an infrared absorption spectrum diagram of a compound.

Claims (1)

[Claims] 1. A product precipitated by mixing berberine or a salt thereof with an aqueous extract of rhubarb in water, which has the following properties: (i) as a tasteless, odorless, yellow and colored powder; Refractory to water, alcohol, chloroform, ether,
Insoluble in benzene, soluble in 50% aqueous acetic acid and 50% aqueous acetone. Furthermore, it does not dissolve in artificial gastric fluid, artificial intestinal fluid, or artificial digestive fluid containing pepsin even when shaken at 37℃ for 30 minutes. (ii) Dissolve 150 mg of this product in 5 ml of N-N-dimethylformamide (PH7.0). The pH of the solution added with 10 ml of distilled water is 5.75. (iii) This product was mixed with N-hydrochloric acid: methanol: water (2:
The maximum ultraviolet absorption wavelength of this product in a solution dissolved in 100:98) and the E ¹ % ₁ cm at that wavelength are λmax: 210nm (501), 268nm (313), 345n
(iv) The infrared absorption spectrum (KBr) of this product is νmax: 3300 (broad), 1600, 1500,
1440, 1275, 1360, 1095, 1025 ^{cm -1} . (v) The reaction of the 50% aqueous acetic acid solution (sample solution) of this product to the color precipitation reagent is as follows: a) Add hydrochloric acid to the sample solution, shake it, and add potassium iodide test solution to the sample solution to form a yellow precipitate; b. When ferric chloride test solution is added, it exhibits a dark blue color, and when left to stand, a black and colored precipitate is produced. c. When Dragendorff test solution is added to the sample solution, a reddish-orange precipitate is produced. (vi) Melting point: 191~ 193℃ (decomposition) (vii) Elemental analysis Empirical formula C ₄₀ H ₃₇ N ₁ O ₁₈ 2H ₂ O C H N Calculated value (%) 56.1 4.8 1.6 Actual value (%) 56.2 4.7 1.7 (viii) Berberine with molecular weight 855.76 system compound. 2. Berberine or a salt thereof is obtained by treating orensis or amberberry in a conventional manner, removing the resin, and adding a mineral acid as an aqueous solution to precipitate the berberine salt, or desalting it to produce berberine. The berberine compound according to claim 1, which substantially contains berberine or a salt thereof. 3. The berberine compound according to claim 1 or 2, wherein the berberine salt is berberine hydrochloride or berberine sulfate. 4 The aqueous extract of rhubarb is treated with berberine or its salt in water, and the resulting precipitate is separated and collected, characterized by the following properties: (i) It is a tasteless, odorless, yellow and colored powder that can be mixed with berberine or its salt in water. , poorly soluble in alcohol, chloroform, ether,
Insoluble in benzene, soluble in 50% aqueous acetic acid and 50% aqueous acetone. Furthermore, it does not dissolve in artificial gastric fluid, artificial intestinal fluid, or artificial digestive fluid containing pepsin even when shaken at 37℃ for 30 minutes. (ii) Dissolve 150 mg of this product in 5 ml of N-N-dimethylformamide (PH7.0). The pH of the solution added with 10 ml of distilled water is 5.75. (iii) This product was mixed with N-hydrochloric acid: methanol: water (2:
The maximum ultraviolet absorption wavelength of this product in a solution dissolved in 100:98) and the E ¹ % ₁ cm at that wavelength are λmax: 210nm (501), 268nm (313), 345n
(iv) The infrared absorption spectrum (KBr) of this product is νmax: 3300 (broad), 1600, 1500,
1440, 1275, 1360, 1095, 1025 ^{cm -1} . (v) The reaction of the 50% aqueous acetic acid solution (sample solution) of this product to the color precipitation reagent is as follows: a) Add hydrochloric acid to the sample solution, shake it, and add potassium iodide test solution to the sample solution to form a yellow precipitate; b. When ferric chloride test solution is added, it exhibits a dark blue color, and when left to stand, a black and colored precipitate is produced. c. When Dragendorff test solution is added to the sample solution, a reddish-orange precipitate is produced. (vi) Melting point: 191~ 193℃ (decomposition) (vii) Elemental analysis Empirical formula C ₄₀ H ₃₇ N ₁ O ₁₈ 2H ₂ O C H N Calculated value (%) 56.1 4.8 1.6 Actual value (%) 56.2 4.7 1.7 (viii) Berberine with molecular weight 855.76 Method for producing system compounds. 5. The production method according to claim 4, wherein the aqueous rhubarb extract is a hot water extract solution of rhubarb as it is. 6. The aqueous extract of rhubarb is obtained by thermally extracting rhubarb using a water-containing lower aliphatic alcohol, evaporating to dryness, and redissolving with water as necessary. Manufacturing method. 7. The production method according to claim 4, wherein the aqueous rhubarb extract is obtained by treating a hot water extract of rhubarb with a lipophilic organic solvent such as ether, benzene, or hexane. 8. An aqueous extract of rhubarb is obtained by heat extracting rhubarb with a water-containing lower aliphatic alcohol and then evaporating to dryness. The manufacturing method according to claim 4, which is obtained by processing with an organic solvent. 9. The manufacturing method according to claim 4, wherein berberine or a salt thereof is extracted from a crude drug containing berberine. 10. The manufacturing method according to claim 7, wherein the herbal drug containing berberine or a salt thereof is Oren or Orbaku. 11. The production method according to any one of claims 4 to 10, wherein the treatment of the aqueous rhubarb extract and berberine or its salt is carried out under heat. 12. The production method according to any one of claims 4 to 11, wherein an aqueous solution of berberine or a salt thereof is added to an aqueous extract of rhubarb.