JPS625887B2 - - 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. hydrochloride, 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 crude drug containing berberine, such as a salt of substantially berberine obtained from Oren, Kaubaku, etc., 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 can be obtained as follows. First, rhubarb (Rheum)
palmatum Linne´, Rheum tangusticum
Maximowice, Rheum careanum Nakai, Rheum
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 fat-soluble organic solvent that is difficult to dissolve in each other and stirring 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 an insoluble part, 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). By mixing these two, a yellow colored precipitate is precipitated. This operation is continued until an excess of one of the raw materials is added and 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% acetone, 50%
Dissolve in 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 E ¹ % ₁ cm at that wavelength are λmax: 210 nm (501), 268 nm.
(313), 345 nm (157), and 420 nm (61). (iv) The infrared absorption spectrum (KBr) of this product is ν
max: 3300 (broad), 1600, 1500, 1440,
1275, 1360, 1095, 1025 ^{cm -1} . (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 prepare 0.5 potassium iodide test solution. When adding ml, a yellow precipitate forms (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
A red-orange precipitate is produced when adding...
(Presence of berberine alkaloids). TLC (thin layer chromatography) also showed a single spot. showing only a single spot on TLC and for a given amount of berberine or its salts.
The fact that the formation of a precipitate due to the addition of an aqueous extract of rhubarb is completed at a certain amount means that this precipitate product has some combination of berberine or its salt and the active ingredient extracted from rhubarb in a certain ratio. This suggests that there is. This substance is a very low-toxic substance that does not cause any abnormalities even when administered orally once to rats at 1 g/kg, and is widely used as a blood pressure lowering agent for essential hypertension, arteriosclerosis, autonomic imbalance, etc. There is. 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.

[Table] As mentioned above, oral administration of this product causes a blood pressure drop in rats of approximately 20% after 1 hour, which continues for 6 hours, and returns to normal after 24 hours. Thus, administration of this product brings about a significant and sustained 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 asked to rest in a supine position for 15 minutes. The systolic and diastolic blood pressures in the left brachial artery were measured, and then 400 mg of this product was orally administered, and the changes in systolic and diastolic blood pressure over the next 1 hour were measured. The results are as follows (measured value 3
(average value measured twice).

[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, an example of producing a compound 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 1 part of boiling water, and the rhubarb water extract was added to the solution while stirring well. At this time, a yellow colored precipitate was deposited. After all the injections have been completed, add a small amount of rhubarb immersion solution prepared in the same manner to the supernatant liquid, and after confirming that no precipitate is formed again, leave it overnight, and remove the supernatant liquid by decanting. , 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. Benzol (1) was added to this liquid, the mixture was shaken well and allowed 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 water-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 obtained precipitate is washed with water, washed until it no longer turns pale pink even when an ammonia test solution is added to the washing solution, and the precipitate is dried at 60°C or less.12.8
g product was 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 the same manner was added to the supernatant liquid, and after confirming that no precipitate had formed, it was left overnight, and the supernatant liquid was decanted. The precipitate was filtered under reduced pressure. The resulting precipitate was washed with water, washed 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 less to weigh 127 g.
products were obtained.

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes in blood pressure values when a compound according to the present invention was administered to Wistar rats (solid line) and when distilled water was administered (blank: dotted line).

Claims (1)

[Scope of Claims] 1. A hypotensive agent comprising, as an active ingredient, a product precipitated by mixing berberine or a salt thereof with an aqueous extract of rhubarb in water. 2 The product has the following properties: (i) It is a tasteless, odorless, yellow and colored powder that is sparingly soluble in water and alcohol, and is soluble in 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 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), 345nm
(157), 420nm (61), (iv) The infrared absorption spectrum (KBr) of this product is ν
max: 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, and add the potassium iodide test solution. When ferric chloride test solution is added to the sample solution, a yellow precipitate is produced. b. When ferric chloride test solution is added to the sample solution, it becomes dark blue, and when left to stand, a black precipitate is produced. c. When Dragendorff test solution is added to the sample solution, it becomes reddish-orange. The antihypertensive agent according to claim 1, which is a berberine compound having the following: 3. The antihypertensive agent according to claim 1 or 2, wherein the salt of berberine is a sulfate or a hydrochloride. 4. Berberine or a salt thereof is obtained by treating orenium or auricularis in a conventional manner, and then deresin is removed to form an aqueous solution, and a mineral acid is added to precipitate the berberine salt, or it is desalted to obtain berberine. The antihypertensive agent according to claim 1 or 2, which substantially contains berberine or a salt thereof. 5. The antihypertensive agent according to any one of claims 1 to 4, which is adapted for oral administration.