JPS6312880B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a novel vitamin P, which has a strong capillary resistance-enhancing effect and is isolated for the first time from fruits of Rutaceae plants, such as lemons and oranges. . According to the Chemistry Dictionary (Kyoritsu Publishing: Edited by the Chemistry Dictionary Editorial Committee, 7413 ), vitamin P is ``citrin, bioflavonoids, factors that prevent permeability, and several compounds that are flavan derivatives. Belongs to...” is written. Below, the present inventors have discovered a substance that has a novel and powerful vitamin P action and whose chemical properties are completely different from those previously thought to be vitamin P (i.e., flavonoid glycosides such as hesperidin and rutin). This article details the process that led to the first isolation of this substance and the confirmation that this substance was true vitamin P. 1936 Saint George of Hungary {Nature 138 , 27 (1937); ibid, 138 , 798
(1936); Deutch Med.Wochenschr. 62 , 1325
(1936); Z.Physiol.chem.247, 258 (1937)} et al.
They discovered that a second factor exists in lemon juice and Hungarian pepper paprica along with ascorbic acid (vitamin C), and the deficiency of this factor is associated with decreased resistance in capillaries [hemorrhagic purpura and plasma protein resistance]. Increased permeability (symptoms similar to sepsis) was observed. They named this active factor vitamin P (also known as citrine). Furthermore, Saint George {Nature 138 , 1057
(1936); ibid, 139 , (1937)} conducted research on its active ingredient and obtained 2 g of vitamin P (citrine) crystals from the rind of a 200 kg lemon, and soon discovered that this was combined with hesperidin, a flavonoid glycoside. It was announced that it was a mixture with a small amount of eriodictin. St. George et al. showed that bleeding symptoms caused by scurvy diet in guinea pigs were recovered by feeding vitamin P (citrine), and vascular purpura in humans was cured.
It was announced that administering vitamin P (citrine) to patients with P. purpura restores capillary resistance. However, as a result of follow-up tests by Saint-George et al. (Bio-chem.J. 31 , 915 (1937)), this was rejected; Nature 140 ,
426 (1937)} also stated that they were not successful in retesting, and came to the conclusion that in order for vitamin P to exert its effects, the coexistence of a small amount of vitamin C is necessary. On the other hand, Batu Haratsuchi {Analyst 67 , 313
(1942) et al. reported that when guinea pigs were fed with scurvy diet plus vitamin C, blood vessels became fragile;
It is said that adding vitamin P will restore the condition, and that it is also possible to biologically measure vitamin P using this method. Since then, vitamin P has been studied by many researchers, including Griffith {Proc.Soc.Exp.
Biol.Med. 55 , 228 (1944)} reported that rutin exhibits more than 10 times the efficacy of crude hesperidin;
Compt. Rend. Soc. Biol. 137 , 1 (1943)} et al. said that esculin (coumarin glycoside) also exhibits remarkable vitamin P efficacy. In addition, vitamin P
There are many reports that the drug is effective when administered to treat bleeding disorders in humans. For example, Scarborav {Lancet, 610 (1938); Bio-chem.J. 33 ,
1400 (1939); Lancet, 644 (1940); Proc.Roy.
Soc.Med.35, 407 (1942)} Cameron et al. {Canad.
MAJ 46 , 548 (1942)} reported that when vitamin P was given to scurvy patients, the resistance of capillaries increased and bleeding stopped; in particular, Scarborav described experimental human vitamin P deficiency and They reported that the main symptoms were decreased capillary resistance and prolonged bleeding time. On the other hand, Majyovskii et al. {J.Pharmacol.Exptl.
Therp. 81 , 1 (1944)] used mice to compare the effectiveness of purified hesperidin with crude hesperidin extracted from lemons and oranges in terms of pulmonary vascular resistance.
It has been observed that purified hesperidin loses its effectiveness. As described above, there has been controversy over the evidence of vitamin P action and efficacy of hesperidin and other flavonoid glycosides through animal experiments. All other research reports have been based on the premise that the chemical substance of vitamin P is mainly flavonoid glycosides such as hesperidin and rutin. In 1950, the Nomenclature Committee of the American Society of Biochemists and the Committee on Nutrition decided to name vitamin P.
It was decided to abolish the name. {Science 112 , 628
(1950)} Now, the present inventors have shown that, as mentioned above, lemon juice and citrine (crude) have clinical effects, and that crude hesveridin increases the hyporesistance of capillaries in Mazziovskiy's experiment. Considering this together, I have come to the conclusion that lemon juice and citrine, which is a crude hesperidin fraction, may contain true vitamin P that exerts vitamin P effects in extremely small amounts in addition to flavonoid glycosides. It is. In order to confirm the existence of true vitamin P, which has a powerful vitamin P action (capillary resistance enhancement action), we conducted extensive research and finally discovered that the physical and chemical properties of crude citrine were completely different from those of flavonoid glycosides. They successfully isolated the vitamin P fraction and completed the present invention. Next, the progress of the fractional isolation will be described in detail. The present inventor first extracted and separated crude hesperidin (citrine) from lemons and oranges in accordance with the report by Mazziovskii. That is, fresh lemons and oranges are crushed with a mixer, extracted with hot ethanol, the extract is concentrated to obtain an ethanol extract, this is extracted with hot isopropanol, the isopropanol-soluble part is concentrated as much as possible, and petroleum ether is added. After cooling, the resulting precipitate was collected, dried, extracted with hot isopropanol, concentrated, cooled in a freezer overnight, and the precipitate deposited was collected and dried to obtain crude hesperidin (citrine). Isolation of true vitamin P according to the present invention from crude hesperidin can be carried out, for example, by the following method. That is, an adsorption method using an appropriate adsorbent (for example, silica gel, alumina, florisil, ion exchange resin, activated carbon, etc.) and an organic solvent that is immiscible with water (for example, lower alkyl halides, acetic esters, alcohols, ketones, etc.) Use extraction methods such as these alone or in appropriate combinations. Preferred embodiments of these will be described in more detail as follows. For example, if crude hesperidin is mixed with chloroform,
Dissolve in a solvent such as ethanol, add a small amount of silica gel, remove the solvent under reduced pressure, and adsorb onto the silica gel. Next, normal hexane, benzene, a mixed solvent of benzene-ether, ether, ether-
Elution is carried out with a mixed solvent such as ethanol, and the obtained vitamin P fraction and eluate are separately concentrated under reduced pressure to precipitate crystals, which are collected and recrystallized from an organic solvent such as ethanol or dioxane to release the vitamin. The P fraction and fractions are obtained as crystals. The vitamin P fraction and the vitamin P fraction have the following physical and chemical properties. [Vitamin P fraction] (1) Rf value of thin layer chromatography 0.28±0.05 Developing solvent Benzene-ethanol (5:1) Thin layer plate Merck, Silica gel 60 Sheet No.
5554 In the present invention, the Rf value of 0.28 refers to the above numerical value under the above measurement conditions. (2) Solubility Soluble in ether, chloroform, pyridine, dioxane, and benzene, and soluble in methanol, ethanol, and acetone. Poorly soluble in water. (3) Color reaction a. No coloration with alkali b. Reddish-purple with sulfuric acid C. No discoloration in ferric chloride reaction. D. Negative reaction with ninhydrin. H. No reduction with Fehling's reagent. F. Negative flavonoid coloration with hydrochloric acid and magnesium (4) Melting point 125 ~158℃ (5) Colorless scaly crystals (recrystallized from ethanol) (6) Elemental analysis values (actual values C: 75.30% H:
11.02% (7) Optical rotation [α] ²⁰ _D −24.8 (C=1 pyridine) (8) Infrared absorption spectrum ν ^KBr _nax cm ^-1 ; 3450,
2930, 2855, 1720, 1465, 1378, 1365, 1305,
1250, 1169, 1130, 1105, 1085, 1055, 1019,
925, 835, 795, (9) Ultraviolet absorption spectrum Shows only terminal absorption. [Note: Hesperidin is λ ^EtOH _nax mμ286, 330]
shows absorption. ] [Vitamin P fraction] (1) Rf value of thin layer chromatography 0.15±0.05 The developing solvent, thin layer plate, and measurement conditions are the same as for the fraction. (2) Solubility Soluble in pyridine, soluble in ethanol, dioxane, ether, chloroform, benzene, acetone, poorly soluble in water (3) Color reaction Same as fraction. (4) Melting point 275-282℃ (decomposed) (5) Colorless crystalline powder (recrystallized from ethanol) (6) Elemental analysis values (actual values, C: 72.63% H:
10.20%) (7) Optical rotation [α] ²⁰ _D −45.2 (C=1, pyridine) (8) Infrared absorption spectrum ν ^KBr _nax cm ^-1 ; 3390,
2925, 2855, 1465, 1455, 1420, 1375, 1365,
1340, 1193, 1156, 1100, 1070, 1050, 1019,
955, 885, 796, 635, 615, 595 (9) Ultraviolet absorption spectrum Shows only terminal absorption. Next, the vitamin P fraction and its biological activity will be described. Method: Capillary resistance enhancement effect 50±0.5mmHg/according to the method of Maziovskii et al.
The degree of pulmonary hemorrhage in mice under 15 sec. of vacuum was evaluated by a scoring method, and the 50% effective dose of the test drug was calculated from the inhibition rate relative to the untreated control group. result;

[Table] As shown in the table above, the vitamin P fraction and fraction showed surprising effects, 29 and 56 times more effective than hesperidin, respectively. The Rutaceae plants used in the present invention refer to plants that botanically belong to the Rutaceae family, and include vitamin P
Anything containing can be used.
To give an example only for the purpose of explanation, Rutaceae is a general term for the genus Citrus, genus Kumquat, genus Citrus, etc. Among them, plants of the genus Citrus, especially Citrus citrus, Yuzu, Citrus citrus, Citrus citrus, Citrus citrus, Citrus citrus, Citrus citrus, Citrus citrus, In addition to mandarin oranges, mandarin oranges, mandarin oranges, mandarin oranges, mandarin oranges, mandarin oranges, sabon, mandarin oranges, mandarin oranges, mandarin oranges, kumquats, lemons, navels, oranges, and grapefruits, those called lemons, navels, oranges, and grapefruits are advantageously used in the present invention. Next, examples of the present invention will be shown, but the present invention is not limited thereto. Example 1 (Production of crude hesperidin) 3.35Kg of fresh lemons were crushed in a mixer and extracted twice with hot ethanol 6. Concentrate the extract
359g of ethanol extract was obtained, extracted 3 times with hot isopropanol 2, concentrated the extract to 500ml, added petroleum ether 2,
Cool to ℃ overnight, remove the formed precipitate, and dry.
22.0 g of a tan powder was obtained. The mother liquor is subjected to the fractionation in Example 2. The powder obtained above was mixed with hot isopropanol.
The extract was extracted three times with
12.9g was obtained. Yield 0.385% (Isolation of vitamin P fraction and fractions) 12.9 g of the crude hesperidin obtained above was dissolved in ethanol, and 30 g of silica gel was added.
The solvent is distilled off under reduced pressure and then it is packed onto a column packed with 100 g of silica gel. This column was mixed with n-hexane, benzene, a mixed solvent of benzene and ether (1:1), ether,
Elute in the order of a mixed solvent of ether and ethanol (1:1). When the ether is distilled off from the fraction eluted with ether, an almost colorless crystalline powder is obtained. When this product was crystallized from ethanol by adding a small amount of activated carbon, 18 mg of vitamin P fraction was obtained as colorless scaly crystals. Yield: 0.00053% (based on fresh lemon) Melting point: 125-158℃ When the solvent is distilled off from the fraction eluted with a mixed solvent of ether and ethanol (1:1), a slightly yellow colored powder is obtained. . A small amount of activated carbon was added to this and recrystallization was repeated three times from dioxane to obtain 320 mg of vitamin P fraction as a colorless crystalline powder. Yield 0.00950% (based on fresh lemon) Melting point 275-282°C (decomposed) Example 2 The petroleum ether-containing mother liquor obtained at the end of the first paragraph of Example 1 is concentrated under reduced pressure to 100 ml. Silica gel 30 to this crude hesperidin
g and the solvent was distilled off under reduced pressure. this thing
Pack onto a column packed with 100 g of silica gel. When this column was subjected to the same elution treatment as in the second paragraph of Example 1, 103 mg (yield 0.00307%) of vitamin P fraction was obtained from the portion eluted with ether, and ether and ethanol (1:1 ) 49mg from the part eluted with mixed solvent
(yield 0.00146%) of vitamin P fraction was obtained.

Claims (1)

[Claims] 1 Crude hesperidin produced from Rutaceae plants by a conventional method is subjected to adsorption treatment using an adsorbent and extraction treatment using an organic solvent immiscible with water, either alone or as appropriate. A method for producing vitamin P, which comprises obtaining a vitamin P fraction and a vitamin P fraction by carrying out in combination. 2. The combination of adsorption treatment and extraction treatment is defined in claim 1, in which crude hesperidin is dissolved in a solvent as necessary, adsorbed on an adsorbent, and then fractionated with an organic solvent that is immiscible with water. The manufacturing method described in Section 1. 3. The manufacturing method according to claim 2, wherein the adsorbent is silica gel, and the organic solvent is ether or an ether-ethanol mixture.