JPH0684394B2 - Method for producing flavone glycoside - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing flavone glycosides as a pharmaceutical agent for improving blood flow disorders.

Conventional technology Ichiyo has been used as a folk medicine for a long time since it has been expected to have expectorant, antitussive, detoxify, cure, urinary control, and nourishing properties, and is also used for the treatment of cancer, pulmonary tuberculosis, octopus, and euphoria. ing. On the other hand, the extract extracted from the yew leaf has effects such as vasodilation, increase in blood flow, prevention of aging of vascular system, especially improvement of peripheral blood flow in brain and skin, improvement of mental symptoms, cerebral arteriosclerosis, It is also used in modern therapy for the treatment of senile blur, diabetic angiopathy, blood circulation disorder, mental disorder, etc.

Currently, the main components of the yew extract, which is used as a medicine in Europe, is quercetin glycoside,
JP-B-49-27323 discloses that they are isoquercetin glycoside, kaempferol-3-rhamnoside-glycoside, luteolin glycoside and sitosterol glycoside. More specifically, for example, regarding kaempferol glycoside, the molecular formula of kaempferol-3-rhamnoglucoside represented by the molecular formula C ₂₇ H ₃₀ O ₁₅ and the heptaacetyl derivative are
It is known that the kaempferol-glucoside represented by C ₃₅ H ₃₄ O ₁₈ is Arzneimitel
tiel rorschung], 18, 537-53 (1968). In recent years, 5,7,3 ', 4'-tetrahydroxy-flavono- 3-0 -α-rhamnopyranosyl-4- O- β-D- (6-trans- p -coumaroyl)-has been newly added to the above extract. Glucopyranoside has been isolated. [Arzteitschrift fur Naturheilverfahren], 2
2, 593-604 (1981)].

Object of the Invention However, not only the pharmacological activity of each flavone glycoside, which is the main component in the above extract, but also the composition and the content thereof are used without being clarified.

The present inventors clarified the active ingredient among the flavone glycosides contained in the leaves of the yew tree, and in the course of investigation for the purpose of utilizing it, in recent years, the flavone glycoside represented by the formula [I] was effective. It was successfully isolated as a component.

(Hereinafter, when R of formula [I] is H, it is described as flavone glycoside [A], and when R is OH, it is described as flavone glycoside [B].) However, the yield is as low as 3% or less and it is expensive. Reversed phase silica gel was used as a filler and required a large amount of solvent.
With normal phase silica gel, the loss of the desired product was even greater. Moreover, good separation was not obtained with polystyrene or polyamide. As a result of repeated studies to solve these problems, the present inventor has developed a method for efficiently separating and purifying flavone glycosides [A] and [B] by using countercurrent distribution chromatography, and the present invention completed.

Structure of the Invention That is, the present invention relates to a method for producing a flavone glycoside represented by the formula [I], which is characterized in that the extract of the leaf of Yew is purified and separated by countercurrent distribution chromatography.

The leaves of the Yew tree used in the production of the present invention can be collected from any of the male tree and female tree as long as it is the Yew tree (Ginkgo bilaba L.), and the tree varieties (for example, the middle (neutral species), Fujikuro (late varieties), Kinbei (early varieties)] and growing regions cannot be limited. The Yew leaves used for extraction may be either green leaves or dry leaves as long as they are green leaves. However, in the case of fresh leaves, it is preferable to immediately dry or start the extraction process after collection in order to minimize changes in the components.

In order to obtain an extract of Yew leaves,
A hydrophilic organic solvent such as water, methanol, ethanol, propanol, butanol, isopropanol, acetone, and methyl ethyl ketone, a mixed solvent thereof, an acetic acid ester, or the like is used, and heat extraction is performed at room temperature or as necessary.

The target flavone glycoside is extracted by this operation,
The obtained extract is a low-polar compound such as chlorophyll, carotenoid, aliphatic hydrocarbon, aliphatic aldehyde or alcohol, polyprenoid, low-polar phenol, biflavone, etc. in addition to the target compound, sugar, protein nucleic acid, condensed tannin, etc. Since it contains a large amount of high-polarity compounds, hexane, pentane, heptane, isooctane, petroleum ether, carbon tetrachloride, after removing the low-polarity compound with a fat-soluble solvent such as chloroform, acetate ester,
It is preferable to extract and separate the medium polar fraction containing the flavone glycoside from the above highly polar compound with methyl ethyl ketone, propanol, butanol or the like. The thus-obtained Yew leaf extract contains the flavone glycosides [A] and [B] of the formula [I] in a proportion of about 10%.

Countercurrent distribution chromatography used in the present invention is 2
A separation method based on the principle of continuous multi-stage extraction of liquid layers,
Specifically, droplet countercurrent distribution chromatography (DCCC),
Examples include rotary multi-stage countercurrent distribution chromatography (RLCCC), spiral countercurrent distribution chromatography (HCCC), and centrifugal liquid-partitioning chromatography (CPC). The Journal of Biochemical and Biophysical
Method (Journal of Biochemical and Biophysical M
ethods, 5 , 105 (1981)].

In the production of the flavone glycosides [A] and [B] of the present invention, countercurrent distribution chromatography is superior to the column method using a packing material in the following points.

1) There is no denaturation of the target substance by the adsorbent and no loss due to adsorption. When a compound having a relatively high polarity such as the flavone glycoside of the present invention is fractionated, the recovery rate is 60% or less by using normal phase silica gel column chromatography.

2) It is not necessary to use an expensive packing material, and the amount of solvent used can be small compared to the column method.

3) It is not necessary to consider the maintenance of the column such as deterioration of the packing material, and it is possible to separate a compound having a considerable polarity range such as a crude plant extract without pretreatment.

In particular, CPC is excellent in that it can treat a large amount of Abies sachalinensis extract in a short time, and the desired flavone glycosides [A] and [B] can be purified in large quantities. The selection of the solvent system to be used and the determination of the optimum ratio in the case of 3 or more components are performed by thin layer chromatography (TLC) on silica gel.

That is, when a silica gel thin-layer chromatograph spotted with the yew extract is used as the developing solvent of the solvent having the lower polarity of the two layers formed, the flavone glycosides [A] and [B] of the present invention It is preferable to use a solvent system in which the spot falls within the range of Rf 0.3-0.7. Examples of the solvent system that can be used in the present invention include ethyl acetate-water, butyl acetate-water, chloroform-methanol-water, ethyl acetate-butanol-water and the like.

EFFECTS OF THE INVENTION According to the present invention, a large amount of flavone glycosides [A] and [B] of the formula [I] can be separated and purified from an extract of Abies sachalinensis.

Examples Examples of the present invention will be shown below.

Example 1 Methanol exudates of 2 kg of fresh yew leaves were suspended in water and extracted with hexane, chloroform and ethyl acetate in this order.
6 g of the ethyl acetate extract are treated with a droplet countercurrent partition chromatograph (DCC-300, Tokyo Rika Kikai) using a solvent system consisting of chloroform / methanol / water (7: 13: 8). Fractionated by elution method was monitored by UV absorption at 315 mm, and 110 mg flavone glycoside [A] and 148 mg
Flavonoid glycoside [B] was obtained. The elution curve is shown in FIG.

Example 2 60 kg of dried yew leaves were heated and refluxed in 345 l of methanol for 3 hours, and the leaves were separated to obtain a methanol solution having a volume of 39 l.
Concentrate until 24 l of water was added to this, and extracted with chloroform 21, 8 l and 2 l three times in total to remove fat-soluble compounds.
The methanol in the aqueous layer is distilled off, and the mixture is extracted three times with 36 l, 24 l and 12 l of methyl ethyl ketone in total. The resulting methyl ethyl ketone solution is concentrated to dryness to obtain 1.7 kg of a crude flavone fraction. Next, 450 g of the crude flavone fraction was continuously extracted with a centrifugal liquid-liquid partition chromatograph (CPC system, Miki Engineering) using butyl acetate as the mobile phase and water as the stationary phase, and the fraction obtained by reverse elution was converted to butanol. The mobile phase and CPC with water as the stationary phase are similarly treated to obtain a normal elution part. Finally, add this to chloroform / methanol / water (7.
9: 7.9: 4.2) and re-treated with CPC using the solvent system, and the reversed elution fraction was fractionated in the same manner as in Example 1 to obtain 9.8 g of flavone glycoside [A] and 24.5 g of flavone glycoside. Body [B] was obtained.

Example 3 5 g of a crude flavone fraction obtained in the same manner as in Example 2 was treated with CPC using a solvent system consisting of chloroform / methanol / water (7.9: 7.9: 4.2), and the reversed elution part was treated as in Example 1. Fractionation was performed in the same manner to obtain 65 mg of flavone glycoside [A] and 92 mg of flavone glycoside [B].

[Brief description of drawings]

FIG. 1 is an outflow curve diagram in Example 1, in which the horizontal axis represents the outflow volume and the vertical axis represents the absorbance (315 nm).

Claims (1)

[Claims] 1. A method for producing a flavone glycoside represented by the formula [I], which comprises purifying and separating an extract of Abies sachalinensis by countercurrent partition chromatography.