阅读说明：本技术 采用高速逆流色谱制备钩藤中喜果苷的方法 (Method for preparing vincoside-lactam in uncaria by adopting high-speed countercurrent chromatography ) 是由 蒋海强 陈振山 田振华 王小明 李运伦 于 2020-07-03 设计创作，主要内容包括：本发明属于化学医药领域,其公开了一种采用高速逆流色谱制备钩藤中喜果苷的方法,其步骤如下：(1)取钩藤,提取得粗提物；(2)将粗提物经萃取得乙酸乙酯萃取物；将乙酸乙酯萃取物过硅胶柱,梯度洗脱,回收洗脱液,干燥,得粗品；将粗品通过高速逆流色谱,一次制备得到式1化合物。本发明的方法可以规模化、高效、快速提纯得到喜果苷。并且制备过程中的流动相和固定相可以反复利用,节约了制备成本。(The invention belongs to the field of chemical medicine, and discloses a method for preparing vincoside-lactam in uncaria by adopting high-speed countercurrent chromatography, which comprises the following steps: (1) taking uncaria, and extracting to obtain a crude extract; (2) extracting the crude extract to obtain an ethyl acetate extract; passing the ethyl acetate extract through a silica gel column, carrying out gradient elution, recovering the eluent, and drying to obtain a crude product; and (3) carrying out high-speed counter-current chromatography on the crude product to prepare the compound shown in the formula 1 in one step. The method can be used for purifying the vincoside lactam in a large scale, high efficiency and fast. And the mobile phase and the stationary phase in the preparation process can be repeatedly utilized, so that the preparation cost is saved.)

1. A method for preparing vincoside-lactam from ramulus Uncariae cum uncis by high-speed countercurrent chromatography comprises the following steps:

the method is characterized by comprising the following steps:

(1) taking uncaria, and extracting to obtain a crude extract;

(2) extracting the crude extract to obtain an ethyl acetate extract;

(3) passing the ethyl acetate extract through a silica gel column, carrying out gradient elution, recovering the eluent, and drying to obtain a crude product;

(4) and (3) carrying out high-speed counter-current chromatography on the crude product to prepare the compound shown in the formula 1 in one step.

2. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 1, wherein the extraction in the step (1) is ethanol cold-leaching extraction.

3. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 2, wherein the mass-volume ratio of uncaria rhynchophylla to ethanol is as follows: 1:10, the unit of mass-to-volume ratio is kg/L.

4. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 1, wherein the step (2) is specifically as follows: dissolving the crude extract in distilled water to obtain suspension, sequentially extracting with n-hexane, ethyl acetate and n-butanol, retaining ethyl acetate extract phase to obtain ethyl acetate extract, and concentrating the ethyl acetate extract under reduced pressure to obtain ethyl acetate extract.

5. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 4, wherein the mass-volume ratio of uncaria rhynchophylla to distilled water is as follows: 1:10, the unit of mass-to-volume ratio is kg/L.

6. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 1, wherein gradient elution in step (3) is divided into 10 components, which are respectively named as Fr.1-Fr.10; and taking Fr.5 to recover the eluent.

7. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography according to claim 6, wherein a mixed elution system of ethyl acetate and dichloromethane is adopted when the ethyl acetate extract passes through a silica gel column, and the volume ratio of the ethyl acetate to the dichloromethane is 100:1, 99:1, 98:2, 97:3, 95:5, 9:1, 4:1, 7:3, 3:2 and 1: 1; the elution system was changed every five column volumes.

8. The method for preparing vincoside-lactam in uncaria rhynchophylla according to claim 1, wherein in step (4), the solvent system adopted by the high-speed countercurrent chromatography is n-butanol: ethyl acetate: methanol: water (4:0.5:1:5, v/v/v/v).

9. The method for preparing vincoside-lactam in uncaria rhynchophylla by high-speed countercurrent chromatography as claimed in claim 8, wherein the high-speed countercurrent chromatography is performed at a rotation speed of 800r/min at a detection wavelength of 254nm at a flow rate of 10ml/min and a temperature of 30 ℃ with the upper phase being a stationary phase and the lower phase being a mobile phase.

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to a method for industrially and rapidly preparing a monomer compound vincoside-lactam from uncaria rhynchophylla.

Background

Uncaria rhynchophylla (Uncaria rhynchophylla), also known as Uncaria gambir, Uncaria oblonga, Araliaceae, Uncaria gambir, etc., is a evergreen vine plant of Uncaria genus. Uncaria rhynchophylla is classified into Uncaria macrophylla Wall, Uncaria tomentosa Wall, Uncaria gambir Wall or Uncaria cannatis Wall. The proper growing environment of uncaria is warm and moist, loose and fertile soil, and the uncaria is planted in Guangdong, Guizhou, Guangxi, Fujian, Jiangxi and the like. The medicinal part of uncaria is usually the branch with hook, which is sweet and bitter in taste and slightly cold in flavor, enters heart meridian and has the function of clearing heat and expelling pathogenic factors. More than 100 chemical components are reported to be separated from uncaria rhynchophylla at present, and comprise alkaloids, flavonoids, triterpenes, glycosides and the like, wherein the content of the alkaloids is particularly rich, such as uncarine, isocoubine, corynoxeine and the like. The pharmacological actions of uncaria are closely related to the chemical components of uncaria, such as diminishing inflammation, relieving pain, reducing blood pressure, resisting cancer, resisting epilepsy and the like.

The rapid separation and purification technology of traditional Chinese medicine components has important significance for understanding the complex material basis of traditional Chinese medicine, controlling important quality and discovering potential active substances. HSCCC can rapidly realize the separation of chemical components of traditional Chinese medicine on the premise of ensuring no sample loss, recyclable solvent system and large sample loading amount. And because the separated substance can be fully contacted with the liquid phase stationary phase, the separation preparation amount of the sample is greatly improved. Is an ideal means for industrially preparing the monomer substance of the traditional Chinese medicine. The characteristic enables the high-speed countercurrent chromatography to be widely applied to the separation research of samples such as traditional Chinese medicines, biological products, foods and the like.

Disclosure of Invention

Based on the prior art, the invention provides a method for preparing vincoside-lactam in uncaria by adopting high-speed countercurrent chromatography, which can be suitable for rapidly and industrially preparing vincoside-lactam compounds in uncaria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing vincoside-lactam from ramulus Uncariae cum uncis by high-speed countercurrent chromatography comprises the following steps:

the method comprises the following steps:

(1) taking uncaria, and extracting to obtain a crude extract;

(2) extracting the crude extract to obtain an ethyl acetate extract;

(4) passing the ethyl acetate extract through a silica gel column, carrying out gradient elution, recovering the eluent, and drying to obtain a crude product;

(4) and (3) carrying out high-speed counter-current chromatography on the crude product to prepare the compound shown in the formula 1 in one step.

Preferably, the extraction in the step (1) is ethanol cold leaching extraction.

Preferably, the mass volume ratio of the uncaria and the ethanol is as follows: 1:10, the unit of mass-to-volume ratio is kg/L.

Preferably, the step (2) is specifically: dissolving the crude extract in distilled water to obtain suspension, sequentially extracting with n-hexane, ethyl acetate and n-butanol, retaining ethyl acetate extract phase to obtain ethyl acetate extract, and concentrating the ethyl acetate extract under reduced pressure to obtain ethyl acetate extract.

Preferably, the mass-volume ratio of the uncaria and the distilled water is 1:10, and the unit of the mass-volume ratio is kg/L.

Preferably, the gradient elution in the step (3) is divided into 10 components which are respectively named as Fr.1-Fr.10; and taking Fr.5 to recover the eluent.

Preferably, when the ethyl acetate extract passes through a silica gel column, a mixed elution system of ethyl acetate and dichloromethane is adopted, and the volume ratio of ethyl acetate to dichloromethane is 100:1, 99:1, 98:2, 97:3, 95:5, 9:1, 4:1, 7:3, 3:2 and 1:1 in sequence; the elution system was changed every five column volumes.

Preferably, in the step (4), the solvent system adopted by the high-speed countercurrent chromatography is n-butanol: ethyl acetate: methanol: water (4:0.5:1:5, v/v/v/v).

Preferably, the upper phase of the high-speed countercurrent chromatography is a stationary phase, the lower phase of the high-speed countercurrent chromatography is a mobile phase, the rotating speed is 800r/min, the flow rate is 10ml/min, the temperature is 30 ℃, and the detection wavelength is 254 nm.

The invention has the beneficial effects that:

the invention takes dried uncaria stem with hooks of uncaria of rubiaceae as raw material, obtains crude product by simple silica gel column chromatographic separation, and obtains high-purity vincoside-lactam by high-speed countercurrent chromatography industrialization. The method can be used for large-scale, high-efficiency and rapid purification to obtain vincoside-lactam; and the mobile phase and the stationary phase in the preparation process can be repeatedly utilized, so that the preparation cost is saved.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 HSCCC chromatogram of vincoside-lactam;

FIG. 2 HSCCC chromatogram of vincoside-lactam (solvent system n-butanol: ethyl acetate: methanol: water (4:0.3:1:5, v/v/v/v));

FIG. 3 HSCCC chromatogram of vincoside-lactam (solvent system n-butanol: ethyl acetate: methanol: water (4:0.7:1:5, v/v/v/v));

FIG. 4 Compound 1 NMR¹H, spectrogram;

FIG. 5 Compound 1 NMR¹³And C, spectrum.

Detailed Description

The invention is further illustrated by the following examples. The following examples must be interpreted to illustrate the invention without limiting it. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.

Homopield TBE-300C high-speed countercurrent chromatography (Shanghai Homopield, Inc.); pujindo TU-1950 ultraviolet spectrophotometer (Beijing Pujindo); model METTLER AE240 electronic balance (mettler-toledo (shanghai) ltd), KQ-250B ultrasonic cleaner (kunshan ultrasonic instruments ltd). The other reagents were analytical grade, and the water was distilled water.