Method for enriching and purifying steroidal alkaloid in fritillaria medicinal material

A technology of steroidal alkaloids and alkaloids, which is applied in the field of enrichment and purification of steroidal alkaloids, can solve the problems of large solvent consumption, obstruction, low recovery rate, etc., achieve fast enrichment methods, overcome low solubility, high Variety effect

Pending Publication Date: 2022-07-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, acid extraction, alkali precipitation and silica gel column chromatography often have low recovery rates, require repeated operations, and consume a lot of solvents; liquid-liquid extraction may cause irreversible structural changes to pH-sensitive components during the pH adjustment process
Ion-exchange resins and macroporous resins are commonly used in resin adsorption methods. Liu et al. used cation-exchange resin columns to optimize the purification process of total alkaloids of Fritillaria chinensis [Liu Yuantao et al. Liaoning Journal of Traditional Chinese Medicine, 2016,43(03):583-586.], Ion exchange resin can selectively adsorb basic compounds, but the dead adsorption is serious, and the eluent with high ionic strength is introduced in the elution process, which makes post-processing difficult and the recovery rate of trace alkaloids is low
Wang et al. used macroporous resins to enrich alkaloids in Fritillaria based on reversed phase mode [Wang DD et al.Biomed ResInt.2014.], but besides steroidal alkaloids, Fritillaria also contains Steroidal saponin components, conventional reversed-phase mode is difficult to achieve high-purity enrichment effect
In addition, steroidal alkaloids are highly hydrophobic and difficult to dissolve in high water phase, which hinders the enrichment of steroidal alkaloids in the traditional reversed phase mode.

Method used

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  • Method for enriching and purifying steroidal alkaloid in fritillaria medicinal material
  • Method for enriching and purifying steroidal alkaloid in fritillaria medicinal material
  • Method for enriching and purifying steroidal alkaloid in fritillaria medicinal material

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preparation example Construction

[0032] Preparation of stationary phase FC8HL: add 10.3g of hydrated silica gel (silica gel water absorption rate 3%) to a 250mL flask, add 100mL of xylene, stir evenly, and then dropwise add 5.5g of 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane (moles: 11 mmol) and 4.1 g of 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane (moles: 12 mmol) mixture, react at 30°C for 3 hours, filter, use dichloromethane and methanol successively , washed with methanol water, methanol, and tetrahydrofuran in a volume ratio of 1:1, and the obtained solid was dried in a drying oven at 80°C for 24 hours to obtain a stationary phase FC8HL, with the following structure:

[0033]

[0034] The content of fluorine-containing alkyl chains per g of silica gel is 1.1 mmol, and the polar group is 1.2 mmol; the stationary phase prepared above is used as a filler to be packed into an FC8HL chromatographic column and applied in the following examples;

[0035] The isosteroidal alkaloids described in the present...

Embodiment approach

[0039] 1. Reference substance preparation: respectively take the isosteroidal alkaloid reference substances fritillin A, fritillin B, fritillin and sambarine, and the steroidal saponin reference substances timosaponin BII, timosaponin AIII and Dioscorea saponin is appropriate amount, accurately weighed, and methanol is added respectively to make a stock solution containing 1000 μg of reference substance per 1 mL; each stock solution is diluted with methanol into a mixed standard solution with a concentration of 10 μg / mL of each component, and passed through a 0.22 μm organic membrane to remove impurities post analysis.

[0040] 2. Analysis conditions:

[0041] Instrument: Agilent 1290 UHPLC-QToF6540

[0042] Chromatographic column: FC8HL (Acchrom-Tech, 4.6×100mm, packing particle size 5μm)

[0043] Flow rate: 1.5mL / min (split)

[0044] Column temperature: 40℃

[0045] Injection volume: 4μL

[0046] Wavelength collection range: 190nm-400nm

[0047] Mass spectrometry param...

Embodiment 2

[0055] Method of implementation

[0056] 1. Sample preparation: The fritillary medicinal material was extracted with 15 times (medicine material g / mL solvent) volume concentration of 90% ethanol for 120 minutes, solid-liquid separation, and the extract was concentrated at 50 °C to 1 / 10 of the volume of the extract. , lyophilized total extract.

[0057] 2. Preparation of the test solution: take an appropriate amount of the total extract of Fritillaria fritillary, add an appropriate amount of methanol, and ultrasonically dissolve it to prepare a solution containing 200 mg of total extract per 1 mL.

[0058] 3. Solid-phase extraction process: Take 20g FC8HL solid-phase extraction (SPE) column, activate and equilibrate with 3 times the column volume of methanol, and load the total extract with 1% of the sample (g / g); use 5 times the column volume The SPE column was rinsed with volume methanol to elute the non-alkaloid component (F1); the SPE column was rinsed with a 20 mM ammoniu...

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Abstract

The invention relates to a method for enriching and purifying steroid alkaloid in fritillaria medicinal materials, and belongs to the technical field of purification of traditional Chinese medicine extracts. According to the method, enrichment and purification of the steroid alkaloids are realized based on the perfluorooctyl stationary phase for the first time, and alkaloid active components in fritillaria medicinal materials can be effectively refined. The method provided by the invention can simply, quickly and efficiently enrich steroid alkaloid active components in the fritillaria medicinal material, provides high-quality and high-purity steroid alkaloid, and has important application significance for further pharmacological research and material fundamental research.

Description

technical field [0001] The invention relates to a method for enriching and purifying steroid alkaloids in medicinal materials of the genus Fritillaria, and belongs to the technical field of purification of traditional Chinese medicine extracts. Background technique [0002] Steroid alkaloids are nitrogen-containing derivatives of natural steroids, and can be divided into three categories: pregnane alkaloids, cyclopregnane alkaloids and cholestane alkaloids according to the steroid skeleton. Cholestane alkaloids can be further divided into cholestanes and isocholestanes, mainly distributed in Solanaceae Solanaceae and Liliaceae Fritillaria and Veratrum. Its parent nucleus is composed of 1,2 benzene fluorene and a nitrogen-containing heterocyclic ring. This kind of alkaloid is an important active ingredient, which has the functions of anti-tumor, immune regulation, anti-virus, antihypertensive, antitussive and so on. Broad pharmacological activity. The steroidal alkaloids co...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/08G01N30/06G01N30/14G01N30/34G01N30/72G01N30/88
CPCG01N30/02G01N30/08G01N30/06G01N30/14G01N30/34G01N30/72G01N30/88G01N2030/062G01N2030/065G01N2030/884
Inventor 梁鑫淼司玮刘艳芳沈爱金俞冬萍郭志谋
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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