A combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine

Abstract

The present invention discloses a combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine. The method uses Sephadex LH-20, a high-performance liquid chromatograph, a high-resolution mass spectrometer, an enrichment column, and a nuclear magnetic resonance spectrometer for combined separation and purification. The method of the present invention uses a high-resolution mass spectrometer to track and lock the target component, and can achieve effective separation of high-abundance components and efficient enrichment and acquisition of target low-abundance components through a simple two-step separation.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a method for efficiently and quickly obtaining low-abundance components of traditional Chinese medicine. Background Art

[0002] The composition of traditional Chinese medicine is extremely complex. Although the easily accessible high-abundance components in traditional Chinese medicine have long been reported in large quantities, they can only partially reflect the efficacy of traditional Chinese medicine. In addition, there are a large number of unknown low-abundance active ingredients hidden in traditional Chinese medicine. It is generally believed that the low-abundance components of traditional Chinese medicine refer to components with a content of less than 0.1% in traditional Chinese medicine extracts. They not only have special structural characteristics and significant pharmacological activities, but are also closely related to the efficacy of traditional Chinese medicine. They have always been an important source of innovative traditional Chinese medicine drugs (such as the anti-tumor drugs vinblastine and vincristine, whose content in Catharanthus roseus is 0.0069% and 0.0013% respectively). However, the research progress on low-abundance active ingredients in traditional Chinese medicine is very slow. The reason lies in the following key scientific issues: due to their extremely low content, low-abundance components are difficult to effectively identify and separate using conventional technical methods.

[0003] Take Corydalis yanhusuo (WTWang), for example. It is the dried tuber of Corydalis yanhusuo (WTWang), a plant of the Papaveraceae family and genus Corydalis. Li Shizhen's Compendium of Materia Medica describes Corydalis yanhusuo as possessing four major benefits: promoting blood circulation, invigorating qi, relieving pain, and promoting bowel movements. The 2015 edition of the Chinese Pharmacopoeia states that Corydalis yanhusuo is pungent, bitter, and warm in nature, and is primarily used to treat chest, flank, abdominal, and wrist pain, chest pain, heart pain, postpartum stasis, and swelling and pain from falls. Tetrahydropalmatine, dehydrocorydaline, coptisine, scaphine, berberine, bamipine, and jatrorrhizine are the main high-abundance components in Corydalis yanhusuo. Numerous studies have shown that, in addition to these high-abundance components, Corydalis yanhusuo also contains numerous low-abundance components, many of which possess potent biological activities.

[0004] Authorized Chinese patent ZL201911278103 discloses an alkaloid dimer component, corydaline A, discovered from Corydalis yanhusuo, which has excellent activity in blocking PD-1 / PD-L1 interactions and can be used to develop PD-1 / PD-L1 interaction inhibitors. However, compared with the high-abundance components in Corydalis yanhusuo, the content of corydaline A in the medicinal material is extremely low, and there is serious interference from high-abundance components and matrices with similar polarity. The disclosed separation methods are extremely complicated, and the amount obtained is also extremely small, which is the main reason that currently limits in-depth research on this component. Most conventional separation methods require the use of multi-step separations such as macroporous resins, silica gel column chromatography, open ODS column chromatography, Sephadex LH-20, and high-performance liquid chromatography in sequence. Not only are the steps cumbersome, but they are also very likely to cause component loss, and the compound is difficult to obtain. How to obtain this compound quickly and efficiently is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine.

[0006] The technical solution of the present invention is summarized as follows:

[0007] A combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine comprises the following steps:

[0008] 1) dissolving the Chinese herbal medicine extract in a solvent, centrifuging, collecting the supernatant, separating the supernatant on a Sephadex LH-20 chromatographic column 1, and then obtaining a fraction containing the target component using the quasi-molecular ion peak of the target component detected by a first high-resolution mass spectrometer 2 as a guide;

[0009] 2) The mobile phase is controlled by the binary pump 4 of the preparative high performance liquid chromatograph 3 and passed into the chromatographic column 6 of the preparative high performance liquid chromatograph. After the mobile phase is equilibrated, the component containing the target component is injected into the chromatographic column of the preparative high performance liquid chromatograph for separation;

[0010] 3) The effluent from the chromatographic column is split into a 95% volume portion and a 5% volume portion through a first three-way valve 14, the 5% volume portion is passed to a second high-resolution mass spectrometer 7 for online detection, and the 95% volume portion is passed to a UV detector 5 of a preparative high-performance liquid chromatograph for detection; the liquid flowing out of the UV detector is controlled to pass into a first waste liquid tank 8 through a second three-way valve 15; guided by the detection of the target component by the UV detector and the quasi-molecular ion peak of the target component by the second high-resolution mass spectrometer, when a target component signal appears, the liquid flowing out of the UV detector is passed through the second three-way valve and then, together with the water pumped into the binary infusion pump 9, is passed into an enrichment column 10 through a third three-way valve 16, so that the target component is enriched on the enrichment column; the effluent from the enrichment column is passed into a second waste liquid tank 11 through a fourth three-way valve 17;

[0011] The flow rate of the water pumped by the binary infusion pump is the same as the flow rate of the 95% volume portion passing into the UV detector of the preparative high performance liquid chromatograph;

[0012] 4) Repeat steps 2) and 3) 10 to 20 times;

[0013] 5) Turn off the preparative HPLC instrument, and control the third three-way valve with a binary infusion pump to pump methanol or acetonitrile through the third three-way valve into the enrichment column at a flow rate of 1 to 4 mL / min to elute the target component. Control the fourth three-way valve to allow the eluate to flow into the collection bottle 12;

[0014] 6) The liquid in the collection bottle is dried to obtain the target component, and a portion of the target component is detected using a nuclear magnetic resonance spectrometer 13 to obtain nuclear magnetic resonance spectrum information to confirm the target component.

[0015] The Chinese herbal medicine extract is preferably Corydalis yanhusuo extract, Coptis chinensis extract, Paeonia lactiflora extract, Litsea cubeba extract, Siegesbeckia ulmoides extract or Periploca odorata extract. Other Chinese herbal medicine extracts may also be used.

[0016] In step 2), the solvent is preferably water, methanol, ethanol, methanol aqueous solution, ethanol aqueous solution, dichloromethane, or a mixed solution of dichloromethane and methanol.

[0017] Preferably, the enrichment column is filled with ODS C 18 Filled chromatographic column.

[0018] Advantages of the present invention:

[0019] The present invention provides a combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine. By tracking and locking the target components with a high-resolution mass spectrometer, the effective removal of high-abundance components and the efficient enrichment and acquisition of target low-abundance components can be achieved through a simple two-step separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of a combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine. DETAILED DESCRIPTION

[0021] The extraction method of the Chinese herbal extract used in the present invention, taking Corydalis yanhusuo extract as an example, is as follows: soak the Chinese herbal extract Corydalis yanhusuo in 10 times the amount of distilled water for 30 minutes, heat and reflux to extract for 1 hour, filter the filtrate, repeat the extraction three times, combine the filtrates, and evaporate to dryness under reduced pressure to obtain the Corydalis yanhusuo extract. Other extracts such as Coptis chinensis extract, Paeonia lactiflora extract, Litsea cubeba extract, Siegesbeckia scoparia extract, or Periploca odorifera extract, as well as other Chinese herbal extracts, can also be obtained by the above extraction method.

[0022] Traditional Chinese medicines such as Corydalis yanhusuo, Coptis chinensis, Paeonia lactiflora, Litsea cubeba, Siegesbeckia scoparia or Perilla frutescens bark meet the standards of the 2020 edition of the Pharmacopoeia.

[0023] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0024] Example 1

[0025] A combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine (see Figure 1 ), including the following steps:

[0026] 1) 30 g of a traditional Chinese medicine extract (taking Corydalis extract as an example) was dissolved in 80 mL of methanol, centrifuged at 5000 rpm for 10 min, and the supernatant was separated on a Sephadex LH-20 column 1 (φ80 mm×1200 mm), eluted with methanol, and 30 mL of each fraction was collected as a fraction. Each fraction was detected by a first high-resolution mass spectrometer 2, and the quasi-molecular ion peaks of the target component (single-charged molecular ion peak with m / z of 671.2388 and doubly-charged molecular ion peak with m / z of 336.1236) detected by the first high-resolution mass spectrometer 2 were used as a guide to obtain a fraction containing the target component (corydalis A);

[0027] 2) The mobile phase (40% by volume acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid)) was controlled by binary pump 4 of preparative HPLC 3 (Waters 2545) (mobile phase flow rate of 6 mL / min) and passed into chromatographic column 6 of the preparative HPLC (chromatographic column: C18 MGII (5 μm, 20 × 250 mm)). After the mobile phase was equilibrated, the fraction containing the target component was injected into the chromatographic column of the preparative HPLC for separation;

[0028] 3) The effluent from the chromatographic column is split into a 95% volume portion and a 5% volume portion through a first three-way valve 14, the 5% volume portion is passed to a second high-resolution mass spectrometer 7 for online detection, and the 95% volume portion is passed to a UV detector 5 of a preparative high-performance liquid chromatograph for detection; the liquid flowing out of the UV detector is controlled to pass into a first waste liquid tank 8 through a second three-way valve 15; guided by the detection of the target component by the UV detector and the quasi-molecular ion peak of the target component (a single-charged molecular ion peak with m / z of 671.2388 and a doubly-charged molecular ion peak with m / z of 336.1236) by the second three-way valve, when the target component signal appears, the liquid flowing out of the UV detector is passed through a second three-way valve and then, together with the water pumped in by the binary infusion pump 9, is passed through a third three-way valve 16 into an enrichment column 10 (the enrichment column 10 is filled with 10 g of ODS C 18 The effluent from the enrichment column is passed through a fourth three-way valve 17 into a second waste liquid tank 11;

[0029] The flow rate of the water pumped by the binary infusion pump is the same as the flow rate of the 95% volume portion passing into the UV detector of the preparative high performance liquid chromatograph;

[0030] 4) Repeat steps 2) and 3) 15 times; (You can also use any one of 10 to 20 times, such as 10, 11, 12, 13, 14, 16, 17, 18, 19, 20 times)

[0031] 5) The preparative HPLC instrument is turned off. By controlling the third three-way valve, the binary infusion pump 9 pumps methanol (or acetonitrile) through the third three-way valve into the enrichment column at a flow rate of 2 mL / min (or any one of 1-4 mL / min, such as 1, 3, or 4 mL / min) to elute the target component. The fourth three-way valve is then controlled to allow the eluate to flow into the collection bottle 12.

[0032] 6) The liquid in the collection bottle was dried to obtain the target component (15.1 mg) with a yield of 0.05%. A portion of the target component was detected by nuclear magnetic resonance spectrometer 13 to obtain nuclear magnetic resonance spectrum information, confirming that the target component was corydaline A.

[0033] Corydaline A is compound 2 in the authorized Chinese patent ZL201911278103 disclosed in "A class of alkaloid dimer compounds and their application in the preparation of PD-1 / PD-L1 pathway inhibitors."

[0034] By adopting the method of Example 1, low-abundance components in traditional Chinese medicines such as Corydalis yanhusuo, Coptidis rhizome, Paeonia lactiflora, Litsea cubeba, Siegesbeckia scoparia, or Periploca odorata can be efficiently prepared.

[0035] Experiments have shown that using water, ethanol, methanol aqueous solution, ethanol aqueous solution, dichloromethane, and a mixed solution of dichloromethane and methanol instead of the methanol of this embodiment, the low-abundance components of traditional Chinese medicine can be separated and purified with the same other methods as this embodiment.

[0036] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, for those skilled in the art, various improvements and modifications may be made to the present invention without departing from the principles of the present invention, and such improvements and modifications are also protected by the claims of the present invention.

Claims

1. A combined separation and purification method for efficiently obtaining low-abundance components of traditional Chinese medicine, characterized by The steps include: 1) dissolving the Chinese herbal medicine extract with a solvent, centrifuging, collecting the supernatant, separating the supernatant through a Sephadex LH-20 chromatographic column (1), and eluting with methanol; then, using the quasi-molecular ion peak of the target component detected by a first high-resolution mass spectrometer (2) as a guide, obtaining a fraction containing the target component; the quasi-molecular ion peak includes a single-charged molecular ion peak with an m / z of 671.2388 and a double-charged molecular ion peak with an m / z of 336.1236; 2) The mobile phase is controlled by a binary pump (4) of a preparative high performance liquid chromatograph (3) and introduced into a chromatographic column (6) of the preparative high performance liquid chromatograph. After the mobile phase is equilibrated, the component containing the target component is injected into the chromatographic column of the preparative high performance liquid chromatograph for separation. The mobile phase is an acetonitrile aqueous solution with a volume concentration of 40%, and the acetonitrile aqueous solution contains 0.1% trifluoroacetic acid. The chromatographic column is C18 MGII. 3) The effluent from the chromatographic column is split into a 95% volume portion and a 5% volume portion through a first three-way valve (14), the 5% volume portion is passed into a second high-resolution mass spectrometer (7) for online detection, and the 95% volume portion is passed into a UV detector (5) of a preparative high-performance liquid chromatograph for detection; the liquid flowing out of the UV detector is controlled to flow into a first waste liquid tank (8) through a second three-way valve (15); guided by the detection of the target component by the UV detector and the quasi-molecular ion peak of the target component by the second high-resolution mass spectrometer, when a target component signal appears, the liquid flowing out of the UV detector is passed through the second three-way valve, and then, together with the water pumped in by the binary infusion pump (9), is passed into an enrichment column (10) through a third three-way valve (16) to enrich the target component on the enrichment column; the effluent from the enrichment column is passed into a second waste liquid tank (11) through a fourth three-way valve (17); the enrichment column is filled with ODS C 18 Chromatographic columns filled with packing materials; The flow rate of the water pumped by the binary infusion pump is the same as the flow rate of the 95% volume portion passing into the UV detector of the preparative high performance liquid chromatograph; 4) Repeat steps 2) and 3) 10 to 20 times; 5) The preparative high performance liquid chromatograph is closed, and the binary infusion pump is used to pump methanol or acetonitrile into the enrichment column through the third three-way valve at a flow rate of 1 to 4 mL / min by controlling the third three-way valve to elute the target component, and the fourth three-way valve is controlled to allow the eluate to flow into the collection bottle (12); 6) collecting the liquid in the bottle, drying it to obtain the target component, taking part of the target component and detecting it with a nuclear magnetic resonance spectrometer (13), obtaining nuclear magnetic resonance spectrum information, and confirming the target component; the Chinese medicine extract is a Corydalis yanhusuo extract; the target component is:

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