A method for separating saposin K from Bupleurum chinense

Abstract

The present application relates to the field of medicine, and specifically discloses a method for separating nepo saponin K from Bupleurum chinense, comprising: extracting Bupleurum chinense with alcohol to obtain extract A; subjecting extract A to silica gel column chromatography, eluting with an eluent composed of solvent I, solvent II and water in a weight ratio of (3-9): 1: 0.1 to obtain target powder B, wherein solvent I is chloroform or ethyl acetate, and solvent II is methanol or ethanol; subjecting target powder B to purification by purification step I or purification step II to obtain nepo saponin K; purification step I: adding target powder B to methanol or ethanol for ultrasonic treatment; purification step II: subjecting target powder B to a first C18 column chromatography, eluting with methanol, and concentrating the eluent to a second C18 column chromatography, eluting with solvent III, wherein solvent III is acetonitrile or methanol. The method of the present application successfully separated nepo saponin K from Bupleurum chinense with a purity of up to 99%.

Description

Technical Field

[0001] The present application relates to the field of medicine, and more specifically, to a method for separating niposide K from Bupleurum chinense. Background Art

[0002] Radix Bupleuri is a herbal medicine included in the 2020 edition of the Chinese Pharmacopoeia. It is the dried root of Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd. of the Umbelliferae family. Radix Bupleuri tastes pungent and bitter, and is slightly cold in nature. It enters the liver, gallbladder, and lung meridians, and has the functions of reconciling the exterior and interior, soothing the liver and relieving depression, raising yang and lifting depression, reducing fever and stopping malaria, calming and relieving pain. It is clinically used for colds and fever, alternating colds and heat, chest and flank pain, irregular menstruation, uterine prolapse, and rectal prolapse. The chemical components reported in Radix Bupleuri mainly include saikosaponins, volatile oils, etc.

[0003] Since there are many varieties of Bupleurum in my country, with a long history of use and a wide range of uses, and the standards vary greatly from place to place, the production, circulation, and use of Bupleurum medicinal materials and pieces on the market are relatively chaotic, and there are many adulterations. In recent years, there have been illegal acts of using Tibetan Bupleurum to impersonate Bupleurum to produce pieces or use it as raw material for Chinese patent medicines. Tibetan Bupleurum is not a pharmacopoeial variety, but because of its high content of some Bupleurosaponin components, it is widely "favored" by Chinese medicine preparation production.

[0004] Tibetan Bupleurum is derived from the dried roots of Bupleurum marginatumvar.stenophyllum, a plant of the Umbelliferae family. It was introduced from Tibet and has been planted in large quantities due to its high yield and high content of saikosaponins. The Guizhou Provincial Quality Standard for Traditional Chinese Medicines and Ethnic Medicinal Materials includes Bupleurum marginatumvar.stenophyllum, which has similar properties and flavors to Bupleurum. Nepasaikosaponin K is a characteristic component of Tibetan Bupleurum, which is not found in Bupleurum. The Pharmacopoeia Commission of the People's Republic of China has issued a supplementary inspection method (draft) for the inspection of Tibetan Bupleurum components in Bupleurum granules. The draft mainly determines whether there is adulteration of Tibetan Bupleurum by detecting whether Bupleurum granules contain Nepasaikosaponin K. The China Food and Drug Inspection Institute has also included Nepasaikosaponin K as a reference substance. Therefore, the preparation of high-purity Nepasaikosaponin K is crucial for the smooth implementation of the above work. However, there is currently no patent on the preparation and separation of Nepasaikosaponin K. Summary of the invention

[0005] In order to successfully separate niposide K with higher purity and to facilitate the determination of whether there is adulteration of medicines and foods with Bupleurum tataricum, the present application provides a method for separating niposide K from Bupleurum tataricum.

[0006] A method for separating saposin K from Bupleurum chinense comprises the following steps:

[0007] (1) extracting dried Bupleurum chinense with alcohol, and concentrating the extract to obtain extract A;

[0008] (2) subjecting the extract A to silica gel column chromatography, using an eluent consisting of solvent I, solvent II and water in a weight ratio of (3-9):1:0.1 for elution, collecting the target portion, and concentrating under reduced pressure to obtain the target powder B;

[0009] The solvent I is chloroform or ethyl acetate, and the solvent II is methanol or ethanol;

[0010] (3) Purifying the target powder B by using purification step I or purification step II to obtain saikosaponin K;

[0011] Purification step I: add the target powder B to methanol or ethanol for ultrasonic treatment, filter, precipitate and dry;

[0012] Purification step II: subjecting the target powder B to a first C18 column chromatography, using methanol for elution, collecting the eluate, concentrating the eluate, subjecting the target powder B to a second C18 column chromatography, using solvent III for elution, collecting the target portion, and concentrating under reduced pressure;

[0013] The solvent III is acetonitrile or methanol.

[0014] By adopting the above technical scheme and the extraction steps of the present application, Nepalese saikosaponin K was successfully extracted and separated from Tibetan Bupleurum. HPLC purity test showed that its HPLC purity could reach more than 99%, which can meet the requirements of Chinese medicine chemical reference material. The obtained Nepalese saikosaponin K can be used as a Chinese medicine chemical reference material, thereby meeting drug testing and research needs.

[0015] Preferably, in step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 3:1:0.1.

[0016] Preferably, in step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 9:1:0.1.

[0017] Preferably, in step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 5:1:0.1.

[0018] By adopting the above technical solution and further optimizing the composition of the eluent, the purity of the finally separated Nepalese saikosaponin K can be guaranteed.

[0019] Preferably, in step (3), the target powder B is purified by purification step II.

[0020] By adopting the above technical scheme and using purification step II to purify the target powder B, the HPLC purity of the finally obtained Nepalese saikosaponin K can be further improved.

[0021] Preferably, during the second C18 column chromatography, 20% v / v to 45% v / v acetonitrile gradient elution is used.

[0022] Preferably, during the second C18 column chromatography, 40% v / v acetonitrile is used for isocratic elution.

[0023] Preferably, during the second C18 column chromatography, a gradient elution of 35% v / v to 75% v / v methanol is used.

[0024] Preferably, during the second C18 column chromatography, 60% v / v methanol is used for isocratic elution.

[0025] In summary, this application has the following beneficial effects:

[0026] 1. The method of the present application successfully extracted and separated Nepalese saposin K from Tibetan Bupleurum with a purity of more than 99%, which can meet the requirements of Chinese medicine chemical reference material raw materials and can be used as a reference material to meet drug testing and research needs.

[0027] 2. The method of the present application has a simple process, convenient preparation and separation operations, low equipment cost, easy production, large single batch processing capacity, stable and reliable process, good reproducibility, and can be mass-produced industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the HPLC spectrum of niposide saponin K obtained in Example 1 of the present application;

[0029] Figure 2 This is the HPLC spectrum of niposide saponin K obtained in Example 2 of the present application;

[0030] Figure 3 This is the HPLC spectrum of niposide saponin K obtained in Example 3 of the present application;

[0031] Figure 4 This is the HPLC spectrum of niposide saponin K obtained in Example 4 of the present application;

[0032] Figure 5 This is the HPLC spectrum of niposide saponin K obtained in Example 5 of the present application;

[0033] Figure 6 This is the HPLC spectrum of niposide saponin K obtained in Example 6 of the present application;

[0034] Figure 7This is the HPLC spectrum of niposide saponin K obtained in Example 7 of the present application;

[0035] Figure 8 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 1 of the present application;

[0036] Fig. 9 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 2 of the present application;

[0037] Fig.10 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 3 of the present application;

[0038] Fig.11 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 4 of the present application;

[0039] Fig.12 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 5 of the present application;

[0040] Fig.13 This is the HPLC spectrum of niposide saponin K obtained in Comparative Example 6 of the present application. DETAILED DESCRIPTION

[0041] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0042] The raw materials used in the examples of the present application are commercially available unless otherwise specified. Example

[0043] Example 1

[0044] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0045] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0046] (2) The extract A was subjected to silica gel column chromatography (filler: silica gel, column bed size: 100*600 mm), and an eluent consisting of chloroform, methanol and water in a weight ratio of 3:1:0.1 was used for elution, and the flow rate through the column was 100 mL / min. The target portion was collected by TLC detection and concentrated under reduced pressure to obtain the target powder B;

[0047] (3) The target powder B was added to ethanol and ultrasonically treated, filtered, and the filter residue was vacuum dried to obtain 13.26 g of niposide saponin K with HPLC purity greater than 99% and a yield of 37.89%.

[0048] Example 2

[0049] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0050] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0051] (2) subjecting the extract A to silica gel column chromatography, using an eluent consisting of chloroform, methanol and water in a weight ratio of 9:1:0.1, with a flow rate of 100 mL / min, collecting the target portion through TLC detection, and concentrating under reduced pressure to obtain the target powder B;

[0052] (3) The target powder B was added to ethanol and ultrasonically treated, filtered, and the filter residue was vacuum dried to obtain 13.86 g of niposide saponin K with HPLC purity greater than 99% and a yield of 39.60%.

[0053] Example 3

[0054] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0055] (2) subjecting the extract A to silica gel column chromatography, using an eluent consisting of ethyl acetate, ethanol and water in a weight ratio of 3:1:0.1, with a flow rate of 100 mL / min, collecting the target portion through TLC detection, and concentrating under reduced pressure to obtain the target powder B;

[0056] (3) The target powder B was added to ethanol and ultrasonically treated, filtered, and the filter residue was vacuum dried to obtain 14.49 g of niposide saponin K with HPLC purity greater than 99% and a yield of 41.4%.

[0057] Example 4

[0058] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0059] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0060] (2) The extract A was subjected to silica gel column chromatography (filler: silica gel, column bed size: 100*600 mm), and an eluent consisting of chloroform, methanol and water in a weight ratio of 3:1:0.1 was used for elution, and the flow rate through the column was 100 mL / min. The target portion was collected by TLC detection and concentrated under reduced pressure to obtain the target powder B;

[0061] (3) The target powder B was subjected to C18 medium-pressure column chromatography (filler: C18, particle size: 35-75 μm, column bed: 100*460 mm), using 40% v / v~75% v / v methanol gradient elution, the flow rate through the column was 100 mL / min, TLC detection and collection of the target portion, and C18 high-pressure preparation (filler: C18, particle size: 5 μm, column bed: 50*250 mm) was performed on the target component, using 20% ​​v / v~45% v / v acetonitrile gradient elution, HPLC detection and collection of the target portion, and concentrated under reduced pressure to obtain 16.41 g of niposide saponin K, with HPLC purity greater than 99% and a yield of 46.89%.

[0062] Example 5

[0063] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0064] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0065] (2) The extract A was subjected to silica gel column chromatography (filler: silica gel, column bed size: 100*600 mm), and an eluent consisting of chloroform, methanol and water in a weight ratio of 3:1:0.1 was used for elution, and the flow rate through the column was 100 mL / min. The target portion was collected by TLC detection and concentrated under reduced pressure to obtain the target powder B;

[0066] (3) The target powder B was subjected to C18 medium-pressure column chromatography, and a methanol gradient elution of 40% v / v to 75% v / v was used to collect the target portion. The target component was subjected to C18 high-pressure preparation (filler: C18, particle size: 5 μm, column bed: 50*250 mm), and a methanol gradient elution of 35% v / v to 75% v / v was used. The target portion was collected by HPLC detection and concentrated under reduced pressure to obtain 4.01 g of niposide saponin K1, with an HPLC purity of more than 99% and a yield of 40.03%.

[0067] Example 6

[0068] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0069] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0070] (2) The extract A was subjected to silica gel column chromatography (filler: silica gel, column bed size: 100*600 mm), and an eluent consisting of chloroform, methanol and water in a weight ratio of 5:1:0.1 was used for elution, and the flow rate through the column was 100 mL / min. The target portion was collected by TLC detection and concentrated under reduced pressure to obtain the target powder B;

[0071] (3) The target powder B was subjected to C18 medium-pressure column chromatography, and a gradient elution of 40% v / v to 75% v / v of methanol was used to collect the target fraction. The target component was subjected to C18 high-pressure preparation (filler: C18, particle size: 5 μm, column bed: 50*250 mm), and isocratic elution of 60% v / v of methanol was used. The target fraction was collected by HPLC detection and concentrated under reduced pressure to obtain 15.26 g of niposide saponin K with an HPLC purity of more than 99% and a yield of 43.60%.

[0072] Example 7

[0073] A method for separating saposin K from Bupleurum chinense, comprising the following steps:

[0074] (1) Take 50 kg of dried Tibetan Bupleurum, add 15 times the amount of 70% ethanol (70% by mass, the same below), soak at room temperature (25°C) for 3 days, concentrate the soaking liquid until there is no alcohol taste, cool it to room temperature, add D101 resin for adsorption, and elute with water and 70% ethanol in sequence. The flow rate through the column is 700 mL / min, collect the 70% ethanol part, and concentrate it to obtain extract A;

[0075] (2) The extract A was subjected to silica gel column chromatography (filler: silica gel, column bed size: 100*600 mm), and an eluent consisting of chloroform, methanol and water in a weight ratio of 5:1:0.1 was used for elution, and the flow rate through the column was 100 mL / min. The target portion was collected by TLC detection and concentrated under reduced pressure to obtain the target powder B;

[0076] (3) The target powder B was subjected to C18 medium-pressure column chromatography, and a methanol gradient elution of 40% v / v to 75% v / v was used to collect the target fraction. The target component was subjected to C18 high-pressure preparation (filler: C18, particle size: 5 μm, column bed: 50*250 mm), and an acetonitrile isocratic elution of 40% v / v was used. The target fraction was collected by HPLC detection and concentrated under reduced pressure to obtain 14.68 g of niposide saponin K with an HPLC purity of more than 99% and a yield of 41.94%.

[0077] Comparative Example

[0078] Comparative Example 1

[0079] A separation method, which differs from Example 1 only in that:

[0080] In step (2), elution is performed using an eluent consisting of chloroform, methanol and water in a weight ratio of 1:1:0.1.

[0081] The HPLC purity of the finally obtained niposide saponin K was 94.4%.

[0082] Comparative Example 2

[0083] A separation method, which differs from Example 1 only in that:

[0084] In step (2), elution is performed using an eluent consisting of chloroform, methanol and water in a weight ratio of 13:1:0.1.

[0085] The HPLC purity of the finally obtained niposide saponin K was 96.7%.

[0086] Comparative Example 3

[0087] A separation method, which differs from Example 4 only in that:

[0088] In step (3), during high pressure preparation, 50% v / v acetonitrile is used for isocratic elution.

[0089] The HPLC purity of the finally obtained niposide saponin K was 95.7%.

[0090] Comparative Example 4

[0091] A separation method, which differs from Example 4 only in that:

[0092] In step (3), during high pressure preparation, 25% v / v methanol is used for isocratic elution.

[0093] The HPLC purity of the finally obtained niposide saponin K was 96.4%.

[0094] Comparative Example 5

[0095] A separation method, which differs from Example 4 only in that:

[0096] In step (3), during high pressure preparation, 80% v / v methanol is used for isocratic elution.

[0097] The HPLC purity of the finally obtained niposide saponin K was 97.3%.

[0098] Comparative Example 6

[0099] A separation method, which differs from Example 4 only in that:

[0100] In step (3), during high pressure preparation, 15% v / v acetonitrile was used for isocratic elution.

[0101] The HPLC purity of the finally obtained niposide saponin K was 96.9%.

[0102] In summary, combined with Figure 1-Figure 13 From the HPLC purity data of Nepalese saikosaponin K obtained in Examples 1-7 and Comparative Examples 1-6, it can be seen that the method of the present application was used to successfully extract and separate Nepalese saikosaponin K from Tibetan Bupleurum, and the purity was relatively high, which could reach more than 99%, and could meet the purity requirements of Chinese medicine chemical reference raw materials. The obtained Nepalese saikosaponin K can be used for drug testing and research needs, and promote the development of the field of traditional Chinese medicine.

[0103] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for isolating saposin K from Bupleurum chinense, characterized in that: The following steps are involved: (1) extracting dried Bupleurum chinense with alcohol, and concentrating the extract to obtain extract A; (2) subjecting the extract A to silica gel column chromatography, using an eluent consisting of solvent I, solvent II and water in a weight ratio of (3-9):1:0.1 for elution, collecting the target portion, and concentrating under reduced pressure to obtain the target powder B; The solvent I is chloroform or ethyl acetate, and the solvent II is methanol or ethanol; (3) Purifying the target powder B by using purification step I or purification step II to obtain saikosaponin K; Purification step I: add the target powder B to methanol or ethanol for ultrasonic treatment, filter, precipitate and dry; Purification step II: subjecting the target powder B to a first C18 column chromatography, using methanol for elution, collecting the eluate, concentrating the eluate, subjecting the target powder B to a second C18 column chromatography, using solvent III for elution, collecting the target portion, and concentrating under reduced pressure; The solvent III is acetonitrile or methanol.

2. The method for separating saposin K from Bupleurum chinense according to claim 1, characterized in that: In step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 3:1:0.

1.

3. The method for separating saposin K from Bupleurum chinense according to claim 1, characterized in that: In step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 9:1:0.

1.

4. The method for separating saposin K from Bupleurum chinense according to claim 1, characterized in that: In step (2), the eluent is composed of chloroform, methanol and water in a weight ratio of 5:1:0.

1.

5. The method for separating saposin K from Bupleurum chinense according to claim 1, characterized in that: In step (3), the target powder B is purified by purification step II.

6. The method for separating saikosaponin K from Bupleurum chinense according to claim 5, characterized in that: During the second C18 column chromatography, 20% v / v to 45% v / v acetonitrile gradient elution was used.

7. The method for separating saposin K from Bupleurum chinense according to claim 5, characterized in that: During the second C18 column chromatography, 40% v / v acetonitrile was used for isocratic elution.

8. The method for separating saposin K from Bupleurum chinense according to claim 5, characterized in that: During the second C18 column chromatography, a gradient elution of 35% v / v to 75% v / v methanol was used.

9. The method for separating saposin K from Bupleurum chinense according to claim 5, characterized in that: During the second C18 column chromatography, 60% v / v methanol was used for isocratic elution.

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