Method for preparing saikoside B1 and saikoside G

Abstract

The invention provides a method for preparing saikoside B1 and saikoside G, and belongs to the field of plant extraction and separation and traditional Chinese medicine waste reutilization. The method comprises the following steps: extracting saikoside from radix bupleuri dregs serving as a selected raw material by adopting an overspeed crushing method, concentrating saponin substances in an extracting solution by utilizing macroporous adsorption resin, and purifying saikoside B1 and saikoside G by utilizing preparative high performance liquid chromatography. According to the method disclosed by the invention, purification of the two saponins is realized, the purity of the obtained saikoside B1 is 98.5%, and the yield is 429.3 mu g / g; the purity of saikosaponin G is 95.2%, and the yield of saikosaponin G is 112.91 [mu] g / g. The resource utilization of the traditional Chinese medicine waste is also realized.

Description

Technical Field

[0001] The present invention provides a method for preparing saikosaponin B 1 The method for preparing saikosaponin G comprises the following steps: extracting the dregs of Radix Bupleuri by ultra-speed crushing, concentrating the extract with a macroporous resin, and purifying saikosaponin by preparative high performance liquid chromatography to obtain saikosaponin B. 1 and saikosaponin G. The invention belongs to the field of plant extraction and separation and recycling of traditional Chinese medicine waste. Background Art

[0002] Bupleurum is one of the commonly used Chinese medicinal materials. It is the dried rhizome of the Umbelliferae plant Bupleurum (Bulpeurum Chinese. DC.) or Bupleurum narrow-leaved (Bulpeurums corzonerifolium. Wild). Bupleurum residue is the residue left after Chinese medicine manufacturers use Bupleurum to extract volatile oils. It is usually treated as waste from Chinese medicine factories, increasing the burden of environmental protection work. Studies have found that the waste Bupleurum residue still contains a variety of biologically active substances, such as saikosaponins and polysaccharides, especially saikosaponins with a high content. In recent years, it has been reported that the use of Bupleurum as an antidepressant drug is due to saikosaponin B 1 etc. and combine with the corresponding targets to exert antidepressant effects. Therefore, using Bupleurum dregs as the source of Bupleurum saponins has very high economic value and is of great significance in saving Chinese medicine resources.

[0003] In existing reports, the extraction of saikosaponins usually adopts immersion method, hot reflux extraction, Soxhlet extraction (SE), microwave-assisted and ultrasonic-assisted extraction, etc. The first three operations of these methods require a long extraction time and need to be heated to the boiling point of the solvent; the latter two methods, ultrasonic and microwave-assisted, can effectively improve production efficiency, but the scale of equipment is still limited. Ultrasonic crushing extraction method uses high-speed shearing to break plant cells, so that the bioactive substances in them are more easily dissolved into the extraction solvent, and can complete the high-yield extraction process in a shorter time, and can easily realize process amplification to reach production scale.

[0004] The present invention focuses on the preparation of saikosaponin B 1 The process development of saikosaponin G and saikosaponin B was carried out based on the ultra-fast crushing of bupleurum dregs to extract the two saikosaponins, and the concentration and purification preparation process parameters were studied. A method for preparing saikosaponin B was designed. 1 The method first uses ethanol aqueous solution as the extraction solvent, and extracts saikosaponin from the bupleurum residue by ultra-speed crushing, then uses D101 macroporous resin to concentrate the saikosaponin in the extract, and then uses preparative high performance liquid chromatography to prepare saikosaponin B. 1 and saikosaponin G, and two saikosaponin samples were obtained.

[0005] The extraction process of the method of the present invention is simple to operate, takes a short time, has few steps, and has a short production cycle. No toxic or harmful reagents are used, and no complicated production equipment is required. High-purity saikosaponin B can be obtained. 1 and saikosaponin G, with low preparation cost and convenient for expanding production scale. Summary of the invention

[0006] The purpose of the present invention is to prepare saikosaponin B 1 and pure products of saikosaponin G. In the preparation process, the raw material selected is the dregs of bupleurum, saikosaponin is extracted by ultra-speed crushing method, and saponin substances in the extract are concentrated by macroporous adsorption resin, and saikosaponin B is finally purified by preparative high performance liquid chromatography. 1 and saikosaponin G. The present invention not only realizes the purification of the two saponins, but also realizes the resource utilization of traditional Chinese medicine waste, and further improves the value of traditional Chinese medicine.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing saikosaponin B 1 The method comprises the following steps:

[0009] (1) grinding the dried Radix Bupleuri residue into powder, passing through a No. 4 sieve, and then extracting the Radix Bupleuri residue powder using an ethanol aqueous solution of a certain concentration and ratio, with the speed of overspeed crushing not exceeding 5000 rpm, and performing solid-liquid separation after extraction for a certain period of time to obtain an extract;

[0010] (2) concentrating the saikosaponin extract in step (1), passing it into a D101 macroporous adsorption resin column for preliminary purification, and eluting it with different ethanol aqueous solutions to obtain a saikosaponin solution with a higher concentration;

[0011] (3) The eluate in step (2) was concentrated, filtered through a 0.22 μm filter membrane, and then purified by preparative high performance liquid chromatography. The preparative chromatographic conditions were as follows: a 21.2×250 mm chromatographic column filled with 5 μm C18 filler, a detection wavelength of 210 nm, acetonitrile (A) and water (B) as the mobile phases, and a gradient of A%: 0-20 min, 35-40; 20-60 min, 40-50; 60-85 min, 50-70; 85-90 min, 70-35; 90-100 min, 35; the retention time of saikosaponin G (45.3-47 min) and saikosaponin B was controlled by the retention time. 1 (48-50 min) Collection: Concentrate each collected solution to obtain saikosaponin B 1 and saikosaponin G, the purity of both saponins is greater than 95%.

[0012] The extraction speed in step (1) is 4000-5000 rpm, the solid-liquid ratio is 1:10-1:80 g / ml, and the extraction time is 2-12 min. After optimization, the powder of bupleurum dregs in step (1) is 7.5 g, the concentration of ethanol water is 60%, the solid-liquid ratio is 1:20 g / ml, the rotation speed is 4500 rpm, the extraction time is 6 min, the extraction number is 1, and the extraction rate of the two saponins is the highest. Bupleurum saponin B 1 The extraction rate of is about 2.124 mg / g, and the extraction rate of saikosaponin G is about 0.513 mg / g.

[0013] Wherein, in step (2), saikosaponin B in the sample solution 1 When the concentration of is about 180μg / ml and the concentration of saikosaponin G is about 40μg / ml, the sample flow rate is 0.5-4ml / min, preferably 1ml / min; the sample volume is 1-9BV, preferably 6BV, and the adsorption rate of D101 macroporous resin can reach 97%. The ethanol concentration of the ethanol aqueous solution in the elution process in step (2) is 70%, the elution flow rate is 1ml / min, the elution volume of the eluent is 1-10BV, preferably 6BV, and saikosaponin B is 0.5-4ml / min, preferably 1ml / min; the sample volume is 1-9BV, preferably 6BV, and the adsorption rate of D101 macroporous resin can reach 97%. 1 The adsorption rate was 96.1%, the elution rate was 89.88%, the content increased from 0.5% before sampling to 23.47%, and the recovery rate was 86.37%; the adsorption rate of saikosaponin G was 88.1%, the elution rate was 93.5%, the content increased from 0.11% before sampling to 5.01%, and the recovery rate was 82.37%.

[0014] The injection volume of the preparative liquid chromatography purification process in step (3) is 40-500 μl, preferably 50 μl, and the separation degree of the chromatographic peaks is high and the reproducibility is good. The flow rate of the preparative chromatography is 10-20 ml / min, preferably 10 ml / min.

[0015] In the above process, saikosaponin B 1 The HPLC analysis conditions for the quantitative analysis of saikosaponin G are as follows:

[0016] C18 chromatographic column (4.6×250mm, 5μm), detection wavelength of 210nm, injection volume of 10μL, column temperature of 30℃, mobile phase of acetonitrile (A) and water (B), flow rate of 1mL / min, the gradient of mobile phase during analysis is shown in the table:

[0017]

[0018] The present invention has achieved the following beneficial results: 1. Designed a method for preparing saikosaponin B 1and the method of saikosaponin G, and the best preparation process was obtained by optimizing the process parameters; 2. The preparation method of the present invention uses ethanol water, acetonitrile water and other solutions as solvents for extraction and separation, and does not involve toxic, harmful, flammable and explosive hazardous chemicals; 3. The preparation method of the present invention is simple to operate, highly efficient and highly repeatable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Preparation of saikosaponin B 1 Process diagram of saikosaponin G

[0020] Figure 2 Preparative HPLC

[0021] Figure 3 Saikosaponin B 1 HPLC chart of

[0022] Figure 4 This is the HPLC chart of the prepared saikosaponin G

[0023] Figure 5 Saikosaponin B 1 Mass spectrum of

[0024] Figure 6 This is the mass spectrum of the prepared saikosaponin G

[0025] Figure 7 Saikosaponin B 1 H NMR spectrum of

[0026] Figure 8 This is the H NMR spectrum of the prepared saikosaponin G DETAILED DESCRIPTION

[0027] In combination with the above description and the accompanying drawings, a method for preparing saikosaponin B of the present invention is described below through specific embodiments. 1 The method of preparing saikosaponin G is further described to enable those skilled in the art to better understand the present invention, but the present invention is not limited thereto.

[0028] The following table shows examples 1-5 of the present invention, which are examples under different conditions of step (1). Examples 1-5 respectively optimize the amount of raw materials input, the ethanol content in the extraction solvent, the number of rotations of ultra-speed extraction, the material-liquid ratio and the extraction time, and obtain the extraction rates of the two saikosaponins under different conditions.

[0029]

[0030]

[0031] The following table is an example of step (2). Examples 6-10 are different parameters when using macroporous adsorption resin for concentration in step (2), including sample volume, sample flow rate, proportion of ethanol in the eluent, elution flow rate and elution volume.

[0032]

[0033] The following table shows examples of different parameters used in the preparative high performance liquid chromatography method in step (3). Examples 11-13 analyze the collected amounts of two target saponins under different injection volume conditions, and obtain different recovery rates and respective purities.

[0034]

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing saikosaponin B1 and saikosaponin G, characterized in that: (1) grinding the dried Radix Bupleuri residue into powder, passing through a No. 4 sieve, and then extracting the Radix Bupleuri residue powder using an ethanol aqueous solution of a certain concentration and ratio, with the speed of overspeed crushing not exceeding 5000 rpm, and performing solid-liquid separation after extraction for a certain period of time to obtain an extract; (2) concentrating the saikosaponin extract in step (1), passing it into a D101 macroporous adsorption resin column for preliminary purification, and eluting it with different ethanol aqueous solutions to obtain a saikosaponin solution with a higher concentration; (3) The eluate in step (2) is concentrated, filtered through a 0.22 μm filter membrane, and then purified by preparative high performance liquid chromatography. The preparative chromatographic conditions are: a 21.2×250 mm chromatographic column filled with 5 μm C18 filler, a detection wavelength of 210 nm, acetonitrile (A) and water (B) as the mobile phases, and a gradient change of A%: 0-20 min, 35-40; 20-60 min, 40-50; 60-85 min, 50-70; 85-90 min, 70-35; 90-100 min, 35; the retention time is controlled by collecting saikosaponin G (45.3-47 min) and saikosaponin B1 (48-50 min): after concentrating each collected solution, saikosaponin B1 and saikosaponin G are obtained, and the purity of the two saponins is greater than 95%.

2. A method for preparing saikosaponin B1 and saikosaponin G according to claim 1, characterized in that: In step (1), the extraction rotation speed is 4000-5000 rpm, preferably 4500 rpm; the extraction solid-liquid ratio is 1:10-1:80 g / ml, preferably 1:20 g / ml; and the extraction time is 2-12 min, preferably 6 min.

3. A method for preparing saikosaponin B1 and saikosaponin G according to claim 1, characterized in that: In step (2), the sample loading flow rate is 0.5-4 ml / min, preferably 1 ml / min; the sample loading volume is 1-9 BV, preferably 6 BV; and the volume of the eluent is 1-10 BV, preferably 6 BV.

4. A method for preparing saikosaponin B1 and saikosaponin G according to claim 1, characterized in that: In step (3), the injection volume is 1-500 μL, preferably 50 μL, the flow rate is 10-20 ml / min, preferably 10 ml / min, the detection wavelength is 210 nm, the mobile phase is acetonitrile (A) and water (B), and the gradient change is A%: 0-20 min, 35-40; 20-60 min, 40-50; 60-85 min, 50-70; 85-90 min, 70-35; 90-100 min, 35; the collection of saikosaponin G (45.3-47 min) and saikosaponin B1 (48-50 min) is controlled by retention time.

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