CN113599404A

CN113599404A - Preparation method of bupleurum extract and bupleurum tenue granules

Info

Publication number: CN113599404A
Application number: CN202111117517.5A
Authority: CN
Inventors: 袁崇均; 张磊; 陈帅; 杨薇; 罗森; 吴瑕; 汤依娜; 周静; 王笳
Original assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Current assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2021-11-05

Abstract

The invention discloses a bupleurum extract, which is prepared by Na-meridian of bupleurum₂CO₃Extracting the resulting extract with an aqueous solution; each 1g of the extract is equivalent to 5-6 g of the raw medicinal material. The invention also discloses a preparation method of the radix bupleuri granules. The saikosaponin a and the saikosaponin d in the finished product prepared by the preparation method of the small bupleurum particles are almost not decomposed, so that the curative effect of the medicine is improved.

Description

Preparation method of bupleurum extract and bupleurum tenue granules

Technical Field

The invention relates to a method for preparing bupleurum extract and bupleurum tenue particles.

Background

The bupleurum tenue particles consist of bupleurum, scutellaria, ginger processed pinellia, codonopsis pilosula, ginger, liquorice and Chinese date, have the effects of relieving exterior syndrome, dissipating heat, soothing liver and harmonizing stomach, are used for treating exogenous diseases and the syndrome of pathogen attacking shaoyang, and have the symptoms of alternating cold and heat, fullness in chest and hypochondrium, inappetence, vexation and vomiting preference and bitter taste and dry throat, are collected in 2020 edition of Chinese pharmacopoeia and are common medicines of Chinese patent medicines for treating cold. At present, the Xiaochaihu granules have approved literature numbers obtained by nearly 100 manufacturers.

In the Xiaochaihu granules, the bupleurum is a monarch drug, and the dosage (150g) in the formula is nearly 3 times (56g) of that of other traditional Chinese medicines, so that the extraction and quality detection of the bupleurum are very important. In the Chinese pharmacopoeia 2020 edition, the quality of small bupleurum particles in the first part is detected by thin-layer chromatography under the item of bupleurum, and the reference solution is bupleurum reference medicinal material, and the two thin-layer chromatographies are consistent. However, in the quality inspection process, the thin-layer chromatograms of a plurality of small bupleurum particles are difficult to be consistent, the thin-layer sample amount of a bupleurum control medicinal material (the sample amount is 1g) in the pharmacopeia is 2 mul, the sample amount of a small bupleurum particle (the converted bupleurum medicinal material amount is 0.9g) detection solution is 2-10 mul, and the difference between the two is about 5 times at most. The reason why the detection does not meet the standard is that the saikosaponin component is decomposed in the preparation process of the small bupleurum particles, and the saikosaponin is rarely decomposed under the preparation condition of the reference solution of the bupleurum Chinese medicine at the moment, so that the thin-layer chromatography of the bupleurum Chinese medicine and the bupleurum Chinese medicine is difficult to be consistent.

Bupleuri radix is dried root of Bupleurum chinense DC and Bupleurum angustifolium Willd of Umbelliferae,has the effects of dispelling heat, relieving fever, dispersing stagnated liver qi, relieving qi stagnation, and invigorating yang. Saikosaponin a, d and saikosaponin are considered as main effective components of bupleurum, and play a very important role in the curative effect of the whole medicine. Research reports that saikosaponin a and saikosaponin d have 13, 28-oxygen ring structure and unstable oxygen ring, and during the extraction process of bupleurum, saikosaponin a and saikosaponin d are changed into saikosaponin b without some pharmacological activity of original saponin or with relatively weakened pharmacological activity under the influence of inherent components of Chinese medicinal materials₁、b₂. At present, no practical reference is available for solving the problem that the effective components of bupleurum are damaged when the bupleurum is used as the raw material of a Chinese medicinal compound preparation, particularly small bupleurum particles.

Disclosure of Invention

In order to solve the above problems, the present invention provides a bupleurum root extract which is Na of bupleurum root₂CO₃An aqueous extract; each 1g of the extract is equivalent to 5-6 g of the raw medicinal material.

Further, said Na₂CO₃The concentration of the aqueous solution is 0.01-0.03% w/v, g/ml, preferably 0.025% w/v, g/ml; the bupleuri radix is coarse powder of bupleuri radix.

Furthermore, each 1g of the extract corresponds to 5.4g of the original medicinal material.

The invention also provides a preparation method of the bupleurum extract, which comprises the following steps:

taking coarse powder of radix bupleuri, adding Na₂CO₃Extracting with water solution, filtering, and concentrating the filtrate.

Further, the extraction is decoction extraction for 2 times, and 5-15 times of v/w, ml/gNa is added for each time₂CO₃Decocting the aqueous solution for 0.5-2 hours, preferably 1 hour.

Further, the Na₂CO₃The concentration of the aqueous solution is 0.01-0.03% w/v, g/ml, preferably 0.025% w/v, g/ml.

Further, the preparation method also comprises the steps of taking the concentrated extract, drying the extract under reduced pressure and crushing the dried extract into fine powder; the reduced pressure drying temperature was 60 ℃.

The invention finally provides a preparation method of the bupleurum tenue particles, which further comprises the following steps:

1) decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water, filtering, and concentrating the filtrate to obtain concentrated solution;

2) soaking rhizoma Pinelliae Preparata and rhizoma Zingiberis recens with 70% ethanol, percolating, collecting percolate, recovering ethanol, adding the concentrated solution of step 1), mixing, concentrating, adding the above bupleuri radix extract and sucrose, granulating, and drying.

Further, the mass-volume ratio of the bupleurum extract, the scutellaria baicalensis, the ginger processed pinellia tuber, the codonopsis pilosula, the ginger, the liquorice and the Chinese date is 27-28 g: 56 g: 56 g: 56g, preferably 27.8 g: 56 g: 56 g: 56g, 56g and 56 g.

Further, the decoction in the step 1) is carried out for 2 times, and each time lasts for 1.5 hours; and/or, in the step 2), soaking for 24 hours, wherein the amount of the collected percolate is 5-6 v/w, ml/g of the sum of the weight of the ginger processed pinellia tuber and the weight of the ginger.

The preparation method of the bupleurum extract of the invention adopts Na with specific concentration₂CO₃The saikosaponin loss in the bupleurum extract obtained by aqueous solution extraction is less, the bupleurum tenue particles prepared by the bupleurum tenue extract are detected by thin-layer chromatography under bupleurum term according to the quality detection of the bupleurum tenue particles in the first part of China pharmacopoeia 2020 edition, and the thin-layer chromatography is consistent with bupleurum control medicinal materials.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 identification thin layer chromatogram of bupleuri radix granule (from left to right, bupleuri radix reference material, comparison group 1 ~ 5 samples)

FIG. 2 thin-layer chromatogram for identification in Experimental example 2 (from left to right, 1. mu.l bupleuri radix extract, 1. mu.l Xiaochaihu granule, 2. mu.l bupleuri radix extract, 2. mu.l Xiaochaihu granule, and 2. mu.l bupleuri radix reference material)

FIG. 3 test example 3 thin-layer identification chromatogram (from left to right: sample (r), Bupleurum root reference drug, sample (r), 2. mu.l, Bupleurum root reference drug 1. mu.l)

FIG. 4 test example 4 thin-layer identification chromatogram (from left to right: sample (r), and radix bupleuri control drug)

FIG. 5 thin-layer identification of Bupleurum scorzonerifolium (from left to right: saikoside a control, Brand 1 Xiaochaihu Keli, example 1 Xiaochaihu Keli, Bupleurum scorzonerifolium reference medicinal materials, Brand 2 Xiaochaihu Keli)

Detailed Description

EXAMPLE 1 preparation of the extract of Bupleurum chinense of the invention

Pulverizing bupleuri radix into coarse powder, adding 0.025% Na₂CO₃Decocting in water for 2 times, 1 hr each time, and Na each time₂CO₃The water solution is 10 times (V/W) of the medicinal materials, filtering, mixing filtrates, concentrating to obtain extract, drying at 60 deg.C under reduced pressure, and pulverizing into fine powder to obtain bupleuri radix extract (brown dry loose powder, 1g is 5.4g of the original medicinal materials).

EXAMPLE 2 preparation of Xiaochaihu granules of the present invention

The formula is as follows: 27.8g of the extract of Bupleurum chinense Pub, 56g of Scutellaria baicalensis Georgi, 56g of pinellia ternata, 56g of Codonopsis pilosula, 56g of ginger, 56g of licorice, and 56g of Chinese date prepared in example 1

Preparation method

1) Decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to obtain concentrated solution;

2) soaking rhizoma Pinelliae Preparata and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate 600ml, recovering ethanol, mixing with the concentrated solution of step 1), concentrating, adding bupleuri radix extract and sucrose, granulating, drying, and making into 1000g

The advantageous effects of the present invention are described below by way of test examples.

Instrument and reagent

The instrument comprises the following steps: agilent HP 1100 high performance liquid chromatograph (Agilent chromatography workstation), Sartonus electronic balance (BP211D model, usa), CQX-25-06 ultrasonic cleaner (shanghai bi-cheng ultrasonic limited), RE 5205 rotary evaporator (shanghai yanglong biochemical instruments factory), vacuum drying oven (shanghai-heng scientific instruments limited), DJ smart pulverizer (shanghai jiu chinese medicine mechanical manufacturing limited).

Materials: radix bupleuri saponin a, d reference substances (Chengdumanst), radix bupleuri, radix Scutellariae, rhizoma Pinelliae preparata, radix Codonopsis, rhizoma Zingiberis recens, radix Glycyrrhizae, fructus Jujubae (from QINGCHUAN Chinese medicinal decoction pieces, LLC), and radix bupleuri reference medicinal materials (China food and drug testing institute).

Reagent: acetonitrile (chromatographically pure), water is high-purity water, and other reagents are analytically pure

Experimental example 1 preparation of Xiaochaihu granules

1. Preparation of Xiaochaihu granules

Comparative group 1

The formula is as follows: 150g of radix bupleuri, 56g of radix scutellariae, 56g of ginger processed pinellia, 56g of radix codonopsitis, 56g of ginger, 56g of liquorice and 56g of Chinese date

The preparation method comprises the following steps: the seven ingredients comprise radix bupleuri (coarse powder), radix Scutellariae, radix Codonopsis, radix Glycyrrhizae, fructus Jujubae and 0.05% Na₂CO₃Decocting with water twice, each for 1.5 hr, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate about 600ml, recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding appropriate amount of sucrose, granulating, drying, and making into 1000 g.

Comparative group 2

The formula is as follows: same comparative group 1

The preparation method comprises the following steps: bupleuri radix (coarse powder), 0.025% Na₂CO₃Decocting with water twice, each time for 1.5 hr, mixing decoctions, filtering, concentrating the filtrateTo a proper amount. Decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate about 600ml, recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding appropriate amount of sucrose, granulating, drying, and making into 1000 g.

Comparative group 3

The formula is as follows: same comparative group 1

The preparation method comprises the following steps: decocting bupleuri radix (coarse powder) in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate about 600ml, recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding appropriate amount of sucrose, granulating, drying, and making into 1000 g.

Comparative group 4

The formula is as follows: same comparative group 1

The preparation method comprises the following steps: decocting bupleuri radix (coarse powder) in water twice for 1.5 hr each time, mixing decoctions, filtering, concentrating the filtrate to appropriate amount, drying under reduced pressure, and pulverizing into fine powder to obtain bupleuri radix dry extract. Decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate about 600ml, recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding the above bupleuri radix dry extract and appropriate amount of sucrose, granulating, drying, and making into 1000 g.

Comparative group 5

The formula is as follows: same comparative group 1

The preparation method comprises the following steps: bupleuri radix (coarse powder), 0.025% Na₂CO₃Decocting with water twice, each for 1.5 hr, mixing decoctions, filtering, concentrating the filtrate to appropriate amount, drying under reduced pressure, and pulverizing into fine powder to obtain bupleuri radix extract. Decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice,each time for 1.5 hours, the decoction liquid is merged and filtered, and the filtrate is concentrated to a proper amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate (about 600 ml), recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding the above bupleuri radix extract and appropriate amount of sucrose, granulating, drying, and making into 1000 g.

2. Detection of

Taking the bupleurum tenue granules prepared in the comparative groups 1-5, and referring to the Thin Layer Chromatography (TLC) detection of bupleurum tenue under the identification item in the first page 605 of the China pharmacopoeia 2020 edition.

Taking 6g of a sample of the comparative group 1, adding 20ml of water, stirring to dissolve, centrifuging, taking supernatant, adding the supernatant to a polyamide column (100-200 meshes, 8g, the inner diameter of the column is 2.5-3 cm, and the column is filled by a wet method), eluting by respectively using 100ml of water, 20% ethanol and 50% ethanol, collecting 50% ethanol eluent, evaporating to dryness, and adding 1ml of methanol to dissolve residues to obtain a sample solution of the comparative group 1. The test solutions of the comparative groups 2-5 were prepared by the same method. Taking 1g of bupleurum root as a reference medicinal material, adding a proper amount of water, decocting for 1.5 hours, filtering, concentrating the filtrate to about 10ml, adding the filtrate on a polyamide column (100-200 meshes, 4g, the inner diameter is 2cm, and the column is filled by a wet method), eluting by 100ml of water and 150ml of 50% ethanol respectively, collecting 50% ethanol eluent, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a reference medicinal material solution. Performing thin layer chromatography (general rule 0502) test, respectively sucking 2 μ l of each of the test solution and the control solution of examples 1-5, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-ethanol-water (12: 2: 1) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution containing 5% p-dimethylaminobenzaldehyde, blowing hot air to the spot to develop color clearly, and inspecting under ultraviolet lamp (365 nm).

3. Results

The specific result is shown in fig. 1, and it can be seen from the figure that TLC of the bupleurum root control drug has only two distinct fluorescent spots, and the comparative groups 1, 3 and 4 have other fluorescent spots, i.e. under the preparation condition, the saikosaponin is partially decomposed, while TLC of the comparative groups 2 and 5 is completely consistent with that of the bupleurum root control drug.

From the above results, it can be seen that Bupleurum root uses weak baseWater (0.025% Na)₂CO₃Aqueous solution) or bupleurum extract, and the TLC under the bupleurum identification item in the quality detection of the prepared small bupleurum particles is qualified, and the preparation process of the small bupleurum particles is optimal, so the comparison groups 2 and 5 are the optimal production processes. With weak alkaline water (0.025% Na)₂CO₃Aqueous solution) decocting bupleuri radix to obtain bupleuri radix extract, and adding bupleuri radix extract to obtain XIAOCHAIHU granule. Thus, comparative example 5 is the best process.

Experimental example 2 quality test of Bupleurum extract and Small Bupleurum particles

1. Preparation of bupleuri radix extract

The Chinese medicinal material is extract of dried root of Bupleurum chinense DC of Umbelliferae.

Pulverizing bupleuri radix into coarse powder, adding 0.025% Na₂CO₃Decocting the water solution for 2 times, each time for 1 hr, with the amount of alkaline water 10 times (V/W) of the medicinal materials, filtering, mixing filtrates, concentrating to obtain extract, drying at 60 deg.C under reduced pressure, and pulverizing into fine powder to obtain bupleuri radix extract (brown dry loose powder, yield 18.5%, 1g is equivalent to 5.4g of the original medicinal materials).

2. Preparation of Xiaochaihu granules

The formula is as follows: 27.8g of radix bupleuri extract, 56g of scutellaria baicalensis, 56g of ginger processed pinellia tuber, 56g of codonopsis pilosula, 56g of ginger, 56g of liquorice and 56g of Chinese date

The preparation method comprises the following steps: decocting Scutellariae radix, radix Codonopsis, Glycyrrhrizae radix and fructus Jujubae in water twice for 1.5 hr each time, mixing decoctions, filtering, and concentrating the filtrate to appropriate amount. Soaking rhizoma Pinelliae and rhizoma Zingiberis recens with 70% ethanol as solvent for 24 hr, percolating, collecting percolate about 600ml, recovering ethanol, mixing with the above concentrated solution, concentrating to appropriate amount, adding bupleuri radix extract 27.8g and sucrose, granulating, drying, and making into 1000 g.

Remarking: 1g of bupleurum extract is equivalent to 5.4g of raw medicinal material, so 150/5.4-27.8 g of bupleurum extract in the bupleurum tenue granules is added

3. TLC identification of bupleurum extract and bupleurum tenue particles

Taking about 20mg of bupleurum extract (which is equivalent to 1g of bupleurum medicinal material, and the calculation is that 1g/5.4g is 0.02g, namely 20mg), adding 20ml of water, stirring to dissolve, centrifuging, taking supernatant, adding the supernatant onto a polyamide column (100-200 meshes, 8g, the inner diameter is 2.5-3 cm, and loading the column by a wet method), respectively eluting with 100ml of water, 20% ethanol and 50% ethanol, collecting 50% ethanol eluent, evaporating to dryness, adding 1ml of methanol into residues to dissolve, and preparing a bupleurum extract test sample solution. Then 6g of Xiaochaihu granules are prepared into a test solution of the Xiaochaihu granules by the same method. Taking 1g of bupleurum root as a reference medicinal material, adding a proper amount of water, decocting for 1.5 hours, filtering, concentrating the filtrate to about 10ml, adding the filtrate on a polyamide column (100-200 meshes, 4g, the inner diameter is 2cm, and the column is filled by a wet method), eluting by 100ml of water and 150ml of 50% ethanol respectively, collecting 50% ethanol eluent, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a reference medicinal material solution. Performing thin layer chromatography (general rule 0502) test, sucking 1-2 μ l of each of the 3 solutions, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-ethanol-water (12: 2: 1) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution containing 5% p-dimethylaminobenzaldehyde, blowing hot air to the spot to develop color clearly, and inspecting under ultraviolet lamp (365 nm).

4. The result of the detection

The specific results are shown in FIG. 2. As can be seen from the figure, the thin layer chromatography of the Bupleurum falcatum extract and the bupleurum tenue granules is completely consistent with that of the Bupleurum tenue reference drug.

Experimental example 3 theoretical study on preparation process of extract from Xiaochaihu granules

Sample (I): 15g of bupleurum (coarse powder) is decocted for two times by adding water, the amount of the water is 10 times (V/W) of the amount of the medicinal materials each time, the decoction is combined and filtered, the filtrate is added with water to about 300ml, and the PH value of the solution is measured by a PHS-3CPH meter (Shanghai Jingke).

Sample 2: 15g of radix bupleuri (coarse powder), 5.6g of scutellaria baicalensis, codonopsis pilosula, liquorice and Chinese date respectively, and 37.4g of the coarse powder, adding water and decocting twice, 1.5 hours each time, wherein the water dosage is 10 times (V/W) of the amount of the medicinal materials, combining the decoction, filtering, adding water into the filtrate to about 750ml, and measuring the pH value of the solution by using a PHS-3C pH meter (Shanghai Jingke).

Sample 3: 15g of radix bupleuri (coarse powder), 0.025% of Na₂CO₃Decocting the water solution twice for 1.5 hr each time, with water amount 10 times (V/W) of the medicinal materials, mixing decoctions, filtering, adding 0.025% Na into the filtrate₂CO₃The aqueous solution was adjusted to about 300ml and the pH of the solution was measured using a PHS-3C pH meter (Shanghai sperm family).

Sample iv: 15g of radix bupleuri (coarse powder), 5.6g of scutellaria baicalensis, codonopsis pilosula, liquorice and Chinese date respectively, 37.4g of radix bupleuri (coarse powder), and 0.05 percent of Na₂CO₃Decocting the water solution twice for 1.5 hr each time, with water amount 10 times (V/W) of the medicinal materials, mixing decoctions, filtering, adding 0.05% Na into the filtrate₂CO₃The aqueous solution was adjusted to about 750ml and the pH of the solution was measured using a PHS-3C pH meter (Shanghai sperm family).

As a result, the pH values were 6.0, 6.4, 7.3 and 7.2, respectively.

Respectively taking 20ml of samples, 50ml of samples, 20ml of samples and 50ml of samples (all equal to 1G of radix bupleuri crude drug), preparing sample test sample solutions 1, 2, 3 and 4 according to the method of the test example 1, preparing radix bupleuri reference drug solution, testing according to a thin-layer chromatography (general rule 0502), sucking 1-2 mu l of the 5 solutions, respectively dropping the solutions on the same silica gel G thin-layer plate, taking ethyl acetate-ethanol-water (12: 2: 1) as a developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution of 5% p-dimethylaminobenzaldehyde, blowing hot air to the spots to be clear, and inspecting under an ultraviolet lamp (365 nm).

The results are shown in FIG. 3, from which it can be seen that sample C, i.e., bupleuri radix, was treated with 0.025% Na₂CO₃Decocting with water solution, and keeping TLC substantially the same as that of bupleuri radix control.

Further analysis of the results gave: because of the inherent acidity and alkalinity of Chinese herbs, most of the original liquid medicine decoction is acidic, for example, the pH of the bupleurum root decoction is 6.0, while the pH of the bupleurum root, pilose asiabell root, baical skullcap root, licorice root and Chinese date decoction is 6.4. The saikosaponin is easy to be chemically changed under acidic condition, i.e. saikosaponin a and saikosaponin d are converted into saikosaponin b₁、b₂During the extraction process, some acidic components in bupleurum are neutralized by weak alkali aqueous solution, so that the stability of saikosaponin can be improved.

Experimental example 4 theoretical study on preparation process of extract from Xiaochaihu granules-

Preparation of a sample I: taking 1g of radix bupleuri (coarse powder), adding 0.05% of Na₂CO₃Decocting an appropriate amount of aqueous solution for 1.5 hours, filtering, concentrating the filtrate to about 10ml, adding the filtrate on a polyamide column (100-200 meshes, 4g, the inner diameter of 2cm, wet column packing), eluting with 100ml of water and 150ml of 50% ethanol respectively, collecting 50% ethanol eluate, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution I.

Preparation of sample 2: taking 1g of radix bupleuri (coarse powder), adding 0.1% of Na₂CO₃Proper amount of aqueous solution, decocting for 1.5 hours, filtering, concentrating the filtrate to about 10ml, adding the filtrate on a polyamide column (100-200 meshes, 4g, the inner diameter is 2cm, wet loading the column), respectively eluting with 100ml of water and 150ml of 50% ethanol, collecting 50% ethanol eluent, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample, namely a ② solution.

Preparing a bupleurum contrast medicinal material sample solution: taking 1g of bupleurum serving as a reference medicinal material, adding a proper amount of water solution, decocting for 1.5 hours, filtering, concentrating the filtrate to about 10ml, adding the filtrate on a polyamide column (100-200 meshes, 4g, the inner diameter of the column is 2cm, and the column is filled by a wet method), eluting by using 100ml of water and 150ml of 50% ethanol respectively, collecting 50% ethanol eluent, evaporating to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a bupleurum serving as a reference medicinal material sample solution.

Testing by thin layer chromatography (general rule 0502), sucking 2 μ l of each of the 3 solutions, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-ethanol-water (12: 2: 1) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution containing 5% p-dimethylaminobenzaldehyde, blowing hot air to the spot to develop color clearly, and inspecting under ultraviolet lamp (365 nm).

The results are shown in FIG. 4, in which the radix bupleuri material is 0.05% Na₂CO₃Aqueous solution, 0.1% Na₂CO₃Decocting in water solution, decomposing saikosaponin, wherein TLC is different from that of bupleuri radix control material, and saikosaponin is decomposed, so that stronger alkaline water solution (0.05% or 0.1% Na)₂CO₃Aqueous solution) for a long time, and can also decompose saikosaponin.

Experimental example 5 theoretical study on preparation process of extract from Xiaochaihu granules

Content determination of saikosaponin a and saikosaponin d in bupleurum medicinal material

Measuring by high performance liquid chromatography (general rule 0512).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 210 nm. The number of theoretical plates is not less than 10000 calculated according to the peak of saikosaponin a.

Preparation of reference solution taking appropriate amount of saikosaponin a reference and saikosaponin d reference, precisely weighing, adding methanol to obtain solution containing saikosaponin a 0.4mg and saikosaponin d 0.5mg per 1ml, and shaking.

Preparation of test solution I about 0.5g of bupleuri radix powder (sieved by a No. four sieve) is precisely weighed, placed in a conical flask with a plug, added with 25ml of methanol solution containing 5% concentrated ammonia test solution, sealed with the plug, ultrasonically treated at 30 ℃ for 30 minutes (power of 200W and frequency of 40kHz), filtered, the container and the medicine residues are washed by 20ml of methanol for 2 times, the washing solution is combined with the filtrate, and the solvent is recovered to be dry. Dissolving the residue with methanol, transferring to 5ml measuring flask, adding methanol to scale, shaking, filtering, and collecting the filtrate.

Sample solution 2 is prepared by taking about 1g of radix bupleuri powder (screened by a sieve with the number four), precisely weighing, adding a proper amount of water, decocting for 2 times, 1.5 hours each time, filtering, concentrating the filtrate to dryness, dissolving the residue in methanol, transferring to a 5ml measuring flask, adding methanol to the scale, shaking up, filtering, and taking the subsequent filtrate.

Preparation of test solution (III) about 1g of bupleuri radix powder (sieved with sieve IV) is precisely weighed, and 0.025% Na is added₂CO₃Decocting the appropriate amount of water solution for 2 times, each for 1.5 hr, filtering, concentrating the filtrate to dryness, dissolving the residue with methanol, transferring to 5ml measuring flask, adding methanol to the scale, shaking, filtering, and collecting the filtrate.

Preparation of test solution (4) about 1g of bupleuri radix powder (sieved with a sieve of four numbers) is precisely weighed and added with 0.1% of Na₂CO₃Decocting the appropriate amount of water solution for 2 times, each for 1.5 hr, filtering, concentrating the filtrate to dryness, dissolving the residue with methanol, transferring to 5ml measuring flask, adding methanol to the scale, shaking, filtering, and collecting the filtrate.

The determination method precisely absorbs 20 mul of the reference solution and 10-20 mul of the test solution respectively, and injects the solution into a liquid chromatograph for determination.

The results of the content measurement are shown in table 1:

TABLE 1 determination of the content of Bupleurum chinense

Sample (I)	Saikosaponin a	Saikosaponin d	Total amount of saikosaponin a and d	Is the content of the sample
					①	0.34％	0.54％	0.88％	100％
②	0.30％	0.42％	0.72％	84％
					③	0.33％	0.48％	0.81％	92％
④	0.29％	0.33％	0.63％	72％

As can be seen from Table 1, the obtained saikosaponin is not lost by ultrasonic extraction in an alkaline alcohol solution, but in industrial mass production, ultrasonic extraction is difficult, and the extract extracted by methanol has potential safety hazard, so water extraction is usually adopted, the cost is saved, and the safety of the drug extract is ensured. However, the radix bupleuri water has 16% decomposition (compared with radix bupleuri ultrasonic extraction), 0.025% Na₂CO₃The water decoction is only 8% decomposed and 0.1% Na₂CO₃The water decoction is decomposed by about 30%. Therefore, the weak base aqueous solution (0.025% Na)₂CO₃Aqueous solution) is preferred.

Test example 6 thin-layer chromatography of Bupleurum chinense under identification of commercially available Xiaochaihu Keli particles

Two kinds of commercially available small bupleuri radix granules (specification 10 g/bag) each 6g are respectively taken, and identified by Thin Layer Chromatography (TLC) under the identification item of small bupleuri radix granules (refer to page 605 of the first part of China pharmacopoeia 2020 edition), which is shown in figure 5 (meanwhile, saikosaponin a is used as a reference).

As can be seen from the figure, the commercial Bupleurum tenue granule bupleurum thin layer identification bupleurum saponin d has been partially or completely decomposed, and the chromatographic behavior is inconsistent with that of the Bupleurum tenue control drug.

In conclusion, the preparation method of the bupleurum tenue granules of the invention is based on the preparation method of the pharmacopoeia,three improvements are made: firstly, the bupleurum is crushed into coarse powder, which is beneficial to the extraction of effective components. ② 0.025 percent of Na used for coarse powder of bupleurum₂CO₃Decocting the aqueous solution, and neutralizing inherent acidic components in the radix bupleuri medicinal material with weak base, thereby being beneficial to the stability of the saikosaponin. ③ 0.025 percent of Na used for coarse powder of bupleurum₂CO₃Decocting in water, filtering decoction, concentrating, drying under reduced pressure, and pulverizing to obtain bupleuri radix extract, which is suitable for storage, and can be added into bupleuri radix according to the original dosage to obtain XIAOCHAIHU granule. The preparation method of the small bupleurum particles completely reserves the effective components (saikosaponin a and d) of bupleurum, ensures that the bupleurum is completely qualified for identification, and improves the clinical curative effect.

Claims

1. A radix bupleuri extract is characterized in that: it is Na of bupleurum₂CO₃An aqueous extract; each 1g of the extract is equivalent to 5-6 g of the raw medicinal material.

2. The extract of claim 1, wherein: the Na is₂CO₃The concentration of the aqueous solution is 0.01-0.03% w/v, g/ml, preferably 0.025% w/v, g/ml; the bupleuri radix is coarse powder of bupleuri radix.

3. The extract of claim 1, wherein: each 1g of the extract is equivalent to 5.4g of the raw medicinal material.

4. A method for preparing the extract of Bupleurum chinense Pursh as claimed in any of claims 1-3, wherein the method comprises the following steps: it comprises the following steps:

5. The method according to claim 4, wherein: the extraction is decoction extraction for 2 times, and 5-15 times of v/w, ml/g Na is added for each time₂CO₃Decocting and extracting the water solution for 0.5 to 2 hours.

6. The production method according to claim 4 or 5, characterized in that: the Na is₂CO₃The concentration of the aqueous solution is 0.01-0.03% w/v, g/ml, preferably 0.025% w/v, g/ml.

7. The method according to claim 4, wherein: the preparation method also comprises the steps of taking the concentrated extract, drying the extract under reduced pressure and crushing the dried extract into fine powder; the reduced pressure drying temperature was 60 ℃.

8. A preparation method of Xiaochaihu granules is characterized by comprising the following steps: it comprises the following steps:

2) soaking rhizoma Pinelliae Preparata and rhizoma Zingiberis recens with 70% ethanol, percolating, collecting percolate, recovering ethanol, adding the concentrated solution of step 1), mixing, concentrating, adding bupleuri radix extract and sucrose of claim 1, granulating, and drying.

9. The method of claim 8, wherein: the mass-volume ratio of the radix bupleuri extract to the scutellaria baicalensis, the ginger processed pinellia tuber, the codonopsis pilosula, the ginger, the liquorice and the Chinese date is 27-28 g: 56 g: 56 g: 56g, 56g and 56 g.

10. The method of claim 8, wherein: decocting for 2 times, 1.5 hours each time; and/or, in the step 2), soaking for 24 hours, wherein the amount of the collected percolate is 5-6 v/w, ml/g of the sum of the weight of the ginger processed pinellia tuber and the weight of the ginger.