CN109358141B - Cassia seed reference extract and preparation method and application thereof - Google Patents
Cassia seed reference extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cassia seed contrast extract and a preparation method and application thereof. The preparation method of the semen cassiae control extract comprises the following steps: (1) loading the alcohol extract of semen Cassiae onto macroporous adsorbent resin column to obtain semi-prepared component I; (2) uniformly mixing the semi-prepared component I with blank silica gel for sample mixing to obtain sample mixing silica gel; (3) filling a blank silica gel for chromatography into a column by a dry method, then filling the sample-mixing silica gel, and loading the sample to obtain a silica gel chromatographic column; then eluting the silica gel chromatographic column with an eluent; (4) and (3) averagely dividing the eluted silica gel chromatographic column into 18-24 parts from the column head to the bottom, respectively adding methanol for elution to obtain methanol eluates, detecting, combining the methanol eluates containing index components, and drying to obtain the cassia seed control extract. The preparation method provided by the invention is simple in process and suitable for large-scale preparation.
Description
Technical Field
The invention relates to a cassia seed comparison extract and a preparation method and application thereof, in particular to a cassia seed naphthopyrone comparison extract and a preparation method and application thereof.
Background
The quality control and evaluation of traditional Chinese medicine is an important scientific problem in the field of traditional Chinese medicine research. The existing important quality control method mainly uses single chemical component as reference substance, and performs quality control on Chinese medicinal materials and compound prescription from qualitative and quantitative aspects. The mode of representing important overall quality by single chemical component cannot really control and evaluate the quality of the traditional Chinese medicine and is not consistent with the overall appearance of the traditional Chinese medicine. The complexity of the traditional Chinese medicine components determines that the quality of the traditional Chinese medicine is difficult to objectively evaluate by using a single component or index component as a contrast, however, the quality evaluation mode of the multi-index component is limited by factors such as high separation difficulty of the traditional Chinese medicine chemical contrast, poor monomer stability, overhigh experiment cost and the like.
The traditional Chinese medicine control extract is a traditional Chinese medicine extract with definite main chemical components and content, good stability and higher purity, the proportion of each chemical component is relatively fixed, the content of index components is calibrated, qualitative and quantitative analysis of a plurality of components can be realized simultaneously, and the price is usually lower than the sum of a plurality of component monomer control products. Therefore, the traditional Chinese medicine contrast extract has lower material cost, the detection procedure is simplified, and the detection cost can be greatly saved.
The naphthopyrone compound is a characteristic component with higher content in cassia seed medicinal materials and has the effects of protecting liver, reducing blood fat and the like; however, the reference substance of the naphthopyrone compound monomer in the cassia seed is very rare, and the price is high or even no supply is provided. Zhoudcour et al disclose a preparation method of a cassia seed naphthopyrone control extract (Zhoudcour et al, research on cassia seed naphthopyrone control extract and application thereof in cassia seed medicinal material quality control, China traditional Chinese medicine J, Vol.42, No. 17, pp.3385-3390, 9 months in 2017), which comprises passing an alcohol extract of cassia seed medicinal material through a macroporous adsorption resin column, eluting with water, a 20% ethanol solution and a 50% ethanol solution in sequence, collecting 50% eluent, and evaporating to dryness to obtain a semi-preparation component I; uniformly mixing the semi-prepared component I and silica gel in a ratio of 3:5, and performing vacuum drying to obtain sample-mixed silica gel; loading the sample-mixed silica gel and blank silica gel at a ratio of 1:13 by a dry method, eluting with chloroform-methanol-water (6.5:3.5:0.6), dividing a dry column into 20 parts after the elution is finished, combining silica gels of 2-7 th and 11-17 th, adding methanol for elution, and evaporating to dryness to obtain a semi-preparation component II; purifying the semi-preparation component II by an ODS column, taking a methanol-acetonitrile (2:1) mixed solution as an A phase and water as a B phase to carry out gradient elution, wherein the elution procedures are 30% A (2 times of column volume), 33% A (1 time of column volume), 36% A (1 time of column volume), 39% A (1 time of column volume), 42% A (1 time of column volume) and 45% A (1 time of column volume), then detecting by a thin-layer chromatography, combining eluents containing index components, and drying to obtain the cassia seed naphthopyrone control extract. The process is quite complex, high in cost, small in single sample preparation amount, time-consuming, labor-consuming, low in operability and not suitable for large-scale production. In addition, the preparation process is mainly rich in three naphthopyrones compounds.
Therefore, it is necessary to establish a method for preparing the cassia seed naphthopyrone control extract, which has simple process and is suitable for large-scale production.
Disclosure of Invention
One object of the present invention is to provide a method for preparing a control extract of cassia seed, which is simple in process and suitable for large-scale preparation. Further, the present invention provides a preparation method capable of preparing a cassia seed control extract containing four naphthopyrone index components.
The invention also aims to provide a cassia seed control extract which has high content of naphthopyrone compounds and contains four naphthopyrone index components.
The invention further aims to provide the application of the cassia seed control extract in controlling the quality of cassia seed medicinal materials.
The purpose of the invention is realized by the following technical scheme.
In one aspect, the invention provides a method for preparing a cassia seed control extract, which comprises the following steps:
(1) loading the alcohol extract of the cassia seed on a macroporous adsorption resin column, sequentially carrying out gradient elution by using a first eluent and a second eluent to respectively obtain a first eluent and a second eluent, collecting the second eluent, and drying to obtain a semi-preparation component I;
wherein the first eluent is 15-30 vol% ethanol water, and the dosage of the first eluent is 2-4 times of the volume of the macroporous adsorption resin column; the second eluent is 40-60 vol% ethanol water, and the dosage of the second eluent is 4-6 times of the volume of the macroporous adsorption resin column;
(2) uniformly mixing the semi-prepared component I and blank silica gel for sample mixing according to the weight ratio of 1: 1.8-2.4 to obtain sample mixing silica gel;
(3) filling a blank silica gel for chromatography into a column by a dry method, compacting the column to ensure that the diameter-height ratio is 1: 14.5-18, then filling the sample-mixing silica gel, and compacting the sample-mixing silica gel to obtain a silica gel chromatographic column; then eluting the silica gel chromatographic column with a third eluent;
in the silica gel chromatographic column, the dosage ratio of the semi-prepared component I to the blank silica gel for chromatography is 1g: 24-28 ml; the third eluent is a solvent system of chloroform, methanol and water with the volume ratio of 6.5:3.5: 0.6; and the volume of the third eluent is 1.2-1.4 times of the volume of the silica gel chromatographic column;
(4) averagely dividing the eluted silica gel chromatographic column into 18-24 parts from the column head to the bottom, adding methanol for elution respectively to obtain methanol eluates, detecting the methanol eluates respectively, combining the methanol eluates containing index components, and drying to obtain the semen cassiae control extract;
wherein the index component is semen Cassiae glycoside B2And cassia seed glycoside C2At least one of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C.
According to the preparation method of the present invention, preferably, in the step (1), the semen cassiae alcohol extract is an extract obtained by extracting a semen cassiae medicinal material with 25-40 vol% ethanol water solution as a solvent; the macroporous adsorption resin column is an AB-8 macroporous adsorption resin column, and the dosage ratio of the semen cassiae alcohol extract to the macroporous adsorption resin in the macroporous adsorption resin column is 1g: 30-70 ml.
According to the preparation method of the invention, preferably, in the step (2), the semi-preparation component I is dissolved in an ethanol water solution and mixed with the blank silica gel for sample mixing, and the concentration of the ethanol water solution is 40-60 vol%.
According to the preparation method provided by the invention, in the step (3), the using amount ratio of the semi-preparation component I to the blank silica gel for chromatography is preferably 1g: 25-26 ml.
According to the preparation method provided by the invention, preferably, in the step (3), the blank silica gel for chromatography is filled into the column by a dry method and compacted, so that the diameter-height ratio is 1: 15-15.5.
According to the preparation method of the present invention, preferably, in the step (3), the volume of the third eluent is 1.25 to 1.35 times that of the silica gel chromatographic column.
According to the preparation method of the present invention, preferably, in the step (4), the methanol eluate is detected by thin layer chromatography, the thin layer plate is a silica gel G plate, and the developing solvent is a solvent system of chloroform, methanol and water in a volume ratio of 6.5:3.5: 0.6.
In another aspect, the present invention provides a control extract of cassia seed comprising cassia seed glycoside B2And cassia seed glycoside C2rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C, wherein the cassia seed glycoside B2And cassia seed glycoside C2The total content of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C is higher than 60 wt%.
The cassia seed control extract according to the invention preferably comprises 6-13 wt% of cassia seed glycoside B26-13 wt% of cassia seed glycoside C211-35 wt% of rubrofusarin-6-O-beta-D-gentiobioside and 18-35 wt% of cassia seed glycoside C.
In another aspect, the invention also provides the application of the cassia seed control extract in controlling the quality of cassia seed medicinal materials.
The preparation method of the cassia seed contrast extract has the advantages of relatively simple process, lower cost, stability, reliability and good reproducibility, and is suitable for large-scale production. The cassia seed control extract of the invention comprises cassia seed glycoside B2And cassia seed glycoside C2Red, redThe four naphthopyrones with definite structures and definite contents of the fusamycin-6-O-beta-D-gentiobioside and the cassia seed glycoside C have higher purity and stable content, can realize qualitative and quantitative analysis of the four components at the same time, and has cost far lower than the sum of the four component monomer reference substances.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
< preparation method >
The preparation method of the cassia seed contrast extract comprises the following steps:
(1) loading the alcohol extract of the cassia seed on a macroporous adsorption resin column, sequentially carrying out gradient elution by using a first eluent and a second eluent to respectively obtain a first eluent and a second eluent, collecting the second eluent, and drying to obtain a semi-preparation component I;
wherein the first eluent is 15-30 vol% ethanol water, and the dosage of the first eluent is 2-4 times of the volume of the macroporous adsorption resin column; the second eluent is 40-60 vol% ethanol water, and the dosage of the second eluent is 4-6 times of the volume of the macroporous adsorption resin column;
(2) uniformly mixing the semi-prepared component I and blank silica gel for sample mixing according to the weight ratio of 1: 1.8-2.4 to obtain sample mixing silica gel;
(3) filling a blank silica gel for chromatography into a column by a dry method, compacting the column to ensure that the diameter-height ratio is 1: 14.5-18, then filling the sample-mixing silica gel, and compacting the sample-mixing silica gel to obtain a silica gel chromatographic column; then eluting the silica gel chromatographic column with a third eluent;
in the silica gel chromatographic column, the dosage ratio of the semi-prepared component I to the blank silica gel for chromatography is 1g: 24-28 ml; the third eluent is a solvent system of chloroform, methanol and water with the volume ratio of 6.5:3.5: 0.6; and the volume of the third eluent is 1.2-1.4 times of the volume of the silica gel chromatographic column;
(4) averagely dividing the eluted silica gel chromatographic column into 18-24 parts from the column head to the bottom, adding methanol for elution respectively to obtain methanol eluates, detecting the methanol eluates respectively, combining the methanol eluates containing index components, and drying to obtain the semen cassiae control extract;
wherein the index component is semen Cassiae glycoside B2And cassia seed glycoside C2At least one of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C.
According to the preparation method, in the step (1), the semen cassiae alcohol extract is an extract obtained by extracting a semen cassiae medicinal material by using an ethanol water solution as a solvent. The concentration of the ethanol aqueous solution may be 25 to 40 vol%, preferably 28 to 35 vol%, and more preferably 30 vol%.
According to a preferred embodiment of the present invention, the semen cassiae alcohol extract is prepared by the following steps: heating and refluxing a cassia seed medicinal material by using 30 vol% ethanol water solution as a solvent for 3 times, wherein the dosage of the solvent is 10 times of that of the cassia seed medicinal material each time, and the extraction time is 2 hours each time, so as to obtain an alcohol extract; and recovering the solvent from the alcohol extract, and drying to obtain the semen cassiae alcohol extract.
According to the preparation method, in the step (1), the dosage ratio of the semen cassiae alcohol extract to the macroporous adsorption resin in the macroporous adsorption resin column is 1g to 30-70 ml, preferably 1g to 40-60 ml, and more preferably 1g to 50 ml. The semen cassiae alcohol extract can be dispersed by water to obtain a sample solution, and then the sample solution is loaded on a macroporous adsorption resin column. The content of the semen cassiae alcohol extract in the sample loading liquid can be 0.01-0.1 g/ml, preferably 0.02-0.08 g/ml, and more preferably 0.05 g/ml. The macroporous adsorption resin column is preferably an AB-8 macroporous adsorption resin column. The diameter-height ratio of the macroporous adsorption resin column can be 1: 6-10, preferably 1: 7-9, and more preferably 1: 8.
According to the preparation method of the invention, in the step (1), the first eluent is preferably 15-25 vol% ethanol water solution, and more preferably 20 vol% ethanol water solution. The dosage of the first eluent is preferably 2.5-3.5 times of the volume of the macroporous adsorption resin column, and more preferably 3 times of the volume of the macroporous adsorption resin column. The second eluent is preferably 45-55 vol% ethanol water solution, and more preferably 50 vol% ethanol water solution. The amount of the second eluent is preferably 4.5-5.5 times, more preferably 5 times of the volume of the macroporous adsorption resin column.
According to the preparation method, in the step (2), the weight ratio of the semi-preparation component I to the blank silica gel for sample mixing is preferably 1: 1.9-2.2, and more preferably 1: 2. The semi-preparation component I can be dissolved in a small amount of ethanol water solution and then mixed with the blank silica gel for sample mixing, so that the sample mixing silica gel which is uniformly mixed can be obtained. The ethanol aqueous solution can be 40-60 vol% ethanol aqueous solution, preferably 45-55 vol% ethanol aqueous solution, and more preferably 50 vol% ethanol aqueous solution.
According to the preparation method, in the step (3), the dosage ratio of the semi-preparation component I to the blank silica gel for chromatography in the silica gel chromatographic column is 1g: 24-28 ml, and preferably 1g: 25-26 ml. In the step (3), the blank silica gel for chromatography is filled into a column by a dry method and compacted, so that the diameter-height ratio is 1: 14.5-18, preferably 1: 14.5-17, and more preferably 1: 15. When the diameter-height ratio is too large, the naphthopyrone compounds of the stigma, in particular the cassia seed glycoside B2And cassia seed glycoside C2Too high a residual rate; when the diameter-height ratio is too small, naphthopyrone compounds of stigma, especially cassia seed glycoside B2And cassia seed glycoside C2Although the residual rate of (2) is low, the amount of silica gel required is increased, and the production cost is increased. When the sample loading amount and the diameter-height ratio of the semi-preparation component I are adopted, the residue rate of the naphthopyrone compound on column heads is small, the required silica gel amount is moderate, and the preparation cost is reduced on the basis of ensuring the stable content of the naphthopyrone component.
According to the preparation method of the present invention, in the step (3), preferably, the volume of the third eluent is 1.25 to 1.35 times, more preferably 1.3 times, the volume of the silica gel chromatographic column.
In the present invention, the blank silica gel for sample mixing and the blank silica gel for chromatography may be the same kind of blank silica gel, but for convenience of understanding, they are expressed as blank silica gel for sample mixing and blank silica gel for chromatography. The particle size of the blank silica gel can be 150-350 meshes, and preferably 200-300 meshes.
According to the preparation method of the present invention, in the step (4), preferably, the silica gel column after elution is divided into 19 to 22 parts, more preferably 20 to 21 parts, on average from the column head to the bottom. And (4) eluting the methanol until the methanol is colorless, so as to obtain methanol eluent. The methanol eluate can be detected by thin layer chromatography, or by high performance liquid chromatography, preferably by thin layer chromatography. The thin layer plate adopted by the thin layer chromatography can be a silica gel G plate, and the developing agent can adopt a solvent system of chloroform, methanol and water with the volume ratio of 6.5:3.5: 0.6. The developer system can convert semen Cassiae glycoside B2And cassia seed glycoside C2The four naphthopyrones of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C are fully unfolded and the separation degree of adjacent components is good.
The preparation method can obtain the cassia seed control extract with high purity of naphthopyrones component under the condition of not carrying out complex purification of ODS (ozone depleting substance) column by accurately adjusting the parameters of the proportion of the semi-prepared component I in the sample-mixing silica gel to the blank silica gel, the dosage ratio of the semi-prepared component I in the silica gel chromatographic column to the blank silica gel, the elution volume of the third eluent and the like, and the cassia seed control extract contains the cassia seed glycoside B2And cassia seed glycoside C2Four naphthopyrones, namely rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C, are more favorable for the quality control of the cassia seed medicinal material. The preparation method has the advantages of relatively simple process, lower material cost and time cost, stability, reliability and good reproducibility, and is suitable for large-scale production.
< Cassia seed control extract >
The cassia seed control extract of the invention contains naphthopyrones component with the content higher than 60 wt%, preferably higher than 62 wt%. Cassia seed glycoside B2And cassia seed glycoside C2The content of the four naphthopyrones components has stronger correlation in the raw medicinal material cassia seed and the contrast extract, and when the content of the index components in the raw medicinal material cassia seed is higher, the content of the index components in the contrast extract prepared by the method of the invention is higherThe content is relatively high.
The cassia seed control extract according to the invention preferably comprises 6-13 wt% of cassia seed glycoside B26-13 wt% of cassia seed glycoside C211-35 wt% of rubrofusarin-6-O-beta-D-gentiobioside and 18-35 wt% of cassia seed glycoside C.
The control extract of the present invention was prepared according to the above-described preparation method.
Compared with the prior art, the cassia seed contrast extract contains four naphthopyrones with definite structures and contents, has high purity and stable index component content, can realize qualitative and quantitative analysis of the four components at the same time, and has the cost far lower than the sum of the plurality of index component monomer contrast products.
< use of cassia seed control extract >
The invention also provides application of the cassia seed control extract in controlling the quality of cassia seed medicinal materials. The cassia seed control extract can be used for detecting the quality of a cassia seed medicinal material. The application comprises the cassia seed glycoside B2And cassia seed glycoside C2Reference extract of semen Cassiae with known content of rubrofusarin-6-O-beta-D-gentiobioside and semen Cassiae glycoside C is used as reference, and semen Cassiae glycoside B in semen Cassiae medicinal material is used as reference2And cassia seed glycoside C2And the content of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C is measured.
The semen Cassiae control extract can be prepared by pre-adding semen Cassiae glycoside B2Reference substance, semen Cassiae glycoside C2Calibrating reference substances, rubrofusarin-6-O-beta-D-gentiobioside reference substance and semen Cassiae glycoside C reference substance as reference substances to determine semen Cassiae glycoside B in the semen Cassiae reference extract2And cassia seed glycoside C2rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C. Multiple batches of the cassia seed control extract can be calibrated simultaneously.
The present invention will be described in detail below with reference to specific examples.
Preparation example 1
Heating and refluxing a cassia seed medicinal material by using 30 vol% ethanol water solution as a solvent for 3 times, wherein the dosage of the solvent is 10 times of that of the cassia seed medicinal material each time, and the extraction time is 2 hours each time, so as to obtain an alcohol extract; and recovering the solvent from the alcohol extract, and drying to obtain the semen cassiae alcohol extract. Adding water into the semen Cassiae alcohol extract, and dispersing to 0.05g/ml to obtain the sample solution.
Loading the AB-8 macroporous adsorption resin into a column to obtain the AB-8 macroporous adsorption resin column with the diameter-height ratio of 1: 8.
Example 1
The cassia seed control extract was prepared as follows:
(1) loading the sample solution of preparation example 1 onto an AB-8 macroporous adsorption resin column of preparation example 1, enabling the dosage ratio of the semen cassiae alcohol extract to the AB-8 macroporous adsorption resin to be 1g to 50ml, sequentially carrying out gradient elution by using a first eluent and a second eluent to respectively obtain a first eluent and a second eluent, collecting the second eluent, and drying to obtain a semi-preparation component I; wherein the first eluent is 20 vol% ethanol water solution, and the dosage of the first eluent is 3 times of the volume of the macroporous adsorption resin column; the second eluent is 50 vol% ethanol water solution, and the dosage of the second eluent is 5 times of the volume of the macroporous adsorption resin column;
(2) dissolving a semi-prepared component I in a small amount of 50 vol% ethanol aqueous solution, and then uniformly mixing the semi-prepared component I with blank silica gel for sample mixing, wherein the weight ratio of the semi-prepared component I to the blank silica gel for sample mixing is 1:2, so as to obtain sample mixing silica gel;
(3) filling a blank silica gel for chromatography into a column by a dry method, compacting the column until the diameter-height ratio is 1:15, then filling the sample-mixing silica gel, and compacting the sample-mixing silica gel to obtain a silica gel chromatographic column; then eluting the silica gel chromatographic column with a third eluent;
wherein in the silica gel chromatographic column, the dosage ratio of the semi-prepared component I to the blank silica gel for chromatography is 1g:26 ml; the third eluent is a solvent system of chloroform, methanol and water with the volume ratio of 6.5:3.5: 0.6; and the volume of the third eluent is 1.3 times of the volume of the silica gel chromatographic column;
(4) the eluted silica gel chromatographic column is arranged from the column head to the column headAnd (3) evenly dividing the bottom into 20 parts of silica gel, respectively adding methanol for elution to obtain methanol eluates, respectively detecting the methanol eluates, combining the methanol eluates containing index components, and drying to obtain the semen cassiae control extract. The index component comprises semen Cassiae glycoside B2And cassia seed glycoside C2rubrofusarin-6-O-beta-D-gentiobioside and/or cassia seed glycoside C.
Example 2
The same batch of cassia seed medicinal material in example 1 was taken, and the cassia seed control extract was obtained in the same manner as in example 1 except that the blank silica gel dry method for chromatography in step (3) was used for packing and compacting the column, the diameter of the column was not changed, and the diameter-height ratio was 1: 17.
Comparative example 1
The same batch of cassia seed medicinal material as in example 1 was taken, and the cassia seed control extract was obtained in the same manner as in example 1 except that the blank silica gel dry method for chromatography in step (3) was used for packing and compacting the column with the diameter of the column unchanged so that the ratio of the diameter to the height was 1: 13.
Experimental example 1
And (3) eluting the column cap silica gel of the embodiment 1-2 and the comparative example 1 with methanol, evaporating to dryness and drying to obtain column cap extract powder. The cassia seed glycosides B in the cassia seed control extracts of the examples 1-2 and the comparative example 1 were respectively detected2And cassia seed glycoside C2Content of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C, and content of cassia seed glycoside B in column extract powder of examples 1-2 and comparative example 12And cassia seed glycoside C2The residual ratio of (2). The residual rate of the components in the stigma extract powder is equal to the amount of the components in the stigma extract powder/the total amount of the components in the sample feeding liquid. The results are shown in Table 1.
TABLE 1
When the ratio of column diameter to height is 1:13, the cassia seed glycoside B in column head2And cassia seed glycoside C2Too high a residual rate; diameter of chromatographic columnWhen the height ratio is 1:15 and 1:17, the cassia seed glycoside B in the stigma2And cassia seed glycoside C2The residual rate of the naphthopyrones is reduced, and the total content of the naphthopyrones is increased. Wherein when the ratio of the column diameter to the column diameter is 1:17, the cassia seed glycoside B in the column head2And cassia seed glycoside C2The ratio of the residual rate to the diameter-height ratio of 1:15 is slightly reduced, but the reduction range is small, and the use amount of silica gel is obviously increased, so that the ratio of the diameter-height ratio of 1:15 is more suitable for industrial production on the whole.
Experimental example 2
In addition, 3 batches of semen cassiae control extract samples are prepared from another semen cassiae medicinal material by the same preparation process as the example 1. Accurately weighing 12mg of each sample, placing the sample in a 50mL measuring flask, adding 30% methanol for ultrasonic dissolution, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane, accurately sucking 10 mu L of sample injection, and performing content measurement by high performance liquid chromatography, wherein the results are shown in Table 2.
TABLE 2
As can be seen from the results in Table 2, the content of different samples is basically stable, and the preparation method is stable and reliable.
Experimental example 3
And taking S1-S10 cassia seeds from different producing areas and batches, respectively preparing 10 batches of cassia seed contrast extract samples by adopting the same preparation process in the example 1, and calculating the yield of the contrast extract according to the medicinal materials. The results of the content measurement by high performance liquid chromatography are shown in Table 3.
TABLE 3
The experimental results in table 3 show that the control extracts prepared from different batches of cassia seed medicinal materials are obviously different, but the total content of 4 index components of the control extract reaches more than 60 wt%, which indicates that the preparation process is stable and feasible, and can be used for preparing the cassia seed control extracts from different production places and batches of medicinal materials.
Experimental example 4
Collecting 34kg of S5 semen Cassiae material, and preparing semen Cassiae control extract according to the preparation method of example 1 to obtain 508.95g of control extract, wherein semen Cassiae glycoside B2And cassia seed glycoside C2The contents of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C are respectively 10.81 wt%, 11.14 wt%, 20.33 wt% and 28.29 wt%, and the total content is 70.57 wt%. The result shows that the process can be used for mass production and is stable.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (8)
1. The preparation method of the cassia seed control extract is characterized by comprising the following steps:
(1) loading the alcohol extract of the cassia seed on a macroporous adsorption resin column, sequentially carrying out gradient elution by using a first eluent and a second eluent to respectively obtain a first eluent and a second eluent, collecting the second eluent, and drying to obtain a semi-preparation component I;
wherein the first eluent is 15-30 vol% ethanol water, and the dosage of the first eluent is 2-4 times of the volume of the macroporous adsorption resin column; the second eluent is 40-60 vol% ethanol water, and the dosage of the second eluent is 4-6 times of the volume of the macroporous adsorption resin column;
(2) uniformly mixing the semi-prepared component I and blank silica gel for sample mixing according to the weight ratio of 1: 1.8-2.4 to obtain sample mixing silica gel;
(3) filling a blank silica gel for chromatography into a column by a dry method, compacting the column to ensure that the diameter-height ratio is 1: 14.5-18, then filling the sample-mixing silica gel, and compacting the sample-mixing silica gel to obtain a silica gel chromatographic column; then eluting the silica gel chromatographic column with a third eluent;
in the silica gel chromatographic column, the dosage ratio of the semi-prepared component I to the blank silica gel for chromatography is 1g: 25-26 ml; the third eluent is a solvent system of chloroform, methanol and water with the volume ratio of 6.5:3.5: 0.6; and the volume of the third eluent is 1.25-1.35 times of the volume of the silica gel chromatographic column;
(4) averagely dividing the eluted silica gel chromatographic column into 18-24 parts from the column head to the bottom, adding methanol for elution respectively to obtain methanol eluates, detecting the methanol eluates respectively, combining the methanol eluates containing index components, and drying to obtain the semen cassiae control extract;
wherein the index component is semen Cassiae glycoside B2And cassia seed glycoside C2At least one of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C.
2. The preparation method according to claim 1, wherein in the step (1), the semen cassiae alcohol extract is an extract obtained by extracting a semen cassiae medicinal material by using 25-40 vol% ethanol water solution as a solvent; the macroporous adsorption resin column is an AB-8 macroporous adsorption resin column, and the dosage ratio of the semen cassiae alcohol extract to the macroporous adsorption resin in the macroporous adsorption resin column is 1g: 30-70 ml.
3. The method according to claim 1, wherein in step (2), the semi-preparative component I is dissolved in an aqueous ethanol solution, and mixed with the blank silica gel for sample mixing, and the concentration of the aqueous ethanol solution is 40 to 60 vol%.
4. The preparation method according to claim 1, wherein in the step (3), a blank silica gel for chromatography is filled into a column by a dry method and compacted to ensure that the diameter-height ratio is 1: 15-15.5.
5. The preparation method according to claim 1, wherein in the step (4), the methanol eluate is detected by thin layer chromatography, the thin layer plate is a silica gel G plate, and the developing solvent is a solvent system of chloroform, methanol and water in a volume ratio of 6.5:3.5: 0.6.
6. The cassia seed control extract prepared by the preparation method of any one of claims 1 to 5, wherein the cassia seed control extract comprises cassia seed glycoside B2And cassia seed glycoside C2rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C, wherein the cassia seed glycoside B2And cassia seed glycoside C2The total content of rubrofusarin-6-O-beta-D-gentiobioside and cassia seed glycoside C is higher than 60 wt%.
7. The cassia seed control extract as claimed in claim 6, which comprises 6-13 wt% of cassia seed glycoside B26-13 wt% of cassia seed glycoside C211-35 wt% of rubrofusarin-6-O-beta-D-gentiobioside and 18-35 wt% of cassia seed glycoside C.
8. Use of the cassia seed control extract as claimed in claim 6 or 7 for controlling the quality of cassia seed medicinal material.
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