CN109358141A - Cassia seed reference extract and its preparation method and application - Google Patents
Cassia seed reference extract and its preparation method and application Download PDFInfo
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- G01N30/02—Column chromatography
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- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/14—Preparation by elimination of some components
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
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Abstract
The invention discloses a kind of cassia seed reference extracts and its preparation method and application.The preparation method of cassia seed reference extract includes: that large pore resin absorption column on cassia seed alcohol extract will partly be prepared components I by (1)；(2) by it is described partly prepare components I with mix sample and be uniformly mixed with blank silica gel, obtain mixing sample silica gel；(3) by chromatography blank silica gel dry column-packing, be reloaded into it is described mix sample silica gel, loading, to obtain silica gel chromatographic column；Then the silica gel chromatographic column is eluted with eluant, eluent；(4) silica gel chromatographic column after elution is divided into 18~24 parts from column cap to bottom, respectively plus methanol affords meoh eluate, and detection merges the meoh eluate containing index components, and it is dry, obtain the cassia seed reference extract.Preparation method simple process of the invention and suitable large scale preparation.
Description
Technical field
The present invention relates to a kind of cassia seed reference extract and its preparation method and application, especially a kind of cassia seed naphtho-
Pyranone reference extract and its preparation method and application.
Background technique
Traditional Chinese medicine quality control and evaluation are the important scientific issues in traditional Chinese medicine research field.Existing important quality control method
It is mainly studied with unification and is divided into reference substance, quality control is carried out to Chinese medicine and compound from qualitative and quantitative angle.It is this with
The mode that single chemical component characterizes important total quality really can not control and evaluate traditional Chinese medicine quality, also with traditional Chinese medicine Overall View
It is not consistent.The complexity of traditional Chinese medicine ingredients determines single component or index components are that control is difficult to objectively evaluate the quality of Chinese medicine,
However, the Quality Evaluation Model of multi-target ingredient again, monomer stability big by traditional Chinese chemical contrast separating difficulty it is poor and
The restriction of the excessively high factor of experimental cost.
Chinese medicine reference extract refers to that main chemical compositions and content are clear, stability is good, the higher traditional Chinese medicine extraction of purity
Object, each chemical component ratio is relatively fixed, and index component content has been demarcated, and may be implemented simultaneously to determine multiple components
Property and quantitative analysis, price are usually less than the summation of multiple constituent monomers reference substances.Therefore, the substance of Chinese medicine reference extract
Cost is relatively low, detects program simplification, can greatly save testing cost.
Aphthopyrans ketone compound is that a kind of content in Cassia obtusifolia L is higher, has the effects that liver protection, reducing blood lipid
Characteristic chemical constituent；But aphthopyrans ketone compound monomer reference substance is very rare in cassia seed, it is expensive even without
Supply.Zhou Deyong etc. discloses preparation method (Zhou Deyong etc. the, " Cassia of a kind of cassia seed aphthopyrans ketone reference extract
Sub- aphthopyrans ketone reference extract research and its application in the control of Cassia obtusifolia L quality ", CHINA JOURNAL OF CHINESE MATERIA MEDICA, the
The phase of volume 42 the 17th, page 3385~3390, in September, 2017), be by large pore resin absorption column on Cassia obtusifolia L alcohol extract, according to
Secondary water, 20% ethanol solution, the elution of 50% ethanol solution, collect after 50% eluent is evaporated and are partly prepared components I；It will be partly
It prepares components I and silica gel and sample is mixed with the ratio uniform of 3:5, be dried in vacuo, obtain mixing sample silica gel；This is mixed into sample silica gel and blank silicon
Glue is eluted with chloroform-methanol-water (6.5:3.5:0.6) with the ratio dry method loading of 1:13, dry column is divided into 20 after the completion of elution
Position merges the silica gel of 2-7,11-17, adds methanol to elute, be evaporated, partly prepared compositionⅱ；Compositionⅱ will partly be prepared to lead to
ODS column purification is crossed, is that B phase carries out gradient elution with water, elution program is with methanol-acetonitrile (2:1) mixed solution for A phase
30%A (2 times of column volumes), 33%A (1 times of column volume), 36%A (1 times of column volume), 39%A (1 times of column volume), (1 times of 42%A
Column volume), 45%A (1 times of column volume), then by thin-layered chromatography examine know, merge the eluent containing index components, do
It is dry, obtain cassia seed aphthopyrans ketone reference extract.The technique is sufficiently complex, at high cost, and single sample preparation amount is few, time-consuming to take
Power, operability is not strong, more unsuitable large-scale production.In addition, the preparation process is mainly enriched with three kinds of aphthopyrans ketones
Close object.
Therefore, it is necessary to establish a kind of simple process and be suitable for the cassia seed aphthopyrans ketone reference extract of mass production
Preparation method.
Summary of the invention
It is an object of the present invention to provide a kind of preparation method of cassia seed reference extract, this method simple processes
And it is suitble to large scale preparation.Further, the present invention provides a kind of preparation method, being capable of cassia seed reference extract obtained
Containing there are four types of aphthopyrans ketone index components.
Another object of the present invention is to provide a kind of cassia seed reference extract, naphthopyrone kind compound contents
Height, and containing there are four types of aphthopyrans ketone index components.
A further object of the present invention is to provide the cassia seed reference extracts for controlling Cassia obtusifolia L quality
Purposes.
The purpose of the present invention is what is be achieved through the following technical solutions.
On the one hand, the present invention provides a kind of preparation method of cassia seed reference extract, includes the following steps:
(1) by large pore resin absorption column on cassia seed alcohol extract, ladder successively is carried out with the first eluant, eluent and the second eluant, eluent
Degree elution, respectively obtains the first eluent and the second eluent, collects second eluent, dry, obtains half preparation component
Ⅰ；
Wherein, first eluant, eluent is 15~30vol% ethanol water, and dosage is the large pore resin absorption column body
2~4 times of long-pending amounts；Second eluant, eluent is 40~60vol% ethanol water, and dosage is the large pore resin absorption column body
4~6 times of long-pending amounts；
(2) by it is described partly prepare components I with mix sample and mixed with blank silica gel according to the ratio that weight ratio is 1:1.8~2.4
Uniformly, it obtains mixing sample silica gel；
(3) it by chromatography blank silica gel dry column-packing and is compacted, compares diameter height for 1:14.5~18, be reloaded into and described mix sample
Silica gel, compacting, to obtain silica gel chromatographic column；Then the silica gel chromatographic column is eluted with third eluant, eluent；
Wherein, in the silica gel chromatographic column, partly preparing components I and the chromatography with the amount ratio of blank silica gel is 1g:24
~28ml；The third eluant, eluent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution；And described
The volume of three eluant, eluents is 1.2~1.4 times of amounts of the silica gel chromatograph column volume；
(4) silica gel chromatographic column after elution is divided into 18~24 parts from column cap to bottom, respectively plus methanol elutes
To meoh eluate, the meoh eluate is detected respectively, and the meoh eluate containing index components is merged, it is dry, it obtains
The cassia seed reference extract；
Wherein, the index components are cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and
At least one of cassia seed glycosides C.
Preparation method according to the present invention, it is preferable that in step (1), the cassia seed alcohol extract refers to Cassia obtusifolia L
The extract obtained using the ethanol water of 25~40vol% as solvent extraction；The large pore resin absorption column is AB-8
Large pore resin absorption column, and the amount ratio of the macroporous absorbent resin in the cassia seed alcohol extract and the large pore resin absorption column
For 1g:30~70ml.
Preparation method according to the present invention, it is preferable that in step (2), the components I that partly prepares is dissolved in ethanol water
In mix sample with described and mixed with blank silica gel, and the concentration of the ethanol water is 40~60vol%.
Preparation method according to the present invention, it is preferable that described partly to prepare components I and chromatography blank silica gel in step (3)
Amount ratio be 1g:25~26ml.
Preparation method according to the present invention, it is preferable that in step (3), by chromatography blank silica gel dry column-packing and it is compacted,
Compare diameter height for 1:15~15.5.
Preparation method according to the present invention, it is preferable that in step (3), the volume of the third eluant, eluent is the silica gel
1.25~1.35 times of amounts of chromatography column volume.
Preparation method according to the present invention, it is preferable that in step (4), the detection of the meoh eluate passes through thin layer color
Spectrum inspection know, lamellae be silica G plate, solvent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution.
On the other hand, the present invention provides a kind of cassia seed reference extract comprising cassiaside B2, cassia seed glycosides C2, it is red
Sickle mycin -6-O- β-D- O-gentibioside and cassia seed glycosides C, and the cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O-
The total content of β-D- O-gentibioside and cassia seed glycosides C are higher than 60wt%.
Cassia seed reference extract according to the present invention, it is preferable that it includes the cassiaside B of 6~13wt%2, 6~
The cassia seed glycosides C of 13wt%2, rubrofusarin -6-O- β-D- the O-gentibioside of 11~35wt% and the Cassia of 18~35wt%
Sub- glycosides C.
In another aspect, the present invention also provides the use that the cassia seed reference extract is used to control Cassia obtusifolia L quality
On the way.
The preparation method technique of cassia seed reference extract of the invention is relatively easy, and cost is lower, reliable and stable, reappears
Property it is good, be suitble to large-scale production.Cassia seed reference extract of the invention includes cassiaside B2, cassia seed glycosides C2, red sickle it is mould
Element -6-O- β-D- O-gentibioside and these four structures of cassia seed glycosides C are clear and the exact aphthopyrans ketones component of content, pure
Higher, stable content is spent, may be implemented to carry out qualitative and quantitative analysis to aforementioned four component simultaneously, and cost is far below above-mentioned
The summation of four constituent monomers reference substances.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but protection scope of the present invention is not limited to
This.
< preparation method >
The preparation method of cassia seed reference extract of the invention includes the following steps:
(1) by large pore resin absorption column on cassia seed alcohol extract, ladder successively is carried out with the first eluant, eluent and the second eluant, eluent
Degree elution, respectively obtains the first eluent and the second eluent, collects second eluent, dry, obtains half preparation component
Ⅰ；
Wherein, first eluant, eluent is 15~30vol% ethanol water, and dosage is the large pore resin absorption column body
2~4 times of long-pending amounts；Second eluant, eluent is 40~60vol% ethanol water, and dosage is the large pore resin absorption column body
4~6 times of long-pending amounts；
(2) by it is described partly prepare components I with mix sample and mixed with blank silica gel according to the ratio that weight ratio is 1:1.8~2.4
Uniformly, it obtains mixing sample silica gel；
(3) it by chromatography blank silica gel dry column-packing and is compacted, compares diameter height for 1:14.5~18, be reloaded into and described mix sample
Silica gel, compacting, to obtain silica gel chromatographic column；Then the silica gel chromatographic column is eluted with third eluant, eluent；
Wherein, in the silica gel chromatographic column, partly preparing components I and the chromatography with the amount ratio of blank silica gel is 1g:24
~28ml；The third eluant, eluent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution；And described
The volume of three eluant, eluents is 1.2~1.4 times of amounts of the silica gel chromatograph column volume；
(4) silica gel chromatographic column after elution is divided into 18~24 parts from column cap to bottom, respectively plus methanol elutes
To meoh eluate, the meoh eluate is detected respectively, and the meoh eluate containing index components is merged, it is dry, it obtains
The cassia seed reference extract；
Wherein, the index components are cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and
At least one of cassia seed glycosides C.
Preparation method according to the present invention, in step (1), the cassia seed alcohol extract refers to Cassia obtusifolia L using ethyl alcohol
The extract that aqueous solution is obtained as solvent extraction.The concentration of the ethanol water can be 25~40vol%, preferably 28
~35vol%, more preferably 30vol%.
A preferred embodiment according to the present invention, the cassia seed alcohol extract the preparation method comprises the following steps: by cassia seed
Medicinal material uses the ethanol water of 30vol% as solvent heating and refluxing extraction 3 times, and each solvent usage is the 10 of Cassia obtusifolia L
It measures again, each extraction time is 2h, obtains alcohol extract；It is dry by the alcohol extract recycling design, obtain the cassia seed alcohol extracting
Object.
Preparation method according to the present invention, in step (1), in the cassia seed alcohol extract and the large pore resin absorption column
Macroporous absorbent resin amount ratio be 1g:30~70ml, preferably 1g:40~60ml, more preferably 1g:50ml.It is described to determine
Pine torch alcohol extract water-dispersible can obtain sample solution, then upper large pore resin absorption column.Cassia seed alcohol extracting in the sample solution
The content of object can be 0.01~0.1g/ml, preferably 0.02~0.08g/ml, more preferably 0.05g/ml.The macropore is inhaled
Attached resin column is preferably AB-8 large pore resin absorption column.The diameter height ratio of the large pore resin absorption column can be 1:6~10, preferably
For 1:7~9, more preferably 1:8.
Preparation method according to the present invention, in step (1), first eluant, eluent is preferably 15~25vol% ethanol water
Solution, more preferably 20vol% ethanol water.The dosage of first eluant, eluent is preferably the large pore resin absorption column body
2.5~3.5 times of long-pending amounts, more preferably 3 times amounts.Second eluant, eluent is preferably 45~55vol% ethanol water, more excellent
It is selected as 50vol% ethanol water.The dosage of second eluant, eluent be preferably the large pore resin absorption column volume 4.5~
5.5 times of amounts, more preferably 5 times amounts.
Preparation method according to the present invention, it is described partly to prepare components I and mix sample blank silica gel according to weight in step (2)
Amount is than being preferably 1:1.9~2.2, more preferably 1:2.It is described partly to prepare after components I is soluble in a small amount of ethanol water
It mixes sample with described and is mixed with blank silica gel, uniformly mixed mix sample silica gel to be conducive to obtain.The ethanol water can be with
For 40~60vol% ethanol water, preferably 45~55vol% ethanol water, more preferably 50vol% ethyl alcohol is water-soluble
Liquid.
Preparation method according to the present invention, it is described partly to prepare components I and chromatography in the silica gel chromatographic column in step (3)
Amount ratio with blank silica gel is 1g:24~28ml, preferably 1g:25~26ml.In step (3), by chromatography blank silica gel
Dry column-packing is simultaneously compacted, and compares diameter height for 1:14.5~18, preferably 1:14.5~17, more preferably 1:15.The high ratio of diameter
When excessive, the aphthopyrans ketone compound of column cap, especially cassiaside B2, cassia seed glycosides C2Residual rate it is excessive；The diameter
Height than it is too small when, the aphthopyrans ketone compound of column cap, especially cassiaside B2, cassia seed glycosides C2Although residual rate drop
It is low, but required silica gel usage amount increases, preparation cost is promoted.Using the above-mentioned applied sample amount for partly preparing components I of the invention
And diameter height than when, aphthopyrans ketone compound is small in column cap residual rate, and required silica gel amount is moderate, is ensuring aphthopyrans
On the basis of ketones component stable content, preparation cost is reduced.
Preparation method according to the present invention, in step (3), it is preferable that the volume of the third eluant, eluent is the silica gel
1.25~1.35 times of amounts of chromatography column volume, more preferably 1.3 times amounts.
It is described to mix sample blank silica gel and chromatography and can be same kind of blank silica gel with blank silica gel in the present invention,
Sample blank silica gel and chromatography blank silica gel are mixed only to facilitate understanding and being stated that.The partial size of the blank silica gel can be with
In 150~350 mesh, preferably 200~300 mesh.
Preparation method according to the present invention, in step (4), it is preferable that by the silica gel chromatographic column after elution from column cap the bottom of to
Portion is divided into 19~22 parts, more preferably 20~21 parts.In step (4), the methanol be eluted to it is colourless, obtain methanol elution
Liquid.The detection of the meoh eluate can be examined by thin-layer chromatography to be known, or is examined and known by high performance liquid chromatography, is preferably passed through
Thin-layer chromatography inspection is known.It can be silica G plate that the lamellae used is known in thin-layer chromatography inspection, solvent can use volume ratio for
6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution.The solvent system can be by cassiaside B2, cassia seed glycosides C2、
Tetra- kinds of aphthopyrans ketones components of rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C are sufficiently spread out and adjacent component
Separating degree it is good.
Preparation method of the invention is by mixing the proportion, the silica gel chromatograph that partly prepare components I and blank silica gel in sample silica gel
The accurate adjusting that the parameters such as the amount ratio of components I and blank silica gel and the elution volume of third eluant, eluent are partly prepared in column, can
It is extracted with obtaining the higher cassia seed control of aphthopyrans ketones component purity without ODS column complicated purification
Object, and contain cassiaside B in the cassia seed reference extract2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside
And tetra- kinds of aphthopyrans ketones components of cassia seed glycosides C, it is more advantageous to the quality control of Cassia obtusifolia L.Preparation method of the invention
Technique is relatively easy, and Material Cost and time cost are lower, reliable and stable, favorable reproducibility, is suitble to large-scale production.
< cassia seed reference extract >
The content containing aphthopyrans ketones component is higher than 60wt% in cassia seed reference extract of the invention, preferably high
In 62wt%.Cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C, these four naphthalenes
And the content of pyranone ingredient correlation in cassia seed raw medicinal material and reference extract is stronger, when used cassia seed medicated
When the content of these index components is higher in material raw material, these indexs in resulting reference extract are prepared using the method for the present invention
The content of ingredient is relatively also higher.
Cassia seed reference extract according to the present invention, it is preferable that it includes the cassiaside B of 6~13wt%2, 6~
The cassia seed glycosides C of 13wt%2, rubrofusarin -6-O- β-D- the O-gentibioside of 11~35wt% and the Cassia of 18~35wt%
Sub- glycosides C.
Reference extract of the invention is prepared according to above-mentioned preparation method.
Compared with the existing technology, cassia seed reference extract of the invention includes that four kinds of structures are clear and the exact naphthalene of content
And pyranone ingredient, purity is higher, and index components content is stablized, and may be implemented simultaneously to carry out four components qualitative and quantitative
Analysis, and cost is far below the summation of above-mentioned multiple indicator component monomer reference substances.
The purposes > of < cassia seed reference extract
The present invention also provides the purposes that the cassia seed reference extract is used to control Cassia obtusifolia L quality.Of the invention
Cassia seed reference extract can be used for detecting the quality of Cassia obtusifolia L.The purposes includes with cassiaside B2, cassia seed glycosides
C2, cassia seed reference extract known to rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C content be control, it is right
Cassiaside B in Cassia obtusifolia L2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C content
It is measured.
The cassia seed reference extract can be in advance with cassiaside B2Reference substance, cassia seed glycosides C2Reference substance, red sickle are mould
Element -6-O- β-D- O-gentibioside reference substance and cassia seed glycosides C reference substance are that control is demarcated, with the determination cassia seed pair
According to cassiaside B in extract2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C content.
The cassia seed reference extract of multiple batches can be demarcated simultaneously.
Below by way of specific embodiment, the present invention is described in detail.
Preparation example 1
Use the ethanol water of 30vol% as solvent heating and refluxing extraction 3 times Cassia obtusifolia L, each solvent usage
For 10 times of amounts of Cassia obtusifolia L, each extraction time is 2h, obtains alcohol extract；It is dry by the alcohol extract recycling design, it obtains
To cassia seed alcohol extract.Add moisture to be dissipated to 0.05g/ml the cassia seed alcohol extract, obtains sample solution.
AB-8 macroporous absorbent resin is filled into column, obtains diameter height than the AB-8 large pore resin absorption column for 1:8.
Embodiment 1
Cassia seed reference extract is prepared with the following method:
(1) by the AB-8 large pore resin absorption column of preparation example 1 on the sample solution of preparation example 1, make cassia seed alcohol extract and AB-
The amount ratio of 8 macroporous absorbent resins is 1g:50ml, successively carries out gradient elution with the first eluant, eluent and the second eluant, eluent, respectively
The first eluent and the second eluent are obtained, second eluent is collected, it is dry, partly prepared components I；Wherein, described
First eluant, eluent is 20vol% ethanol water, and dosage is 3 times of amounts of the large pore resin absorption column volume；Described second washes
De- agent is 50vol% ethanol water, and dosage is 5 times of amounts of the large pore resin absorption column volume；
(2) components I will be partly prepared to be dissolved in a small amount of 50vol% ethanol water, then with to mix sample blank silica gel mixed
Close uniformly, wherein partly prepare components I and it is described to mix sample with the weight ratio of blank silica gel be 1:2, obtain mixing sample silica gel；
(3) by chromatography blank silica gel dry column-packing and be compacted, compare diameter height for 1:15, be reloaded into it is described mix sample silica gel,
Compacting, to obtain silica gel chromatographic column；Then the silica gel chromatographic column is eluted with third eluant, eluent；
Wherein, in the silica gel chromatographic column, partly preparing components I and the chromatography with the amount ratio of blank silica gel is 1g:
26ml；The third eluant, eluent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution；And the third
The volume of eluant, eluent is 1.3 times of amounts of the silica gel chromatograph column volume；
(4) silica gel chromatographic column after elution is divided into 20 parts of silica gel from column cap to bottom, respectively plus methanol elutes
To meoh eluate, the meoh eluate is detected respectively, and the meoh eluate containing index components is merged, it is dry, it obtains
The cassia seed reference extract.The index components include cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- rough gentian
Bioside and/or cassia seed glycosides C.
Embodiment 2
Example 1 in addition to chromatography blank silica gel dry column-packing in step (3) and is pressed with a batch of Cassia obtusifolia L
Real, column diameter is constant, compares diameter height for 1:17, other are same as Example 1, obtain cassia seed reference extract.
Comparative example 1
Example 1 in addition to chromatography blank silica gel dry column-packing in step (3) and is pressed with a batch of Cassia obtusifolia L
Real, column diameter is constant, compares diameter height for 1:13, other are same as Example 1, obtain cassia seed reference extract.
Experimental example 1
The column cap silica gel of Examples 1 to 2, comparative example 1 is eluted with methanol, is evaporated, it is dry, obtain column cap medicinal extract powder.
Cassiaside B in the cassia seed reference extract of Examples 1 to 2, comparative example 1 is detected respectively2, cassia seed glycosides C2, rubrofusarin-
In the content and Examples 1 to 2 of 6-O- β-D- O-gentibioside and cassia seed glycosides C, the column cap medicinal extract powder of comparative example 1 certainly
Pine torch glycosides B2, cassia seed glycosides C2Residual rate.Ingredient in the residual rate of ingredient=column cap medicinal extract powder in column cap medicinal extract powder
The total amount of ingredient in amount/sample solution.Experimental result is shown in Table 1.
Table 1
When chromatographic column diameter height compares for 1:13, cassiaside B in column cap2, cassia seed glycosides C2Residual rate it is excessive；Work as chromatography
When column diameter height compares for 1:15 and 1:17, cassiaside B in column cap2, cassia seed glycosides C2Residual rate reduce, aphthopyrans ketone at
The total content divided improves.Wherein, when chromatographic column diameter height compares for 1:17, cassiaside B in column cap2, cassia seed glycosides C2Residual rate ratio
Diameter height compares slightly to be reduced for 1:15, but fall is smaller, and silica gel usage amount significantly increases, thus sees the high ratio of diameter on the whole
It is more suitable for industrial production for 1:15.
Experimental example 2
Cassia obtusifolia L separately is taken, 3 batches of cassia seed reference extracts are prepared using preparation process same as Example 1 respectively
Sample.Precision weighs each sample 12mg, is placed in 50mL measuring bottle, adds 30% methanol ultrasonic dissolution, and constant volume shakes up, and 0.45 μm micro-
The filtration of hole filter membrane, precision draw 10 μ L sample introductions, and high performance liquid chromatography carries out assay, the results are shown in Table 2.
Table 2
By 2 result of table as it can be seen that different sample room contents are basicly stable, preparation method is reliable and stable.
Experimental example 3
The Cassia obtusifolia L for separately fetching S1~S10 from different sources, batch, using the identical preparation process of embodiment 1
10 batches of cassia seed reference extract samples are prepared respectively, calculate reference extract yield by medicinal material.High performance liquid chromatography carries out
Assay the results are shown in Table 3.
Table 3
Table 3 the experimental results showed that, have obviously by reference extract prepared by raw material of the Cassia obtusifolia L of different batches
Difference, but 4 index components total contents of reference extract reach 60wt% or more, illustrate that this stable preparation process is feasible,
It can be used for different sources, batch medicinal material carries out the preparation work of cassia seed reference extract.
Experimental example 4
The Cassia obtusifolia L 34kg for taking S5 prepares cassia seed reference extract according to the preparation process of embodiment 1, obtains pair
According to extract 508.95g, wherein cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and cassia seed
The content of glycosides C is respectively 10.81wt%, 11.14wt%, 20.33wt%, 28.29wt%, total content 70.57wt%.As a result
Show that technique of the invention can be used for being prepared on a large scale production, and process stabilizing.
Present invention is not limited to the embodiments described above, without departing from the essence of the present invention, this field skill
Any deformation, improvement, the replacement that art personnel are contemplated that each fall within the scope of the present invention.
Claims (10)
1. a kind of preparation method of cassia seed reference extract, which is characterized in that the preparation method includes the following steps:
(1) by large pore resin absorption column on cassia seed alcohol extract, gradient successively is carried out with the first eluant, eluent and the second eluant, eluent and is washed
It is de-, the first eluent and the second eluent are respectively obtained, second eluent is collected, it is dry, partly prepared components I；
Wherein, first eluant, eluent is 15~30vol% ethanol water, and dosage is the large pore resin absorption column volume
2~4 times of amounts；Second eluant, eluent is 40~60vol% ethanol water, and dosage is the large pore resin absorption column volume
4~6 times of amounts；
(2) by it is described partly prepare components I with mix sample and be uniformly mixed with blank silica gel according to the ratio that weight ratio is 1:1.8~2.4,
It obtains mixing sample silica gel；
(3) it by chromatography blank silica gel dry column-packing and is compacted, compares diameter height for 1:14.5~18, be reloaded into and described mix sample silicon
Glue, compacting, to obtain silica gel chromatographic column；Then the silica gel chromatographic column is eluted with third eluant, eluent；
Wherein, in the silica gel chromatographic column, partly prepare components I and the chromatography with the amount ratio of blank silica gel be 1g:24~
28ml；The third eluant, eluent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water dicyandiamide solution；And the third
The volume of eluant, eluent is 1.2~1.4 times of amounts of the silica gel chromatograph column volume；
(4) silica gel chromatographic column after elution is divided into 18~24 parts from column cap to bottom, respectively plus methanol affords first
Alcohol eluen detects the meoh eluate respectively, and the meoh eluate containing index components is merged, dry, obtains described
Cassia seed reference extract；
Wherein, the index components are cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and Cassia
At least one of sub- glycosides C.
2. preparation method according to claim 1, which is characterized in that in step (1), the cassia seed alcohol extract refers to certainly
The extract that pine torch medicinal material is obtained using the ethanol water of 25~40vol% as solvent extraction；The macroporous absorbent resin
Column is AB-8 large pore resin absorption column, and the macroporous absorbent resin in the cassia seed alcohol extract and the large pore resin absorption column
Amount ratio be 1g:30~70ml.
3. preparation method according to claim 1, which is characterized in that in step (2), the components I that partly prepares is dissolved in
It in ethanol water, and mixes sample with described and is mixed with blank silica gel, and the concentration of the ethanol water is 40~60vol%.
4. preparation method according to claim 1, which is characterized in that described partly to prepare components I and chromatography in step (3)
Amount ratio with blank silica gel is 1g:25~26ml.
5. the preparation method according to claim 4, which is characterized in that in step (3), chromatography blank silica gel dry method is filled
Column is simultaneously compacted, and compares diameter height for 1:15~15.5.
6. preparation method according to claim 5, which is characterized in that in step (3), the volume of the third eluant, eluent is
1.25~1.35 times of amounts of the silica gel chromatograph column volume.
7. preparation method according to claim 1, which is characterized in that in step (4), the detection of the meoh eluate is logical
Cross thin-layer chromatography inspection know, lamellae be silica G plate, solvent be volume ratio be 6.5:3.5:0.6 chloroform, first alcohol and water it is molten
Agent system.
8. cassia seed reference extract made from described in any item preparation methods according to claim 1~7, which is characterized in that
The cassia seed reference extract includes cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and certainly
Pine torch glycosides C, and the cassiaside B2, cassia seed glycosides C2, rubrofusarin -6-O- β-D- O-gentibioside and cassia seed glycosides C it is total
Content is higher than 60wt%.
9. cassia seed reference extract according to claim 8, which is characterized in that the cassia seed reference extract includes
The cassiaside B of 6~13wt%2, 6~13wt% cassia seed glycosides C2, 11~35wt% rubrofusarin -6-O- β-D- rough gentian
The cassia seed glycosides C of bioside and 18~35wt%.
10. the purposes that cassia seed reference extract according to claim 8 or claim 9 is used to control Cassia obtusifolia L quality.
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