CN106496299B - The preparation method of high concentration Cinobufacini extract - Google Patents
The preparation method of high concentration Cinobufacini extract Download PDFInfo
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- CN106496299B CN106496299B CN201610794722.8A CN201610794722A CN106496299B CN 106496299 B CN106496299 B CN 106496299B CN 201610794722 A CN201610794722 A CN 201610794722A CN 106496299 B CN106496299 B CN 106496299B
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Abstract
This case is related to the preparation method of high concentration Cinobufacini extract, comprising: by the vacuum drying of toad skin, crushes, obtains toad skin powder；Toad skin powder is placed in enzymatic vessel, distilled water is added, after mixing evenly, salt acid for adjusting pH is added to acidity, pepsin stirring, heat preservation enzymatic hydrolysis is added in heating；Sodium hydroxide is added and adjusts pH to neutrality, 30min is boiled in heating, is centrifuged, takes supernatant, obtain cinobufagin extracting solution；Through adsorbing resin adsorption after cinobufagin extracting solution is cooled to 40 DEG C, using linear gradient elution method, first absorption resin is eluted with 60% ethyl alcohol, then absorption resin is eluted with 80% ethyl alcohol, collect eluent；Eluent is concentrated under reduced pressure into no alcohol taste, obtains Cinobufacini extract.Toad skin is decomposed in this case by using pepsin, to release cinobufagin, and improves the rate of transform of active principle and the content of Cinobufacini extract indoles alkaloid by absorption resin adsorption and gradient elution, improves extraction efficiency.
Description
Technical field
The present invention relates to a kind of extracting technique of Chinese medicine, and in particular to a kind of preparation method of high concentration Cinobufacini extract.
Background technique
Cinobufagin derives from bufo gargarizans Cantor (Bufo bufo gargarizans Cantor) or Bufo melanostictus (Bufo
Melanostictus Schneider) skin it is isolated by extracting, be in two kind new medicines voluntarily developed of China and country
Medicine protects kind.
Cinobufagin has different degrees of inhibiting effect to kinds of tumor cells, can induce liver about cinobufagin in recent years
The apoptosis of the tumour cells such as cancer, gastric cancer inhibits the research of oncogene expression more.Cinobufagin have inhibit growth of tumour cell and
Stimulate the double action of immune system.Clinical practice proves that cinobufagin can increase patient to the tolerance of radiotherapy, chemotherapy, improves
The sensibility of tumour cell improves curative effect, mitigates the dosage of radiotherapy, chemotherapy, improves the life quality of tumor patient, extends existence
Phase.It has been widely used on the clinical treatment of antitumor, treatment chronic hepatitis B and other diseases at present.
Cinobufagin injection, oral solution, tablet are the Chinese medicine protection kind in China, and principle active component is indoles
Alkaloid, bufotenine, toad quaternary amine, bufothionine and dehydrobufotenine etc., wherein by indoles in the national drug standards
Quality index of alkaloid (using in terms of the serotonin) content as cinobufagin.Cinobufagin extraction process reported at present mainly has
Water extraction and alcohol precipitation method, ethanol extract from water precipitation, but the research and development of these techniques all encounter bottleneck, therefore how to explore a kind of novel highly concentrated
The preparation method for spending Cinobufacini extract, becomes research tendency instantly.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of systems of high concentration Cinobufacini extract
Preparation Method improves the purpose of production efficiency to reach raising active constituent content.
Technical solution of the present invention is summarized as follows:
A kind of preparation method of high concentration Cinobufacini extract comprising following steps:
S1: it by the vacuum drying of toad skin, crushes, obtains toad skin powder；
S2: the toad skin powder is placed in enzymatic vessel, and distilled water is added, and after mixing evenly, salt acid for adjusting pH is added to acid
Property, pepsin stirring, heat preservation enzymatic hydrolysis is added in heating；
S3: sodium hydroxide is added and adjusts pH to neutrality, 30min is boiled in heating, is centrifuged, takes supernatant, obtains cinobufagin extraction
Liquid；
S4: it is first used through absorption resin adsorption using linear gradient elution method after the cinobufagin extracting solution is cooled to 40 DEG C
60% ethyl alcohol elutes absorption resin, then is eluted with 80% ethyl alcohol to absorption resin, collects eluent；
S5: eluent is concentrated under reduced pressure into no alcohol taste, obtains Cinobufacini extract.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein vacuum drying condition is in S1
65 DEG C, -0.09MPa, drying time 2-5h.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein the addition weight of distilled water in S2
It is 5-10 times of toad skin powder.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein the addition weight of pepsin in S2
Amount is the 0.5%-2% of toad skin powder.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein centrifugal rotational speed is 2000- in S3
4000 turns/min, centrifugation time 2-10min.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein the condition that is concentrated under reduced pressure in S5 is 50
℃、-0.08MPa。
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein in S4, by volume ratio in terms of,
The dosage of 60% ethyl alcohol is 2-5 times of cinobufagin extracting solution.
Preferably, the preparation method of the high concentration Cinobufacini extract, wherein in S4, by volume ratio in terms of,
The dosage of 80% ethyl alcohol is 2-5 times of cinobufagin extracting solution.
The beneficial effects of the present invention are: toad skin is decomposed in this case by using pepsin, so that cinobufagin is released, and
The rate of transform and Cinobufacini extract indoles alkaloid of active principle are improved by absorption resin adsorption and gradient elution
Content reduces production cost, improves extraction efficiency.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, to enable those skilled in the art referring to specification
Text can be implemented accordingly.
1, this case scheme
(1) toad skin is placed in 65 DEG C, -0.09MPa, is crushed after dry 5h, obtains toad skin powder；
(2) 100g toad skin powder is placed in enzymatic vessel, the distilled water of 10 times of weight is added, after mixing evenly, hydrochloric acid is added
Solution adjusts pH to 2.0, is heated to 40 DEG C, and 1g pepsin is added and is sufficiently stirred, and 40 DEG C of heat preservations digest 1h；
(3) sodium hydroxide solution is added and adjusts pH to 7.0,30min is boiled in heating, and 4000 turns/min is centrifuged 5min, takes
Clear liquid obtains cinobufagin extracting solution；
(4) it when cinobufagin extracting solution being cooled to 40 DEG C or less, is adsorbed through macroporous absorbent resin, using the side of gradient elution
Method, first with the ethyl alcohol of 60% (V/V) of 3 times of cinobufagin extracting liquid volumes to absorption resin elute, then with 3 times China toads
85% (V/V) ethyl alcohol of plain extracting liquid volume elutes absorption resin, collects eluent；
(5) eluent is concentrated under reduced pressure into no alcohol taste under conditions of 50 DEG C, -0.08MPa, obtains Cinobufacini extract；
(6) the toad skin of different batches is taken to be repeated 5 times experiment, according to the content of indoles alkaloid in 3.2 measurement extracting solutions；
Simultaneously according to the content of indoles alkaloid in 3.3 measurement toad skin medicinal materials.
2, traditional water decoction extraction method
Dry maxima skin 100g is taken, is cleaned, water 500g~600g is added, is decocted secondary, 45 minutes first times, second 30 minutes,
Collecting decoction, filtration, it is 1.1~1.2 (85 ± 5 DEG C) that filtrate, which is concentrated into relative density, is cooled to 40 DEG C, ethyl alcohol is added to make alcohol content
It up to 60% (V/V), stirs evenly, stands 24 hours, take supernatant recycling ethyl alcohol to no alcohol taste, ethyl alcohol is added to make alcohol content up to 85% (V/
V), stir evenly, stand 48 hours, filtration recycles ethyl alcohol to no alcohol taste, obtains Cinobufacini extract.
The toad skin of different batches is taken to be repeated 5 times experiment, according to the content of indoles alkaloid in 3.2 measurement extracting solutions.Together
When according to 3.3 measurement toad skin medicinal materials in indoles alkaloid content.
3, detection method (national drug standards)
3.1 standard curves:
Serotonin hydrochloride reference substance 15.03mg is taken, sets in 100ml measuring bottle, scale is dissolved and be diluted to purified water,
It shakes up, precision measures 10ml, sets in 50ml measuring bottle, adds purified water to scale, shake up, obtain reference substance solution.
Precision measures reference substance solution 1.0,2.0,3.0,4.0,5.0ml, is set in 10ml measuring bottle respectively, adds 15% pair of diformazan
Aminobenzaldehyde hydrochloric acid (2 → 3) solution 5ml, shakes up, and adds purified water to scale, shakes up, be placed at room temperature for 35min, according to spectrophotometric
Method (" Chinese Pharmacopoeia " 2010 editions, annex VB) measures absorbance at 555nm wavelength, is with absorbance using purified water as blank
Ordinate, corresponding concentration are abscissa, draw canonical plotting.The regression equation of curve obtained is Y=0.0389X+0.0276,
R=0.9994 shows indoles alkaloid in 0.139~0.608 μ gml-1Linear relationship is good in concentration range.
Indoles alkaloid assay in 3.2 cinobufagin medicinal extract:
Precision measurement cinobufagin medicinal extract is appropriate, and centrifugation is diluted to suitable multiple, then by defined method under 3.1, certainly
" adding 15% paradime thylaminobenzaldehyde hydrochloric acid (2 → 3) solution " rises, and trap is measured in accordance with the law, by standard curve regression equation meter
Calculate the content for obtaining test solution indoles alkaloid.
Indoles alkaloid assay in 3.3 toad skin medicinal materials:
It is crushed after taking toad skin medicinal material drying, crosses 20 meshes, weigh toad skin dry powder 4g, chlorination imitates 30ml, ultrasonic extraction
15min, filtration.Filter residue adds methanol 50ml, ultrasonic extraction 15min, and filtration, filtrate is evaporated, and filter residue, which is dissolved in water, to be diluted to suitably
Concentration, then by defined method under 3.1, from " adding 15% paradime thylaminobenzaldehyde hydrochloric acid (2 → 3) solution ", in accordance with the law
Trap is measured, the content of indoles alkaloid in toad skin medicinal material is obtained by standard curve regression equation calculation.
4, preparation method compares
1 this case scheme of table
| Serial number | The content (mg) of indoles alkaloid in 100g raw material extracting solution | The rate of transform (%) |
| 1 | 51.62 | 53.22 |
| 2 | 53.12 | 51.57 |
| 3 | 49.76 | 56.54 |
| 4 | 50.54 | 52.64 |
| 5 | 51.22 | 55.08 |
| Average value | 51.25 | 53.72 |
2 traditional water decoction of table boils method
By Tables 1 and 2 as it can be seen that this case scheme is remarkably improved the content of Cinobufacini extract indoles alkaloid, significantly
Improve the rate of transform of active principle.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details.
Claims (8)
1. a kind of preparation method of high concentration Cinobufacini extract, which comprises the following steps:
S1: it by the vacuum drying of toad skin, crushes, obtains toad skin powder；
S2: the toad skin powder is placed in enzymatic vessel, and distilled water is added, and after mixing evenly, salt acid for adjusting pH is added to acidity, adds
Pepsin stirring, heat preservation enzymatic hydrolysis is added in heat；
S3: sodium hydroxide is added and adjusts pH to neutrality, 30min is boiled in heating, is centrifuged, takes supernatant, obtain cinobufagin extracting solution；
S4: through adsorbing resin adsorption after the cinobufagin extracting solution is cooled to 40 DEG C, using linear gradient elution method, first with 60%
Ethyl alcohol elutes absorption resin, then is eluted with 80% ethyl alcohol to absorption resin, collects eluent；
S5: eluent is concentrated under reduced pressure into no alcohol taste, obtains Cinobufacini extract.
2. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that be dried in vacuo in S1
Condition be 65 DEG C, -0.09MPa, drying time 2-5h.
3. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that distilled water in S2
It is added 5-10 times that weight is toad skin powder.
4. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that pepsin in S2
Addition weight be toad skin powder 0.5%-2%.
5. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that centrifugal rotational speed in S3
Turn/min, centrifugation time 2-10min for 2000-4000.
6. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that be concentrated under reduced pressure in S5
Condition is 50 DEG C, -0.08MPa.
7. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that in S4, with volume
Than to count, the dosage of 60% ethyl alcohol is 2-5 times of cinobufagin extracting solution.
8. the preparation method of high concentration Cinobufacini extract according to claim 1, which is characterized in that in S4, with volume
Than to count, the dosage of 80% ethyl alcohol is 2-5 times of cinobufagin extracting solution.
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| CN101396373B (en) * | 2007-09-29 | 2011-04-06 | 四川省中医药科学院 | Cinobufacini extract and preparation method thereof |
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