CN105596373B - A kind of extraction purification from bilein contains the total extract method of chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid - Google Patents
A kind of extraction purification from bilein contains the total extract method of chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid Download PDFInfo
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- CN105596373B CN105596373B CN201510845226.6A CN201510845226A CN105596373B CN 105596373 B CN105596373 B CN 105596373B CN 201510845226 A CN201510845226 A CN 201510845226A CN 105596373 B CN105596373 B CN 105596373B
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses extraction purification in a kind of bilein contain chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid total extract method, its method for extraction and purification mainly includes: taking a certain amount of ox courage cream powder, it is impregnated and (ox courage cream: ethyl alcohol=1: 2), is extracted in ultrasonic extractor with 95% ethyl alcohol；Ethyl acetate is added in extracting solution and places 30min precipitating, filters, concentration is freeze-dried spare.A certain amount of processed macroreticular resin is weighed in glass chromatography column, 40% ethyl alcohol of bilein extract freeze-drying powder is dissolved, is then slowly added into lysate in analysis column, elution filtering, concentration is freeze-dried to obtain product.High using ultrasonic extraction bilein recovery rate, extraction time is shorter, and technological operation is easy.Present invention employs Amberlyst process to isolate and purify bilein extract, and what this method was separated, and purified, being enriched with makes good use of, and facilitates the extraction purification of chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid.
Description
Technical field
The present invention relates to drug ingedients to extract field, particularly, is related to extraction purification in a kind of bilein and contains goose deoxidation
Cholic acid, ursodesoxycholic acid, hyodesoxycholic acid total extract method.
Background technique
Fel bovis is the gallbladder of bovid ox Bostaurus domesticus Gmelin or buffalo Bubalus L.
Juice.Fel bovis it is medicinal, first recorded in one book of Shennong's Herbal of Han dynasty, be classified as middle product, later age book on Chinese herbal medicine also when have discussion.This product
Bitter is cold in nature, has and clears liver and improve vision, normalizing the secretion and discharge of bile, the efficacy of removing toxicity for detumescence effect, is widely used in treatment the hot hot eyes of liver swelling and pain, and children are frightened
Wind, carbuncle sore tumefacting virus and constipation, the diseases such as quench one's thirst.Bilein (dry bile) is anaesthetic special medicinal material, the entitled crow of anaesthetic and benevolence-again
Su Su has the effect of calming down association's day, callus, removing toxic substances, improving eyesight.It is sick, significant in efficacy with toxication that Mongolian medicine is clinically used for association's day.At present
Think to mainly contain the ingredients such as bilirubin, bile acid, cholesterol, inorganic elements, albumen and amino acid in bilein.
Contain in fel bovis there are many Cholic acids chemical component, is deoxygenated including chenodeoxycholic acid, ursodesoxycholic acid and pig
Cholic acid, but its content is lower.
Chenodeoxycholic acid clinically not only with the drug of dissolving cholesterol class calculus and correction saturation bile, also has aobvious
Land relieving asthma, be anti-inflammatory, antibechic and the effects of eliminating the phlegm.Chenodeoxycholic acid is also the synthesis material of expensive ursodesoxycholic acid, they
It is all the active drug for treating disease of biliary tract.
Ursodesoxycholic acid has cell membrane stability effect, has detoxication to hepatotoxicity substance, while it is low to increase liver
The intake of density lipoprotein to increase the output of liver cell lipid, while increasing fatty acid oxidation or triglyceride conversion, delays
Solve hepatic cell fattydegeneration.
Hyodesoxycholic acid has the effects that reducing blood lipid, antispastic, eliminating the phlegm, is the important component of artificial ox sulphur, clinically can be used
In treatment hyperlipemia, tracheitis and the indigestion as caused by disease in the liver and gallbladder, to bacillus pertussis, corynebacterium diphtheriae, golden yellow
Staphylococcus also has inhibiting effect.
Extracting in the prior art not from bilein has chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid always to mention
Object technical matters is taken to report.
1. the total extract that the present invention only prepares chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid from bilein；
What is mainly utilized is macroreticular resin；
2. these macroreticular resins were only applied in our study, others is not yet reported that；
3. the advantage of macroreticular resin is mainly the compound of gathering trace, resin is mainly utilized in our study
Enrichment and purification.
Summary of the invention
It is an object of the present invention to provide extraction purifications in a kind of bilein to contain chenodeoxycholic acid, ursodeoxycholic
The method of sour, hyodesoxycholic acid total extract.
Particularly, the extraction purification from bilein contain chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid it is total
The method of extract is the working principle for effectively being extracted using macroreticular resin, effectively removing the gred, be effectively enriched with, effectively purifying；It utilizes
The quality-high and inexpensive ethyl alcohol without concentration reaches the purifying of last total extract；Total extract after purification can carry out anaesthetic
The reform of preparation formulation is studied, and specific steps include:
(1) 95% EtOH Sonicate of bilein extracts (supersonic frequency 50-80Hz): taking a certain amount of bilein that certain volume is added
95% ethyl alcohol (material ratio 1: 2) ultrasonic extraction 3 times, each 1h, each time after ultrasonic extraction, appropriate second is added on extracting solution
Acetoacetic ester places 30min precipitating, uses filtered on buchner funnel after waiting precipitating completely, precipitating is again plus the continuation of 95% ethyl alcohol mentions for the second time
It takes, above method is extracted three times.Finally merge filtered fluid.
(2) recycling concentration: recycling concentration filtrate is concentrated into no ethanol flavor.
(3) be freeze-dried: concentrate freeze-drying obtains bilein freeze-dried powder.
(4) 40% EtOH Sonicate (supersonic frequency 50-80Hz) of bilein freeze-dried powder dissolves, spare.
(5) macroporous absorbent resin isolates and purifies: weighing 1kg processing resin in glass chromatography column, is slowly added to above-mentioned molten
Liquid is solved, successively with 20%, 40%, 60%, 80% ethanol elution, filtering, 60% and 80% ethanol eluate of collection, concentration 60%
With 80% ethanol eluate, it is freeze-dried 60% and 80% ethyl alcohol concentrate.
Particularly, a variety of macroreticular resins have been used in this experiment, including D101 type, D001 type, HPD-100 type,
HPD-750 type, HPD-722 type, SIP1300 type, X-5 type.
Particularly, resin is by pretreatment: 95% appropriate amount of ethanol of resin is impregnated for 24 hours, soaked resin is filled into pillar,
First 95% ethyl alcohol rinses, and is then rinsed with purified water, rinses until white opacity do not occur and not having ethanol flavor, then with 5%
Hydrochloric acid and 5% sodium hydroxide rush pillar, respectively rush 5 column volumes, and last 5~6 column volume purified water is rinsed to neutral spare.
Attached drawing summary description
Attached drawing 1 gives a kind of specific extraction purification separation process figure of anaesthetic compound of the present invention.
Attached drawing 2 shows that 60% and 80% bilein freeze-dried powder of the invention is identified by TLC and goes containing chenodeoxycholic acid, bear
The thin layer figure of oxycholic acid and hyodesoxycholic acid.It is compareed respectively with chenodeoxycholic acid, ursodesoxycholic acid and hyodesoxycholic acid standard items
Contain chenodeoxycholic acid, ursodesoxycholic acid and hyodesoxycholic acid in 60% and 80% bilein freeze-dried powder.The knot of Fig. 2 thin layer identification
Fruit, which shows to contain in 60% and 80% bilein freeze-dried powder, compares condition with chenodeoxycholic acid, ursodesoxycholic acid and hyodesoxycholic acid
Same point (identical Rf value) and identical color reaction.
Thin layer identifies specific implementation method:
Solvent: toluene-glacial acetic acid-water (19.5: 10: 0.3) ratio is configured to solvent, and adds a drop formic acid.
Color developing agent: 10% ethanol solution of sulfuric acid color developing agent: the sulfuric acid solution that pharmacopeia measures 2.85ml is abided by, is diluted with ethanol to
50ml is made.
Specific embodiment
Embodiment 1
600g bilein is weighed, is put into triangular flask, 95% ethyl alcohol of 1.2L is added, is covered, is put into ultrasonic wave and extracts,
30 DEG C of ultrasonic temperature, time 30min, 1h is extracted every time, is extracted 3 times, it is on extracting solution plus appropriate after each ultrasonic extraction
Ethyl acetate places 30min precipitating, waits and filters after extracting liquid precipitate, finally merges filtered fluid, and recycling concentration is freeze-dried
496g bilein is spare.
496g bilein freeze-dried powder adds appropriate 40% EtOH Sonicate to dissolve, the D101 resin 1kg that will be handled well in chromatographic column,
It is slowly added to 40% ethyl alcohol bilein solution, absorption parsing elution is collected 60% and 80% ethanol eluate, filtered, and concentration is cold
Dry product is lyophilized.60% ethanol eluate freeze-dried powder 29.897g, 80% ethanol eluate freeze-dried powder 21.182g.
60% and 80% bilein freeze-dried powder is identified by TLC deoxygenates gallbladder containing chenodeoxycholic acid, ursodesoxycholic acid and pig
Acid, wherein chenodeoxycholic acid, ursodesoxycholic acid and hyodesoxycholic acid are enriched in 60% bilein freeze-dried powder.
Embodiment 2
Pharmacodynamic study is carried out to 60% and 80% bilein freeze-dried powder, further confirms Mongolian medicinal preparation or anaesthetic prescription
In, the pharmacological action of the symptoms such as treatment cholelithiasis, hyperlipidemia, indigestion, wherein with the freeze-drying of 60% ethyl alcohol position bilein
Powder carries out for pharmacodynamic study.
(1) to the influence of bile secretion
Using biliary canulization method, taking weight is 180-200g male rat 16, is randomly divided into two groups, i.e. blank control group
And administration group, each 8 male rats, blank group give stomach-filling and give physiological saline sodium carboxymethylcellulose suspension, administration group fills
The 60% ethyl alcohol position bilein freeze-dried powder of stomach 100mg/kg 30 minutes after medicine, is anaesthetized with 20% urethane, and bile duct is inserted in operation,
Collect two hours choleresis.
Experimental result illustrates that 60% ethyl alcohol position bilein freeze-dried powder has significant choleretic effect compared with control group, can obviously increase
The secretory volume for adding bile, experimental result is shown in (tables 1).
The influence that (table 1) 60% ethyl alcohol position bilein freeze-dried powder secretes rat bile
(2) to the influence of serum cholesterol
Using one sulfuric acid development process of high-speed rail, take weight 18-22g mouse 16, be randomly divided into two groups, i.e., blank control group and
Administration group, each 8 mouse, blank group give stomach-filling and give physiological saline sodium carboxymethylcellulose suspension, administration group stomach-filling
The 60% ethyl alcohol position bilein freeze-dried powder of 300mg/kg, successive administration 4 days.5 hours after last time is administered, eyeball is extractd
Blood is taken, after preparing serum, measures its cholesterol concentration.
The result shows that there is significant difference between administration group and control group, illustrate 60% ethyl alcohol position bilein freeze-dried powder
Effect is substantially reduced to serum cholesterol.Experimental result is shown in (tables 2)
Influence of (table 2) the 60% ethyl alcohol position bilein freeze-dried powder to mice serum cholesterol concentration
Claims (4)
1. in a kind of bilein extraction purification contain chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid total extract side
Method, it is characterised in that comprising steps of
(1) 95% EtOH Sonicate of bilein extracts, 50~80Hz of supersonic frequency: taking a certain amount of bilein that certain volume 95% is added
Ethyl alcohol, material ratio 1: 2 ultrasonic extraction 3 times, each 1h, add appropriate acetic acid second each time on extracting solution after ultrasonic extraction
Ester places 30min precipitating, uses filtered on buchner funnel after waiting precipitating completely, and precipitating is again plus 95% ethyl alcohol continues second of extraction, with
Upper method is extracted three times, and filtered fluid is finally merged；
(2) recycling concentration: recycling concentration filtrate is concentrated into no ethanol flavor；
(3) be freeze-dried: concentrate freeze-drying obtains bilein freeze-dried powder；
(4) 40% EtOH Sonicate of bilein freeze-dried powder, 50~80Hz of supersonic frequency dissolution are spare；
(5) macroporous absorbent resin isolates and purifies: 1kg processing resin is weighed in glass chromatography column, is slowly added to above-mentioned lysate,
Successively with 20%, 40%, 60%, 80% ethanol elution, filtering, 60% and 80% ethanol eluate of collection, 60% He of concentration
80% ethanol eluate is freeze-dried 60% and 80% ethyl alcohol concentrate；
Wherein, the resin is D101 type resin.
2. the method according to claim 1, wherein the method for processing resin are as follows: impregnate for 24 hours 95% appropriate amount of ethanol of resin, will soak
The resin dress pillar soaked, first 95% ethyl alcohol rinses, and is then rinsed with purified water, and rinsing to there is not white opacity does not have ethyl alcohol
Until taste, pillar then is rushed with 5% hydrochloric acid and 5% sodium hydroxide, respectively rushes 5 column volumes, last 5~6 column volume purified water punching
It is washed till neutral spare.
3. according to claim 1-2 any methods preparations containing chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid it is total
Extract.
4. Mongolian medicinal preparation made of total extract according to claim 3, it is characterised in that dosage form include pill, soft capsule,
Capsule, granule, injection, tablet, cataplasm, ointment, gelling agent, skin-permeable and control-released plaster, spray.
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| CN201510845226.6A CN105596373B (en) | 2015-11-27 | 2015-11-27 | A kind of extraction purification from bilein contains the total extract method of chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid |
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| CN201510845226.6A CN105596373B (en) | 2015-11-27 | 2015-11-27 | A kind of extraction purification from bilein contains the total extract method of chenodeoxycholic acid, ursodesoxycholic acid, hyodesoxycholic acid |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781350A (en) * | 2010-02-02 | 2010-07-21 | 浙江大学 | Method for purifying ursodeoxycholic acid by mixed solvent |
| CN102552325A (en) * | 2010-12-27 | 2012-07-11 | 福建归真堂药业股份有限公司 | Bear gall extract, preparation method thereof and application thereof to preparation of fatty liver treatment medicament |
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2015
- 2015-11-27 CN CN201510845226.6A patent/CN105596373B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781350A (en) * | 2010-02-02 | 2010-07-21 | 浙江大学 | Method for purifying ursodeoxycholic acid by mixed solvent |
| CN102552325A (en) * | 2010-12-27 | 2012-07-11 | 福建归真堂药业股份有限公司 | Bear gall extract, preparation method thereof and application thereof to preparation of fatty liver treatment medicament |
Non-Patent Citations (1)
| Title |
|---|
| 猪胆粉中结合型胆甾酸的化学成分及生物活性研究;何姣;《中国博士学位论文全文数据库 医药卫生科技辑》;20121115(第11期);第14页第5-6行 |
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