CN104013972B - A kind of preparation method of hydrophilic belulinic acid Betulinic acid preparation - Google Patents
A kind of preparation method of hydrophilic belulinic acid Betulinic acid preparation Download PDFInfo
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Abstract
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation, relates to the preparation method of a kind of belulinic acid Betulinic acid preparation.The present invention is to solve that belulinic acid Betulinic acid is insoluble in water, and the problem limiting its application in terms of pharmaceutical preparation.Method: one, 6 tolysulfonyl beta cyclodextrin；Two, 6 diethylenetriamine chain beta cyclodextrin；Three, diethylenetriamine bridging beta cyclodextrin dimer；Four, 6 diethylenetriamine chain beta cyclodextrins or diethylenetriamine bridging beta cyclodextrin dimer are weighed in sample cell, add distilled water to dissolve, add belulinic acid Betulinic acid, after sonic oscillation, continue vibration on the oscillator, centrifugal, take supernatant aqueous phase cellulose filter membrane to filter, again filtrate is dried, obtains white powder, be hydrophilic belulinic acid Betulinic acid preparation.The present invention can be effectively improved belulinic acid Betulinic acid dissolubility in water, thus solves a belulinic acid Betulinic acid difficult problem in terms of biological preparation.The present invention is used for preparing belulinic acid Betulinic acid preparation.
Description
Technical field
The present invention relates to the preparation method of a kind of belulinic acid Betulinic acid preparation.
Background technology
Belulinic acid Betulinic acid (betulinicacid), has another name called betulic acid, is the compound of a kind of pentacyclic triterpene, is widely present in birch
In bark and other plant, it is possible to prepared by betulinol synthesis, molecular weight 456.71g/mol, colorless needle crystals, molecule
Formula: C30H48O3, molecular structural formula:
Belulinic acid Betulinic acid, as natural bioactive substance, has the effect such as malaria, antiinflammatory.Research finding, belulinic acid Betulinic acid is to people
Class black cancer has good antitumaous effect, and the disease such as lymphatic cancer, carcinoma of prostate is also had good therapeutic effect.Birch
Wood acid is because having the effect that antitumor increases, and there is also newly at anti-acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)) aspect
Mechanism of action, caused the extensive concern of research worker.Belulinic acid Betulinic acid does not only have many medical mechanisms, but also
Do not synthesize the toxicity of quasi drugs, the most do not synthesize the drug resistance of quasi drugs, so belulinic acid Betulinic acid has in terms of medicament research and development
DEVELOPMENT PROSPECT widely.
But owing to belulinic acid Betulinic acid is insoluble in water, limit its application in terms of pharmaceutical preparation.
Cyclodextrin (Cyclodextrin is called for short CD) is a kind of cyclic oligomer sugar compounds, has particularly ring-shaped hollow circle
Cylindrical structure, internal diameter 0.7～0.8nm can form clathrate with enclosed molecule (such as medicine) under certain condition.Conventional β-ring is stuck with paste
Essence has the loop configuration being made up of 7 glucose molecules, appropriately processed with medicine after, pharmaceutical pack can be wrapped in its ring
Shape structure is formed ultra micro capsule clathrate, the dissolubility of medicine, stability can be increased.But this dissolving in water of beta-schardinger dextrin-
Degree is not very big, so the efficiency of inclusion medicine also ratio is relatively low.
Summary of the invention
The present invention is to solve that belulinic acid Betulinic acid is insoluble in water, and the problem limiting its application in terms of pharmaceutical preparation, it is provided that one
Plant the preparation method of hydrophilic belulinic acid Betulinic acid preparation.
The preparation method of hydrophilic belulinic acid Betulinic acid preparation of the present invention, sequentially includes the following steps:
One, weigh 40～60g beta-schardinger dextrin-s to join in 400～500mL distilled water, obtain cyclodextrin solution, take 6～7g
NaOH is dissolved in 20～25mL distilled water, obtains NaOH solution, and the NaOH solution of preparation is added dropwise to cyclodextrin
In solution, obtain transparent and homogeneous solution, take 7～10g p-methyl benzene sulfonic chlorides and be dissolved in 30～40mL acetonitriles, must mix molten
Liquid, is added dropwise to mixed solution in transparent and homogeneous solution, is stirred at room temperature reaction 2～4h, and sucking filtration is precipitated,
Filtrate is preserved at 0～5 DEG C 12h, then sucking filtration must precipitate, the precipitation vacuum drying 12～16h that twice is obtained, obtain
White solid, with absolute ethanol washing white solid 3～5 times, uses dehydrated alcohol 50～80mL every time, obtains 6-to toluene
Sulphonyl-beta-schardinger dextrin-；
Two, weigh 1.5～3g6-tolysulfonyl-beta-schardinger dextrin-, 20～30mgKI and 0.5～1.0mL diethylenetriamine is molten
In the 5～8mL N-Methyl pyrrolidone being dried, under the conditions of 70～80 DEG C, stirring reaction 4～7h, obtains yellow solution,
Being cooled to room temperature, yellow solution is poured into precipitation white precipitate in 100～150mL dehydrated alcohol, collected by suction white is sunk
Form sediment, wash with 100～150mL dehydrated alcohol and 50～80mL ether respectively, be vacuum dried to obtain 1.3～2.5g product A,
Being dissolved with 8～12mL distilled water by product A, carry out eluting with hydrogen ion exchange resin post, eluant is followed successively by 400～500
The ammonia of mL distilled water, 400～500mL1mol/L, the eluent collecting ammonia part concentrates, is vacuum dried, and obtains product
Thing B, is dissolved in 20～30mL distilled water by product B, and decompression, concentrate drying obtains 6-diethylenetriamine chain-beta-schardinger dextrin-；
Three, 2.6～3.9g6-tolysulfonyl-beta-schardinger dextrin-and 1.2～2.4g6-diethylenetriamine chains-beta-schardinger dextrin-are weighed,
It is dissolved in 80～100mLN, in dinethylformamide, at N2Reaction stirring 2～3 under protection and 75～85 DEG C of condition of water bath heating
My god, reaction solution being concentrated into 20～30mL, is then added in 300～400mL acetone produce precipitation, sucking filtration is also used
Acetone cyclic washing precipitates 3～5 times, then precipitation is dissolved in 10～15mL distilled water and filters with aqueous phase cellulose filter membrane,
Filtrate is dried to obtain diethylenetriamine bridging-beta-schardinger dextrin-dimer；
Four, clathrate is produced: weigh 6-diethylenetriamine chain-beta-schardinger dextrin-or the 0.7～1.0g divinyl three of 0.5～0.7g
Amine bridging-beta-schardinger dextrin-dimer, in sample cell, adds 5～6mL distilled water and makes it dissolve, add 30～40mg
Belulinic acid Betulinic acid, after sonic oscillation 20～60min, continues vibration 6～7 days on the oscillator, be placed on afterwards in high speed centrifuge with
10000～12000rpm are centrifuged 15～30min, take supernatant aqueous phase cellulose filter membrane and filter, then filtrate are dried,
Obtain white powder, be hydrophilic belulinic acid Betulinic acid preparation.
Described in step 2, resin column specification is: 25 × 450mm, 724 weak-acid cation-exchange resins, and height filled by resin
Degree is 250mm.
Beneficial effects of the present invention:
Cyclodextrin derivatization, the not only high degree of the cyclodextrin after derivatization are added dissolubility own by the present invention, but also
The inclusion to medicine can be promoted.The cyclodextrin derivative utilizing synthesis carries out inclusion to belulinic acid Betulinic acid, it is possible to be effectively improved belulinic acid Betulinic acid
Dissolubility in water, thus solve a belulinic acid Betulinic acid difficult problem in terms of biological preparation.
Accompanying drawing explanation
Fig. 1 is the hydrophilic of belulinic acid Betulinic acid, 6-diethylenetriamine chain-beta-schardinger dextrin-, the two physical mixture and embodiment 1 preparation
The infrared spectrum of type belulinic acid Betulinic acid preparation；Fig. 2 is belulinic acid Betulinic acid, 6-diethylenetriamine chain-beta-schardinger dextrin-, the two physical mixture
X-ray diffractogram with the hydrophilic belulinic acid Betulinic acid preparation of embodiment 1 preparation；Fig. 3 is belulinic acid Betulinic acid standard specimen mass spectrum；Fig. 4
Hydrophilic belulinic acid Betulinic acid preparation for embodiment 1 preparation takes off the mass spectrum of thing of contracting for fixed output quotas；Fig. 5 is belulinic acid Betulinic acid standard specimen UPLC figure；
Fig. 6 is the UPLC figure that hydrophilic belulinic acid Betulinic acid preparation prepared by embodiment 1 takes off thing of contracting for fixed output quotas；Fig. 7 is belulinic acid Betulinic acid, divinyl
Triamine bridging beta-schardinger dextrin-dimer, the two physical mixture and hydrophilic belulinic acid Betulinic acid preparation infrared of embodiment 2 preparation
Spectrogram；Fig. 8 is that belulinic acid Betulinic acid, diethylenetriamine bridging beta-schardinger dextrin-dimer, the two physical mixture and embodiment 2 are made
The X-ray diffractogram of standby hydrophilic belulinic acid Betulinic acid preparation；Fig. 9 is the de-bag of hydrophilic belulinic acid Betulinic acid preparation of embodiment 2 preparation
The mass spectrum of product；Figure 10 is the UPLC figure that hydrophilic belulinic acid Betulinic acid preparation prepared by embodiment takes off thing of contracting for fixed output quotas.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes appointing between each detailed description of the invention
Meaning combination.
Detailed description of the invention one: the preparation method of present embodiment hydrophilic belulinic acid Betulinic acid preparation, sequentially includes the following steps:
One, weigh 40～60g beta-schardinger dextrin-s to join in 400～500mL distilled water, obtain cyclodextrin solution, take 6～7g
NaOH is dissolved in 20～25mL distilled water, obtains NaOH solution, and the NaOH solution of preparation is added dropwise to cyclodextrin
In solution, obtain transparent and homogeneous solution, take 7～10g p-methyl benzene sulfonic chlorides and be dissolved in 30～40mL acetonitriles, must mix molten
Liquid, is added dropwise to mixed solution in transparent and homogeneous solution, is stirred at room temperature reaction 2～4h, and sucking filtration is precipitated,
Filtrate is preserved at 0～5 DEG C 12h, then sucking filtration must precipitate, the precipitation vacuum drying 12～16h that twice is obtained, obtain
White solid, with absolute ethanol washing white solid 3～5 times, uses dehydrated alcohol 50～80mL every time, obtains 6-to toluene
Sulphonyl-beta-schardinger dextrin-；
Two, weigh 1.5～3g6-tolysulfonyl-beta-schardinger dextrin-, 20～30mgKI and 0.5～1.0mL diethylenetriamine is molten
In the 5～8mL N-Methyl pyrrolidone being dried, under the conditions of 70～80 DEG C, stirring reaction 4～7h, obtains yellow solution,
Being cooled to room temperature, yellow solution is poured into precipitation white precipitate in 100～150mL dehydrated alcohol, collected by suction white is sunk
Form sediment, wash with 100～150mL dehydrated alcohol and 50～80mL ether respectively, be vacuum dried to obtain 1.3～2.5g product A,
Being dissolved with 8～12mL distilled water by product A, carry out eluting with hydrogen ion exchange resin post, eluant is followed successively by 400～500
The ammonia of mL distilled water, 400～500mL1mol/L, the eluent collecting ammonia part concentrates, is vacuum dried, and obtains product
Thing B, is dissolved in 20～30mL distilled water by product B, and decompression, concentrate drying obtains 6-diethylenetriamine chain-beta-schardinger dextrin-；
Three, 2.6～3.9g6-tolysulfonyl-beta-schardinger dextrin-and 1.2～2.4g6-diethylenetriamine chains-beta-schardinger dextrin-are weighed,
It is dissolved in 80～100mLN, in dinethylformamide, at N2Reaction stirring 2～3 under protection and 75～85 DEG C of condition of water bath heating
My god, reaction solution being concentrated into 20～30mL, is then added in 300～400mL acetone produce precipitation, sucking filtration is also used
Acetone cyclic washing precipitates 3～5 times, then precipitation is dissolved in 10～15mL distilled water and filters with aqueous phase cellulose filter membrane,
Filtrate is dried to obtain diethylenetriamine bridging-beta-schardinger dextrin-dimer；
Four, clathrate is produced: weigh 6-diethylenetriamine chain-beta-schardinger dextrin-or the 0.7～1.0g divinyl three of 0.5～0.7g
Amine bridging-beta-schardinger dextrin-dimer, in sample cell, adds 5～6mL distilled water and makes it dissolve, add 30～40mg
Belulinic acid Betulinic acid, after sonic oscillation 20～60min, continues vibration 6～7 days on the oscillator, be placed on afterwards in high speed centrifuge with
10000～12000rpm are centrifuged 15～30min, take supernatant aqueous phase cellulose filter membrane and filter, then filtrate are dried,
Obtain white powder, be hydrophilic belulinic acid Betulinic acid preparation.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: weigh 2g6-in step 2 to first
Benzene sulfonyl-beta-schardinger dextrin-, 25mgKI and 0.57mL diethylenetriamine are dissolved in the N-Methyl pyrrolidone that 5mL is dried.
Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one or two: resin column described in step 2
Specification is: 25 × 450mm, 724 weak-acid cation-exchange resins, and resin packed height is 250mm.Other with tool
Body embodiment one or two is identical.
Detailed description of the invention four: present embodiment is unlike one of detailed description of the invention one to three: weigh 2.6 in step 3
G6-tolysulfonyl-beta-schardinger dextrin-and 2.4g6-diethylenetriamine chain-beta-schardinger dextrin-, be dissolved in 80mLN, N-dimethyl methyl
In amide.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to four: weigh 0.6 in step 4
The 6-diethylenetriamine chain-beta-schardinger dextrin-of g or 0.8g diethylenetriamine bridging-beta-schardinger dextrin-dimer are in sample cell.Its
It is identical with one of detailed description of the invention one to four.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: add in step 4
35mg belulinic acid Betulinic acid.Other is identical with one of detailed description of the invention one to five.
Detailed description of the invention seven: present embodiment is unlike one of detailed description of the invention one to six: dry described in step 4
Dry employing freeze-drying, particularly as follows: be placed on filtrate in freezer dryer, at-50 DEG C of precoolings 6～8h, 30～200
24～36h it are vacuum dried under Pa.Other is identical with one of detailed description of the invention one to six.
Embodiment 1:
The preparation method of the present embodiment hydrophilic belulinic acid Betulinic acid preparation, sequentially includes the following steps:
One, weigh 60g beta-schardinger dextrin-to join in 500mL distilled water, obtain cyclodextrin solution, take 6.5gNaOH molten
In 20mL distilled water, obtain NaOH solution, the NaOH solution of preparation is added dropwise in cyclodextrin solution,
To transparent and homogeneous solution, take 10g p-methyl benzene sulfonic chloride and be dissolved in 30mL acetonitrile, obtain mixed solution, by mixed solution
Being added dropwise in transparent and homogeneous solution, be stirred at room temperature reaction 2h, sucking filtration is precipitated, and filtrate is protected at 5 DEG C
Deposit 12h, then sucking filtration must precipitate, the precipitation vacuum drying 12h that twice is obtained, obtain white solid, wash with dehydrated alcohol
Wash white solid 4 times, use dehydrated alcohol 60mL every time, obtain 6.4g6-tolysulfonyl-beta-schardinger dextrin-；
Two, weigh 2g6-tolysulfonyl-beta-schardinger dextrin-, 25mgKI and 0.57mL diethylenetriamine is dissolved in 5mL and does
In dry N-Methyl pyrrolidone, under the conditions of 70 DEG C, stirring reaction 4h, obtains yellow solution, is cooled to room temperature, will
Yellow solution pours precipitation white precipitate, collected by suction white precipitate in 100mL dehydrated alcohol into, uses 100mL respectively
Dehydrated alcohol and the washing of 50mL ether, be vacuum dried to obtain 1.7g product A, is dissolved by product A 10mL distilled water,
Carrying out eluting with hydrogen ion exchange resin post, eluant is followed successively by the ammonia of 400mL distilled water, 500mL1mol/L,
The eluent collecting ammonia part concentrates, is vacuum dried, and obtains product B, product B is dissolved in 20mL distilled water, subtracts
Pressure, concentrate drying obtains 1.4g6-diethylenetriamine chain-beta-schardinger dextrin-；
Three, produce clathrate: weigh the 6-diethylenetriamine chain-beta-schardinger dextrin-of 0.5g in sample cell, add 5mL and steam
Distilled water makes it dissolve, and adds 30mg belulinic acid Betulinic acid, after sonic oscillation 30min, continues vibration 7 days on the oscillator,
It is centrifuged 15min with 10000rpm in being placed on high speed centrifuge afterwards, takes supernatant aqueous phase cellulose filter membrane and filter, then will
Filtrate is dried, and obtains white powder, is hydrophilic belulinic acid Betulinic acid preparation.
It is dried described in step 3 and uses freeze-drying, particularly as follows: filtrate be placed in freezer dryer ,-50 DEG C of pre-coolings
Freeze 6h, under 200Pa, be vacuum dried 24h.
Utilize infrared, mass spectrum, thermogravimetric analysis, X-ray diffraction and nuclear magnetic resonance, NMR to characterize, and utilize super effect liquid phase pair
Clathrate takes off the solution after bag and carries out the detection of belulinic acid Betulinic acid content.Hydrophilic birch prepared by the present embodiment is recorded through super effect liquid phase
In acid supplement, belulinic acid Betulinic acid content is 329.75 μ g/mL.
Fig. 1 be belulinic acid Betulinic acid, 6-diethylenetriamine chain-beta-schardinger dextrin-, the two physical mixture and the present embodiment prepare hydrophilic
The infrared spectrum of type belulinic acid Betulinic acid preparation.In Fig. 1, a is belulinic acid Betulinic acid, and b is 6-diethylenetriamine chain-beta-schardinger dextrin-, and c is two
Person's physical mixture, d is hydrophilic belulinic acid Betulinic acid preparation.Can be seen that belulinic acid Betulinic acid and 6-diethylenetriamine chain-beta-schardinger dextrin-are each
Having the infrared signature absworption peak of self, the spectrogram of physical mixture has additivity, shows as the superposition of two components, and hydrophilic
Type belulinic acid Betulinic acid preparation is different, the C=O stretching vibration absworption peak (1686.34cm of belulinic acid Betulinic acid association carboxylic acid-1) and C-O stretch
Contracting vibration peak (1235.71cm-1) disappear, show that belulinic acid Betulinic acid, by inclusion, defines clathrate.
Fig. 2 be belulinic acid Betulinic acid, 6-diethylenetriamine chain-beta-schardinger dextrin-, the two physical mixture and the present embodiment prepare hydrophilic
The X-ray diffractogram of type belulinic acid Betulinic acid preparation.In Fig. 2, a is belulinic acid Betulinic acid, and b is 6-diethylenetriamine chain-beta-schardinger dextrin-, c
For the two physical mixture, d is hydrophilic belulinic acid Betulinic acid preparation.Belulinic acid Betulinic acid and 6-diethylenetriamine chain-beta-schardinger dextrin-each have
Have visibly different diffraction maximum, in physical mixture spectrogram the diffraction maximum of existing belulinic acid Betulinic acid have again 6-diethylenetriamine chain-β-
The diffraction maximum of cyclodextrin, and hydrophilic belulinic acid Betulinic acid preparation is different from physical mixture, the Partial Feature diffraction maximum of belulinic acid Betulinic acid disappears,
Show really to define new thing phase, i.e. define clathrate.
Fig. 3 is belulinic acid Betulinic acid standard specimen mass spectrum, and Fig. 4 is the matter that hydrophilic belulinic acid Betulinic acid preparation prepared by the present embodiment takes off thing of contracting for fixed output quotas
Spectrogram, it can be seen that in belulinic acid Betulinic acid standard substance figure, stable existence mass-to-charge ratio is the molecular ion peak of 454.8, at hydrophilic birch
Acid supplement take off thing of contracting for fixed output quotas exists 455.0 molecular ion peak and intensity big, illustrate de-thing of contracting for fixed output quotas contains belulinic acid Betulinic acid.
Fig. 5 is belulinic acid Betulinic acid standard specimen UPLC figure, and Fig. 6 is that hydrophilic belulinic acid Betulinic acid preparation prepared by the present embodiment takes off thing of contracting for fixed output quotas
UPLC schemes, and the de-belulinic acid Betulinic acid integrated peak areas contracted for fixed output quotas in thing is 74407, is brought into belulinic acid Betulinic acid standard curve linear equation A=
In 6705.25C (μ g/mL)-14037.09 (correlation coefficient 0.9997), obtaining concentration is 13.19ppm, is computed
Understand belulinic acid Betulinic acid dissolubility in water and increase to 329.75 μ g/mL.
Embodiment 2:
The preparation method of the present embodiment hydrophilic belulinic acid Betulinic acid preparation, sequentially includes the following steps:
One, weigh 50g beta-schardinger dextrin-to join in 500mL distilled water, obtain cyclodextrin solution, take 6gNaOH and be dissolved in
In 20mL distilled water, obtain NaOH solution, the NaOH solution of preparation is added dropwise in cyclodextrin solution, obtains
Transparent and homogeneous solution, takes 8g p-methyl benzene sulfonic chloride and is dissolved in 30mL acetonitrile, obtain mixed solution, by mixed solution dropwise
Joining in transparent and homogeneous solution, be stirred at room temperature reaction 3h, sucking filtration is precipitated, and filtrate preserves at 5 DEG C 12h,
Sucking filtration must precipitate again, the precipitation vacuum drying 14h obtained twice, obtains white solid, solid by absolute ethanol washing white
Body 3 times, uses dehydrated alcohol 50mL every time, obtains 4.5g6-tolysulfonyl-beta-schardinger dextrin-；
Two, weigh 2g6-tolysulfonyl-beta-schardinger dextrin-, 30mgKI and 0.6mL diethylenetriamine is dissolved in 5mL and does
In dry N-Methyl pyrrolidone, under the conditions of 75 DEG C, stirring reaction 7h, obtains yellow solution, is cooled to room temperature, will
Yellow solution pours precipitation white precipitate, collected by suction white precipitate in 100mL dehydrated alcohol into, uses 100mL respectively
Dehydrated alcohol and the washing of 50mL ether, be vacuum dried to obtain 1.7g product A, is dissolved by product A 10mL distilled water,
Carrying out eluting with hydrogen ion exchange resin post, eluant is followed successively by the ammonia of 400mL distilled water, 400mL1mol/L,
The eluent collecting ammonia part concentrates, is vacuum dried, and obtains product B, product B is dissolved in 20mL distilled water, subtracts
Pressure, concentrate drying obtains 1.5g6-diethylenetriamine chain-beta-schardinger dextrin-；
Three, weigh 2.6g6-tolysulfonyl-beta-schardinger dextrin-and 2.4g6-diethylenetriamine chain-beta-schardinger dextrin-, be dissolved in 80mL
In DMF, at N2Under protection and 80 DEG C of condition of water bath heating, reaction stirring 3 days, concentrate reaction solution
To 20mL, being then added in 300mL acetone produce precipitation, sucking filtration also precipitates 5 times with acetone cyclic washing, then will
Precipitation be dissolved in 15mL distilled water and with aqueous phase cellulose filter membrane filtration, filtrate be dried 3.8g diethylenetriamine bridging-
Beta-schardinger dextrin-dimer；
Four, produce clathrate: weigh the diethylenetriamine bridging-beta-schardinger dextrin-dimer of 0.7g in sample cell, add 5
ML distilled water makes it dissolve, and adds 40mg belulinic acid Betulinic acid, after sonic oscillation 40min, continues vibration 7 on the oscillator
My god, it is centrifuged 30min with 10000rpm in being placed on high speed centrifuge afterwards, takes supernatant aqueous phase cellulose filter membrane and filter,
Again filtrate is dried, obtains white powder, be hydrophilic belulinic acid Betulinic acid preparation.
It is dried described in step 4 and uses freeze-drying, particularly as follows: filtrate be placed in freezer dryer ,-50 DEG C of pre-coolings
Freeze 6h, under 200Pa, be vacuum dried 24h.
Utilize infrared, mass spectrum, thermogravimetric analysis, X-ray diffraction and nuclear magnetic resonance, NMR to characterize, and utilize super effect liquid phase pair
Clathrate takes off the solution after bag and carries out the detection of belulinic acid Betulinic acid content.Hydrophilic birch prepared by the present embodiment is recorded through super effect liquid phase
In acid supplement, belulinic acid Betulinic acid content is 336 μ g/mL.
Fig. 7 is prepared by belulinic acid Betulinic acid, diethylenetriamine bridging beta-schardinger dextrin-dimer, the two physical mixture and the present embodiment
The infrared spectrum of hydrophilic belulinic acid Betulinic acid preparation.In Fig. 7, a is belulinic acid Betulinic acid, and b is diethylenetriamine bridging beta-schardinger dextrin-two
Aggressiveness, c is the two physical mixture, and d is hydrophilic belulinic acid Betulinic acid preparation.The spectrogram that can be seen that physical mixture is birch
Acid and the superposition of diethylenetriamine bridging beta-schardinger dextrin-dimer absworption peak, the C=O remaining belulinic acid Betulinic acid association carboxylic acid stretches
Vibration absorption peak (1685.65cm-1) and C-O stretching vibration peak (1236.35cm-1), and hydrophilic belulinic acid Betulinic acid preparation spectrum
Scheme significantly different, there is no the two characteristic absorption peak of belulinic acid Betulinic acid, show that belulinic acid Betulinic acid, by inclusion, defines clathrate.
Fig. 8 is prepared by belulinic acid Betulinic acid, diethylenetriamine bridging beta-schardinger dextrin-dimer, the two physical mixture and the present embodiment
The X-ray diffractogram of hydrophilic belulinic acid Betulinic acid preparation.In Fig. 8, a is belulinic acid Betulinic acid, and b is that diethylenetriamine bridging β-ring is stuck with paste
Essence dimer, c is the two physical mixture, and d is hydrophilic belulinic acid Betulinic acid preparation.As can be seen from the figure belulinic acid Betulinic acid and diethyl
Alkene triamine bridging beta-schardinger dextrin-dimer each has visibly different diffraction maximum, and physical mixture spectrogram is the folded of two components
Add, physical mixture remains the characteristic diffraction peak of belulinic acid Betulinic acid, and the feature diffraction of belulinic acid Betulinic acid in hydrophilic belulinic acid Betulinic acid preparation
Peak disappears, and illustrates to define new thing phase, i.e. clathrate.
Fig. 9 is the mass spectrum that hydrophilic belulinic acid Betulinic acid preparation prepared by the present embodiment takes off thing of contracting for fixed output quotas, it can be seen that belulinic acid Betulinic acid standard
In product figure, stable existence mass-to-charge ratio is the molecular ion peak of 454.8, exists in the de-thing of contracting for fixed output quotas of hydrophilic belulinic acid Betulinic acid preparation
The molecular ion peak of 455.2 and intensity are big, illustrate in de-thing of contracting for fixed output quotas containing belulinic acid Betulinic acid.
Figure 10 is the UPLC figure that hydrophilic belulinic acid Betulinic acid preparation prepared by the present embodiment takes off thing of contracting for fixed output quotas, the de-birch in thing of contracting for fixed output quotas
Acid integrated peak areas be 76094, be brought into belulinic acid Betulinic acid standard curve linear equation A=6705.25C (μ g/mL)-
In 14037.09 (correlation coefficienies 0.9997), obtaining concentration is 13.44ppm, is computed understanding belulinic acid Betulinic acid and dissolves in water
Degree increases to 336.00 μ g/mL.
Claims (7)
1. the preparation method of a hydrophilic belulinic acid Betulinic acid preparation, it is characterised in that the method sequentially includes the following steps:
One, weigh 40～60g beta-schardinger dextrin-s to join in 400～500mL distilled water, obtain cyclodextrin solution, take 6～7g
NaOH is dissolved in 20～25mL distilled water, obtains NaOH solution, and the NaOH solution of preparation is added dropwise to cyclodextrin
In solution, obtain transparent and homogeneous solution, take 7～10g p-methyl benzene sulfonic chlorides and be dissolved in 30～40mL acetonitriles, must mix molten
Liquid, is added dropwise to mixed solution in transparent and homogeneous solution, is stirred at room temperature reaction 2～4h, and sucking filtration is precipitated, will
Filtrate preserves 12h at 0～5 DEG C, then sucking filtration must precipitate, the precipitation vacuum drying 12～16h obtained twice, obtains white
Solid, with absolute ethanol washing white solid 3～5 times, uses dehydrated alcohol 50～80mL every time, obtain 6-tolysulfonyl-
Beta-schardinger dextrin-；
Two, weigh 1.5～3g 6-tolysulfonyl-beta-schardinger dextrin-, 20～30mg KI and 0.5～1.0mL diethylenetriamine is molten
In the 5～8mL N-Methyl pyrrolidone being dried, under the conditions of 70～80 DEG C, stirring reaction 4～7h, obtains yellow solution,
It is cooled to room temperature, yellow solution is poured into 100～150mL dehydrated alcohol separate out white precipitate, collected by suction white precipitate,
Wash with 100～150mL dehydrated alcohol and 50～80mL ether respectively, be vacuum dried to obtain 1.3～2.5g product A, by product
A dissolves with 8～12mL distilled water, carries out eluting with hydrogen ion exchange resin post, and eluant is followed successively by 400～500mL steamings
Distilled water, 400～the ammonia of 500mL 1mol/L, the eluent collecting ammonia part concentrates, is vacuum dried, and obtains product B,
Product B is dissolved in 20～30mL distilled water, and decompression, concentrate drying obtains 6-diethylenetriamine chain-beta-schardinger dextrin-；
Three, 2.6～3.9g 6-tolysulfonyl-beta-schardinger dextrin-and 1.2～2.4g 6-diethylenetriamine chains-beta-schardinger dextrin-are weighed,
It is dissolved in 80～100mL DMFs, at N2Reaction stirring 2～3 under protection and 75～85 DEG C of condition of water bath heating
My god, reaction solution is concentrated into 20～30mL, is then added in 300～400mL acetone produce precipitation, sucking filtration with third
Ketone cyclic washing precipitates 3～5 times, then precipitation is dissolved in 10～15mL distilled water and filters with aqueous phase cellulose filter membrane, filter
Liquid is dried to obtain diethylenetriamine bridging-beta-schardinger dextrin-dimer；
Four, produce clathrate: weigh 0.7～1.0g diethylenetriamine bridging-beta-schardinger dextrin-dimer in sample cell, add
5～6mL distilled water make it dissolve, and add 30～40mg belulinic acid Betulinic acid, after sonic oscillation 20～60min, on the oscillator
Continue vibration 6～7 days, in being placed on high speed centrifuge afterwards, be centrifuged 15～30min with 10000～12000rpm, take supernatant and use
Aqueous phase cellulose filter membrane filters, then filtrate is dried, and obtains white powder, is hydrophilic belulinic acid Betulinic acid preparation.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 1, it is characterised in that claim in step 2
Take 2g 6-tolysulfonyl-beta-schardinger dextrin-, 25mg KI and 0.57mL diethylenetriamine and be dissolved in the N-methyl that 5mL is dried
In ketopyrrolidine.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 1 and 2, it is characterised in that step 2
Described resin column specification is: 25 × 450mm, 724 weak-acid cation-exchange resins, and resin packed height is 250mm.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 3, it is characterised in that claim in step 3
Take 2.6g 6-tolysulfonyl-beta-schardinger dextrin-and 2.4g 6-diethylenetriamine chain-beta-schardinger dextrin-, be dissolved in 80mL N, N-diformazan
In base Methanamide.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 4, it is characterised in that claim in step 4
Take 0.8g diethylenetriamine bridging-beta-schardinger dextrin-dimer in sample cell.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 5, it is characterised in that add in step 4
Enter 35mg belulinic acid Betulinic acid.
The preparation method of a kind of hydrophilic belulinic acid Betulinic acid preparation the most according to claim 6, it is characterised in that described in step 4
It is dried and uses freeze-drying, particularly as follows: filtrate is placed in freezer dryer, at-50 DEG C of precoolings 6～8h, 30～200
24～36h it are vacuum dried under Pa.
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