CN105816883B - A kind of probiotics folate-targeted carrier and preparation method thereof loading anticancer drug curcumin - Google Patents
A kind of probiotics folate-targeted carrier and preparation method thereof loading anticancer drug curcumin Download PDFInfo
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- CN105816883B CN105816883B CN201610076508.9A CN201610076508A CN105816883B CN 105816883 B CN105816883 B CN 105816883B CN 201610076508 A CN201610076508 A CN 201610076508A CN 105816883 B CN105816883 B CN 105816883B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
Abstract
The invention discloses a kind of probiotics targeting vectors and preparation method thereof for loading anticancer drug curcumin.Curcumin is connected with the spore surface of bacillus coagulans by esterification first, synthesis obtains curcumin-probiotics carrier (CUR-Spore)；Upper folic acid is connected by esterification again, the probiotics folate-targeted carrier (CUR-Spore-FA) of load curcumin is successfully prepared.The target medicine carrier can be applied to the treatment of colon cancer well, have targeting, and can reduce the toxic side effect of drug.Meanwhile there are also protective effects for curcumin for bacillus coagulans in the carrier medicament, can be resistant to gastric acid and be smoothly through stomach, are colonized in intestines and sprout release curcumin in enteron aisle to treat colon cancer.The present invention is to improve the utilization rate of curcumin, establishes the targeted delivery system of anticancer drug and the treatment applied to colon cancer is laid a solid foundation, and provides a kind of new mode for the treatment of colon cancer.
Description
Technical field
The invention belongs to pharmaceutical technology fields.More particularly, to a kind of probiotics leaf for loading anticancer drug curcumin
Sour targeting vector and preparation method thereof.
Background technique
Cancer has become a big threat of human health.If colorectal cancer (Colorectal cancer, CRC) is wherein
A kind of high-incidence malignant tumour of the mankind, disease incidence is in rising trend always, and is had risen in global Cancer Mortality
Three.With the change of living habit and dietary structure, the lipid material of people's intake increases, and the colon cancer morbidity in China is in
Rise year by year trend.Colon cancer has become the public health issue of very severe, seriously threatens the health and life of the people
Life, greatly hinders Chinese society expanding economy, and the further investigation of etiology and pathogenesis and treatment method to colon cancer is especially aobvious
It is important.Treatment for cancers such as colon cancers, other than traditional operation, radiotherapy and chemotherapy.Due to the multicomponent of natural drug,
Multiple target point, too many levels, multi-pathway effect, make the importance it was recognized that " going back to nature ", native compound it is antitumor
Research has gradually become the hot spot of clinical antitumor agents research.
Curcumin is a kind of natural polyphenol extracted from the rhizome of herbaceous plant turmeric, be turmeric chief active at
Point.In some countries in Asia, curcumin be widely used as always treatment inflammation, asthma, the drug of diseases such as liver dysfunction,
Food color and flavoring agent.In addition, curcumin also plays anti-inflammatory, anti-oxidant, antitumor and change in many tumour cells
Learn prevention effect.Many inside and outside experiments also confirm that curcumin can inhibit tumour growth, substantially reduce the number of knurl, mitigate more
Damage of the kind carcinogenic factor to body.Curcumin has very strong apoptosis induction ability, and inducing cell apoptosis is its antitumor work
Important mechanisms are related to the biological process of a series of complex, and the way different by intraor extracellular number of ways and signal point
Form that diameter is showed is different, has intersection between them, has overlapping, constitute intricate regulated and control network.To sum up, turmeric
Plain broad-spectrum anti-tumor activity has good tolerance and lower toxicity.However, curcumin poorly water-soluble leads to its biology benefit
Expenditure is low, these hinder application of the curcumin as drug candidate.
Recent studies indicate that carrying out prodrug design to curcumin precursor structure is a kind of effective method.Curcumin
Prodrug design mainly carries out esterification modification to its phenolic hydroxyl group with the small molecule containing carboxyl or macromolecular carrier, by the way that phenol is transformed
On the one hand hydroxyl enhances the electronics transfer of enol structure, protect phenolic hydroxyl group and eliminate electron delocalization in structure, on the other hand pass through
The small molecule of connection or the property of macromolecular carrier can improve curcumin dissolubility, improve stability, extend half-life period,
Bioavilability is improved, enhances bioactivity, while there is certain controlled release, slow release effect.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of existing natural anti-cancer drugs curcumin application and technologies not
Foot provides a kind of probiotics targeting vector that can load anticancer drug curcumin, by the natural drug turmeric with antitumaous effect
Element is connected with the coat protein on bacillus coagulans surface, folic acid, and CUR-Spore-FA carrier is prepared.And by pair
CUR-Spore-FA carrier is characterized, release rule of the research curcumin in simulation gastro-intestinal Fluid, the results showed that of the invention
CUR-Spore-FA carrier both can guarantee curcumin can in enteron aisle stable release and hardly discharged in gastric juice, for improve
Natural anti-cancer drugs-curcumin utilization rate, establishes the targeted delivery system of anticancer drug and the treatment applied to colon cancer is beaten
Lower solid foundation had not only solved the problems, such as that natural anti-cancer drugs-curcumin element availability was low but also reduces treatment colon cancer
Toxic side effect, and make the treatment of colon cancer that there is targeting, opened up a kind of new approach for the treatment of colon cancer.
The object of the present invention is to provide a kind of probiotics targeting vectors for loading anticancer drug curcumin.
Another object of the present invention is to provide the preparation of the probiotics folate-targeted carrier of above-mentioned load anticancer drug curcumin
Method.
Another object of the present invention is to provide answering for the probiotics folate-targeted carrier of above-mentioned load anticancer drug curcumin
With.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of probiotics folate-targeted carrier loading anticancer drug is first by chemically reacting anticancer drug and coagulating
The spore surface connection of bacillus is tied, synthesis obtains anticancer drug-probiotics carrier；It is connected then through esterification
The probiotics folate-targeted carrier of load natural anti-cancer drugs is prepared in folic acid.
Preferably, the anticancer drug is curcumin.
Preferably, the chemical reaction is esterification.
It i.e. preferably, is logical first the present invention provides a kind of probiotics targeting vector for loading anticancer drug curcumin
Over-churning reaction connects curcumin with the spore surface of bacillus coagulans, and synthesis obtains curcumin-probiotics carrier (CUR-
Spore)；Upper folic acid is connected then through esterification, the probiotics leaf of load natural anti-cancer drugs curcumin is prepared
Sour targeting vector (CUR-Spore-FA).
It is further preferred that described connect anticancer drug with the spore surface of bacillus coagulans is to pass through esterification
Natural anti-cancer drugs (curcumin) are connected on probiotics Bacillus coagulans spore outer layer capsid protein.
In addition, a kind of preparation method for the probiotics folate-targeted carrier for loading anticancer drug curcumin, including walk as follows
It is rapid:
S1. it is anti-esterification to occur using the carboxyl of hydroxyl and Bacillus coagulans spore coat protein in curcumin structure
It answers, building obtains curcumin-probiotics carrier (CUR-Spore)；
Esterification occurs for the carboxyl in the hydroxyl and folic acid on curcumin-probiotics carrier that S2. step S1 is obtained, will
Folic acid is connected in the albumen of spore coat albumen of bacillus coagulans, obtains the probiotics folic acid of load natural anti-cancer drugs
Targeting vector (CUR-Spore-FA).
Wherein it is preferred to which the specific method of step S1 is:
S11. Bacillus coagulans spore powder is mixed with methylene chloride, ice salt bath is to 0 DEG C；
S12. curcumin, DMAP and N, N- dicyclohexylcarbodiimide, ultrasonic vibration is added in the system of step S11 again
Uniformly, ice-water bath the reaction was continued 20~30h；
S13. the system of step S12 is centrifuged 5~15min through 8000~12000rpm, then passes through dehydrated alcohol and steaming respectively
Distilled water washes away unreacted curcumin, is dried in vacuo to get curcumin-probiotics carrier (CUR-Spore) is arrived；
Preferably, the specific method of step S2 is:
S21. curcumin-probiotics carrier (CUR-Spore) and methylene chloride are mixed, ice salt bath is to 0 DEG C；
S22. it is equal that folic acid, DMAP and N, N- dicyclohexylcarbodiimide, ultrasonic vibration is added in the system of step S21 again
Even, ice-water bath the reaction was continued 20~30h, centrifuge washing removes unreacted folic acid, and is freeze-dried to get natural anti-to load
The probiotics folate-targeted carrier (CUR-Spore-FA) of cancer drug.
It is highly preferred that the mass volume ratio of Bacillus coagulans spore powder and methylene chloride described in step S11 be 0.01~
0.2g:5ml；Curcumin described in step S12, DMAP and N, the mass ratio of N- dicyclohexylcarbodiimide be 5~15:2~4:2~
4。
It is highly preferred that curcumin described in step S21-probiotics carrier (CUR-Spore) and methylene chloride quality volume
Than for 0.01~0.2g:20ml；The mass ratio of step S22 folic acid, DMAP and N, N- dicyclohexylcarbodiimide is 30~50:2
~4:2~4.
It is highly preferred that the mass ratio of the Bacillus coagulans spore powder and curcumin is 2~5:1；Curcumin-the benefit
The mass ratio of raw bacterium carrier (CUR-Spore) and folic acid is 1:2~5.
In addition, it is further preferred that the ice-water bath time that the reaction was continued described in step S12 and step S22 is for 24 hours.
Preferably, centrifugation described in S13 is 10000rpm centrifugation 10min.
Preferably, dehydrated alcohol described in S13 and distillation water washing are washings 3 times.
It is highly preferred that the mass volume ratio of Bacillus coagulans spore powder and methylene chloride described in step S11 is 0.1g:
5ml；Curcumin described in step S12, DMAP and N, the mass ratio of N- dicyclohexylcarbodiimide are 10:3:3.
It is highly preferred that curcumin described in step S21-probiotics carrier (CUR-Spore) and methylene chloride quality volume
Than for 0.1g:20ml；The mass ratio of step S22 folic acid, DMAP and N, N- dicyclohexylcarbodiimide is 40:3:3.
It is highly preferred that the mass ratio of the Bacillus coagulans spore powder and curcumin is 4:1；The curcumin-is prebiotic
The mass ratio of bacterium carrier (CUR-Spore) and folic acid is 1:4.
Finally, the probiotics folate-targeted carrier of above-mentioned load anticancer drug (curcumin) is in terms of preparing anticancer drug
Using also within protection scope of the present invention.
Preferably, the anticancer drug is the drug of anti-colorectal carcinoma.
Natural anti-cancer drugs curcumin is innovatively connected to by the present invention by chemical reaction can be colonized in enteron aisle to people
On the outer layer capsid protein of the beneficial probiotics Bacillus coagulans spore of body, while CUR-Spore carrier connects folic acid structure again
Target medicine carrier (CUR-Spore-FA) is built, and carries out material characterization, the characterizing method of use has: FTIR spectrum
(FTIR), Raman spectrum (Raman), scanning electron microscope etc., it was demonstrated that vector construction success.Meanwhile it being investigated gemma and simulating
Gastro-intestinal Fluid in sprouting and Drug-Release Behavior, using the determined by ultraviolet spectrophotometry carrier simulation gastro-intestinal Fluid in
Release, it was demonstrated that curcumin can in enteron aisle stable release and hardly discharged in gastric juice, which can be applied to well
The treatment of colon cancer is established the targeted delivery system of anticancer drug and is answered to improve natural anti-cancer drugs-curcumin utilization rate
Treatment for colon cancer is laid a solid foundation, and provides a kind of new mode for the treatment of colon cancer.
Moreover, bacillus coagulans (Bacillus coagulans) used in the present invention are used as a kind of probiotics, tool
Have the advantages that it is anti-inflammatory, enhance human immunity, resist antigen, exclude foreign matter；In addition, bacillus coagulans are in addition to having general benefit
Outside raw bacterium has the advantage that, there are also itself unique advantages, such as: resistance is strong, high temperature high voltage resistant, easily stores, anti-gastric acid, gemma
It can be colonized and be sprouted in enteron aisle and in enteron aisle, improve intestine microenvironment etc..
The present invention passes through inhibiting tumor cell verification experimental verification, the results show that the load anticancer drug turmeric that the present invention is successfully prepared
The probiotics folate-targeted carrier of element can not only carry anticancer drug curcumin and reach lesion, additionally it is possible to play medicine with targeting
The effect of object sustained release, and effect of the anticancer drug curcumin to cancer cell has been successfully reserved, there is suppression well to cancer cell
Production is used and curative effect.
The invention has the following advantages:
Natural anti-cancer drugs curcumin is connected to that can be colonized in enteron aisle beneficial to human body by the present invention by chemical reaction
Probiotics Bacillus coagulans spore outer layer capsid protein on, while CUR-Spore carrier connects folic acid again, successfully constructs
A kind of target medicine carrier (CUR-Spore-FA) reduces the toxic side effect for the treatment of colon cancer drug, and has targeting
Property.
Meanwhile in the carrier medicament bacillus coagulans for anticancer drug curcumin there are also a kind of protective effect, make it
It is resistant to gastric acid and is smoothly through stomach, be colonized in intestines and sprout release curcumin in enteron aisle and treat colon cancer, the carrier energy
The treatment for being applied to colon cancer well establishes the targeting of anticancer drug to improve natural anti-cancer drugs-curcumin utilization rate
Transportation system and treatment applied to colon cancer are laid a solid foundation, and provide a kind of new mode for the treatment of colon cancer.
Detailed description of the invention
Fig. 1 is schematic diagram prepared by CUR-Spore and CUR-Spore-FA.
Fig. 2 is the standard curve of curcumin.
Fig. 3 is using fourier transform infrared spectroscopy to the infrared spectrum (FTIR) of respective reaction object and Characterization of The Products；A
Gemma (Spore), B curcumin (Curcumin), C gemma-curcumin (CUR-Spore), D gemma-curcumin-folic acid (CUR-
Spore-FA)。
Fig. 4 is using Raman spectrometer to the Raman map of respective reaction object and product；A gemma (Spore), B curcumin
(Curcumin), C gemma-curcumin (CUR-Spore), D gemma-curcumin-folic acid (CUR-Spore-FA).
Fig. 5 is scanning electron microscope (SEM) photograph；A gemma (Spore), B curcumin (Curcumin), C gemma-curcumin (CUR-
Spore), D gemma-curcumin-folic acid (CUR-Spore-FA).
The charging ratio and the release figure in simulation gastro-intestinal Fluid that Fig. 6 is curcumin；A is that probiotics gemma loads curcumin
Charging ratio；B is the audio-visual picture of curcumin release liquid in simulation gastro-intestinal Fluid of probiotics gemma load；C is negative for probiotics gemma
The curcumin of load in simulate the gastric juice scheme for 24 hours by release；D is that the curcumin of probiotics gemma load discharges for 24 hours in simulated intestinal fluid
Figure.
Fig. 7 is gemma activity identification figure；A is the spread plate of bacillus coagulans Spore；B is the painting of CUR-Spore carrier
The plate of cloth；C is the plate of CUR-Spore-FA carrier coating.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
There is used main agents in following embodiment: curcumin, methylene chloride, 4-dimethylaminopyridine (DMAP),
Mono- dicyclohexylcarbodiimide of N, N (DCC), pepsin, dehydrated alcohol are purchased from up to fast Biotechnology Co., Ltd.Pancreatin
For GIBCOBRL Products.Gemma, which closes swallow Puritanism obtained from South China Normal University's Life Science College, awards laboratory cultures.
There is used key instrument in following embodiment: German LEO company field emission scanning electron microscope: LEO1530VP,
Sigma32184 high speed freezing centrifuge, Medical Instruments factory of Jintan City of Jiangsu Province 78-1 magnetic stirring apparatus, HV-85 high pressure sterilization
Device, aseptic operating platform, Guangzhou Ke Qiao experimental technique equipment Co., Ltd thermostat water bath, shaking table, ultrasonic disperse instrument, ultraviolet spectrometry
Photometer, ice machine etc..
Embodiment 1 loads the preparation of the probiotics folate-targeted carrier of natural anti-cancer drugs curcumin
The schematic diagram of CUR-Spore and CUR-Spore-FA preparation is as shown in Fig. 1.
1, curcumin-probiotics carrier (CUR-Spore) preparation
Esterification structure occurs using the hydroxyl in curcumin structure and the carboxyl of Bacillus coagulans spore coat protein
Build curcumin probiotics carrier.Specific reaction is as follows:
(1) 0.4g Bacillus coagulans spore powder is added in 50ml beaker, takes 20ml methylene chloride to be added, ice salt bath is extremely
0℃。
(2) 0.1g curcumin, mono- dicyclohexylcarbodiimide (DCC) of 30mg DMAP and 30mg N, N, ultrasonic vibration is added
Uniformly, ice-water bath the reaction was continued for 24 hours.
(3) 10000rpm is centrifuged 10 minutes, collects curcumin-probiotics carrier, dehydrated alcohol and distilled water wash 3 respectively
The secondary carrier for washing away unreacted curcumin vacuum drying and having obtained CUR-Spore.
2, curcumin-probiotics-folate-targeted carrier (CUR-Spore-FA) preparation
The carboxyl in hydroxyl and folic acid on the connection carrier of bacillus coagulans and curcumin is by esterification, by leaf
Acid is connected in the albumen of spore coat albumen.Specific reaction is as follows:
(1) above-mentioned 0.1g curcumin probiotics carrier is added in 50ml beaker, takes 20ml methylene chloride to be added, ice salt bath
To 0 DEG C.
(2) 0.4g folic acid is added, mono- dicyclohexylcarbodiimide (DCC) of 30mg DMAP and 30mg N, N, ultrasonic vibration is equal
Even, the reaction was continued for ice-water bath for 24 hours.
(3) centrifuge washing removes unreacted folic acid, and being freeze-dried is CUR-Spore-FA.
The characterization of 2 support C UR-Spore-FA of embodiment
1, the standard curve of curcumin is as shown in Fig. 2.
The curcumin solution light absorption value for measuring various concentration at 420nm by ultraviolet spectrophotometry, to obtain
The standard curve of Fig. 2, the R of Fig. 22=0.997 reached 0.99 grade or more therefore this standard can use.But standard curve
Use scope be 0~5.5 μ g/ml.
2, infrared spectroscopy detects
(1) Spore, CUR-Spore, CUR-Spore-FA are dried, are then placed in mortar, is added certain
The KBr of amount, grinding uniformly makes mixture be ground to granularity less than 2 μm, in case stray light effects, are put into drying machine later and carry out
It is dried, mixture is pressed into transparent sheet, upper machine measurement with the pressure of 10MPa or so on hydraulic press.
(2) as shown in Fig. 3, A is gemma (Spore) to infared spectrum (FTIR) in figure, and B is curcumin (Curcumin), C
It is gemma-curcumin-folic acid (CUR-Spore-FA) for gemma-curcumin (CUR-Spore), D.
Spectrogram A has strong absworption peak representative-OH at 3434.18；There are strong absworption peak, representative-C=O, bud at 1648.69
Just there are these groups in spore.There is the characteristic group beta diketone key of curcumin in B.A, C, the D amount of-OH and-C=O in C that compares increase
There is beta diketone key in more and C, illustrates there is curcumin to connect in C.Because there is phenolic hydroxyl group on curcumin, carbonyl and beta diketone key.Cause
It is that esterification occurs at phenyl ring, and D is 821.50 compared with C, 893.47 etc. the peaks having more are the cyclosubstituted positions of benzene
Illustrate there is folic acid to connect.
3, Raman spectrum detects
By Spore, CUR-Spore, CUR-Spore-FA is dried, and then takes suitable sample in load glass respectively
On piece, upper machine testing.
Raman map is as shown in Fig. 4, and A is gemma (Spore) in figure, and B is curcumin (Curcumin), and C is gemma-ginger
Flavine (CUR-Spore), D are gemma-curcumin-folic acid (CUR-Spore-FA).
Spectrogram A has strong absworption peak representative-COOH at 2200, and having absorption peak to represent in esters gemma at 1650 has this
A little groups illustrate that A is gemma.Map B has the characteristic group beta diketone key of curcumin at 1628, it was demonstrated that B figure is exactly reactant ginger
Flavine.Carboxylic group on map C gemma disappears, and ester bond occur may be that gemma is reacted with curcumin, therefore CUR-
Spore vector construction is successful.There is the characteristic group beta diketone key of curcumin on map D, it was demonstrated that there is curcumin to connect.
4, scanning electron microscope (SEM) photograph is as shown in Fig. 5, and A is gemma (Spore) in figure, and B is curcumin (Curcumin), and C is bud
Spore-curcumin (CUR-Spore), D are gemma-curcumin-folic acid (CUR-Spore-FA).
It can be seen that gemma is rod-shaped and size is μm grade；It can be seen that having granular turmeric on gemma in C figure
Element connects；D figure gemma-curcumin-folic acid scanning electron microscope (SEM) photograph can see that gemma structure is less complete, we guess possibility
It is the number integralities for destroying gemma relatively of centrifugation, in order to verify whether also gemma connects after upper curcumin and folic acid more
It is active that we observe gemma sprouting situation to these sample spread plates.It was found that still have on plate gemma sprout illustrate connect
Gemma or active after upper curcumin and folic acid.
In 3 support C UR-Spore-FA of embodiment probiotics gemma load curcumin charging ratio and simulation gastro-intestinal Fluid in
Release
1, the measurement of the curcumin charging ratio of probiotics gemma load
UV spectrophotometer measuring: consulting literatures learn that the characteristic absorption peak of curcumin is 426nm
(1) specific step is as follows for the standard curve of drafting curcumin:
1. the preparation of curcumin stock solution
It accurately weighs curcumin 1mg to be placed in a beaker, after adding appropriate dehydrated alcohol to dissolve, is transferred to the volumetric flask of 10mL
In, then with dehydrated alcohol it is settled to scale, it is spare as curcumin stock solution.
2. measure 0.1 from stock solution respectively, 0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5mL is respectively placed in
In 9 10mL volumetric flasks, it is settled to scale with dehydrated alcohol, is shaken up.Using dehydrated alcohol as blank control, measured at 420nm
Absorbance, and with the average value (A) of each concentration absorbance three times for ordinate, concentration (c) is abscissa, and it is bent to draw calibration
Line.
(2) measurement of the curcumin charging ratio of probiotics gemma load
1. preparing the concentration of curcumin in the solution before CUR-Spore can amount (W), the concentration of solution according to curcumin
(V), concentration (W1)=W/V × 100%. of curcumin before loading are calculated by formula
2. solution (solution including centrifuge washing) can measure its suction at 420nm after preparing CUR-Spore reaction
Light value calculates the concentration (W2) of curcumin in solution according to standard curve.
3. calculating the charging ratio of curcumin according to formula (DG)=(W1-W2)/W1 × 100%
2, release measurement of the curcumin of probiotics gemma load in simulation gastro-intestinal Fluid
(1) configuration of artificial simulation gastric juices
The dense HCl of 1.8ml is weighed, 2g pepsin and a certain amount of water is added, adjusts pH value after completely dissolution to 1.3 constant volumes
200ml, as artificial simulation gastric juices[21]。
(2) configuration of artificial simulation intestinal juice
Weigh 1.36g KH2PO4It is dissolved in about 200ml water, separately weighs 2g trypsase and be dissolved in water, be settled to 200ml,
NaOH adjusts PH to 7.6, as artificial simulation intestinal juice.
(3) measurement of the curcumin release rate of probiotics gemma load
A certain amount of Spore and CUR-Spore-FA are added separately in simulation gastro-intestinal Fluid, surveyed respectively every two hours
Determine the light absorption value at solution 420nm, draw release figure of the curcumin of probiotics gemma load in simulation gastro-intestinal Fluid, determination is released
Put rule and its optimal release time.Release of the Spore in simulation gastro-intestinal Fluid is as control.
3, experimental result
The charging ratio of curcumin and simulation gastro-intestinal Fluid in release figure it is as shown in Fig. 6, in figure, A be probiotics gemma
Load the charging ratio of curcumin；B is the audio-visual picture of curcumin release liquid in simulation gastro-intestinal Fluid of probiotics gemma load；C is
The curcumin of probiotics gemma load in simulate the gastric juice scheme for 24 hours by release；D is the curcumin of probiotics gemma load in simulation intestines
Release is schemed for 24 hours in liquid.
A figure in Fig. 6 is using the charging ratio of spectrophotometry measurement curcumin, and measurement reaction front and back solution is in 420nm
The light absorption value at place calculates the concentration of curcumin by the standard curve of curcumin；It is average by calculating to determine four charging ratios
Value draws A figure with error line, wherein W1 is represented be grafted before curcumin concentration, W2 represents curcumin in the solution dense after grafting
The charging ratio of degree therefore curcumin should be DG=(W1-W2)/W1 × 100%.Figure A shows that the charging ratio of curcumin is up to
98.17%, and error is smaller, illustrates that the connection of curcumin is very successfully.
B figure in Fig. 6 is the audio-visual picture of probiotics gemma load curcumin release liquid in simulation gastro-intestinal Fluid, wherein a1Table
Show release figure of the simple gemma (Spore) in simulate the gastric juice, a2Indicate simple gemma (Spore) in simulated intestinal fluid
Release figure；b1Indicate release figure of (CUR-Spore) carrier in simulate the gastric juice, b2Indicate (CUR-Spore) carrier in simulation intestines
Release figure in liquid；c1Indicate the release figure of (CUR-Spore--FA) in simulate the gastric juice, c2Indicate that (CUR-Spore--FA) exists
Release figure in simulate the gastric juice.Because curcumin discharged in intestinal juice be yellow liquid, compare a1, a2, b1, b2, c1, c2Simulation
Release figure in gastro-intestinal Fluid can intuitively find out b2, c2Obviously compare b1, c1It is yellow.That is CUR-Spore and CUR-Spore-FA are in mould
Release rate in quasi- intestinal juice is significantly larger than simulate the gastric juice；Spore is as control.Therefore it can intuitively find out that curcumin is being simulated
Release rate in intestinal juice is much higher than simulate the gastric juice.
C figure in Fig. 6 is release figure of the probiotics gemma load curcumin in simulate the gastric juice for 24 hours, and wherein a is CUR-
Release figure of the Spore carrier in simulate the gastric juice, b are release figure of the Spore in simulate the gastric juice as control, exclude gemma and exist
Release liquid interference similar with curcumin in simulate the gastric juice；D figure is the release figure of curcumin in simulated intestinal fluid for 24 hours, and wherein a is
Release figure of the CUR-Spore carrier in simulated intestinal fluid, b are release figure of the Spore in simulated intestinal fluid liquid as control.C, D
Figure relatively can significantly find out that release of the curcumin in simulated intestinal fluid will be significantly larger than in gastric juice and can exclude bud
The influence of spore.From D figure it can be seen that curcumin 8h release rate highest and be all within one to eight hours smoothly to discharge.Its
In, some reductions of the amount of curcumin in simulated intestinal fluid later by eight hours, the reason is that spore surface is rough and uneven in surface with gemma
Sprouting amount increase adsorbed release curcumin reduce the light absorption value of curcumin.On the whole, curcumin is released in intestinal juice
Putting is that comparison is stable, has certain slow release effect.
The active identification of gemma in 4 support C UR-Spore-FA of embodiment
1, in order to verify connect upper curcumin and folic acid after gemma on CUR-Spore and GUR-Spore-FA carrier whether
Also it is active we done microorganism plate coating experiment, the specific steps are as follows:
(1) configuration beef-protein medium formula it is as follows: beef extract 3g/L, peptone 10g/L, sodium chloride 10g/L,
Agar 25g/L, PH to 7~7.4 is adjusted.
(2) Spore, CUR-Spore, the CUR-Spore-FA of 1mg are weighed respectively, and Spore sterile water is diluted 1011
Times, by CUR-Spore and CUR-Spore-FA respectively with sterile water dilution 108Times, then with coating beef extract-peptone plate.
(3) flat-plate inverted is seated in 37 DEG C of constant incubators, the sprouting situation of overnight incubation second day observation gemma is simultaneously
It takes pictures.
2, gemma activity identification figure is as shown in Fig. 7, and in figure, A is the spread plate of bacillus coagulans Spore；B is
The plate of CUR-Spore carrier coating；C is the plate of CUR-Spore-FA carrier coating.
In Fig. 7, A figure is sprouting situation of the Spore on beef high protein peptone plate, it can be seen that although having diluted
1011Times, but there are many gemma to sprout；B figure is sprouting situation of the CUR-Spore on beef extract-peptone plate, although
Dilute 108Times, but there are also more gemma to sprout；C figure is sprouting of the CUR-Spore-FA on beef extract-peptone plate
Situation dilutes 108Still there is gemma sprouting after times.
By B in Fig. 7, C is it is apparent that CUR-Spore and CUR-Spore-FA is carried after connecting upper curcumin and folic acid
Gemma on body is still active.Although gemma number is reduced on CUR-Spore-FA carrier, but have a large amount of
Gemma is active, and the release of anticancer drug curcumin can be finally reached to the effect for the treatment of colon cancer by the sprouting of gemma.
Claims (9)
1. a kind of probiotics folate-targeted carrier for loading anticancer drug, which is characterized in that be that will be resisted by chemical reaction first
Cancer drug is connected with the spore surface of bacillus coagulans, and synthesis obtains anticancer drug-probiotics carrier；Then through esterification
The probiotics folate-targeted carrier of load anticancer drug is prepared in folic acid on reaction forming；The anticancer drug is curcumin.
2. loading the probiotics folate-targeted carrier of anticancer drug according to claim 1, which is characterized in that the chemistry is anti-
It should be esterification.
3. loading the probiotics folate-targeted carrier of anticancer drug according to claim 1, which is characterized in that described by anticancer
It is that anticancer drug curcumin is connected to probiotics by esterification to coagulate that drug is connected with the spore surface of bacillus coagulans
It ties on bacillus spore outer layer capsid protein.
4. a kind of preparation method for the probiotics folate-targeted carrier for loading anticancer drug curcumin, which is characterized in that including such as
Lower step:
S1. esterification, structure occurs using the carboxyl of hydroxyl and Bacillus coagulans spore coat protein in curcumin structure
It builds to obtain curcumin-probiotics carrier；
Esterification occurs for the carboxyl in the hydroxyl and folic acid on curcumin-probiotics carrier that S2. step S1 is obtained, by folic acid
It is connected in the albumen of spore coat albumen of bacillus coagulans, obtains the probiotics folic acid target of load anticancer drug curcumin
To carrier.
5. preparation method according to claim 4, which is characterized in that the specific method of step S1 is:
S11. Bacillus coagulans spore powder is mixed with methylene chloride, ice salt bath is to 0 DEG C；
S12. it is equal that curcumin, DMAP and N, N- dicyclohexylcarbodiimide, ultrasonic vibration is added in the system of step S11 again
It is even, ice-water bath the reaction was continued 20~30h；
S13. the system of step S12 is centrifuged 5~15min through 8000~12000 rpm, then passes through dehydrated alcohol and distilled water respectively
Unreacted curcumin is washed away, is dried in vacuo to get curcumin-probiotics carrier is arrived；
The specific method of step S2 is:
S21. curcumin-probiotics carrier and methylene chloride are mixed, ice salt bath is to 0 DEG C；
S22. folic acid, DMAP and N, N- dicyclohexylcarbodiimide is added in the system of step S21 again, ultrasonic vibration is uniform,
Ice-water bath the reaction was continued 20~30h, centrifuge washing remove unreacted folic acid, and are freeze-dried to get to load anticancer drug
The probiotics folate-targeted carrier of curcumin.
6. preparation method according to claim 5, which is characterized in that Bacillus coagulans spore powder and two described in step S11
The mass volume ratio of chloromethanes is 0.01~0.2g:5ml；Curcumin described in step S12, DMAP and N, N- dicyclohexyl carbon two are sub-
The mass ratio of amine is 5~15:2~4:2~4.
7. preparation method according to claim 5, which is characterized in that curcumin described in step S21-probiotics carrier and dichloro
The mass volume ratio of methane is 0.01~0.2g:20ml；The matter of step S22 folic acid, DMAP and N, N- dicyclohexylcarbodiimide
Amount is than being 30~50:2~4:2~4.
8. preparation method according to claim 5, which is characterized in that the matter of the Bacillus coagulans spore powder and curcumin
Amount is than being 2~5:1；The mass ratio of curcumin-probiotics carrier and folic acid is 1:2~5.
9. preparation method system described in the probiotics folate-targeted carrier of load anticancer drug or claim 5 described in claim 1
Application of the probiotics folate-targeted carrier of standby obtained load anticancer drug curcumin in terms of preparing anticancer drug.
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| CN107998404B (en) * | 2017-12-09 | 2021-05-04 | 浙江大学 | Folic acid targeting vector loaded with anticancer drug and preparation method and application thereof |
| CN110812493A (en) * | 2019-11-22 | 2020-02-21 | 郑州大学 | Preparation method and application of self-contained nano generator pharmaceutical composition based on probiotic spores |
| CN112438996B (en) * | 2020-12-09 | 2021-08-17 | 郑州大学 | Preparation method and application of probiotic spore capsid based nano material and probiotic composition |
| CN112656765B (en) * | 2020-12-28 | 2022-07-26 | 黑龙江大学 | Preparation method of active targeting anticancer drug nanoparticles |
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