CN105816883A - Probiotics folic acid targeting carrier carrying anti-cancer medicament curcumin and preparation method thereof - Google Patents
Probiotics folic acid targeting carrier carrying anti-cancer medicament curcumin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a probiotics targeting carrier carrying an anti-cancer medicament curcumin and a preparation method thereof. The preparation method includes connecting the curcumin with spore surface of bacillus coagulans by means of an esterification reaction to synthesize and obtain curcumin-probiotics carrier (CUR-Spore); and then connecting the carrier with folic acid by means of the esterification reaction to successfully prepare and obtain probiotics folic acid targeting carrier carrying curcumin (CUR-Spore-FA). The targeting medicament carrier can be used to treat colorectal carcinoma, has targeting, and can reduce the toxic and side effect of the medicament. Moreover, the bacillus coagulans can protect the curcumin in the carrier medicament, so that the carrier drug can tolerate gastric acid to get through the stomach smoothly. The carrier drug can be colonization in the intestine and germination in the intestine to release curcumin for treating colorectal carcinoma. The preparation method improves the utilization rate of curcumin, establishes a targeting conveying system of the anti-cancer medicament, can be used for treating colorectal carcinoma, and provides a novel mode of treating colorectal carcinoma.
Description
Technical field
The invention belongs to pharmaceutical technology field.More particularly, to a kind of probiotic bacteria folate-targeted carrier loading cancer therapy drug curcumin and preparation method thereof.
Background technology
What cancer had become human health one threatens greatly.If colorectal cancer (Colorectal cancer, CRC) is one of which mankind malignant tumor occurred frequently, its sickness rate is the most in rising trend, has risen to the 3rd in the Cancer Mortality of the whole world.Along with living habit and the change of dietary structure, the lipid material that people take in increases, and the colon cancer sickness rate of China is in ascendant trend year by year.Colon cancer has become the severeest public health issue, and the health and lives of the people in serious threat, greatly hinders Chinese society expanding economy, and the etiology and pathogenesis of colon cancer and the further investigation of Therapeutic Method are shown important especially.For the treatment of the cancers such as colon cancer, in addition to traditional operation, radiotherapy are with chemotherapy.Due to the multicomponent of natural drug, Mutiple Targets, too many levels, multi-pathway effect, making people recognize the importance of " going back to nature ", the antitumor research of native compound the most progressively becomes the focus of clinical antitumor agents research.
Curcumin is a kind of natural polyphenol extracted from the rhizome of herbaceous plant Rhizoma Curcumae Longae, for the main active of Rhizoma Curcumae Longae.Some countries in Asia, curcumin is widely used as treating the medicine of disease, food color and the flavoring agents such as inflammation, asthma, liver dysfunction always.Additionally, curcumin also plays antiinflammatory, antioxidation, antitumor and elemental abundances in many tumor cells.Many experiment in vivo and vitro also confirm that curcumin can suppress tumor growth, substantially reduce the number of tumor body, alleviate the infringement to body of the multiple carcinogenic factor.Curcumin has the strongest apoptosis induction ability, inducing cell apoptosis is the important mechanisms of its antitumor action, relate to the biological process of series of complex, and divided the form that different approach showed different by intraor extracellular number of ways and signal, intersection is had between them, there is overlap, constitute intricate regulated and control network.Sum it up, curcumin broad-spectrum anti-tumor activity, there is good toleration and relatively low toxicity.But, curcumin poorly water-soluble causes its bioavailability low, and these hinder the curcumin application as drug candidate.
Recent studies indicate that, it is a kind of effective method that curcumin precursor structure carries out prodrug design.Curcumin prodrug is mainly designed to, with the little molecule containing carboxyl or macromolecular carrier, its phenolic hydroxyl group is carried out esterification and modifies; on the one hand the electron transfer of enol structure is strengthened by transformation phenolic hydroxyl group; protection phenolic hydroxyl group also eliminates electron delocalization in structure; on the other hand can improve the dissolubility of curcumin, raising stability, prolongation half-life by the character of the little molecule connected or macromolecular carrier, improve bioavailability; strengthen biological activity, there is certain controlled release, slow release effect simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is the defect and technical deficiency overcoming existing natural anti-cancer drugs curcumin to apply, a kind of probiotic bacteria targeting vector loading cancer therapy drug curcumin is provided, coat protein, the folic acid of the natural drug curcumin and Bacillus coagulans surface with antitumaous effect are connected, prepare CUR-Spore-FA carrier.And by CUR-Spore-FA carrier is characterized, research curcumin release rule in simulation gastro-intestinal Fluid, result show the CUR-Spore-FA carrier of the present invention both can guarantee that curcumin can release stable in intestinal and discharge hardly in gastric juice, for improving the utilization rate of natural anti-cancer drugs-curcumin, the targeted delivery system setting up cancer therapy drug and the treatment being applied to colon cancer are laid a solid foundation, not only solve the low problem of the plain availability of natural anti-cancer drugs-curcumin but also decrease the toxic and side effects for the treatment of colon cancer, and the treatment making colon cancer has targeting, a kind of new approach has been opened up in treatment for colon cancer.
It is an object of the invention to provide a kind of probiotic bacteria targeting vector loading cancer therapy drug curcumin.
Another object of the present invention is to provide the preparation method of the probiotic bacteria folate-targeted carrier of above-mentioned load cancer therapy drug curcumin.
Another object of the present invention is to provide the application of the probiotic bacteria folate-targeted carrier of above-mentioned load cancer therapy drug curcumin.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of probiotic bacteria folate-targeted carrier loading cancer therapy drug, is to first pass through chemical reaction to be connected by the spore surface of cancer therapy drug and Bacillus coagulans, and synthesis obtains cancer therapy drug-probiotic bacteria carrier；Connect upper folic acid then through esterification, prepare the probiotic bacteria folate-targeted carrier of load natural anti-cancer drugs.
Preferably, described cancer therapy drug is curcumin.
Preferably, described chemical reaction is esterification.
The most preferably, the invention provides a kind of probiotic bacteria targeting vector loading cancer therapy drug curcumin, be to first pass through esterification to be connected by the spore surface of curcumin and Bacillus coagulans, synthesis obtains curcumin-probiotic bacteria carrier (CUR-Spore)；Connect upper folic acid then through esterification, prepare probiotic bacteria folate-targeted carrier (CUR-Spore-FA) of load natural anti-cancer drugs curcumin.
It is further preferred that described connection by the spore surface of cancer therapy drug and Bacillus coagulans is to be connected on probiotic bacteria Bacillus coagulans spore outer layer capsid protein by natural anti-cancer drugs (curcumin) by esterification.
It addition, the preparation method of a kind of probiotic bacteria folate-targeted carrier loading cancer therapy drug curcumin, comprise the steps:
S1. utilize the carboxyl generation esterification of the hydroxyl in curcumin structure and Bacillus coagulans spore coat protein, build and obtain curcumin-probiotic bacteria carrier (CUR-Spore)；
S2. the hydroxyl on curcumin-probiotic bacteria carrier that step S1 obtains and the carboxyl generation esterification in folic acid, folic acid is connected in the albumen of spore coat albumen of Bacillus coagulans, obtains loading probiotic bacteria folate-targeted carrier (CUR-Spore-FA) of natural anti-cancer drugs.
Wherein it is preferred to, the concrete grammar of step S1 is:
S11. being mixed with dichloromethane by Bacillus coagulans spore powder, cryosel is bathed to 0 DEG C；
Adding curcumin, DMAP and N, N-dicyclohexylcarbodiimide the most again in the system of step S11, ultrasonic vibration is uniform, and ice-water bath continues reaction 20～30h；
S13. the system of step S12 is centrifuged 5～15min through 8000～12000rpm, then washes away unreacted curcumin respectively through dehydrated alcohol and distilled water wash, vacuum drying, i.e. obtains curcumin-probiotic bacteria carrier (CUR-Spore)；
Preferably, the concrete grammar of step S2 is:
S21. curcumin-probiotic bacteria carrier (CUR-Spore) and dichloromethane being mixed, cryosel is bathed to 0 DEG C；
Folic acid, DMAP and N is added the most again in the system of step S21, N-dicyclohexylcarbodiimide, ultrasonic vibration is uniform, ice-water bath continues reaction 20～30h, centrifuge washing removes unreacted folic acid, and lyophilization, i.e. obtain loading probiotic bacteria folate-targeted carrier (CUR-Spore-FA) of natural anti-cancer drugs.
It is highly preferred that the mass volume ratio of Bacillus coagulans spore powder and dichloromethane described in step S11 is 0.01～0.2g:5ml；Curcumin, DMAP and N described in step S12, the mass ratio of N-dicyclohexylcarbodiimide is 5～15:2～4:2～4.
It is highly preferred that the mass volume ratio of curcumin described in step S21-probiotic bacteria carrier (CUR-Spore) and dichloromethane is 0.01～0.2g:20ml；Step S22 folic acid, DMAP and N, the mass ratio of N-dicyclohexylcarbodiimide is 30～50:2～4:2～4.
It is highly preferred that the mass ratio of described Bacillus coagulans spore powder and curcumin is 2～5:1；The mass ratio of described curcumin-probiotic bacteria carrier (CUR-Spore) and folic acid is 1:2～5.
It addition, it is further preferred that it is 24h that ice-water bath described in step S12 and step S22 continues the time of reaction.
Preferably, centrifugal described in S13 is that 10000rpm is centrifuged 10min.
Preferably, dehydrated alcohol described in S13 and distilled water wash are washings 3 times.
It is highly preferred that the mass volume ratio of Bacillus coagulans spore powder and dichloromethane described in step S11 is 0.1g:5ml；Curcumin, DMAP and N described in step S12, the mass ratio of N-dicyclohexylcarbodiimide is 10:3:3.
It is highly preferred that the mass volume ratio of curcumin described in step S21-probiotic bacteria carrier (CUR-Spore) and dichloromethane is 0.1g:20ml；Step S22 folic acid, DMAP and N, the mass ratio of N-dicyclohexylcarbodiimide is 40:3:3.
It is highly preferred that the mass ratio of described Bacillus coagulans spore powder and curcumin is 4:1；The mass ratio of described curcumin-probiotic bacteria carrier (CUR-Spore) and folic acid is 1:4.
Finally, the application in terms of preparing cancer therapy drug of the probiotic bacteria folate-targeted carrier of above-mentioned load cancer therapy drug (curcumin), also within protection scope of the present invention.
Preferably, described cancer therapy drug is the medicine of anti-colorectal carcinoma.
Natural anti-cancer drugs curcumin is innovatively connected to can be colonizated on the outer layer capsid protein of the intestinal probiotic bacteria Bacillus coagulans spore useful to human body by the present invention by chemical reaction, CUR-Spore carrier connects folic acid again and builds target medicine carrier (CUR-Spore-FA) simultaneously, and carry out material characterization, the characterizing method used has: FTIR spectrum (FTIR), Raman spectrum (Raman), scanning electron microscope etc., it was demonstrated that vector construction success.Simultaneously, it is investigated spore sprouting in the gastro-intestinal Fluid of simulation and Drug-Release Behavior, utilize the release in simulation gastro-intestinal Fluid of this carrier of determined by ultraviolet spectrophotometry, prove curcumin can release stable in intestinal and discharge hardly in gastric juice, this carrier can well be applied to the treatment of colon cancer, for improving the utilization rate of natural anti-cancer drugs-curcumin, the targeted delivery system setting up cancer therapy drug and the treatment being applied to colon cancer are laid a solid foundation, and the treatment for colon cancer provides a kind of new mode.
And, bacillus coagulans used in the present invention (Bacillus coagulans), as a kind of probiotic bacteria, has antiinflammatory, strengthens the advantages such as body immunity, opposing antigen, eliminating foreign body；Additionally, bacillus coagulans in addition to there is general probiotic bacteria and have the advantage that, the advantage also having self uniqueness, such as: strong stress resistance, high temperature high voltage resistant, easily storage, anti-gastric acid, spore can be sprouted in intestinal and in intestinal in field planting, improves intestine microenvironment etc..
The present invention is through inhibiting tumor cell verification experimental verification, result shows, the probiotic bacteria folate-targeted carrier of the load cancer therapy drug curcumin that the present invention is successfully prepared, cancer therapy drug curcumin can not only be carried targeting and arrive focus, the effect of medicament slow release can also be played, and successfully reserved the effect to cancerous cell of the cancer therapy drug curcumin, cancerous cell is had good inhibiting effect and curative effect.
The method have the advantages that
Natural anti-cancer drugs curcumin is connected to can be colonizated on the outer layer capsid protein of the intestinal probiotic bacteria Bacillus coagulans spore useful to human body by the present invention by chemical reaction, CUR-Spore carrier connects folic acid more simultaneously, successfully construct a kind of target medicine carrier (CUR-Spore-FA), decrease the toxic and side effects for the treatment of colon cancer drug, and there is targeting.
Simultaneously; in this carrier medicament, Bacillus coagulans also has a kind of protective effect for cancer therapy drug curcumin; gastric acid can be tolerated and be smoothly through stomach; it is colonizated in intestinal and usually treats colon cancer in intestinal sprouting release Rhizoma Curcumae Longae; this carrier can well be applied to the treatment of colon cancer; for improving the utilization rate of natural anti-cancer drugs-curcumin, the targeted delivery system setting up cancer therapy drug and the treatment being applied to colon cancer are laid a solid foundation, and the treatment for colon cancer provides a kind of new mode.
Accompanying drawing explanation
Fig. 1 is schematic diagram prepared by CUR-Spore and CUR-Spore-FA.
Fig. 2 is the standard curve of curcumin.
Fig. 3 is the infrared spectrum (FTIR) utilizing fourier transform infrared spectroscopy to respective reaction thing and Characterization of The Products；A spore (Spore), B curcumin (Curcumin), C spore-curcumin (CUR-Spore), D spore-curcumin-folic acid (CUR-Spore-FA).
Fig. 4 is the Raman collection of illustrative plates utilizing Raman spectrometer to respective reaction thing and product；A spore (Spore), B curcumin (Curcumin), C spore-curcumin (CUR-Spore), D spore-curcumin-folic acid (CUR-Spore-FA).
Fig. 5 is scanning electron microscope (SEM) photograph；A spore (Spore), B curcumin (Curcumin), C spore-curcumin (CUR-Spore), D spore-curcumin-folic acid (CUR-Spore-FA).
Fig. 6 be curcumin charging ratio and simulation gastro-intestinal Fluid in release figure；A is the charging ratio of probiotic bacteria spore load curcumin；B is the audio-visual picture that the curcumin of probiotic bacteria spore load discharges liquid in simulation gastro-intestinal Fluid；C is curcumin 24h release figure in simulated gastric fluid of probiotic bacteria spore load；D is curcumin 24h release figure in simulated intestinal fluid of probiotic bacteria spore load.
Fig. 7 is spore activity identification figure；A is the spread plate of Bacillus coagulans Spore；B is the flat board that CUR-Spore is support coating；C is the flat board that CUR-Spore-FA is support coating.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but the present invention is not limited in any form by embodiment.Unless stated otherwise, the present invention uses reagent, method and apparatus are the art conventional reagent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are commercial.
Main agents used in following example has: curcumin, dichloromethane, DMAP (DMAP), N, N mono-dicyclohexylcarbodiimide (DCC), pepsin, and dehydrated alcohol is purchased from Da Su bio tech ltd.Pancreatin is GIBCOBRL Products.Spore is derived from South China Normal University's Life Science College pass swallow Puritanism and awards laboratory cultures.
Key instrument used in following example has: LEO company of Germany field emission scanning electron microscope: LEO1530VP, Sigma32184 High speed refrigerated centrifuge, Medical Instruments factory of Jintan City of Jiangsu Province 78-1 magnetic stirring apparatus, HV-85 autoclave, aseptic operating platform, Guangzhou Ke Qiao experimental technique equipment company limited thermostat water bath, shaking table, ultrasonic disperse instrument, ultraviolet spectrophotometer, ice machine etc..
Embodiment 1 loads the preparation of the probiotic bacteria folate-targeted carrier of natural anti-cancer drugs curcumin
Schematic diagram prepared by CUR-Spore and CUR-Spore-FA is as shown in Figure 1.
1, the preparation of curcumin-probiotic bacteria carrier (CUR-Spore)
The carboxyl generation esterification utilizing the hydroxyl in curcumin structure and Bacillus coagulans spore coat protein builds curcumin probiotic bacteria carrier.Concrete reaction is as follows:
(1) being added in 50ml beaker by 0.4g Bacillus coagulans spore powder, take 20ml dichloromethane and add, cryosel is bathed to 0 DEG C.
(2) adding 0.1g curcumin, 30mg DMAP and 30mg N, N mono-dicyclohexylcarbodiimide (DCC), ultrasonic vibration is uniform, and ice-water bath continues reaction 24h.
(3) 10000rpm is centrifuged 10 minutes, collects curcumin-probiotic bacteria carrier, and dehydrated alcohol and distilled water wash respectively 3 times and wash away the vacuum drying of unreacted curcumin and i.e. obtained the carrier of CUR-Spore.
2, the preparation of curcumin-probiotic bacteria-folate-targeted carrier (CUR-Spore-FA)
Bacillus coagulans passes through esterification with the hydroxyl on the connection carrier of curcumin with the carboxyl in folic acid, is connected to by folic acid in the albumen of spore coat albumen.Concrete reaction is as follows:
(1) being added in 50ml beaker by above-mentioned 0.1g curcumin probiotic bacteria carrier, take 20ml dichloromethane and add, cryosel is bathed to 0 DEG C.
(2) adding 0.4g folic acid, 30mg DMAP and 30mg N, N mono-dicyclohexylcarbodiimide (DCC), ultrasonic vibration is uniform, and ice-water bath continues reaction 24h.
(3) centrifuge washing removes unreacted folic acid, and lyophilization is CUR-Spore-FA.
The sign of embodiment 2 support C UR-Spore-FA
1, the standard curve of curcumin is as shown in Figure 2.
At 420nm, measured the curcumin solution light absorption value of variable concentrations by ultraviolet spectrophotometry, thus draw the standard curve of Fig. 2, the R of Fig. 22=0.997 reached more than 0.99 grade therefore this standard can use.But the range of standard curve is 0～5.5 μ g/ml.
2, infrared spectrum detection
(1) by Spore, CUR-Spore, CUR-Spore-FA is dried process, it is then placed in mortar, adds a certain amount of KBr, grind and uniformly make mixture be ground to granularity less than 2 μm, in order to avoid stray light effects, putting into afterwards in drying machine and be dried process, with the pressure of about 10MPa, mixture is pressed into transparent sheet on hydraulic press, upper machine measures.
(2) infared spectrum (FTIR) is as shown in Figure 3, in figure, A is spore (Spore), B be curcumin (Curcumin), C be spore-curcumin (CUR-Spore), D be spore-curcumin-folic acid (CUR-Spore-FA).
Spectrogram A has strong absworption peak representative-OH at 3434.18；At 1648.69, there are strong absworption peak, representative-C=O, spore just has these groups.B has the characteristic group beta diketone key of curcumin.A, C, the D amount of-OH and-C=O in C that compares increases and has beta diketone key in C, illustrates have curcumin to connect in C.Because there being phenolic hydroxyl group on curcumin, carbonyl and beta diketone key.Because esterification occurs at phenyl ring, and compared with D with C, 821.50,893.47 etc. the peaks having more are that the substituted position description of phenyl ring has folic acid to connect.
3, Raman spectrum detection
By Spore, CUR-Spore, CUR-Spore-FA are dried process, take appropriate sample the most respectively on microscope slide, upper machine testing.
Raman collection of illustrative plates as shown in Figure 4, in figure A be spore (Spore), B be curcumin (Curcumin), C be spore-curcumin (CUR-Spore), D be spore-curcumin-folic acid (CUR-Spore-FA).
Spectrogram A has strong absworption peak representative-COOH at 2200, and having absworption peak to represent in esters spore at 1650 has these groups, illustrates that A is spore.Collection of illustrative plates B has the characteristic group beta diketone key of curcumin at 1628, it was demonstrated that B figure is exactly reactant curcumin.Carboxylic group on collection of illustrative plates C spore disappears, and occurs in that ester bond is probably spore and reacts with curcumin, and therefore CUR-Spore vector construction is successful.The characteristic group beta diketone key of curcumin is had, it was demonstrated that have curcumin to connect on collection of illustrative plates D.
4, scanning electron microscope (SEM) photograph is as shown in Figure 5, in figure A be spore (Spore), B be curcumin (Curcumin), C be spore-curcumin (CUR-Spore), D be spore-curcumin-folic acid (CUR-Spore-FA).
Can be seen that spore is shaft-like and size is μm level；C figure can be seen that and on spore, have granular curcumin to connect；The scanning electron microscope (SEM) photograph of D figure spore-curcumin-folic acid is it can be seen that spore structure is the most complete, our guess is probably the relatively integrity destroying spore of centrifugal number of times more, in order to verify spore connect upper curcumin and folic acid after the most active we these sample spread plates are observed spore sprouting situation.Find that the explanation still having spore to sprout on flat board connects and go up after curcumin and folic acid spore or activated.
The curcumin charging ratio that in embodiment 3 support C UR-Spore-FA, probiotic bacteria spore loads and the release in simulation gastro-intestinal Fluid
1, the mensuration of the curcumin charging ratio of probiotic bacteria spore load
UV spectrophotometer measuring: consulting literatures learns that the characteristic absorption peak of curcumin is 426nm
(1) standard curve drawing curcumin specifically comprises the following steps that
1. the preparation of curcumin storing solution
Accurately weigh curcumin 1mg to be placed in beaker, after adding appropriate anhydrous alcohol solution, be transferred in the volumetric flask of 10mL, then be settled to scale with dehydrated alcohol, standby as curcumin storing solution.
From storing solution, measure 0.1 the most respectively, 0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5mL be respectively placed in 9 10mL volumetric flasks, be settled to scale with dehydrated alcohol, shake up.With dehydrated alcohol as blank, at 420nm, measure absorbance, and with the meansigma methods (A) of each three absorbances of concentration as vertical coordinate, concentration (c) is abscissa, draw calibration curve.
(2) mensuration of the curcumin charging ratio of probiotic bacteria spore load
1. before preparing CUR-Spore, curcumin concentration in the solution can calculate concentration (W1)=W/V × 100%. of curcumin before loading according to the amount (W) of curcumin, the concentration (V) of solution by formula
2. after preparing CUR-Spore reaction, solution (including the solution of centrifuge washing) can measure its light absorption value at 420nm, calculates the concentration (W2) of curcumin in solution according to standard curve.
3. the charging ratio of curcumin is calculated according to formula (DG)=(W1-W2)/W1 × 100%
2, the release in simulation gastro-intestinal Fluid of the curcumin of probiotic bacteria spore load measures
(1) configuration of artificial simulation gastric juices
Weighing the dense HCl of 1.8ml, add 2g pepsin and a certain amount of water, after fully dissolving, regulation pH value is to 1.3 constant volume 200ml, is 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 trypsin and be dissolved in water, be settled to 200ml, NaOH and regulate PH to 7.6, be artificial simulation intestinal juice.
(3) mensuration of the curcumin release rate of probiotic bacteria spore load
It is added separately to a certain amount of Spore and CUR-Spore-FA simulate in gastro-intestinal Fluid, the light absorption value at solution 420nm is measured respectively every two hours, the curcumin of drafting probiotic bacteria spore load release figure in simulation gastro-intestinal Fluid, determines release rule and optimal release time thereof.Spore release in simulation gastro-intestinal Fluid is as comparison.
3, experimental result
As shown in Figure 6, in figure, A is the charging ratio of probiotic bacteria spore load curcumin for the charging ratio of curcumin and the release figure in simulation gastro-intestinal Fluid；B is the audio-visual picture that the curcumin of probiotic bacteria spore load discharges liquid in simulation gastro-intestinal Fluid；C is curcumin 24h release figure in simulated gastric fluid of probiotic bacteria spore load；D is curcumin 24h release figure in simulated intestinal fluid of probiotic bacteria spore load.
A figure in Fig. 6 is the charging ratio utilizing spectrophotometry curcumin, measures solution light absorption value at 420nm before and after reacting and is calculated the concentration of curcumin by the standard curve of curcumin；Determine four charging ratios and draw A figure by calculating meansigma methods and error line, the concentration of curcumin before wherein W1 represents grafting, it should be DG=(W1-W2)/W1 × 100% that W2 represents the charging ratio of the curcumin therefore curcumin of concentration in the solution after being grafted.Figure A shows that the charging ratio of curcumin is up to 98.17%, and error is less, illustrates that the connection of curcumin is the most successful.
B figure in Fig. 6 is the audio-visual picture that probiotic bacteria spore load curcumin discharges liquid in simulation gastro-intestinal Fluid, wherein a1Represent simple spore (Spore) the release figure in simulated gastric fluid, a2Represent simple spore (Spore) the release figure in simulated intestinal fluid；b1Represent (CUR-Spore) carrier release figure in simulated gastric fluid, b2Represent (CUR-Spore) carrier release figure in simulated intestinal fluid；c1Represent (CUR-Spore--FA) release figure in simulated gastric fluid, c2Represent (CUR-Spore--FA) release figure in simulated gastric fluid.Because it is the liquid of yellow that curcumin discharges in intestinal juice, contrast a1, a2, b1, b2, c1, c2Release figure in simulation gastro-intestinal Fluid can find out b intuitively2, c2Substantially than b1, c1Yellow.I.e. CUR-Spore and CUR-Spore-FA release rate in simulated intestinal fluid is significantly larger than simulated gastric fluid；Spore is as comparison.Therefore can find out that curcumin release rate in simulated intestinal fluid is far above simulated gastric fluid intuitively.
C figure in Fig. 6 is that probiotic bacteria spore loads curcumin release figure of 24h in simulated gastric fluid, wherein a is CUR-Spore carrier release figure in simulated gastric fluid, b is that Spore release figure in simulated gastric fluid discharges, as comparison, eliminating spore, the interference that liquid is similar to curcumin in simulated gastric fluid；D figure is curcumin release figure of 24h in simulated intestinal fluid, and wherein a is CUR-Spore carrier release figure in simulated intestinal fluid, and b is that Spore release figure in simulated intestinal fluid liquid is as comparison.C, D figure relatively can significantly find out that curcumin release in simulated intestinal fluid to be significantly larger than in gastric juice and can get rid of the impact of spore.From D figure, can be seen that curcumin is the highest at the release rate of 8h and within one to eight hours, be all release smoothly.Wherein, to after eight hours in simulated intestinal fluid the amount of curcumin some reduce, reason is that spore surface is rough and uneven in surface has adsorbed the curcumin of release make the light absorption value of curcumin reduce along with the sprouting amount of spore increases.On the whole, it is that comparison is stable that curcumin discharges in intestinal juice, has certain slow release effect.
The qualification of spore activity in embodiment 4 support C UR-Spore-FA
1, in order to verify connect upper curcumin and folic acid after spore on CUR-Spore and GUR-Spore-FA carrier the most active we done the flat board coating of microorganism and tested, specifically comprise the following steps that
(1) configuration beef-protein medium formula is as follows: Carnis Bovis seu Bubali cream 3g/L, peptone 10g/L, sodium chloride 10g/L, agar 25g/L, regulation PH to 7～7.4.
(2) weigh Spore, CUR-Spore, the CUR-Spore-FA of 1mg respectively, and Spore sterilized water is diluted 1011Times, CUR-Spore and CUR-Spore-FA is diluted 10 with sterilized water respectively8Times, then with coating beef extract-peptone flat board.
(3) being seated in by flat-plate inverted in 37 DEG C of constant incubators, overnight incubation is observed the sprouting situation of spore for second day and takes pictures.
2, spore activity identification figure is as shown in Figure 7, and in figure, A is the spread plate of Bacillus coagulans Spore；B is the flat board that CUR-Spore is support coating；C is the flat board that CUR-Spore-FA is support coating.
In Fig. 7, A figure is Spore sprouting situation on beef high protein peptone flat board, it can be seen that although having diluted 1011Times, but have many spore to sprout；B figure is CUR-Spore sprouting situation on beef extract-peptone flat board, although dilute 108Times, but the most more spore is sprouted；C figure is CUR-Spore-FA sprouting situation on beef extract-peptone flat board, dilutes 108Spore is still had to sprout after Bei.
By B in Fig. 7, C it is apparent that after connecting upper curcumin and folic acid the spore on CUR-Spore and CUR-Spore-FA carrier the most active.Although spore number has reduced on CUR-Spore-FA carrier, but there is substantial amounts of spore active, by the sprouting of spore, the release of cancer therapy drug curcumin can be finally reached the effect for the treatment of colon cancer.
Claims (10)
1. the probiotic bacteria folate-targeted carrier loading cancer therapy drug, it is characterised in that being to first pass through chemical reaction to be connected by the spore surface of cancer therapy drug and Bacillus coagulans, synthesis obtains cancer therapy drug-probiotic bacteria carrier；Connect upper folic acid then through esterification, prepare the probiotic bacteria folate-targeted carrier of load natural anti-cancer drugs.
Load the probiotic bacteria folate-targeted carrier of cancer therapy drug the most according to claim 1, it is characterised in that described cancer therapy drug is curcumin.
Load the probiotic bacteria folate-targeted carrier of cancer therapy drug the most according to claim 1, it is characterised in that described chemical reaction is esterification.
Load the probiotic bacteria folate-targeted carrier of cancer therapy drug the most according to claim 1, it is characterized in that, described connection by the spore surface of cancer therapy drug and Bacillus coagulans is to be connected on probiotic bacteria Bacillus coagulans spore outer layer capsid protein by natural anti-cancer drugs curcumin by esterification.
5. the preparation method of the probiotic bacteria folate-targeted carrier loading cancer therapy drug curcumin, it is characterised in that comprise the steps:
S1. utilize the carboxyl generation esterification of the hydroxyl in curcumin structure and Bacillus coagulans spore coat protein, build and obtain curcumin-probiotic bacteria carrier；
S2. the hydroxyl on curcumin-probiotic bacteria carrier that step S1 obtains and the carboxyl generation esterification in folic acid, be connected to folic acid in the albumen of spore coat albumen of Bacillus coagulans, obtains loading the probiotic bacteria folate-targeted carrier of natural anti-cancer drugs.
Preparation method the most according to claim 5, it is characterised in that the concrete grammar of step S1 is:
S11. being mixed with dichloromethane by Bacillus coagulans spore powder, cryosel is bathed to 0 DEG C；
Adding curcumin, DMAP and N, N-dicyclohexylcarbodiimide the most again in the system of step S11, ultrasonic vibration is uniform, and ice-water bath continues reaction 20～30h；
S13. the system of step S12 is centrifuged 5～15min through 8000～12000 rpm, then washes away unreacted curcumin respectively through dehydrated alcohol and distilled water wash, vacuum drying, i.e. obtains curcumin-probiotic bacteria carrier；
The concrete grammar of step S2 is:
S21. curcumin-probiotic bacteria carrier and dichloromethane being mixed, cryosel is bathed to 0 DEG C；
Adding folic acid, DMAP and N, N-dicyclohexylcarbodiimide the most again in the system of step S21, ultrasonic vibration is uniform, ice-water bath continues reaction 20～30h, centrifuge washing removes unreacted folic acid, and lyophilization, i.e. obtains loading the probiotic bacteria folate-targeted carrier of natural anti-cancer drugs.
Preparation method the most according to claim 6, it is characterised in that the mass volume ratio of Bacillus coagulans spore powder and dichloromethane described in step S11 is 0.01～0.2g:5ml；Curcumin, DMAP and N described in step S12, the mass ratio of N-dicyclohexylcarbodiimide is 5～15:2～4:2～4.
Preparation method the most according to claim 6, it is characterised in that the mass volume ratio of curcumin described in step S21-probiotic bacteria carrier and dichloromethane is 0.01～0.2g:20ml；Step S22 folic acid, DMAP and N, the mass ratio of N-dicyclohexylcarbodiimide is 30～50:2～4:2～4.
Preparation method the most according to claim 6, it is characterised in that the mass ratio of described Bacillus coagulans spore powder and curcumin is 2～5:1；The mass ratio of curcumin-probiotic bacteria carrier and folic acid is 1:2～5.
10. described in claim 1, load the application in terms of preparing cancer therapy drug of the probiotic bacteria folate-targeted carrier of the load cancer therapy drug curcumin that preparation method described in the probiotic bacteria folate-targeted carrier of cancer therapy drug or claim 5 prepares.
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