CN106420880B - A kind of barren wort total chromocor enzymolysis product and its preparation method and application - Google Patents
A kind of barren wort total chromocor enzymolysis product and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of barren wort total chromocor enzymolysis products, it is to generate barren wort total chromocor by immobilization glusulase enzymatic hydrolysis, the enzymolysis product has anti-tumor activity more better than barren wort total chromocor prototype and resolvase enzymolysis product, and has preferable oral absorptivity.The invention also discloses the preparation method of the enzymolysis product, the method digests barren wort total chromocor using immobilised enzymes, and the glusulase and immobilization carrier joint efficiency of selection are high, stability is good, digests high conversion rate, and immobilised enzymes can also be recycled, it can economize on resources, realize green production.The application that the invention further relates to enzymolysis products in antitumor, the barren wort total chromocor enzymolysis product obtained according to technical solution provided by the invention has significant inhibited proliferation to typeⅡ pneumocyte, hepatoma Hep G 2 cells, MCF-7 Breast Cancer Cell, cervical cancer Hela cells, can be used for the treatment of lung cancer, liver cancer, breast cancer, cervical carcinoma.
Description
Technical field
The present invention relates to a kind of barren wort total chromocor enzymolysis product, which is that barren wort total chromocor is passed through immobilization snail
Ox enzyme enzymatic hydrolysis generates.The invention further relates to the preparation method of the enzymolysis product and in the application of anti-tumor aspect, belong to medicine
Technical field.
Background technique
Herba Epimedii continues to use history in existing thousand as traditional kidney-nourishing tcm drug, civil, has kidney-replenishing, and strengthening the bones and muscles is dispelled
The effect of rheumatism, clinic are usually used in treating impotence and seminal emission, and muscles and bones is withered soft, the diseases such as rheumatic arthralgia.
Herba Epimedii mainly contains flavones ingredient, mainly there is icariin, Epimedin A, Epimedin B, epimedin C and the precious leaves of pulse plants
The flavonoid glycosides such as glycosides I (structure is shown in Fig. 1).Modern pharmacological studies have shown that epimedium flavone glycosides has good bioactivity, except having
Outside anti-osteoporosis activity, also there is good antitumor action.Document report epimedium flavone constituents are to respiratory system
Lung cancer tumor has certain killing ability to digestive system liver cancer and gastric cancer, to hematological system leukaemia cell, can control
The growth of tumour cell promotes the death of tumour cell, inhibits Tumor angiogenesis and can effectively adjust special in body
Different, non-specific immunocyte, inhibits the immunologic escape of tumour cell, to play antitumor action.
This seminar early-stage study shows oral epimedium flavone glycosides, and prototype absorption is less, needs by enteron aisle enzyme enteron aisle
The hydrolysis of bacterium is absorbed in the form of secondary glycosides or aglycon and plays curative effect.Therefore it finds in vitro and fits epimedium flavone glycosides
The method preferably digested obtains the secondary glycosides that activity is higher and is easy to absorb, and has preferable antitumor action to further research and development
New drug is of great significance applied to clinic.Currently, have the more research about external enzymatic hydrolysis epimedium flavone prototype glycosides, but
These methods are mostly that icariin is digested using resolvase, such as utilize beta-glucosidase, cellulase, glusulase or aspergillus
The research for the induction enzymatic conversion icariin that mould generates.But resolvase is very sensitive to the microenvironment of reaction system, any pH,
The change of temperature can make its activity be affected, and be difficult to be recycled, spend cost high, and it is raw to be unfavorable for large-scale industry
It produces.Due to other than containing icariin ingredient, also containing the more flavones ingredient of other three kinds of contents: towards the leaves of pulse plants in Herba Epimedii
Determine A, Epimedin B and epimedin C.Therefore, icariin is extracted from epimedium herb merely to be digested again, not only need numerous
Trivial separating-purifying step, and the effective component in epimedium herb cannot make full use of, and be unfavorable for the rationalization of natural resources of Chinese medicinal materials
Using.
Summary of the invention
An object of the present invention is to provide a kind of barren wort total chromocor enzymolysis product, which is by barren wort total chromocor
It is generated by immobilization glusulase enzymatic hydrolysis, which has than barren wort total chromocor prototype and resolvase enzymolysis product more
Good anti-tumor activity, and there is preferable oral absorptivity, the utilization rate to epimedium active constituent is improved, medicine is realized
The comprehensive utilization in aptitude source, and immobilised enzymes used in product preparation can be recycled, and can economize on resources, and realize that green is raw
It produces.
For foregoing invention purpose, the present invention the following technical schemes are provided:
A kind of barren wort total chromocor enzymolysis product, the enzymolysis product digest barren wort total chromocor by immobilization glusulase
It obtains, the glusulase is fixed by amino mesoporous silicon.
Wherein, the preparation method of the barren wort total chromocor are as follows: Herba Epimedii is measured 40%~90% with 6~40 (m/v) times
Ethyl alcohol is concentrated after using circumfluence distillation, is further purified through Flavonoids by Macroporous Adsorption Resin, obtains mass content and is not less than 60%
Barren wort total chromocor.Wherein, icariin in barren wort total chromocor, Epimedin A, Epimedin B, epimedin C and baohuoside Ⅰ is total
Mass content is not less than 30% (HPLC is measured).
A kind of preferred amino mesoporous silicon the preparation method comprises the following steps: immersing silica dissolved with 3- aminopropyl-triethoxy silicon
In the toluene of alkane, magnetic agitation 800rmin-1, under 90 DEG C of high temperature, react 12h；Reactant is taken out, ultrasound is washed in toluene
10min is washed, is dried in vacuo 2h at 60 DEG C.
Further, the fixing means of the glusulase are as follows: glusulase and the mesoporous silicon carrier of amino are placed in 5.0 phosphorus of pH
It is reacted in acid buffering salting liquid, magnetic agitation is reacted at room temperature；Kept dry after distillation water washing later.
Wherein, the glusulase and the mesoporous silicon carrier dosage of amino are mass ratio 1:1~1:4, preferably 1:1.Glusulase with
The joint efficiency of amino mesoporous silicon is incremented by between mass ratio 1:1~1:4, and the combination of glusulase and mesoporous silicon is imitated when mass ratio 1:4
Rate reaches maximum, can reach 90% or more, but consider preparation cost, and the mass ratio of final choice glusulase and mesoporous silicon is 1:1,
Both reduced the dosage of mesoporous silicon carrier, effective joint efficiency can also be kept, with this condition, glusulase and amino mesoporous silicon
Joint efficiency can reach 89.63%.
Further, enzymatic hydrolysis condition is preferred are as follows: amino mesoporous silicon immobilization glusulase and barren wort total chromocor addition contain
In the buffer salt solution of 40% volume fraction DMSO, buffer salt solution pH 5.0, amino mesoporous silicon immobilization glusulase and excessive sheep
Leaves of pulse plants general flavone mass ratio is 1:1, is reacted under the conditions of 50 DEG C of temperature, and 2h is reacted.
Almost without epimedium aglucone in enzymolysis product of the present invention, enzymolysis product is mainly by arrow leaves of pulse plants glycosides A；Arrow leaves of pulse plants glycosides
B；Rhamnopyranosyl icariside I I；Precious leaves of pulse plants glycosides I composition；Its composition ratio is about arrow leaves of pulse plants glycosides A: arrow leaves of pulse plants glycosides B: the excessive sheep of rhamnopyranosyl
The leaves of pulse plants time glycosides II: precious leaves of pulse plants glycosides I is 1.5:2.0:1.0:5.8.
The second object of the present invention is to provide a kind of barren wort total chromocor enzyme solution, and specific technical solution is to use
Immobilization glusulase digests barren wort total chromocor, wherein the immobilization carrier of the glusulase selects amino mesoporous silicon.
Barren wort total chromocor, the combination effect of glusulase and carrier are digested using immobilization snail enzyme method of the present invention
Rate is high, and the advantageous pH range of reaction is broadening, also has better adaptability to reaction temperature, and stability is good, and digests conversion
Rate is high.
In addition, further requirement of the present invention protects above-mentioned barren wort total chromocor enzymolysis product in the preparation of antitumor drugs
Using.
Immobilization glusulase of the present invention is to barren wort total chromocor enzymolysis product, than barren wort total chromocor prototype and trip
There is better anti-tumor activity from enzyme enzymolysis product, and there is preferable oral absorptivity.
Barren wort total chromocor is digested using amino mesoporous silicon immobilization snail enzyme method of the present invention, immobilised enzymes is not only
The advantages of remaining resolvase also has and is easy to separate with substrate, repeats to recycle convenient for recycling, suitable Reaction conditions range
Width can reduce production cost, be suitable for the advantages such as industrialized production.And the joint efficiency of glusulase and the mesoporous silicon carrier of amino
Height, stability is good, and digests high conversion rate.In addition, The invention also achieves to the Huang in Herba Epimedii in addition to icariin
The effective use of ketones component, the general flavone ingredient extracted in Herba Epimedii are digested, and epimedium herb effective component is improved
Utilization rate realizes the comprehensive utilization of herb resource.Importantly, barren wort total chromocor is through the fixed glusulase of amino mesoporous silicon
Product of the enzymolysis product than prototype compound and through resolvase enzymatic hydrolysis has better anti-tumor activity, researches and develops as raw material anti-
Tumour new drug has preferable clinical value.
Detailed description of the invention
Fig. 1 is the chemical structure schematic diagram of Main Flavonoids contained by barren wort total chromocor and enzymolysis product；
Fig. 2 is SEM characterization schematic diagram of amino mesoporous silicon immobilization glusulase (amplification factor is 8.1mm × 50.0k)；
Fig. 3 varies with temperature schematic diagram with respect to enzyme activity and conversion ratio for amino mesoporous silicon immobilization glusulase；Wherein, A is
Opposite enzyme activity, B are conversion ratio, (N=3,*P < 0.05,**P<0.01)；
Fig. 4 changes schematic diagram with storage time with respect to enzyme activity and transformation efficiency for amino mesoporous silicon immobilization glusulase；Its
Middle A is opposite enzyme activity；B be conversion ratio (N=3)；
Fig. 5 is that amino mesoporous silicon immobilization glusulase digests barren wort total chromocor optimum reaction conditions investigation schematic diagram；Its
In, A is using enzyme activity as the optimal reaction pH of index；B is using conversion ratio as the optimal reaction pH of index；C is using enzyme activity as index
Optimal reactive temperature；D is using conversion ratio as the optimal reactive temperature of index；E is using enzyme activity as the most suitable enzyme-to-substrate of index
Mass ratio；F is using conversion ratio as the most suitable enzyme-to-substrate mass ratio of index；
Fig. 6 is the HPLC map of barren wort total chromocor and its enzymolysis product；Wherein, A is mixed reference substance solution；B is excessive
Sheep leaves of pulse plants general flavone enzymolysis product, wherein 1 digests general flavone product for free glusulase, 2 be amino mesoporous silicon immobilization glusulase
Digest general flavone product；A. Epimedin A；B. Epimedin B；C. epimedin C；D. icariin；E. arrow leaves of pulse plants glycosides A；F. arrow leaves of pulse plants glycosides B；
G. rhamnopyranosyl icariside I I；H. precious leaves of pulse plants glycosides I；I. epimedium aglucone；
Fig. 7 is barren wort total chromocor and its immobilised enzymes enzymolysis product to A549, HepG2, MCF-7, Hela cytosis
The inhibiting rate comparison schematic diagram of 48h；Wherein, A is A549 cell；B is HepG2 cell；C is MCF-7 cell；D is Hela cell；
(n=6, *P < 0.05,**P<0.01)；
Fig. 8 is thin to A549, HepG2, MCF-7, Hela for the product of resolvase and immobilised enzymes enzymatic hydrolysis barren wort total chromocor
The inhibiting rate comparison schematic diagram of born of the same parents' effect 48h；Wherein, A is MCF-7 cell；B is Hela cell；C is HepG2 cell；D is
A549 cell；(n=6, *P < 0.05,**P<0.01)。
Specific embodiment
Below with reference to example, invention is further described in detail, but the scope of the present invention is not appointed by these examples
What is limited.
Selected hydrolase information in the embodiment of the present invention: (> 300U/mg, Shanghai source leaf biotechnology are limited for glusulase
Company), unless otherwise specified, various raw material/preparations used in the present invention are commercially available known product.
Embodiment 1
The present embodiment provides a kind of preferred embodiments using the method for the present invention enzymatic hydrolysis barren wort total chromocor, digest to the present invention
Method does further specific descriptions.
1. the preparation of barren wort total chromocor
Herba Epimedii is measured into 40%~90% ethyl alcohol using being concentrated after circumfluence distillation, through macroporous absorption with 6~40 (m/v) times
Resin method is further purified, and obtains the barren wort total chromocor that mass content is not less than 60%.
Barren wort total chromocor main component is icariin, Epimedin A, Epimedin B, epimedin C and precious leaves of pulse plants glycosides I, and
Icariin, Epimedin A, Epimedin B, epimedin C and precious leaves of pulse plants glycosides I gross mass content are not less than 30%.
2. the preparation and characterization of the mesoporous silicon carrier of amino
Silica is immersed dissolved in the toluene of 3- aminopropyl triethoxysilane (C=50mM), magnetic agitation
800r·min-1, under 90 DEG C of high temperature, react 12h.Reactant is taken out, in toluene, supersound washing 10min, at 60 DEG C, very
The dry 2h of sky is saved backup in 4 DEG C of refrigerators to get the mesoporous silicon sample of amino.
Using the surface knot of Hitachi S-3400 scanning electron microscope (SEM) observation amino mesoporous silicon immobilised enzymes
Structure.Take amino mesoporous silicon sample powder to be fixed with solid conduction adhesive tape, after gold-plated, operating voltage 15kV, under vacuum condition into
Row characterization, is shown in Fig. 2.It prepares resulting amino mesoporous silicon to visually observe, appearance is the uniform powder of white.(SEM) prepared by observation
Amino mesoporous silicon appearance rounding, surface have more gap structure, and the porous structure aperture of amino mesoporous silicon is more uniform, benefit
In the fixed efficiency for improving enzyme.
3. the preparation of amino mesoporous silicon immobilization glusulase
Glusulase is weighed according to the mass ratio of 1:1 and the mesoporous silicon carrier of amino is placed in conical flask in right amount, in 5.0 phosphorus of pH
It is reacted in acid buffering salting liquid, at room temperature magnetic agitation 500rmin-1, react 10h.Then it distills water washing 3~5 times, in 37
Vacuum drying is placed in 4 DEG C of refrigerators and saves backup to get amino mesoporous silicon immobilization glusulase under the conditions of DEG C.
4. the measurement of amino mesoporous silicon immobilization glusulase zymologic property
(1) measurement of amino mesoporous silicon immobilization snail enzyme heat stability
It weighs appropriate free and amino mesoporous silicon immobilization glusulase and is placed in different temperatures (50,60,70,80,90,100
DEG C) under the conditions of place 1h after, then under conditions of temperature is 50 DEG C and pH is 5.0, concentration of substrate 0.5mgmL-1, enzyme and bottom
Amount of substance ratio is 1:1, digests barren wort total chromocor, is sampled after reacting 1h, and methanol is added and terminates and reacts, HPLC measurement, as a result
See Fig. 3.The enzyme activity of amino mesoporous silicon immobilised enzymes is above resolvase, and in extreme temperature conditions, the two has significant difference,
The thermal stability of amino mesoporous silicon immobilization glusulase is higher than free glusulase.
(2) measurement of amino mesoporous silicon immobilization glusulase reusing
The mesoporous silicon-immobilized glusulase of changing surely of appropriate amino is weighed under conditions of temperature is 50 DEG C and pH is 5.0, concentration of substrate
0.5mg·mL-1, enzyme-to-substrate mass ratio is 1:1, digests barren wort total chromocor, is sampled after reacting 1h, and methanol is added and terminates instead
It answers, HPLC measurement.Above-mentioned amino mesoporous silicon immobilization glusulase is separated from enzymatic hydrolysis system, after distilled water sufficiently washs 3 times,
It reacts at identical conditions, HPLC measurement repeats the above steps, and investigates access times to amino mesoporous silicon immobilization glusulase
The influence of enzyme activity.Recycling amino mesoporous silicon immobilization glusulase 5 times after, enzyme activity still be positively retained at 60% with
On；Compared with resolvase, amino mesoporous silicon immobilised enzymes has preferable reusable property, reduces production cost.
(3) measurement of amino mesoporous silicon immobilization glusulase storage-stable
Amino mesoporous silicon immobilization glusulase is placed under the conditions of 4 DEG C and is saved, is sampled 1 time weekly, is turned according to above-mentioned condition
Change barren wort total chromocor, HPLC measurement calculates the enzyme activity of immobilization glusulase, METHOD FOR CONTINUOUS DETERMINATION 6 weeks, investigates amino mesoporous silicon and fix
The storage-stable for changing glusulase, is as a result shown in Fig. 4.After amino mesoporous silicon immobilization glusulase saves 6 weeks under the conditions of 4 DEG C, enzyme
Vigor can still retain about 80%, have preferable storage-stable.
5. amino mesoporous silicon immobilization glusulase digests barren wort total chromocor
(1) optimal reaction pH is investigated
Appropriate free and amino mesoporous silicon immobilization glusulase is weighed, under the conditions of 50 DEG C of temperature, respectively at different pH value
Barren wort total chromocor is digested in the phosphate buffer of (2.0,3.0,4.0,5.0,6.0,7.0,8.0) condition, concentration of substrate is
0.5mg·mL-1, enzyme-to-substrate mass ratio is 1:1, is sampled after reacting 1h, and addition methanol, which terminates, reacts, HPLC measurement, as a result
See Fig. 5 (A, B).Free and amino mesoporous silicon immobilization glusulase enzyme activity and enzymolysis efficiency change with the increase of pH, are in
The trend of existing first increases and then decreases；The optimal reaction pH of free glusulase enzymatic hydrolysis barren wort total chromocor extract is 6.0, at this time its
Enzyme activity and the efficiency for digesting barren wort total chromocor extract are maximum；It is always yellow that amino mesoporous silicon immobilization glusulase digests Herba Epimedii
The optimal reaction pH of ketone extract is 5.0, its enzyme activity and the efficiency of enzymatic hydrolysis barren wort total chromocor extract are maximum at this time.Separately
Outside, under the conditions of extreme pH, the enzyme activity and enzymolysis efficiency of amino mesoporous silicon immobilization glusulase are significantly higher than free snail
Enzyme shows glusulase after immobilization, and the optimum pH range of enzyme reaction broadens.
(2) optimal reactive temperature is investigated
Appropriate free and amino mesoporous silicon immobilization glusulase is weighed, in the phosphate buffer that pH value is 5.0, respectively
Barren wort total chromocor, concentration of substrate 0.5mgmL are digested under the conditions of 4,25,37,50,60,70 DEG C of temperature-1, enzyme-to-substrate
Mass ratio is 1:1, is sampled after reacting 1h, and addition methanol terminates reaction, as a result Fig. 5 (C, D) is shown in HPLC measurement.It is free and
The enzyme activity and enzymolysis efficiency of amino mesoporous silicon immobilization glusulase change with the increase of temperature, and first increases and then decreases is presented
Trend；When free and amino mesoporous silicon immobilization glusulase digests barren wort total chromocor extract under the conditions of 50 DEG C, the two
Enzyme activity and the equal highest of enzymolysis efficiency, therefore, the optimal reactive temperature that the two digests barren wort total chromocor extract is 50 DEG C.With
Free glusulase is compared, and amino mesoporous silicon immobilization glusulase enzyme activity and enzymolysis efficiency variation at a temperature of differential responses is smaller,
Under the conditions of 25,37,60,70 DEG C, enzyme activity and enzymolysis efficiency are obviously higher than free glusulase.The experimental results showed that snail
Enzyme is particularly suited for the various enzymatic hydrolysis conversion reactions under condition of different temperatures after immobilization.
(3) most suitable substrate is than investigating
Appropriate free and amino mesoporous silicon immobilization glusulase is weighed, in optimal reactive temperature and optimal reaction pH value condition
Lower enzymatic hydrolysis barren wort total chromocor, concentration of substrate 0.5mgmL-1, enzyme-to-substrate mass ratio is respectively 1:2,1:1,2:1,3:1,
4:1 is sampled after reacting 1h, and addition methanol terminates reaction, as a result Fig. 5 (E, F) is shown in HPLC detection.Free glusulase with it is excessive
When sheep leaves of pulse plants general flavone mass ratio is 1:2, enzyme activity and the equal highest of enzymolysis efficiency, with the increasing of enzyme and barren wort total chromocor mass ratio
Add, enzyme activity and enzymolysis efficiency gradually decrease；Amino mesoporous silicon immobilization glusulase is being 1:1 with barren wort total chromocor mass ratio
When, enzyme activity and enzymolysis efficiency highest.Therefore, it is 1 that the glusulase that dissociates, which digests the most suitable enzyme-to-substrate mass ratio of barren wort total chromocor:
2, the most suitable enzyme-to-substrate mass ratio that amino mesoporous silicon immobilization glusulase digests barren wort total chromocor extract is 1:1.
6. amino mesoporous silicon immobilization glusulase digests barren wort total chromocor in differential responses system
Since solubility of the barren wort total chromocor in aqueous phase reactions system is poor, the Herba Epimedii being precipitated in reaction system is total
Flavones is unfavorable for the progress of conversion reaction, and therefore, we are using amino mesoporous silicon immobilization glusulase by DMSO (organic phase)
Barren wort total chromocor extract is digested in the diphasic system formed with the phosphoric acid buffers saline solution (water phase) of pH5.0.Different
The volume fraction of DMSO is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% in system,
Barren wort total chromocor extract is dissolved in above-mentioned system to being saturated, barren wort total chromocor extract in different systems is investigated and dissolves
Maximum and different systems in amino mesoporous silicon immobilization snail enzymatic conversion barren wort total chromocor the case where.The results are shown in Table 1 ( N=3), the most suitable diphasic system of amino mesoporous silicon immobilization snail enzymatic conversion barren wort total chromocor is 40%
5.0 phosphate buffer solution of pH (water phase) of DMSO (organic phase) and 60%.Herein in most suitable diphasic system, Herba Epimedii is always yellow
Concentration of the ketone in system is significantly increased than simple water phase, meanwhile, amino mesoporous silicon immobilization glusulase can keep effective
Enzyme activity is 87.86 ± 3.09%.
The optimization of 1 amino mesoporous silicon immobilization glusulase biphasic reaction system of table
7. the stability that amino mesoporous silicon immobilization glusulase digests barren wort total chromocor in diphasic system
Amino mesoporous silicon immobilization glusulase and barren wort total chromocor extract are added by 40%DMSO and 60%pH 5.0
Buffer salt solution composition biphasic reaction system in, the two mass ratio be 1:1.It is reacted under the conditions of 50 DEG C of temperature, respectively at
It reacts 1h and 2h to be measured by sampling, in triplicate, measures reaction stability.After reacting 1h, the life of barren wort total chromocor enzymolysis product
It is 54.71 ± 0.62% (n=3) at rate, after reacting 2h, the production rate of barren wort total chromocor enzymolysis product is 84.30 ±
0.98% (n=3).Measurement result is reliable and stable, and reaction of the amino mesoporous silicon immobilization glusulase in most suitable diphasic system is steady
It is qualitative good.
8. the HPLC of barren wort total chromocor enzymolysis product is measured
Appropriate free and amino mesoporous silicon immobilization glusulase is weighed, under the conditions of 50 DEG C of temperature, respectively at 6.0 He of pH
Barren wort total chromocor, concentration of substrate 0.5mgmL are digested in 5.0 phosphate buffer-1, enzyme-to-substrate mass ratio is respectively
Methanol termination reaction is added after reacting 2h in 1:2 and 1:1, and centrifuging and taking supernatant after vortex filters to get test solution, HPLC
Measurement map is shown in Fig. 6.The result shows that obtained by free glusulase and amino mesoporous silicon immobilization glusulase enzymatic hydrolysis barren wort total chromocor
There are significant differences for the composition and ratio of product, wherein arrow leaves of pulse plants glycosides A, the arrow leaves of pulse plants glycosides B and rhamnopyranosyl Herba Epimedii being converted into
Glycosides II no significant difference, the two convert main difference place as the ratio of precious leaves of pulse plants glycosides and epimedium aglucone.Dissociate the excessive of glusulase
Sheep leaves of pulse plants general flavone enzymolysis product is mainly by arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B, rhamnopyranosyl icariside I I, treasured leaves of pulse plants glycosides I and Herba Epimedii
Aglycon composition, its ratio be 1.2:2.1:1.0:2.6:3.0；And the barren wort total chromocor enzyme of amino mesoporous silicon immobilization glusulase
Solution product does not have epimedium aglucone, is mainly made of arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B, rhamnopyranosyl icariside I I and treasured leaves of pulse plants glycosides I,
Its ratio be 1.5:2.0:1.0:5.8.
Embodiment 2
This example demonstrates that using the hydrolysis result of other immobilization carriers enzymatic hydrolysis barren wort total chromocor.
The present embodiment compared digesting excessive sheep using the amino mesoporous silicon immobilised enzymes of barium alginate, microballoon and embodiment 1
The hydrolysis result of leaves of pulse plants general flavone.The results are shown in Table 2:
The immobilization glusulase of the different enzyme technique for fixing preparations of table 2 compares
As it can be seen that there is higher joint efficiency, more compared to other immobilization carriers using amino mesoporous silicon immobilization glusulase
Good stability and higher conversion ratio.
Embodiment 3
This example demonstrates that the anti-tumor activity of enzymolysis product of the present invention.
Precision weighs the resolvase enzymolysis product and immobilised enzymes of barren wort total chromocor and the method acquisition using embodiment 1
Each 2.65mg of enzymolysis product is dissolved in 25 μ L DMSO respectively, is prepared into mother liquor.Mother liquor is not exclusively cultivated with RPMI-1640 again
Based sols dilute 100 times, and then equimultiple is diluted to different dosing concentration again: 4.0,17.0,66.0,265.0,1060.0 μ g
mL-1.MCF-7, A549, HepG2 and Hela cell of logarithmic growth phase are inoculated in 96 orifice plates respectively, and 20 μ are added in culture afterwards for 24 hours
The final concentration of L medical fluid to be measured, medical fluid distinguishes 0.4,1.7,6.6,26.5,106.0 μ gmL-1.6 multiple holes are arranged in each sample,
And the incomplete culture medium blank group of not drug containing is set, continue after cultivating 48h, MTT solution (5.0mgmL is added in every hole-1)
20 μ L gently inhale supernatant in abandoning hole after cultivating 4h, then the DMSO of 150 μ L of every hole addition, and microoscillator oscillation 5~
10min measures the absorbance value A (OD value) in every hole using microplate reader at measurement wavelength 490nm.Calculate the cell of each medical fluid
Growth inhibition ratio (Inhibiton rate) using Graphpad software data processing, and calculates each compound IC50, test number
Mean ± standard deviation (Mean ± SD) records accordingly.Inhibiting rate (Inhibiton rate) calculation formula:
Barren wort total chromocor and its enzymolysis product act on the inhibiting rate knot of MCF-7, A549, HepG2 and Hela cell 48h
Fruit be shown in Table respectively 3-6 (n=6,)。
The inhibiting rate of 3 barren wort total chromocor of table and its enzymolysis product to MCF-7 cytosis 48h
The inhibiting rate of 4 barren wort total chromocor of table and its enzymolysis product to A549 cytosis 48h
The inhibiting rate of 5 barren wort total chromocor of table and its enzymolysis product to HepG2 cytosis 48h
The inhibiting rate of 6 barren wort total chromocor of table and its enzymolysis product to Hela cytosis 48h
IC50Calculated result be shown in Table 7 (n=6,)。
The IC of 7 barren wort total chromocor of table and its enzymolysis product to A549, HepG2, MCF-7, Hela cell50
Inhibiting rate is shown in Fig. 7 and Fig. 8 to administration concentration mapping results.Barren wort total chromocor and its enzymolysis product to MCF-7,
Certain concentration dependent is presented in the inhibiting rate of A549, HepG2 and Hela cell；The cell of barren wort total chromocor enzymolysis product
Toxic action is significantly stronger than barren wort total chromocor prototype glycosides, and the cytotoxicity of immobilised enzymes enzymolysis product is significantly stronger than resolvase enzyme
Solve product；Barren wort total chromocor and its enzymolysis product are most strong to the effect of MCF-7 cell, and Hela, HepG2 take second place, and A549 is most weak.
Claims (11)
1. a kind of enzymolysis product of barren wort total chromocor, which is characterized in that the enzymolysis product is digested by immobilization glusulase
Barren wort total chromocor obtains, and the immobilization carrier of the glusulase selects amino mesoporous silicon.
2. barren wort total chromocor enzymolysis product according to claim 1, which is characterized in that the system of the barren wort total chromocor
Preparation Method are as follows: Herba Epimedii is measured into 40% ~ 90% ethyl alcohol using being concentrated after circumfluence distillation, through macroporous absorption tree with 6 ~ 40(m/v) times
Rouge method is further purified, and obtains the barren wort total chromocor that mass content is not less than 60%.
3. barren wort total chromocor enzymolysis product according to claim 1, which is characterized in that excessive in the barren wort total chromocor
Sheep leaves of pulse plants glycosides, Epimedin A, Epimedin B, epimedin C and precious leaves of pulse plants glycosidesGross mass content be not less than 30%.
4. barren wort total chromocor enzymolysis product according to claim 1, which is characterized in that the preparation of the amino mesoporous silicon
Method are as follows: immerse silica dissolved in the toluene of 3- aminopropyl triethoxysilane, 800 rmin of magnetic agitation-1,
Under 90 DEG C of high temperature, 12h is reacted；Reactant is taken out, 10 min of supersound washing in toluene is dried in vacuo 2 h at 60 DEG C.
5. barren wort total chromocor enzymolysis product according to claim 1, which is characterized in that the fixing means of the glusulase
Are as follows: glusulase and the mesoporous silicon carrier of amino are placed in 5.0 phosphate buffered saline solution of pH and reacted, magnetic agitation is reacted at room temperature；
Kept dry after distillation water washing later.
6. barren wort total chromocor enzymolysis product according to claim 5, which is characterized in that the glusulase and amino are mesoporous
Silicon carrier dosage is mass ratio 1:1 ~ 1:4.
7. barren wort total chromocor enzymolysis product according to claim 6, which is characterized in that the glusulase and amino are mesoporous
Silicon carrier dosage is mass ratio 1:1.
8. barren wort total chromocor enzymolysis product according to claim 1, which is characterized in that enzymatic hydrolysis condition are as follows: amino is situated between
The buffer salt solution of the pH 5.0 containing 40% volume fraction DMSO is added in hole silicon immobilization glusulase and barren wort total chromocor
In, amino mesoporous silicon immobilization glusulase and barren wort total chromocor mass ratio are 1:1, are reacted under the conditions of 50 DEG C of temperature, reaction 2
h。
9. barren wort total chromocor enzymolysis product according to claim 1-8, which is characterized in that product main component
It is arrow leaves of pulse plants glycosides A: arrow leaves of pulse plants glycosides B for arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B, rhamnopyranosyl icariside I I and precious leaves of pulse plants glycosides I, composition ratio:
Rhamnopyranosyl icariside I I: precious leaves of pulse plants glycosides I=1.5:2.0:1.0:5.8.
10. a kind of barren wort total chromocor enzyme solution, which is characterized in that barren wort total chromocor is digested using immobilization glusulase,
The immobilization carrier of the glusulase selects amino mesoporous silicon.
11. the described in any item barren wort total chromocor enzymolysis product application in preparations of anti-tumor drugs of claim 1-8.
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