CN106420880A - Herba epimedii total flavonoid enzymatic product and preparing method and application thereof - Google Patents
Herba epimedii total flavonoid enzymatic product and preparing method and application thereof Download PDFInfo
- Publication number
- CN106420880A CN106420880A CN201610749179.XA CN201610749179A CN106420880A CN 106420880 A CN106420880 A CN 106420880A CN 201610749179 A CN201610749179 A CN 201610749179A CN 106420880 A CN106420880 A CN 106420880A
- Authority
- CN
- China
- Prior art keywords
- glusulase
- barren wort
- total chromocor
- wort total
- immobilization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
- A61K36/296—Epimedium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to a herba epimedii total flavonoid enzymatic product. Herba epimedii total flavonoid is generated through immobilized helicase, and the enzymatic product has higher antineoplastic activity than the herba epimedii total flavonoid archetype and free enzyme enzymatic product and has high oral absorptivity. The invention further discloses a preparing method of the enzymatic product. The immobilized enzyme is used for enzymolysis of herba epimedii total flavonoid, selected helicase is high in combining efficiency with a fixed carrier and high in stability, the enzymolysis conversion rate is large, the immobilized enzyme can be used circularly, resources can be saved, and green production is achieved. The invention further relates to application of the enzymatic product in antitumous effect, and the herba epimedii total flavonoid enzymatic product obtained through the technical scheme has a remarkable anti-proliferative effect on the human lung cancer cell A549, liver cancer HepG2 cell, breast cancer MCF-7 cell, cervical cancer Hela cell and can be used for treating the lung cancer, liver cancer, breast cancer and cervical cancer.
Description
Technical field
The present invention relates to a kind of barren wort total chromocor enzymolysis product, this product is that barren wort total chromocor is passed through immobilization snail
Ox enzyme enzymolysis produces.The invention still further relates to the preparation method of this enzymolysis product and the application in anti-tumor aspect, belong to medicine
Technical field.
Background technology
Barrenwort as traditional kidney-nourishing tcm drug, continues to use existing history in thousand among the people, has a kidney-replenishing, strengthening the bones and muscles, dispels
The effect of rheumatism, clinic is usually used in treating impotence and seminal emission, and muscles and bones is withered soft, the disease such as arthralgia due to wind-dampness.
Barrenwort mainly contains flavones ingredient, mainly has 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 research shows, epimedium flavone glycosides has good biologically active, except having
Outside anti-osteoporosis activity, also there is good antitumor action.Document report epimedium flavone constituents are to respiratory system
Lung cancer tumor, to digestive system liver cancer and cancer of the stomach, to hematological system leukaemia, there is certain killing ability, can control
The growth of tumour cell, promotes the death of tumour cell, and suppression Tumor angiogenesis simultaneously can adjust special in body effectively
Different, non-specific immunocyte, the immunologic escape of suppression tumour cell, thus play antitumor action.
This seminar early-stage Study shows, oral epimedium flavone glycosides, and prototype absorption is less, needs through enteron aisle enzyme enteron aisle
The hydrolysis of bacterium, is absorbed in the form of secondary glycosides or aglycon and is played curative effect.Therefore find in vitro and epimedium flavone glycosides is fitted
The method of suitable enzymolysis, it is higher and be easy to the secondary glycosides absorbing to obtain activity, has preferable antitumor action to research and development further
New drug is applied to clinical significant.At present, the existing more research with regard to external enzymolysis epimedium flavone prototype glycosides, but
Mostly these methods are to digest icariin using resolvase, such as using beta-glucosidase, cellulase, glusulase or aspergillus
The research of the induction enzymatic conversion icariin that mould produces.But resolvase is very sensitive to the microenvironment of reaction system, any pH,
The change of temperature all can make its activity be affected, and be difficult to recycle, spend cost high, is unfavorable for that large-scale industry is given birth to
Produce.The flavones ingredient more due in addition to containing icariin composition, also containing other three kinds of contents in barrenwort：Towards the leaves of pulse plants
Determine A, Epimedin B and epimedin C.Therefore, extract merely icariin from epimedium herb to be digested again, not only need numerous
Trivial separating-purifying step, and the active ingredient in epimedium herb can not make full use of, and is unfavorable for the rationalization of natural resources of Chinese medicinal materials
Application.
Content of the invention
An object of the present invention is to provide a kind of barren wort total chromocor enzymolysis product, and this product is by barren wort total chromocor
Produced by immobilization glusulase enzymolysis, this enzymolysis product has than barren wort total chromocor prototype and resolvase enzymolysis product more more
Good antitumor activity, and have preferable oral absorptivity, improves the utilization rate to epimedium active constituent it is achieved that medicine
The comprehensive utilization in aptitude source, and product preparation immobilised enzymes used can recycle, and can economize on resources, realize green raw
Produce.
For foregoing invention purpose, the present invention provides technical scheme below：
A kind of barren wort total chromocor enzymolysis product, described enzymolysis product digests barren wort total chromocor by immobilization glusulase
Obtain, described glusulase is fixed by amino mesoporous silicon.
Wherein, the preparation method of described barren wort total chromocor is：By barrenwort 6～40 (m/v) times amount 40%～90%
Ethanol concentrates after adopting circumfluence distillation, is further purified through Flavonoids by Macroporous Adsorption Resin, obtains mass content and be not less than 60%
Barren wort total chromocor.Wherein, in barren wort total chromocor icariin, Epimedin A, Epimedin B, epimedin C and baohuoside Ⅰ total
Mass content is not less than 30% (HPLC records).
A kind of preparation method of preferred amino mesoporous silicon is：Silica is immersed dissolved with 3- aminopropyl-triethoxy silicon
In the toluene of alkane, magnetic agitation 800r min-1, under 90 DEG C of high temperature, react 12h；Take out reactant, ultrasonic in toluene wash
Wash 10min, be vacuum dried 2h at 60 DEG C.
Further, the fixing means of described glusulase is：Glusulase and the mesoporous silicon carrier of amino are placed in pH 5.0 phosphorus
React in acid buffering salting liquid, magnetic agitation reaction under room temperature；Kept dry after distillation water washing afterwards.
Wherein, described glusulase and amino mesoporous silicon carrier consumption are mass ratio 1：1～1：4, preferably 1：1.Glusulase with
The joint efficiency of amino mesoporous silicon is in mass ratio 1：1～1：It is incremented by between 4, mass ratio 1：When 4, glusulase and the combination of mesoporous silicon are imitated
Rate reaches maximum, can reach more than 90%, but considers preparation cost, and final choice glusulase is 1 with the mass ratio of mesoporous silicon：1,
Both decreased the consumption of mesoporous silicon carrier, also can keep effective joint efficiency, with this understanding, glusulase and amino mesoporous silicon
Joint efficiency can reach 89.63%.
Further, enzymatic hydrolysis condition is preferably：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, react under 50 DEG C of temperature conditionss, react 2h.
Almost there is no epimedium aglucone, its enzymolysis product is mainly by arrow leaves of pulse plants glycosides A in enzymolysis product of the present invention；Arrow leaves of pulse plants glycosides
B；Rhamanopyranosyl icariside I I；Precious leaves of pulse plants glycosides I composition；Its proportion of composing is about arrow leaves of pulse plants glycosides A：Arrow leaves of pulse plants glycosides B：The excessive sheep of rhamanopyranosyl
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 concrete technical scheme is to adopt
Immobilization glusulase digests barren wort total chromocor, and the immobilization carrier of wherein said 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 more preferable adaptability, good stability to reaction temperature, and digests conversion
Rate is high.
Additionally, further requirement of the present invention protects above-mentioned barren wort total chromocor enzymolysis product in preparing antineoplastic
Application.
Immobilization glusulase of the present invention to barren wort total chromocor enzymolysis product, than barren wort total chromocor prototype and trip
From enzyme enzymolysis product, there is more preferable antitumor activity, 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 advantage remaining resolvase, is also had and is easy to be separated with substrate, is easy to recovery and repeats to recycle, suitable Reaction conditions range
Wide, it is possible to decrease the advantage such as production cost, suitable industrialized production.And glusulase and the joint efficiency of the mesoporous silicon carrier of amino
Height, good stability, and digest high conversion rate.In addition, The invention also achieves to the Huang in addition to icariin in barrenwort
The effectively utilizes of ketones component, the general flavone composition extracting in barrenwort is digested, and improves epimedium herb active ingredient
Utilization rate is it is achieved that the comprehensive utilization of herb resource.Importantly, barren wort total chromocor fixes glusulase through amino mesoporous silicon
The product than prototype compound and through resolvase enzymolysis for the enzymolysis product has more preferable antitumor activity, anti-as raw material research and development
Tumour new drug has preferable clinical value.
Brief description
Fig. 1 is the chemical constitution schematic diagram of Main Flavonoids and enzymolysis product contained by barren wort total chromocor；
Fig. 2 is that the SEM of amino mesoporous silicon immobilization glusulase characterizes schematic diagram (multiplication factor is 8.1mm × 50.0k)；
Fig. 3 varies with temperature schematic diagram for amino mesoporous silicon immobilization glusulase relative to enzyme activity and conversion ratio；Wherein, A is
Enzyme activity relatively, B is conversion ratio, (N=3,*P<0.05,**P<0.01)；
Fig. 4 be amino mesoporous silicon immobilization glusulase relative to enzyme activity and transformation efficiency with storage time change schematic diagram；Its
Middle A is relative enzyme activity；B be conversion ratio (N=3)；
Fig. 5 is that amino mesoporous silicon immobilization glusulase enzymolysis barren wort total chromocor optimum reaction conditionses investigate schematic diagram；Its
In, A is the optimal reaction pH with enzyme activity as index；B is the optimal reaction pH with conversion ratio as index；C is with enzyme activity as index
Optimal reactive temperature；D is the optimal reactive temperature with conversion ratio as index；E is the most suitable enzyme-to-substrate with enzyme activity as index
Mass ratio；F is the most suitable enzyme-to-substrate mass ratio with conversion ratio as index；
Fig. 6 is the HPLC collection of illustrative plates 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 is free glusulase enzymolysis general flavone product, and 2 is amino mesoporous silicon immobilization glusulase
Enzymolysis 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. rhamanopyranosyl icariside I I；H. precious leaves of pulse plants glycosides I；I. epimedium aglucone；
Fig. 7 is for 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 enzymolysis barren wort total chromocor
Born of the same parents act on the inhibiting rate comparison schematic diagram of 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
With reference to example, the present invention is described in further detail, but the scope of the present invention is not appointed by these examples
What limits.
Selected hydrolase information in the embodiment of the present invention：Glusulase (>300U/mg, Shanghai source leaf biotechnology is limited
Company), unless otherwise specified, the various raw material/preparations used by the present invention are commercially available known product.
Embodiment 1
The present embodiment provides a kind of employing the inventive method to digest the preferred embodiment of barren wort total chromocor, and the present invention is digested
Method is done and is specifically described further.
1. the preparation of barren wort total chromocor
Barrenwort is adopted with 6～40 (m/v) times amount 40%～90% ethanol and concentrates after circumfluence distillation, through macroporous absorption
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 of the mesoporous silicon carrier of amino and sign
Silica is immersed in the toluene dissolved with 3- aminopropyl triethoxysilane (C=50mM), magnetic agitation
800r·min-1, under 90 DEG C of high temperature, react 12h.Take out reactant, in toluene, supersound washing 10min, at 60 DEG C, very
Sky is dried 2h, obtains final product the mesoporous silicon sample of amino, saves backup in 4 DEG C of refrigerators.
Observe the surface knot of amino mesoporous silicon immobilised enzymes using Hitachi S-3400 SEM (SEM)
Structure.Take amino mesoporous silicon sample powder solid conduction adhesive tape to fix, gold-plated after, operating voltage 15kV, enter under vacuum condition
Row characterizes, and sees Fig. 2.The amino mesoporous silicon preparing gained visually observes, and outward appearance is white homogeneous powder.(SEM) observe preparation
Amino mesoporous silicon outward appearance rounding, surface has more gap structure, and the loose structure aperture of amino mesoporous silicon is more homogeneous, profit
In the fixed efficiency improving enzyme.
3. the preparation of amino mesoporous silicon immobilization glusulase
According to 1：1 mass ratio is weighed glusulase and is placed in right amount in conical flask, in pH 5.0 phosphorus with the mesoporous silicon carrier of amino
React in acid buffering salting liquid, magnetic agitation 500r min under room temperature-1, react 10h.Then distillation water washing 3～5 times, in 37
It is vacuum dried under the conditions of DEG C, obtain final product amino mesoporous silicon immobilization glusulase, be placed in 4 DEG C of refrigerators and save backup.
4. the mensure of amino mesoporous silicon immobilization glusulase zymologic property
(1) mensure of amino mesoporous silicon immobilization snail enzyme heat stability
Weigh in right amount dissociate and amino mesoporous silicon immobilization glusulase be placed in different temperatures (50,60,70,80,90,100
DEG C) under the conditions of place 1h after, be then concentration of substrate 0.5mg mL under conditions of 50 DEG C and pH are 5.0 in temperature-1, enzyme and bottom
Amount of substance ratio is 1：1, digest barren wort total chromocor, sample after reaction 1h, add methyl alcohol terminating reaction, HPLC measures, 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 heat endurance of amino mesoporous silicon immobilization glusulase is higher than free glusulase.
(2) mensure of amino mesoporous silicon immobilization glusulase reusing
Weigh the mesoporous silicon-immobilized glusulase of surely changing of appropriate amino under conditions of temperature is 5.0 for 50 DEG C and pH, concentration of substrate
0.5mg·mL-1, enzyme-to-substrate mass ratio is 1：1, digest barren wort total chromocor, sample after reaction 1h, add methyl alcohol to terminate anti-
Should, HPLC measures.Above-mentioned amino mesoporous silicon immobilization glusulase is separated from enzymatic hydrolysis system, after distilled water fully washs 3 times,
React at identical conditions, HPLC measures, repeat the above steps, investigate access times to amino mesoporous silicon immobilization glusulase
The impact of enzyme activity.After recycling amino mesoporous silicon immobilization glusulase 5 times, its enzyme activity be still positively retained at 60% with
On；Compared with resolvase, amino mesoporous silicon immobilised enzymes has preferably repeatable usability, reduces production cost.
(3) mensure of amino mesoporous silicon immobilization glusulase storage-stable
Preserve under the conditions of amino mesoporous silicon immobilization glusulase is placed in 4 DEG C, sample 1 time weekly, turn according to above-mentioned condition
Change barren wort total chromocor, HPLC measures, calculate the enzyme activity of immobilization glusulase, METHOD FOR CONTINUOUS DETERMINATION 6 weeks, investigate amino mesoporous silicon and fix
Change the storage-stable of glusulase, result is shown in Fig. 4.After amino mesoporous silicon immobilization glusulase preserves 6 weeks under the conditions of 4 DEG C, enzyme
Vigor still can retain about 80%, has preferable storage-stable.
5. amino mesoporous silicon immobilization glusulase enzymolysis barren wort total chromocor
(1) optimal reaction pH investigates
Weigh and dissociate in right amount and amino mesoporous silicon immobilization glusulase, under 50 DEG C of temperature conditionss, respectively at different pH value
Barren wort total chromocor is digested, concentration of substrate is in the phosphate buffer of (2.0,3.0,4.0,5.0,6.0,7.0,8.0) condition
0.5mg·mL-1, enzyme-to-substrate mass ratio is 1：1, sample after reaction 1h, all add methyl alcohol terminating reaction, HPLC measures, result
See Fig. 5 (A, B).The free enzyme activity with amino mesoporous silicon immobilization glusulase and enzymolysis efficiency all change with the increase of pH, are in
The trend of existing first increases and then decreases；The optimal reaction pH that free glusulase digests barren wort total chromocor extract is 6.0, now its
The efficiency of enzyme activity and enzymolysis barren wort total chromocor extract is all maximum；Amino mesoporous silicon immobilization glusulase enzymolysis barrenwort is always yellow
The optimal reaction pH of ketone extract is 5.0, and now the efficiency of its enzyme activity and enzymolysis barren wort total chromocor extract is all maximum.Separately
Outward, under the conditions of extreme pH, the enzyme activity of amino mesoporous silicon immobilization glusulase and enzymolysis efficiency are higher than significantly all free snail
Enzyme, shows glusulase after immobilization, and the optimum pH scope of enzyme reaction broadens.
(2) optimal reactive temperature is investigated
Weigh and dissociate in right amount and amino mesoporous silicon immobilization glusulase, pH value be 5.0 phosphate buffer in, respectively
Digest barren wort total chromocor under 4,25,37,50,60,70 DEG C of temperature conditionss, concentration of substrate is 0.5mg mL-1, enzyme-to-substrate
Mass ratio is 1:1, sample after reaction 1h, all add methyl alcohol terminating reaction, HPLC measures, and result is shown in Fig. 5 (C, D).Free and
The enzyme activity of amino mesoporous silicon immobilization glusulase and enzymolysis efficiency all change with the increase of temperature, assume first increases and then decreases
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 of the two enzymolysis 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 change at a temperature of differential responses is less,
Under the conditions of 25,37,60,70 DEG C, its enzyme activity and enzymolysis efficiency are obviously higher than free glusulase.Test result indicate that, snail
Enzyme is particularly suited for the various enzymolysis conversion reactions under condition of different temperatures after immobilization.
(3) the most suitable substrate is than investigation
Weigh and dissociate in right amount and amino mesoporous silicon immobilization glusulase, in optimal reactive temperature and optimal reaction pH value condition
Lower enzymolysis barren wort total chromocor, concentration of substrate is 0.5mg mL-1, enzyme-to-substrate mass ratio is respectively 1：2,1：1,2：1,3：1,
4：1, sample after reaction 1h, all add methyl alcohol terminating reaction, HPLC detects, result is shown in Fig. 5 (E, F).Free glusulase with excessive
Sheep leaves of pulse plants general flavone mass ratio is 1：When 2, enzyme activity and the equal highest of enzymolysis efficiency, with the increasing of enzyme and barren wort total chromocor mass ratio
Plus, its enzyme activity and enzymolysis efficiency are gradually lowered；Amino mesoporous silicon immobilization glusulase is being 1 with barren wort total chromocor mass ratio：1
When, enzyme activity and enzymolysis efficiency highest.Therefore, the most suitable enzyme-to-substrate mass ratio that free glusulase digests barren wort total chromocor is 1：
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
Because solubility in aqueous phase reactions system for the barren wort total chromocor is poor, the barrenwort separating out in reaction system is total
Flavones is unfavorable for the carrying out of conversion reaction, and therefore, we utilize amino mesoporous silicon immobilization glusulase by DMSO (organic phase)
Barren wort total chromocor extract is digested in the diphasic system forming with the phosphoric acid buffers saline solution (aqueous phase) of pH5.0.Different
In system, the volume fraction of DMSO is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
Barren wort total chromocor extract is dissolved in saturation in above-mentioned system, investigates barren wort total chromocor extract dissolving in different systems
Maximum and different systems in amino mesoporous silicon immobilization snail enzymatic conversion barren wort total chromocor situation.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%
DMSO (organic phase) and 60% pH 5.0 PBS (aqueous phase).In the most suitable diphasic system of here, barrenwort is always yellow
The simple aqueous phase of concentration ratio in system for the ketone significantly increases, and meanwhile, amino mesoporous silicon immobilization glusulase can keep effective
Enzyme activity is 87.86 ± 3.09%.
The optimization of table 1 amino mesoporous silicon immobilization glusulase biphasic reaction system
7. amino mesoporous silicon immobilization glusulase digests the stability of barren wort total chromocor in diphasic system
Amino mesoporous silicon immobilization glusulase and barren wort total chromocor extract add by 40%DMSO and 60%pH 5.0
Buffer salt solution composition biphasic reaction system in, the two mass ratio be 1:1.React under 50 DEG C of temperature conditionss, respectively at
Reaction 1h and 2h is measured by sampling, and in triplicate, measures reaction stability.After reaction 1h, the life of barren wort total chromocor enzymolysis product
One-tenth rate is 54.71 ± 0.62% (n=3), after reaction 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 in the most suitable diphasic system for the amino mesoporous silicon immobilization glusulase is steady
Qualitative good.
8. the HPLC of barren wort total chromocor enzymolysis product measures
Weigh and dissociate in right amount and amino mesoporous silicon immobilization glusulase, under 50 DEG C of temperature conditionss, respectively at pH 6.0 He
Barren wort total chromocor is digested, concentration of substrate is 0.5mg mL in 5.0 phosphate buffer-1, enzyme-to-substrate mass ratio is respectively
1：2 and 1：1, add methyl alcohol terminating reaction, centrifuging and taking supernatant after vortex, filtration after reaction 2h, obtain final product need testing solution, HPLC
Measure collection of illustrative plates and see Fig. 6.Result shows, free glusulase and amino mesoporous silicon immobilization glusulase digest barren wort total chromocor gained
There is significant difference in the composition of product and ratio, wherein, arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B and the rhamanopyranosyl barrenwort time that are converted into
Glycosides II no significant difference, is the ratio of precious leaves of pulse plants glycosides and epimedium aglucone in place of the two conversion main difference.Free glusulase excessive
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, rhamanopyranosyl icariside I I, precious leaves of pulse plants glycosides I and barrenwort
Aglycon forms, and its ratio is 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 up of arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B, rhamanopyranosyl icariside I I and precious leaves of pulse plants glycosides I,
Its ratio is 1.5：2.0：1.0：5.8.
Embodiment 2
This example demonstrates that digest the hydrolysis result of barren wort total chromocor using other immobilization carriers.
The present embodiment compared for 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.Result is as shown in table 2：
The immobilization glusulase of the different enzyme technique for fixing preparation of table 2 compares
It can be seen that, other immobilization carriers are compared using amino mesoporous silicon immobilization glusulase there is higher joint efficiency, more
Good stability and the conversion ratio of Geng Gao.
Embodiment 3
This example demonstrates that the antitumor activity of enzymolysis product of the present invention.
Precision weighs barren wort total chromocor and the resolvase enzymolysis product obtaining using the method for embodiment 1 and immobilised enzymes
The each 2.65mg of enzymolysis product, is dissolved in respectively in 25 μ L DMSO, is prepared into mother liquor.Again mother liquor is not exclusively cultivated with RPMI-1640
Based sols dilute 100 times, and then equimultiple is diluted to different dosing concentration again：4.0、17.0、66.0、265.0、1060.0μg·
mL-1.MCF-7, A549, the HepG2 and Hela cell in growth period of taking the logarithm is inoculated in 96 orifice plates respectively, adds 20 μ after culture 24h
L liquid to be measured, final concentration 0.4,1.7,6.6,26.5, the 106.0 μ g mL respectively of liquid-1.Each sample arranges 6 multiple holes,
And the incomplete culture medium blank group of not drug containing is set, after continuing culture 48h, every hole adds MTT solution (5.0mg mL-1)
20 μ L, gently inhale after culture 4h and abandon supernatant in hole, and then every hole adds the DMSO of 150 μ L, and microoscillator vibration 5～
10min, is measuring, using ELIASA, the absorbance A (OD value) measuring every hole at wavelength 490nm.Calculate the cell of each liquid
Growth inhibition ratio (Inhibiton rate), using Graphpad software data processing, and calculates each compound IC50, test number
Mean ± standard deviation (Mean ± SD) record according to this.Inhibiting rate (Inhibiton rate) computing 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 table 3 barren wort total chromocor and its enzymolysis product inhibiting rate to MCF-7 cytosis 48h
The table 4 barren wort total chromocor and its enzymolysis product inhibiting rate to A549 cytosis 48h
The table 5 barren wort total chromocor and its enzymolysis product inhibiting rate to HepG2 cytosis 48h
The table 6 barren wort total chromocor and its enzymolysis product inhibiting rate to Hela cytosis 48h
IC50Result of calculation be shown in Table 7 (n=6,).
The table 7 barren wort total chromocor and its enzymolysis product IC 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,
The inhibiting rate of A549, HepG2 and Hela cell all assumes certain concentration dependent；The cell of barren wort total chromocor enzymolysis product
Toxic action is significantly stronger than barren wort total chromocor prototype glycosides, and the CDCC of immobilised enzymes enzymolysis product is significantly stronger than resolvase enzyme
Solution product；Barren wort total chromocor and its enzymolysis product are the strongest to the effect of MCF-7 cell, and Hela, HepG2 take second place, and A549 is the weakest.
Claims (10)
1. a kind of enzymolysis product of barren wort total chromocor is it is characterised in that described enzymolysis product is digested by immobilization glusulase
Barren wort total chromocor obtains, and the immobilization carrier of described glusulase selects amino mesoporous silicon.
2. barren wort total chromocor enzymolysis product according to claim 1 is it is characterised in that the system of described barren wort total chromocor
Preparation Method is：By barrenwort with 6 ~ 40（m/v）Times amount 40% ~ 90% ethanol concentrates, through macroporous absorption tree after adopting circumfluence distillation
Fat 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 is it is characterised in that excessive in described barren wort total chromocor
Sheep leaves of pulse plants glycosides, Epimedin A, the gross mass content of Epimedin B, epimedin C and precious leaves of pulse plants glycosides I are not less than 30%.
4. barren wort total chromocor enzymolysis product according to claim 1 is it is characterised in that the preparation of described amino mesoporous silicon
Method is：Silica is immersed in the toluene dissolved with 3- aminopropyl triethoxysilane, magnetic agitation 800 r min-1,
Under 90 DEG C of high temperature, react 12h；Take out reactant, supersound washing 10 min in toluene, be vacuum dried 2 h at 60 DEG C.
5. barren wort total chromocor enzymolysis product according to claim 1 is it is characterised in that the fixing means of described glusulase
For：Glusulase and the mesoporous silicon carrier of amino are placed in reaction in pH 5.0 PBS, magnetic agitation reaction under room temperature；
Kept dry after distillation water washing afterwards.
6. barren wort total chromocor enzymolysis product according to claim 4 is it is characterised in that described glusulase is mesoporous with amino
Silicon carrier consumption is mass ratio 1：1~1：4, preferably 1：1.
7. barren wort total chromocor enzymolysis product according to claim 1 is it is characterised in that enzymatic hydrolysis condition is：Amino is situated between
Hole silicon immobilization glusulase and the buffer salt solution of the barren wort total chromocor pH 5.0 containing 40% volume fraction DMSO for the addition
In, amino mesoporous silicon immobilization glusulase and barren wort total chromocor mass ratio are 1：1, react under 50 DEG C of temperature conditionss, react 2
h.
8. the barren wort total chromocor enzymolysis product according to any one of claim 1-7 is it is characterised in that product main component
For arrow leaves of pulse plants glycosides A, arrow leaves of pulse plants glycosides B, rhamanopyranosyl icariside I I and precious leaves of pulse plants glycosides I, its proportion of composing is arrow leaves of pulse plants glycosides A：Arrow leaves of pulse plants glycosides B：
Rhamanopyranosyl icariside I I：Precious leaves of pulse plants glycosides I=1.5：2.0：1.0：5.8.
9. a kind of barren wort total chromocor enzyme solution is it is characterised in that digest barren wort total chromocor, institute using immobilization glusulase
The immobilization carrier stating glusulase selects amino mesoporous silicon.
10. application in preparing antineoplastic for the barren wort total chromocor enzymolysis product described in any one of claim 1-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610749179.XA CN106420880B (en) | 2016-08-29 | 2016-08-29 | A kind of barren wort total chromocor enzymolysis product and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610749179.XA CN106420880B (en) | 2016-08-29 | 2016-08-29 | A kind of barren wort total chromocor enzymolysis product and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106420880A true CN106420880A (en) | 2017-02-22 |
| CN106420880B CN106420880B (en) | 2019-04-30 |
Family
ID=58181630
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610749179.XA Active CN106420880B (en) | 2016-08-29 | 2016-08-29 | A kind of barren wort total chromocor enzymolysis product and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106420880B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995829A (en) * | 2017-05-12 | 2017-08-01 | 南京林业大学 | A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone |
| CN107164436A (en) * | 2017-05-12 | 2017-09-15 | 南京林业大学 | Application of the β glucuroides in conversion barren wort total chromocor prepares precious glycosides I suddenly |
| CN112176008A (en) * | 2020-10-13 | 2021-01-05 | 烟台大学 | Enzymolysis method for efficiently and quickly preparing icariside II |
| CN115894585A (en) * | 2022-12-23 | 2023-04-04 | 中国药科大学 | Preparation method of epimedium extract, and epimedium extract and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760487A (en) * | 2009-08-18 | 2010-06-30 | 江苏省中医药研究院 | Preparation method of epimedium aglycone |
| CN104127466A (en) * | 2013-08-23 | 2014-11-05 | 江苏省中医药研究院 | Oral enteric-coated preparation of total flavonoids of herba epimedii and application thereof |
-
2016
- 2016-08-29 CN CN201610749179.XA patent/CN106420880B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760487A (en) * | 2009-08-18 | 2010-06-30 | 江苏省中医药研究院 | Preparation method of epimedium aglycone |
| CN104127466A (en) * | 2013-08-23 | 2014-11-05 | 江苏省中医药研究院 | Oral enteric-coated preparation of total flavonoids of herba epimedii and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 彭静 等: "固定化β-葡萄糖苷酶和蜗牛酶生物转化淫羊藿苷效率的比较研究", 《药学学报》 * |
| 彭静 等: "固定化蜗牛酶同时生物转化淫羊藿中4种黄酮苷", 《中成药》 * |
| 彭静 等: "微乳化-凝胶法制备微球固定化蜗牛酶并生物转化淫羊藿苷研究", 《中草药》 * |
| 高霞 等: "淫羊藿总黄酮的生物转化过程分析", 《中国中药杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995829A (en) * | 2017-05-12 | 2017-08-01 | 南京林业大学 | A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone |
| CN107164436A (en) * | 2017-05-12 | 2017-09-15 | 南京林业大学 | Application of the β glucuroides in conversion barren wort total chromocor prepares precious glycosides I suddenly |
| CN106995829B (en) * | 2017-05-12 | 2021-03-30 | 南京林业大学 | Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method |
| CN112176008A (en) * | 2020-10-13 | 2021-01-05 | 烟台大学 | Enzymolysis method for efficiently and quickly preparing icariside II |
| CN115894585A (en) * | 2022-12-23 | 2023-04-04 | 中国药科大学 | Preparation method of epimedium extract, and epimedium extract and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106420880B (en) | 2019-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106420880A (en) | Herba epimedii total flavonoid enzymatic product and preparing method and application thereof | |
| CN104277134A (en) | Preparation method and application of dictyophora indusiata polysaccharide-zinc chelate with antitumor activity | |
| CN103784468B (en) | A kind of production method of Fructus Hippophae polysaccharide microcapsules | |
| CN108191931B (en) | Three new compounds in Xinjiang peony and health care and medical application thereof | |
| CN102309694B (en) | Method for extraction and purification of corn silk general flavone, and use of corn silk general flavone | |
| CN105566271A (en) | Biflavone compound and application thereof to preparation of medicine for treating cancer | |
| Li et al. | Semen Sojae Preparatum as a traditional Chinese medicine: manufacturing technology, bioactive compounds, microbiology and medicinal function | |
| CN105481880B (en) | A kind of noval chemical compound and its application | |
| CN102030813B (en) | Preparation method and application of yellow ginger zingiberensis saponin having high-efficiency anti-cancer activity | |
| CN107669686A (en) | Calycosin derivative is preparing the application in treating ER negative breast cancer drugs | |
| CN101508693A (en) | Xylogen like flavonoid compounds, method of preparing the same and pharmaceutical use | |
| CN1977885A (en) | Antihepatitis medicinal composition | |
| CN103610682B (en) | The preparation method of 3 Alpha-hydroxy-30-olive-12,20 (29)-diene-28-acid and preparing the application in antitumor drug | |
| CN103655559B (en) | Horned artemisia ester alkali compounds is preparing the application in anti-breast cancer medicines | |
| CN109363044A (en) | A kind of functional beverage and preparation method containing water-soluble capejasmine extract | |
| CN100590119C (en) | Antineoplastic compound of red pineapple flower alkali A, preparation method and application thereof | |
| CN108653209A (en) | A kind of new type natural Tea Flower derives the preparation and its application of lipid nanometer vesica | |
| CN111744439B (en) | Hypsizygus marmoreus polysaccharide chelated zinc microcapsule and preparation method thereof | |
| CN106176873B (en) | Capsella bursa-pastoris total alkaloid extract and preparation method and application thereof | |
| CN103420971B (en) | Antitumor compounds derived from G. nujiangnsis, and preparation method and application thereof | |
| CN104436201A (en) | Preparation of dual-hepatic-targeting long-circulation gypenoside liposome and preparation method of liposome | |
| CN103417528B (en) | New application of isojacareubin | |
| CN108403775B (en) | Gorgon fruit shell extract for inhibiting cancer cell proliferation as well as preparation method and application thereof | |
| CN103169660B (en) | Prepared ribavirin lipidosome oral emulsion with high encapsulation efficiency | |
| CN104337807B (en) | Nanometer liposome of load paclitaxel and sensitizer and preparation method thereof and application altogether |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |