CN109966476A - Three kinds of ingredient collaboration enhancing FGF2 of radix scutellariae promote the purposes of cell Proliferation - Google Patents

Three kinds of ingredient collaboration enhancing FGF2 of radix scutellariae promote the purposes of cell Proliferation Download PDF

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CN109966476A
CN109966476A CN201910337252.6A CN201910337252A CN109966476A CN 109966476 A CN109966476 A CN 109966476A CN 201910337252 A CN201910337252 A CN 201910337252A CN 109966476 A CN109966476 A CN 109966476A
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fgf2
compound
scutelloside
skullcap root
baical skullcap
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CN109966476B (en
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蒋世翠
金春燕
李东浩
郭建鹏
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Yanbian University
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Abstract

The invention discloses Baical Skullcap root P.E-FGF2 compounds, it is the compound that Baical Skullcap root P.E and FGF2 hatch acquisition altogether, the Baical Skullcap root P.E is scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides or wogonoside, and the compound of acquisition is FGF2- scutelloside compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound or FGF2- wogonoside compound；The hatching are as follows: dissolve Baical Skullcap root P.E with the methanol of 5 ~ 20 times of weight；FGF2 target protein is dissolved in PBS buffer solution；FGF2 PBS solution and Baical Skullcap root P.E methanol solution, mixing；Hatch 25 ~ 35min, freeze-drying at 36 ~ 38 DEG C；FGF2- scutelloside compound, FGF2- qroxylin A -7-0- β-application of D-Glucose aldehydic acid glycosides compound and/or FGF2- wogonoside compound in terms of promoting cell Proliferation.

Description

Three kinds of ingredient collaboration enhancing FGF2 of radix scutellariae promote the purposes of cell Proliferation
Technical field
The invention belongs to technical field of molecular biology, and in particular to three kinds of ingredient collaboration enhancing FGF2 of radix scutellariae promote cell Proliferation Purposes.
Background technique
The complicated multiplicity of traditional Chinese medicine ingredients, develops that practicable modern Chinese herbal medicine active constituent quickly screen and identification is that Chinese medicine shows For the key of research and development.The screening of bioactive ingredients is the important content in traditional Chinese medicine research field.Traditional Chemical Decomposition, Structural Identification, screening active ingredients mode to activity guiding Chemical Decomposition there are targets indefinite, cumbersome, heavy workload, period Long, active constituent many deficiencies such as easy to be lost during the separation process.Modern pharmacology research shows drug and large biological molecule The affine first step for being it and playing a role of (such as enzyme, receptor, DNA, RNA).We have developed in a kind of quickly screening Chinese medicine The method of bioactive ingredients: multicell electrophoretic separation system is simultaneously combined high performance liquid chromatography tandem mass spectrum technology.The technology can be Physiological status meets the Separation of Proteins of solution state quickly that target molecule-bioactive molecule is compound in target molecule affine technolog Object is separated from incubation solution, reduces the requirement of complex dissociation in separation process.The advantage of multicell electrophoresis is: 1) having There is simplicity is quick, action target spot is clear, target spot is not necessarily to label, screens list target spot multicomponent, screens multiple target point multicomponent etc.；2) more Room, multidimensional, external " visual ", dynamic analysis active constituent and target spot synergistic effect and variation；3) from folk prescription or complicated prescription The unknown active constituent with known targeted integration is screened in active constituent, while finding to promote the combination effect of principal component and target spot The active constituent answered.The technology compared with existing screening technique, have it is easy quickly, action target spot is clear, target spot without label, Without immobilization, screen the advantages such as single target spot multicomponent, screening multiple target point multicomponent.Research report: it is separated by electrophoresis based on multicell System is 7.4 in pH, and it is multiple that enzyme-active constituent is isolated from the solution after hatching under conditions of voltage 8V, disengaging time 15min Object is closed, and is combined high performance liquid chromatography tandem mass spectrum technology and qualitative analysis is carried out to the active constituent filtered out, and filter out seven Potential α-the glucosidase inhibitor of kind.
FGF2 can promote cell division proliferation, participate in including a variety of groups of mammary gland of mouse epithelium, lung epithelial, salivary gland epithelia etc. Knit lateral configuration.FGF2 has expression in breast ductal epithelial cells, musculoepithelia cell and acinar epithelial cells.FGF2 Closely related, controllable mammary gland of mouse conduit extension, cell Proliferation and cream during promotion mammogenesis are formed with mammary gland branch Galandular epithelium expansion.
Radix scutellariae (Scutellaria baicalensis Georgi) be medicinal plant radix scutellariae root, have reduce phlegm and internal heat it is dry Wet, Qinghuo Jiedu, miscarriage prevention, adjusting is immune, prevents and treats the effects of diabetes, anti-inflammatory anti-apoptotic.Correlative study shows the antibacterial of radix scutellariae And antivirus action is stronger, contained by baicalein and scutelloside external a variety of Gram-positives and negative bacterium can be effectively suppressed, And can be to causing characteristic of disease dermatophyte to play effective inhibiting effect, furthermore scutelloside and baicalein are in antitumor, antiallergy, anti- Atherosclerosis and to liver and central nervous system protection in also have certain effect.The a variety of flavones being rich in radix scutellariae Class compound scutelloside and baicalein, wogonoside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside have one Fixed anti-tumor activity, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides have antioxidation.Therefore, pharmacology action is wide General and development prospect is wide, has biggish research application space.
Summary of the invention
Object of the present invention is to the associations to solve synergistic effect and Chinese medicine and protein drug administering drug combinations between traditional Chinese medicine ingredients The problem of same-action, and three kinds of ingredient collaboration enhancing FGF2 of radix scutellariae application for promoting cell Proliferation is provided.
Baical Skullcap root P.E-FGF2 compound, it is the compound that Baical Skullcap root P.E and FGF2 hatch acquisition altogether, the Huang A kind of reed mentioned in ancient books extract is scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides or wogonoside, and the compound of acquisition is FGF2- Scutelloside compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound or FGF2- wogonoside compound；
The hatching are as follows:
1) Baical Skullcap root P.E is taken, Baical Skullcap root P.E is dissolved with the methanol of its 5 ~ 20 times of weight, obtains methanol solution；
2) FGF2 target protein is dissolved in PBS buffer solution, obtains FGF2 PBS solution；
3) FGF2 PBS solution and Baical Skullcap root P.E methanol solution, mixing；
4) hatch 25 ~ 35min at 36 ~ 38 DEG C, vacuum freeze drying obtains Baical Skullcap root P.E-FGF2 compound；
The concentration of PBS buffer solution described in step 2 is 10 mM, pH value 6.8；
FGF2 PBS solution and Baical Skullcap root P.E methanol solution described in step 3), 1:1 is mixed by volume；
Step 4) hatches 30min at 37 DEG C.
FGF2- scutelloside compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound and/or FGF2- Application of the wogonoside compound in terms of promoting cell Proliferation.
Detailed description of the invention: the present invention is based on principle of electrophoresis, in conjunction with the affine principle of target molecule, according to large biological molecule Both sexes research and development.In the physiological state by the Separation of Proteins of solution state, meet in target molecule affine technolog quickly by target point Son-bioactive molecule compound is separated from incubation solution, reduces the requirement of complex dissociation in separation process.With letter Just quickly, action target spot is clear, target spot without label, screen the advantages such as single target spot multicomponent, screening multiple target point multicomponent；It is more Room, multidimensional, external " visual ", dynamic analysis active constituent and target spot synergistic effect and variation；It is living from folk prescription or complicated prescription Property ingredient in screening and the unknown active constituent of known targeted integration, while finding to promote the combination effect of principal component and target spot Active constituent.
The present invention provides Baical Skullcap root P.E-FGF2 compounds, it is that Baical Skullcap root P.E hatches answering for acquisition with FGF2 altogether Close object, the Baical Skullcap root P.E be scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides or wogonoside, acquisition Compound is FGF2- scutelloside compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound or the FGF2- Chinese Scutelloside compound；The hatching are as follows: 1) Baical Skullcap root P.E is taken, dissolves Baical Skullcap root P.E with the methanol of its 5 ~ 20 times of weight, Obtain methanol solution；2) FGF2 target protein is dissolved in PBS buffer solution, obtains FGF2 PBS solution；3) FGF2 PBS solution and Huang A kind of reed mentioned in ancient books extract methanol solution, mixing；4) hatch 25 ~ 35min at 36 ~ 38 DEG C, vacuum freeze drying obtains Baical Skullcap root P.E- FGF2 compound；FGF2- scutelloside compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound and/or Application of the FGF2- wogonoside compound in terms of promoting cell Proliferation.Cell experiment the result shows that, Baical Skullcap root P.E-FGF2 is multiple It is obvious that conjunction object is administered alone better effect to the value-added effect ratio FGF2 of cell.
Detailed description of the invention
The total ion scan chromatogram of Fig. 1；
The total ion scan chromatography enlarged drawing of Fig. 2；
Negative receiving chamber MRM mode segmentation detection chromatogram under Fig. 3 14V voltage；
Fig. 4 electrophoresis Hou Fu receiving chamber MRM segmentation detection chromatogram；
3 kinds of active constituent structural formula schematic diagrames of Fig. 5；(1): scutelloside, (2): qroxylin A -7-0- β-D-Glucose aldehydic acid Glycosides, (3): wogonoside；
Fig. 6 FGF2 and radix scutellariae active constituent combination schematic diagram；
Fig. 7 radix scutellariae active material administration concentration the selection result；BC: scutelloside, WGS: wogonoside, OXS: qroxylin A -7- 0- β-D-Glucose aldehydic acid glycosides；
Fig. 8 FGF2Administration concentration the selection result；
Fig. 9 drug and FGF2Be administered alone and 24 h of cooperativing medicine-feeding after cell proliferation result；
Figure 10 drug and FGF2Be administered alone and 48 h of cooperativing medicine-feeding after cell proliferation result.
Specific embodiment
The preparation of 1 radix scutellariae extracting solution of embodiment
Pharmacy buys dry baikal skullcap root decoction pieces, removes impurity, dry radix scutellariae is ground into uniform powder, weighs the addition of 2 g powder 20 mL methanol carry out ultrasonic extraction, extract 3 times, 20 min, combining extraction liquid after filtering are crossed 0.22 μm of filter membrane, blown every time Nitrogen concentration drying, ultrapure water back dissolving cross 0.22 μm of filter membrane, are saved at 4 DEG C until using.
Embodiment 2 screens the ingredient in radix scutellariae extracting solution in conjunction with FGF2
1, the preparation of FGF2- radix scutellariae active constituent compound
FGF2 freeze-dried powder is dissolved in 10 mM pH6.8 ammonium acetate buffers, obtains FGF2 ammonium acetate solution；It is molten by FGF2 ammonium acetate Liquid: radix scutellariae extracting solution (v:v)=1:1 proportion, mixing form FGF2- radix scutellariae extracting solution mixed liquor；Hatch 30min at 37 DEG C, Make the active constituent in radix scutellariae extracting solution in conjunction with FGF2, forms FGF2- radix scutellariae active constituent compound；
2, multicell is separated by electrophoresis
FGF2- radix scutellariae extracting solution mixed liquor after hatching is as sample solution, and radix scutellariae extracting solution is as contrast solution, respectively in fact Test group and control group；Using multicell electrophoretic techniques, device dedicated electrophoretic separation with reference to described in CN104307368A patent of invention Device；The Sample Room of electrophoretic apparatus is made of two cellulose acetate films, aperture≤0.2 μm；The both ends of cellulose acetate film Respectively positive electrode chamber and negative electricity pole room are added buffer solution in positive electrode chamber and negative electricity pole room, make positive electrode chamber and negative electrode The liquid level of room is identical as the height of sample in Sample Room, and no liquid level is poor；Specific steps are as follows:
1) cellulose acetate film cut and ultrafiltration membrane are immersed in buffer solution 5 minutes, soak film completely；
2) film surface is cleaned with ultrapure water wash bottle, then is impregnated with clean buffer solution；
3) cellulose acetate film and ultrafiltration membrane each 2 are cut according to polytetrafluoroethylene (PTFE) partition；
4) totally 3 polytetrafluoroethylene (PTFE) partitions (thickness) put a piece of cellulose acetate film (/) among every 2 partitions, such as: thick partition/ Thick partition/thickness partition；
5) put among totally 2 polytetrafluoroethylene (PTFE) partitions (thin), thin partition and thick partition a piece of ultrafiltration membrane (), such as: thin partition Thick partition/thickness partition/thickness partition thin partition；
6) step 4) and 5) combine after, be placed between two slot electrodes, set upper clip tighten, it is to note that film keep formation state, can not Deform film；
7) a little vaseline is smeared in bottom, glues on the glass sheet, it is ensured that leakproofness is good；
8) assembled separation system is linked with buffer system, and regulating switch makes buffer smooth flow；
9) 100 μ L buffer solutions are respectively put in two receiving chambers, with injection needle by 100 μ L sample solution or contrast solution, are added to electrophoresis In the Sample Room of device；
10) electrode chamber is put into slot electrode, applies voltage 12V, time 15min；
11) after electrophoresis, take out in each room that solution is in 1.5mL centrifuge tube, for use；
3, the dissociation of FGF2- radix scutellariae active constituent compound
With the solution in 600 μ L methanol dissociation receiving chamber, 1200rpm centrifuging and taking supernatant；Supernatant is concentrated to 100 μ L, point It is to be detected that it is not transferred to sample injection bottle etc.；The solution of remaining each room is dissociated with same procedure, as laboratory reference；
The qualitative analysis of radix scutellariae active constituent of the embodiment 3 in conjunction with FGF2
1, the solution in receiving chamber after embodiment 2 dissociates, carries out HPLC high performance liquid chromatography-tandem mass spectrum and nuclear magnetic resonance Spectrum Analysis；
Chromatographic condition:
Chromatographic column: the mm of C18(4.6 mm × 150,5 μm)
Mobile phase: 0.1% aqueous formic acid (A), 0.1% formic acid acetonitrile solution (B)
Sample volume: 5 μ L
Flow velocity: 0.5 mL min-1
Gradient elution process:
Time (min) Mobile phase B (%)
0 0
7 0
10 25
20 40
30 65
40 80
Mass Spectrometry Conditions:
Electric spray ion source, negative ion mode, using full scan mode；Spray pressure power: 30 psi；Dry gas (N2) flow velocity: 13.0 L/min, dry temperature degree: 300 DEG C；Capillary voltage: 2500 V, capillary outlet voltage: 135 V；Mass number is swept Retouch range: 100-600 m/z.
Experimental group and the total ion scan chromatogram of control group and enlarged drawing difference are as shown in Figure 1, 2；As can be seen from the figure There are 3 kinds of active constituents and FGF in radix scutellariae2In conjunction with and migrate and cause negative receiving chamber；Retention time is shown in Table 1；Identified, radix scutellariae extracts The active constituent of number 1,2,3 is respectively scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, the Chinese in the negative receiving chamber of liquid Scutelloside shows that scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside can be with FGF2In conjunction with formation phase The compound answered；
2, MRM detects (the more reaction detections of mass spectrum)
Experimental group and control group carry out MRM detection respectively, carry out the mass spectrum point of the negative receiving chamber solution under 14 V voltage conditions Analysis, MRM mode segmentation detection chromatogram such as Fig. 3；The FGF from figure2The negative receiving chamber of radix scutellariae extracting solution and the negative reception of radix scutellariae extracting solution Total ion chromatogram comparison in room illustrates to have in radix scutellariae extracting solution 3 active constituents and FGF2 to have preliminary in conjunction with simultaneously contrast standard product Qualitative 3 active constituents out are respectively scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside.
The solution in receiving chamber being separated by electrophoresis using multicell carries out MRM segmentation detection as sample；Concrete operations It is as follows: to have initially set up scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside three kinds of active constituents MRM detection method, method information are shown in Table 2；Negative receiving chamber MRM segmentation detection chromatogram is shown in Fig. 4, the results showed that 3 filtered out Active constituent is determined as scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside；The structural formula of active constituent It is detailed in Fig. 5；
3, the combination of FGF2 and radix scutellariae active constituent
Using Autodock software to three kinds of compounds and FGF2Computer fitting is carried out, the bound site of small molecule and albumen is investigated Point and hydrogen bond number；
A. FGF2 and scutelloside binding site (Fig. 6 A): ASN-27:27 asparagines (2 hydrogen bonds)；ARG-120:120 The arginine (1 hydrogen bond) of position；LYS-125:125 lysine (1 hydrogen bond)；LYS-135:135 lysine (2 Hydrogen bond)；
B. FGF2 and qroxylin A -7-0- β-D-Glucose aldehydic acid binding site (Fig. 6 B): ASN-27:27 asparagines (5 hydrogen bonds)；ARG-120:120 arginine (2 hydrogen bonds)；LYS-135:135 lysine (1 hydrogen bond)；
C. FGF2 and wogonoside binding site (Fig. 6 C): ASN-27:27 asparagines (1 hydrogen bond): ARG-120: 120 arginine (2 hydrogen bonds)；LYS-125:125 lysine (1 hydrogen bond)；LYS-135:135 lysine (2 A hydrogen bond)；
The combination of FGF2 and 3 kinds of active constituents are with Hydrogenbond；Scutelloside and wogonoside binding site are just the same, But hydrogen bond number is different；Qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides lacks on 125 lysine (LYS-125) Hydrogen bond.
The quantitative analysis of radix scutellariae active constituent of the embodiment 4 in conjunction with FGF2
1, BCA protein concentration test experience
FGF is measured using BCA method2Protein concentration in radix scutellariae active constituent compound；
Particular content: detecting specification configuration standard curve sample according to BCA protein concentration, by standard curve sample and through more 20 μ L are added toward 96 orifice plates in sample after the electrophoresis of room, and BCA working solution is added, and 60 DEG C of 30 min of hatching are existed using microplate reader It is checked at light absorption value 562nm.
The regression equation of BCA standard curve: y=0.11107+0.00117X, R2=0.9999；Protein concentration is 0.5 mg/ mL；
2, standard curve is made
The assessment of the HPLC-MS/MS experiment porch range of linearity and sensitivity weighs scutelloside, qroxylin A -7-0- β-respectively The standard items of D-Glucose aldehydic acid glycosides and wogonoside are respectively 1 mg, and 0.5 mg, 0.5 mg are dissolved in 5 mL methanol solutions, It takes 50 μ L to move in 5 mL volumetric flasks, that is, S6 concentration samples in table 2 is made.With S6 sample doubling dilution at S1-S6 gradient concentration Sample, for standard curve to be determining and linear relationship verifying.Machine examination on S1 concentration level Sample Dilution to lower concentration gradient Survey, measure signal-to-noise ratio (S/N), until S/N10:1. is in 5 samples of parallel processing, meet precision (RSD) less than 20%, then this When concentration of specimens be instrument Monitoring lower-cut.
HPLC-MS/MS platform standard curve is to be automatically processed to provide by workstation software, and cardinal principle is according to inspection S1~S6 series of concentrations of survey and corresponding peak area draw normal concentration-normal concentration area curve, and calculated curve Equation and correlation coefficient r.
Regression equation see the table below 4；
3, three kinds of ingredients and FGF2In conjunction with the quantitative analysis of radix scutellariae active constituent and combine ratio calculate
According to embodiment 2 and embodiment 3 obtains and FGF2In conjunction with three kinds of ingredients, the standard curve in 4, carries out in conjunction with the embodiments Quantitative analysis；Three kinds of scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside active constituent Component molars are dense The specific measuring method of degree is that MRM is detected FGF2The negative receiving chamber of+radix scutellariae extracting solution and the negative receiving chamber of radix scutellariae extracting solution and system The standard curve finished calculates active constituent molar ratio using quantitation software；It the results are shown in Table 5
, it can be deduced that scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides, wogonoside and FGF2It is small after protein binding Ratio between molecule is 3:1:1；
5 FGF of embodiment2The preparation of radix scutellariae active constituent compound
1、FGF2Scutelloside compound
Scutelloside standard items are taken, scutelloside standard items is dissolved with the methanol of its 10 times of weight, obtains scutelloside methanol solution；FGF2 Target protein is dissolved in 10 mM pH6.8 PBS buffer solution, obtains FGF2PBS solution；By FGF2PBS solution: scutelloside methanol solution (v:v)=1:1 proportion, mixing form FGF2Scutelloside mixed liquor；Hatch 30min at 37 DEG C, makes in scutelloside methanol solution Scutelloside and FGF2In conjunction with formation FGF2Scutelloside compound；By the mixed liquor vacuum freeze drying after hatching to get arriving FGF2Scutelloside compound；
2、FGF2Qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound
The same FGF of preparation method2Scutelloside compound is identical；
3、FGF2Wogonoside compound
The same FGF of preparation method2Scutelloside compound is identical.
6 FGF of embodiment2The preparation of radix scutellariae active constituent compound
1、FGF2Scutelloside compound
Scutelloside standard items are taken, scutelloside standard items is dissolved with 100% dimethyl sulphoxide solution of its 10 times of weight, obtains radix scutellariae Glycosides dimethyl sulphoxide solution；FGF2Target protein is dissolved in 10 mM pH6.8 PBS buffer solution, obtains FGF2PBS solution；By FGF2Second Acid ammonium solution: scutelloside dimethyl sulphoxide solution (v:v)=1:1 proportion, mixing form FGF2Scutelloside mixed liquor；At 37 DEG C Lower hatching 30min makes scutelloside and FGF in scutelloside dimethyl sulphoxide solution2In conjunction with formation FGF2Scutelloside compound； By the mixed liquor vacuum freeze drying after hatching to get arrive FGF2Scutelloside compound.
2、FGF2Qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound
The same FGF of preparation method2Scutelloside compound is identical；
3、FGF2Wogonoside compound
The same FGF of preparation method2Scutelloside compound is identical.
7 three kinds of ingredients of embodiment and FGF2, which are administered in combination, promotees cell proliferation experiment
1. the screening of administration concentration
Three kinds of active material scutellosides (BC), qroxylin A -7- have been determined according to qualitative and quantitative analysis during embodiment 2-4 0- β-D-Glucose aldehydic acid glycosides (OXS), wogonoside (WGS), while determining scutelloside: qroxylin A: the ratio of wogonoside Concentration screening is administered for 3:1:1；The administration concentration of FGF2 is screened simultaneously；
Specific step is as follows:
1) NIH3T3 cell is adjusted to concentration with the DMEM containing 10% FBS is 2*104A/mL is inoculated in 96 orifice plates, every hole 100ul；
2) in the 5%CO of concentration2Under, cultivated for 24 hours in 37 DEG C of constant incubators after, compound group be added serum-free DMEM it is diluted Compound: the concentration of compound is as follows in Fig. 7: compound monomer gradient administration, administration concentration are as follows: 200 μM, 100 μM, 50 μM, 25μM,12.5μM,6.25μM,3.125μM,1.5625μM,0.78125μM；Fig. 8 FGF2 group administration concentration be 200 ng/mL, 100 ng/mL、50 ng/mL、25 ng/mL、12.5 ng/mL、6.25 ng/mL、3.125 ng/mL、1.5625 ng/mL、 0.7825 ng/mL；
3) it sets up cell-free blank control group (serum-free DMEM is only added), (the serum-free DMEM dilution of cell free drug control group Various acute drugs) and cell controls group (only be added serum-free DMEM), be all provided with 5 multiple holes；Experiment is repeated 3 times；
4) after cultivating 48h, MTT is added is being under the microscope after cultivating 48h in every hole 20ul, 5%CO2,37 DEG C of constant incubators No formation purple is Filamentous or circle crystallizes, and 100ulDMSO is added, after gently shaking 10min, with microplate reader under 562nm wavelength, Detect each hole OD value；
As a result see Fig. 7,8；As shown in fig. 7, the ingredient 3 of radix scutellariae three active constituent individually a compound be administered when to cell to thin Born of the same parents determine compound administration concentration at 50-1.5625 μM without increment effect is promoted；As can be known from Fig. 8, when FGF2 is administered alone, FGF2Administration concentration be 50ng/mL, proliferation is best.
2. being administered in combination the effect of proliferation function
On the basis of above-mentioned administration concentration the selection result, three kinds of compounds and FGF2 are according to the compound screened in embodiment 4 Ratio 3:1:1 be administered in combination；The administration concentration range of three kinds of compounds is administered in 30-1.13 μM of gradient, and FGF2 administration is dense Spend the fixed administration of 50ng/mL；Specific step is as follows:
1) NIH3T3 cell is adjusted to concentration with the DMEM containing 10% FBS is 2*104A/mL is inoculated in 96 orifice plates, every hole 100ul；
2) in the 5%CO of concentration2Under, cultivated for 24 hours in 37 DEG C of constant incubators after, compound group be added serum-free DMEM it is diluted Compound: compound concentration is as follows in Fig. 9, Figure 10:
N: serum-free DMEM only blank control group: is added；
BC: it is followed successively by 30.00 μM, 15.00 μM, 7.50 μM, 3.75 μM of scutelloside from left to right；
WGS: it is followed successively by 9.56 μM, 4.78 μM, 2.39 μM, 1.295 μM of wogonoside from left to right；
OXS: be followed successively by from left to right qroxylin A -7-0- β -9.04 μM of D-Glucose aldehydic acid glycosides, 4.52 μM, 2.26 μM, 1.13 μM；
FGF2: serum-free DMEM dilution is added, makes 50 ng/mL of FGF2 concentration；
FGF2+BC: it is followed successively by the FGF2 of (1) scutelloside=30.00 μM, 50 ng/mL, (2) scutelloside=15.00 from left to right μM, the FGF2 of 50 ng/mL, (3) scutelloside=7.50 μM, 50 ng/mL FGF2, (4) scutelloside=3.75 μM, 50 ng/ The FGF2 of mL；
FGF2+WGS: being followed successively by FGF2, wogonoside=9.56 μM of (1) 50 ng/mL from left to right, (2) 50 ng/mL's FGF2, wogonoside=4.78 μM, the FGF2 of (3) 50 ng/mL, wogonoside=2.39 μM, the FGF2 of (4) 50 ng/mL, Wogonoside=1.295 μM；
FGF2+OXS: be followed successively by from left to right FGF2, the qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides of (1) 50 ng/mL= 9.04 μM, FGF2, the qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=4.52 μM of (2) 50 ng/mL, (3) 50 ng/mL FGF2, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=2.26 μM, FGF2, the qroxylin A -7- of (4) 50 ng/mL 0- β-D-Glucose aldehydic acid glycosides=1.13 μM；
BC+WGS+OXS: it is followed successively by (1) scutelloside=30.00 μM, wogonoside=9.56 μM, qroxylin A -7- from left to right 0- β-D-Glucose aldehydic acid glycosides=9.04 μM, (2) scutelloside=15.00 μM, wogonoside=4.78 μM, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=4.52 μM, (3) scutelloside=7.50 μM, wogonoside=2.39 μM, qroxylin A -7-0- β-D- Glucuronide=2.26 μM, (4) scutelloside=3.75 μM, wogonoside=1.295 μM, the Portugal qroxylin A -7-0- β-D- Grape glycuronide=1.13 μM；
FGF2+BC+WGS+OXS: it is followed successively by FGF2, scutelloside=30.00 μM, the wogonoside of (1) 50 ng/mL from left to right =9.56 μM, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=9.04 μM, the FGF2 of (2) 50 ng/mL, scutelloside= 15.00 μM, wogonoside=4.78 μM, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=4.52 μM, (3) 50 ng/mL FGF2, scutelloside=7.50 μM, wogonoside=2.39 μM, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides=2.26 μ FGF2, scutelloside=3.75 μM, wogonoside=1.295 μM, the qroxylin A -7-0- β-D- grape of M, (4) 50 ng/mL Glycuronide=1.13 μM；
3) it sets up cell-free blank control group (serum-free DMEM is only added), (the serum-free DMEM dilution of cell free drug control group Various acute drugs) and cell controls group (only be added serum-free DMEM), be all provided with 5 multiple holes；Experiment is repeated 3 times；
4) after cultivating 48h, MTT, every hole 20ul, 5%CO is added2, after cultivating 48h in 37 DEG C of constant incubators, it is being under the microscope No formation purple is Filamentous or circle crystallizes, and 100ul DMSO is added, after gently shaking 10min, with microplate reader in 562nm wavelength Under, detect each hole OD value；
As a result as shown in Fig. 9,10, cell is acted on almost without promotion increment when three compounds of radix scutellariae are administered alone, but 3 When kind active constituent cooperativing medicine-feeding, has to cell and promote value-added effect；Three compounds respectively with FGF2In conjunction with Yi Jisan A compound and FGF2Joint, when administration, all compare FGF to the value-added effect of cell2It is administered alone better effect obviously and is administered 24 It is significant that h and 48 h compares 48 h cell proliferation function and effect；Conclusion: according to being administered after the ratio of combination, promotion increases in cell Value effect.

Claims (5)

1. Baical Skullcap root P.E-FGF2 compound, it is the compound that Baical Skullcap root P.E and FGF2 hatch acquisition altogether, the radix scutellariae Extract is scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides or wogonoside, and the compound of acquisition is FGF2- yellow A kind of reed mentioned in ancient books glycosides compound, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides compound or FGF2- wogonoside compound.
2. Baical Skullcap root P.E-FGF2 compound according to claim 1, it is characterised in that: the hatching are as follows:
1) Baical Skullcap root P.E is taken, Baical Skullcap root P.E is dissolved with the methanol of its 5 ~ 20 times of weight, obtains methanol solution；
2) FGF2 target protein is dissolved in PBS buffer solution, obtains FGF2 PBS solution；
3) FGF2 PBS solution and Baical Skullcap root P.E methanol solution, mixing；
4) hatch 25 ~ 35min at 36 ~ 38 DEG C, vacuum freeze drying obtains Baical Skullcap root P.E-FGF2 compound；
The Baical Skullcap root P.E is scutelloside, qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides or wogonoside.
3. Baical Skullcap root P.E-FGF2 compound according to claim 2, it is characterised in that:
The concentration of PBS buffer solution described in step 2 is 10 mM, pH value 6.8；
FGF2 PBS solution and Baical Skullcap root P.E methanol solution described in step 3), 1:1 is mixed by volume.
4. Baical Skullcap root P.E-FGF2 compound according to claim 3, it is characterised in that: step 4) is hatched at 37 DEG C 30min。
5. FGF2- scutelloside compound described in claim 1, FGF2- qroxylin A -7-0- β-D-Glucose aldehydic acid glycosides are multiple Close the purposes of object and/or FGF2- wogonoside compound in terms of promoting cell Proliferation.
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