CN109966476B - Application of three components of scutellaria baicalensis to synergistic enhancement of FGF2 cell proliferation promotion - Google Patents
Application of three components of scutellaria baicalensis to synergistic enhancement of FGF2 cell proliferation promotion Download PDFInfo
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Abstract
The invention discloses a scutellaria baicalensis extract-FGF 2 compound, which is a compound obtained by incubating scutellaria baicalensis extract and FGF2 together, wherein the scutellaria baicalensis extract is baicalin, oroxylin A-7-0-beta-D-glucuronide or wogonoside, and the obtained compound is an FGF 2-baicalin compound, FGF 2-oroxylin A-7-0-beta-D-glucuronide compound or FGF 2-wogonoside compound; the hatching is as follows: dissolving the scutellaria baicalensis extract by using 5-20 times of methanol by weight; dissolving FGF2 target protein in PBS buffer; mixing FGF2 PBS solution and Scutellariae radix extract methanol solution; incubating at 36-38 ℃ for 25-35 min, and freeze-drying; the application of FGF 2-baicalin complex, FGF 2-oroxylin A-7-0-beta-D-glucuronide complex and/or FGF 2-wogonoside complex in promoting cell proliferation.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an application of three components of scutellaria in synergistic enhancement of FGF2 to promote cell proliferation.
Background
The Chinese medicinal components are complex and various, and the feasible development and rapid screening and identification of the active ingredients of the modern Chinese medicaments are the key points of the modern research and development of the Chinese medicaments. The screening of bioactive components is an important content in the field of traditional Chinese medicine research. The traditional chemical separation, structure identification and activity screening mode to activity-oriented chemical separation have the defects of unclear target, complex operation, large workload, long period, easy loss of active ingredients in the separation process and the like. Modern pharmacological studies have shown that the affinity of drugs for biological macromolecules (e.g. enzymes, receptors, DNA, RNA, etc.) is the first step in their action. We have developed a method for rapid screening of bioactive components in Chinese herbs: the multi-chamber electrophoresis separation system is connected in parallel by a high performance liquid chromatography tandem mass spectrometry technology. The technology can separate the protein in a solution state in a physiological state, meets the requirement of quickly separating a target molecule-active molecule compound from an incubation solution in a target molecule affinity technology, and reduces the dissociation requirement of the compound in the separation process. The advantages of multi-compartment electrophoresis are: 1) the method has the advantages of simplicity, convenience, rapidness, clear action target, no need of marking the target, screening of single-target multi-component, screening of multi-target multi-component and the like; 2) multi-chamber, multi-dimensional, in vitro 'visible', dynamic analysis of the synergy and changes of the active ingredients and the target; 3) screening unknown active ingredients combined with known targets from single-component or complex-component active ingredients, and finding active ingredients promoting the combined effect of the known ingredients and the targets. Compared with the existing screening technology, the technology has the advantages of simplicity, convenience, rapidness, definite action target, no need of marking and immobilizing the target, screening of single-target-point multi-component, screening of multi-target-point multi-component and the like. The research reports that: separating enzyme-active component compound from the incubated solution based on a multi-chamber electrophoresis separation system under the conditions of pH 7.4, voltage 8V and separation time 15min, and qualitatively analyzing the screened active components by high performance liquid chromatography-tandem mass spectrometry technology, and screening seven potential alpha-glucosidase inhibitors.
FGF2 can promote cell division and proliferation, and is involved in the branch morphogenesis of various tissues including mouse mammary gland epithelium, lung epithelium, salivary gland epithelium, etc. FGF2 was expressed in mammary duct epithelial cells, myoepithelial cells, and acinar epithelial cells. FGF2 is closely related to branch formation of mammary gland, can control elongation of mammary duct of mouse, and promote cell proliferation and mammary epithelial expansion in mammary gland development process.
Baikal skullcap root (Scutellaria baicalensis Georgi) Is the root of medicinal plant scutellaria, and has the functions of removing heat and drying dampness, clearing away fire and toxic material, preventing miscarriage, regulating immunity, preventing and treating diabetes, resisting inflammation and resisting apoptosis, etc. Related researches show that the scutellaria baicalensis has strong antibacterial and antiviral effects, the baicalein and the baicalein contained in the scutellaria baicalensis can effectively inhibit various gram-positive and gram-negative bacteria in vitro and can effectively inhibit disease-causing dermatophytes, and the baicalein also have certain effects on resisting tumors, resisting allergy, resisting atherosclerosis and protecting livers and central nervous systems. The Scutellariae radix is rich in various flavonoid compounds including baicalin and baicalein, wogonoside and oroxylin A-7-0-beta-D-glucuronide, and wogonoside with certain antitumor effectThe active oroxylin A-7-0-beta-D-glucuronide has an antioxidant effect. Therefore, the compound has wide pharmacological application and wide development prospect, and has larger research and application space.
Disclosure of Invention
The invention aims to solve the problems of the synergistic effect between the traditional Chinese medicine components and the synergistic effect of the combined administration of the traditional Chinese medicine and the protein medicine, and provides the application of the three components of the scutellaria baicalensis for synergistically enhancing the FGF2 to promote cell proliferation.
The scutellaria baicalensis extract-FGF 2 compound is a compound obtained by incubating scutellaria baicalensis extract and FGF2, wherein the scutellaria baicalensis extract is baicalin, oroxylin A-7-0-beta-D-glucuronide or wogonoside, and the obtained compound is FGF 2-baicalin compound, FGF 2-oroxylin A-7-0-beta-D-glucuronide compound or FGF 2-wogonoside compound;
the hatching is as follows:
1) dissolving the scutellaria baicalensis extract by using methanol which is 5-20 times of the weight of the scutellaria baicalensis extract to obtain a methanol solution;
2) dissolving FGF2 target protein in PBS buffer solution to obtain FGF2 PBS solution;
3) mixing FGF2 PBS solution and Scutellariae radix extract methanol solution;
4) incubating at 36-38 ℃ for 25-35 min, and performing vacuum freeze drying to obtain a scutellaria baicalensis extract-FGF 2 compound;
the concentration of the PBS buffer solution in the step 2) is 10 mM, and the pH value is 6.8;
mixing the FGF2 PBS solution and the scutellaria baicalensis extract methanol solution in the step 3) according to the volume ratio of 1: 1;
step 4) incubation at 37 ℃ for 30 min.
The application of FGF 2-baicalin complex, FGF 2-oroxylin A-7-0-beta-D-glucuronide complex and/or FGF 2-wogonoside complex in promoting cell proliferation.
Detailed description of the invention: the invention is developed based on the principle of electrophoresis and the principle of target molecule affinity according to the amphipathy of biological macromolecules. The protein in a solution state is separated in a physiological state, so that the requirement of quickly separating a target molecule-active molecule compound from an incubation solution in a target molecule affinity technology is met, and the dissociation of the compound in the separation process is reduced. The method has the advantages of simplicity, convenience, rapidness, definite action target, no need of marking the target, screening of single-target multi-component, screening of multi-target multi-component and the like; multi-chamber, multi-dimensional, in vitro 'visible', dynamic analysis of the synergy and changes of the active ingredients and the target; screening unknown active ingredients combined with known targets from single-component or complex-component active ingredients, and finding out active ingredients promoting the combination effect of the known ingredients and the targets.
The invention provides a scutellaria baicalensis extract-FGF 2 compound, which is a compound obtained by co-hatching scutellaria baicalensis extract and FGF2, wherein the scutellaria baicalensis extract is baicalin, oroxylin A-7-0-beta-D-glucuronide or wogonoside, and the obtained compound is FGF 2-baicalin compound, FGF 2-oroxylin A-7-0-beta-D-glucuronide compound or FGF 2-wogonoside compound; the hatching is as follows: 1) dissolving the scutellaria baicalensis extract in methanol which is 5-20 times of the weight of the scutellaria baicalensis extract to obtain methanol solution; 2) dissolving FGF2 target protein in a PBS buffer solution to obtain FGF2 PBS solution; 3) mixing FGF2 PBS solution and Scutellariae radix extract methanol solution; 4) incubating at 36-38 ℃ for 25-35 min, and performing vacuum freeze drying to obtain a scutellaria baicalensis extract-FGF 2 compound; the application of FGF 2-baicalin complex, FGF 2-oroxylin A-7-0-beta-D-glucuronide complex and/or FGF 2-wogonoside complex in promoting cell proliferation. Cell experiment results show that the proliferation effect of the scutellaria baicalensis extract-FGF 2 compound on cells is better and more obvious than that of the single administration of FGF 2.
Drawings
FIG. 1 Total ion scan chromatogram;
FIG. 2 is an enlarged view of a total ion scanning chromatogram;
the negative receiving chamber MRM mode segmented detection chromatogram at 314V voltage;
FIG. 4 is a graph of a negative receptor MRM sectional assay chromatogram after electrophoresis;
FIG. 53 schematic representation of active ingredient structural formula; (1): baicalin, (2): oroxylin A-7-0-beta-D-glucuronide (3): wogonoside;
FIG. 6 is a schematic representation of the binding of FGF2 to active principles of Scutellaria baicalensis Georgi;
FIG. 7 shows the screening results of the dosing concentration of the active substance of Scutellaria baicalensis Georgi; BC: baicalin, WGS: wogonoside, OXS: oroxylin A-7-0-beta-D-glucuronide;
FIG. 8FGF 2 The result of screening the administration concentration of (1);
FIG. 9 drugs and FGF 2 Cell proliferation results after 24h of single administration and synergistic administration;
FIG. 10 drugs and FGF 2 Cell proliferation results after 48h of single and co-administration.
Detailed Description
Example 1 preparation of Scutellaria baicalensis Georgi extract
Purchasing dry radix Scutellariae decoction pieces in pharmacy, removing impurities, grinding dry radix Scutellariae into uniform powder, weighing 2 g of powder, adding 20 mL of methanol, performing ultrasonic extraction for 3 times (20 min each time), filtering, mixing extractive solutions, filtering with 0.22 μm filter membrane, blowing nitrogen, concentrating, blow-drying, dissolving with ultrapure water, filtering with 0.22 μm filter membrane, and storing at 4 deg.C until use.
Example 2 screening of Scutellaria baicalensis Georgi extract for binding to FGF2
1. Preparation of FGF 2-radix scutellariae active ingredient compound
Dissolving FGF2 lyophilized powder in 10 mM ammonium acetate buffer solution with pH6.8 to obtain FGF2 ammonium acetate solution; mixing FGF2 ammonium acetate solution and Scutellariae radix extractive solution (v: v) =1:1 to obtain FGF 2-Scutellariae radix extractive solution mixture; incubating at 37 deg.C for 30min to combine the active ingredient in Scutellariae radix extractive solution with FGF2 to form FGF 2-Scutellariae radix active ingredient complex;
2. multi-compartment electrophoretic separation
Taking the incubated FGF 2-radix Scutellariae extract mixed solution as a sample solution, taking the radix Scutellariae extract as a control solution, and respectively taking the radix Scutellariae extract as an experimental group and a control group; the device adopts a multi-chamber electrophoresis technology, and refers to a special electrophoresis separation device described in the patent of CN104307368A invention; the sample chamber of the electrophoresis device consists of two cellulose acetate membranes, and the aperture of the sample chamber is less than or equal to 0.2 mu m; the two ends of the cellulose acetate membrane are respectively provided with a positive electrode chamber and a negative electrode chamber, and buffer solution is added into the positive electrode chamber and the negative electrode chamber, so that the liquid level height of the positive electrode chamber and the negative electrode chamber is the same as the height of a sample in the sampling chamber, and no liquid level difference exists; the specific operation steps are as follows:
1) soaking the cut cellulose acetate membrane and the ultrafiltration membrane in a buffer solution for 5 minutes to completely soak the membranes;
2) washing the surface of the membrane by using an ultrapure water washing bottle, and soaking the membrane by using a clean buffer solution;
3) cutting 2 cellulose acetate membranes and 2 ultrafiltration membranes according to the polytetrafluoroethylene spacers;
4) a total of 3 polytetrafluoroethylene spacers (thick) with one cellulose acetate membrane (/) in between every 2 spacers, for example: thick spacer/thick spacer;
5) a total of 2 polytetrafluoroethylene spacers (thin) with an ultrafiltration membrane (e.g.: thin spacer \ thick spacer/thick spacer \ thin spacer;
6) after the steps 4) and 5) are combined, the electrode is placed between two electrode grooves, a clamping piece is sleeved on the electrode grooves, and attention is paid to the following steps: the film is kept in a flat state and cannot be deformed;
7) a little vaseline is smeared at the bottom and is stuck on the glass sheet, so that good sealing property is ensured;
8) the assembled separation system is linked with a buffer system, and a switch is adjusted to enable the buffer solution to flow stably;
9) 100 mu L of buffer solution is respectively placed in the two receiving chambers, and 100 mu L of sample solution or control solution is added into the sample chamber of the electrophoresis device by using a syringe needle;
10) placing the electrode chamber into an electrode groove, and applying voltage of 12V for 15 min;
11) after electrophoresis is finished, taking out the solution in each chamber to a 1.5mL centrifuge tube for later use;
3. dissociation of FGF 2-Scutellaria baicalensis active ingredient complex
The solution in the receiving chamber was dissociated with 600. mu.L of methanol, and the supernatant was centrifuged at 1200 rpm; concentrating the supernatant to 100 μ L, transferring to sample bottles, and detecting; the solution in other chambers is dissociated by the same method and is used as an experimental reference;
example 3 qualitative analysis of Scutellaria baicalensis active ingredient binding to FGF2
1. Example 2 the dissociated solution in the receiving chamber was subjected to HPLC-tandem mass spectrometry and nmr spectroscopy;
chromatographic conditions are as follows:
a chromatographic column: c18 (4.6 mm X150 mm, 5 μm)
Mobile phase: 0.1% formic acid aqueous solution (A), 0.1% formic acid acetonitrile solution (B)
Sample introduction amount: 5 μ L
Flow rate: 0.5 mL min -1
Gradient elution procedure:
time (min) mobile phase B (%)
0 0
7 0
10 25
20 40
30 65
40 80
Mass spectrum conditions:
an electrospray ion source adopts a negative ion mode and adopts a full scanning mode; spray gas pressure: 30 psi; drying gas (N) 2 ) Flow rate: 13.0L/min, drying gas temperature: 300 ℃; capillary voltage: 2500V, capillary exit voltage: 135V; mass number scan range: 100-.
The total ion scanning chromatograms and enlarged images of the experimental group and the control group are respectively shown in figures 1 and 2; it can be seen from the figure that there are 3 active ingredients and FGF in the scutellaria 2 Binds and migrates to the negative receiving chamber; the retention times are shown in table 1; the identification shows that the active ingredients numbered 1, 2 and 3 in the negative receiving chamber of Scutellariae radix extractive solution are baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside, respectively, and indicate that baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside can be mixed with FGF 2 Binding to form a corresponding complex;
2. MRM detection (Mass Spectrometry multiple reaction detection)
Respectively carrying out MRM detection on the experimental group and the control group, carrying out mass spectrometry of the solution in the negative receiving chamber under the condition of 14V voltage, and carrying out MRM mode segmented detection chromatogram as shown in figure 3; from the figure, FGF 2 Comparing the total ion chromatograms of the negative receiving chamber of the radix scutellariae extracting solution and the negative receiving chamber of the radix scutellariae extracting solution, which shows that 3 active ingredients in the radix scutellariae extracting solution are combined with FGF2, and comparing the standard product to preliminarily determine that the 3 active ingredients are baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside respectively.
Using the solution in the receiving chamber obtained by multi-chamber electrophoretic separation as a sample to carry out MRM sectional detection; the specific operation is as follows: firstly, MRM detection methods of three active ingredients of baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside are established, and the method information is shown in a table 2; the negative acceptance chamber MRM sectional detection chromatogram is shown in figure 4, and the result shows that the 3 screened active ingredients are determined to be baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside; the structural formula of the active ingredient is shown in figure 5;
3. combination mode of FGF2 and active ingredients of scutellaria baicalensis
Using Autodock software to react three compounds with FGF 2 Performing computer fitting, and inspecting the binding sites and hydrogen bond number of the small molecules and the proteins;
a. FGF2 binding site to baicalin (fig. 6A): ASN-27: asparagine at position 27 (2 hydrogen bonds); ARG-120: arginine at position 120 (1 hydrogen bond); LYS-125: lysine at position 125 (1 hydrogen bond); LYS-135: lysine at position 135 (2 hydrogen bonds);
b. FGF2 and oroxylin a-7-0- β -D-glucuronic acid binding site (fig. 6B): ASN-27: asparagine at position 27 (5 hydrogen bonds); ARG-120: arginine at position 120 (2 hydrogen bonds); LYS-135: lysine at position 135 (1 hydrogen bond);
c. FGF2 and wogonoside binding site (fig. 6C): ASN-27: asparagine at position 27 (1 hydrogen bond): ARG-120: arginine at position 120 (2 hydrogen bonds); LYS-125: lysine at position 125 (1 hydrogen bond); LYS-135: lysine at position 135 (2 hydrogen bonds);
the combination of FGF2 and 3 active ingredients is hydrogen bond combination; baicalin and wogonoside have the same binding site, but have different hydrogen bonds; oroxylin A-7-0-beta-D-glucuronide lacks a hydrogen bond at lysine position 125 (LYS-125).
Example 4 quantitative analysis of active ingredient of Scutellaria baicalensis Georgi bound to FGF2
1. BCA protein concentration detection assay
FGF assay Using BCA method 2 -protein concentration in the scutellaria active ingredient complex;
the specific content is as follows: preparing a standard curve sample according to the BCA protein concentration detection instruction, adding 20 mu L of the standard curve sample and the sample subjected to multi-chamber electrophoresis into a 96-well plate, adding a BCA working solution, incubating for 30min at 60 ℃, and detecting at a light absorption value of 562nm by using a microplate reader.
Regression equation for BCA standard curve: y =0.11107+0.00117X, R2= 0.9999; the protein concentration is 0.5 mg/mL;
2. making a standard curve
And (3) evaluating the linear range and the sensitivity of an HPLC-MS/MS experiment platform, weighing 1 mg, 0.5 mg and 0.5 mg of standard substances of baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside respectively, dissolving in 5mL of methanol solution, taking 50 mu L, transferring to a 5mL volumetric flask, and preparing S6 concentration samples in the table 3. Samples diluted by the S6 sample multiple ratio to S1-S6 gradient concentration are used for standard curve determination and linear relation verification. The S1 concentration level sample is diluted to a lower concentration gradient and is subjected to machine detection, and the signal-to-noise ratio (S/N) is measured until the ratio of S/N10: 1. when 5 samples are processed in parallel, the precision (RSD) is less than 20%, and the sample concentration at the moment is the detection lower limit of the instrument.
The standard curve of the HPLC-MS/MS platform is given by automatic processing of workstation software, and the main principle is to draw a standard concentration-standard concentration area curve according to the detected S1-S6 series concentrations and corresponding peak areas and calculate a curve equation and a correlation coefficient r.
The regression equation is shown in Table 4 below;
3. three components with FGF 2 Quantitative analysis of combined active components of radix Scutellariae and calculation of combination ratio
Preparation of the peptide with FGF according to example 2 and example 3 2 The three components combined, combined with the standard curve in example 4, were subjected to quantitative analysis; the specific determination method of the molar concentration of three active ingredients including baicalin, oroxylin A-7-0-beta-D-glucuronide and wogonoside comprises detecting FGF with MRM 2 Calculating the molar ratio of the active ingredients by using quantitative software in the scutellaria baicalensis extracting solution negative receiving chamber, the scutellaria baicalensis extracting solution negative receiving chamber and the prepared standard curve; the results are shown in Table 5
Obtaining baicalin, oroxylin A-7-0-beta-D-glucuronide, wogonoside and FGF 2 The ratio of the small molecules after protein combination is 3:1: 1;
example 5 FGF 2 Preparation of-scutellaria active ingredient complex
1、FGF 2 -baicalin complexes
Dissolving the baicalin standard substance with methanol 10 times the weight of the baicalin standard substance to obtain baicalin methanol solution; FGF (fibroblast growth factor) 2 The target protein is dissolved in 10 mM PBS buffer (pH6.8) to obtain FGF 2 PBS solution; according to FGF 2 PBS solution baicalin methanol solution (v: v) =1:1 preparationMixing to form FGF 2 -baicalin mixed liquor; incubating at 37 deg.C for 30min to allow baicalin and FGF in the baicalin methanol solution 2 Bind to form FGF 2 -a baicalin complex; vacuum freeze drying the incubated mixed solution to obtain FGF 2 -a baicalin complex;
2、FGF 2 -oroxylin A-7-0-beta-D-glucuronide complex
Preparation method is the same as FGF 2 -baicalin complexes are the same;
3、FGF 2 -wogonoside complex
Preparation method is the same as FGF 2 -baicalin complexes are the same.
Example 6 FGF 2 Preparation of-scutellaria active ingredient complex
1、FGF 2 -baicalin complexes
Dissolving the baicalin standard substance with 10 times of 100% dimethyl sulfoxide solution to obtain baicalin dimethyl sulfoxide solution; FGF (fibroblast growth factor) 2 The target protein is dissolved in 10 mM PBS buffer (pH6.8) to obtain FGF 2 PBS solution; according to FGF 2 Ammonium acetate solution and baicalin dimethyl sulfoxide solution (v: v) =1:1 ratio, mixing to form FGF 2 -baicalin mixed liquor; incubating at 37 deg.C for 30min to allow baicalin and FGF in baicalin dimethyl sulfoxide solution 2 Bind to form FGF 2 -a baicalin complex; freeze-drying the incubated mixed solution in vacuum to obtain FGF 2 -baicalin complexes.
2、FGF 2 -oroxylin A-7-0-beta-D-glucuronide complex
Preparation method is the same as FGF 2 -baicalin complexes are the same;
3、FGF 2 -wogonoside complex
Preparation method is the same as FGF 2 -baicalin complexes are the same.
EXAMPLE 7 cell proliferation-promoting assay with three Components in combination with FGF2
1. Screening of dosing concentration
Three active substances, Baicalin (BC), oroxylin A-7-0-beta-D-glucuronide (OXS), Wogonin (WGS), were determined according to qualitative and quantitative analysis in the course of examples 2-4, while baicalin: oroxylin A: screening the administration concentration of the wogonoside at a ratio of 3:1: 1; screening the administration concentration of FGF 2;
the method comprises the following specific steps:
1) NIH3T3 cells were adjusted to a concentration of 2 x 10 with DMEM containing 10% FBS 4 One per mL, inoculated in a 96-well plate, each well of 100 ul;
2) at a concentration of 5% CO 2 After 24h of incubation in a 37 ℃ incubator, the compound group was supplemented with serum-free DMEM diluted compounds: the concentrations of the compounds in figure 7 are as follows: compound monomer was administered in a gradient at the following concentrations: 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M; FIG. 8 shows that the FGF2 group was administered at a concentration of 200 ng/mL, 100 ng/mL, 50ng/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) setting a cell-free blank control group (only adding serum-free DMEM), a cell-free drug control group (various concentrations of drugs diluted by the serum-free DMEM), and a cell control group (only adding the serum-free DMEM), wherein 5 multiple wells are respectively arranged; the experiment was repeated 3 times;
4) after culturing for 48h, adding MTT (methyl thiazolyl tetrazolium) with 20ul of CO2 in each hole, culturing for 48h in a constant-temperature incubator at 37 ℃, observing whether purple filamentous or circular crystals are formed under a mirror, adding 100ul of DMSO, slightly shaking for 10min, and detecting the OD (optical density) value of each hole by using an enzyme-labeling instrument at the wavelength of 562 nm;
the results are shown in FIGS. 7 and 8; as shown in figure 7, when 3 active ingredients of the three components of the scutellaria baicalensis are independently administered, the compound has no effect of promoting proliferation of cells, and the administration concentration of the compound is determined to be 50-1.5625 mu M; as can be seen from FIG. 8, FGF2, when administered alone, was 2 The administration concentration of (3) is 50ng/mL, and the cell proliferation promoting effect is the best.
2. Effect of Combined proliferation
Based on the results of the above-mentioned administration concentration screening, three compounds were co-administered with FGF2 in a ratio of 3:1:1 of the compound screened in example 4; the three compounds are administered in a gradient of 30-1.13 mu M, and FGF2 is administered at a fixed concentration of 50 ng/mL; the method comprises the following specific steps:
1) NIH3T3 cells were adjusted to a concentration of 2 x 10 with DMEM containing 10% FBS 4 One per mL, inoculated in a 96-well plate, each well of 100 ul;
2) at a concentration of 5% CO 2 After incubation in a 37 ℃ incubator for 24h, the compound group was supplemented with serum-free DMEM diluted compounds: the compound concentrations in fig. 9 and 10 are as follows:
n: blank control group: serum-free DMEM alone was added;
BC: baicalin 30.00 mu M, baicalin 15.00 mu M, baicalin 7.50 mu M and baicalin 3.75 mu M are sequentially arranged from left to right;
WGS: 9.56 μ M, 4.78 μ M, 2.39 μ M and 1.295 μ M of wogonoside from left to right;
and (4) OXS: oroxylin A-7-0-beta-D-glucuronide is 9.04 mu M, 4.52 mu M, 2.26 mu M and 1.13 mu M in sequence from left to right;
FGF 2: adding serum-free DMEM for dilution so that the concentration of FGF2 is 50 ng/mL;
FGF2+ BC: (1) baicalin =30.00 μ M, 50ng/mL FGF2, (2) baicalin =15.00 μ M, 50ng/mL FGF2, (3) baicalin =7.50 μ M, 50ng/mL FGF2, (4) baicalin =3.75 μ M, 50ng/mL FGF 2;
FGF2+ WGS: from left to right, (1) 50ng/mL FGF2, wogonoside =9.56 μ M, (2) 50ng/mL FGF2, wogonoside =4.78 μ M, (3) 50ng/mL FGF2, wogonoside =2.39 μ M, (4) 50ng/mL FGF2, wogonoside =1.295 μ M;
FGF2+ OXS: from left to right in sequence, (1) 50ng/mL FGF2, oroxylin a-7-0- β -D-glucuronide =9.04 μ M, (2) 50ng/mL FGF2, oroxylin a-7-0- β -D-glucuronide =4.52 μ M, (3) 50ng/mL FGF2, oroxylin a-7-0- β -D-glucuronide =2.26 μ M, (4) 50ng/mL FGF2, oroxylin a-7-0- β -D-glucuronide =1.13 μ M;
BC + WGS + OXS: (1) baicalin =30.00 μ M, wogonoside =9.56 μ M, oroxylin a-7-0- β -D-glucuronide =9.04 μ M, (2) baicalin =15.00 μ M, wogonoside =4.78 μ M, oroxylin a-7-0- β -D-glucuronide =4.52 μ M, (3) baicalin =7.50 μ M, wogonoside =2.39 μ M, oroxylin a-7-0- β -D-glucuronide =2.26 μ M, (4) baicalin =3.75 μ M, wogonoside =1.295 μ M, oroxylin a-7-0- β -D-glucuronide =1.13 μ M;
FGF2+ BC + WGS + OXS: from left to right, (1) 50ng/mL FGF2, baicalin = 30.00. mu.M, wogonoside = 9.56. mu.M, oroxylin A-7-0-beta-D-glucuronide = 9.04. mu.M, (2) FGF2 at 50ng/mL, baicalin =15.00 μ M, wogonoside =4.78 μ M, oroxylin a-7-0- β -D-glucuronide =4.52 μ M, (3) FGF2 at 50ng/mL, baicalin =7.50 μ M, wogonoside =2.39 μ M, oroxylin a-7-0- β -D-glucuronide =2.26 μ M, (4) FGF2, baicalin =3.75 μ M, wogonoside =1.295 μ M, oroxylin a-7-0- β -D-glucuronide =1.13 μ M at 50 ng/mL;
3) setting a cell-free blank control group (only adding serum-free DMEM), a cell-free drug control group (various concentrations of drugs diluted by the serum-free DMEM), and a cell control group (only adding the serum-free DMEM), wherein 5 multiple wells are respectively arranged; the experiment was repeated 3 times;
4) after 48h incubation, MTT was added at 20 ul/well, 5% CO 2 Culturing in a constant-temperature incubator at 37 ℃ for 48 hours, observing whether purple filamentous or circular crystals are formed under a mirror, adding 100ul DMSO, slightly shaking for 10min, and detecting the OD value of each hole by using an enzyme-labeling instrument at a wavelength of 562 nm;
as shown in fig. 9 and 10, the three compounds of scutellaria have little effect on promoting proliferation of cells when administered alone, but 3 active ingredients have effect on promoting proliferation of cells when administered in combination; three compounds being each independently of FGF 2 Binding of three compounds with FGF 2 In combination, the effect on cell proliferation upon administration is greater than that of FGF 2 The single administration effect is better and obvious, and the effect of the single administration for 24 hours and 48 hours on cell proliferation is obvious compared with 48 hours; and (4) conclusion: administration after the binding ratio, forThe cells have proliferation promoting effect.
Claims (6)
1. The scutellaria baicalensis extract-FGF 2 compound is a compound obtained by incubating scutellaria baicalensis extract and FGF2, wherein the scutellaria baicalensis extract is baicalin, oroxylin A-7-0-beta-D-glucuronide and/or wogonoside; the hatching is as follows:
1) dissolving Scutellariae radix extract in methanol to obtain methanol solution;
2) dissolving FGF2 target protein in a PBS buffer solution to obtain FGF2 PBS solution;
3) mixing FGF2 PBS solution and Scutellariae radix extract methanol solution to obtain mixed solution;
4) incubating at 36-38 ℃ for 25-35 min, and performing vacuum freeze drying to obtain a scutellaria baicalensis extract-FGF 2 compound;
the mixed solution in the step 3):
FGF 2-baicalin mixture a: the concentration of the baicalin is 7.5-15 mu M;
or FGF 2-wogonoside mixture B: the concentration of the wogonoside is 2.39-4.78 mu M;
or FGF 2-oroxylin a-7-0- β -D-glucuronide complex C: oroxylin A-7-0-beta-D-glucuronide 2.26-4.52 mu M;
or FGF 2-Scutellaria baicalensis mixed solution D: baicalin concentration is 3.75-30.00 mu M, wogonoside concentration is 1.295-9.56 mu M, and oroxylin A-7-0-beta-D-glucuronide concentration is 1.13-9.04 mu M;
the concentration of FGF2 is 50 ng/mL.
2. The scutellaria baicalensis extract-FGF 2 complex of claim 1, wherein: the compound A: baicalin concentration 7.5 μ M, complex B: wogonoside concentration 2.39 μ M, complex C: oroxylin A-7-0-beta-D-glucuronide 2.26 mu M or complex D: baicalin concentration is 3.75-7.5 mu M, wogonoside concentration is 1.295-2.39 mu M, and oroxylin A-7-0-beta-D-glucuronide concentration is 1.13-2.26 mu M.
3. The scutellaria baicalensis extract-FGF 2 complex of claim 2, wherein: the compound D: baicalin concentration of 7.5. mu.M, wogonoside concentration of 2.39. mu.M and oroxylin A-7-0-beta-D-glucuronide concentration of 2.26. mu.M.
4. The Scutellaria baicalensis extract-FGF 2 complex of claim 3, wherein:
the concentration of the PBS buffer solution in the step 2) is 10 mM, and the pH value is 6.8;
mixing the FGF2 PBS solution and the scutellaria baicalensis extract methanol solution in the step 3) according to the volume ratio of 1: 1.
5. The Scutellaria baicalensis extract-FGF 2 complex of claim 4, wherein: step 4) incubation at 37 ℃ for 30 min.
6. The application of scutellaria baicalensis extract-FGF 2 compound in promoting cell proliferation; the cell is an NIH3T3 cell cultured in vitro;
adding the complex of claim 1 to serum-free DMEM and returning to the volume of the mixture in step 3).
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| 黄芩苷抑制肝癌HepG-2细胞增殖的实验研究;张学武等;《时珍国医国药》;20081231;第19卷(第9期);全文 * |
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