Use of three components of baical skullcap root for synergistically enhancing cell proliferation promoted by FGF1

Abstract

The invention discloses a baical skullcap root extract-FGF1 compound. The baical skullcap root extract-FGF1 compound is a compound obtained by co-incubating a baical skullcap root extract and FGF1, wherein the baical skullcap root extract is baicalin, oroxylin A-7-O-beta-D-glucuronide or wogonoside, and the obtained compound is an FGF1-baicalin compound, an FGF1-oroxylin A-7-O-beta-D-glucuronide compound or an FGF1-wogonoside compound. The incubating comprises the following steps: 1) dissolving the baical skullcap root extract with methanol, performing blow-drying with nitrogen, and performingre-dissolving with water; and dissolving FGF1 target protein in a PBS buffer solution, performing mixing with the baical skullcap root extract aqueous solution, performing incubating at 36-38 DEG C for 25-35 min, and performing vacuum freeze-drying. The invention further discloses an application of the FGF1-baicalin compound, the FGF1-oroxylin A-7-O-beta-D-glucuronide compound or the FGF1-wogonoside compound in the aspect of promoting cell proliferation.

Description

technical field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to the use of three components of Scutellaria baicalensis synergistically enhancing FGF1 to promote the proliferation of NIH3T3 cells. Background technique

[0002] The screening of bioactive components is an important content in the field of traditional Chinese medicine research. Traditional chemical separation, structure identification, activity screening mode, and activity-oriented chemical separation have many shortcomings such as unclear goals, cumbersome operations, heavy workload, long cycle, and easy loss of active components during the separation process. Modern pharmacological studies have shown that the affinity between drugs and biomacromolecules (such as enzymes, receptors, DNA, RNA, etc.) is the first step in their action. We developed a method for rapid screening of bioactive components in traditional Chinese medicine: a multi-chamber electrophor...

Examples

Embodiment 1

[0028] Example 1 Preparation of Radix Scutellariae Extract

[0029] Purchase dried scutellaria baicalensis slices at the pharmacy, remove impurities, grind the dried scutellaria baicalensis into a uniform powder, weigh 2 g of the powder and add 20 mL of methanol for ultrasonic extraction, 20 min each time, extract 3 times, combine the extracts, and pass the suspension to 0.22 μm filter membrane, blow nitrogen to concentrate to dryness, redissolve in ultrapure water, pass through a 0.22 μm filter membrane and store at 4 °C until use.

Embodiment 2

[0030] Example 2 Screening of active ingredients that bind to FGF1 in Scutellaria baicalensis extract

[0031] 1. Preparation of FGF1-Scutellaria baicalensis active ingredient complex

[0032] Dissolve 1 mg of FGF1 lyophilized powder in 1 ml of ultrapure water to obtain 1 mg / ml of FGF1 aqueous solution, dilute 1 mg / ml of FGF1 with 10 mM pH4 ammonium acetate buffer to 0.1 mg / ml, take 95 ul of FGF1 and 20 mg / ml 5 ul of Scutellaria baicalensis extract was mixed to form 100 ul of FGF1-Scutellaria baicalensis active ingredient mixture; incubate at 37 degrees Celsius for 30 min to make the active ingredients in Scutellaria baicalensis extract combine with FGF1 to form a FGF1-Scutellaria baicalensis active ingredient complex.

[0033] 2. Multi-chamber electrophoresis separation

[0034] After hatching, the FGF1-scutellaria baicalensis active ingredient mixed solution is used as the sample solution, and the scutellaria baicalensis extract is used as the control solution, and is divid...

Embodiment 3

[0037] Example 3 Qualitative analysis of active components of Scutellaria baicalensis combined with FGF1

[0038] 1. The solution in the receiving chamber after the dissociation of embodiment 2 is subjected to high performance liquid phase tandem mass spectrometry analysis;

[0039] Chromatographic conditions:

[0040] Chromatographic column: Tnature C18 reverse chromatographic column (4.6 mm×150mm, 5μm)

[0041] Mobile phase: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution;

[0042] Flow rate: 0.5mL / min;

[0043] Injection volume: 5 μL;

[0044] The gradient elution program is as follows: 0-10 min, 25%-40%B; 10-20 min, 40%-65%B; 20-30 min, 65%-80%B;

[0045] Mass Spectrometry Conditions:

[0046] Mass Spectrometry Conditions Electrospray ionization source, negative ion mode, full scan mode; spray gas pressure: 30 psi; drying gas (N2) flow rate: 13.0 L / min, drying gas temperature: 300°C; capillary voltage: 2500...

technical field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to the use of three components of Scutellaria baicalensis synergistically enhancing FGF1 to promote the proliferation of NIH3T3 cells. Background technique

[0002] The screening of bioactive components is an important content in the field of traditional Chinese medicine research. Traditional chemical separation, structure identification, activity screening mode, and activity-oriented chemical separation have many shortcomings such as unclear goals, cumbersome operations, heavy workload, long cycle, and easy loss of active components during the separation process. Modern pharmacological studies have shown that the affinity between drugs and biomacromolecules (such as enzymes, receptors, DNA, RNA, etc.) is the first step in their action. We developed a method for rapid screening of bioactive components in traditional Chinese medicine: a multi-chamber electrophor...

Examples

Embodiment 1

[0028] Example 1 Preparation of Radix Scutellariae Extract

[0029] Purchase dried scutellaria baicalensis slices at the pharmacy, remove impurities, grind the dried scutellaria baicalensis into a uniform powder, weigh 2 g of the powder and add 20 mL of methanol for ultrasonic extraction, 20 min each time, extract 3 times, combine the extracts, and pass the suspension to 0.22 μm filter membrane, blow nitrogen to concentrate to dryness, redissolve in ultrapure water, pass through a 0.22 μm filter membrane and store at 4 °C until use.

Embodiment 2

[0030] Example 2 Screening of active ingredients that bind to FGF1 in Scutellaria baicalensis extract

[0031] 1. Preparation of FGF1-Scutellaria baicalensis active ingredient complex

[0032] Dissolve 1 mg of FGF1 lyophilized powder in 1 ml of ultrapure water to obtain 1 mg / ml of FGF1 aqueous solution, dilute 1 mg / ml of FGF1 with 10 mM pH4 ammonium acetate buffer to 0.1 mg / ml, take 95 ul of FGF1 and 20 mg / ml 5 ul of Scutellaria baicalensis extract was mixed to form 100 ul of FGF1-Scutellaria baicalensis active ingredient mixture; incubate at 37 degrees Celsius for 30 min to make the active ingredients in Scutellaria baicalensis extract combine with FGF1 to form a FGF1-Scutellaria baicalensis active ingredient complex.

[0033] 2. Multi-chamber electrophoresis separation

[0034] After hatching, the FGF1-scutellaria baicalensis active ingredient mixed solution is used as the sample solution, and the scutellaria baicalensis extract is used as the control solution, and is divid...

Embodiment 3

[0037] Example 3 Qualitative analysis of active components of Scutellaria baicalensis combined with FGF1

[0038] 1. The solution in the receiving chamber after the dissociation of embodiment 2 is subjected to high performance liquid phase tandem mass spectrometry analysis;

[0039] Chromatographic conditions:

[0040] Chromatographic column: Tnature C18 reverse chromatographic column (4.6 mm×150mm, 5μm)

[0041] Mobile phase: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution;

[0042] Flow rate: 0.5mL / min;

[0043] Injection volume: 5 μL;

[0044] The gradient elution program is as follows: 0-10 min, 25%-40%B; 10-20 min, 40%-65%B; 20-30 min, 65%-80%B;

[0045] Mass Spectrometry Conditions:

[0046] Mass Spectrometry Conditions Electrospray ionization source, negative ion mode, full scan mode; spray gas pressure: 30 psi; drying gas (N2) flow rate: 13.0 L / min, drying gas temperature: 300°C; capillary voltage: 2500...

Images