阅读说明：本技术 一种从山橿中提取银松素的提取方法及其应用 (Extraction method for extracting pinosylvin from lindera reflexa hemsl and application of extraction method ) 是由 陈随清 段懿哲 付宇航 孙孝亚 于 2021-07-29 设计创作，主要内容包括：本发明涉及从山橿中提取银松素的提取方法及其应用,可有效解决幽门螺杆菌的治疗用药问题,其解决的技术方案是,提取方法包括以下步骤：1)提取山橿提取物溶液；2)提取山橿总黄酮部位；3)提取银松素；本发明为探索山橿中发挥抑制幽门螺杆菌的有效成分,分离提取得到山橿中主要的化合物,并进行抑菌实验,得到对幽门螺杆菌有较好抑制作用的活性成分银松素,是从山橿中提取银松素的提取方法及其应用上的创新。(The invention relates to an extraction method for extracting pinosylvin from lindera reflexa hemsl and application thereof, which can effectively solve the problem of treatment medication of helicobacter pylori and adopts the technical scheme that the extraction method comprises the following steps: 1) extracting a lindera reflexa hemsl extract solution; 2) extracting total flavone part of lindera reflexa Hemsl; 3) extracting pinosylvin; the invention provides an extraction method for extracting pinosylvin from lindera reflexa Hemsl and innovation on application thereof, which aims to explore effective components in lindera reflexa Hemsl for inhibiting helicobacter pylori, separate and extract main compounds in lindera reflexa Hemsl, and perform an antibacterial experiment to obtain the active component pinosylvin with a good inhibition effect on helicobacter pylori.)

1. An extraction method for extracting pinosylvin from lindera reflexa Hemsl is characterized by comprising the following steps:

1) lindera reflexa hemsl extract solution

Taking crushed lindera reflexa Hemsl medicinal materials, performing ultrasonic extraction on the crushed lindera reflexa Hemsl medicinal materials respectively by using ethanol with the volume concentration of 70% which is 12 times that of the lindera reflexa Hemsl for 3 times, 1h each time, combining extracting solutions obtained in three times, concentrating under reduced pressure to obtain concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into lindera reflexa Hemsl extract solution which contains 0.05mg of crude drugs per 1mL for standby;

2) part for extracting total flavonoids of lindera reflexa hemsl

Soaking macroporous adsorption resin with ethanol with volume concentration of 95% overnight, performing gradient elution with distilled water until no alcohol smell exists, performing wet column packing, packing lindera reflexa hemsl extract solution, eluting with distilled water to remove impurities, eluting with macroporous adsorption resin with volume of 4 times and ethanol with concentration of 70%, collecting eluent, concentrating under reduced pressure and drying to obtain lindera reflexa hemsl total flavone part;

3) extracting pinosylvin

Taking 0.5g of dried lindera reflexa total flavonoids, precisely weighing, placing in a conical flask with a plug, precisely adding 100ml of methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, taking the subsequent filtrate, and packing the subsequent filtrate in a column.

2. The method of claim 1, comprising the steps of:

1) lindera reflexa hemsl extract solution

Taking 20g of crushed lindera reflexa hemsl medicinal materials, performing ultrasonic extraction on the crushed lindera reflexa hemsl medicinal materials respectively by using ethanol with the volume concentration of 70% which is 12 times that of the lindera reflexa hemsl for 3 times, 1h each time, combining extracting solutions obtained in three times, concentrating under reduced pressure to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution which contains 0.05mg (0.05 mg/mL) of crude drugs per 1mL for later use;

2) part for extracting total flavonoids of lindera reflexa hemsl

Soaking macroporous adsorption resin with ethanol with volume concentration of 95% overnight, performing gradient elution with distilled water until no alcohol smell exists, performing wet column packing, packing lindera reflexa hemsl extract solution, eluting with distilled water to remove impurities, eluting with ethanol with volume concentration of 70%, collecting eluate, concentrating under reduced pressure, and drying to obtain lindera reflexa hemsl total flavone part;

3) extracting pinosylvin

Taking 0.5g of dried lindera reflexa total flavonoids, precisely weighing, placing in a conical flask with a plug, precisely adding 100ml of methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, taking the subsequent filtrate, and packing the subsequent filtrate in a column.

3. The method for extracting pinosylvin from lindera reflexa Hemsl according to claim 1 or 2, wherein the column chromatography conditions are as follows: YMC-Pack ODS-A column: 250 multiplied by 20mml.D.S-5 μm,12nm, gradient elution of mobile phase methanol (A) -water (B), 0-35 min, 80% -85% A; 35-45 min, 85% -100% A; 45-55 min, 100% A; 55-60 min, 100-80% A; 60-65 min, 80% A, detection wavelength 297nm, flow rate 7mL/min, sample injection amount 500 μ L, collecting chromatographic peak with retention time of 11.535min, concentrating under reduced pressure, and drying to obtain pinosylvin.

4. Use of pinosylvin extracted by the extraction method according to any one of claims 1-2 in the preparation of a medicament for the treatment of helicobacter pylori.

Technical Field

The invention relates to the field of biomedicine, in particular to an extraction method for extracting pinosylvin from lindera reflexa hemsl and application thereof.

Background

Lindera reflexa Hemsl (Lindera reflexa Hemsl.), Lindera reflexa Hemsl, Lauraceae (Lauraceae) Lindera plant, deciduous shrub or small arbor, which is often grown under mountain slope forests or in shrubs, is distributed in regions of Dabie mountain in Henan, Hubei, Hunan, Anhui, Jiangxi, Guizhou, Zhejiang and the like. Lindera reflexa has a long history of use among folks in Henan, is commonly used for treating stomach diseases such as chronic gastritis and gastric ulcer, and is recorded in the Chinese medicinal material standard of Henan province (1993 edition). Currently, researches on lindera reflexa Hemsl mainly comprise separation and extraction of chemical components, screening of effective components, pharmacological activity researches and the like, wherein the lindera reflexa Hemsl total flavonoids are found to be main active sites of the lindera reflexa Hemsl and have the effects of analgesia, anti-inflammation, antioxidation and the like.

Helicobacter pylori is a gram-negative helicoidal bacterium, which was first discovered in the human stomach by the Australian university Barry Marshall and Robin Warren in 1982, and since then, it was studied for several decades, which is the only microorganism species that humans now find to survive in the stomach, world health organization in 1994 classified helicobacter pylori as a class I carcinogen, and about 50% of the world were infected with helicobacter pylori, because of the abuse of antibiotics, single antibiotic drugs such as clarithromycin, metronidazole and the like cannot eradicate helicobacter pylori in the stomach, meta-analysis results show that the standard triple scheme (proton pump inhibitor plus two antibiotics) in China has the helicobacter pylori eradication rate lower than 80%, and the quadruple therapy (proton pump inhibitor plus two antibiotics and bismuth agent) is commonly used for treating the helicobacter pylori. With the increasing year by year of the drug resistance of helicobacter pylori to antibiotics, the search for new safe and effective anti-helicobacter pylori drugs is of great significance.

Experiments show that lindera reflexa has a certain inhibition effect on helicobacter pylori, but the lindera reflexa is only limited to coarsely divided parts such as petroleum ether parts, water parts, alkaloid parts, n-butanol parts and the like, and effective active ingredients are not screened, so that the ingredients exerting the inhibition effect are not clear at once, and the researches are to be researched to find out the key ingredients exerting the curative effect of lindera reflexa and how to exert the medicinal value of lindera reflexa to the maximum, and meanwhile, a new treatment scheme is provided for treating helicobacter pylori, which is a main problem concerned by technicians in the field.

Disclosure of Invention

In view of the above situation, in order to solve the defects of the prior art, the present invention aims to provide an extraction method for extracting pinosylvin from lindera reflexa hemsl and an application thereof, which can effectively solve the problem of drug administration for the treatment of helicobacter pylori.

The technical scheme of the invention is that the constitutional formula of the pinosylvin (trans-3, 5-dihydroxy stilbene) is as follows:

the extraction method comprises the following steps:

1) lindera reflexa hemsl extract solution

Taking crushed lindera reflexa Hemsl medicinal materials, performing ultrasonic extraction on the crushed lindera reflexa Hemsl medicinal materials respectively by using ethanol with the volume concentration of 70% which is 12 times that of lindera reflexa Hemsl for 3 times, each time for 1h, combining extracting solutions obtained in three times, performing reduced pressure concentration to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa Hemsl extract solution which contains 0.05mg (0.05 mg/mL) of crude drugs per 1mL for standby;

2) part for extracting total flavonoids of lindera reflexa hemsl

Soaking macroporous adsorption resin with ethanol with volume concentration of 95% overnight, performing gradient elution with distilled water until no alcohol smell exists, performing wet column packing, packing lindera reflexa hemsl extract solution, eluting with distilled water to remove impurities, eluting with macroporous adsorption resin with volume of 4 times and ethanol with concentration of 70%, collecting eluent, concentrating under reduced pressure and drying to obtain lindera reflexa hemsl total flavone part;

3) extracting pinosylvin

Taking 0.5g of dried lindera reflexa total flavonoids, precisely weighing, placing in a conical flask with a plug, precisely adding 100ml of methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, taking the subsequent filtrate, and packing the subsequent filtrate in a column.

The column chromatography conditions are as follows: YMC-Pack ODS-A chromatographic column (250 x 20mml. D.S. -5 μm,12nm), gradient elution with mobile phase methanol (A) -water (B) (0-35 min, 80-85% A; 35-45 min, 85-100% A; 45-55 min, 100% A; 55-60 min, 100-80% A; 60-65 min, 80% A), detection wavelength of 297nm, flow rate of 7mL/min, sample introduction amount of 500 μ L, collection of chromatographic peak with retention time of 11.535min, reduced pressure concentration and drying to obtain pinosylvin.

The invention provides an extraction method for extracting pinosylvin from lindera reflexa Hemsl and innovation on application thereof, which aims to explore effective components in lindera reflexa Hemsl for inhibiting helicobacter pylori, separate and extract main compounds in lindera reflexa Hemsl, and perform an antibacterial experiment to obtain the active component pinosylvin with a good inhibition effect on helicobacter pylori.

Drawings

Fig. 1 is a liquid chromatogram of the total flavonoids lindera reflexa extract part of the invention.

FIG. 2 is a 1H-NMR (inMeOD 500M) chart of pinosylvin according to the present invention.

FIG. 3 is a 1C-NMR (inMeOD 500M) chart of pinosylvin according to the present invention.

FIG. 4 is a graph of urease activity versus absorbance standard according to the present invention.

Detailed Description

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Example 1

In the specific implementation of the invention, the extraction method comprises the following steps:

1) lindera reflexa hemsl extract solution

Taking 20g of crushed lindera reflexa hemsl medicinal materials, performing ultrasonic extraction on the crushed lindera reflexa hemsl medicinal materials respectively by using ethanol with the volume concentration of 70% which is 12 times that of the lindera reflexa hemsl for 3 times, 1h each time, combining extracting solutions obtained in three times, concentrating under reduced pressure to obtain a concentrated solution, and adding water into the concentrated solution to dilute the concentrated solution into a lindera reflexa hemsl extract solution which contains 0.05mg (0.05 mg/mL) of crude drugs per 1mL for later use;

2) part for extracting total flavonoids of lindera reflexa hemsl

Soaking macroporous adsorption resin with ethanol with volume concentration of 95% overnight, performing gradient elution with distilled water until no alcohol smell exists, performing wet column packing, packing lindera reflexa hemsl extract solution, eluting with distilled water to remove impurities, eluting with macroporous adsorption resin with volume of 4 times and ethanol with concentration of 70%, collecting eluent, concentrating under reduced pressure and drying to obtain lindera reflexa hemsl total flavone part;

3) extracting pinosylvin

Taking 0.5g of dried lindera reflexa total flavonoids, precisely weighing, placing in a conical flask with a plug, precisely adding 100ml of methanol, weighing, ultrasonically treating for 10min, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, taking the subsequent filtrate, and packing the subsequent filtrate in a column.

The compound pinosylvin in lindera reflexa hemsl is obtained by separation and extraction, and has good inhibition effect on helicobacter pylori proved by an antibacterial experiment, and relevant test data are as follows:

1. bacteriostasis experiment of pinosylvin:

1.1. experimental materials:

helicobacter pylori (ATCC 43504) was purchased from the Guangdong province collection of microorganisms. Defibered sheep blood and fetal calf serum are purchased from Solarbio, columbia agar powder and brain heart extract powder are purchased from OXOID, urease is purchased from Shanghai-derived leaf Biotech limited, and other chemical reagents are analytically pure.

1.2 Experimental content:

experiment for inhibiting helicobacter pylori by using 1.2.1 lindera reflexa Hemsl

Weighing appropriate amount of Columbia agar powder and brain heart infusion powder, dissolving in double distilled water, autoclaving at 121 deg.C for 15min, cooling to about 45 deg.C, rapidly adding appropriate amount of sterile defibered sheep blood, mixing, pouring into sterile culture dish, and packaging.

The bacteria concentration was adjusted to 0.5 using a turbiditube (bacteria concentration about 1.5X 10)⁸mL), sucking a proper amount of the bacterial liquid on the surface of a culture dish, and uniformly coating. Respectively dripping lindera reflexa Hemsl extract dissolved with dimethyl sulfoxide (DMSO), pinosylvin and clarithromycin liquid medicine on blank sterile drug sensitive paper (diameter 6mm) to obtain medicated paper sheet, sticking the medicated paper sheet on the surface of culture medium coated with uniform bacterial liquid, and culturing at 37 deg.C with 5% O₂，15％CO₂，80％N₂Culturing for 48-72 h in an anaerobic workstation. And measuring the diameter of the inhibition zone.

1.2.2 Effect of lindera reflexa Hemsl on helicobacter pylori urease activity:

preparing a Bertholot color developing solution: solution A: 0.2892g of sodium nitroferricyanide and 11.2073g of sodium salicylate are precisely weighed and dissolved in 100mL of PBS-EDTA buffer solution, and the solution is stored at 4 ℃ in the dark for later use. And B, liquid B: 18.0003g of sodium hydroxide is precisely weighed and dissolved in 90mL of PBS-EDTA buffer solution, 24mL of sodium hypochlorite is added and mixed evenly, and the mixture is stored at 4 ℃ in the dark for standby.

Establishing a giant bean urease activity standard curve: taking 5 centrifuge tubes, respectively adding 100 mu L of megastigmaa urease solution (0.128, 0.064, 0.032, 0.016 and 0.008U/mL) and 100 mu L of urea solution (50mM) with different concentrations, shaking and mixing uniformly, reacting for 20min at room temperature in a dark place, immediately and sequentially adding 100 mu L of Bertholt color development liquid A liquid and 100 mu L of Bertholt color development liquid B liquid, shaking and mixing uniformly, and standing for 10min at room temperature in a dark place for color development. The mixture was pipetted in an amount of 250. mu.L each, transferred to a 96-well plate, and the absorbance was measured at a wavelength of 635 nm. The absorbance was plotted on the ordinate and the enzyme activity (U/mL) on the abscissa to prepare a standard curve of megastigma urease activity.

Adjusting the turbidity of the bacterial liquid to 3-4 (the bacterial concentration is about 1 multiplied by 10)⁹Per mL), a pipette is used for sucking 0.5mL of bacterial liquid, transferring the bacterial liquid into a 10mL centrifuge tube, adding 4.5mL of liquid culture medium for mixing, then respectively adding 10 MuL of lindera reflexa Hemsl extract liquid medicine, pinosylvin, clarithromycin (positive control) and DMSO (blank control) to obtain bacterial liquid culture medium containing different drugs, culturing for 1h in an anaerobic workstation, transferring the bacterial liquid culture medium into a 10mL centrifuge tube, freezing and centrifuging (4 ℃, 5000g and 15min), washing 3 times with PBS buffer solution repeatedly, adding 2mL of PBS buffer solution, storing in a refrigerator at-20 ℃, taking out after 10h, unfreezing at room temperature, carrying out ultrasonic vibration and ice bath for 3min, freezing and centrifuging (4 ℃, 15000g and 10min), taking supernatant and mixing uniformly with glycerol-PBS buffer solution with the same volume, taking 100 MuL of mixed liquid of each centrifuge tube, adding 100 MuL of urea solution (50mM) for mixing, reacting for 20min at room temperature in a dark place, and sequentially adding 100 mu L of Bertholt color development liquid A and 100 mu L of Bertholt color development liquid B, uniformly mixing, standing at room temperature in a dark place for 10min for color development, respectively absorbing 250 mu L of mixed liquid, transferring the mixed liquid into a 96-well plate, detecting the absorbance of the mixed liquid at the wavelength of 635nm, substituting the absorbance into a standard curve to obtain the activity of the helicobacter pylori urease under the action of the medicament, and comparing a blank control with a positive control.

2.3 results of the experiment

The inhibition effect of different drugs on helicobacter pylori is detected by adopting a drug sensitive paper sheet method, and the diameters of inhibition zones are shown in table 1.

TABLE 1 results of zone diameter inhibition of H.pylori by different drugs: (n＝3)

As can be seen from the results, pinosylvin has a better inhibitory effect on helicobacter pylori.

Drawing a standard curve of urease activity-absorbance (635nm), wherein the absorbance (Y) of the reaction solution is increased along with the increase of the urease activity (X), which shows that the urease activity and the absorbance of the reaction solution have a certain linear relationship, as shown in figure 4. Y is 6.4809X +0.5645, and the correlation coefficient r is 0.9931.

The results of the urease activity and relative inhibition experiments are shown in Table 2.

TABLE 2 urease activity and relative inhibition

The result shows that lindera reflexa total flavone extract and pinosylvin both have certain inhibiting effect on urease activity by taking clarithromycin as a control.

The compound pinostrobin in lindera reflexa obtained by separation and extraction has a good inhibition effect on helicobacter pylori, the medicinal value of lindera reflexa is effectively brought into the maximum, a new treatment scheme is provided for treating the helicobacter pylori, the method is an extraction method for extracting the pinostrobin from lindera reflexa and innovation in application of the extraction method, and the method has good economic and social benefits.