CN108853127B - Method for extracting 3 '-O-benzoyl salicin from populus tomentosa leaves and application of 3' -O-benzoyl salicin - Google Patents

Method for extracting 3 '-O-benzoyl salicin from populus tomentosa leaves and application of 3' -O-benzoyl salicin Download PDF

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CN108853127B
CN108853127B CN201810951367.XA CN201810951367A CN108853127B CN 108853127 B CN108853127 B CN 108853127B CN 201810951367 A CN201810951367 A CN 201810951367A CN 108853127 B CN108853127 B CN 108853127B
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CN108853127A (en
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付雪艳
董琳
郭东燕
黄�俊
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Abstract

The invention discloses application of 3' -O-benzoyl salicin in preventing and treating malignant tumors and inflammations, and belongs to the technical field of medicine application. Experiments prove that the compound 3' -O-benzoyl salicin which can resist inflammation and inhibit the growth of tumor cells exists in Chinese medicinal material Chinese white poplar leaves, and has obvious effects of inhibiting the growth of tumor cells and diminishing inflammation under the concentration of 250-500 mu g/ml. The invention also discloses a method for extracting the 3 ' -O-benzoyl salicin from the hairy poplar leaves, which comprises the steps of pretreatment, extraction, concentration and extraction on the hairy poplar leaves, the 3 ' -O-benzoyl salicin is obtained by extraction and enrichment, a constant-temperature extraction environment is adopted in the process, the back mixing efficiency of a solvent and the hairy poplar leaves is improved, the extraction efficiency is further improved, and the yield of the 3 ' -O-benzoyl salicin is improved.

Description

Method for extracting 3 '-O-benzoyl salicin from populus tomentosa leaves and application of 3' -O-benzoyl salicin
Technical Field
The invention belongs to the technical field of medicine application, and particularly relates to a method for extracting 3 '-O-benzoyl salicin from Chinese white poplar leaves and application of the 3' -O-benzoyl salicin.
Background
The literature states that 3 '-O-benzoyl salicin (chaenomelidin) exists in the trunk, bark and leaves of the willow, but is often ignored or prepared into mixed extracts with other compounds to study the pharmaceutical properties of the salix, because the content of the 3' -O-benzoyl salicin is low. With the further research on the salicin derivatives in modern medicine, the salicin derivatives are synthesized industrially in a large amount, and the pharmaceutical effect of 3' -O-benzoyl salicin is neglected more and more. However, the 3' -O-benzoyl salicin widely exists in poplar such as Chinese white poplar, especially Chinese white poplar leaf, the source of raw materials is wide, and the separation and purification of effective components in Chinese white poplar leaf and the research of medicinal value thereof greatly promote the application of Chinese white poplar leaf in the field of traditional Chinese medicine. Generally, the scholars study the medicinal value of the composition extracted from the leaves of populus tomentosa, which results in the lack of research on the medicinal value of 3 '-O-benzoyl salicin, and cannot clearly show the effect of 3' -O-benzoyl salicin in treating the disease and the cytotoxicity exhibited thereby.
In the prior art, a multifunctional extraction tank is generally used for extracting effective components of the populus tomentosa, a rotary evaporator is generally used for evaporation and concentration, the extraction efficiency is low, the final yield of target compounds is low, and the target compounds with low content originally contained in traditional Chinese medicinal materials cannot be effectively separated out, so that serious waste is caused. In a laboratory, due to low extraction efficiency and low target compound yield, the subsequent experimental research is affected by the shortage of raw materials.
Disclosure of Invention
In view of the above, the present invention provides a method for extracting 3' -O-benzoyl salicin from Populus tomentosa leaves.
The invention also discloses application of the 3' -O-benzoyl salicin in preventing and treating malignant tumors.
The invention also discloses application of the 3' -O-benzoyl salicin in preventing and treating inflammation.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for extracting the 3' -O-benzoyl salicin from the Chinese white poplar leaves comprises the following steps:
pretreatment: cleaning Chinese medicinal material white poplar leaves, crushing, and screening particles with 50-200 meshes;
extraction: putting pretreated white poplar leaves into a multifunctional extraction tank for a laboratory, adding 70% ethanol water solution according to a preset liquid medicine ratio, and extracting for 3 hours at a preset extraction temperature;
concentration: filtering the crude liquid, and concentrating at a predetermined concentration temperature by evaporation until no alcohol smell is produced;
and (3) extraction: extracting the concentrated solution generated by concentration by using petroleum ether at a preset extraction ratio and extraction temperature, concentrating a petroleum ether extract phase to obtain a petroleum ether part, extracting a petroleum ether raffinate phase by using dichloromethane at a preset extraction ratio and extraction temperature, concentrating a dichloromethane extract phase to obtain a dichloromethane part, separating the dichloromethane part by sequentially passing through a normal-pressure chromatographic column, a silica gel column and an ODS column, and removing impurities to obtain 3' -O-benzoyl salicin;
wherein in the step of extraction, the preset liquid-medicine ratio is 1:10, and the preset extraction temperature is 70-90 ℃;
in the step of concentration, the preset concentration temperature is 55-65 ℃;
in the step of extraction, the preset extraction temperature is 20-40 ℃, and the preset extraction ratio is 1: 3-1: 5.
Application of 3' -O-benzoyl salicin extracted from Chinese white poplar leaves in preparing medicine for preventing and treating malignant tumor.
Application of 3' -O-benzoyl salicin extracted from Chinese white poplar leaves in preparing medicaments for preventing and treating inflammation.
According to the technical scheme, the 3 ' -O-benzoyl salicin extracted from the Chinese white poplar leaves is applied to preventing or treating malignant tumors or inflammations, and has the advantages that experiments show that the 3 ' -O-benzoyl salicin obtains unexpected effects in pathological tests of tumor cell growth inhibition and anti-inflammation, the inhibition rate of the 3 ' -O-benzoyl salicin on colon cancer tumor cells reaches over 70 percent, the inhibition rate of the 3 ' -O-benzoyl salicin on stomach cancer tumor cells exceeds 50 percent, and the anti-inflammation activity of the 3 ' -O-benzoyl salicin is particularly obvious under high dose. Meanwhile, the 3' -O-benzoyl salicin is extracted and enriched from the Chinese white poplar leaves, so that the waste Chinese white poplar leaves are reasonably utilized, the raw material source is wide, and the process is simple. In the process of extracting and enriching the 3 ' -O-benzoyl salicin from the Chinese white poplar leaves, a constant-temperature extraction method is adopted, a proper extraction temperature is selected, constant-temperature concentration and extraction are adopted, and the extraction efficiency of the 3 ' -O-benzoyl salicin and the yield of the 3 ' -O-benzoyl salicin are improved to the maximum extent.
Drawings
FIG. 1 is a flow chart of the extraction and preparation process of 3' -O-benzoyl salicin based on white poplar leaf.
FIG. 2 is a flow chart of methylene chloride partial isolation of Populus tomentosa leaves.
FIG. 3 is a bar graph of the anti-inflammatory activity of 3' -O-benzoyl salicin.
FIG. 4 is a bar graph of 3' -O-benzoyl salicin inhibiting the growth of gastric cancer tumor cells.
FIG. 5 is a bar graph of 3' -O-benzoyl salicin inhibition of colon cancer tumor cell growth.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples.
The 3' -O-benzoyl salicin exists in Chinese medicinal material Chinese white poplar leaves, and the structural formula is as follows:
Figure GDA0002635299770000041
the 3' -O-benzoyl salicin is white needle crystal, which is dissolved in methanol.
Referring to fig. 3, the inventors found in experiments that 3 '-O-benzoyl salicin is present in a small amount in aspen leaves or other willow leaves, but plays a positive and effective role in inhibiting tumor cell growth and diminishing inflammation, and the experiments demonstrated that 3' -O-benzoyl salicin has excellent anti-inflammatory activity at a concentration of 62.5 to 500 μ g/ml, excellent activity in inhibiting tumor cell growth at a concentration of 250 to 500 μ g/ml, and particularly has an inhibition rate of 50% or more as a drug for inhibiting the growth of tumor cells of gastric cancer, and an inhibition rate of 70% or more as a drug for inhibiting the growth of tumor cells of intestinal cancer, and is weak in cytotoxicity.
The content of the 3 ' -O-benzoyl salicin in the Chinese white poplar leaves is low, and in the process of extraction and enrichment, the extraction efficiency of the 3 ' -O-benzoyl salicin and the final yield of the 3 ' -O-benzoyl salicin need to be continuously improved from various aspects.
Referring to fig. 1, in a preferred embodiment, a constant temperature extraction atmosphere and a constant temperature extraction separation atmosphere with a constant extraction ratio are adopted, so that the extraction efficiency of extracting 3 '-O-benzoyl salicin from populus tomentosa leaves is effectively improved, and the yield of 3' -O-benzoyl salicin is also effectively improved. For example, the following production process flow is adopted:
pretreatment: cleaning Chinese medicinal material white poplar leaves, crushing, and screening particles with 50-200 meshes;
extraction: putting pretreated white poplar leaves into a multifunctional extraction tank for a laboratory, adding 70% ethanol water solution according to a preset liquid medicine ratio, and extracting for 3 hours at a preset extraction temperature;
concentration: filtering the crude liquid, and concentrating at a predetermined concentration temperature by evaporation until no alcohol smell is produced;
and (3) extraction: extracting the concentrated solution generated by concentration by using petroleum ether at a preset extraction ratio and extraction temperature, concentrating a petroleum ether extract phase to obtain a petroleum ether part, extracting a petroleum ether raffinate phase by using dichloromethane at a preset extraction ratio and extraction temperature, concentrating a dichloromethane extract phase to obtain a dichloromethane part, separating the dichloromethane part by sequentially passing through a normal-pressure chromatographic column, a silica gel column and an ODS column, and removing impurities to obtain 3' -O-benzoyl salicin;
wherein in the step of extraction, the preset liquid-medicine ratio is 1:10, and the preset extraction temperature is 70-90 ℃;
in the step of concentration, the preset concentration temperature is 55-65 ℃;
in the step of extraction, the preset extraction temperature is 20-40 ℃, and the preset extraction ratio is 1: 3-1: 5.
Preferably, in the step "extraction", the predetermined liquid-drug ratio is 1:10, and the predetermined extraction temperature is 75 ℃.
Preferably, in the step "concentration", the predetermined concentration temperature is 55 ℃.
Preferably, in the step of "extraction", the predetermined extraction temperature is 25 ℃ and the predetermined extraction ratio is 1: 3.
The inventor also unexpectedly finds that a certain vacuum degree is beneficial to the separation efficiency of the 3 ' -O-benzoyl salicin from the Chinese white poplar leaves, in the embodiment, the inventor selects the pressure of 65 KPaA-95 KPaA (surface pressure, the same below), namely the vacuum degree of 6 KPaA-36 KPaA to separate and extract the 3 ' -O-benzoyl salicin from the Chinese white poplar leaves, the final yield of the 3 ' -O-benzoyl salicin is obviously improved, and simultaneously, the extraction efficiency is effectively improved.
The following experiments specifically describe the method for extracting 3' -O-benzoyl salicin from Chinese white poplar leaves and the application of Chinese white poplar leaves as a medicine for inhibiting the growth of tumor cells and as an anti-inflammatory medicine.
Extraction and enrichment of 3' -O-benzoyl salicin
1. Taking 11.5kg of the white poplar leaves, cleaning, crushing, screening 50-200-mesh powder particles, adding 3kg of the powder particles into a multifunctional extraction tank for a laboratory, adding 30kg of 70% ethanol aqueous solution (the liquid-drug ratio is 1:10), and extracting at the extraction temperature of 70 ℃ for 3 h/time.
Concentrating all the crude liquid obtained after extraction at 55 deg.C until there is no alcohol smell to obtain concentrated product as total extract of white poplar leaf.
Taking the total extract part, firstly using petroleum ether (analysis purity) to perform standing extraction at the extraction temperature of 25 ℃, wherein the extraction ratio (extraction liquid: extractant, the same below) is 1:3, and after the extraction is finished, concentrating the extract phase at the extraction temperature of 55 ℃ to obtain the petroleum ether part of the populus tomentosa leaves; standing and extracting the raffinate phase with dichloromethane (analytically pure) at the same extraction temperature at an extraction ratio of 1:3, and concentrating the extract phase at the same temperature to obtain dichloromethane part of the white poplar leaf; performing standing extraction on the raffinate phase by using ethyl acetate (analytically pure) at the same temperature, wherein the extraction ratio is 1:4, and after the extraction is finished, concentrating the extract phase at the same temperature to obtain an ethyl acetate part of the populus tomentosa leaves; standing and extracting the raffinate phase with n-butanol (analytically pure) at the same temperature at an extraction ratio of 1:5, and concentrating the extract phase at the same temperature to obtain n-butanol fraction of the populus tomentosa leaves; concentrating the raffinate phase at the same temperature to obtain the water part of the white poplar leaf.
Respectively refrigerating the obtained total extract, petroleum ether part, dichloromethane part, ethyl acetate part, n-butanol part and water part at-4 deg.C.
In the experiment, the extract content of the total extract extracted from the white poplar leaves is 2.496kg, and the extract yield is 21.70%. The extraction rate of dichloromethane was 26.8%, the extraction rate of ethyl acetate was 7.43%, and the extraction rate of n-butanol was 44.2%.
2. In experiment 1, 11.5kg of the white poplar leaves are taken, washed, crushed and screened to obtain 50-200-mesh powder particles, 3kg of the powder particles are added into a multifunctional extraction tank for a laboratory, 30kg of 70% ethanol aqueous solution is added (the ratio of the medicinal solution to the medicinal solution is 1:10), and extraction is carried out at the extraction temperature of 80 ℃ for 3 h/time. Other conditions are the same.
In the experiment, the extract content of the total extract is 2.412Kg, and the extract yield of the total extract is 20.97%. Wherein, the extraction rate of dichloromethane is 26.5 percent, the extraction rate of ethyl acetate is 7.38 percent, and the extraction rate of n-butanol is 18.03 percent.
3. In experiment 2, 11.5kg of the white poplar leaves are taken, washed, crushed and screened to obtain 50-200-mesh powder particles, 3kg of the powder particles are added into a multifunctional extraction tank for a laboratory, 30kg of 70% ethanol aqueous solution is added (the ratio of the medicinal solution to the medicinal solution is 1:10), and extraction is carried out at the extraction temperature of 90 ℃ for 3 h/time. Other conditions are the same.
In the experiment, the extract content of the total extract is 2.375Kg, and the extract yield of the total extract is 20.65%. Wherein, the extraction rate of dichloromethane is 26.7 percent, the extraction rate of ethyl acetate is 7.46 percent, and the extraction rate of n-butanol is 17.75 percent.
In the experiment, the extract can be obtained clearly, the constant-temperature extraction environment improves the extract yield of the total extract, and the yield of the target compound 3' -O-benzoyl salicin is indirectly improved. In the experiment, the constant-temperature extraction environment is beneficial to the back mixing efficiency of the extraction solvent and the poplar leaf material, and the extract yield of the total extract is improved, but in the process of raising the temperature, the extraction solvent is gasified quickly, so that a large amount of foam is generated in the extraction tank, and the experimental effect is influenced. Therefore, the extraction temperature of 70-90 ℃ is selected, and the target compound 3' -O-benzoyl salicin is obtained to the maximum extent under the condition of not influencing the extraction efficiency.
4. In experiment 1, all crude liquid obtained after the completion of extraction was concentrated at a concentration temperature of 65 ℃ until no alcohol smell was observed, and the obtained concentrated product was a total extract of populus tomentosa leaves. Other conditions were unchanged.
In the experiment, the extract content of the total extract is 2.458Kg, and the extract yield of the total extract is 21.37%. Wherein, the extraction rate of dichloromethane is 26.7 percent, the extraction rate of ethyl acetate is 1.58 percent, and the extraction rate of n-butanol is 17.95 percent.
5. In experiment 1, firstly, petroleum ether (analysis purity) is used for standing extraction at the extraction temperature of 40 ℃, the extraction ratio is 1:3, and after extraction is finished, an extract phase is concentrated at the concentration temperature of 55 ℃ to obtain the petroleum ether part of the populus tomentosa leaves; standing and extracting the raffinate phase with dichloromethane (analytically pure) at the same extraction temperature at an extraction ratio of 1:3, and concentrating the extract phase at the same temperature to obtain dichloromethane part of the white poplar leaf; performing standing extraction on the raffinate phase by using ethyl acetate (analytically pure) at the same temperature, wherein the extraction ratio is 1:4, and after the extraction is finished, concentrating the extract phase at the same temperature to obtain an ethyl acetate part of the populus tomentosa leaves; standing and extracting the raffinate phase with n-butanol (analytically pure) at the same temperature at an extraction ratio of 1:5, and concentrating the extract phase at the same temperature to obtain n-butanol fraction of the populus tomentosa leaves; concentrating the raffinate phase at the same temperature to obtain the water part of the white poplar leaf. Other conditions were unchanged.
In the experiment, the extract content of the total extract is 2.490Kg, and the extract yield of the total extract is 21.65%. Wherein, the extraction rate of dichloromethane is 25.2 percent, the extraction rate of ethyl acetate is 1.32 percent, and the extraction rate of n-butanol is 17.02 percent.
From experiments 4 and 5, it is clear that the concentration temperature, the extraction temperature and the extraction ratio all have a certain influence on the yield of the target compound, and the proper concentration temperature is selected to ensure that the target compound is not deteriorated or gasified, thereby being beneficial to further improving the yield of the target compound. The extraction temperature and the extraction ratio are the most important factors influencing the extraction process, the yield is reduced due to the fact that the target compound is dissolved in the raffinate phase in an excessive amount due to the excessively high extraction ratio and the excessively high temperature, the target compound cannot be completely extracted into the extraction phase due to the excessively low extraction ratio and the excessively low extraction temperature, and the extraction efficiency is reduced.
7. In experiment 2, the extraction atmosphere of slight negative pressure was adopted, the pressure was 65KPaA, and the other conditions were unchanged.
In the experiment, the extract content of the total extract is 2.896Kg, and the extract yield of the total extract is 25.18%. Wherein, the extraction rate of dichloromethane is 26.1 percent, the extraction rate of ethyl acetate is 1.35 percent, and the extraction rate of n-butanol is 17.32 percent.
8. In experiment 2, the extraction atmosphere of slight negative pressure is adopted, the pressure is 85KPaA, and other conditions are unchanged.
In the experiment, the extract content of the total extract is 2.760Kg, and the extract yield of the total extract is 24.00%. Wherein, the extraction rate of dichloromethane is 25.8 percent, the extraction rate of ethyl acetate is 1.29 percent, and the extraction rate of n-butanol is 18.01 percent.
9. In experiment 2, the extraction atmosphere of slight negative pressure is adopted, the pressure is 95KPaA, and other conditions are unchanged.
In the experiment, the extract content of the total extract is 2.572Kg, and the extract yield of the total extract is 22.36%. Wherein, the extraction rate of dichloromethane is 26.2 percent, the extraction rate of ethyl acetate is 1.32 percent, and the extraction rate of n-butanol is 17.65 percent.
Experiments 7 to 9 show that the vacuum degree is improved along with the reduction of the pressure, the material exchange rate of the extraction reaction is continuously enhanced, particularly, the boiling point of the extraction solvent is reduced along with the reduction of the pressure, the reaction disturbance is obvious, the efficiency of extracting the target compound from the Chinese white poplar leaves is increased, and the yield of the target compound is also obviously improved. As the pressure is further reduced, a large amount of foam appears in the reaction tank, which greatly affects the extraction efficiency of the reaction.
The above experiment prepared a total extract of populus tomentosa leaves, a petroleum ether fraction, a methylene chloride fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction. By extracting at constant temperature, the extraction efficiency is improved, the extraction solvent is fully contacted with the white poplar leaves, and the back mixing is carried out, so that the extraction yield is improved to a certain extent. And the extraction efficiency of the 3 '-O-benzoyl salicin and the final yield of the 3' -O-benzoyl salicin are improved to the maximum extent by constant-temperature concentration and constant-temperature extraction.
Secondly, separating the dichloromethane part of the white poplar leaf to obtain the target compound 3' -O-benzoyl salicin
Referring to fig. 2, the methylene chloride fraction of populus tomentosa leaves was separated to obtain 6 compounds. The separation process is as follows:
taking a proper amount of the methylene dichloride part extract of the populus tomentosa leaves, adding a proper amount of methanol to dissolve the extract in an ultrasonic cleaning machine, adding 100-200 meshes of silica gel which is equal to the extract after all the extract is dissolved in the methanol, mixing the mixture with a sample, drying the mixture in a drying oven, and uniformly grinding the mixture. Then according to the silica gel: sample 20: 1, and then performing gradient elution by using a dichloromethane-methanol system (50: 1-0: 1). The eluates were then loaded in 250ml Erlenmeyer flasks and, depending on TLC, fractions of similar composition were combined to give E1-E5 in total of 5 fractions.
The E2 fraction is passed through ODS column (elution gradient, 20% -100% methanol) to obtain E2-2, and then is passed through silica gel column chromatography (elution gradient, dichloromethane: methanol system: 25: 1-0: 1) to obtain compound 2 and compound 3.
Through modern nuclear magnetic spectrum identification, the compound 2 is: 3' -O-benzoyl salicin (chaenomelidin).
In the above process, the yield of 3 '-O-benzoyl salicin relative to dichloromethane portion of white poplar leaf was 0.0021%, that is, 2.1mg of 3' -O-benzoyl salicin was isolated per 100g of dichloromethane portion extract.
Anti-inflammatory activity experiment of tri, 3' -O-benzoyl salicin
1. Experimental Material
Material Source
RAW264.7 cells Shanghai cell bank of Chinese academy of sciences
DMEM medium Hyclone Co
Fetal bovine serum Products of Gibco Corp
0.25% Trypsin BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.
Double-antibody of penicillin-streptomycin BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.
Cell Counting Kit-8 Biyuntian Biotech Co Ltd
PBS (phosphate buffer solution) powder Beijing China fir Jinqiao Biotech Co Ltd
DMSO (Dimethyla mock) Beijing Mengyimei Biotech Co., Ltd
2. Main instrument
Thermo371 carbon dioxide constant temperature incubator Shanghai Bajiu industries Ltd
Super clean bench Suzhou jin Jing depuration plant Co Ltd
SX-500 high-pressure sterilizing pot Shanghai Lai Rui scientific instruments GmbH
High-speed centrifugal machine HC3518 Kyoto Innovation, Mijia division of the great Kyoto, K.K.
-20 ℃ low-temperature refrigerator Qingdao Haier Co Ltd
-80 ℃ low-temperature refrigerator SANYO Inc
Liquid transfer device Gilson P type pipettor Co
Cell culture plate and cell culture dish Costa corporation
Microscope OLYMPUS Inc
The head of the RNase-free gun,EP pipe Axygen Scientific Inc
Enzyme-linked immunosorbent assay (ELISA) instrument Finland Lebo microplate reader Co Ltd
Electric heating constant temperature water bath Shanghai Sensin laboratory instruments Ltd
Electronic precision balance Shanghai Aohaus instruments Ltd
3. Experimental methods
Heat source removal treatment of experimental articles:
cleaning plastic experimental articles such as a sample adding gun head, an EP tube, a cell culture bottle and the like, irradiating at 60 ℃ (1x106Gray for 30 minutes), and removing a heat source. Glass experimental articles such as beakers, test tubes, glass culture bottles and the like are cleaned, dried for 2 hours at 300 degrees, and cooled, and then the openings of the bottles are sealed by tin foil paper for later use. Other experimental articles are sterilized by autoclaving according to the conventional experiment.
Preparing a reagent:
cell cryopreservation solution: is a DMEM (dulbecco's modified eagle medium) high-sugar culture solution containing 10% Fetal Bovine Serum (FBS) and 10% DMSO.
LPS (lipopolysaccharide) formulation: precisely weighing 1mgLPS and 1ml DMEM high-glucose culture solution containing 10% fetal calf serum, mixing, and shaking to obtain mother liquor.
Preparing monomers: placing each monomer compound in a 1.5EP tube, adding 1ml DMSO to prepare a mother solution, and shaking and mixing uniformly.
D-Hanks liquid: weighing KCl 0.4g, NaCl 8g and Na2HPO4 0.133g，NaHCO30.35g，KH2PO40.06g of phenol red and 0.02g of phenol red are sequentially dissolved in about 700ml of double distilled water, and the volume is determined to be 1000ml, fully and uniformly mixing, subpackaging into bottles, tightly wrapping, then putting into a pressure cooker, and autoclaving at 122 ℃ for 40 min.
Recovery of RAW264.7 cells:
the vial containing RAW264.7 was taken out from the liquid nitrogen tank, immediately placed in warm water at 37 ℃ and rapidly thawed. Then the cell suspension is transferred into a centrifuge tube which is added with a proper amount of DMEM high-sugar culture solution containing 10% FPS, 100U/ml penicillin and 100 mu g/ml streptomycin in advance, a plug is covered, the DMEM high-sugar culture solution is added after the centrifugation for 5min at 1000rpm after the balancing, the supernatant is removed, the DMEM high-sugar culture solution is added, the cells are resuspended, and then the cells are inoculated into a culture flask.
RAW264.7 cell culture:
RAW264.7 cells are cultured by an adherence method, the culture solution is DMEM high-glucose culture solution containing 10 percent of FPS, 100U/ml of penicillin and 100 mu g/ml of streptomycin, and the culture condition is 5 percent of CO2At the temperature of 37 ℃ and relative saturation humidity, placing the culture bottle under an inverted display mirror to observe the growth condition of the cells, and giving appropriate liquid change according to the growth condition of the cells until the cells are fused to 80-90 percent according to the ratio of 1:4, passage.
RAW264.7 cells frozen:
taking RAW264.7 with good growth state, washing with D-Hanks solution for 1 time, adding D-Hanks solution to soak cells for 2min, discarding the solution, adding complete culture solution, blowing the bottom surface of a cell culture flask with a dropper, centrifuging at 1000rpm for 5min to collect cells, adding 2ml of cell cryopreservation solution, re-suspending the cells, transferring into a cryopreservation tube, screwing down a tube cover, and marking the cell name and the cryopreservation date. And (3) putting the freezing tube into a refrigerator with the temperature of 20 ℃ below zero for 2h, then transferring the tube into a refrigerator with the temperature of 80 ℃ below zero for 24h, and finally transferring the tube into liquid nitrogen for long-term storage.
4. Cell modeling and grouping
The compound 3' -O-benzoyl salicin separated from the white poplar leaves is respectively dissolved in DMSO, and is prepared into the drug concentration of 500 mug/ml by DMEM without FBS according to the experimental requirements: then, according to the double reduction dilution method, 1ml of 500 mu g/ml is diluted to 2ml to obtain 250 mu g/ml of medicine, and the medicine is sequentially diluted to obtain 125 mu g/ml, 62.5 mu g/ml and 31.25 mu g/ml for standby.
Taking mouse Raw264.7 cells, sucking residual culture medium in a culture bottle, washing twice by using PBS buffer solution, adding trypsin-EDTA digestive solution, placing in an incubator for 10 minutes, digesting adherent cells, adding a proper amount of complete culture medium, centrifuging the cells, sucking waste liquid, adding a proper amount of complete culture medium, and uniformly mixing the cells. After counting, the cell concentration was adjusted to 1X 105 cells/mL, and the cells were plated in a 96-well plate at 100. mu.l/well. At 37 ℃ 5% CO2After 24 hours of incubation in the cell incubator, residual medium was aspirated from the 96-well plate and 100. mu.l of 2. mu.g/ml LPS solution (complete medium preparation) was added. At 37 ℃ 5% CO2Culturing for 24 hours in a cell culture box to obtain the Raw264.7 inflammation model.
Taking the LPS to induce Raw264.7 inflammatory cells, adding the medicine to be tested for anti-inflammatory activity, dividing into six groups of 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 31.25 μ g/ml and blank control, setting four multiple wells in each group, and testing at 37 deg.C and 5% CO2After the cells are cultured in the cell culture box for 24 hours, 10 mul of CCK-8 detection reagent is added into each hole, after the cells are cultured in the cell culture box for 2.5 hours, an enzyme-labeling instrument is used for detecting the absorbance OD value of each hole under the wavelength of 450nm, and the inhibition rate and the cell survival rate of the cells are calculated.
In the experiment, the 3 ' -O-benzoyl salicin has good anti-inflammatory activity, particularly under the dosage of 62.5-500 mu g/ml, the anti-inflammatory activity is obvious, the anti-inflammatory activity of the 3 ' -O-benzoyl salicin is in direct proportion to the dosage, the higher the dosage is, the stronger the anti-inflammatory activity is, no obvious cytotoxicity is found under the concentration, and the 3 ' -O-benzoyl salicin has the potential of being used as an anti-inflammatory drug.
Tetra, 3' -O-benzoyl salicin for inhibiting growth of gastric cancer tumor cells
Taking gastric cancer cells of mice, sucking residual culture medium in a culture bottle, washing twice by using PBS buffer solution, adding trypsin-EDTA digestive solution, placing in an incubator for 10 minutes, digesting adherent cells, adding a proper amount of complete culture medium, centrifuging the cells, sucking waste liquid, adding a proper amount of complete culture medium, and uniformly mixing the cells. After counting, the cell concentration was adjusted to 1X 105 cells/mL and the cell concentration was measuredSeeded in 96-well plates at 100. mu.l cells per well. At 37 ℃ 5% CO2Culturing for 24h in a cell culture box, sucking out residual culture medium in a 96-well plate after 24h, adding the 3' -O-benzoyl salicin to be tested into six groups of 500 mu g/ml, 250 mu g/ml, 125 mu g/ml, 62.5 mu g/ml, 31.25 mu g/ml and blank control, setting four multiple wells in each group, and culturing at 37 ℃ and 5% CO2After culturing for 24h in the cell culture box, adding 10 mul of CCK-8 detection reagent into each hole, culturing for 2.5h in the cell culture box, detecting the absorbance OD value of each hole under the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the inhibition rate of the cells.
In the experiment, the 3 ' -O-benzoyl salicin can be clearly obtained to have good effect of inhibiting the growth of the gastric cancer tumor cells, particularly, under the dosage of 250-500 mu g/ml, the activity of inhibiting the growth of the gastric cancer tumor cells is obvious, the anticancer activity of the 3 ' -O-benzoyl salicin is in direct proportion to the dosage, the higher the dosage is, the stronger the anticancer activity is, no obvious cytotoxicity is found under the concentration, and the 3 ' -O-benzoyl salicin has the potential of being used as a medicine for inhibiting the growth of the gastric cancer tumor cells.
Five, 3' -O-benzoyl salicin for inhibiting growth of intestinal cancer tumor cells
Taking mouse intestinal cancer cells, sucking residual culture medium in a culture bottle, cleaning twice by using PBS buffer solution, adding trypsin-EDTA digestive solution, placing in an incubator for 10 minutes, digesting the adherent cells, adding a proper amount of complete culture medium, centrifuging the cells, sucking waste liquid, adding a proper amount of complete culture medium, and uniformly mixing the cells. After counting, the cell concentration was adjusted to 1X 105 cells/mL, and the cells were plated in a 96-well plate at 100. mu.l/well. At 37 ℃ 5% CO2Culturing for 24h in a cell culture box, sucking out residual culture medium in a 96-well plate after 24h, adding the 3' -O-benzoyl salicin to be tested into six groups of 500 mu g/ml, 250 mu g/ml, 125 mu g/ml, 62.5 mu g/ml, 31.25 mu g/ml and blank control, setting four multiple wells in each group, and culturing at 37 ℃ and 5% CO2After culturing for 24h in the cell culture box, adding 10 mul of CCK-8 detection reagent into each hole, culturing for 2.5h in the cell culture box, detecting the absorbance OD value of each hole under the wavelength of 450nm by using an enzyme-labeling instrument, and calculatingInhibition rate of cells.
In the experiment, the 3 ' -O-benzoyl salicin can be clearly obtained to have good effect of inhibiting the growth of the intestinal cancer tumor cells, particularly, under the dosage of 62.5-500 mu g/ml, the anti-cancer activity is obvious, the anti-cancer activity of the 3 ' -O-benzoyl salicin is in direct proportion to the dosage, the higher the dosage is, the stronger the anti-cancer activity is, no obvious cytotoxicity is found under the concentration, and the 3 ' -O-benzoyl salicin has the potential of being used as a medicament for inhibiting the growth of the intestinal cancer tumor cells.
In the experiment, 3 '-O-benzoyl salicin can be clearly obtained to have positive effect in inhibiting the growth of gastric cancer and intestinal cancer tumor cells, and the same experiment proves that 3' -O-benzoyl salicin can also play positive effect in inhibiting the growth of other tumor cells, such as breast cancer, lung cancer, esophageal cancer, liver cancer and uterine cancer tumor cells.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (4)

1. A method for extracting 3' -O-benzoyl salicin from Chinese white poplar leaves is characterized by comprising the following steps:
pretreatment: cleaning Chinese medicinal material white poplar leaves, crushing, and screening particles with 50-200 meshes;
extraction: putting pretreated white poplar leaves into a multifunctional extraction tank for a laboratory, adding 70% ethanol water solution according to a preset liquid medicine ratio, and extracting for 3 hours at a preset extraction temperature;
concentration: filtering the crude liquid, and concentrating at a predetermined concentration temperature by evaporation until no alcohol smell is produced;
and (3) extraction: extracting the concentrated solution generated by concentration by using petroleum ether at a preset extraction ratio and extraction temperature, concentrating a petroleum ether extract phase to obtain a petroleum ether part, extracting a petroleum ether raffinate phase by using dichloromethane at a preset extraction ratio and extraction temperature, concentrating a dichloromethane extract phase to obtain a dichloromethane part, separating the dichloromethane part by sequentially passing through a normal-pressure chromatographic column, a silica gel column and an ODS column, and removing impurities to obtain 3' -O-benzoyl salicin;
wherein in the step of extraction, the preset liquid-medicine ratio is 1:10, and the preset extraction temperature is 70-90 ℃;
in the step of concentration, the preset concentration temperature is 55-65 ℃;
in the step of extraction, the preset extraction temperature is 25-40 ℃, and the preset extraction ratio is 1: 3-1: 5.
2. The method for extracting 3' -O-benzoyl salicin from leaves of Populus tomentosa as claimed in claim 1, wherein the predetermined extraction temperature in the step of "extracting" is 70 ℃.
3. The method for extracting 3' -O-benzoyl salicin from Chinese white poplar leaves as claimed in claim 2, wherein in the step of "extracting", the predetermined extraction ratio is 1:3 and 1:3, respectively, and the predetermined extraction temperature is 25 ℃.
4. The method for extracting 3' -O-benzoyl salicin from leaves of Populus tomentosa as claimed in claim 3, wherein the step of "concentrating" is performed at a predetermined concentration temperature of 55 ℃.
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* Cited by examiner, † Cited by third party
Title
Cytotoxic phenolic compounds in leaf buds of populous tremuloides;pichette,andre 等;《Canadian Journal of Chemistry》;20101231;第88卷(第2期);第104-110页 *
杨树酚苷成分及其活性方面的研究;于雪莹等;《国土与自然资源研究》;20151231(第3期);第77-84页 *
银中杨树叶化学成分研究;吕伟强等;《天然产物研究与开发》;20151231;第25卷;第620-623页 *
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