CN111808150B - Method for separating compounds from bergenia purpurascens on large scale and method for detecting oxidation resistance - Google Patents
Method for separating compounds from bergenia purpurascens on large scale and method for detecting oxidation resistance Download PDFInfo
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- CN111808150B CN111808150B CN202010876173.5A CN202010876173A CN111808150B CN 111808150 B CN111808150 B CN 111808150B CN 202010876173 A CN202010876173 A CN 202010876173A CN 111808150 B CN111808150 B CN 111808150B
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Abstract
The invention relates to a preparation method of effective ingredients of traditional Chinese medicines, and particularly provides a method for separating compounds from bergenia crassifolia on a large scale and an oxidation resistance detection method. The method for separating the compounds from the bergenia purpurascens on a large scale comprises the following steps: extracting bergenia purpurascens to obtain extract, and performing countercurrent chromatography to obtain monomer components including beta-arbutin, bergenin, 6-O-galloyl arbutin, gallic acid, 11-O-galloyl bergenin, and (-) -epicatechin 3-O-gallate. The method solves the problems that in the prior art, one compound is often separated at one time, the efficiency is low, and the high-speed counter-current chromatography is not applied to the compound extraction and separation of bergenia.
Description
Technical Field
The invention relates to a preparation method of effective ingredients of traditional Chinese medicines, and particularly provides a method for separating compounds from bergenia crassifolia on a large scale and an oxidation resistance detection method.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Bergenia purpurascens (Saxifragaceae) is a perennial herb, and is located in Himalayan region with altitude of 2700 and 4700 m. In the Pakistan Giltett area, the plant has been used as an herbal medicine for the treatment of diabetes. It has been reported that a herb containing bergenia purpurascens can treat acne, diabetes, obesity and viral infections. In addition, it has been reported that the plant can be used for treating kidney stones, wounds, etc., and has antifungal, antioxidant, antibacterial and immunoregulatory effects. The main chemical components reported by bergenia include bergenin or its derivative terpenes, flavonoids, etc.
High-speed counter-current chromatography is a continuous high-efficiency and rapid liquid-liquid partition chromatography separation technology without any solid support developed in the last 30 years, and avoids various problems of easy dead adsorption, loss, denaturation and the like of a sample caused by a solid support or a carrier. When other liquid chromatography is used for preparative separation, the distribution efficiency is obviously reduced, the solvent consumption is high, the high-speed counter-current chromatography ensures higher peak resolution, the separation amount is large, the sample is not lost, the recovery rate is high, the separation environment is mild, and the solvent is saved. The high-speed counter-current chromatography can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, and the method is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like.
The method for separating and purifying the compounds of the medicinal materials such as the Japanese ardisia herb and the like by high-speed countercurrent chromatography is provided in the prior art, but the inventor finds that the efficiency is lower because one compound is often separated at one time by adopting the high-speed countercurrent chromatography in the prior art, and the requirement on operators is higher because the parameters are required to be changed again for separating one compound. And the high-speed countercurrent chromatography in the prior art is not applied to the extraction and separation of the compounds of bergenia.
Disclosure of Invention
Aiming at the problems that in the prior art, a compound is often separated by a high-speed countercurrent chromatography at one time, the efficiency is low, and the high-speed countercurrent chromatography is not applied to the extraction and separation of the compound of the bergenia crassifolia, the disclosure mainly provides a method which has the advantages of low preparation cost, simple and convenient operation, high efficiency and capability of separating and preparing the monomer compound with the purity of more than 95 percent from the bergenia crassifolia in large batches.
In one or more embodiments of the present disclosure, a method for bulk isolation of a compound from bergenia cordifolia is provided, comprising the steps of: extracting bergenia purpurascens to obtain extract, and performing countercurrent chromatography to obtain monomer components including beta-arbutin, bergenin, 6-O-galloyl arbutin, gallic acid, 11-O-galloyl bergenin and (-) -epicatechin 3-O-gallate.
The present disclosure is oneOr in some embodiments, the method for detecting the oxidation resistance of the compound separated from the bergenia crassifolia is used for treating the cells to be detected with hydrogen peroxide, respectively treating the cells to be detected with the compound separated from any one of the methods for separating the compound from the bergenia crassifolia on a large scale, and respectively detecting the oxidation resistance of the compound separated from the bergenia crassifolia on a H scale2O2And detecting the activity of the cells before and after treatment by 6 different monomers, thereby judging the oxidation resistance of 6 monomer compounds.
In one or some embodiments of the disclosure, the application of 6-O-galloyl arbutin in preparing a medicament for preventing or treating oxidative diseases is provided.
In one or some embodiments of the disclosure, the application of 6-O-galloyl arbutin in preparing a medicament for preventing or treating oxidative diseases is provided.
One or some of the above technical solutions have the following advantages or beneficial effects:
1) the method adopts the combination of the high-speed counter-current chromatography and the ultraviolet chromatography, discharges materials according to the peak value of the ultraviolet chromatography, and separates six compounds at one time, thereby greatly improving the separation efficiency of the compounds, avoiding the situation that a large number of parameters are required to be changed in each separation of the high-speed counter-current chromatography, and feeding materials again in each separation of one compound, and reducing the operation difficulty while improving the separation efficiency.
2) The method disclosed by the invention is used for separating the bergenia purpurascens which is cheap and easy to obtain, and the separated compound is high in purity and wide in application range.
3) The disclosure further provides six separated compounds, beta-arbutin (1) and 6-O-galloyl arbutin (3) have significant protective effect on cell viability, and 6-O-galloyl arbutin especially resists H at high dose2O2The resulting oxidative damage has a very significant effect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1 is a flowchart of the idea of examples 1 and 3.
FIG. 2 is a diagram of the countercurrent chromatography separation of crude extract of bergenia cordifolia in example 1.
FIG. 3 is a high performance liquid chromatogram of the separation of monomers from crude bergenia purpurascens extract and countercurrent chromatography in example 1.
FIG. 4 is a graph of cell viability test of crude bergenia purpurascens extract and monomers of example 3.
FIG. 5 is the antioxidant test chart of crude extract and monomer of bergenia cordifolia in example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
In one or more embodiments of the present disclosure, a method for bulk isolation of a compound from bergenia cordifolia is provided, comprising the steps of: extracting bergenia purpurascens to obtain extract, and performing countercurrent chromatography to obtain monomer components including beta-arbutin, bergenin, 6-O-galloyl arbutin, gallic acid (4), 11-O-galloyl bergenin, and (-) -epicatechin 3-O-gallate.
Preferably, the operation of obtaining the extract solution from bergenia purpurascens comprises the following steps: soaking rhizoma Seu herba Bergeniae, extracting with solvent repeatedly, mixing all extractive solutions, and rotary evaporating to dryness to obtain crude rhizoma Seu herba Bergeniae.
Preferably, the extractant is ethanol.
More preferably, the ethanol mass fraction is 80%.
Preferably, the extraction time is 24 h.
Preferably, the number of repetitions is 3.
Preferably, rotary evaporation is carried out at 40 ℃.
Preferably, the extract is stored in a refrigerator at 4 ℃ for use.
Preferably, the countercurrent chromatographic separation comprises the following steps: dissolving the crude bergenia purpurascens product in the upper and lower phases of the solvent phase, respectively, filling the stationary phase into a chromatographic separation column by using a high-speed liquid chromatograph, setting the flow velocity of the mobile phase, feeding after achieving hydrodynamic balance, receiving 6 monomer components according to an ultraviolet spectrogram, and finally separating 6 monomers.
Preferably, a solvent system is prepared from a two-phase solvent system of tert-butyl methyl ether/n-butanol/methanol/water, the solvent system is placed in a separating funnel, the mixture is shaken up and then is kept stand for layering, the upper phase and the lower phase are separated after the mixture is balanced for a period of time, and crude bergenia crassifolia is taken and respectively dissolved in the upper phase and the lower phase for standby.
Preferably, a high-speed counter-current chromatograph is adopted, the sample injection valve is firstly in a sample injection state, the chromatographic separation column is filled with the stationary phase at a certain flow rate by a pump, and the pump is stopped.
Preferably, the speed controller is started to enable the chromatographic separation column of the high-speed flow chromatograph to rotate forwards, the flow velocity of the mobile phase is set after a certain rotating speed is reached, the mobile phase is pumped, after the fluid dynamic balance is reached, a well-dissolved sample is injected into a sample injection valve of the countercurrent chromatograph by an injector, the sample injection valve is rotated to be in a column connection state, the sample enters the chromatographic separation column, then 6 monomer components are received according to a detector ultraviolet spectrogram, and finally 6 monomers are separated.
Preferably, the volume ratio of the tert-butyl methyl ether to the n-butanol to the methanol to the water is 1:3:1: 5.
Preferably, when the forward rotation speed of the chromatographic separation column of the high-speed flow chromatograph reaches 800 revolutions per minute, the flow velocity of the mobile phase is set.
Further preferably, the flow rate of the mobile phase is set to 2.0 mL/min.
Preferably, after separation of the 5 th peak, the 6 th peak is ejected quickly.
Preferably, the purity of 6 monomers separated is checked by high performance liquid chromatography.
Preferably, the liquid phase conditions are: waters Symmetry C18column (5column ymme 250mm, i.d.), uv detection wavelength 254nm, flow rate: 1.0mL/min, sample size: 10 mu L of the solution; the mobile phase adopts water (A) and acetonitrile (B), 0-5min,88%A;5-15min,88%-70%A;15-20min,70%A;20-21min,70%-88%A;21-25min,12%B。
In one or more embodiments of the present disclosure, a method for detecting oxidation resistance of a compound isolated from bergenia crassifolia is provided, wherein H is2O2Treating test cells with a compound isolated from any of the above methods for isolating compounds from bergenia purpurascens on a large scale, and treating test cells with a compound isolated from bergenia purpurascens on a large scale, respectively2O2And detecting the activity of the cells before and after treatment by 6 different monomers, thereby judging the oxidation resistance of 6 monomer compounds.
Preferably, the cell is a HepG2 cell.
Preferably, three different H's are provided2O2The concentration of the test sample was measured for cells, and 6 different monomers were treated for three different H2O2The cells at the concentration are processed.
Preferably, H2O2The concentrations were 25, 50 and 100. mu.g/mL, respectively.
In one or some embodiments of the disclosure, the application of beta-arbutin in preparing a medicine or health product for improving cell viability is provided.
In one or some embodiments of the disclosure, the application of 6-O-galloyl arbutin in preparing a medicament for preventing or treating oxidative diseases is provided.
Preferably, the application of 6-O-galloyl arbutin in preparing the reagent for improving the survival rate of HepG2 cells in the oxidative environment.
Preferably, the oxidative disease comprises diabetes, cardiovascular and cerebrovascular diseases, neurodegenerative diseases and cancer.
Preferably, the antioxidant drugs comprise powder, paste, combined drug and tablet.
Example 1
This example provides a method for bulk isolation of compounds from bergenia purpurascens.
1. Extraction of samples
Soaking rhizoma Seu herba Bergeniae (collected from Pakistan) 5kg, extracting with 2 × 12L 80% ethanol for 24 hr, and repeating for 3 times. All extracts were combined and rotary evaporated to dryness at 40 ℃ to give 256g of crude bergenia purpurascens, which was further purified in a refrigerator at 4 ℃.
2. Preparation of monomer components by countercurrent chromatography
Preparing a solvent system from a two-phase solvent system of tert-butyl methyl ether/n-butanol/methanol/water (1:3:1:5, v/v) according to the solvent ratio, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, taking 1.0g of crude product, and dissolving the crude product in 5mL of lower phase and 5mL of lower phase for later use. A high-speed counter-current chromatograph is used, which is composed of plunger pump, sample valve, ultraviolet detector, recorder and chromatographic separation column (spiral column with capacity of 300mL formed by winding polytetrafluoroethylene tubes in multiple layers). Starting a speed controller to enable a chromatographic separation column of a high-speed flow chromatograph to rotate forwards, setting the flow speed of a mobile phase to be 2.0mL/min when the rotating speed reaches 800 revolutions per minute, starting pumping the mobile phase, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector after the hydrodynamic balance is achieved, rotating the sample injection valve to be in a column connection state, enabling the sample to enter the chromatographic separation column, and then receiving components I to V in the figure 2 according to a detector ultraviolet spectrogram. After peak V was isolated, peak VI was rapidly ejected and finally six high purity monomers of peak I (compound 1, 35mg), peak II (compound 2, 76mg), peak III (compound 3, 21mg), peak IV (compound 4, 20mg), peak V (compound 5, 42mg) and peak VI (compound 6, 52mg) were isolated as shown in fig. 2; the purity was over 95% as determined by liquid chromatography, see FIG. 3.
And (3) analyzing the separated substances by using high performance liquid chromatography, wherein the liquid phase conditions are as follows: waters Symmetry C18column (5 μm,4.6mm × 250mm, i.d.), uv detection wavelength 254nm, flow rate: 1.0mL/min, sample size: 10 mu L of the solution; the mobile phase adopts water (A) and acetonitrile (B), 0-5min, 88% A; 5-15min, 88% -70% A; 15-20min, 70% A; 20-21min, 70% -88% A; 21-25min, 12% B.
And (3) structural identification: respectively measuring MS and NMR spectra of the separated ginkgolic acid component monomers by using an Agilent 6520Q-TOF mass spectrometer and a Bruker AV-400MHz nuclear magnetic resonance spectrometer, wherein the obtained data are as follows:
compound 1: ESI-MS (negative ion mode) m/z 271.0526.1H-NMR(MeOH-d4,400MHz)δ:3.69(1H,dd,J=2.54,11.5Hz,H-6'a),3.87(1H,d,J=11.9Hz,H-6'b),3.37(1H,m,H-4'),3.41(1H,m,H-2'),3.40(1H,m,H-5'),3.45(1H,m,H-3'),4.72(1H,d,J=7.0Hz,H-1'),6.96(2H,d,J=8.8Hz,H-2,H-6),6.68(2H,d,J=8.8Hz,H-3,H-5).13C-NMR(MeOH-d4,100MHz)δ:62.6(CH2C-6'),71.5(CH, C-4'),75.0(CH, C-2'),78.0(CH, C-5'),78.1(CH, C-3'),103.7(CH, C-1'),116.6(CH, C-2, C-6),119.4(CH, C-3, C-5),152.4(C-1),153.8 (C-4). Identified as beta-arbutin.
Compound 2: ESI-MS (negative ion mode) m/z 327.0367.1H-NMR(MeOH-d4,400MHz)δ:3.90(3H,s,H-12),3.64-3.72(2H,m,H-11),3.43(1H,m,H-3),4.95(1H,d,J=10.4Hz,,H-10b),4.00-4.08(2H,m,H-4,H-4a),3.81(1H,m,H-2),7.08(1H,s,H-7).13C-NMR(MeOH-d4,100MHz)δ:60.9(OCH3,C-12),62.7(CH2C-11),71.9(CH, C-3),74.2(CH, C-10b),75.6(CH, C-4),81.4(CH, C-4a),83.0(CH, C-2),111.0(CH, C-7),117.3(C-10a),119.4(C-6a),142.3(C-9),149.4(C-10),152.3(C-8),165.8 (C-6). Identified as bergenin.
Compound 3: ESI-MS (negative ion mode) m/z 425.3755.1H-NMR(MeOH-d4,400MHz)δ:4.57(1H,dd,J=1.2,11.4Hz,Glc-H-6a),4.43(1H,dd,J=6.8,11.7Hz,Glc-H-6b),3.41-3.49(1H,m,Glc-H-4),3.67-3.71(1H,m,Glc-H-5),3.41-3.49(1H,m,Glc-H-2),3.41-3.49(1H,m,Glc-H-3),4.69(1H,d,J=6.94Hz,Glc-H-1),7.11(2H,s,H-2',H-6'),6.92(2H,d,J=8.7Hz,H-2,H-6),6.61(2H,d,J=8.7Hz,H-3,H-5).13C-NMR(MeOH-d4,100MHz)δ:64.9(CH2Glc-6),71.8(CH, Glc-4),75.0(CH, Glc-5),75.6(CH, Glc-2),78.0(CH, Glc-3),103.9(CH, Glc-1),110.3(CH, C-2', C-6'),116.7(CH, C-2, C-6),119.5(CH, C-3, C-5),121.4(C-1'),139.9(C-4'),146.6(C-3', C-5'),152.4(C-4),153.9(C-1),168.2(CO, C-7 '). Identified as 6-O-galloyl arbutin.
Compound 4: ESI-MS (negative ion mode) m/z 480.1H-NMR(MeOH-d4,400MHz)δ:colorless solid(MeOH),ESI-MS(negative ion mode)m/z:169.0270.1H-NMR(MeOH-d4,400MHz)δ:7.05(2H,s,H-2,H-6).13C-NMR(MeOH-d4100MHz) delta 110.3(2CH, C-2, C-6),122.5(C-1),139.4(C-OH, C-4),146.3(2C-OH, C-3, C-5),170.7(COOH, C-7). Identified as gallic acid.
Compound 5: ESI-MS (negative ion mode) m/z 479.0358.1H-NMR(MeOH-d4,400MHz)δ:3.90(3H,s,H-12),4.40(1H,dd,J=12.2,6.7Hz,H-11a),4.54(1H,d,J=7.5Hz,H-11b),3.53-3.58(1H,m,H-3),5.01(1H,d,J=10.5Hz,H-10b),3.84-3.88(1H,m,H-4),3.94-3.98(1H,m,H-2),4.09-4.14(1H,m,H-4a),7.09(2H,s,H-2',6'),7.07(1H,s,H-7).13C-NMR(MeOH-d4,100MHz)δ:61.0(OCH3,C-12),64.6(CH2C-11),71.8(CH, C-3),74.3(CH, C-10b),75.4(CH, C-4),81.3(CH, C-2),82.8(CH, C-4a),110.3(CH, C-2',6'),111.2(CH, C-7),117.0(C-10a),119.4(C-6a),121.0(C-1'),140.1(C-9),142.3(C-4'),146.5(C-3',5'),149.3(C-10),152.3(C-8),165.7(CO, C-6),168.2(CO, C-7 '). Identified as 11-O-galloyl bergenin.
Compound 6: ESI-MS (negative ion mode) m/z 441.0598.1H-NMR(MeOH-d4,400MHz)δ:2.85(1H,dd,J=5.00,16.55Hz,H-4α),2.72(1H,dd,J=5.9,16.6Hz,H-4β),5.33-5.38(1H,m,H-3),5.07(1H,d,J=5.8,H-2),5.96(1H,distorted s,H-6),5.97(1H,distorted s,H-8),6.97(2H,s,H-2”,6”),6.93(1H,d,J=1.3Hz,H-2'),6.72(1H,m,H-5'),6.84(1H,m,H-6').13C-NMR(MeOH-d4,100MHz)δ:24.3(CH2C-4),71.1(CH, C-3),79.3(CH, C-2),95.6(CH, C-6),96.5(CH, C-8),99.6(C-4a),110.1(CH, C-2',6 "), 114.4(CH, C-2'),116.2(CH, C-5'),119.2(CH, C-6'),121.4 (C-1"), 131.5(C-1'),139.8(C-4 "), 146.1(C-3',4'),146.3 (C-3', 5"), 156.4(C-8a),157.6(C-5),158.0(C-7),167.5(C-7 "). Identified as (-) -epicatechin 3-O-gallate.
Example 2
This example provides K at different solvent compositionsDThe value is obtained.
TABLE 1K of the target Compounds of different solvent systemsDValue of
The selection of a suitable two-phase solvent system for countercurrent chromatographic separation is a very critical step, and a suitable two-phase solvent system can successfully complete the separation experiment. In this study, a number of biphasic mixed solvent systems including two, three or four solvent mixtures were tested in different proportions. A good solvent system can provide a desired partition coefficient (K) for the target compound in the mixtureD) The value is obtained. KDThe values describe the ratio of solute distribution between two-phase solvent systems in equilibrium with each other. The biphasic solvent system studied in our experiments included n-hexane/ethyl acetate/methanol/water (1:9:1:9, v/v), ethyl acetate/n-butanol/water (1:4:5, v/v). In addition, different ratios of the biphasic solvent system tert-butyl methyl ether/n-butanol/methanol/water (1:3:1:5, 2:2:1:5 and 3:1:1:5, v/v) were investigated. As shown in Table 1, K for most compounds when using a biphasic n-hexane/ethyl acetate/methanol/water (1:9:1:9, v/v) solvent systemDAll values are less than 0.5 and are therefore unsuitable for countercurrent chromatographic separation. In the case of an ethyl acetate/n-butanol/water (1:4:5, v/v) solvent system, K was foundDThe range of values is greater than the suggested value and is therefore also disadvantageous for a better separation. In addition, other solvent systems were used, such as tert-butyl methyl ether/n-butanol/methanol/water (2:2:1:5 and 3:1:1:5, v/v), which resulted in better solvent systems than previously mentioned, but only for a few target compounds. Furthermore, another solvent system, i.e. tert-butyl methyl ether/n-butanol/methanol/water (1:3:1:5, v/v) was found to be most suitable, since the K of the target componentDThe value is very feasible.
Example 3
This example provides a cellular activity test against oxidation of 6 monomeric compounds as described in example 1.
The cytotoxicity of the crude extracts and monomeric compounds, i.e., β -arbutin (1), bergenin (2), 6-O-galloyl arbutin (3), gallic acid (4), 11-O-galloyl bergenin (5) and (-) -epicatechin 3-O-gallate (6), was determined in HepG2 (human hepatoma cell line) cells according to MTT analysis.
First, HepG2 cells were cultured in DMEM supplemented with penicillin (100U/mL)/streptomycin (100. mu.g/mL) and 10% FBS. Cells were then incubated at 5% CO2Incubate in the presence of 37 ℃. Trypsin solution was used to digest HepG2 cells in the logarithmic growth phase. The cell density was then adjusted to 5X 10 with the culture medium5and/mL. Cells were incubated at 37 ℃ and 5% CO in a volume of 100. mu.L/well2Seeded into 96-well cell culture plates. Thereafter, the seed cells were treated with the test sample at an appropriate concentration for 24 hours. The wavelength of absorbance was kept at 570nm to determine cell viability. The effect of the components on cell viability was calculated using the given formula: cell viability (%) ═ treatment sample a570nm/untreated sample A570nmX 100%. HepG2 cells treated with gallic acid (4) at a concentration of 100. mu.g/mL decreased rapidly. In the case of HepG2 cells treated with the compound β -arbutin (1), cell viability was slightly increased at all concentrations. In the case of treatment with compounds 2, 4 and 5, a slight increase in HepG2 cell viability was observed at concentrations of 25 and 50 μ g/mL, see figure 4.
Hydrogen peroxide (H)2O2) Being an important ROS, it easily attacks cell membranes and reacts with iron ions in cells, and thus can be used for evaluating oxidative damage. In this assay, cells were used at 200. mu. M H2O2And treating for 6 h. As shown in fig. 2, the cell viability was approximately 43.15% compared to the control group. To investigate H in HepG2 cells by the test samples2O2Influence of induced oxidative damage of cells, we used three different concentrations (25, 50 and 100. mu.g/mL) of the test samples, i.e., crude bergenia purpurascens and compounds 1-3, 5, 6. For compound 4, HepG2 cells were treated with three concentrations of 12.5, 25 and 50. mu.g/mL. Cell viability was reduced (p) in samples of 25. mu.g/mL (bergenia crassifolia, 1, 2 and 4)<0.01). No significant change was observed in HepG2 cells treated with crude bergenia, compounds 2, 4 and 6 at 50. mu.g/mL. HepG2 cells were treated with 50, 100. mu.g/mL of compounds 1 and 5 in the crude extract and compound 6, 100. mu.g/mL, respectivelyIn the case of (2), an increase in cell viability was observed. But with H2O2The viability of the cells treated with Compound 3 (50 and 100. mu.g/mL) was significantly improved (p) compared to the treated cells (200. mu.g/mL)<0.05)。
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Claims (15)
1. A method for separating compounds from bergenia crassifolia on a large scale is characterized by comprising the following steps: extracting rhizoma Seu herba Bergeniae to obtain extract, and performing countercurrent chromatography to obtain monomer componentsβArbutin, bergenin, 6-OGalloyl arbutin, gallic acid, 11-OGalloyl bergenin, (-) -epicatechin 3-O-a gallic acid ester;
the countercurrent chromatographic separation comprises the following steps: preparing a solvent system from a two-phase solvent system of tert-butyl methyl ether/n-butanol/methanol/water, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, taking a crude bergenia purpurascens product, respectively dissolving the crude bergenia purpurascens product in the upper phase and the lower phase of a solvent phase, filling a chromatographic separation column with a stationary phase by adopting a high-speed counter-current chromatograph, setting the flow rate of a mobile phase, feeding after achieving hydrodynamic balance, receiving 6 monomer components according to an ultraviolet spectrogram, and finally separating 6 monomers;
the volume ratio of tert-butyl methyl ether to n-butanol to methanol to water is 1:3:1: 5.
2. The method for mass separation of compounds from bergenia cordifolia as claimed in claim 1, wherein the step of obtaining extract from bergenia cordifolia comprises the steps of: soaking rhizoma Seu herba Bergeniae, extracting with the extractant repeatedly, mixing all extractive solutions, and rotary evaporating to dryness to obtain crude rhizoma Seu herba Bergeniae.
3. The method for mass separation of compounds from bergenia cordifolia as claimed in claim 2, wherein the extractant is ethanol.
4. A process for the bulk isolation of a compound from bergenia cordifolia as claimed in claim 3, wherein the ethanol is present in an amount of 80% by weight.
5. A process for bulk isolation of a compound from bergenia cordifolia as claimed in claim 2, wherein the extraction time is 24 hours.
6. A process for bulk isolation of a compound from bergenia cordifolia as claimed in claim 2, wherein the number of repetitions is 3.
7. A process for bulk isolation of a compound from bergenia cordifolia as claimed in claim 2, wherein the rotary evaporation is performed at 40 ℃.
8. The method for mass isolation of compounds from bergenia cordifolia as claimed in claim 1, wherein the extract is stored in a refrigerator at 4 ℃ for use.
9. The method of mass separation of chemical compounds from bergenia cordifolia as claimed in claim 1, wherein the high speed countercurrent chromatography is adopted, the sample injection valve is first set in the sample injection state, the stationary phase is pumped to flow rate to fill the chromatographic separation column, and the pump is stopped.
10. The method for mass separation of chemical compounds from bergenia cordifolia as claimed in claim 1, wherein the speed controller is turned on to rotate the chromatographic separation column of the high speed countercurrent chromatograph forward, and after reaching a certain rotation speed, the flow rate of the mobile phase is set, and the pump of the mobile phase is started, and after reaching the fluid dynamic equilibrium, the dissolved sample is injected into the sample injection valve of the countercurrent chromatograph by the injector, and the sample injection valve is rotated to a connection state, so that the sample enters the chromatographic separation column, and then 6 monomer components are received according to the ultraviolet spectrogram of the detector, and finally 6 monomers are separated.
11. The method for mass separation of chemical compounds from bergenia cordifolia as claimed in claim 1, wherein the mobile phase flow rate is set when the forward rotation speed of the chromatographic separation column of the high-speed flow chromatograph reaches 800 rpm.
12. A method for bulk isolation of a compound from bergenia cordifolia as claimed in claim 1, wherein the mobile phase flow rate is set to 2.0 mL/min.
13. A process for bulk isolation of a compound from bergenia cordifolia as claimed in claim 1, wherein after separation of the 5 th peak, the 6 th peak is rapidly ejected.
14. The method for mass separation of chemical compounds from bergenia cordifolia as claimed in claim 1, wherein the purity of the separated 6 monomers is measured by high performance liquid chromatography.
15. A process for bulk isolation of a compound from bergenia cordifolia as claimed in claim 14, wherein the liquid phase conditions are: waters Symmetry C18column, ultraviolet detection wavelength 254nm, flow rate: 1.0mL/min, sample size: 10 mu L of the solution; the mobile phase adopts water (A) and acetonitrile (B), 0-5min, 88% A; 5-15min, 88% -70% A; 15-20min, 70% A; 20-21min, 70% -88% A; 21-25min, 12% B.
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CN107722080A (en) * | 2017-11-08 | 2018-02-23 | 长沙爱扬医药科技有限公司 | A kind of method that ursin is extracted in the leaf from purple bergenia herb |
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Non-Patent Citations (3)
Title |
---|
alfa-Glucosidase and a-amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata, Haw.);Megh Raj Bhandari,等;《Food Chemistry》;20081231;第106卷;第249-250页 * |
GALLOYLARBUTIN AND OTHER POLYPHENOLS FROM BERGENIA PURPURASCENS;CHEN XIN-MIN,等;《Phytochemistry》;19871231;第26卷(第2期);第515-516页 * |
岩白菜叶的化学成分研究;张姗姗;《东南大学工程硕士学位论文》;20180415;摘要、第9-11页 * |
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