CN113387998A - Preparation method of dioscin in fenugreek - Google Patents
Preparation method of dioscin in fenugreek Download PDFInfo
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Abstract
The invention provides a preparation method of dioscin in fenugreek, belonging to the technical field of separation and purification. The invention takes fenugreek as a raw material, adopts a foam separation method combined with a preparative high performance liquid chromatography (P-HPLC) method to separate and purify dioscin in the fenugreek, establishes a preparation method of dioscin monomer, and qualitatively determines the obtained monomer compound to be the dioscin by carrying out structure determination. The result shows that the method has the characteristics of simple operation, short separation period, high efficiency and the like, is suitable for separating and preparing the dioscin in the fenugreek, and provides theoretical basis and technical reference for separation and purification and large-scale production of the dioscin in the fenugreek.
Description
Technical Field
The invention relates to the technical field of separation and purification, and particularly relates to a preparation method of dioscin in fenugreek.
Background
Fenugreek (Trigonella foenum-graeeum, L.), also known as Ruta graveolens and Medicago sativa, is an annual herb with extremely high viability, is a subfamily Papilionaceae of Leguminosae, originates in southern Europe, Western Asia and Mediterranean regions, and has been used as a spice and a flavoring agent since ancient times. The fenugreek seeds contain rich chemical components, such as galactomannan, steroidal saponins, flavonoids, alkaloids, triterpenes and coumarins, and the pharmacological action is very wide. The steroid saponins can reduce the content of serum total cholesterol and inhibit the absorption of cholesterol, and are intermediates for synthesizing steroid hormone drugs, the content of the steroid saponins in fenugreek is about 6.5%, and the dioscin is the main chemical component of the steroid saponins. Therefore, dioscin has received wide attention worldwide as a main component of fenugreek steroid saponins.
At present, the traditional methods for purifying dioscin mainly comprise a macroporous resin adsorption method, a membrane separation method, a high-speed counter-current chromatography method and a column chromatography method, but the methods have the problems of long time consumption and complicated separation and purification steps.
Disclosure of Invention
The invention aims to provide a method for preparing dioscin in fenugreek, which has the characteristics of simple operation, short separation period, high efficiency and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of dioscin in fenugreek, which comprises the following steps:
mixing the fenugreek powder with an alcohol solvent, and extracting to obtain an extracting solution;
performing foam separation on the extracting solution to obtain a dioscin crude product;
and separating and purifying the dioscin crude product by using preparative high performance liquid chromatography to obtain the dioscin.
Preferably, the alcohol solvent comprises an ethanol solution; the mass concentration of the ethanol solution is 60-80%; the mass ratio of the fenugreek powder to the volume of the alcohol solvent is 1g (20-40) mL.
Preferably, the extraction pressure is 300-400 MPa, and the extraction time is 6-12 min.
Preferably, after the extraction, the method further comprises the steps of sequentially carrying out reduced pressure filtration and concentration on the extracted feed liquid.
Preferably, the foam separation conditions are as follows: the sample loading concentration is 0.01-0.05 mg/mL, the temperature is 25-45 ℃, the gas velocity is 350-550 mL/min, and the liquid loading amount is 250-450 mL.
Preferably, the foam separation conditions are as follows: the sample loading concentration is 0.03mg/mL, the temperature is 28 ℃, the gas velocity is 450mL/min, and the liquid loading amount is 400 mL.
Preferably, the chromatographic conditions of the preparative high performance liquid chromatography comprise: mobile phase: water and acetonitrile; acetonitrile with the volume fraction of 5-15% is 0-5 min; 5-15 min, and 15-45% acetonitrile by volume fraction; 15-35 min, and the volume fraction is 45-100% of acetonitrile; the flow rate is 10 mL/min; the sample volume was 5 mL.
Preferably, the chromatographic conditions of the preparative high performance liquid chromatography further comprise: a chromatographic column: HederaODS-2 column, 20X 250mm, 10 μm; the detection wavelength is 208 nm.
The invention provides a preparation method of dioscin in fenugreek, which comprises the following steps: mixing the fenugreek powder with an alcohol solvent, and extracting to obtain an extracting solution; performing foam separation on the extracting solution to obtain a dioscin crude product; and separating and purifying the dioscin crude product by using preparative high performance liquid chromatography to obtain the dioscin. The invention takes fenugreek as raw material, and adopts a foam separation method combined with a preparative high performance liquid chromatography (P-HPLC) method to separate and purify dioscin in the fenugreek, the dioscin extracting solution has more impurities and low product purity, the dioscin belongs to spirostane saponins, the spirostane steroid saponin is composed of spirostane aglycone and a sugar chain, the sugar chain has stronger polarity (water solubility), and the lipophilicity of the aglycone part is better, so the steroid saponin has good foaming performance; and then further purifying and collecting the dioscin crude product obtained by separation by adopting preparative high performance liquid chromatography, wherein the purification effect is good, and the method has the characteristics of simple operation, short separation period, high efficiency and the like.
Drawings
FIG. 1 is a graph showing the effect of loading concentration on foam separation of dioscin;
FIG. 2 is a graph of the effect of temperature on foam separation of dioscin;
FIG. 3 is a graph showing the effect of air velocity on foam separation of dioscin;
FIG. 4 is a graph showing the effect of liquid loading on foam separation of dioscin;
FIG. 5 is a graph showing the effect of the interaction of two factors, namely loading concentration-temperature, loading concentration-gas velocity and loading concentration-liquid loading amount, on foam separation of dioscin;
FIG. 6 is a graph showing the effect of the interaction of two factors, temperature-air velocity, temperature-liquid loading capacity and air velocity-liquid loading capacity, on the foam separation of dioscin;
FIG. 7 is a graph showing the effect of the interaction of two factors, temperature-loading concentration, gas velocity-loading concentration and liquid loading amount-loading concentration, on the foam separation of dioscin;
FIG. 8 is a graph showing the effect of the interaction of two factors, namely air velocity-temperature, liquid loading amount-temperature and liquid loading amount-air velocity, on the foam separation of dioscin;
FIG. 9 is a P-HPLC chart of the dioscin standard sample (a), the test sample (b) and the negative sample (c);
FIG. 10 is an HPLC chart of the dioscin sample (a), the test sample (b) and the negative sample (c);
fig. 11 is an infrared spectrum of the dioscin sample and the dioscin sample prepared in example 1;
FIG. 12 is a mass spectrum of the dioscin sample prepared in example 1;
FIG. 13 is a NMR spectrum of a dioscin sample prepared in example 1;
fig. 14 is a nuclear magnetic resonance carbon spectrum of the dioscin sample prepared in example 1.
Detailed Description
The invention provides a preparation method of dioscin in fenugreek, which comprises the following steps:
mixing the fenugreek powder with an alcohol solvent, and extracting to obtain an extracting solution;
performing foam separation on the extracting solution to obtain a dioscin crude product;
and separating and purifying the dioscin crude product by using preparative high performance liquid chromatography to obtain the dioscin.
In the present invention, unless otherwise specified, all the starting materials required for the preparation are commercially available products well known to those skilled in the art.
The invention mixes the fenugreek powder with an alcohol solvent for extraction to obtain an extracting solution. In the invention, the preparation method of the fenugreek powder is preferably to crush and dry fenugreek seeds in sequence to obtain the fenugreek powder; the present invention does not specifically limit the pulverization process and the particle size of the pulverized material, and the pulverization may be carried out according to a process known in the art. In the invention, the drying mode is preferably drying, the drying temperature is preferably 50 ℃, and the drying time is preferably drying to constant weight of the material.
In the present invention, the alcohol solvent preferably comprises an ethanol solution; the mass concentration of the ethanol solution is preferably 60-80%, and more preferably 71%; the mass ratio of the fenugreek powder to the volume of the alcohol solvent (namely the material-liquid ratio) is preferably 1g (20-40) mL, and more preferably 1g (30) mL.
The process for mixing the fenugreek powder and the alcohol solvent is not particularly limited, and the materials can be uniformly mixed according to the process well known in the art.
In the invention, the pressure of the extraction is preferably 300-400 MPa, more preferably 360MPa, the time is preferably 6-12 min, and more preferably 563 s. In the extraction process, dioscin in the fenugreek powder is extracted by an alcohol solvent.
After the extraction is finished, the invention preferably further comprises the steps of carrying out reduced pressure suction filtration and concentration on the obtained feed liquid in sequence. The process of the reduced pressure filtration is not particularly limited in the present invention, and may be performed according to a process known in the art. In the present invention, the concentration is preferably performed by a rotary evaporator, and it is preferable that the solution obtained by vacuum filtration is concentrated until no alcohol smell is generated.
After the extracting solution is obtained, the extracting solution is subjected to foam separation to obtain a dioscin crude product. The foam separation column used in the foam separation is not particularly limited in the present invention, and any commercially available foam separation column known in the art may be used; in the embodiment of the invention, the device is a circulating water constant-temperature foam separation column.
In the present invention, the conditions for the foam separation are preferably: the sample loading concentration is 0.01-0.05 mg/mL, the temperature is 25-45 ℃, the gas velocity is 350-550 mL/min, the liquid loading amount is 250-450 mL, and the preferable formula is as follows: the sample loading concentration is 0.03mg/mL, the temperature is 28 ℃, the gas velocity is 450mL/min, and the liquid loading amount is 400 mL.
After the foam separation is completed, the invention preferably carries out freeze drying on the obtained dioscin crude product, adds methanol to dissolve the obtained powder substance and carries out subsequent chromatographic separation. The amount of the methanol is not particularly limited, and the powdery substance can be completely dissolved.
After obtaining the dioscin crude product, the invention adopts preparative high performance liquid chromatography to separate and purify the dioscin crude product to obtain the dioscin. In the present invention, the preparative hplc apparatus is preferably available from jiangsu hanbang technologies ltd.
In the present invention, the chromatographic conditions of the preparative high performance liquid chromatography preferably include: mobile phase: water and acetonitrile; acetonitrile with the volume fraction of 5-15% is 0-5 min; 5-15 min, and acetonitrile with the volume fraction of 15% -45%; 15-35 min, and the volume fraction is 45% -100% of acetonitrile; the flow rate is 10 mL/min; the sample injection amount is5 mL; preferably, the method further comprises the following steps: a chromatographic column: hedera ODS-2 column, 20X 250mm, 10 μm; the detection wavelength is 208 nm.
After the separation and purification are completed, the invention preferably carries out evaporation concentration on the obtained purified product, removes an organic phase, carries out evaporation concentration and carries out freeze drying to obtain the dioscin. The process of evaporative concentration and freeze-drying is not particularly limited in the present invention and may be performed according to a process well known in the art.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Crushing fenugreek seeds, drying in a 50 ℃ oven to constant weight, placing 1g of the obtained fenugreek powder and 30mL of 71% ethanol solution in a polyethylene bottle, processing 563s under 360MPa, performing vacuum filtration on the obtained feed liquid, concentrating the obtained solution by using a rotary evaporator until no alcohol smell exists, performing foam separation on the obtained stock solution in a circulating water constant-temperature foam separation column under the conditions of sample loading concentration of 0.03mg/mL, temperature of 28 ℃, gas speed of 450mL/min and liquid loading capacity of 400mL, freeze-drying the obtained dioscin crude product, adding methanol into the obtained powder for dissolving, shaking uniformly, and performing separation and purification on a preparative high performance liquid chromatography, wherein the chromatographic conditions are as follows: a chromatographic column: hedera ODS-2 column, 20X 250mm, 10 μm; the detection wavelength is 208 nm; mobile phase: water and acetonitrile; acetonitrile with the volume fraction of 5-15% is 0-5 min; 5-15 min, and 15-45% acetonitrile by volume fraction; 15-35 min, and the volume fraction is 45-100% of acetonitrile; the flow rate is 10 mL/min; the sample amount is 5mL, after separation, the organic phase is collected, evaporated, concentrated and freeze-dried to obtain the dioscin.
Example 2
The difference from example 1 is that: the liquid loading amount was 350mL, the gas flow rate was 500mL/min, and the temperature was 30 ℃ for foam separation, and the sample loading concentrations were 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, and 0.05mg/mL in this order.
Example 3
The difference from example 1 is that: the liquid loading amount was 350mL, the gas flow rate was 500mL/min, and the sample loading concentration was 0.04mg/mL, and the temperatures were 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ in this order.
Example 4
The only difference from example 1 is: the foam separation is carried out under the conditions that the liquid loading amount is 350mL, the temperature is 30 ℃, and the sample loading concentration is 0.04mg/mL, and the gas velocity is 350mL/min, 400mL/min, 450mL/min, 500mL/min and 550mL/min in sequence.
Example 5
The only difference from example 1 is: the foam separation was carried out at a gas flow rate of 500mL/min, a temperature of 30 ℃ and a loading concentration of 0.04mg/mL, and the liquid contents were 250mL, 300mL, 350mL, 400mL and 450mL in this order.
Evaluation and testing
1. Evaluation index of foam separation of dioscin in fenugreek
Measuring the concentration of the dioscin in the fenugreek by adopting a vanillin-perchloric acid color development method, and calculating the recovery rate E and the enrichment ratio R of the dioscin in the fenugreek according to the formulas (1) and (2) respectively.
In the formula: rhof、ρs-mass concentration of dioscin in the foam layer and residual layer, μ g/mL, V0、Vs、VfThe volumes of the stock solution, the residual layer, and the foam layer, in that order, mL.
2. Calculating the recovery rate E and the enrichment ratio R of the dioscin in the fenugreek prepared in the examples 2-5 according to the formulas (1) and (2), and respectively obtaining results shown in the figures 1-4;
FIG. 1 is a graph showing the effect of loading concentration on foam separation of dioscin, and as shown in FIG. 1, the enrichment ratio gradually decreases with increasing concentration, and the recovery ratio gradually increases with increasing concentration. When the concentration of the solution is lower, the foam stability is poorer, the viscosity of the solution is lower, and the foam liquid-holding rate is lower, so that the phenomena of high enrichment ratio and low recovery rate are presented. Along with the continuous increase of solution concentration foam stability improves gradually, the viscosity of solution constantly increases simultaneously, and the foam entrainment rate becomes high, and difficult backward flow of liquid consequently the rate of recovery risees gradually.
Fig. 2 is a graph showing the effect of temperature on foam separation of dioscin, and as shown in fig. 2, the enrichment ratio is increased and then decreased with the increase of temperature, and the recovery rate is continuously decreased. When the temperature is lower, the solution viscosity and the foam stability are high, and the foam has higher liquid retention rate, so the recovery rate is higher. When the temperature is higher, the foam liquid holding rate is reduced, the content of the dioscin carried by the foam layer is reduced, and therefore the recovery rate is reduced.
FIG. 3 is a graph showing the effect of air velocity on foam separation of dioscin; as shown in FIG. 3, the enrichment ratio decreases with increasing gas velocity, and the recovery ratio increases and then decreases. When the gas velocity is low, the retention time of the foam in the separation column is long, the bubbles are sufficiently gathered and discharged, and the carried dioscin is less, so that the enrichment ratio is high and the recovery rate is low. When the gas velocity is increased, the foam generation rate and the foam rising rate are increased, and the liquid holding rate is high, so that the recovery rate is increased.
Fig. 4 is a graph showing the effect of the liquid loading amount on the foam separation of dioscin, and as shown in fig. 4, when the liquid loading amount is small, the retention time of the foam in the separation column is long, and dioscin carried in the foam flows back to the stock solution to a large extent, so that the enrichment ratio is increased. When the liquid loading amount increases, the liquid holdup of the foam is high, and the entrained liquid in the foam is blown out before the backflow, so that the recovery rate increases.
3. Box-Behnken response surface optimization test
1) According to the results of fig. 1-4, the sample concentration (a), the temperature (B), the gas velocity (C) and the liquid loading amount (D) are taken as factors, the dioscin extraction yield is taken as an index, an optimization scheme is designed by adopting a response surface Box-Behnken center combined test, and the selection of four factors and three levels is shown in table 1.
TABLE 1 factor level design Table
Selecting concentration (A), temperature (B), gas velocity (C) and liquid loading amount (D) to carry out four-factor three-level Box-Behnken response surface experiment design, wherein the response surface experiment design and results are shown in Table 2.
Table 2 response surface experimental design and results
2) Establishment and inspection of regression model
After performing multiple linear regression fitting on the table 2, a regression equation is obtained and variance analysis is performed on the equation, and the variance analysis result is shown in tables 3 and 4.
Enrichment ratio (E) (+ 1.85-0.83A +0.15B-0.52C-0.21D-0.040AB +0.32AC +0.21AD-0.017BC +0.070BD +0.080 CD-1.083E-003A)2-0.050B2+0.23C2+0.50D2；
Recovery (R) ═ 89.13+2.69A +1.96B +0.87C +1.19D-0.54AB-3.02AC-4.24AD +2.62BC-6.70BD-5.33CD-1.50A2-6.80B2-1.89C2-3.11D2。
TABLE 3 Linear regression Table of enrichment ratios
Note: "x" indicates extreme significance (P < 0.01); "" indicates significance (0.01 < P < 0.05); not significant (P > 0.1)
TABLE 4 Linear regression table of recovery
As can be seen from tables 3 and 4, the F values in the enrichment ratio and the recovery rate model reach 42.34 and 11.27 respectively, which reach significant levels, while the R value of the enrichment ratio reaches2At 0.9769, R of recovery20.9185, corrected determination coefficient R of the enrichment ratio and recovery after correction2 Adj0.9539 and 0.8370 respectively, the results show that the model fits well with practical experiments, and that 94.92% of response values can be explained for the enrichment ratio and 91.85% of response values can be explained for the recovery ratio. The F value of the mismatching term of the enrichment ratio is 1.56, the P value is 0.3544, the probability of the F value of the mismatching term caused by errors is 35.44%, the F value of the mismatching term of the recovery ratio is 1.33, the P value is 0.4218, the probability of the F value of the mismatching term caused by errors is 42.18%, and the models of E and R both indicate that the mismatching term is not obvious to pure errors, show that the correlation between the predicted value and the value measured under the actual condition is relatively good, and the test error is small, so the method can be used for prediction in a design range. As can also be seen from tables 3 and 4, for the enrichment ratio model: AD. C2All are significant, A, B, C, D, AC, D2The factor is extremely remarkable, and according to the magnitude of the F value, the main effect relationship of the factor is as follows: a > C > D>B; for the recovery model: A. AD, BD, CD, B2、D2The factor is extremely obvious, B, AC and BC are obvious, and according to the magnitude of the F value, the main effect relationship of the factors is as follows: a > B > D > C.
3) Response surface analysis and determination of optimal process conditions
The preparation parameters of the dioscin in the fenugreek are optimized by using Design-expert 8.0.6 software, interaction of the two factors is researched, the obtained results are shown in figures 5-8 respectively, and the optimal purification process parameters of the dioscin foam separation obtained by calculation of figures 5-8 are as follows: the sample loading concentration is 0.03mg/mL, the temperature is 28.36 ℃, the gas velocity is 450mL/min, the liquid loading amount is 400mL, under the condition, the recovery rate of the dioscin is 88.31%, and the enrichment ratio is 3.65.
In order to verify and predict the reliability of the regression model of the enrichment ratio and the recovery rate, from the actual situation, the optimal conditions are adjusted as follows: the sample loading concentration is 0.03mg/mL, the temperature is 28 ℃, the gas velocity is 450mL/min, and the liquid loading amount is 400 mL. Under the conditions (i.e. the conditions of example 1), the recovery rate of the obtained dioscin calculated according to the formulas (1) and (2) is 86.57%, the enrichment ratio is 3.82, and is close to the predicted value (the recovery rate is 88.31%, the enrichment ratio is 3.65) calculated by adopting Design-Expert 8.0.6 software, which indicates that the optimal process conditions obtained by the Design-Expert 8.0.6 Design software are feasible.
4. Chromatography analysis
1) Preparing a standard substance solution of 124 mug/mL with methanol from 0.0062g of dioscin standard substance; a proper amount of standard solution is taken to be scanned in a full-wave band by an ultraviolet spectrophotometer, and the result shows that 208nm can be used as the optimal chromatographic separation wavelength.
Dissolving dioscin prepared in example 1 in methanol to obtain a test solution (concentration of 62. mu.g/mL);
respectively sampling the sample solution, the standard solution and the negative sample (pure methanol) into preparative high performance liquid chromatogram, and obtaining a P-HPLC chromatogram shown in figure 9; wherein a is a standard sample, b is a test sample, and c is a negative sample; as shown in fig. 9, the peak-off time of the standard sample is 15.62min, the peak-off time of the sample is 15.57min, and the negative sample only contains a solvent peak, which indicates that the pure product obtained by preparative high performance liquid chromatography separation of the present invention is dioscin.
2) Receiving sample components flowing out of the sample and the sample in section 4 in the peak time period by using a container, sequentially performing freeze drying and methanol redissolution, and respectively determining the samples by using a high performance liquid chromatograph, wherein pure methanol is used as a negative sample, and the conditions of the high performance liquid chromatograph are as follows: a chromatographic column: hedera ODS-2 column (4.6X 250mm, 10 μm), mobile phase: water (a) and acetonitrile (B); gradient elution was performed: 0-5 min, 5-15% of B; 5-15 min, 15-45% of B; 15-35 min, 45-100% B, and the detection wavelength is 208 nm; the sample injection amount is 20 mu L; the flow rate is 0.8 mL/min; the column temperature is 28 ℃; the results are shown in FIG. 10, where a is the standard, b is the test sample, and c is the negative sample; as shown in fig. 10, the peak-off time of the sample was 23.26min, and the peak-off time of the sample was 23.03min, which indicates that the sample obtained by preparative hplc purification was dioscin.
5. Fourier Infrared Spectroscopy (FI-IR) determination
Appropriate amount of dioscin sample and dioscin sample prepared in example 1 were mixed with KBr, ground into powder and pressed into thin sheets, and then Nicolet iS50 type FT-IR was used as the whole band (4000--1) Scanning, and obtaining the result shown in figure 11; as can be seen from FIG. 11, the dioscin extraction sample is substantially consistent with the standard map, and the standard map and the sample are 3859-3738cm-1Has absorption at the position, the absorption peak is-OH stretching vibration absorption peak, 3355-3428cm-1The treated standard product and the sample both have wider absorption peaks at 2930cm-1Has a strong absorption peak which is a C-H stretching vibration peak on a six-membered ring. This is obtained by comparing the two peaks in the graph: 1380cm-1Is represented by-CH3An absorption peak; 1600--11300-1000cm out-of-plane bending vibration with absorption peak of C-H-1Is the strong absorption vibration peak of C-O. The infrared spectrogram analysis shows that the patterns of the dioscin sample prepared in the example 1 are basically consistent with those of the standard product, which indicates that the purification method is good and the extract has high purity.
6. High Resolution Mass Spectrometry (HRMS) assay
After dissolving the dioscin sample prepared in example 1 in methanol, the solution was dissolved in solvent (Aglient 7250)&The JEOL-JMS-T100 LPACuTOF high resolution mass spectrometer measures the mass spectrum, and the obtained result is shown in figure 12; as can be seen from FIG. 12, a molecular ion peak of m/z 891.47128 appears in the sample, which is nearly identical to the previous experimental results (the research results of Shimahong, etc. indicate that the dioscin can obtain an excimer ion peak of m/z 891.9 after primary full scan, Shimahong, Rishucary, Cao Xizhen, etc.. the LC-MS-n analysis of in vivo metabolites after intravenous injection of methylprotodioscin to rats [ J-MS-n analysis]Analytical test journal 2014,33(09): 1010-. After the structural formula of the dioscin is drawn in Chemdraw software, the dioscin is subjected to simulation analysis to obtain the mass-to-charge ratio of 868.48, the mass-to-charge ratio which is closest to the dioscin is 869.48952 as can be known from a mass spectrogram 12 of a sample, and the molecular charge amount corresponding to the mass-to-charge ratio is calculated to be 1 according to a main peak and adjacent peaks, so that the molecular charge amount is equal to that of a target compound [ M + H ]]+The molecular weight of (c) corresponds to.
7. Measurement of Nuclear magnetic resonance carbon Spectroscopy (13C-NMR) and Hydrogen Spectroscopy (1H-NMR)
Dissolving the dioscin sample prepared in the example 1 in deuterium-enriched methanol, and measuring the dioscin sample by using a BRUKERAVANCE400 nuclear magnetic resonance spectrometer, wherein the obtained result is shown in figures 13-14;
as shown in fig. 13, the nuclear magnetic hydrogen spectrum data of dioscin: apart from the solvent peaks, no significant signal was found in the 5.41, 5.23, 4.59, 4.53, 4.51, 4.44, 4.43,4.41, 4.39, 4.17, 4.15, 4.14, 4.13, 3.95, 3.94, 3.94, 3.91, 3.85, 3.83, 3.80, 3.69, 3.67, 3.65, 3.64, 3.61, 3.59, 3.57, 3.54, 3.52, 3.48, 3.46, 3.45, 3.44, 3.43, 3.42, 3.40, 3.39, 3.37, 3.34, 3.33, 3.33, 2.48, 2.46, 2.34, 2.32, 2.29, 2.05, 2.01, 1.94, 1.93, 1.88, 1.81, 1.70, 1.79, 1.70, 1.9, 1.9.9.9, 1.9, 1.9.9, 1.9.9.9.9.9.9.9.9.9.9, 1.9.9.9, 1.9.9, 1.9, 1.9.9.9.9.9.9.9.9, 1.9.9.9.9.9.9.9.9.6, 1..
As shown in fig. 14, the nuclear magnetic carbon spectrum data of dioscin: 141.89(5), 122.63(6), 110.58(22), 103.00(Rh 103.00-1), 103.00(Rh 103.00-1), 100.46(Glc-1), 103.00 (16), 103.00 (Glc-2), 103.00 (Glc-3), 103.00 (3/Glc-5), 103.00 (Glc-4), 103.00(Rh 103.00-4), 73.72(Rh 103.00-4), 103.00(Rh 103.00-103.00), 103.00(Rh 103.00-3), 72.17(Rh 103.00-2), 70.66(Rh 103.00-5), 69.77(Rh 103.00-5), 103.00 (26), 103.00 (Glc-6), 103.00 (17), 57.80(14), 103.00 (9), 103.00 (20), 41.42(13), 103.00 (12), 39.50(4), 103.00 (1), 103.00 (10), 33.18(23), 33.80 (32), 30.32 (32), 30.19.32 (31.32), 30.19), 30.42 (19.19), 30.32 (19), 31.42 (19), 31.32 (19), 31.42 (19), 17.99(27), 17.87(Rh2-6), 17.50(Rh1-6), 16.78(21), 14.90 (18). Substantially all carbons in the molecular structure can find a corresponding signal in the map.
As can be seen from fig. 13 to 14, the carbon atom and the hydrogen atom in the dioscin prepared in example 1 can find corresponding signals in the carbon spectrogram (fig. 14) and the hydrogen spectrogram (fig. 13) of the sample, and the mass spectrogram (fig. 12) is combined to illustrate that the sample extracted and purified by the method of the present invention is dioscin.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The preparation method of the dioscin in fenugreek is characterized by comprising the following steps:
mixing the fenugreek powder with an alcohol solvent, and extracting to obtain an extracting solution;
performing foam separation on the extracting solution to obtain a dioscin crude product;
and separating and purifying the dioscin crude product by using preparative high performance liquid chromatography to obtain the dioscin.
2. The method of claim 1, wherein the alcohol solvent comprises an ethanol solution; the mass concentration of the ethanol solution is 60-80%; the mass ratio of the fenugreek powder to the volume of the alcohol solvent is 1g (20-40) mL.
3. The method according to claim 1, wherein the pressure of the extraction is 300 to 400MPa, and the time is 6 to 12 min.
4. The method according to claim 1, further comprising, after the extracting, subjecting the extracted material solution to suction filtration under reduced pressure and concentration in this order.
5. The method of claim 1, wherein the conditions for foam separation are: the sample loading concentration is 0.01-0.05 mg/mL, the temperature is 25-45 ℃, the gas velocity is 350-550 mL/min, and the liquid loading amount is 250-450 mL.
6. The method of claim 5, wherein the conditions for foam separation are: the sample loading concentration is 0.03mg/mL, the temperature is 28 ℃, the gas velocity is 450mL/min, and the liquid loading amount is 400 mL.
7. The method of claim 1, wherein the chromatographic conditions of preparative high performance liquid chromatography comprise: mobile phase: water and acetonitrile; acetonitrile with the volume fraction of 5-15% is 0-5 min; 5-15 min, and 15-45% acetonitrile by volume fraction; 15-35 min, and the volume fraction is 45-100% of acetonitrile; the flow rate is 10 mL/min; the sample volume was 5 mL.
8. The method according to claim 1 or 7, wherein the chromatographic conditions of preparative high performance liquid chromatography further comprise: a chromatographic column: hedera ODS-2 column, 20X 250mm, 10 μm; the detection wavelength is 208 nm.
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