CN111116700A - A kind of extraction, separation and purification method of diosgenin from chrysanthemum leaf - Google Patents
A kind of extraction, separation and purification method of diosgenin from chrysanthemum leaf Download PDFInfo
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- CN111116700A CN111116700A CN201911165111.7A CN201911165111A CN111116700A CN 111116700 A CN111116700 A CN 111116700A CN 201911165111 A CN201911165111 A CN 201911165111A CN 111116700 A CN111116700 A CN 111116700A
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- mobile phase
- methanol
- dioscorea
- diosgenin
- column chromatography
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- 238000000605 extraction Methods 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 37
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 title claims abstract description 24
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 238000000926 separation method Methods 0.000 title claims abstract description 20
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 title claims abstract description 17
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Abstract
本发明公开了一种菊叶薯蓣皂苷的提取与分离提纯方法。所述总皂苷包括薯蓣皂苷、甲基原薯蓣皂苷、皂苷Pb和原皂苷Pb，具体提取方法为：菊叶薯蓣的块茎洗净，自然晾干后切片，然后冻干、粉碎成粉末，并过筛，然后将菊叶薯蓣冻干粉采用乙醇浸泡提取，并进行微波处理，即可得到总皂苷的提取液。本发明以微波辅助提取技术来提取菊叶薯蓣中的总皂苷，通过优化微波提取条件，实现菊叶薯蓣中总皂苷的高效提取，总皂苷得率为8.93mg/g；同时通过采用特定固定相的柱层析，以及流动相的梯度洗脱，分离得到4种甾体皂苷，不仅其得率高，其含量分别为61.1mg/g、4.5mg/g、39.9mg/g和0.84mg/g，而且分离过程简单、快速，分离效果好，分离得到的皂苷纯度高。
The invention discloses a method for extracting, separating and purifying diosgenin from chrysanthemum leaves. Described total saponins include diosgenin, methyl diosgenin, saponin Pb and protosaponin Pb, and the specific extraction method is as follows: the tubers of Dioscorea chrysanthemum are washed, dried naturally, sliced, then freeze-dried, pulverized into powder, and subjected to filtration. Then, the lyophilized powder of Dioscorea japonica is soaked and extracted with ethanol, and subjected to microwave treatment to obtain the extract of total saponins. The invention uses microwave-assisted extraction technology to extract the total saponins in Dioscorea chrysanthemum, and realizes the efficient extraction of total saponins in Dioscorea chrysanthemum by optimizing the microwave extraction conditions, and the total saponin yield is 8.93 mg/g; at the same time, by using a specific stationary phase Column chromatography and gradient elution of mobile phase, four kinds of steroidal saponins were separated and obtained, not only their yields were high, but their contents were 61.1mg/g, 4.5mg/g, 39.9mg/g and 0.84mg/g respectively , and the separation process is simple and fast, the separation effect is good, and the saponin obtained by separation is high in purity.
Description
Technical Field
The invention relates to the technical field of separation and extraction of saponin in dioscorea composita, and more particularly relates to a method for extracting, separating and purifying dioscorea composita saponin.
Background
Dioscorea composita Hemsl, native to Mexico, has high content of diosgenin and starch, and is an important plant resource for producing diosgenin in America. The steroid saponin component in the dioscorea composita is stable, the yield is high, the planting advantages are obvious, and the comprehensive utilization value is high. The utilization technologies of the dioscorea composita at different levels are researched, the processing system of the dioscorea composita is perfected, the utilization channels of dioscorea composita resources are increased, the comprehensive utilization efficiency of the resources is improved, and technical and theoretical supports are provided for promoting the comprehensive utilization and industrialization of the dioscorea composita resources.
Dioscin is a main active ingredient in Chinese herbal medicines for treating cardiovascular and cerebrovascular diseases, has activity in aspects of antibiosis, immunity enhancement, lipid reduction, tumor resistance, virus resistance and the like, and is directly used as a medicine for treating the cardiovascular and cerebrovascular diseases; the pharmaceutical composition is used as a raw material medicine for improving coronary insufficiency, is approved to be put on the market at home, and has wide market prospect. The analysis of the dioscin components shows that the content of the total saponin in the dioscin is about 10% (W/D), and the content of the dioscin is the highest; in per gram of dry matter of the raw materials, the content of the dioscin reaches 61.4mg/g, and the development value is high.
The basic process of utilizing natural effective components includes extraction and preparation. According to different research purposes and characteristics of raw materials, different strategies and means are adopted. The tuber raw material has complex components, wherein the plant structural components and the stored substances are more, the extraction obstruction is large, and the saponin polarity is large, so the traditional method has low saponin extraction efficiency and more extraction impurities. The microwave is an electromagnetic wave with the frequency of 0.3-300 GHz and the wavelength of 30 cm-1 mm, is used as an energy source for auxiliary heating, drying, sterilization and the like, is used for auxiliary extraction in recent years, becomes a novel efficient extraction method, and has the characteristics of short time, high speed, uniform heating, energy conservation, high efficiency and easiness in control compared with the traditional extraction technology. The natural active substances contain a certain amount of impurities after extraction, and in order to further improve the purity and value of the product, the extracted substances are often required to be purified and prepared. The purification preparation has different complexity according to different sources of the extract, and different methods are adopted; the steroid saponin has high polarity, and most of the components in the impurities are polar substances. Macroporous resin has strong surface adsorption capacity and molecular sieve effect, and is often used for separation and preparation of saponin.
However, at present, there is no record on how to extract the total saponins and the dioscin from the dioscorea composita at a high yield, and detailed research is needed.
Disclosure of Invention
The invention aims to provide a method for extracting total saponins from dioscorea composita. The invention extracts the total saponin in the dioscorea composita by a microwave-assisted extraction technology, obtains a simple, rapid and clean extraction method by optimizing microwave extraction conditions and an extraction solvent, and has high extraction rate of the total saponin, namely 8.93 mg/g.
The invention also aims to provide a method for separating and purifying dioscin from dioscorea composita.
The above object of the present invention is achieved by the following scheme:
a method for extracting total saponins from Dioscorea composita, wherein the total saponins comprise dioscin, methyl protodioscin, saponin Pb and protosaponin Pb, and the specific extraction method comprises the following steps: washing tuber of Dioscorea composita, naturally air drying, slicing, freeze drying, pulverizing into powder, sieving, soaking Dioscorea composita freeze-dried powder in ethanol, extracting, and microwave treating to obtain total saponin extract.
Preferably, the mass concentration of the ethanol is 70%; the ratio of the material to the liquid in the ethanol soaking process is 1: 16; the power of the microwave is 340W.
The invention also provides a preparation method of dioscin in dioscorea composita, which comprises the following steps:
s1, washing tubers of dioscorea composita, naturally airing, slicing, freeze-drying, crushing into powder, and sieving;
s2, soaking and extracting dioscorea composita freeze-dried powder by using ethanol to obtain an ethanol extracting solution; then adding a n-butanol aqueous solution for extraction to obtain an ethanol aqueous solution and a n-butanol aqueous solution;
s3, separating the ethanol water solution obtained in the step S2 by adopting column chromatography, wherein a mobile phase is methanol, and performing gradient elution to obtain a W1-W4 fraction;
s4, separating the fraction W1 by adopting column chromatography, wherein mobile phases are dichloromethane and methanol, and performing gradient elution to obtain two fractions W11 and W12;
s5, further separating the fraction W11 by column chromatography, wherein mobile phases are dichloromethane and methanol, and performing gradient elution to obtain 57;
s6, further separating the fraction W12 by column chromatography, wherein mobile phases are dichloromethane and methanol, and performing gradient elution to obtain 4;
s7, carrying out liquid phase separation on the n-butanol aqueous solution obtained in the step S2, and carrying out gradient elution on a mobile phase which is a methanol aqueous solution to obtain B1-B4 fractions;
s8, separating the fraction B2 by adopting column chromatography, and performing gradient elution on dichloromethane and methanol serving as mobile phases to obtain 5;
s9, separating the fraction B3 by adopting column chromatography, and carrying out gradient elution on dichloromethane and methanol serving as mobile phases to obtain the compound 3.
Preferably, in step S2, the ethanol mass concentration is 90%, and the ethanol soaking extraction process is performed 3 times.
Preferably, in step S2, the volume ratio of n-butanol to water is 1: 1.
Preferably, in step S3, the stationary phase of column chromatography is Sephadex LH 20.
Preferably, in step S4, the stationary phase of column chromatography is silica gel, and the gradient elution process is as follows: the mobile phase starts from pure dichloromethane, and the volume ratio of methanol is increased in turn until the mobile phase is pure methanol.
Preferably, in step S5, the stationary phase of column chromatography of fraction W11 is silica gel; the mobile phase starts from pure dichloromethane, and the volume ratio of methanol is increased in turn until the mobile phase is pure methanol.
Preferably, in step S6, the stationary phase of column chromatography of fraction W12 is silica gel; the mobile phase starts from pure dichloromethane, and the volume ratio of methanol is increased in turn until the mobile phase is pure methanol.
Preferably, in step S8, the stationary phase of column chromatography of fraction B2 is silica gel; the mobile phase starts from pure dichloromethane, and the volume ratio of methanol is increased in turn until the mobile phase is pure methanol.
Preferably, in step S9, the stationary phase of column chromatography of fraction B3 is silica gel; the mobile phase starts from pure dichloromethane, and the volume ratio of methanol is increased in turn until the mobile phase is pure methanol.
Compared with the prior art, the invention has the following beneficial effects:
the invention extracts the total saponin in the dioscorea composita by a microwave-assisted extraction technology, realizes the high-efficiency extraction of the total saponin in the dioscorea composita by optimizing microwave extraction conditions, and the yield of the total saponin is 8.93 mg/g;
because the polarities of the 4 steroid saponins in the dioscorea composita are relatively close and the separation is difficult to be good, the invention adopts column chromatography of a specific stationary phase and gradient elution of a mobile phase to separate and obtain 4 steroid saponins, namely dioscin, methylprotodioscin, saponin Pb and protosaponin Pb, the yield is high, the contents of the steroid saponins are respectively 61.1mg/g, 4.5mg/g, 39.9mg/g and 0.84mg/g, the separation process is simple and rapid, the separation effect is good, and the purity of the saponins obtained by separation is high.
Drawings
FIG. 1 is an absorption curve of dioscin color development.
Fig. 2 is a standard curve of dioscin.
Fig. 3 is a graph of the effect of microwave power on extraction efficiency.
FIG. 4 is a graph showing the effect of ethanol concentration on saponin extraction yield.
FIG. 5 shows the effect of feed liquid ratio on saponin extraction yield.
FIG. 6 is a graph of the response curve of the total saponins extracted from Dioscorea composita.
Figure 7 is a flow chart of steroid saponin separation.
Figure 8 is an HPLC profile of a steroid saponin standard.
FIG. 9 is HPLC chromatogram of dioscin.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Dioscorea composita Hemsl, 2 years old, available from Hippocampus Huake Biotech Co., Ltd, of south China agricultural university, was identified as Dioscorea composita Dioscorea (Dioscorea) Dioscorea (Dioscorea composita) Dioscorea by energy plant resources and utilization focus laboratory. Cleaning tuber, naturally drying, slicing, freeze drying, pulverizing, and storing in a dryer.
The reagents and apparatus used in the following tests are shown in tables 1 and 2, respectively.
TABLE 1 reagents used
TABLE 2 Experimental instruments adopted
| Name of instrument | Model number | Manufacturer of the product |
| Circulating water vacuum pump | SHZ-3 | Shanghai Yangrong |
| Rotary evaporator | IKA |
German IKA/moxa-moxibustion |
| Preparative chromatography system | CHEETAH | Tianjin Bonnajiel |
| High performance liquid chromatography | LC-20A | Shimadzu |
| Ultraviolet visible light photometer | UV-2600 | Shimadzu |
| Ultraviolet detector | SPD-20A | Shimadzu |
| Evaporation light detector | ELSD-10A | Shimadzu |
| Nuclear magnetic resonance spectrometer | AVANCEⅢHD | Bruker, Switzerland |
| Mass spectrum detector | LCMS-8050 | Shimadzu |
| Constant temperature water bath kettle | B-260 | Shanghai Yangrong |
| High-speed centrifugal machine | Centrifuge 5920R, refrigerated | Eppendorf |
| Automatic fraction collector | BSZ-160 | Shanghai horizontal duty |
| Ultrapure water system | simplicity | Milipore USA |
| Air pump | PM200 | huaaobio |
| Analytical balance (thousandth) | BL-120F | Setra |
| Ultrasonic cleaning machine | CC-4112-01 | Sub-tachyphylaxis |
| Automatic Soxhlet extractor | SER158/6 | VELP |
Example 1 isolation of Total saponins from Dioscorea composita
The main saponins in dioscorea composita are 4 kinds of saponins, namely dioscin, methyl protodioscin, saponin Pb and protosaponin Pb, so the total saponins in dioscorea composita are limited to the 4 kinds of saponins, and the following example is a high-yield method for obtaining the 4 kinds of saponins by optimizing the separation process.
1. Alcohol extract of dioscorea composita
Weighing 960.0g Dioscorea composita freeze-dried powder, placing in 5.0L conical flask, adding 3.0L 90% ethanol, soaking at room temperature for 24h, extracting with the same volume of 90% ethanol for three times, and filtering to obtain extractive solution.
2. Determination of saponin content:
(1) the determination is carried out by adopting a saponin perchloric acid color development method, and the specific process is as follows: placing 200 μ L of the extractive solution in a glass test tube with a plug by using a 200 μ L pipette, volatilizing the solvent at 70 deg.C, and cooling to room temperature; adding 5mL perchloric acid, fully and uniformly mixing, and carrying out water bath at 70 ℃ for 20 min; cooling in ice bath for 5 min; the absorbance was measured at the maximum absorption wavelength with an ultraviolet-visible spectrophotometer.
(2) Determination of the absorption Curve
The steroid saponin can generate a series of color reaction with strong acid under the anhydrous condition, and the content of the steroid saponin can be determined according to the absorption of visible light or ultraviolet of a color substance. In this experiment, dioscin is used as a standard for color reaction, and after perchloric acid color development, the maximum absorption peak is at 414nm (as shown in figure 1), so 414nm is selected as the absorption wavelength of total saponin measurement.
(3) Establishment of a Standard Curve
Taking 0.0625mL, 0.125mL, 0.25mL, 0.5mL and 1.0mL of dioscin standard solution with the concentration of 1.0mg/mL respectively in a 10mL test tube with a plug, using perchloric acid as a blank after color development according to a color development method, measuring absorbance at 414nm, and drawing a standard curve (shown in figure 2), wherein the linear concentration range of the dioscin is 31.2 mu g/mL-2.0 mg/mL, and the linear regression curve is that y is 0.000206x + 0.003753; coefficient of correlation R20.9998. The experiment takes the dioscin after color development as a standard, and the calculated value of the total saponin is the equivalent of the dioscin.
(4) Microwave extraction single factor analysis
The essence of solvent extraction of natural products is a mass transfer process of solute transfer from a solid phase to a liquid phase, the theoretical basis is diffusion, and the process generally includes three processes of solvent permeation, solute resolution and dissolution and diffusion; the factors influencing the extraction process mainly include solvent and extraction conditions. In the microwave-assisted extraction, the main factors influencing the extraction efficiency comprise three factors of a solvent, microwave power and a feed-liquid ratio; in order to obtain the maximum extraction efficiency, three factors for extracting the total saponin in the dioscorea composita by microwave are optimized. Firstly, performing single factor analysis on the three factors to determine an optimization range; then, the three factors are optimized through response surface analysis, and the optimal extraction condition is obtained.
1. Influence of microwave power on total saponin extraction
In the microwave extraction process, polar substances, particularly water molecules in the material absorb energy, are partially vaporized to generate impact pressure, so that the microstructure in the material is damaged, and the processes of three extraction processes are accelerated; however, disruption of excessively high microstructure can alter the raw material matrix properties (e.g., starch gelatinization, etc.), and can reduce the efficiency of extraction. The microwave power of 100W, 200W, 300W, 400W and 500W is examined, and other conditions are the conditions in the microwave-assisted extraction of Curcuma rhizome-Curcuma rhizome in the reference literature, wherein the material-liquid ratio is 1:10, the ethanol concentration is 80%, and the extraction time is 5 min.
The experimental result is shown in fig. 3, and it can be seen from the graph that, at low microwave power, the total saponin extraction yield of dioscorea composita is improved along with the increase of microwave power, and reaches a maximum of 6.24mg/g at 300W, which corresponds to that the higher the microwave power, the higher the effect of polar molecules for rapidly absorbing energy is, the destruction of the microstructure of dioscorea composita powder is promoted, the penetration of ethanol in the raw material and the dissolution and diffusion of saponin are accelerated, so that the total saponin extraction yield can be rapidly improved in unit time. When the microwave power is more than 300W, the extraction yield tends to be flat and slightly reduced; the reason is that the dioscorea composita contains a large amount of starch, polar molecules, particularly water, cause too high temperature due to too high microwave power after absorbing microwave energy, and further cause gelatinization and dissolution of starch, so that the original microstructure collapses, and the positive phase effect of high-power microwave on improving the dissolution and diffusion speed of saponin is weakened to a certain extent.
2. Influence of ethanol concentration on Total Saponin extraction
In the natural product extraction process, the main influence factors of the extraction efficiency are three factors of materials, solvents and extraction conditions; wherein the solvent determines the extractionIf the solvent is sufficient, the components extracted by different solvents are different. When selecting the solvent, the structure and physical and chemical properties of the extract generally need to be considered, and the solvent can dissolve the ineffective components to the maximum extent and dissolve the ineffective components to the low extent. The obtained four steroidal saponins have molecular polar surface area of 200A2In the above, the polarity of the molecule is large, so ethanol is selected as the extraction solvent. Ethanol as a semi-polar solvent, which is intermediate between polar and non-polar solvents, is used to adjust the polarity by mixing with water to obtain the best extraction solvent. In the experiment, ethanol with different concentrations is used as an extraction solvent, the material-liquid ratio is 1:10, the microwave power is 200W, and the extraction time is 5 min.
The experimental results are shown in fig. 4, and it is known that the ethanol concentration is increased from 50% to 70%, the total saponin extraction yield is increased, and the highest ethanol concentration is reached when the ethanol concentration is 70%; the solubility of the saponin in the dioscorea composita is improved along with the increase of the concentration of ethanol, and is the maximum in 70% ethanol; when the ethanol concentration is further increased, the extraction rate of the total saponins is rapidly reduced, wherein the reduction range of the extraction yield of the saponins with 90% of the ethanol concentration is the largest. It is believed that the solubility of the solvent is the main factor affecting the extraction yield, whereas the solubility of the saponin can also be described by the extraction yield; the saponin in the dioscorea composita is biased to polarity in an ethanol water system through an extraction yield curve.
3. Influence of feed liquid ratio on total saponin extraction
The feed-liquid ratio is the ratio of the solvent usage amount to the raw materials in the extraction process, determines the effective concentration of the final extracted target substance, and influences the subsequent solvent recovery. Selecting 70% ethanol, 200W microwave power, 15ml extraction volume, 5min extraction time, and setting material-liquid ratio of 1:5, 1:10, 1:20, 1:30, 1:40 respectively; the results of the experiment are shown in FIG. 5.
The comparison shows that the extraction yield of the total saponins is highest when the ratio of the feed to the liquid is 1:20, and when the ratio of the feed to the liquid is too small, the total saponins cannot be fully infiltrated and dissolved under the influence of the saturated solubility of the solvent, so that the extraction yield is lower; when the ratio of the material to the liquid is more than 20, the extraction rate is already gentle and is not increased any more, and compared with the total saponin concentration in the extracting solution, the concentration of the total saponin in the extracting solution is reduced, and the relative extraction efficiency of the solvent is reduced.
4. Microwave extraction response surface optimization
According to a single-factor result of microwave extraction of the total saponins of dioscorea composita, in order to explore an optimal microwave-assisted extraction process, the microwave power (W), the feed-liquid ratio and the ethanol concentration are used as objects to be investigated, a Box-Behnken response surface method with 3-factor 3 level is adopted to optimize the total saponins extraction process of dioscorea composita, and the extraction factor level (Table 3); extraction process parameters experimental arrangement and results (table 4).
TABLE 3 level chart for extracting experimental design factors
Table 4 experimental design and results
The experimental results were subjected to data processing using "Design Expert 11.0" experimental software, taking the total saponin extraction yield as an evaluation index (dependent variable), performing multiple linear regression analysis and binomial equation fitting on each factor, and performing F-test on each coefficient in the equation, the results being shown in table 5.
TABLE 5 analysis of model variance
| Sources of variance | Sum of squares | Degree of freedom | Mean square error | F-value | p-value | |
| Model | 5.41 | 9 | 0.6015 | 18.17 | 0.0005 | significant |
| A | 1.98 | 1 | 1.98 | 59.81 | 0.0001 | |
| B | 0.1922 | 1 | 0.1922 | 5.81 | 0.0468 | |
| C | 0.0968 | 1 | 0.0968 | 2.92 | 0.1310 | |
| AB | 0.0169 | 1 | 0.0169 | 0.5105 | 0.4980 | |
| AC | 0.1089 | 1 | 0.1089 | 3.29 | 0.1126 | |
| BC | 0.0004 | 1 | 0.0004 | 0.0121 | 0.9156 | |
| A2 | 1.69 | 1 | 1.69 | 51.04 | 0.0002 | |
| B2 | 0.1342 | 1 | 0.1342 | 4.05 | 0.0840 | |
| C2 | 0.9440 | 1 | 0.9440 | 28.52 | 0.0011 | |
| Residual error | 0.2317 | 7 | 0.0331 | |||
| Missimilitude term | 0.2270 | 3 | 0.0757 | 64.69 | 0.0008 | significant |
| Pure error | 0.0047 | 4 | 0.0012 | |||
| Cor Total | 5.65 | 16 |
After the Design Expert 11.0 software is processed and fitted, the obtained multivariate binomial regression equation is as follows:
Y＝-20.02+-0.0026375×A+0.31275×B+0.76925×C-0.00044×AB+0.0003725×AC+0.00595×BC-2.45e-05×A2-0.0172×B2-0.007125×C2. The F value of the model is 7.56, the P value is less than 0.0500, the model is obvious, the mismatching term is 0.227 and is not obvious, and the equation is well fitted.
For the relationship between independent variables and dependent variables as shown in fig. 6, it can be seen that the optimal conditions are: power 340W; the material-liquid ratio is 1: 16; ethanol concentration 70%, theoretical saponin yield: 8.93mg/g, the extraction yield was measured to be 8.82mg/g ± 0.04mg/g (n ═ 3) according to the optimum conditions for the simulation, and the deviation was 1.23%, and it was found that the actually observed value was relatively close to the predicted value.
Example 2
The main steroid saponins in dioscorea composita are as follows: dioscin, methylprotodioscin, saponin Pb and protosaponin Pb, and in the following examples, these 4 steroid saponins are mainly used as research targets.
1. Extraction and separation of steroid saponin of dioscorea composita
Weighing 960.0g Dioscorea composita freeze-dried powder, placing in 5.0L conical flask, adding 3.0L 90% ethanol, soaking at room temperature for 24h, extracting with the same volume of 90% ethanol for three times, and filtering to obtain extractive solution. The extract was concentrated at 40 ℃ by a rotary evaporator until no alcohol smell was observed, and then separated according to the flow of FIG. 7.
In fig. 7, among the finally separated substances, 3 represents dioscin; 5 represents methyl protodioscin; 4 represents saponin Pb; 7 contains protopanasaponin Pb.
Dissolving and separating the concentrated substance of the ethanol extracting solution obtained by extraction by using a normal butanol aqueous solution with the volume ratio of 1:1, and extracting to obtain an ethanol aqueous solution and a normal butanol aqueous solution;
concentrating an ethanol water solution, and separating by column chromatography, wherein a stationary phase is Sephadex LH20, and a mobile phase is methanol to obtain W1-W44 fractions; then separating the fraction W1 by column chromatography, wherein the mobile phase is dichloromethane and methanol, performing gradient elution, starting from pure dichloromethane, sequentially increasing the volume ratio of methanol until the mobile phase is pure methanol, and obtaining two fractions W11 and W12; separating the fraction W11 by column chromatography, gradient eluting with dichloromethane and methanol as mobile phase, sequentially increasing the volume ratio of methanol from pure dichloromethane until the mobile phase is pure methanol to obtain methylprotodioscin and protosaponin Pb; and further separating the fraction W12 by column chromatography, wherein the mobile phases are dichloromethane and methanol, performing gradient elution, and sequentially increasing the volume ratio of the methanol from the pure dichloromethane to the mobile phase which is pure methanol to obtain the saponin Pb.
Concentrating an n-butanol aqueous solution, separating by column chromatography, and performing gradient elution to obtain B1-B4 fractions, wherein the stationary phase is RpC18, and the mobile phase is a methanol aqueous solution; separating fraction B2 by column chromatography, gradient eluting with dichloromethane and methanol as mobile phase, sequentially increasing the volume ratio of methanol from pure dichloromethane until the mobile phase is pure methanol to obtain methyl protodioscin; and separating the fraction B3 by column chromatography, wherein the mobile phase is dichloromethane and methanol, performing gradient elution, and the volume ratio of methanol is sequentially increased from pure dichloromethane to pure methanol to obtain dioscin.
In the above separation process, the volume of the mobile phase solvent varies depending on the species of the substances in the fractions.
2. Establishment of method for measuring steroid saponin in dioscorea composita
Preparing a saponin standard solution: accurately weighing the separated steroid saponin standard substance, dissolving in a small amount of methanol solution, and diluting to constant volume with a 10mL volumetric flask to prepare a standard solution.
Establishment of HPLC detection conditions:
(1) establishment of Detector Condition
The ultraviolet absorption of the steroid saponin is weak, the response value on an ultraviolet detector commonly used by HPLC is low, and the detection is insensitive when quantitative analysis is carried out. Evaporative light detector (ELSD), a universal type detector, differs from uv and fluorescence detectors in that the response of the ELSD is independent of the optical properties of the sample, and any sample with a volatility lower than that of the mobile phase can be detected without being affected by its functional groups. The ELSD response is proportional to the mass of the sample and can be used to determine the purity of the sample or to detect an unknown. The evaporative light scattering detector has higher sensitivity than a differential refractive detector, is insensitive to temperature change, has stable base line, and is suitable for being combined with gradient elution liquid chromatography. Most of the steroid saponins have similar polarity, are difficult to separate in HPLC isoconcentration elution, and mostly adopt a gradient elution mode. Principle of evaporative light scattering detector; atomizing a mobile phase to form aerosol, adding an evaporative hot solvent into a drift tube, and detecting non-volatile solute particles in a light scattering detection pool.
The response value of an Evaporative Light-scattering Detector (ELSD) is proportional to the mass of a sample, and thus can be used to determine the purity of the sample or detect an unknown substance, which is a general-purpose Detector; because the ultraviolet absorption of the steroid saponin is very weak, the ELSD is selected as a detector for separating the steroid saponin in the dioscorea composita. The evaporation temperature is optimized mainly, and the temperature with the best response to the steroid saponin is determined.
(2) Optimization of chromatographic conditions
① flow phase ratio
The detector is Shimadzu ELSD-10A, (Gain 6; 46 deg.C; atomizing gas N)2 Pressure 350 MPa); the chromatographic column is Shimazu-GL intersdustain C18 (30X 150mm, S/N8 AR 55067); the mobile phase is acetonitrile water, the elution mode is gradient elution, the flow rate is 0.4mL/min, and the sample volume is 10 mu L. The retention times of the four saponins (Dioscin, protodioscin, saponin Pb Asperin, and protosaponin Pb asperoide) in different elution systems are shown in table 6.
TABLE 6 Retention time T of four steroidal saponins in different concentrations in acetonitrilem(min)
The retention times of the four saponins were increased with decreasing acetonitrile ratio, and decreased when the acetonitrile ratio was below 30%, indicating that the retention times were affected by the flow rate of the mobile phase and the solubility of the sample in the mobile phase, in addition to the column packing of the chromatography column. When the acetonitrile proportion is 10%, the solubility of the four saponins in the mobile phase is low, and the retention time of the saponins on a chromatographic column is influenced; when 70% and 90% acetonitrile is used as a mobile phase, the distribution of the saponin between the mobile phase and a fixed phase is proper, so that the saponin has a remarkable retaining effect, and the concentration of 30% acetonitrile is optimal;
when the concentration of acetonitrile is 70% and 90%, the retention capacity of the stationary phase is weak due to strong polarity of saponin; thus, the retention time of the saponin on the column is very short and at this concentration of mobile phase the saponin is more prone to partition in the mobile phase.
② HPLC gradient elution of four saponins
Considering the retention time of the four steroidal saponins in different acetonitrile concentrations, the gradient elution program is set to 10-10-80-10-10 (acetonitrile concentration) time of 0-5-35-45-50(min), 4 standards are used, other conditions are the same, the chromatogram is shown in fig. 8, and the retention time of the four steroidal saponins is respectively shown in the figure: peak No. 4 is dioscin (T)m32.06 min); peak 3 is asperin (T)m31.43 min); peak 2 is protodioscin (T)m23.80 min); peak No. 1 is asperosaside (T)m21.67 min). The four saponins can be completely separated under the gradient elution condition, the peaks do not overlap, and the separation degree R is>1.5 meets the detection requirements of production and experiments.
(3) Methodology study
① creation of Standard Curve
In order to be able to carry out quantitative analysis of the four steroidal saponins, a standard curve was established using the four standards using the HPLC method defined above, and the results are shown in table 7. Linear regression analysis using origin established the linear equation, R20.9999, all have significant linear dependence.
TABLE 7 Standard curves for the four steroidal saponins
| Saponin | Linear equation of equations | Linear Range (mg/mL) |
| dioscin | y＝6,806,562.83x-92,551.50；R2＝0.9999 | 1.03～0.064 |
| asperin | y＝4,375,481.47x-25,081.71；R2＝0.9999 | 0.96～0.06 |
| 25-O-protodioscin | y＝3,454,774.12x+111,404.04；R2＝0.9999 | 1.0～0.0625 |
| asperoside | y＝3,733,218.53x-32,150.56；R2＝0.9999 | 1.33～0.08 |
② precision and accuracy
The sample was injected 6 times repeatedly under the HPLC chromatographic conditions described above, and the accuracy and precision of the detection method were analyzed, and the results are shown in Table 8.
Accuracy and precision of the method of Table 8
The concentration of the four steroid saponins is accurately measured to be 0.2mg/mL respectively, and the precision is expressed by Relative Standard Deviation (RSD) after sample introduction is repeated for 6 times; and the average relative error is used to measure the accuracy of the method. From the results, the RSD and average relative error of the four saponins are within 5.0%, and the precision and the accuracy are good.
(4) Result of separation
The steroid saponin components in the dioscorea composita are analyzed by the method, and 4 steroid saponin compounds are obtained by adopting Sephadex LH-20, ODS column chromatography and silica gel column chromatography step by step.
The structures of the 4 steroid saponins are shown in the following structural formula and are respectively identified as Dioscin (ygy 03) ((3 β, 25R) -spirost-5-en-3-yl O-6-deoxy- α -L-mannopyranosyl by various spectral data
- (1 → 2) -O- [6-deoxy- α -L-mannopyranosyl- (1 → 4) ] - β -D-Glucopyranoside), methylprotodioscin (25-O-protopyoscin, ygy05) β -D-Glucopyranoside, (3 β, 22 α, 25R) -26- (β -D-glucopyranosyloxy) -22-methoxyfurost-5-en-3-ylO-6-deoxy- α -L-mannopyranosyl
- (1 → 2) -O- [6-deoxy- α -L-mannopyranosyl- (1 → 4) ] -, saponin Pb (asperin, ygy04) (3 β, 25R) -Spirost-5-en-3-yl-6-deoxy- α -L-mannopyranosyl- (1 → 2) -O- [ O-6-deoxy- α -L-
mannopyranosyl- (1 → 4) -6-deoxy- α -L-mannopyranosyl- (1 → 4) ] - β -D-glucopyranoside, pro-saponin Pb (ygy 07) (3 β, 22 α, 25R) -26- (β -D-Glucopyranosyloxy) -22-
hydroxyfurost-5-en-3-yl O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-[O-6-deoxy-α-L
-mannopyranosyl-(1→4)-6-deoxy-α-L-mannopyranosyl-(1→4)]-β-D-glucopyranoside。
Structural formula of compound
Dioscorea opposita saponin
Methyl protodioscin
Saponin Pb
Prosaponin Pb
Structural analysis of 4 steroid saponins
(1) Dioscin (compound ygy-03) as white needle crystal, ESI-MS m/z: 870.1[ M + H]+，887.1[M+NH4]+，892.1[M+Na]+，908[M+K]+, relative molecular mass 869, molecular formula C45H72O16The structure (FIG. 2.2) was estimated from the NMR spectrum data.1In the H NMR (solvent: deuterated pyridine, 400MHz) spectrum, delta 0.71(3H, d, J ═ 3.6Hz, CH) appears on aglycone in high field3-27)，δ0.84(3H，s，CH3-18)，δ1.06(3H，s，CH3-19)，δ1.14(3H，d，J＝6.8Hz，CH3-21) four methyl groups, and δ 5.34(1H, s, CH-5) olefinic hydrogens. In addition, in delta 1.63(3H, d, J ═ 6.0Hz, CH)3-6(rha)) and δ 1.77(3H, d, J ═ 6.0Hz, CH)36(rha)) methyl groups on the two rhamnoses, indicating that the glycosidic chain has 2 rhamnoses, a hydrogen signal on the low field at δ 5.86(1H, s) and δ 6.40(1H, s) of the anomeric carbons of the two glycosidic chains.13In a C NMR (solvent: deuterated pyridine, 400MHz) spectrum, the signals of the terminal carbon atoms of the sugar units are delta 103.23, 102.38 and 100.62, and the glycosidic chain is trisaccharide; delta 109.61 position C-22, the aglycone is spirostanol aglycone; delta. 141.14 and 122.15 indirectly demonstrate a double bond at C-5. Specific nuclear magnetic data are shown in table 9.
TABLE 9 Nuclear magnetic data (Δ, ppm,400M) for the compound ygy-03 aglycone part
Glycoside chain of Compound ygy-03 (Table 10), by1H-1HCOSY (correction spectroscopy) and HMBC (1H detected conjugated multiple bond correlation), wherein the connection mode Glc (inner) is connected with C-3 of aglycone, C-3 is associated with C-1 of glucose on HMBC spectrogram, and aglycone connection is confirmed; rha (1 → 4) and Rha (1 → 2) are mainly confirmed by HMBC map association; and inside the sugar moleculeCarbon is located through1H-1Correlation between HCOSY and HMBC patterns, and correlation between carbon and hydrogen by HSQC (hydrogen and carbon dissociation) pattern, dioscin ((3 β, 25R) -spirot-5-en-3-ylO-6-deoxy- α -L-mannopyranosyl- (1 → 2) -O- [6-deoxy- α -L-mannopyranosyl- (1 → 4) in the literature]- β -D-Glucopyranoside) (Huang et al, 2009).
TABLE 10 Nuclear magnetic data (δ, ppm,400M) of the glycoside chain portion of Compound ygy-03
(2) Methylodioscin (compound ygy-04) white needle crystal, ESI-MS m/z: 1033.2[ M + NH4]+，1038.15[M+Na]+，1054.15[M+K]+Relative molecular mass of 1015 and molecular formula of C51H82O20And determining the structure by combining NMR spectrum data. The key signals of the aglycone are shown,1h NMR (solvent: deuterated pyridine, 400MHz) spectra showed that the aglycone moiety had four methyl groups, delta 0.71(3H, d, J ═ 3.6Hz, CH)3-27)，δ0.84(3H，s，CH3-18)，δ1.06(3H，s，CH3-19)，δ1.14(3H，d，J＝6.8Hz，CH3-21), olefinic hydrogen is δ 5.34(1H, s, CH-5); in addition, in13In the C NMR (solvent: deuterated pyridine, 400MHz) spectrum, the delta of 4 methyl groups is respectively as follows: 16.9(CH3-18), 19.9(CH3-19), 15.6(CH3-21), 17.9(CH 3-27); double bond high field δ 122.3 (CH-5); four quaternary carbons: δ 37.7(C-10), δ 141.3(C-5), δ 41.0(C-13), δ 109.8 (C-22); δ 67.4(CH-26) indicates spirostanes; delta. 78.6(CH-3) indicates the oxygen carbon of the aglycone. Identifying homonuclear correlation by HSQC, attributing hydrocarbon, and performing heteronuclear correlation on HMBC spectrogram and1H-1HCOSY spectrogram, structure identification is completed, and the sapogenin part is diosgenin.
Glycoside chain signalling (Table 11) in1In the H NMR (solvent: deuterated pyridine, 400MHz) spectrum, except for the hydrogen signal of sapogenin, the rest is about 4.0 of low field, three methyl signals are delta 1.60, delta 1.61 and delta 1.79, the methyl signals are on three rhamnoses, and delta 4.97, delta 5.85, delta 6.30 and delta 6.41 on the low field are the hydrogen signals of anomeric carbon of sugar.13In the C NMR (solvent: deuterated pyridine, 400MHz) spectrum, 4 delta 100.7, 102.5, 102.6 and 103.6 indicate that the glycosidic chain is tetrasaccharide; by passing1H-1HCOSY and HMBC confirmation, wherein the connection mode glc (inner) is connected with C-3 of aglycone, C-3 is associated with C-1 of glucose on the HMBC spectrogram, and the aglycone connection is confirmed; rha' (1 → 4), Rha (1 → 2) and Rha (1 → 4) are mainly confirmed by HMBC map association; while the carbon localization within the sugar molecule is by1H-1The correlation between HCOSY and HMBC patterns is confirmed, and the assignment of carbon and hydrogen is confirmed by the correlation between hydrogen and carbon on the HSQC pattern, namely the compound is asperin (3 β, 25R) -Spirost-5-en-3-ylO-6-deoxy- α -L-mannopyranosyl- (1 → 2) -O- [ O-6-deoxy- α -L-mannopyranosyl- (1 → 4) -6-deoxy- α -L-mannopyranosyl- (1 → 4)]-β-D-glucopyranoside asperin(Xiao et al.,2012)。
TABLE 11 Nuclear magnetic data (Δ, ppm,400M) for the glycoside chain portion of Compound ygy-04
(3) Saponin Pb (compound ygy-05) white needle crystals, ESI-MS m/z: 1072.2[ M + Na ]]+，1088.15[M+K]+, relative molecular mass 1063, molecular formula C52H86O22(ii) a The structure was determined by combining the NMR spectroscopic data (tables 12 and 13).1In the H NMR (solvent: deuterated pyridine, 400MHz) spectrum, delta 0.82(3H, s, CH) appears in the aglycon at a high field3-18)，δ1.02(3H，d，J＝6.8Hz,CH3-27)，δ1.05(3H，s，CH3-19)，δ1.21(3H，d，J＝6.8Hz，CH3-21) four methyl groups, and a methoxy group at C-22, δ 3.27(3H), indicating that the F ring of the saponin isRing opened, alkene hydrogen delta 5.34(1H, s, CH-5). In addition, in13In the C NMR (solvent: deuterated pyridine, 400MHz) spectrum, the delta of five methyl groups is respectively as follows: 16.6(CH3-18), 19.7(CH3-19), 16.6(CH3-21), 17.5(CH3-27), 47.6(CH3-28), 47.6 are typical methoxy groups; double bond high field δ 122.3 (CH-5); four quaternary carbons: delta 37.5(C-10), delta 141.5(C-5), delta 41.5(C-13), delta 113.0(C-22) (different from diosgenin, according to furostanol sapogenin); delta.75.5 (CH-26) indicates furostane. In addition, the homonuclear correlation is confirmed through HSQC, the hydrocarbon is assigned, and then the heteronuclear correlation HMBC spectrogram sum is carried out1H-1Determining the structure of the HCOSY spectrogram; wherein the chemical shift at the 26-carbon, δ 75.5(CH-26), and the hydrogen at the δ 105.3 carbon are heteronuclear remotely related to each other, indicating linkage to the sugar.
In the nuclear magnetic signal (table 13) in the glycoside chain part, 2 δ 1.64(3H, d, J ═ 6.0Hz, CH) are present in high field3-6(rha)) and δ 1.78(3H, d, J ═ 6.0Hz, CH)36(rha)) two methyl groups, indicating that the glycosidic chain has 2 rhamnoses, δ 5.86(1H, s), δ 6.40(1H, s), δ 4.86(1H, s) and 4.96(1H, s) hydrogen signals at the anomeric carbon of the glycosidic chain at low field. In that13In a C NMR (solvent: deuterated pyridine, 400MHz) spectrum, four sugar end group carbon signals delta 103.2, 102.3, 100.6 and 105.3 exist, which indicate that the compound is tetrasaccharide saponin; by passing1H-1HCOSY and HMBC confirmation, wherein the connection mode glc (inner) is connected with C-3 of aglycone, C-3 is associated with C-1 of glucose on HMBC spectrogram, C-26 is associated with glucose, and the confirmed compound is methylprotodioscin (Protodioscin, ygy05) β -D-Glucopyranoside, (3 β, 22 α, 25R) -26- (β -D-glucopyranosyloxy) -22-methoxyfurost-5-en-3-ylO-6-deoxy- α -L-mannopyranosyl- (1 → 2) -O- [6-deoxy- α -L-mannopyranosyl- (1 → 4)]-(Cheng et al.,2003)。
TABLE 12 Nuclear magnetic data (delta, ppm,400M) for the compound ygy-05 aglycone part
TABLE 13 Nuclear magnetic data (delta, ppm,400M) for the glycoside chain portion of Compound ygy-05
(4) Compound ygy-07 white needle crystals, ESI-MS m/z: 1218.35[ M + Na ]]+，1234.3[M+K]+, relative molecular mass 1195, molecular formula C58H98O26The aglycone part and the signal are the same as those of the compound ygy-05, and the NMR data are the same as those reported in the literature, and the structure is the protosaponin Pb (ygy 07) (3 β, 22 α, 25R) -26- (β -D-Glucopyranosyloxy) -22-Hydroxyfurost
-5-en-3-yl O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-[O-6-deoxy-α-L-
Mannopyranosyl-(1→4)-6-deoxy-α-L-mannopyranosyl-(1→4)]-β-D-glucopyranoside(Hirai et al.,2008)。
(5) Analysis of content of 4 steroidal saponins in Dioscorea composita
①② determination of steroid saponin content
According to the HPLC method determined above, the steroid saponins in Dioscorea composita are measured, the chromatogram is shown in FIG. 9, and the contents of the four steroid saponins are calculated according to the standard curve (Table 14).
TABLE 14 content of four steroidal saponins in Dioscorea composita
| Saponin | Dioscorea opposita saponin | Saponin Pb | Methyl protodioscin | Prosaponin Pb |
| Content (mg/g) | 61.14±0.96 | 39.89±0.72 | 4.46±0.13 | 0.84±0.08 |
| Ratio (%) | 57.5 | 37.5 | 4.2 | 0.8 |
The ratio is the ratio of each saponin to total saponin
In the chromatogram, the separation effect of each saponin is obvious, wherein the impurities with strong water solubility are about 2.5min, and the other impurities are about 21min and 23.0 min. The peak area integral calculation of the four saponins is carried out to obtain that the content of dioscin (dioscin) in the total saponins is the highest (accounting for 57.5 percent of the content of the 4 total saponins), the saponin asperin is used, the content of the other two furostanol saponins is low, the furostanol saponins can be influenced by an extraction method and can be easily converted into spirostanol saponins, and therefore the content of the furostanol saponins is not high.
The contents of four steroid saponins in the dioscorea composita are measured as follows: dioscin (dioscin)61.1mg/g, saponin Pb (asperin)39.9mg/g, methylprotodioscin (25-O-Protodioscin)4.5mg/g and protosaponin Pb (asperosaide) 0.84 mg/g.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
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