CN101891796A - Preparation method of high-purity soyasaponin monomers - Google Patents
Preparation method of high-purity soyasaponin monomers Download PDFInfo
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Abstract
The invention provides a preparation and detection method of high-purity soyasaponin monomers, comprising the following steps: (1) using soybean germ powder to prepare a soyasaponin crude extract; (2) carrying out fractionation on soyasaponin and soybean isoflavone by using gel chromatography; (3) purifying and separating the monomer soyasaponins by using preparative liquid chromatography; (4) detecting the soyasaponin monomers by using liquid chromatography, an evaporative light scattering detector and a diode array detector; and (5) distinguishing classes of the soyasaponin monomers by using a liquid chromatography-mass spectrometry (LC-MS) method. In the invention, the fractionation on the soyasaponin and the soybean isoflavone is successfully carried out, the purified soyasaponin monomers are prepared and a detection baseline is relatively stable, thereby the soyasaponin is favorable in detection effect; quantitative detection results are more correct by using the diode array detector and the evaporative light scattering detector; and the soyasaponin Aa and Ab in class A, the soyasaponin Ba and Bb in class B and the soyasaponin Be in class E are successfully separated and prepared, the purity is high and peak areas of various samples are up to above 95%.
Description
Technical field
The present invention relates to a kind of preparation method of biological technical field, especially a kind of preparation method who can be used as the quantitative analysis of soybean saponin and each monomer saponin and set up the high-purity soyasaponin monomers of examination criteria foundation.
Technical background
Glucosides such as 2% saponin(e and isoflavones are arranged in the soybean, and wherein saponin(e accounts for 0.5%.That soybean saponin has is anticancer, regulate immunologic function, reduce cholesterol in serum content, prevent and treat cardiovascular disorder, antibiotic, antiviral, protect multiple physiological effect and good pharmacological actions such as liver, fat-reducing, can be developed into protective foods or medicine, it still is a kind of important whipping agent, emulsifying agent, stablizer and flavor-improving agent simultaneously, and market potential is huge.
Japan's scholar Beichuan merit and big Kubo one be different according to aglycon very, soybean saponin is divided into A, B, E and DDMP organizes.It is as follows that soybean saponin is divided into the structural formula of A group:
It is as follows that soybean saponin is divided into the structural formula of B, E and DDMP group:
Simultaneously, table 1 has been listed A group, B group, E group soybean saponin and DDMP unit structure figure and corresponding molecular weight thereof, for the analysis of subsequent experimental.
Table 1 A group, B group, E group soybean saponin and DDMP unit structure figure and corresponding molecular weight thereof
| The monomer title | Molecular structure | Molecular weight |
| Aa | glc(1→2)gal(1→2)glcUA(1→3)A(22←1)ara(3←1)xyl(2，3，4-tri-O-acetyl) | 1364 |
| Ab | glc(1→2)gal(1→2)glcUA(1→3)A(22←1)ara(3←1)glc(2，3，4，6-tetra-O-acetyl) | 1436 |
| Ac | rha(1→2)gal(1→2)glcUA(1→3)A(22←1)ara(3←1)glc(2，3，4，6-tetra-O-acetyl) | 1420 |
| Ad | glc(1→2)ara(1→2)glcUA(1→3)A(22←1)ara(3←1)glc(2，3，4，6-tetra-O-acetyl) | 1390 |
| Ae | gal(1→2)glcUA(1→3)A(22←1)ara(3←1)xyl(2，3，4-tri-O-acetyl) | 1202 |
| Af | gal(1→2)glcUA(1→3)A(22←1)ara(3←1)glc(2，3，4，6-tetra-O-acetyl) | 1274 |
| Ag | ara(1→2)glcUA(1→3)A(22←1)ara(3←1)xyl(2，3，4-tri-O-acetyl) | 1172 |
| Ah | ara(1→2)glcUA(1→3)A(22←1)ara(3←1)glc(2，3，4，6-tetra-O-acetyl) | 1244 |
| Ba | glc(1→2)gal(1→2)glcUA(1→3)B | 958 |
| Bb | rha(1→2)gal(1→2)glcUA(1→3)B | 942 |
| Bc | rha(1→2)ara(1→2)glcUA(1→3)B | 912 |
| Bb’ | gal(1→2)glcUA(1→3)B | 796 |
| Bc’ | ara(1→2)glcUA(1→3)B | 766 |
| Bd | glc(1→2)gal(1→2)glcUA(1→3)E | 956 |
| Be | rha(1→2)gal(1→2)glcUA(1→3)E | 940 |
| αg | glc(1→2)gal(1→2)glcUA(1→3)B(22→2)DDMP | 1084 |
| βg | rha(1→2)gal(1→2)glcUA(1→3)B(22→2)DDMP | 1068 |
| βa | rha(1→2)ara(1→2)glcUA(1→3)B(22→2)DDMP | 1038 |
| γg | gal(1→2)glcUA(1→3)B(22→2)DDMP | 922 |
| γa | ara(1→2)glcUA(1→3)B(22→2)DDMP | 892 |
In many cases, as meticulous pharmacy, the contrast of experimental standard product etc., need to obtain the monomer of a certain class soybean saponin, to obtain more accurate experimental result, the preparation of therefore studying soyasaponin monomers seems very necessary.
Find Chinese patent application number by prior art documents: 200810200834.1, title: the preparation method of a kind of high-purity soybean saponin A and B, this technology claims: the preparation detection method.This technology relates to the method that a kind of high speed adverse current chromatogram prepares high-purity soybean saponin A and B, obtains the monomer of soyasaponin monomers A and soybean saponin B.Soybean saponin A that this invention makes and soybean saponin B can reach quite high purity, and this preparation method is easy and simple to handle, separation efficiency is high, fractional dose is big, the rate of recovery is high and favorable reproducibility.But, this invention isolating to be highly purified soybean A saponins and soybean B saponins, promptly realize be soybean saponin component from, still can not effectively obtain the separation of highly purified each monomer saponin.Other patent is as Chinese patent application: CN101278729A, CN1923845A, CN1245811A, CN1315323, CN1327983A, CN1590385A, CN1683362A, Japanese Patent JP2003-171393A etc.These patents all are the methods that suitability for industrialized production is rich in the soybean saponin mixture.Because soybean saponin is overlapped with the soybean isoflavones polarity of coexistence with it, this causes certain difficulty for the separation and purification of soyasaponin monomers.Academicly, domestic scientist is at the separation method of being devoted to the highly purified soyasaponin monomers of research and inquirement rational and effective.
In addition, the method of the analyzing and testing soybean saponin of bibliographical information is a lot, as thin-layer chromatography optical density(OD) (quantitatively) method, diode array spectrophotometry, thin-layer chromatography colorimetry, vapor-phase chromatography, high performance liquid chromatography, HPLC-MS coupling technique, capillary electrophoresis technique, colorimetry etc.Because the application popularization of HPLC, in recent years, relatively Chang Yong soybean saponin analyzing and testing is to use HPLC.But since saponins component uv-absorbing signal a little less than, usually with 205nm as detecting wavelength, and this is because " end " absorption effect of solvent, has caused the difficulty of HPLC analyzing and testing soybean saponin.Although utilization photodiode array detector (PDA) is arranged at present, but be difficult to solve the difficulty that accurate quantification is analyzed.Simultaneously, because the shortage of proper standard object of reference has also strengthened the difficulty that effective quantitative analysis detects soybean saponin.Current, although the check and analysis national standard promulgation of soybean saponin is arranged, more or less there is certain deficiency in every respect in domestic corresponding soybean saponin check and analysis method.Its basic reason is the shortage of soyasaponin monomers reference material, causes carrying out accurate quantitative analysis to soybean saponin.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, the invention provides a kind of preparation method of high-purity soyasaponin monomers.The present invention is after the macroporous adsorbent resin Solid-Phase Extraction prepares soybean saponin and isoflavones crude extract, by gel chromatography molecule chromatography portions from soybean saponin and isoflavones, the separation of liquid phantom preparing chromatogram method obtains the soybean saponin pure monomer, and liquid chromatograph mass spectrography is made effective detection and judgement to the soyasaponin monomers kind.
The present invention is achieved by the following technical solutions:
The present invention is raw material with soybean saponin content than the soybean germ of horn of plenty, after degreasing, with the extraction of 70% volume percent aqueous ethanolic solution, obtains soybean saponin and soybean isoflavones crude extract through the macroporous adsorbent resin Solid-Phase Extraction more earlier; Then, use gel chromatography molecule chromatography portions from soybean saponin and soybean isoflavones; At last, adopt liquid chromatography, light scattering detector and diode-array detector to detect soyasaponin monomers, and adopt the method for liquid chromatograph mass spectrography to differentiate the soyasaponin monomers kind, adopt preparative liquid chromatography to separate to obtain 〉=95.0% highly purified monomer soybean saponin.
Preparation method of the present invention comprises that step is as follows:
1. utilize soybean germ powder to prepare soybean saponin and soybean isoflavones crude extract;
2. the gel chromatography portions is from soybean saponin and soybean isoflavones;
3. adopt preparative liquid chromatography purifies and separates monomer soybean saponin;
4. adopt liquid chromatography, light scattering detector and diode-array detector to detect soyasaponin monomers;
5. adopt the method for liquid chromatograph mass spectrography to obtain highly purified monomer soybean saponin.
The soybean saponin crude extract of step described in 1., its preparation method is:
A, degreasing: in the Soxhlet extraction plant, carry out about the about 3h of degreasing extracting with sherwood oil (30-60 ℃), take out the back and volatilize sherwood oil in the ventilation naturally.
B, solvent extraction: the soybean germ powder of degreasing is dissolved in about 70% aqueous ethanolic solution by the liquid material that adopts 10: 1 than (mL/g), place on the shaking table, in 25 ℃ of water-baths, rock about the about 4h of lixiviate, or be aided with the ultrasonic pretreatment 45min that frequency is 20KHz, filter, rotary evaporation reclaims ethanol.
C, resin cation (R.C.) impurity elimination: the soybean saponin extracting solution added be equipped with in the adsorption column of acidulous cation resin, flow velocity 0.5-2.0BV/h, effluent liquid be upper prop 3 times repeatedly, 100ml 15% methanol aqueous solution wash-out; Rotary evaporation reclaims methyl alcohol and gets elutriant.
D, Solid-Phase Extraction: above-mentioned soybean saponin is proposed the elutriant adding be equipped with in the adsorption column of macroporous adsorbent resin, under the room temperature, flow rate control is at 0.5-2.0BV/h; The deionized water of 1-5BV, under the room temperature, flow rate control is at 0.5-2.0BV/h wash-out impurity; 1-5BV 〉=95% lower alcohol 2.0-5.0BV/h flushing adsorption column under room temperature, rotary evaporation reclaims lower alcohol and gets elutriant; The elutriant freeze-drying obtains soybean saponin and soybean isoflavones crude extract powder.
The gel chromatography of step described in 2., portions from the technical qualification of soybean saponin and soybean isoflavones is: saponin(e and soybean isoflavones crude extract powder 1g 2.0-3.0mL dissolve with methanol, moving phase are that methyl alcohol, sampling volume are that 200-500 μ L, flow rate of mobile phase are that 0.5-0.8mL/min, diode array absorption detecting wavelength are 215nm and 295nm (monitoring DDMP soybean saponin).
This step portions from the principle of soybean saponin and soybean isoflavones is because the molecular structure of soybean saponin and soybean isoflavones is different, thereby carry out when flowing through gel resin with moving phase size-exclusion or molecule chromatography obtain component from.
The 3. described preparative liquid chromatography analysis condition of step is: the C18 reverse-phase chromatography prepares separator column, and temperature is 30 ℃; Sample concentration is that 1g saponin(e sample is dissolved in 2ml 50% (V/V) methanol aqueous solution, and sampling volume is 100 μ L; Moving phase is A:0.001% (V/V) acetic acid aqueous solution, B:0.001% (V/V) acetate acetonitrile solution; Flow rate of mobile phase is 5mL/min; Detector is PDA diode-array detector (200nm-800nm, 215nm are supervisory wavelength); The HPLC gradient elution, the gradient program sees the following form.The elutriant of fraction collection retention time 〉=40min; Rotary evaporation reclaims acetonitrile, and the freeze-drying concentrated solution obtains.
HPLC gradient elution program
Annotate: 10 → 30min in the table, 30 → 80min, the change in concentration of 80 → 90min in three time periods is all along with the time linear change.The liquid phase chromatography of the employing of step described in 4. is meant: the analytical separator column of C18 reverse-phase chromatography; Sample concentration: 80mg saponin(e sample is dissolved in 10ml 1: 9 (V/V) acetonitrile solution; Sampling volume: 20 μ l; Moving phase: A:0.001% (V/V) acetic acid aqueous solution, B:0.001% (V/V) acetate acetonitrile solution; Flow rate of mobile phase: 1ml/min; Detector: evaporat light scattering (40 ℃ of temperature, air pressure 2.3bar) PDA diode-array detector (205nm, 295nm); The HPLC gradient elution, the gradient program sees the above table.
The liquid chromatograph mass spectrography of step described in 5. is used for promptly that (Electronic SprayIon/Mass Spectrometry obtains highly purified monomer soybean saponin thereby ESI/MS) obtain the soyasaponin monomers molecular weight by electrospray ionization mass spectrometry.
The present invention utilizes light scattering detector of the prior art (Evaportive light Scattering Detector, ELSD), improved its detection method commonly used, the present invention has eliminated the difficult point that is common in traditional HPLC detection method, be different from diode array and fluorimetric detector, the response of ELSD of the present invention does not rely on the optical characteristics with sample, and any volatility is lower than the sample of moving phase all can be detected, is not subjected to the influence of its functional group.The response value of ELSD is directly proportional with the quality of sample, thereby can be used for the purity of working sample or detect unknown material, has brought into play more further advantage for the detection of HPLC, has brought bigger facility.The present invention detects any sample that volatility is lower than moving phase, and does not need sample to contain chromophoric group, and its remolding sensitivity differential refraction detector height is insensitive to temperature variation, and baseline stability is fit to the gradient elution liquid phase chromatograph joint used.
Advantage of the present invention is: the present invention has improved liquid chromatograph mass spectrography technology of the prior art, utilize gel chromatography successfully to soybean isoflavones and soybean saponin carried out portions from; Utilize preparative high performance liquid chromatography to prepare the purifying soyasaponin monomers; Adopt light scattering detector, it is relatively stable to detect baseline, fine for the detection effect of soybean saponin, by photodiode array detection and light scattering detector and usefulness, can make the detection by quantitative result more accurate; Success separates and has prepared soybean saponin A a, the Ab in the category-A, soybean saponin Ba, Bb in the category-B, and the soybean saponin Be in the E class, purity is higher, and each sample peak area reaches more than 95%.It with liquid chromatograph mass spectrography to the high score of complex sample from ability, have highly selective, highly sensitive and can provide relative molecular mass and structural information to combine with MS, obtained in fields such as pharmaceutical analysis, food analysis and environmental analyses using widely.
The present invention provides the foundation for the quantitative analysis of the overall saponin(e of soybean and each monomer saponin, also has great importance for the analyzing and testing establishment of standard of soybean saponin.
Description of drawings
Fig. 1 soybean saponin sample composition reversed-phase HPLC evaporat light scattering and photodiode array detect chromatogram contrast figure;
The gel chromatography separation figure of Fig. 2 soybean saponin and soybean isoflavones;
Fig. 3 ELSD detector is crossed each sample HPLC analysis of control color atlas behind the gel chromatography;
The HPLC of Fig. 4 soyasaponin monomers prepares separating spectrum (detecting wavelength 215nm);
5 preparative chromatographies of Fig. 5 ELSD detector HPLC analyzing and testing are collected sample chromatogram figure;
The ESI/MS collection of illustrative plates of Fig. 6 soyasaponin monomers Aa, Ab, Ba, Bb, Be.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment one
(1) utilize soybean germ powder to prepare the soybean saponin crude extract
A, degreasing: take by weighing soybean germ powder 20g, with sherwood oil (30-60 ℃) degreasing 3hr, taking-up places ventilating kitchen to volatilize naturally.
B, solvent extraction: 20g degreasing germ meal,, filter than (mL/g) lixiviate 4hr by 10: 1 liquid material with 70% aqueous ethanolic solution 200ml, rotary evaporation recovery ethanol gets filtrate.
C, Solid-Phase Extraction: will macroporous adsorbent resin (AB-8 be housed the adding of above-mentioned soybean saponin lixiviate filtrate, Tianjin Chemical Plant of Nankai Univ.) adsorption column (Φ 30mm * 800mm, resin bed floor height 550mm) in, under the room temperature, flow rate control is at 1.5BV/h process macroporous adsorbent resin bed; The deionized water of 5BV, under the room temperature, flow rate control is removed impurity such as sugar (phenol sulfuric acid process checking sugar whether flush away) at the 1.5BV/h flush away; 4BV 〉=90% methyl alcohol 5.0BV/h flushing adsorption column under room temperature, rotary evaporation reclaims methyl alcohol and gets elutriant; The elutriant freeze-drying obtains soybean saponin and soybean isoflavones crude extract powder.
D, resin cation (R.C.) impurity elimination: get powder after the above-mentioned drying and be dissolved in the 50mL50% methanol aqueous solution, add adsorption column (15mm * 300mm that acidulous cation resin (Amberlite FPC 22) is housed, resin bed floor height 200mm) in, flow velocity 2.0BV/h, effluent liquid is upper prop 3 times repeatedly, 100ml 50% methanol aqueous solution wash-out; Rotary evaporation reclaims methyl alcohol and gets elutriant, reclaims the solvent postlyophilization and gets the soybean saponin crude extract.
(2) the gel chromatography portions is from soybean saponin and soybean isoflavones
Sample is prepared: take by weighing the above-mentioned soybean saponin crude extract that obtains, the ratio that is dissolved in the pure methyl alcohol of 2ml according to the 1.00g crude extract is dissolved, and the solution after the dissolving is stand-by as sample, promptly joins promptly and uses; Gel resin is Sephadex LH-20; Chromatographic column: AmerishamBiosciences (300mm * 25mm); Temperature: room temperature; Moving phase: pure methyl alcohol (chromatographically pure); Sampling volume: 300 μ l; Flow rate of mobile phase: 0.5ml/min; Uv-absorbing detects wavelength: 215nm (monitoring saponin(e) and 254nm (monitoring isoflavones), 295nm (monitoring DDMP soybean saponin).As shown in Figure 2, be supervisory wavelength with 215nm and 295nm respectively, collect the elutriant at 1-6 branch peak.The component HPLC of its branch analyzes collection of illustrative plates as shown in Figure 3.
(3) preparative liquid chromatography purifies and separates monomer soybean saponin
Above-mentioned branch separated portion and be the component of soybean saponin through the HPLC qualitative analysis, 1g sample concentration sample is dissolved in 2ml50% (V/V) methanol aqueous solution; Sampling volume is 100 μ L; Separator column is Alltima C18 (250mm * 10.0mm, 10 μ m); Temperature is 30 ℃; Flow rate of mobile phase is 5mL/min; Moving phase is A:0.001% (V/V) acetic acid aqueous solution, B:0.001% (V/V) acetate acetonitrile solution; Detector is PDA diode-array detector (200nm-800nm, 215nm are supervisory wavelength); The HPLC gradient elution.Prepare the HPLC color atlas as shown in Figure 4, collection of illustrative plates as shown in Figure 5 for 5 high-purity soyasaponin monomers sample look HPLC analyzing and testing (ELSD detector) that preparative chromatography is collected.
(4) liquid chromatography, light scattering detector and diode-array detector detect soyasaponin monomers
Analytical column: Aquasil RP C18 (150mm * 4.6mm, 3 μ m); Sample concentration: 80mg saponin(e sample is dissolved in 10ml1: 9 (V/V) acetonitrile: the aqueous solution; Sampling volume: 20 μ l; Moving phase: A:0.001% (V/V) acetic acid aqueous solution, B:0.001% (V/V) acetate acetonitrile solution; Flow rate of mobile phase: 1ml/min; Detector: evaporat light scattering (45 ℃ of temperature, air pressure 2.3bar) PDA diode-array detector (205nm, 295nm); The HPLC gradient elution is shown in the same table of elution program.HPLC analysis mode color atlas as shown in Figure 1.
(5) method of liquid chromatograph mass spectrography obtains highly purified monomer soybean saponin:
Liquid phase chromatogram condition is with (4) operation, mass spectroscopy condition: API+/MS; Ion source (Turbo Spray); Helium: 10.00L/min; Spray voltage: 5500V; Temperature: 500 ℃; Quasi-molecular ions sweep limit: 150-1500.Liquid chromatography-mass spectrography is analyzed collection of illustrative plates, as shown in Figure 6.
The soybean saponin high-purity monomer of preparation is through interpretation of mass spectra result such as following table.
Table 3 example one preparation gained soyasaponin monomers sample purity
| Sample number | Sample retention time (min) | Sample peak area percentage ratio (%) | [M+H]+ | ?[M+Na]+ | Saponin(e kind LC/MS identifies |
| 1 | 62.256 | 96.73 | 943 | 965 | Bb |
| 2 | 61.360 | 95.85 | 943 | 965 | |
| 3 | 64.786 | 96.87 | 941 | 963 | |
| 4 | 52.353 | 96.24 | 1437 | 1459 | |
| 5 | 51.876 | 97.32 | 1437 | 1459 | |
| 6 | 52.807 | 95.89 | 1437 | 1459 | Ab |
| 7 | 51.149 | 97.56 | 1365 | 1387 | Aa |
| 8 | 62.563 | 98.32 | 943 | 965 | Bb |
| 9 | 62.109 | 98.08 | 943 | 965 | |
| 10 | 62.143 | 97.68 | 943 | 965 | Bb |
Embodiment two
(1) utilize soybean germ powder to prepare the soybean saponin crude extract
A, degreasing: with example one.
B, solvent extraction: 20g degreasing germ meal,, filter than (mL/g) the ultrasonic pretreatment 45min that frequency is 20KHz that is aided with by 10: 1 liquid material with 70% aqueous ethanolic solution 200ml, rotary evaporation reclaims ethanol and gets filtrate.
C, Solid-Phase Extraction: the adsorption column (Φ 30mm * 800mm that is equipped with macroporous adsorbent resin (Amberlite XAD-2) adding of above-mentioned soybean saponin lixiviate filtrate, resin bed floor height 550mm) in, under the room temperature, flow rate control is at 1.5BV/h process macroporous adsorbent resin bed; The deionized water of 5BV, under the room temperature, flow rate control is removed impurity such as sugar (phenol sulfuric acid process checking sugar whether flush away) at the 2.0BV/h flush away; 4BV 〉=90% ethanol 2.0-5.0BV/h flushing adsorption column under room temperature, rotary evaporation reclaims ethanol and gets elutriant; The elutriant freeze-drying obtains soybean saponin and soybean isoflavones crude extract powder.
D, resin cation (R.C.) impurity elimination: with example one.
(2) the gel chromatography portions is from soybean saponin and soybean isoflavones
Sample is prepared: with example one.
(3) preparative liquid chromatography purifies and separates monomer soybean saponin
Above-mentioned branch separated portion and be the component of soybean saponin through the HPLC qualitative analysis, 1g sample concentration sample is dissolved in 2ml 1: 9 (V/V) acetonitrile: the aqueous solution; Sampling volume is 100 μ L; Separator column is Alltima C18 (250mm * 10.0mm, 10 μ m); Temperature is 30 ℃; Flow rate of mobile phase is 5mL/min; Moving phase is A:0.001% (V/V) acetic acid aqueous solution, B:0.001% (V/V) acetate acetonitrile solution; Detector is PDA diode-array detector (200nm-800nm, 215nm are supervisory wavelength); The HPLC gradient elution.
(4) liquid chromatography, light scattering detector and diode-array detector detect soyasaponin monomers
With example one.
(5) method of liquid chromatograph mass spectrography obtains highly purified monomer soybean saponin:
With example one.
The soybean saponin high-purity monomer of preparation is through interpretation of mass spectra result such as following table.
Table 4 example two preparation gained soyasaponin monomers sample purities
| Sample number | Sample retention time (min) | Sample peak area percentage ratio (%) | [M+H]+ | [M+Na]+ | Saponin(e kind LC/MS identifies |
| 1 | 62.712 | 99.67 | 943 | 965 | |
| 2 | 62.781 | 98.75 | 943 | 965 | |
| 3 | 65.948 | 99.88 | 941 | 963 | |
| 4 | 52.457 | 98.20 | 1365 | 1387 | |
| 5 | 52.511 | 98.88 | 1365 | 1387 | |
| 6 | 53.070 | 99.59 | 1437 | 1459 | Ab |
| 7 | 54.122 | 98.86 | 1421 | 1443 | Ac |
| 8 | 62.221 | 99.92 | 943 | 965 | Bb |
| 9 | 62.394 | 99.53 | 943 | 965 | |
| 10 | 61.992 | 99.86 | 943 | 965 | Bb |
| 11 | 58.301 | 99.95 | 959 | 981 | Ba |
Claims (7)
1. the preparation method of a high-purity soyasaponin monomers, it is characterized in that, is raw material with soybean saponin content than the soybean germ of horn of plenty, after degreasing, extract with 70% volume percent aqueous ethanolic solution earlier, obtain soybean saponin and soybean isoflavones crude extract through the macroporous adsorbent resin Solid-Phase Extraction again;
Then, use gel chromatography molecule chromatography portions from soybean saponin and soybean isoflavones;
At last, adopt liquid chromatography, light scattering detector and diode-array detector to detect soyasaponin monomers, and adopt the method for liquid chromatograph mass spectrography to differentiate the soyasaponin monomers kind, adopt preparative liquid chromatography to separate to obtain 〉=95.0% highly purified monomer soybean saponin.
2. the preparation method of high-purity soyasaponin monomers according to claim 1 is characterized in that, comprises that step is as follows:
1. utilize soybean germ powder to prepare soybean saponin and soybean isoflavones crude extract;
2. the gel chromatography portions is from soybean saponin and soybean isoflavones;
3. adopt preparative liquid chromatography purifies and separates monomer soybean saponin;
4. adopt liquid chromatography, light scattering detector and diode-array detector to detect soyasaponin monomers;
5. adopt the method for liquid chromatograph mass spectrography to obtain highly purified monomer soybean saponin.
3. according to the preparation method of claim 1 or 2 described high-purity soyasaponin monomers, it is characterized in that, soybean saponin and the soybean isoflavones crude extract of step described in 1., its preparation method is:
A, degreasing: in the Soxhlet extraction plant, carry out about the about 3h of degreasing extracting with sherwood oil (30-60 ℃), take out the back and volatilize sherwood oil in the ventilation naturally;
B, solvent extraction: the soybean germ powder of degreasing is mL/g by the liquid material ratio that adopted 10: 1, be dissolved in 70% aqueous ethanolic solution approximately, place on the shaking table, in 25 ℃ of water-baths, rock about the about 4h of lixiviate, or be aided with the ultrasonic pretreatment 45min that frequency is 20KHz, filter, rotary evaporation reclaims ethanol;
C, resin cation (R.C.) impurity elimination: the soybean saponin extracting solution added be equipped with in the adsorption column of acidulous cation resin, flow velocity 0.5-2.0BV/h, effluent liquid be upper prop 3 times repeatedly, 100ml 15% methanol aqueous solution wash-out; Rotary evaporation reclaims methyl alcohol and gets elutriant;
D, Solid-Phase Extraction: above-mentioned soybean saponin is proposed the elutriant adding be equipped with in the adsorption column of macroporous adsorbent resin, under the room temperature, flow rate control is at 0.5-2.0BV/h; The deionized water of 1-5BV, under the room temperature, flow rate control is at 0.5-2.0BV/h wash-out impurity; 1-5BV 〉=95% lower alcohol 2.0-5.0BV/h flushing adsorption column under room temperature, rotary evaporation reclaims lower alcohol and gets elutriant; The elutriant freeze-drying obtains soybean saponin and soybean isoflavones crude extract powder.
4. the preparation method of high-purity soyasaponin monomers according to claim 1, it is characterized in that, the gel chromatography of step described in 2., portions from the technical qualification of soybean saponin and soybean isoflavones is: saponin(e and soybean isoflavones crude extract powder 1g 2.0-3.0mL dissolve with methanol, moving phase is that methyl alcohol, sampling volume are that 200-500 μ L, flow rate of mobile phase are that 0.5-0.8mL/min, diode array absorption detecting wavelength are 215nm and 295nm, monitoring DDMP soybean saponin.
5. the preparation method of high-purity soyasaponin monomers according to claim 1 is characterized in that, the 3. described preparative liquid chromatography analysis condition of step is: the C18 reverse-phase chromatography prepares separator column, and temperature is 30 ℃; Sample concentration is that 1g saponin(e sample is dissolved in 2ml 50% volume percent methanol aqueous solution, and sampling volume is 100 μ L; Moving phase is A:0.001% volume percent acetic acid aqueous solution, B:0.001% volume percent acetate acetonitrile solution; Flow rate of mobile phase is 5mL/min; Detector is the PDA diode-array detector; The HPLC gradient elution, the elutriant of fraction collection retention time 〉=40min; Rotary evaporation reclaims acetonitrile, and the freeze-drying concentrated solution obtains.
6. the preparation method of high-purity soyasaponin monomers according to claim 1 is characterized in that, the liquid phase chromatography of the employing of step described in 4. is meant: the analytical separator column of C18 reverse-phase chromatography; Sample concentration: 80mg saponin(e sample is dissolved in 1: 9 volume percent acetonitrile solution of 10ml; Sampling volume: 20 μ l; Moving phase: A:0.001% volume percent acetic acid aqueous solution, B:0.001% volume percent acetate acetonitrile solution; Flow rate of mobile phase: 1ml/min; Detector: evaporat light scattering PDA diode-array detector, HPLC gradient elution.
7. the preparation method of high-purity soyasaponin monomers according to claim 1, it is characterized in that, the liquid chromatograph mass spectrography of step described in 5. obtains highly purified monomer soybean saponin thereby obtain the soyasaponin monomers molecular weight by electrospray ionization mass spectrometry.
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| CN103340920A (en) * | 2013-07-17 | 2013-10-09 | 河南中医学院 | Preparation method and application of fructus podophylli total flavone with multidrug resistance reversing activity |
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| WO2020213672A1 (en) * | 2019-04-16 | 2020-10-22 | 花王株式会社 | Method of predicting soybean yield |
| JP2020174553A (en) * | 2019-04-16 | 2020-10-29 | 花王株式会社 | Method of predicting soybean yield |
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| WO2020213672A1 (en) * | 2019-04-16 | 2020-10-22 | 花王株式会社 | Method of predicting soybean yield |
| JP2020174553A (en) * | 2019-04-16 | 2020-10-29 | 花王株式会社 | Method of predicting soybean yield |
| JP2020174668A (en) * | 2019-04-16 | 2020-10-29 | 花王株式会社 | Method of predicting soybean yield |
| JP7244338B2 (en) | 2019-04-16 | 2023-03-22 | 花王株式会社 | Soybean yield prediction method |
| CN110108831A (en) * | 2019-06-14 | 2019-08-09 | 山东师范大学 | A kind of the thin-layer chromatography solvent and detection method of soybean isoflavone glycoside from soybean isoflavones |
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| CN116903572A (en) * | 2023-07-14 | 2023-10-20 | 东明盛源生物科技有限公司 | Process for producing soybean isoflavone products with various purities |
| CN116903572B (en) * | 2023-07-14 | 2024-03-12 | 东明盛源生物科技有限公司 | Process for producing soybean isoflavone products with various purities |
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