CN112724112B - Separation and purification method of stevia rebaudiana - Google Patents

Separation and purification method of stevia rebaudiana Download PDF

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CN112724112B
CN112724112B CN202011219059.1A CN202011219059A CN112724112B CN 112724112 B CN112724112 B CN 112724112B CN 202011219059 A CN202011219059 A CN 202011219059A CN 112724112 B CN112724112 B CN 112724112B
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stevia rebaudiana
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CN112724112A (en
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傅青
金郁
柯燕雄
张欢欢
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East China University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/94—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D301/00—Preparation of oxiranes
    • C07D301/32—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Abstract

The invention relates to a separation and purification method of stevia rebaudiana, which is used for extracting basic substances of the stevia rebaudiana. The separation and purification method of the stevia rebaudiana comprises the step of carrying out reverse phase chromatography and supercritical fluid chromatography separation and purification on the stevia rebaudiana so as to extract the compound.

Description

Separation and purification method of stevia rebaudiana
Technical Field
The invention relates to the field of separation and purification of natural products, in particular to a separation and purification method of stevia rebaudiana and an obtained stevia rebaudiana basic substance.
Background
Stevia rebaudiana (Stevia rebaudiana bertoni, s.rebaudiana), also known as Stevia rebaudiana leaves, is a perennial plant of the Stevia genus of the family compositae. Stevia rebaudiana is native to the northeast high land of paraguay, south america, and in 1977, china introduced stevia rebaudiana from japan and planted in a large scale. Because diterpene glycoside substances in the stevia rebaudiana have the characteristics of high sweetness, difficult absorption and low calorie, the stevia rebaudiana is an ideal sweetener and can be used for preventing obesity and diabetes. Besides being used as a sweetening agent, pharmacological research shows that the stevia rebaudiana has a plurality of activities of oxidation resistance, tumor resistance, inflammation resistance, blood pressure reduction, bacteriostasis, virus resistance and the like. In order to further clarify the pharmacological effects of stevia, it is important to study the material basis.
The basic research of stevia rebaudiana bertoni is mainly based on the separation of chemical components of stevia rebaudiana bertoni, and currently, the known chemical components of stevia rebaudiana bertoni comprise stevioside, flavonoid, phenolic acid, volatile oil and the like. Because the stevia rebaudiana has complex composition, large content difference of different types of components and similar structure of the same type of components, the separation difficulty is increased. The separation and purification work of the system is developed based on the modern separation technology, and the method has important significance for further understanding the chemical composition of the stevia rebaudiana and developing and utilizing stevia rebaudiana resources.
Disclosure of Invention
The invention aims to provide a separation and purification method of stevia rebaudiana, which is used for separating and extracting compounds in the stevia rebaudiana so as to provide a foundation for the development of the stevia rebaudiana. 4 novel compounds are obtained by the separation and purification method, can be used for basic research of stevia rebaudiana and are expected to be applied to the food industry or the pharmaceutical industry.
According to an aspect of the present invention, there is provided a method for separating and purifying stevia. The method for separating and purifying the stevia rebaudiana comprises the step of separating and purifying the stevia rebaudiana by reverse phase chromatography and supercritical fluid chromatography to extract the basic substances of the stevia rebaudiana.
In some embodiments, the method for separating and purifying stevia comprises the steps of: treating stevia rebaudiana mother liquor sugar by a recrystallization method to obtain a mother liquor sugar crude product; subjecting the crude mother liquor sugar to reverse phase chromatographic separation to obtain a plurality of crude components; subjecting the crude fraction to supercritical fluid chromatographic separation to obtain a plurality of fine fractions; and subjecting the fine fraction to reverse phase chromatographic separation to obtain a stevia rebaudiana basic substance.
In some embodiments, in the step of recrystallizing the stevia rebaudiana mother liquor sugar, the stevia rebaudiana mother liquor sugar is recrystallized using anhydrous methanol as a solvent.
In some embodiments, the feed-to-liquid ratio of stevia mother liquor sugar to the anhydrous methanol is: 120g of stevia mother liquor sugar is added to 1L of anhydrous methanol.
In some embodiments, in the reverse phase chromatographic separation step, the separation is performed using an octadecyl-bonded stationary phase; the mobile phase is methanol and water or acetonitrile and water.
In some embodiments, in the step of performing supercritical fluid chromatography separation of the crude component, the stationary phase is C4 and the mobile phase is supercritical CO 2 Methanol is used as modifier, and gradient elution is carried out.
In some embodiments, the stevia rebaudiana basic substance has a structure represented by formulas i-1 to i-4 below:
Figure BDA0002761438650000021
wherein, the name of the compound is:
formula i-1:2,7, 10-tetramethyl-1-oxapipro [4.5] deca-3,6-dien-8-one;
formula i-2:
(E)-4-(7-hydroxy-5-(hydroxymethyl)-1a,2a,5-trimethyldecahydronaphtho[2,3-b]oxiren-2-yl)but-3-en-2-one；
formula i-3:
5-((4,5-dihydroxy-6-(hydroxymethyl)-2-((3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-3-yl)oxy)-5,7,7-trimethyl-4,5,6,7-tetrahydro-1H-inden-1-one；
formula i-4:6- (4-allyl-2-methoxyphenoxy) -5-amino-2- (hydroxymethyl) tetrahydro-2H-pyran-3,4-diol (i-4)
In some embodiments, the method for separating and purifying the stevia rebaudiana by chemical combination specifically comprises the following steps:
step 1, processing stevia rebaudiana mother liquor sugar by adopting a recrystallization method, weighing the stevia rebaudiana mother liquor sugar, placing the stevia rebaudiana mother liquor sugar into a dry and clean container, adding anhydrous methanol, ultrasonically stirring and dissolving, standing for 24 hours at room temperature, and filtering to obtain a mother liquor sugar crude product.
And 2, dissolving the mother liquor sugar crude product by pure water, adopting reverse phase chromatography, separating by using a C18 stationary phase, taking methanol and water as a mobile phase, eluting under a step gradient condition, and collecting three sections of components which are sequentially marked as Fr.1-Fr.3.
Step 3, dissolving Fr.1 with pure methanol, and separating with supercritical fluid chromatography, wherein the stationary phase is C4, and the mobile phase is supercritical CO 2 Methanol is used as modifier, and gradient elution is carried out. 5 fractions were collected by peak and scored as Fr.1-1 to Fr.1-5.
Step 4 Fr.1-1 is dissolved with pure water, reversed phase chromatography is adopted, C18 stationary phase is used for separation, and a mobile phase is methanol and water, and isocratic elution is carried out on 30% methanol to obtain a compound i-1.
Step 5 dissolving Fr.1-2 in pure water, separating by reverse phase chromatography using C18 stationary phase, eluting with methanol and water at 45% methanol isocratic to obtain compounds i-2 and Fr.1-2-1.
Step 6 Fr.1-3 is dissolved by pure water, reversed phase chromatography is adopted, C18 stationary phase is used for separation, the mobile phase is acetonitrile and water, 18% acetonitrile isocratic elution is carried out, and the compound i-3 is obtained.
Step 7 Fr.1-2-1 is dissolved in pure water, and the mixture is separated by reversed phase chromatography using a C18 stationary phase, the mobile phase is acetonitrile and water, and the isocratic elution is carried out with 28% acetonitrile, thus obtaining the compound i-4.
Stevia rebaudiana is widely used as a sweetening agent in food industries such as drinks, dairy products, cakes, candies and the like. Meanwhile, the stevia rebaudiana has various pharmacological activities and has a certain prospect in the development of new drugs. Therefore, the novel compounds in the stevia rebaudiana found by the separation and purification method of the application have great significance for deeply understanding the chemical composition of the stevia rebaudiana and developing and utilizing stevia rebaudiana resources.
In the present invention, a recrystallized mother liquor of stevia is subjected to reverse phase chromatographic separation and purification to obtain 4 unrecited compounds, and further employed 1 H NMR、 13 And C NMR, two-dimensional nuclear magnetic spectrum and high-resolution mass spectrum are used for carrying out structure identification on the obtained compound, so that the molecular structure of the compound is deduced, and a certain material basis is provided for quality control and commercial development of stevia rebaudiana.
Drawings
FIG. 1 reversed phase preparative chromatography of crude mother liquor sugars;
FIG. 2Fr.1, supercritical fluid chromatography preparation diagram;
FIG. 3Fr.1-1 to Fr.1-3, reversed phase preparation;
FIG. 4 preparation of Compound i-1 1 A HNMR map;
FIG. 5 preparation of Compound i-1 13 A CNMR map;
FIG. 6 COSY diagram of compound i-1;
FIG. 7 HMQC plot of compound i-1;
FIG. 8 HMBC plot of compound i-1;
FIG. 9 preparation of Compound i-2 1 A HNMR map;
FIG. 10 preparation of Compound i-2 13 A CNMR map;
FIG. 11 COSY diagram for compound i-2;
FIG. 12 HMQC plot of compound i-2;
FIG. 13 HMBC plot of compound i-2;
FIG. 14 NOESY plot of Compound i-2
FIG. 15 of Compound i-3 1 A HNMR map;
FIG. 16 preparation of Compound i-3 13 A CNMR map;
FIG. 17 COSY diagram for compound i-3;
FIG. 18 HMQC plot of compound i-3;
FIG. 19 HMBC plot 1 (chemical shifts δ 1.0-7.6) for compound i-3;
FIG. 20 HMBC chart 2 (chemical shifts δ 3.1-4.6) for compound i-3;
FIG. 21 HMBC plot 3 (chemical shifts δ 0.9-2.6) for compound i-3;
FIG. 22 of Compound i-4 1 A HNMR map;
FIG. 23 preparation of Compound i-4 13 A CNMR map;
FIG. 24 COSY diagram of compound i-4;
FIG. 25 HMQC plot of compound i-4;
FIG. 26 HMBC plot 1 (chemical shifts δ 3.0-7.4) for compound i-4;
FIG. 27 HMBC pattern 2 (chemical shifts. Delta. 3.2-4.1) for compound i-4.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
Example 1 method for separating and purifying four Compounds from stevia rebaudiana Bertoni
In this embodiment, a method for separating and purifying stevia includes the steps of: treating stevia rebaudiana mother liquor sugar by a recrystallization method to obtain a mother liquor sugar crude product; subjecting the crude mother liquor sugar to reverse phase chromatographic separation to obtain a plurality of crude components; subjecting the crude fraction to supercritical fluid chromatographic separation to obtain a plurality of fine fractions; and subjecting the fine fraction to reverse phase chromatographic separation to obtain a stevia rebaudiana basic substance.
Specifically, in this embodiment, the method for separating and purifying stevia rebaudiana by chemical combination comprises the steps of:
step of processing stevia rebaudiana mother liquor sugar by recrystallization method
Recrystallizing the stevia rebaudiana mother liquor sugar by using anhydrous methanol as a solvent to obtain a mother liquor sugar crude product; wherein the feed-liquid ratio of the stevia rebaudiana mother liquor sugar to the anhydrous methanol is as follows: adding 120g of stevia mother liquor sugar into 1L of anhydrous methanol;
carrying out reversed phase chromatographic separation on the crude product of the mother liquor sugar
Performing separation by using an octadecyl bonded stationary phase, taking methanol and water as mobile phases, and performing gradient elution under a step gradient condition to obtain a plurality of crude components;
step of performing supercritical fluid chromatographic separation on crude component
The stationary phase is C4, and the mobile phase is supercritical CO 2 Methanol is used as a modifier, gradient elution is carried out, and a plurality of fine components are obtained by collecting according to peaks; and (c) a second step of,
a step of subjecting the fine fraction to reversed phase chromatographic separation
And (3) separating by using an octadecyl bonding stationary phase, wherein a mobile phase is methanol and water or acetonitrile and water, and isocratic elution is carried out by using 30-45% of methanol or 15-30% of acetonitrile to obtain the stevia rebaudiana basic substance.
In this embodiment, taking 20g of mother liquid sugar of stevia rebaudiana as an example, the separation and purification steps are as follows:
step 1, processing stevia rebaudiana mother liquor sugar by a recrystallization method: accurately weighing 20g of stevia rebaudiana mother liquor sugar, placing in a dry and clean container, adding anhydrous methanol according to a material-liquid ratio (w/v, g/L) of 120, ultrasonically stirring for dissolving, standing at room temperature for 24h, filtering, and freeze-drying filtrate to obtain a crude product of the mother liquor sugar. A total of 300g of mother liquor sugar was processed to give 135g of lyophilized powder.
And 2, dissolving the crude mother liquor sugar by using pure water, and dissolving with the aid of ultrasonic waves to prepare a sample solution with the concentration of 400 mg/mL. 300g of C18 filler (60 mu m) is taken, the ethanol homogenate is filled into a 50mm DAC column, and the length and the height of the measurement column are 250mm; mobile phase composition: a is water, B is methanol; an elution mode: 0-15min,60% B;15.01-30min,80% B;30.01-40min,100% by weight B; sample introduction volume: 20mL; flow rate: 70.0mL/min. Column temperature: room temperature; the detection wavelength is 227nm. Three-stage components Fr.1 (6.37 g), fr.2 (103.80 g) and Fr.3 (3.22 g) were obtained, and the chromatogram was shown in FIG. 1. In this example, the component Fr.1 is taken as an example for further explanation. It will be appreciated by those skilled in the art that the same subsequent processing steps can also be employed for components Fr.2 and Fr.3.
Step 3 the component Fr.1 was dissolved in pure methanol at a concentration of 70mg/mL. Separation was performed using a column uniformity C4 (250 × 20mm, i.d.,5 μm), mobile phase composition: a is CO 2 B is methanol; and (3) an elution mode: 0-4.2min,15-30% by weight, at a flow rate of 52mL/min;4.2-5.2min,30-50% by weight, and the flow rate is 52-40mL/min;5.2-10min,50% B, flow rate of 40mL/min; a back pressure of 130bar; sample introduction volume: 1mL; column temperature: 25 ℃; the detection wavelength is 227nm. Collecting according to peak to obtain 5 components, recording as Fr.1-1-Fr.1-5, and chromatogram shown in figure 2. In this example, the components Fr.1-1 to Fr.1-3 are taken as examples for further explanation. It will be appreciated by those skilled in the art that the same subsequent processing steps can also be employed for the components Fr.1-4 and Fr.1-5.
Step 4 Fr.1-1 was dissolved in pure water and separated using column C18 (250X 20mm i.d.,10 μm). The mobile phase composition is as follows: water and methanol, flow rate: 19mL/min, column temperature: the detection wavelength is 227nm at room temperature. 0-23min, and 30% methanol elution to obtain compound i-1 (4.05 mg), the chromatogram is shown in FIG. 3a.
Step 5 Fr.1-2 was dissolved in pure water and separated using column C18 (250X 20mm i.d.,10 μm). Mobile phase composition: water and methanol, flow rate: 19mL/min, column temperature: the detection wavelength is 227nm at room temperature. 0-27min, eluting with 45% methanol to obtain compound i-2 (3.86 mg) and component Fr.1-2-1, and obtaining chromatogram shown in FIG. 3b.
Step 6 Fr.1-3 was dissolved in pure water and separated using column C18 (250X 20mm i.d.,10 μm). Mobile phase composition: water and acetonitrile, flow rate: 19mL/min, column temperature: room temperature, detection wavelength 227nm.0-25min,18% acetonitrile elution to give compound i-3 (2.23 mg), chromatogram shown in FIG. 3c.
Step 7 Fr.1-2-1 was dissolved in pure water and separated using column C18 (250X 20mm i.d.,10 μm). The mobile phase composition is as follows: water and acetonitrile, flow rate: 19mL/min, column temperature: the detection wavelength is 227nm at room temperature. 0-12min,28% acetonitrile elution to give compound i-4 (6.24 mg), chromatogram shown in FIG. 3d.
Example 2 identification of four Compounds in stevia
In this example, a method is provided for identifying four monomeric compounds in stevia prepared as in example 1, using modern spectroscopic techniques such as 1 H NMR、 13 The four monomeric compounds obtained in example 1 were subjected to structural identification by C NMR, two-dimensional nuclear magnetic spectrum, and high-resolution mass spectrometry.
Compound i-1: ESI-MS m/z:207.14[ 2 ], [ M + H ]] + Molecular formula C 13 H 18 O 2 . Bonding of 1 H-NMR spectrum (FIG. 4), 13 The C-NMR spectrum (FIG. 5), COSY spectrum (FIG. 6), HMQC spectrum (FIG. 7) and HMBC spectrum (FIG. 8) confirmed that compound i-1 was: 2,7, 10-tetramethyl-1-oxaspiro [4.5]deca-3,6-dien-8-one。
Compound i-2: ESI-MS m/z:309.20[ 2 ], [ M + H ]] + Molecular formula C 18 H 28 O 4 . By passing 1 H-NMR spectrum (FIG. 9), 13 The C-NMR spectrum (FIG. 10), COSY spectrum (FIG. 11), HMQC spectrum (FIG. 12), HMBC spectrum (FIG. 13) and NOESY spectrum (FIG. 14) confirmed that compound i-2 was:
(E)-4-(7-hydroxy-5-(hydroxymethyl)-1a,2a,5-trimethyldecahydronaphtho[2,3-b]oxiren-2-yl)but-3-en-2-one。
compound i-3: ESI-MS m/z of 517.27[ 2 ] M + H] + Molecular formula C 24 H 36 O 12 Is combined with 1 H-NMR spectrum (FIG. 15), 13 The C-NMR spectrum (FIG. 16), COSY spectrum (FIG. 17), HMQC spectrum (FIG. 18) and HMBC spectrum (FIG. 19-FIG. 21) confirmed that compound i-3 was:
5-((4,5-dihydroxy-6-(hydroxymethyl)-2-((3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methoxy)tetrahydro-2H-pyran-3-yl)oxy)-5,7,7-trimethyl-4,5,6,7-tetrahydro-1H-inden-1-one。
compound i-4: ESI-MS m/z 344.17[ 2 ] M + H +H 2 O] + Molecular formula C 16 H 23 NO 6 In combination with 1 H-NMR spectrum (FIG. 22), 13 The C-NMR spectrum (FIG. 23), COSY spectrum (FIG. 24), HMQC spectrum (FIG. 25) and HMBC spectrum (FIG. 26 and FIG. 27) confirmed that compound i-4 was:
6-(4-allyl-2-methoxyphenoxy)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol。
although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.

Claims (2)

1. A method for separating and purifying stevia rebaudiana basic substances is characterized by comprising the following steps:
recrystallizing the stevia rebaudiana mother liquor sugar by using anhydrous methanol as a solvent, and filtering to obtain a mother liquor sugar crude product;
dissolving the crude mother liquor sugar with pure water, and separating by using first reverse phase chromatography; the first reverse phase chromatography comprises: separating with C18 stationary phase, eluting with methanol and water as mobile phase under gradient condition, and collecting three sections of components, which are sequentially marked as Fr.1, fr.2 and Fr.3; the C18 stationary phase is an octadecyl bonded stationary phase;
dissolving Fr.1 with pure methanol, and separating by supercritical fluid chromatography; the supercritical fluid chromatography comprises: the stationary phase is C4, and the mobile phase is supercritical CO 2 Gradient eluting with methanol as modifier, and collecting according to peak to obtain 5 components denoted as Fr.1-1, fr.1-2, fr.1-3, fr.1-4 and Fr.1-5;
dissolving Fr.1-1 with pure water, and separating by second reversed phase chromatography; the second reverse phase chromatography comprises: separating by using a C18 stationary phase, wherein a mobile phase is methanol and water, and isocratic elution is carried out by adopting 30% methanol to obtain a compound i-1;
dissolving Fr.1-2 with pure water, and separating by reverse phase chromatography for the third time; the third reversed phase chromatography comprises: separating with C18 stationary phase, wherein the mobile phase is methanol and water, and isocratic eluting with 45% methanol to obtain compounds i-2 and Fr.1-2-1;
dissolving Fr.1-3 with pure water, and separating by fourth reversed phase chromatography; the fourth reverse phase chromatography comprises: separating by using a C18 stationary phase, wherein a mobile phase is acetonitrile and water, and performing isocratic elution by using 18% acetonitrile to obtain a compound i-3;
dissolving Fr.1-2-1 in pure water, and separating by fifth reversed phase chromatography; the fifth reverse phase chromatography comprises: separating by using a C18 stationary phase, wherein a mobile phase is acetonitrile and water, and isocratic elution is carried out by adopting 28% acetonitrile to obtain a compound i-4;
the stevia rebaudiana basic substance is the compound i-1 and the compound i-4;
the structure of the compound i-1 is shown as the formula i-1:
Figure FDA0003948247360000011
the structure of the compound i-4 is shown as the formula i-4:
Figure FDA0003948247360000012
2. the method for separating and purifying a stevia rebaudiana basic substance as set forth in claim 1, wherein a feed-to-liquid ratio of said stevia rebaudiana mother liquor sugar to said anhydrous methanol is: 120g of mother liquor sugar of stevia rebaudiana Bertoni was added to 1 liter of anhydrous methanol.
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