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

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

A kind of 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 method for separation and purification of stevia rebaudiana and the obtained stevia rebaudiana base material.

Background technique

Stevia (Stevia rebaudiana Bertoni, S.rebaudiana), also known as stevia leaves, is a perennial plant of the genus Stevia in the family Asteraceae. Stevia is native to the northeast highlands of Paraguay in South America. In 1977, my country introduced stevia from Japan and planted it on a large scale. Because the diterpene glycosides in Stevia rebaudiana have the characteristics of high sweetness, not easy to be absorbed, and low calorie, so Stevia rebaudiana is an ideal sweetener, which can be used to prevent obesity and diabetes. In addition to being a sweetener, pharmacological studies have shown that stevia has many activities such as antioxidant, anti-tumor, anti-inflammatory, hypotensive, antibacterial and antiviral. In order to further clarify the pharmacological effects of stevia, it is very important to study its material basis.

The research on the material basis of stevia is mainly based on the separation of its chemical components. At present, the known chemical components in stevia include steviol glycosides, flavonoids, phenolic acids and volatile oils. Due to the complex composition of Stevia rebaudiana, the content of different types of components varies greatly, and the structure of similar components is similar, which increases the difficulty of separation. Carrying out systematic separation and purification work based on modern separation technology is of great significance for further understanding of the chemical composition of stevia rebaudiana and the development and utilization of stevia rebaudiana resources.

Contents of the invention

The object of the present invention is to provide a method for separation and purification of stevia, which is used for separating and extracting compounds in stevia, so as to provide a basis for the development of stevia. Four new compounds were obtained through the separation and purification method described in this application, which can be used in the basic research of stevia rebaudiana, and are expected to be applied in the food industry or the pharmaceutical industry.

According to one aspect of the present invention, a method for separating and purifying Stevia is provided. The separation and purification method of stevia is to perform reverse phase chromatography and supercritical fluid chromatography to separate and purify stevia to extract the basic substance of stevia.

In some embodiments, the method for separating and purifying stevia rebaudiana comprises the steps of: treating stevia mother liquor sugar with recrystallization method to obtain crude mother liquor sugar; performing reverse-phase chromatography on the mother liquor crude sugar to obtain multiple crude groups separation; performing supercritical fluid chromatography separation on the coarse component to obtain multiple fine components; and performing reverse phase chromatography separation on the fine component to obtain the stevia base material.

In some embodiments, in the step of recrystallizing the stevia mother liquor, anhydrous methanol is used as a solvent to recrystallize the stevia mother liquor.

In some embodiments, the solid-liquid ratio of the stevia mother liquor to the anhydrous methanol is: 120 g of stevia mother liquor is added to 1 liter of anhydrous methanol.

In some embodiments, in the reversed-phase chromatographic separation step, an octadecyl bonded stationary phase is used for separation; the mobile phase is methanol and water, or acetonitrile and water.

In some embodiments, in the step of separating crude components by supercritical fluid chromatography, the stationary phase is C4, the mobile phase is supercritical CO ₂ , methanol is a modifier, and gradient elution is performed.

In some embodiments, the stevia base material has the structures shown in the following formulas i-1 to i-4:

Wherein, the name of above-mentioned compound is:

Formula i-1: 2,7,10,10-tetramethyl-1-oxaspiro[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 compound separation and purification method in Stevia rebaudiana specifically includes the following steps:

Step 1: adopt the recrystallization method to process the mother liquor of stevia rebaudiana, weigh the mother liquor of stevia rebaudiana, put it in a dry and clean container, add anhydrous methanol, stir and dissolve it ultrasonically, let it stand at room temperature for 24 hours, and obtain the crude sugar of mother liquor after filtration.

Step 2 Dissolve the crude sugar in mother liquor with pure water, use reverse phase chromatography, use C18 stationary phase to separate, methanol and water as mobile phase, step gradient condition elution, collect three sections of components, and record them as Fr.1-Fr in turn .3.

In step 3, Fr.1 is dissolved in pure methanol, separated by supercritical fluid chromatography, the stationary phase is C4, the mobile phase is supercritical CO ₂ , methanol is used as a modifier, and gradient elution is performed. Collect according to the peaks to obtain 5 components, denoted as Fr.1-1～Fr.1-5.

Step 4 Dissolve Fr.1-1 in pure water, use reverse phase chromatography, use C18 stationary phase to separate, mobile phase is methanol and water, 30% methanol isocratic elution, to obtain compound i-1.

Step 5 Dissolve Fr.1-2 in pure water, use reverse phase chromatography, use C18 stationary phase to separate, mobile phase is methanol and water, 45% methanol isocratic elution, to obtain compound i-2 and Fr.1- 2-1.

Step 6 Dissolve Fr.1-3 in pure water, use reverse phase chromatography, use C18 stationary phase to separate, mobile phase is acetonitrile and water, 18% acetonitrile isocratic elution, to obtain compound i-3.

Step 7 Dissolve Fr.1-2-1 in pure water, use reverse phase chromatography, use C18 stationary phase to separate, mobile phase is acetonitrile and water, 28% acetonitrile isocratic elution, to obtain compound i-4.

Stevia is widely used as a sweetener in food industries such as beverages, dairy products, pastries and candies. At the same time, stevia has a variety of pharmacological activities, and has certain prospects in the development of new drugs. Therefore, the above-mentioned new compounds in Stevia rebaudiana discovered based on the separation and purification method of the present application are of great significance for in-depth understanding of the chemical composition of Stevia rebaudiana and the development and utilization of stevia rebaudiana resources.

In the present invention, the recrystallized Stevia rebaudiana mother liquor was separated and purified by reverse phase chromatography to obtain 4 unrecorded compounds, which were further analyzed by ¹ H NMR, ¹³ C NMR, two-dimensional nuclear magnetic spectrum, and high-resolution mass spectrometry. The obtained compound was identified through structural identification, and the molecular structure of the compound was deduced to provide a certain material basis for the quality control and commercial development of stevia.

Description of drawings

The reverse phase preparation chromatographic separation of Fig. 1 mother liquor sugar crude product;

The supercritical fluid chromatography preparation diagram of Fig. 2Fr.1;

Figure 3 Fr.1-1 ~ Fr.1-3 reverse phase preparation diagram;

The ¹ HNMR figure of Fig. 4 compound i-1;

The ¹³ CNMR figure of Fig. 5 compound i-1;

Figure 6 COZY diagram of compound i-1;

The HMQC figure of Fig. 7 compound i-1;

The HMBC figure of Fig. 8 compound i-1;

The ¹ HNMR figure of Fig. 9 compound i-2;

The ¹³ CNMR figure of Fig. 10 compound i-2;

Figure 11 COZY diagram of compound i-2;

The HMQC figure of Fig. 12 compound i-2;

The HMBC figure of Fig. 13 compound i-2;

Figure 14 NOESY plot of compound i-2

The ¹ HNMR figure of Fig. 15 compound i-3;

¹³ CNMR figure of Fig. 16 compound i-3;

Figure 17 COZY diagram of compound i-3;

The HMQC figure of Fig. 18 compound i-3;

Figure 19 HMBC Figure 1 of compound i-3 (chemical shift δ1.0～7.6);

Figure 20 HMBC Figure 2 of compound i-3 (chemical shift δ3.1-4.6);

Figure 21 HMBC Figure 3 of compound i-3 (chemical shift δ0.9～2.6);

The ¹ HNMR figure of Fig. 22 compound i-4;

¹³ CNMR figure of Fig. 23 compound i-4;

Figure 24 COZY diagram of compound i-4;

The HMQC figure of Fig. 25 compound i-4;

Figure 26 HMBC Figure 1 of compound i-4 (chemical shift δ3.0～7.4);

Fig. 27 HMBC diagram 2 of compound i-4 (chemical shift δ3.2-4.1).

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention.

Example 1. Four compound separation and purification methods in Stevia rebaudiana

In this embodiment, a separation and purification method of Stevia rebaudiana is provided, comprising the steps of: treating the mother liquor sugar with recrystallization method to obtain a crude mother liquor sugar; performing reverse-phase chromatographic separation on the mother liquor sugar crude product to obtain multiple Coarse fractions; performing supercritical fluid chromatography on the coarse fractions to obtain a plurality of fine fractions; and performing reversed-phase chromatography on the fine fractions to obtain the stevia base material.

Specifically, in this embodiment, the method for separating and purifying compounds in Stevia rebaudiana includes the steps of:

Steps of processing stevia mother liquor sugar by recrystallization method

Using anhydrous methanol as a solvent to recrystallize the stevia mother liquor sugar to obtain a crude mother liquor sugar; wherein, the solid-liquid ratio of the stevia mother liquor sugar to the anhydrous methanol is: every 1 liter of anhydrous methanol is added 120g stevia mother liquid sugar;

Carrying out the step of reverse phase chromatographic separation to described mother liquid sugar crude product

Octadecyl bonded stationary phase is used for separation, methanol and water are used as mobile phase, and step gradient conditions are used for elution to obtain multiple crude components;

Steps for Supercritical Fluid Chromatography Separation of Crude Components

The stationary phase is C4, the mobile phase is supercritical CO ₂ , methanol is used as a modifier, and gradient elution is performed, collecting by peaks to obtain multiple fine components; and,

Procedure for Reversed-Phase Chromatographic Separation of Fine Components

The octadecyl bonded stationary phase is used for separation, the mobile phase is methanol and water or acetonitrile and water, and isocratic elution is performed with 30% to 45% methanol or 15% to 30% acetonitrile to obtain the basic substance of stevia rebaudiana.

In this embodiment, 20g of stevia mother liquid sugar is specifically taken as an example to carry out separation and purification, and the specific steps are as follows:

Step 1 Recrystallization method to process stevia mother liquor sugar: accurately weigh 20g stevia mother liquor sugar and place it in a dry and clean container, add anhydrous methanol according to the ratio of material to liquid (w/v, g/L) 120:1, and ultrasonically stir to dissolve , stand at room temperature for 24h, filter, and freeze-dry the filtrate to obtain crude sugar in mother liquor. A total of 300 g of sugar mother liquor were processed to obtain 135 g of freeze-dried powder.

Step 2 Dissolve the crude sugar in the mother liquor with pure water, and ultrasonically assist the dissolution to prepare a sample solution with a concentration of 400 mg/mL. Take 300g of C18 filler (60μm), put ethanol homogenate on a 50mm DAC column, measure the column length and height to 250mm; mobile phase composition: A is water, B is methanol; elution method: 0-15min, 60% B; 15.01- 30min, 80%B; 30.01-40min, 100%B; injection volume: 20mL; flow rate: 70.0mL/min. Column temperature: room temperature; detection wavelength 227nm. The three-stage components Fr.1 (6.37g), Fr.2 (103.80g) and Fr.3 (3.22g) were obtained. The chromatogram is shown in Figure 1. In this embodiment, the component Fr.1 is taken as an example for further description. Those skilled in the art will understand that the same subsequent processing steps can also be used for components Fr.2 and Fr.3.

Step 3 Dissolve component Fr.1 in pure methanol at a concentration of 70 mg/mL. Use chromatographic column Unitary C4 (250×20mm, id, 5μm) to separate, mobile phase composition: A is CO ₂ , B is methanol; elution method: 0-4.2min, 15-30% B, flow rate 52mL/min; 4.2 -5.2min, 30-50% B, flow rate 52-40mL/min; 5.2-10min, 50%B, flow rate 40mL/min; back pressure 130bar; injection volume: 1mL; column temperature: 25°C; detection wavelength: 227nm. Collect according to the peaks to obtain 5 components, denoted as Fr.1-1-Fr.1-5, the chromatogram is shown in Figure 2. In this embodiment, components Fr.1-1 to Fr.1-3 are taken as examples for further description. Those skilled in the art can understand that the same subsequent processing steps can also be used for the components Fr.1-4 and Fr.1-5.

Step 4 Dissolve Fr.1-1 in pure water and separate using chromatographic column C18 (250×20 mm i.d., 10 μm). Mobile phase composition: water and methanol, flow rate: 19mL/min, column temperature: room temperature, detection wavelength 227nm. After 0-23 min, 30% methanol was eluted to obtain compound i-1 (4.05 mg). The chromatogram is shown in Figure 3a.

Step 5 Dissolve Fr.1-2 in pure water and separate using chromatographic column C18 (250×20 mm i.d., 10 μm). Mobile phase composition: water and methanol, flow rate: 19mL/min, column temperature: room temperature, detection wavelength 227nm. 0-27min, eluted with 45% methanol to obtain compound i-2 (3.86 mg) and component Fr.1-2-1, the chromatogram is shown in Figure 3b.

Step 6: Dissolve Fr.1-3 in pure water and separate using chromatographic column C18 (250×20 mm i.d., 10 μm). Mobile phase composition: water and acetonitrile, flow rate: 19mL/min, column temperature: room temperature, detection wavelength 227nm. 0-25 min, eluted with 18% acetonitrile to obtain compound i-3 (2.23 mg), the chromatogram is shown in Figure 3c.

Step 7 Dissolve Fr.1-2-1 in pure water and separate using chromatographic column C18 (250×20 mm i.d., 10 μm). Mobile phase composition: water and acetonitrile, flow rate: 19mL/min, column temperature: room temperature, detection wavelength 227nm. After 0-12 min, 28% acetonitrile was eluted to obtain compound i-4 (6.24 mg). The chromatogram is shown in Figure 3d.

Example 2. Identification of four compounds in Stevia rebaudiana

In this example, the identification method of the four monomeric compounds in Stevia rebaudiana prepared in Example 1 is provided, using modern spectral techniques such as ¹ H NMR, ¹³ C NMR, two-dimensional nuclear magnetic spectrum, and high-resolution mass spectrometry. The structures of the four monomeric compounds obtained in 1 were identified.

Compound i-1: ESI-MS m/z: 207.14[M+H] ⁺ , molecular formula C ₁₃ H ₁₈ O ₂ . ^{Compound i-} 1 is: 2,7,10,10-tetramethyl-1-oxaspiro[4.5]deca-3,6-dien-8-one.

Compound i-2: ESI-MS m/z: 309.20[M+H] ⁺ , molecular formula C ₁₈ H ₂₈ O ₄ . Through ¹ H-NMR spectrum (Figure 9), ¹³ C-NMR spectrum (Figure 10), COZY spectrum (Figure 11), HMQC spectrum (Figure 12), HMBC spectrum (Figure 13) and NOESY spectrum (Fig. 14) determine that compound i-2 is:

(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: 517.27[M+H] ⁺ , molecular formula C ₂₄ H ₃₆ O ₁₂ , combined with ¹ H-NMR spectrum (Figure 15), ¹³ C-NMR spectrum (Figure 16) , COZY spectrogram (Fig. 17), HMQC spectrogram (Fig. 18) and HMBC spectrogram (Fig. 19-Fig. 21) determine that compound i-3 is:

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[M+H+H ₂ O] ⁺ , molecular formula C ₁₆ H ₂₃ NO ₆ , combined with ¹ H-NMR spectrum (Figure 22), ¹³ C-NMR spectrum (Fig. 23), COZY spectrogram (Fig. 24), HMQC spectrogram (Fig. 25) and HMBC spectrogram (Fig. 26 and Fig. 27) determine that compound i-4 is:

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 aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments, or perform equivalent replacements for some of the technical features. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (2)

1. a method for separation and purification of stevia basic substance, characterized in that, the method for separation and purification of said stevia basic substance comprises the steps: Using anhydrous methanol as a solvent to recrystallize the mother liquor sugar of stevia rebaudiana, and obtain the crude product of mother liquor sugar after filtering; The mother liquor sugar crude product was dissolved in pure water, and separated by reversed-phase chromatography for the first time; the reversed-phase chromatography for the first time included: using C18 stationary phase separation, using methanol and water as mobile phases, and washing with step gradient conditions. Take off, collect three sections of components, which are sequentially recorded as Fr.1, Fr.2 and Fr.3; the C18 stationary phase is an octadecyl bonded stationary phase; Dissolve Fr.1 in pure methanol and separate by supercritical fluid chromatography; the supercritical fluid chromatography includes: the stationary phase is C4, the mobile phase is supercritical CO ₂ , methanol is used as a modifier, and gradient elution is performed according to The peaks were collected to obtain 5 components, which were denoted as Fr.1-1, Fr.1-2, Fr.1-3, Fr.1-4 and Fr.1-5; Dissolve Fr.1-1 in pure water, and use the second reverse phase chromatography to separate; the second reverse phase chromatography includes: use C18 stationary phase separation, mobile phase is methanol and water, using 30% methanol, etc. degree elution to obtain compound i-1; Dissolve Fr.1-2 in pure water, and use the third reverse phase chromatography to separate; the third reverse phase chromatography includes: use C18 stationary phase separation, mobile phase is methanol and water, using 45% methanol, etc. degree elution to obtain compound i-2 and Fr.1-2-1; Dissolve Fr.1-3 in pure water, and use the fourth reverse phase chromatography to separate; the fourth reverse phase chromatography includes: use C18 stationary phase separation, mobile phase is acetonitrile and water, using 18% acetonitrile, etc. degree elution to obtain compound i-3; Dissolve Fr.1-2-1 in pure water and separate by reverse phase chromatography for the fifth time; said reverse phase chromatography for the fifth time includes: separation with C18 stationary phase, mobile phase is acetonitrile and water, and 28% Acetonitrile was eluted isocratically to obtain compound i-4; The stevia base material is the compound i-1 and the compound i-4; The structure of the compound i-1 is shown in formula i-1:

The structure of the compound i-4 is shown in formula i-4:

2. the separation and purification method of stevia base material as claimed in claim 1, is characterized in that, the solid-liquid ratio of described stevia mother liquor sugar and described anhydrous methanol is: add 120g sweetener in every 1 liter of anhydrous methanol Chrysanthemum mother liquid sugar.

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