CN111423480B

CN111423480B - Rebaudioside E crystal form X, preparation method and application thereof

Info

Publication number: CN111423480B
Application number: CN202010299790.3A
Authority: CN
Inventors: 夏咏梅; 王婉洁; 程双武; 沈洁; 胡学一; 樊晔; 刘湘
Original assignee: Jiangnan University
Current assignee: Dongtai Haorui Biological Technology Co ltd
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2022-03-04
Anticipated expiration: 2040-04-16
Also published as: CN111423480A

Abstract

The invention discloses a rebaudioside E crystal form X, a preparation method and application thereof, and belongs to the field of chemical pharmacy. The technology provided by the invention can be used. According to the invention, the rebaudioside-E crystal with the crystal form X is obtained by using low-carbon alcohol water as a solvent and utilizing rubusoside for solubilization and crystallization, so that the difficulty that the rebaudioside-E crystal is difficult to obtain crystals in a large amount with high efficiency due to low solubility of rebaudioside-E in an alcohol water solvent is overcome. The rebaudioside E crystal with the crystal form X is characterized by using technical means such as XRD, DSC, TGA, polarization microscopy and the like, and the rebaudioside E crystal with the crystal form X is found to have the advantages of high crystallinity, good stability and the like. The preparation method provided by the invention is simple, easy to operate and good in reproducibility, and can stably obtain the target crystal in batches.

Description

Rebaudioside E crystal form X, preparation method and application thereof

Technical Field

The invention relates to a rebaudioside E crystal form X, a preparation method and application thereof, and belongs to the field of chemical pharmacy.

Background

Stevioside, as a high sweetener, is widely applied to beverages, soy sauce, various marine foods, pickles, candies, baked foods and the like, has pharmaceutical activity, and has the effects of reducing blood sugar, resisting tumors, preventing decayed teeth, reducing blood pressure, resisting inflammation, resisting oxidation and the like in medical clinical practice. The stevioside has non-fermentation property, does not influence the coagulability and viscosity of food, is not easy to mildew, does not cause Maillard reaction in the processing process, and cannot be utilized by microorganisms, so that the shelf life of the stevioside product can be prolonged, and the stevioside product is easy to store and transport.

Rebaudioside E is an isomer of rebaudioside a, with similar polarity, and is not easily separable. Because rebaudioside E is present in stevia at low levels, its research and utilization is inadequate. Rebaudioside E was isolated and characterized from stevia rebaudiana Bertoni in 1977 by the saharan rules et al, whose structure is shown in formula (I) below. US patent No. US 20190269162 a1 discloses the use of rebaudioside E as an additive in orally consumable products, but the rebaudioside E used is polymorphic or amorphous rebaudioside E.

The same compound with different crystal forms has obvious difference in solubility, dissolution rate, melting point, density, hardness, appearance, bioavailability and the like, thereby affecting the stability and bioavailability of the compound. Patent CN 105037458A discloses multiple crystalline forms of rebaudioside D. Patent US 20130267693 Al discloses four crystalline forms of rebaudioside B. The research on the polymorphism of the drug has become an essential and important part before the pharmaceutical technology and the new drug preparation are determined. However, the crystal structure and crystallization method of rebaudioside E are rarely reported.

In general, when crystallization is performed by a method of concentration after dissolution, if the solubility of the target substance in a solvent is too low, it is difficult to obtain a large amount of crystals. Regarding rebaudioside E, when the inventors prepared crystals from a rebaudioside E powder having a purity of 95% obtained by spray drying as a raw material, the inventors found that the solubility of rebaudioside E in water at 25 ℃ was only 0.034% (i.e., 0.34mg/mL), the highest solubility at room temperature in an aqueous ethanol solution was also only about 0.38% (i.e., 3.8mg/mL), and the solubility in an aqueous 95% ethanol solution at 65 ℃ was also not sufficient for 1% (i.e., 10mg/mL) (see fig. 5 and 6 of the specification); too low solubility makes it difficult to obtain large amounts of rebaudioside E crystals using a post-dissolution concentration method.

Disclosure of Invention

[ problem ] to

The invention aims to solve the technical problem of improving the solubility of rebaudioside E in a solvent for crystallization so as to realize the batch preparation of the rebaudioside E crystal form X with high crystallinity and high stability.

[ solution ]

The invention provides a rebaudioside E crystal with a crystal form X, wherein the structure of the rebaudioside E is shown in a formula I, and an X-ray powder diffraction (XRD) pattern of the crystal form X has characteristic peaks at the following 2 theta +/-0.1 degrees: 5.45, 9.95, 12.85, 16.06, 18.72, 19.27, and 22.23.

In an X-ray powder diffraction pattern of the crystal form X measured by using Cu-Ka rays, the 2 theta +/-0.1 DEG angle value, the interplanar spacing d and the relative intensity of diffraction peaks have the characteristics shown in the following table 1.

TABLE 1

More specifically, the differential scanning calorimetry analysis of the rebaudioside E crystal with the crystal form X has characteristic heat absorption and release peaks in the ranges of 50-150 ℃ and 180-200 ℃.

The present invention also provides a method of preparing the rebaudioside E crystal having form X comprising the steps of:

firstly, adding rebaudioside E into a rubusoside solution at a temperature of 5-10 ℃ lower than the boiling point of a solvent, and stirring to obtain a suspension solution; then, filtering the suspension solution while the suspension solution is hot to respectively obtain filter residue and supernatant; and stirring the supernatant at the speed of 20-60rpm, cooling to-5-25 ℃, filtering and collecting precipitated white solid, washing the crystal with the same solvent at the temperature of 5-25 ℃, and drying the washed crystal to obtain the rebaudioside E crystal with the crystal form X.

More specifically, the filtration can adopt the modes of centrifugation, suction filtration, plate-and-frame filter pressing and the like.

More specifically, the purity (mass percentage) of the rubusoside is 50% -100%. If the impurity-containing rubusoside is used, the rest components are stevioside derived from stevia rebaudiana, wherein the mass content of rebaudioside A is respectively not more than 5% of the total mass of the rubusoside, and the mass content of the steviosides is not more than 5% of the total mass of other stevioside.

More specifically, in the rubusoside solution, the total mass concentration of rubusoside and other stevioside is 0.1% -5%, the mass of the added rebaudioside E in the suspension solution is 20-50 times of the total mass of rubusoside and other stevioside, and the final mass concentration of the added rebaudioside E in the suspension solution is not more than 80% at most.

More specifically, the dry matter purity of rebaudioside E is 70% to 100% when preparing a suspension solution.

More specifically, the solvent is one or a mixture of two or more of water, methanol, ethanol, isopropanol and propanol.

More specifically, when the supernatant is cooled under stirring, the cooling rate is 1-3 ℃/min.

More specifically, the drying may be vacuum drying or evaporation drying.

It is a further object of the present invention to provide the use of the novel rebaudioside E crystals, including as a sweetener to a food and beverage product, or as a flavoring agent to a medicament.

[ advantageous effects ]

The invention takes the rubusoside as the cosolvent to improve the solubility of the rebaudioside E. In view of the use of rebaudioside E, the added co-solvent must be safe, edible, preferably homogeneous with rebaudioside E, yet be easily separated from rebaudioside E during subsequent crystallization despite its similar structure. The rubusoside can greatly help dissolve rebaudioside E compared with other stevioside; under certain conditions, the rubusoside and the rebaudioside E can be separated through crystallization subsequently due to the large difference in polarity between the rubusoside and other steviol glycosides (especially rebaudioside A and steviosides).

The preparation method of the rebaudioside E crystal with the crystal form X can overcome the difficulty that the solubility of rebaudioside E in an alcohol-water solvent is too low, so that the rebaudioside E crystal is difficult to obtain crystals in a large scale with high efficiency. Under the participation of rubusoside, the product can be prepared in batch in common low-carbon alcohol water, and has high crystallinity, high stability, simple process and easy operation.

Drawings

FIG. 1 is an X-ray powder diffraction (XRD) pattern of rebaudioside E crystals of crystalline form X obtained in example 1.

FIG. 2 is a Differential Scanning Calorimetry (DSC) plot of rebaudioside E crystals of form X obtained in example 1 versus a control.

FIG. 3 is a thermogravimetric analysis (TGA) of the rebaudioside E crystal form X obtained in example 1 versus the crystal obtained in control example.

FIG. 4 is a polarization microscope photomicrograph of the rebaudioside E crystals with the crystal form X obtained in example 1 and the crystals obtained in comparative examples one to five; (a) the method comprises the following steps Rebaudioside E crystals in a crystalline form X; (b) the method comprises the following steps Comparing the first comparison example; (c) the method comprises the following steps Comparative example two; (d) the method comprises the following steps Comparative example three; (e) the method comprises the following steps Comparative example four; (f) the method comprises the following steps Control example five.

FIG. 5 is the solubility of rebaudioside E in aqueous ethanol at room temperature.

FIG. 6 is a graph of rebaudioside E solubility in ethanol and 95% ethanol versus temperature.

FIG. 7 is an HPLC plot of rebaudioside E crystals with form X obtained in example 1.

Detailed Description

The raw material rebaudioside E used in the following examples was isolated from conventional isolated steviol glycosides by column chromatography or adsorption on multi-stage macroporous resins followed by continuous elution.

Example one

1g rubusoside with a purity of 95% was dissolved in 100mL of 90% aqueous ethanol and heated to 70 ℃. 65g of rebaudioside E with a purity of 95% was slowly added to the above solution, and the mixture was stirred at 70 ℃ to obtain a suspension solution. Then filtering while the solution is hot to obtain supernatant and filter residue respectively. The supernatant was stirred at 20rpm and cooled to 4 ℃ at 2 ℃/min to precipitate a white solid. And (3) carrying out suction filtration to collect precipitated white solid, washing the crystal by using a 90% ethanol aqueous solution at 10 ℃, and then carrying out vacuum drying on the crystal at 30 ℃ to obtain 30g of rebaudioside-E crystal with the crystal form X, wherein a polarization micrograph is shown in figure 4 a.

Example two

2g rubusoside with a purity of 95% was dissolved in 100mL of 90% aqueous methanol and heated to 58 ℃. 55g of rebaudioside E with a purity of 90% was slowly added to the above solution, and stirred to obtain a suspension solution. Then filtering while the solution is hot to obtain supernatant and filter residue respectively. The supernatant was stirred at 50rpm and cooled to 10 ℃ at 3 ℃/min to precipitate a white solid. And filtering and collecting separated white solid, washing the crystal by using 90% methanol water solution at 10 ℃, and then volatilizing and drying the crystal at 50 ℃ to obtain 35g of rebaudioside-E crystal with the crystal form X.

Comparison example 1

At 58 ℃, 2g of rebaudioside E with a purity of 95% was added to 100mL of 90% aqueous methanol, stirred at 50rpm and cooled to 10 ℃ at a rate of 3 ℃/min, and filtered to obtain a white solid. The white solid was washed with 90% aqueous methanol at 10 ℃ and dried under vacuum at 25 ℃ to give 78mg of rebaudioside E crystals. The polarization micrograph of the resulting rebaudioside E is shown in fig. 4 b.

Comparative example two

2g of rebaudioside E, 95% pure, was added to a 45% aqueous solution of 100mL propanol at 60 deg.C, stirred for 2h and filtered to give a clear solution. Cooling the clear solution to 10 ℃ to separate out white solid; filtration, washing of the crystals with a 45% aqueous propanol solution at 10 ℃ and vacuum drying at 30 ℃ gave 80mg rebaudioside E crystals as a white solid. The polarization micrograph of the resulting rebaudioside E is shown in fig. 4 c.

EXAMPLE III

3g rubusoside with a purity of 75% was dissolved in 100mL of 95% aqueous ethanol and heated to 70 ℃. 80g of rebaudioside E with a purity of 80% was slowly added to the above solution, and stirred to obtain a suspension solution. Then filtering while the solution is hot to obtain supernatant and filter residue respectively. The supernatant was stirred at 40rpm and cooled to 0 ℃ at 1 ℃/min to precipitate a white solid. And filtering and collecting the precipitated white solid, washing the white solid by using a 95% ethanol water solution at 25 ℃, and then drying the crystal in vacuum at 30 ℃ to obtain the rebaudioside E crystal with the crystal form X.

Comparative example three

At 60 ℃, adding 10g of rebaudioside E with the purity of 90% into 100mL of 95% ethanol aqueous solution, stirring for 2h, filtering to obtain a clear solution, cooling the clear solution to 0 ℃, precipitating a white solid, filtering, washing the white solid with 25 ℃ 95% ethanol aqueous solution, and then drying the crystal at 30 ℃ in vacuum, wherein a crystal polarization micrograph of the rebaudioside E is shown in fig. 4 d.

Comparative example four

At 60 ℃, 10g of rebaudioside E with a purity of 90% was added to 100mL of 95% aqueous ethanol solution containing 1g of tween 80, stirred for 2h and filtered to obtain a clear solution, the clear solution was cooled to 25 ℃, a white solid was precipitated, the white solid was filtered while being washed with 25 ℃ aqueous ethanol solution with a purity of 95%, and a crystal polarization micrograph of the rebaudioside E obtained was vacuum-dried at 30 ℃ as shown in fig. 4E.

Example four

1g rubusoside with 70% purity was dissolved in 100mL 80% aqueous propanol and heated to 75 ℃. 30g of rebaudioside E with a purity of 90% was slowly added to the above solution, and stirred to obtain a suspension solution. Then filtering while the solution is hot to obtain supernatant and filter residue respectively. The supernatant was stirred at 40rpm and cooled to 25 ℃ at 2 ℃/min to precipitate a white solid. And filtering and collecting precipitated white solid, washing the crystal by using 80% propanol water solution at 25 ℃, and then drying the crystal in vacuum at 30 ℃ to obtain the rebaudioside E crystal with the crystal form X.

Comparative example five

At room temperature, 5g of rebaudioside E with a purity of 90% was added to 100mL of acetone, stirred for 12 hours, filtered, and the filtrate was allowed to stand at room temperature to slowly volatilize the solution to obtain a white solid. The white solid was dried under vacuum at 25 ℃ and the crystal polarization micrograph of the resulting rebaudioside E is shown in fig. 4 f.

X-ray powder diffraction analysis (XRD), Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA), polarization microscope photograph, solubility measurement, and the like were performed on the rebaudioside E crystal having the crystal form X prepared in example 1.

XRD analysis: the detection is carried out by using an X-ray photoelectron spectrometer model D8 of Bruker AXS GmbH, Germany at room temperature, and the scanning is carried out from 5 degrees to 60 degrees by using an X-ray 2 theta angle scanning with the scanning speed of 2 degrees/min. The band of the XRD diffraction pattern of the specific crystalline form is characteristic. While the relative intensities of the diffraction peaks are not characteristic of the crystal in question, the positions of a set of characteristic peaks are characteristic of a given crystal. Fig. 1 shows that the XRD spectrum shows that the obtained rebaudioside E crystal with the crystal form X has good crystallinity.

DSC analysis: the measurement was carried out by a differential scanning calorimeter model 204F1 manufactured by German Steady instruments manufacturing Co., Ltd, the atmosphere was nitrogen gas, and the heating rate was 10K/min. The analysis results are shown in FIG. 2.

TG analysis: the detection is carried out by adopting a TGA/DSC1/1100SF thermogravimetric analyzer of Meiteler-Toritoduo International trade (Shanghai) Co., Ltd., the temperature range is 30-600 ℃, the scanning speed is 10K/min, and the purging gas is 20mL/min of nitrogen. The analysis results are shown in FIG. 3.

Polarization micrograph: experiments were carried out using an Axio Imager A2POL model hot stage polarizing microscope from Call, Zeiss, Inc., at a test magnification of 500. The analysis result is shown in a polarizing micrograph of fig. 4, and the rebaudioside E crystal with the crystal form X prepared in the above example is a long crystal and has good morphological characteristics.

And (3) measuring the solubility: adding a proper amount of solvent into a round-bottom flask, and then slowly adding the rebaudioside E crystal with the crystal form X until turbidity appears. Stirring for 2h in a constant-temperature water bath, standing for 24h, taking supernatant, filtering with a 0.22 μm filter membrane, and analyzing the rebaudioside E content in the solution with a high performance liquid chromatograph. The analysis conditions of the high performance liquid chromatography are calculated by referring to GB8270(GB8270-2014 food safety national standard food additive stevioside) and using rebaudioside A standard yeast.

The HPLC profile of rebaudioside E crystals with form X (fig. 7) indicated that the crystals contained no rubusoside (retention time should be around 9 min).

The rebaudioside E crystal with the crystal form X can be used as a sweetening agent to be applied to food, beverage and medicines.

Example five sweet drink

A sweet drink is composed of the following raw materials: 3g of citric acid, 4g of xylitol, 1.5g of iso-VC sodium, 20g of high fructose corn syrup, 20g of maltodextrin, 2g of water-soluble dietary fiber, 1g of sodium hexametaphosphate and 1g of rebaudioside E crystal with the crystal form X.

Example six A sweet potato product

A potato dessert, which consists of the following raw materials: 250g of potato, 20g of raisin, 20g of red date and 0.5g of rebaudioside E crystal with the crystal form X.

Example seven Green sweetener

A green sweetener, which consists of the following raw materials: 20g of sugarcane powder, 20g of cyclodextrin and 1g of rebaudioside E crystal with the crystal form X.

Example eight Children's cold-treating medicine

A medicine for treating children cold is prepared from the following raw materials: 80g of acetaminophen, 7g of pseudoephedrine hydrochloride, 0.3g of chlorpheniramine maleate, 20g of starch, 8g of sodium carboxymethyl cellulose, 3g of magnesium stearate and 8g of rebaudioside E crystal with the crystal form X.

Claims

1. A method for preparing rebaudioside E crystals, comprising the steps of:

firstly, adding rebaudioside E into a rubusoside solution at a temperature of 5-10 ℃ lower than the boiling point of a solvent, and stirring to obtain a suspension solution; then, filtering the suspension solution while the suspension solution is hot to respectively obtain filter residue and supernatant; stirring the supernatant at the speed of 20-60rpm, cooling to-5-25 ℃, filtering and collecting precipitated white solid, washing the crystal with the same solvent at the temperature of 5-25 ℃, and drying the washed crystal to obtain rebaudioside E crystal;

the structure of the rebaudioside E is shown in a formula I, the rebaudioside E crystal has a crystal form X,

the X-ray powder diffraction pattern of the crystal form X has characteristic peaks at the following 2 theta +/-0.1 degrees: 5.45, 9.95, 12.85, 16.06, 18.72, 19.27, and 22.23.

2. The method of claim 1, wherein the rubusoside is 50% to 100% pure.

3. The method of claim 1, wherein the total mass concentration of rubusoside and other steviol glycosides in the rubusoside solution is 0.1% -5%, and the mass of the added rebaudioside E in the suspension solution is 20-50 times the total mass of rubusoside and other steviol glycosides, but the final mass concentration of the added rebaudioside E in the suspension solution is no more than 80% at the most.

4. The method of claim 1 or 3, wherein the dry matter purity of rebaudioside E used in preparing the suspension solution is 70% -100%.

5. The method according to any one of claims 1-3, wherein the solvent of the rubusoside solution is one or a mixture of two or more of water, methanol, ethanol, isopropanol, and propanol.

6. The method according to claim 4, wherein the solvent of the rubusoside solution is one or a mixture of two or more of water, methanol, ethanol, isopropanol, and propanol.

7. The method according to claim 1, wherein the form X has the following characteristics in 2 θ ± 0.1 ° angle value, interplanar spacing d and relative intensity of diffraction peaks in an X-ray powder diffraction pattern measured by using Cu-Ka radiation:

8. the method of claim 7, wherein the differential scanning calorimetry analysis of the rebaudioside E crystal form X has characteristic endothermic and exothermic peaks in the ranges of 50-150 ℃ and 180-200 ℃.