Preparation method of atractylodes 5 oligosaccharide pentasaccharide

Abstract

The application provides a preparation method of a radix morindae officinalis oligosaccharide 5 polysaccharide, which comprises the following steps: acetylating a sample containing the radix morindae officinalis oligosaccharide 5 polysaccharide, then performing chromatography, and finally deacetylating; the sample containing the radix morindae officinalis oligosaccharide 5 polysaccharide is subjected to the steps of 'acetylation, separation, deacetylation and purification', the non-polar group of the sample is increased, the chemical properties of the radix morindae officinalis oligosaccharide 5 polysaccharide and corresponding impurities with the same polarity are changed, so that the polarities of the two compounds are increased, then the two compounds are separated through chromatography to obtain a single acetylated sample, the target object is deacetylated, the impurity peaks with a separation degree less than 1 from the five oligosaccharides in the original sample are successfully removed, and the radix morindae officinalis oligosaccharide 5 polysaccharide sample without impurity peak interference is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant extract preparation, in particular to a preparation method of Morinda officinalis oligosaccharide 5 polysaccharide. BACKGROUND

[0002] Morinda officinalis oligosaccharide 5 polysaccharide is mainly isolated from the dried roots of Morinda officinalis How of Rubiaceae. Pharmacological studies show that Morinda officinalis oligosaccharide has obvious antidepressant activity, and 5 polysaccharide is one of the main components. Its 1F-fruit furan glycosyl nystose has a molecular formula of C 30 H 52 O 26 , a molecular weight of 828.72, and a structural formula as shown in Figure 1 According to literature research, Morinda officinalis oligosaccharide 5 polysaccharide has direct application of high-pressure preparation liquid phase for separation and preparation (see the article "Separation and preparation of Morinda officinalis oligosaccharide 5 polysaccharide"). However, according to the previous research, it is found that there is an impurity peak with a separation degree less than 1 in the sample prepared by directly using a medium-pressure preparation liquid chromatography for Morinda officinalis oligosaccharide extract (see Figure 2 ), and the impurity peak cannot be directly and effectively separated by using various chromatographic columns, which affects the true content of Morinda officinalis oligosaccharide 5 polysaccharide. SUMMARY

[0003] Therefore, the technical problem to be solved by the present application is to overcome the defects of high impurity content and low purity of the prepared Morinda officinalis oligosaccharide 5 polysaccharide in the prior art, so as to provide a preparation method of Morinda officinalis oligosaccharide 5 polysaccharide, which has low impurity content and high purity.

[0004] To this end, the present application provides the following technical solution:

[0005] A preparation method of Morinda officinalis oligosaccharide 5 polysaccharide, comprising the following steps: acetylating a sample containing Morinda officinalis oligosaccharide 5 polysaccharide, then performing chromatography, and finally deacetylating.

[0006] Optionally, in the acetylation step, pyridine is used to dissolve the sample containing Morinda officinalis oligosaccharide 5 polysaccharide, and an excess of acetic anhydride is added and stirred.

[0007] Optionally, the stirring conditions are as follows: temperature 5-50℃, stirring speed 5-200rpm, and stirring time 3-48 hours.

[0008] Optionally, in the chromatography step, the reaction solution obtained after acetylation is evaporated under reduced pressure, dissolved in tetrahydrofuran, and mixed with silica gel, and petroleum ether and acetone are used as eluents to perform silica gel column chromatography to obtain acetylated Morinda officinalis oligosaccharide 5 polysaccharide.

[0009] Optionally, the solvent used for the dissolving is a small polarity solvent

[0010] Optionally, the solvent used for the dissolving is tetrahydrofuran or DMSO.

[0011] Optionally, the volume ratio of the eluent petroleum ether 60℃-90℃ and acetone is (10-2)∶2;

[0012] Optionally, the volume ratio of the eluent petroleum ether 60℃-90℃ and acetone is 3.5∶2.

[0013] Optionally, the silica gel is silica gel H.

[0014] Optionally, in the deacetylation step, tetrahydrofuran is added to the eluent after the chromatography, an excess of alkaline solution is added, heated to the reflux of tetrahydrofuran, then liquid-liquid separation is performed, the water layer is neutralized to neutral, and dried.

[0015] Optionally, the heating to the reflux of tetrahydrofuran is 3-48 hours;

[0016] Optionally, the heating to the reflux of tetrahydrofuran is 12 hours.

[0017] Optionally, the alkaline solution includes NaOH or KOH;

[0018] Optionally, the concentration of the alkaline solution is 1-20% by mass;

[0019] Optionally, the concentration of the alkaline solution is 5%.

[0020] Optionally, a step of purifying the deacetylated Radix Panacis Quinquefolii oligosaccharide 5-glycan sample obtained by the deacetylation step is included;

[0021] Optionally, the deacetylated Radix Panacis Quinquefolii oligosaccharide 5-glycan sample is purified by medium pressure preparative liquid chromatography, and the chromatographic conditions of the medium pressure preparative liquid chromatography are as follows: octadecyl-bonded silica gel, mobile phase is 0-10% methanol by volume, isocratic elution, flow rate is 10-25 ml / min, column temperature is 20-40℃;

[0022] Optionally, in the medium pressure preparative liquid chromatography, the chromatographic column filler is YMC*GEL ODS-A-HG 12nm S-50μm;

[0023] Optionally, the sample obtained after the deacetylated Radix Notoginseng oligosaccharide 5 polysaccharide sample is purified by the medium-pressure preparative liquid chromatography is purified by high-pressure preparative liquid chromatography, and the high-pressure preparative liquid chromatography has the following conditions: an octadecyl-bonded silica C18 preparative column, a differential detector, a mobile phase of methanol and water in a volume ratio of 0-10:100-90, a flow rate of 3-10 ml / min, an isocratic elution mode, and a column temperature of 20-40 DEG C.

[0024] The technical scheme of the present application has the following advantages:

[0025] 1. The preparation method of the Radix Notoginseng oligosaccharide 5 polysaccharide provided by the present application comprises the following steps: acetylating a sample containing the Radix Notoginseng oligosaccharide 5 polysaccharide, then performing chromatography, and finally deacetylating; the steps of "acetylating, separating, and deacetylating" are performed on the sample containing the Radix Notoginseng oligosaccharide 5 polysaccharide, the non-polar groups of the sample are increased, the chemical properties of the Radix Notoginseng oligosaccharide 5 polysaccharide and the corresponding impurities with the same polarity are changed, so that the polarities of the two compounds are increased, the two compounds are separated by chromatography at this time to obtain a single acetylated sample, the target substance is deacetylated, the impurity peaks with a separation degree less than 1 from the five oligosaccharides in the original sample are successfully removed, and a Radix Notoginseng oligosaccharide 5 polysaccharide sample without impurity peak interference is obtained, and the prepared Radix Notoginseng oligosaccharide 5 polysaccharide has low impurities and high purity.

[0026] 2. The preparation method of the Radix Notoginseng oligosaccharide 5 polysaccharide provided by the present application combines the medium-pressure preparation method, the chemical method, and the high-pressure preparative liquid method, can obtain a purer sample, obtain a sample with a purity greater than 99%, and greatly improve the preparation quantity of the finished product, so that the batch production purpose is more easily achieved. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present application or the technical scheme in the prior art, the drawings needed in the following description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0028] Figure 1 is the structure formula of 1F- fructofuranosyl neokestose in the background art;

[0029] Figure 2 is the amino column determination result of the sample obtained by directly using the medium-pressure preparative liquid chromatography to prepare the Radix Notoginseng oligosaccharide extract in Embodiment 1 of the present application;

[0030] Figure 3 is the octadecyl-bonded silica column determination result of the sample obtained by directly using the medium-pressure preparative liquid chromatography to prepare the Radix Notoginseng oligosaccharide extract in Embodiment 1 of the present application;

[0031] Figure 4 The results of the amino column determination of the sample of Radix Morindae Officinalis oligosaccharide 5 polysaccharide prepared by medium pressure preparative chromatography in Step 3 in Example 1 of the present application are shown in Table 1.

[0032] Figure 5 The results of the Radix Morindae Officinalis oligosaccharide 5 polysaccharide prepared by high pressure preparative chromatography in Step 3 in Example 1 of the present application are shown in Table 2. DETAILED DESCRIPTION

[0033] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application or the protection afforded. Any product derived from the application or from the combination of the application with other prior art features, which is the same as or similar to the application, falls within the scope of the present application.

[0034] If the specific experimental steps or conditions are not mentioned in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art. If the reagents or instruments are not mentioned by the manufacturer, they are conventional reagent products that can be obtained by purchase.

[0035] The crude Radix Morindae Officinalis oligosaccharide extract used in the following examples was prepared as follows: Radix Morindae Officinalis medicinal materials were extracted with 3-14 times the amount of water for 1-6 times, each time for 0.5-3 hours, the extract was combined, filtered, and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.40. The extract was passed through an activated carbon column and eluted with 5-50 times the amount of 0-20% ethanol by volume based on the weight of the medicinal materials. The eluate was discarded, and then the extract was eluted with 5-50 times the amount of 20-95% ethanol by volume based on the weight of the medicinal materials. The eluate was collected, concentrated under reduced pressure to a relative density of 1.10-1.40, and dried to obtain the product.

[0036] Example 1

[0037] A preparation method of Radix Morindae Officinalis oligosaccharide 5 polysaccharide, comprising:

[0038] 1. The crude extract of oligosaccharides from Radix Morindae Officinalis is separated by medium pressure preparative liquid chromatography. The medium pressure preparative liquid chromatography conditions are as follows: the column (the packing material is YMC*GEL ODS-A-HG 12nm S-50μm), the mobile phase is pure water, isocratic elution, the flow rate is 17ml / min, the sample loading amount is 20g (the sample solution is prepared by diluting the crude extract of oligosaccharides from Radix Morindae Officinalis with the mobile phase and then filtering through a filter membrane with a pore size of 0.45μm, and the filtrate is the sample solution), and the column temperature is 30℃. The sample yield of the crude extract of oligosaccharides from Radix Morindae Officinalis separated by medium pressure preparative liquid chromatography is about 6.25%. The sample obtained above is determined by an amino column, and the determination conditions are as follows: a differential detector, the volume ratio of acetonitrile to water is (68:32), the water phase contains a constant volume percentage of eluent, the column temperature is 40℃, the flow rate is 1ml / min, and the chromatogram is shown in Fig. 1. The sample obtained above is determined by an octadecyl-bonded silica gel column, and the determination results are shown in Fig. 2. The purity of the sample obtained above is about 92%, and the sample only contains pentasaccharides and impurity peaks with a separation degree less than 1 which are to be removed. Figure 2 . Figure 3 .

[0039] 2. The sample containing pentasaccharides from Radix Morindae Officinalis oligosaccharides obtained in step 1 is collected, pyridine (10ml) is added to dissolve the sample (200mg) containing pentasaccharides from Radix Morindae Officinalis oligosaccharides, an excess of acetic anhydride (1.5ml) is added, and stirring is performed at room temperature for about 24 hours. The stirring conditions are as follows: stirring at 25℃ for 24h, the stirring speed is 100r / min, and the reaction progress is detected by thin layer chromatography. After complete acetylation, the obtained reaction solution is evaporated under reduced pressure, dissolved in a small amount of tetrahydrofuran (2ml), and mixed with silica gel H (the mass ratio of silica gel H to the sample is 1:1). The sample is loaded, eluted with petroleum ether (60℃-90℃) and acetone (the volume ratio is 3.5:2) as the eluent, the eluate is collected, and acetylated pentasaccharides from Radix Morindae Officinalis oligosaccharides are obtained by silica gel H column chromatography. The acetylated pentasaccharides from Radix Morindae Officinalis oligosaccharides are dissolved in an appropriate amount of tetrahydrofuran (10ml), an excess of 5% NaOH (10ml) solution is added, and heated to reflux for 12 hours. After the reaction is completed, the reaction solution is placed in a separatory funnel, the water layer containing NaOH is separated, neutralized to neutral with 5% hydrochloric acid, and evaporated to obtain deacetylated pentasaccharides from Radix Morindae Officinalis oligosaccharides.

[0040] 3. The deacetylated Panax oligosaccharide 5-glycan obtained in step 2 is purified by medium pressure preparative liquid chromatography. The medium pressure preparative liquid chromatography conditions are as follows: octadecylsilane-bonded silica gel C18 preparative column (YMC*GEL ODS-A-HG 12 nm S-50 μm), mobile phase is pure water, isocratic elution, flow rate is 17 ml / min, column temperature is 30°C; sample loading amount is 20 g (sample solution: the deacetylated Panax oligosaccharide 5-glycan is diluted with the mobile phase, and then filtered through a filter membrane with a pore size of 0.45 μm, and the filtrate is the sample solution), column temperature is 30°C. The above medium pressure preparative chromatography is used to remove sodium chloride and pigments, and a Panax oligosaccharide 5-glycan sample is obtained, and the yield is about 50%. The Panax oligosaccharide 5-glycan sample obtained above is determined by an amino column. The determination conditions are as follows: differential detector, mobile phase is acetonitrile and water in a volume ratio of (68:32), the water phase contains 0.1% triethylamine by volume, isocratic elution, column temperature is 40°C, and flow rate is 1 ml / min. The determination results are shown in Table 1. Figure 4 The impurity peaks with a separation degree less than 1 from the five oligosaccharides in the original sample are successfully removed, and the purity is about 96%.

[0041] 4. The Panax oligosaccharide 5-glycan sample obtained in step 3 is purified by high pressure preparative chromatography. The chromatography conditions are as follows: octadecylsilane-bonded silica gel C18 preparative column, differential detector, mobile phase is methanol and water in a volume ratio of (5:95), flow rate is 6 ml / ml, isocratic elution mode, and column temperature is about 25°C. A Panax oligosaccharide 5-glycan sample with a purity greater than 99% is obtained, and the yield of this step is about 90%. The chromatogram is shown in Table 2. Figure 5 .

[0042] Example 2

[0043] A preparation method of a Panax oligosaccharide 5-glycan, comprising:

[0044] 1. A crude Panax oligosaccharide extract is separated by medium pressure preparative liquid chromatography. The medium pressure preparative liquid chromatography conditions are as follows: chromatographic column (filler is YMC*GEL ODS-A-HG 12 nm S-50 μm), mobile phase is pure water, isocratic elution, flow rate is 16 ml / min, sample loading amount is 19.5 g (sample solution: the crude Panax oligosaccharide extract is diluted with the mobile phase, and then filtered through a filter membrane with a pore size of 0.45 μm, and the filtrate is the sample solution), and column temperature is 30°C.

[0045] 2. Collect the sample containing the acetylated panax oligosaccharide 5-mer obtained in step 1, dissolve the sample (195 mg) in pyridine (11 ml), add excess acetic anhydride (1.7 ml), stir at room temperature for about 22 hours, the stirring condition is 5 °C, stirring speed is 200 r / min, and the reaction progress is monitored by thin layer chromatography. After complete acetylation, the reaction solution is evaporated under reduced pressure, dissolved in a small amount of tetrahydrofuran (1.8 ml), and then mixed with silica gel H (the mass ratio of silica gel H to the sample is 1:1), loaded, eluted with petroleum ether (60-90 °C) and acetone (volume ratio of 10:2) as eluent, collected the eluate, and then purified by silica gel H column chromatography to obtain the acetylated panax oligosaccharide 5-mer. The acetylated panax oligosaccharide 5-mer is dissolved in an appropriate amount of tetrahydrofuran (11 ml), an excess of 1% NaOH (5 ml) solution is added, and heated to reflux for 3 hours. After the reaction is completed, the reaction solution is placed in a separatory funnel, the water layer containing NaOH is separated, neutralized to neutral with 5% hydrochloric acid, and evaporated to obtain the deacetylated panax oligosaccharide 5-mer.

[0046] 3. Purify the deacetylated panax oligosaccharide 5-mer obtained in step 2 by medium pressure preparative liquid chromatography, and the medium pressure preparative liquid chromatography conditions are as follows: octadecyl-bonded silica gel C18 preparative column, chromatographic column (filler is YMC*GELODS-A-HG 12nm S-50μm), mobile phase is 10% methanol (volume percentage), isocratic elution, flow rate is 25 ml / min, loading amount is 20 g (the sample solution is prepared by diluting the deacetylated panax oligosaccharide 5-mer with the mobile phase, and then filtering through a filter membrane with a pore size of 0.45 μm, and the filtrate is the sample solution), and the column temperature is 32 °C. The above medium pressure preparative chromatography is used to remove sodium chloride and pigments to obtain a panax oligosaccharide 5-mer sample.

[0047] 4. Purify the panax oligosaccharide 5-mer sample obtained in step 3 by high pressure preparative chromatography, and the chromatography conditions are as follows: octadecyl-bonded silica gel C18 preparative column, differential detector, mobile phase is methanol and water (volume ratio is 10:90), flow rate is 3 ml / ml, isocratic elution mode, and the column temperature is about 23 °C. The high pressure preparative chromatography is used to obtain a panax oligosaccharide 5-mer with a purity of more than 99%.

[0048] Example 3

[0049] A method for preparing a panax oligosaccharide 5-mer, comprising:

[0050] 1. The crude extract of oligosaccharides from Radix Morindae Officinalis is separated by medium pressure preparative liquid chromatography. The medium pressure preparative liquid chromatography conditions are as follows: a chromatographic column (the packing material is YMC*GEL ODS-A-HG 12nm S-50μm), the mobile phase is pure water, isocratic elution, the flow rate is 17ml / min, the sample loading amount is 20g (the sample solution is prepared by diluting the crude extract of oligosaccharides from Radix Morindae Officinalis with the mobile phase, and then filtering through a filter membrane with a pore size of 0.45μm, and the filtrate is the sample solution), and the column temperature is 30°C.

[0051] 2. The sample containing 5-mer oligosaccharides from Radix Morindae Officinalis obtained in step 1 is collected, pyridine (12ml) is added to dissolve the sample containing 5-mer oligosaccharides from Radix Morindae Officinalis (205mg), an excess amount of acetic anhydride (1.8ml) is added, and stirring is carried out for about 48 hours under the conditions of stirring at 50°C and a stirring speed of 5 revolutions / min. The reaction progress is monitored by thin layer chromatography. After complete acetylation, the obtained reaction solution is evaporated under reduced pressure, dissolved in a small amount of tetrahydrofuran (2ml), and then mixed with silica gel H (the mass ratio of silica gel H to the sample is 1:1), loaded, eluted with petroleum ether (60°C-90°C) and acetone (the volume ratio is 2:2) as the eluent, and the eluate is collected. The acetylated 5-mer oligosaccharides from Radix Morindae Officinalis are obtained by silica gel H column chromatography. The acetylated 5-mer oligosaccharides from Radix Morindae Officinalis are dissolved in an appropriate amount of tetrahydrofuran (10ml), an excess amount of 20% NaOH (5ml) is added, and heating is carried out to reflux the tetrahydrofuran for 48 hours. After the reaction is completed, the reaction solution is placed in a separatory funnel, the water layer containing NaOH is separated, neutralized to neutral with 5% hydrochloric acid, and evaporated to obtain deacetylated 5-mer oligosaccharides from Radix Morindae Officinalis.

[0052] 3. The deacetylated 5-mer oligosaccharides from Radix Morindae Officinalis obtained in step 2 are purified by medium pressure preparative liquid chromatography. The medium pressure preparative liquid chromatography conditions are as follows: an octadecylsilane-bonded silica gel C18 preparative column (YMC*GEL ODS-A-HG 12nm S-50μm), the mobile phase is pure water, isocratic elution, the flow rate is 10ml / min, the sample loading amount is 20g (the sample solution is prepared by diluting the deacetylated 5-mer oligosaccharides from Radix Morindae Officinalis with the mobile phase, and then filtering through a filter membrane with a pore size of 0.45μm, and the filtrate is the sample solution), and the column temperature is 20°C.

[0053] 4. The 5-mer oligosaccharides from Radix Morindae Officinalis obtained in step 3 are purified by high pressure preparative chromatography. The chromatography conditions are as follows: an octadecylsilane-bonded silica gel C18 preparative column, a differential detector, the mobile phase is pure water, the flow rate is 10ml / min, isocratic elution, and the column temperature is about 35°C. A sample with a purity of greater than 99% is obtained.

[0054] Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. A preparation method of a Panax oligosaccharide 5 glycan, characterized in that, The method comprises the following steps: acetylating a sample containing Radix Panacis oligosaccharide 5 polysaccharide, then performing chromatography, and finally deacetylating; In the chromatography step, the reaction solution obtained after acetylation is evaporated under reduced pressure, dissolved with a small-polarity solvent, and then subjected to silica gel column chromatography with petroleum ether and acetone as eluents to obtain acetylated Radix Panacis oligosaccharide 5 polysaccharide; the volume ratio of the eluents petroleum ether 60-90 DEG C and acetone is (10-2):2; The sample obtained after the deacetylated Radix Panacis oligosaccharide 5 polysaccharide sample is purified by the medium-pressure preparative liquid chromatography is subjected to high-pressure preparative liquid chromatography purification; In the medium-pressure preparative liquid chromatography, the chromatographic conditions are as follows: octadecylsilane-bonded silica gel, and the mobile phase is 0-10% methanol by volume, and isocratic elution; In the high-pressure preparative liquid chromatography, the chromatographic conditions are as follows: octadecylsilane-bonded silica gel C18 preparative column, differential detector, and the mobile phase is methanol and water with a volume ratio of 0-10:100-90, and isocratic elution.

2. The method of claim 1, wherein the preparation of the ginseng oligosaccharide 5-mer is characterized by, In the acetylation step, the sample containing Radix Panacis oligosaccharide 5 polysaccharide is dissolved in pyridine, and excess acetic anhydride is added and stirred.

3. The method of claim 2, wherein the preparation of the ginseng oligosaccharide 5-mer is characterized by, The stirring conditions are as follows: a temperature of 5-50 DEG C, a stirring speed of 5-200 r / min, and a stirring time of 3-48 hours.

4. The method of claim 1, wherein the preparation of the ginseng oligosaccharide 5-mer is characterized by, In the chromatography step, the solvent used for dissolving is tetrahydrofuran or DMSO.

5. The method according to claim 4, wherein the volume ratio of the eluents petroleum ether 60-90 DEG C and acetone is 3.5:

2. The silica gel is silica gel H.

6. The method of claim 4, wherein the preparation of the ginseng oligosaccharide 5 polysaccharide is characterized by, In the deacetylation step, the eluate after chromatography is dissolved in tetrahydrofuran, an excess of an alkaline solution is added, heated to tetrahydrofuran reflux, and then subjected to liquid-liquid separation, the water layer is neutralized to neutral, and dried.

7. The method of preparing Panax oligosaccharide 5-glycan according to any one of claims 1-6, characterized in that, The heating to tetrahydrofuran reflux is performed for 3-48 hours.

8. The method of claim 7, wherein the preparation of the ginseng oligosaccharide 5-mer is characterized by, 9. The method according to claim 8, wherein the heating to tetrahydrofuran reflux is performed for 12 hours. The alkaline solution comprises NaOH or KOH.

11. The method according to claim 7, wherein the concentration of the alkaline solution is 1-20% by mass. 10.The method of claim 7, wherein the preparation of the ginseng oligosaccharide 5-mer is characterized by, 12. The method according to claim 11, wherein the concentration of the alkaline solution is 5%. In the chromatographic conditions of the medium-pressure preparative liquid chromatography, the flow rate is 10-25 ml / min, and the column temperature is 20-40 DEG C. In the medium-pressure preparative liquid chromatography, the chromatographic column filler is YMC*GEL ODS-A-HG 12nm S-50μm. In the chromatographic conditions of the high-pressure preparative liquid chromatography, the flow rate is 3-10 ml / min, and the column temperature is 20-40 DEG C. ​ 13. The method of preparing Panax oligosaccharide 5-glycan according to any one of claims 1-6, characterized in that, ​ ​ ​

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