Method for ultrasonic-assisted aqueous two-phase extraction of fructus aurantii polysaccharide

Abstract

The invention discloses an ultrasonic-assisted aqueous two-phase extraction method for fructus aurantii polysaccharide. An ultrasonic-assisted aqueous two-phase system (ATPS) is adopted for extracting polysaccharide components in traditional Chinese medicine fructus aurantii. A single factor experiment is adopted to determine the two-phase components of the ultrasonic-assisted aqueous two-phase, the mass fraction of the two-phase components, the optimal material-liquid ratio, the temperature, the time and other conditions; and the extraction process conditions are optimized by using a response surface test, and the rapid and effective extraction method based on the ultrasonic-assisted aqueous two-phase system is established. Results show that in ATPS, the mass fraction of the organic phase ethanol is 27.5% (w / w), the mass fraction of the inorganic phase ammonium sulfate is 17% (w / w), the material-liquid ratio is 1: 50g / g, the extraction time is 20 min, the extraction temperature is 40 DEG C, and the yield is 10.9%.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of chitosan polysaccharide, and relates to a method for separating and purifying chitosan polysaccharide by using an ultrasonic-assisted organic solvent-inorganic salt two-phase aqueous system. Background Art

[0002] 1. Citrus aurantium is a traditional Chinese medicinal material, which can relieve cough and asthma, treat symptoms of poor digestive function and abdominal distension and pain, etc. The polysaccharide compounds in Citrus aurantium have a variety of biological activities: such as antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, hypolipidemic and hypoglycemic effects. The study of Citrus aurantium polysaccharide not only helps to reveal its specific mechanism and application in the field of traditional Chinese medicine, but also provides a scientific basis for the development of new natural medicines, so its research is of great significance. At present, the extraction method of Citrus aurantium polysaccharide is mainly hot water extraction and ethanol precipitation. However, in the existing technology, the extraction of Citrus aurantium polysaccharide with too high hot water not only has a very low yield, but also contains a lot of impurities such as proteins, pigments, and flavonoids in the extracted Citrus aurantium polysaccharide. It brings many difficulties to the subsequent purification. At the same time, during the hot water extraction of Citrus aurantium polysaccharide, a large amount of flavonoids, proteins, and pigments will be produced in the Citrus aurantium polysaccharide. In order to remove these impurities, the use of chloroform and n-butanol organic solutions is involved. These chemical reagents are toxic and harmful reagents, which pose certain safety hazards to the human body. Activated carbon is used to remove pigments, but its selectivity is extremely poor, and its efficiency in adsorbing pigments is not high, and it will also cause a large loss of Citrus aurantium polysaccharides.

[0003] 2. The environment in the aqueous two-phase system is relatively mild, usually carried out at room temperature or lower temperatures, without the need for organic solvents or high temperatures, which helps to protect the structure and biological activity of polysaccharides. The aqueous two-phase system has good selectivity for different substances, and can effectively separate the target polysaccharides from impurities such as proteins, pigments, and lipids, thereby improving the purity of polysaccharide extraction. Aqueous two-phase extraction can be carried out directly in the aqueous phase, without the need for complex equipment and operations, and compared with organic solvent extraction, it reduces safety and environmental pollution issues. Due to the smaller losses in aqueous phase extraction, aqueous two-phase extraction can provide a higher polysaccharide recovery rate while ensuring its functional characteristics. Aqueous two-phase extraction is mainly based on water and a small amount of high molecular weight substances, avoiding the use of toxic organic solvents, and therefore is more in line with the requirements of green chemistry and has less impact on the environment. The mild environment of this method can effectively protect the biological activity of polysaccharides and is not prone to degradation or loss of function. Therefore, it is very necessary to study a method that is efficient, mild, green, and can also achieve preliminary separation and purification. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting chitosan polysaccharide by using an ultrasonic-assisted organic solvent-inorganic salt aqueous two-phase system.

[0005] The purpose of the present invention is to provide a method for extracting chitosan polysaccharide by using an ultrasonic-assisted organic solvent-inorganic salt aqueous two-phase body.

[0006] The object of the present invention is achieved by a method for ultrasonic-assisted two-phase extraction of chitosan polysaccharide, characterized in that it comprises the following steps:

[0007] (1) In a 3000 ml beaker, add 1070 g H2O and weigh 340 g (NH4)2SO4 and mix until there is no precipitation;

[0008] (2) Slowly add 550 g of anhydrous ethanol, shake well and let stand for 2 hours to form an ammonium sulfate-ethanol aqueous two-phase;

[0009] (3) Finally, add 40 g of Citrus aurantium powder and shake gently without disrupting the two phases;

[0010] (4) At 40°C, under ultrasonic power of 300 W, ultrasonic time: 20 min;

[0011] (5) Gently aspirate the upper phase liquid, collect the lower phase liquid, and filter it statically with filter paper;

[0012] (6) Concentrate to 150-200 ml using a rotary evaporator. Salt will precipitate out. Filter again to obtain a concentrated solution.

[0013] (7) taking the concentrated solution and adding it into a 200Da dialysis bag, dialyzing it in pure water to obtain a dialysate;

[0014] (8) The dialysate was concentrated to 50 ml of concentrated solution, and the polysaccharide content was measured by phenol-sulfuric acid method. Then, 80 wt % ethanol was added, and its mass was 4-5 times the mass of the concentrated solution to obtain precipitated polysaccharide, which was placed in a -4°C refrigerator for precipitation for 24 h;

[0015] (9) The precipitated polysaccharide is centrifuged and freeze-dried to obtain Citrus aurantium polysaccharide.

[0016] Specifically, the technical solution of the present invention is summarized as follows:

[0017] 1. Determination of two phases

[0018] 20g of the two-phase aqueous system consists of 35% organic phase ethanol solution and 15% inorganic salt solutions of (NH4)2SO4, NaH2PO4, Na2HPO4, and Na2CO3, respectively, dissolved and made up with deionized water. Stir and shake with a glass rod to make the inorganic salt and deionized water form the aqueous phase, and then let it stand for the two phases to separate.

[0019] 2. Single factor experimental results

[0020] In order to optimize the extraction process of Fructus Aurantii Immaturus and obtain a higher polysaccharide extraction yield, a single factor experiment was carried out using ethanol-ammonium sulfate ATPS as the extraction system under the same ultrasonic power (300 W) to investigate the effects of solid-liquid ratio, extraction temperature, extraction time and mass fraction of the two phases on the polysaccharide yield.

[0021] (1) Material-liquid ratio

[0022] The experimental control extraction temperature was 40℃, the time was 20min, the mass fraction of (NH4)2SO4 was 15%, and the mass fraction of ethanol was 35%. Five groups of parallel experiments were set at a solid-liquid ratio of 1:20, 1:30, 1:40, 1:50, and 1:60 to measure the yield of polysaccharides.

[0023] (2) Extraction temperature

[0024] The experimental control of the solid-liquid ratio of Citrus aurantium polysaccharide extraction was 1:50, the time was 20 min, the (NH4)2SO4 mass fraction was 15%, and the ethanol mass fraction was 35%. The temperatures were set at 40, 50, 60, 70, and 80°C respectively, and five groups of parallel experiments were conducted to measure the yield of polysaccharides.

[0025] (3) Extraction time

[0026] In the experiment, the same solid-liquid ratio (1:50) was used for ultrasonic treatment at 300 W power in ethanol-ammonium sulfate ATPS at 40°C, and the extraction was carried out after 10 min, 20 min, 30 min, 40 min and 50 min respectively.

[0027] (4) (NH4)2SO4 mass fraction

[0028] Five parallel experiments were set with (NH4)2SO4 mass fractions of 15%, 17%, 19%, 21%, and 23% for control, and the ethanol mass fraction was controlled at 35%, the solid-liquid ratio was 1:50, the time was 20 minutes, and the temperature was 40°C. The yield of Citrus aurantium polysaccharide extracted with (NH4)2SO4 inorganic solution of different mass fractions was determined.

[0029] (5) Investigation of ethanol mass fraction

[0030] Five parallel experiments were set up with ethanol mass fractions of 25%, 27.5%, 30%, 32.5%, and 35% for comparison, and the (NH4)2SO4 mass fraction was controlled at 17%, the solid-liquid ratio was 1:50, the time was 20min, and the temperature was 40°C. The yield of Citrus aurantium polysaccharide extracted with ethanol organic phase of different mass fractions was determined.

[0031] By setting four different salt ion-ethanol aqueous two-phase systems in the experiment and comparing the extraction yields of Citrus aurantium polysaccharides under the same ultrasonic power (300W), it was found that the ammonium sulfate-ethanol aqueous two-phase system had the highest polysaccharide extraction yield. According to the single factor experiment, the optimal extraction conditions were determined to be 27.5% (w / w) ethanol mass fraction and 17% (w / w) ammonium sulfate mass fraction in the aqueous two-phase system, 1:50 (g / g) solid-liquid ratio, 40°C extraction temperature and 20 min extraction time.

[0032] Beneficial effects of the invention: The invention establishes a rapid and effective extraction method based on an ultrasound-assisted two-phase aqueous system. The results show that in ATPS, under the conditions of 27.5% (w / w) of organic phase ethanol, 17% (w / w) of inorganic phase ammonium sulfate, 1:50 g / g of solid-liquid ratio, 20 min of extraction time, and 40°C of extraction temperature, the extraction yield of chitosan polysaccharide is 48.66 mg / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the two-aqueous phase diagram of the ultrasound-assisted two-aqueous phase extraction of chitosan polysaccharide according to the present invention.

[0034] Figure 2 This is a diagram of the ultrasound-assisted two-phase aqueous extraction of Chitosan polysaccharide described in the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments and drawings, so that those skilled in the art can implement the invention with reference to the description. However, the present invention is not limited to the following embodiments, and the conditions used in the embodiments can be adjusted according to specific circumstances. The substances and their abbreviations in the examples are as follows:

[0036] 1. Determination of two phases

[0037] 20g of the two-phase aqueous system consists of 35% organic phase ethanol solution and 15% inorganic salt solutions of (NH4)2SO4, NaH2PO4, Na2HPO4, and Na2CO3, respectively, dissolved and filled with deionized water. Stir and shake with a glass rod to make the inorganic salt and deionized water form the aqueous phase, and then stand for the two phases to separate; finally confirmed: ethanol-ammonium sulfate ATPS is the extraction system.

[0038] 2. Single factor experimental results

[0039] In order to optimize the extraction process of Fructus Aurantii Immaturus and obtain a higher polysaccharide extraction rate, a single factor experiment was conducted with ethanol-ammonium sulfate ATPS as the extraction system under the same ultrasonic power (300W) to investigate the effects of solid-liquid ratio, extraction temperature, extraction time and mass fraction of the two phases on the polysaccharide yield.

[0040] (1) Material-liquid ratio

[0041] The experimental control extraction temperature was 40℃, the time was 20min, the mass fraction of (NH4)2SO4 was 15%, and the mass fraction of ethanol was 35%. The solid-liquid ratio (mass ratio) of 15% (NH4)2SO4 and 35% ethanol was set at 1:20, 1:30, 1:40, 1:50, and 1:60 respectively to measure the yield of polysaccharides in five groups of parallel experiments (see Table 1). It can be seen from the table that the solid-liquid ratio (mass ratio) of 1:50 has a better extraction effect;

[0042] Table 1 shows the effect of material-liquid ratio

[0043]

[0044] (2) Extraction temperature

[0045] The experimental control of the solid-liquid ratio of the polysaccharide of Citrus aurantium was 1:50, the time was 20min, the mass fraction of (NH4)2SO4 was 15%, and the mass fraction of ethanol was 35%. The temperatures were set at 40, 50, 60, 70, and 80 °C, and five groups of parallel experiments were conducted to measure the yield of polysaccharides (see Table 2). It can be seen from the table that the extraction temperature is 40 °C and the extraction effect is better:

[0046] Table 2 shows the effect of temperature

[0047]

[0048] (3) Extraction time

[0049] The experiment took the same solid-liquid ratio (1:50) and carried out ultrasonic treatment at 300W power in ethanol-ammonium sulfate ATPS at 40℃. The yield of polysaccharides was measured after extraction for 10min, 20min, 30min, 40min and 50min respectively (see Table 3). It can be seen from the table that the extraction effect is better when the extraction time is 20min;

[0050] Table 3 shows the determination of extraction time

[0051]

[0052] (4) (NH4)2SO4 mass fraction

[0053] Five parallel experiments were set at 15%, 17%, 19%, 21%, and 23% (NH4)2SO4 mass fractions for comparison, and the ethanol mass fraction was controlled at 35%, the solid-liquid ratio was 1:50, the time was 20 minutes, and the temperature was 40°C. The yield of Citrus aurantium polysaccharide extracted with (NH4)2SO4 inorganic solution of different mass fractions (see Table 4) shows that the extraction effect of (NH4)2SO4 mass fraction of 17% is better.

[0054] Table 4 Determination of (NH4)2SO4 mass fraction

[0055]

[0056] (5) Investigation of ethanol mass fraction

[0057] Five parallel experiments with ethanol mass fractions of 25%, 27.5%, 30%, 32.5%, and 35% were set for comparison, and the (NH4)2SO4 mass fraction was controlled to be 17%, the solid-liquid ratio of (NH4)2SO4 and ethanol was 1:50, the time was 20min, and the temperature was 40°C. The yield of Citrus aurantium polysaccharide extracted from the organic phase with ethanol of different mass fractions (see Table 5) shows that the extraction effect is better when the ethanol mass fraction is 27.5%.

[0058] Table 5 Determination of ethanol mass fraction

[0059]

[0060] By setting four different salt ion-ethanol aqueous two-phase systems in the experiment, the extraction yields of Citrus aurantium polysaccharides were compared under the same ultrasonic power (300W), and it was found that the ammonium sulfate-ethanol aqueous two-phase system had the highest polysaccharide extraction yield; according to the single factor experiment, the optimal extraction conditions were determined to be 27.5% (w / w) ethanol mass fraction and 17% (w / w) ammonium sulfate mass fraction in the aqueous two-phase system, 17% (NH4)2SO4 mass fraction and 27.5% alcohol mass fraction, a solid-liquid ratio of 1:50 (g / g), an extraction temperature of 40°C, and an extraction time of 20 min.

[0061] Best Mode

[0062] (1) 2000 g of the system was prepared according to the optimal extraction conditions, with a solid-liquid ratio (Fructus Aurantii Immaturus: aqueous two-phase system) (g / g), specifically: 1:50 (Fructus Aurantii Immaturus powder added 40 g), 17% (NH4)2SO4 (340 g), 27.5% ethanol (550 g), and ddH2O (1070 g).

[0063] (2) In a 3000ml beaker, add 1070g ddH2O and weigh 340g (NH4)2SO4 and mix until there is no precipitation.

[0064] (3) Slowly add 550g of anhydrous ethanol, shake well and let stand for 2 hours.

[0065] (4) Finally, add Citrus aurantium powder (40 g) and shake gently without disrupting the two phases.

[0066] (5) At a temperature of (40°C) and an ultrasonic power of (300W), the ultrasonic time is: 20 minutes.

[0067] (6) Gently aspirate the upper phase liquid, collect the lower phase liquid, and filter it statically using filter paper.

[0068] (7) Use a rotary evaporator to concentrate to about 150-200 ml. Salt will precipitate out. Filter again to obtain a concentrated solution.

[0069] (8) Take the concentrated solution and add it to a dialysis bag (200Da) and dialyze it in pure water for 24 hours (half of it is enough. After 1 hour, observe whether the dialysis bag is full. If so, release some of it to prevent the concentration of the dialysate from being too high and the dialysis bag from rupturing) to obtain the dialysate;

[0070] (9) The dialysate was concentrated to about 50 ml, and the polysaccharide content was measured by the phenol-sulfuric acid method. Then, 4-5 times 80 wt % ethanol was added to obtain the precipitated polysaccharide, and the precipitated polysaccharide was placed in a -4°C refrigerator for 24 h.

[0071] (10) The precipitated polysaccharide was centrifuged (4000 r / min) for 10 min and freeze-dried to obtain Citrus aurantium polysaccharide.

Claims

1. A method for extracting chitosan polysaccharide from chitosan assisted by ultrasonic two-phase extraction, characterized in that: The steps include: (1) In a 3000ml beaker, add 1070g H2O and weigh 340g (NH4)2SO4 and mix until there is no precipitation; (2) Slowly add 550 g of anhydrous ethanol, shake well and let stand for 2 hours to form an ammonium sulfate-ethanol aqueous two-phase; (3) Finally, add 40 g of Citrus aurantium powder and shake gently without disrupting the two phases. (4) At 40°C, under ultrasonic power of 300 W, ultrasonic time: 20 min; (5) Gently aspirate the upper phase liquid, collect the lower phase liquid, and filter it statically with filter paper; (6) Use a rotary evaporator to concentrate to 150-200 ml. Salt will precipitate out. Filter again to obtain a concentrated solution. (7) Take the concentrated solution and add it to a 200Da dialysis bag, dialyze it in pure water to obtain dialysate; (8) The dialysate was concentrated to 50 ml of concentrated solution, and the polysaccharide content was measured by phenol-sulfuric acid method. Then, 80 wt% ethanol was added, which was 4-5 times the mass of the concentrated solution, to obtain precipitated polysaccharide, and placed in a -4 °C refrigerator for precipitation for 24 h. (9) The precipitated polysaccharide is centrifuged and freeze-dried to obtain Citrus aurantium polysaccharide.

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