Method for two-aqueous-phase extraction of peony pod polysaccharide

Abstract

The method comprises the following steps: mixing alkali salt and water, and adding ethanol into the obtained mixed solution, so as to obtain two aqueous phases; adding peony pod powder into the two aqueous phases, performing ultrasonic extraction, and separating to obtain a lower-phase material containing polysaccharide; according to the method, two liquid phases are formed by two insoluble aqueous solutions under certain conditions through an aqueous two-phase extraction method, the system provides a unique microenvironment for solute, and due to the unique physicochemical properties of the system, a target compound can show remarkable distribution difference between the two liquid phases; therefore, efficient and selective extraction and separation are realized.

Description

Technical Field

[0001] The invention relates to the technical field of natural product polysaccharide extraction, and in particular to a method for extracting peony fruit pod polysaccharide by using an aqueous two-phase method. Background Art

[0002] Peony is a deciduous shrub belonging to the Ranunculaceae family. It is also known as deer leek, white velvet, wood peony, etc. It is a unique woody flower and medicinal plant in my country. It was first seen in the "Shennong Bencao Jing". The medicinal part is the root bark. It has a long history. Many classics record that it has the effects of clearing heat and cooling blood. Modern pharmacological research shows that the bark of peony is rich in polysaccharides and has many important effects on human health, including regulating immunity and anti-oxidation. Polysaccharides have broad application prospects in the fields of medicine, health products and food. They can be used to treat chronic diseases, as ingredients in health products and food additives.

[0003] Peonies have high ornamental, medicinal and edible values, but their non-medicinal parts are often wasted. Recent studies have shown that these parts are also rich in active ingredients, and the seed oils of Paeonia suffruticosa and Paeonia rubra have been listed as new resource foods, promoting large-scale production of seed oils.

[0004] Every year, when peony seeds are harvested, branches, leaves and pods are separated and removed, which results in a huge waste of resources. During the production process, peony pods and seeds are often harvested together, and then separated after drying, which still results in a large number of peony pods. These pods are often discarded, which can cause environmental pollution and can also be used as low-value fuel by local farmers.

[0005] Peony pods are rich in polysaccharides, opening up new prospects for their application.

[0006] However, there are still some limitations in the extraction methods of peony pod polysaccharides. Although traditional water extraction and enzymatic hydrolysis methods can achieve the extraction of polysaccharides to a certain extent, there is still room for improvement in extraction efficiency, polysaccharide purity, ease of operation and environmental impact. The latest research is to use polyethylene glycol to extract peony pod polysaccharides. Although it improves the extraction rate, the cost of the extraction solution is high and it has certain pollution.

[0007] Therefore, developing an efficient, environmentally friendly, and easy-to-operate polysaccharide extraction method is of great significance for improving the extraction efficiency and purity of peony pod polysaccharides and promoting their further development and utilization. Summary of the invention

[0008] In order to achieve the above object, a method for extracting peony pod polysaccharides by two-phase aqueous phase method is provided, which not only improves the extraction rate, but also is easy to operate, shortens the extraction time compared with the traditional water extraction method, and is easy to obtain the extraction reagent compared with the polyethylene glycol extraction method, reduces the extraction cost, and is more environmentally friendly and healthier for the human body. The technical solution adopted by the present invention is: A method for extracting peony pod polysaccharides using an aqueous two-phase method, comprising the following steps: Mixing an alkali salt and water, and adding ethanol to the mixed solution to obtain a two-phase aqueous solution; Peony pod powder is added into the aqueous two-phase system, and ultrasonic extraction and separation are performed to obtain a lower phase material containing polysaccharides.

[0009] Further optimized, the alkali salt is dipotassium hydrogen phosphate.

[0010] According to further optimization, the mass fraction of the alkaline salt solution is 14% to 24%, and the mass fraction of the ethanol is 26% to 36%.

[0011] Further optimization, the mass fraction of the alkaline salt solution is 18%, and the mass fraction of the ethanol is 30%.

[0012] Further optimized, the preparation method of the peony pod powder is: the dried peony pods are crushed into powder in a small plant crusher, and the powder is sieved through 80 meshes to obtain a pretreated material.

[0013] Further optimization, the mass ratio of the peony pod powder to the aqueous two-phase is 1 g:20-120 mg.

[0014] After further optimization, the mass ratio of the peony pod powder to the aqueous two-phase is 1 g:60 mg.

[0015] Further optimization, the temperature of the ultrasonic extraction is 25-60°C, and the time is 10-60 min.

[0016] Further optimization was performed, and the temperature of the ultrasonic extraction was 50°C and the time was 50 min.

[0017] The beneficial effects of the present invention are: 1. The two-phase aqueous extraction method uses two immiscible aqueous solutions to form two liquid phases under certain conditions. This system not only provides a unique microenvironment for the solute, but also, due to its unique physical and chemical properties, enables the target compound to show significant distribution differences between the two liquid phases, thereby achieving efficient and selective extraction and separation; 2. As an innovative separation technology, ultrasound-assisted aqueous two-phase extraction has shown great application potential in many fields such as natural product development, biomedicine, food science and environmental engineering. Compared with traditional methods such as organic solvent extraction, water distillation or ion exchange, the advantages of ultrasound-assisted aqueous two-phase extraction are: (1) Environmentally friendly: Since an aqueous solution system is used, the use of organic solvents is greatly reduced, which reduces pollution to the environment and harm to the human body, and is in line with the current concept of green chemistry and sustainable development; (2) High efficiency and energy saving: The process is usually carried out at room temperature and pressure, without the need for high temperature and high pressure, which reduces energy consumption. At the same time, due to the large interface area between the two phases and the high mass transfer efficiency, the extraction speed is fast and the cycle is short; (3) High-purity extraction: By precisely controlling the two phases The composition and conditions of the aqueous two-phase extraction technology can effectively control the distribution coefficient of the target substance, achieve high-purity extraction, reduce subsequent purification steps, and reduce costs; (4) Wide applicability: It is not only suitable for the extraction of small molecule compounds, such as alkaloids, polysaccharides, proteins, enzymes and other bioactive substances, but can also effectively process natural mixtures containing complex components, such as Chinese herbal medicine extracts, showing good versatility and flexibility; (5) Easy to scale up and integrate: The process parameters of the aqueous two-phase extraction technology are easy to control, which is convenient for the transformation from small-scale laboratory research to large-scale industrial production. At the same time, it is easy to integrate with other separation technologies such as membrane separation and chromatography to form a more efficient extraction and purification process; With the deepening of basic research on the aqueous two-phase system, its application range has been further broadened, and the extraction efficiency and selectivity have been improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a curve chart showing the effect of adding different amounts of alkali solution on the polysaccharide extraction rate; Figure 2 It is a curve chart showing the effect of adding different amounts of ethanol on the polysaccharide extraction rate; Figure 3 It is a curve diagram showing the effect of different ultrasonic extraction temperatures on the polysaccharide extraction rate; Figure 4 It is a curve chart showing the effect of adding different amounts of peony pod powder on the polysaccharide extraction rate; Figure 5 This is a curve chart showing the effect of different ultrasonic extraction times on polysaccharide extraction rate. DETAILED DESCRIPTION

[0019] In order to more clearly understand the above-mentioned purposes, features and advantages of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the following embodiments.

[0020] A method for extracting peony pod polysaccharides using an aqueous two-phase method, characterized in that the steps are as follows: The preparation method of the peony pod powder comprises the following steps: crushing the dried peony pod into powder in a small plant crusher, and sieving the powder through 80 meshes to obtain a pre-treated material; Mixing 14% to 24% by mass of dipotassium hydrogen phosphate and water, and adding 26% to 36% by mass of ethanol to the resulting mixed solution to obtain a two-phase aqueous solution; Peony pod powder is added to the aqueous two-phase, and ultrasonic extraction is performed at a temperature of 25-60°C for 10-60 min and then separated. The obtained lower phase material contains polysaccharides, wherein the mass ratio of peony pod powder to the aqueous two-phase is 1 g:20-120 mg.

[0021] Further optimization was performed, the mass fraction of the alkaline salt solution was 22%, the mass fraction of the ethanol was 30%, the mass ratio of peony pod powder to the aqueous two-phase was 1 g:60 mg, the temperature of ultrasonic extraction was 50 °C, and the time was 50 min.

[0022] Example 1 As shown in Table 1, 4.2 g, 4.8 g, 5.4 g, 6 g, 6.6 g, and 7.2 g of potassium bicarbonate and 16.8 mL, 16.2 mL, 15.6 mL, 15 mL, 14.4 mL, and 13.8 mL of water were added to different 50 mL centrifuge tubes, and then 11.4 mL of ethanol was added, mixed and allowed to stand for stratification, and mixed to form a two-phase system containing 14%, 16%, 18%, 20%, 22%, and 24% of potassium hydrogen phosphate and 30% of ethanol by mass, respectively, with a total mass of 30 g; then 0.5 g of peony pod powder was added, and the resulting mixture was ultrasonically extracted at 50 ° C and 900 W for 50 min, and the precipitate was removed by centrifugation to separate the upper phase and the lower phase. The lower phase solution was taken, and the polysaccharides in the lower phase were quantitatively determined. The results are shown in Figure 1 .Depend on Figure 1 It can be seen that the optimal dosage of potassium hydrogen phosphate is 18%.

[0023] Table 1 shows the reaction conditions for adding different amounts of alkali solution and water Example 2 As shown in Table 2, 4.8 g of potassium bicarbonate and 17.4 mL, 16.8 mL, 16.2 mL, 15.6 mL, 15 mL, and 14.4 mL of water were added to different 50 mL centrifuge tubes and mixed to form a potassium hydrogen phosphate aqueous solution. Then, 9.8 mL, 10.7 mL, 11.4 mL, 12.2 mL, 12.9 mL, and 13.7 mL of ethanol were added, and the mass fractions were 26%, 28%, 30%, 32%, 34%, and 36%, respectively. The mixture was mixed and allowed to stand for stratification to form a two-phase aqueous solution with a total mass of 30 g. Then, 0.5 g of peony pod powder was added, and the resulting mixture was ultrasonically extracted at 50 ° C and 900 W for 50 min. The precipitate was removed by centrifugation to separate the upper phase and the lower phase. The volume of the lower phase was measured, and the polysaccharides in the lower phase were quantitatively determined. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that the optimal amount of ethanol mass fraction is 30%.

[0024] Table 2 shows the reaction conditions for adding different amounts of water and ethanol Example 3 4.8 g of potassium bicarbonate and 16.2 mL of water were added to different 50 mL centrifuge tubes, mixed to form a potassium dihydrogen phosphate aqueous solution, and then 11.4 mL of ethanol (mass fraction of 30%) was added, mixed and allowed to stand to separate, forming a two-phase aqueous solution with a total mass of 30 g; then 0.5 g of peony pod powder was added, and the resulting mixture was subjected to 900W ultrasonic extraction at 25℃, 30℃, 35℃, 40℃, 50℃, and 60℃ for 50 minutes, and the precipitate was removed by centrifugation to separate the upper and lower phases. The volume of the lower phase was measured, and the polysaccharides in the lower phase were quantitatively determined. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that the optimum temperature is 50°C.

[0025] Example 4 Add 4.8g of potassium bicarbonate and 16.2 mL of water to different 50mL centrifuge tubes, mix well to form a potassium hydrogen phosphate aqueous solution, then add 11.4mL of ethanol (mass fraction of 30%), mix well and let stand to separate, forming a two-phase water solution with a total mass of 30g; then add 0.25g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 1g, 1.25g, and 1.5g of peony pod powder, respectively, and extract the resulting mixture at 50℃ and 900W ultrasonic extraction for 50min, remove the precipitate by centrifugation, and separate the upper phase and the lower phase. Measure the volume of the lower phase, and quantitatively determine the polysaccharides in the lower phase. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that the optimal ratio of solid to liquid is 1:60 g.

[0026] Example 5 Add 4.8g of potassium bicarbonate and 16.2mL of water to different 50mL centrifuge tubes, mix well to form a potassium hydrogen phosphate aqueous solution, then add 11.4mL of ethanol (mass fraction of 30%), mix well and let stand to separate, forming a two-phase water solution with a total mass of 30g; then add 0.5g of peony pod powder, and extract the resulting mixture by ultrasonic extraction at 50℃ for 10min, 20min, 30min, 40min, 50min, and 60min, respectively, and then remove the precipitate by centrifugation to separate the upper and lower phases. Measure the volume of the lower phase, and quantitatively determine the polysaccharides in the lower phase. The results are shown in Figure 5 .Depend on Figure 5 It can be seen that the optimal ultrasonic extraction time is 50 min.

[0027] The above shows and describes the main features, methods of use, basic principles and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements according to actual conditions, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for extracting peony pod polysaccharides using an aqueous two-phase method, characterized in that: Here are the steps: Mixing an alkali salt and water, and adding ethanol to the mixed solution to obtain a two-phase aqueous solution; Peony pod powder is added into the aqueous two-phase system, and ultrasonic extraction and separation are performed to obtain a lower phase material containing polysaccharides.

2. The method for extracting peony pod polysaccharides by aqueous two-phase extraction as claimed in claim 1, characterized in that: The alkaline salt is dipotassium hydrogen phosphate.

3. The method for extracting peony pod polysaccharides by aqueous two-phase extraction as claimed in claim 1, characterized in that: The mass fraction of the alkaline salt solution is 14% to 24%, and the mass fraction of the ethanol is 26% to 36%.

4. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 3, characterized in that: The mass fraction of the alkaline salt solution is 18%, and the mass fraction of the ethanol is 30%.

5. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 1, characterized in that: The preparation method of the peony pod powder comprises the following steps: crushing the dried peony pod into powder in a small plant crusher, and sieving the powder through 80 meshes to obtain a pre-treated material.

6. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 1, characterized in that: The mass ratio of the peony pod powder to the aqueous two-phase is 1 g:20-120 mg.

7. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 6, characterized in that: The mass ratio of the peony pod powder to the aqueous two-phase is 1 g:60 mg.

8. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 1, characterized in that: The temperature of the ultrasonic extraction is 25-60° C., and the time is 10-60 min.

9. The method for extracting peony pod polysaccharides using an aqueous two-phase method as claimed in claim 1, characterized in that: The temperature of the ultrasonic extraction is 50°C and the time is 50 min.

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