Method for separating and purifying homogeneous polysaccharide from thorn-free roxburgh rose fruits

Abstract

The invention discloses a method for separating and purifying homogeneous polysaccharide from thorn-free roxburgh rose fruits, and relates to the technical field of extraction and purification of natural polysaccharide. The method comprises the following steps: degreasing a thorn-free roxburgh rose fruit material by using heating reflux absolute ethyl alcohol, and removing impurities such as pigments and small molecular compounds; obtaining crude polysaccharide by adopting a water extraction and alcohol precipitation method, and then dialyzing by utilizing a dialysis bag; and carrying out decoloration treatment by using macroporous resin, filtering by using a filter membrane, and purifying by using gel to obtain the homogeneous polysaccharide. The purity identification result shows that the high performance gel permeation chromatography result of the homogeneous polysaccharide obtained through purification by the method shows a single symmetric peak, the purity can reach 99.2%, and the homogeneous polysaccharide does not contain protein. According to the method, a technical route for separating and purifying the homogeneous polysaccharide from the thorn-free roxburgh rose fruits is established for the first time, and the method has the advantages of reliability, high safety, low cost, high purity and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural polysaccharide extraction and purification technology, and particularly relates to a method for separating and purifying homogeneous polysaccharides from thornless prickly pear fruit. Background Technology

[0002] Thornless prickly pear (Rosa roxburghii f.eseiosa Ku) is a variant of the prickly pear. It is undemanding in terms of environment, easy to cultivate, and possesses advantages such as drought resistance, tolerance to poor soil, easy propagation, and ability to prevent soil erosion. Its thornless peel facilitates harvesting and processing. Mature thornless prickly pear fruits are yellowish-brown, oblate or conical in shape, with a smooth peel free of hard thorns. The fruit appears smoother, more delicate, rounder, and fuller, with the flesh color indistinguishable from the peel. Thornless prickly pears have high antioxidant activity and are rich in antioxidants such as ascorbic acid, flavonoids, soluble sugars, polyphenols, and anthocyanins. They also contain vitamin P, carotene, vitamin B1, and 16 kinds of amino acids, making them a "nutritious and precious fruit" for disease prevention and health maintenance.

[0003] Currently, the development and utilization of thornless prickly pear fruit resources are limited. In order to clarify the relationship between its structure and function, it is very important to separate and purify the homogeneous active components in crude polysaccharides and further develop thornless prickly pear. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a novel method for separating and purifying homogeneous polysaccharides from thornless prickly pear fruit. The novel method includes steps such as hot water extraction, alcohol precipitation, dialysis, macroporous resin decolorization, and gel column chromatography, and features low cost, simple operation, and high purity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] One objective of this invention is to provide a method for isolating and purifying homogeneous polysaccharides from thornless prickly pear fruit, the method comprising the following steps:

[0007] Step 1: Take thornless prickly pear fruit, remove seeds, dry, crush, add anhydrous ethanol, heat under reflux 2-3 times, filter, dry, and obtain pretreated material;

[0008] Step 2: Take the pretreated material and extract it with pure water 2-3 times at a material-to-liquid ratio of 1:(18-22). Then combine the concentrated extracts and use a gradient alcohol precipitation method. Add anhydrous ethanol and make its volume percentages 38-42%, 58-62%, and 78-82% respectively. Precipitate at 3-5℃ for 5.5-6.5 hours. Collect the precipitate by centrifugation and freeze-dry to obtain thornless prickly pear crude polysaccharides RREK-F40, RREK-F60 and RREK-F80 respectively.

[0009] Step 3: First, take 4-6 g of RREK-F40 and dissolve it in 180-220 mL of pure water. Centrifuge to remove insoluble matter, then dialyze in pure water for 45-50 h until there is no obvious color in the pure water for 3.5-4.5 h. Finally, freeze dry, weigh, and dissolve in a polysaccharide solution with a concentration of 25 mg / mL. Repeat the above operation and dialyze again for 1-3 days.

[0010] Step 4: Dynamic adsorption and decolorization of the dialysis polysaccharide, elution with pure water, then combining the eluents, concentrating and lyophilizing.

[0011] Step 5: Dissolve the decolorized sample in pure water, filter through a 0.4-0.5 μm filter membrane, load the sample onto a gel column, elute with pure water, collect in 10 ml / tube, develop color using the phenol-sulfuric acid method, combine the elution peaks, concentrate, and lyophilize to obtain the homogeneous polysaccharide.

[0012] Furthermore, the thornless prickly pear fruit mentioned in step one is a mature and fresh thornless prickly pear fruit; the drying temperature is 35-45℃.

[0013] Furthermore, the heating reflux temperature in step one is 65-75℃, and the time is 2.5-3.5 h.

[0014] Furthermore, the material-to-liquid ratio mentioned in step two is a weight-to-volume ratio; the extraction time for each pure water extraction is 2.5-3.5 h; and the concentration standard is to concentrate to one-fifth to one-third of the original volume.

[0015] Furthermore, the specific steps of the gradient alcohol precipitation method described in step two include: first, adding anhydrous ethanol to a volume ratio of 38-42%, precipitating at 3-5°C for 5.5-6.5 hours, centrifuging and freeze-drying the precipitate to obtain RREK-F40; then, adding anhydrous ethanol to the supernatant concentrate to a volume ratio of 58-62%, refrigerating, separating the precipitate, concentrating and freeze-drying to obtain RREK-F60; finally, continuing to add anhydrous ethanol to the supernatant to a volume ratio of 78-82%, sealing and storing for 5.5-6.5 hours, taking the precipitate, concentrating and freeze-drying to obtain RREK-F80.

[0016] In step two of this invention, the extraction temperature is 80°C, the alcohol precipitation concentration is 40%, and the extraction rate is 12.74%.

[0017] Furthermore, the centrifugation conditions described in step three are 7500-8500 r for 8-12 min.

[0018] Furthermore, the dialysis bag used in step three has a molecular weight cutoff of 3000-4000 Da and a flat width of 40-50 mm.

[0019] Furthermore, the decolorization in step four utilizes D101 macroporous resin with a column size of 6*40 cm, a polysaccharide loading concentration of 4-6 mg / mL, and a loading volume of 180-220 mL; the standard for elution with pure water is that the absorbance value of the eluent measured by the phenol-sulfuric acid method is less than 0.1 when it is subjected to color development by an enzyme-linked immunosorbent assay (ELISA) reader.

[0020] Furthermore, the gel column mentioned in step five is a G-200 gel column with a specification of 3×100 cm.

[0021] The second objective of this invention is to provide the application of any of the above-described methods for isolating and purifying homogeneous polysaccharides from thornless prickly pear fruit in the development and utilization of thornless prickly pear resources and the preparation of related products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention utilizes hot water extraction, ethanol precipitation, dialysis, macroporous resin decolorization, and gel column chromatography to extract, separate, and purify polysaccharides from thornless prickly pear fruit, obtaining homogeneous polysaccharides. It establishes for the first time a complete technical route for the extraction and purification of homogeneous polysaccharides from thornless prickly pear fruit, which is of great significance for the development and utilization of thornless prickly pear fruit resources. Furthermore, this invention has the advantages of being simple and easy to implement, low in cost, high in safety, and producing high purity polysaccharides. Attached Figure Description

[0024] Figure 1 The elution curve of the thornless prickly pear polysaccharide G-200 gel column in Example 1 is shown.

[0025] Figure 2 This is an image of the homogeneous polysaccharide from Example 1.

[0026] Figure 3 This is a graph showing the purity and molecular weight determination of the homogeneous polysaccharide in Example 1.

[0027] Figure 4 This is the elution curve of G-200 polysaccharide from thornless prickly pear fruit without dialysis pretreatment in Comparative Example 1.

[0028] Figure 5 The chromatograms are of the elution peaks in Comparative Example 1.

[0029] Figure 6 The elution curve of DEAE-52 polysaccharide from thornless prickly pear fruit without dialysis pretreatment is shown in Comparative Example 2.

[0030] Figure 7 The chromatograms of the elution peaks of RREK-F40-DEAE-H2O, 0.2M, and 0.4M NaCl in Comparative Example 2 are shown. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0032] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0033] Example 1

[0034] This embodiment provides a novel method for isolating and purifying homogeneous polysaccharides from thornless prickly pear fruit. The specific implementation steps are as follows:

[0035] S1 Take mature, fresh, thornless prickly pear fruit, remove the seeds, dry at 40℃, pulverize, add anhydrous ethanol and heat under reflux at 70℃ for 3 h to defatting and remove small molecule compounds such as pigments. Repeat twice and dry to constant weight to obtain pretreated thornless prickly pear fruit powder.

[0036] S2. Take the powder processed in S1 and extract it with pure water at 80℃ for 3 h at a material-to-liquid ratio of 1:20 (w / v), twice. Combine the filtrates and concentrate to one-quarter of the original volume. Use a gradient alcohol precipitation method, slowly adding anhydrous ethanol to make the anhydrous ethanol account for 40% of the volume, and precipitate at 4℃ for 6 h. Collect the precipitate by centrifugation and freeze-dry to obtain thornless prickly pear crude polysaccharide RREK-F40, with an extraction rate of 12.74%. Concentrate the supernatant to half volume, add anhydrous ethanol, and stir continuously until the anhydrous ethanol volume ratio is 60%. After refrigeration, separate the precipitate, concentrate and freeze-dry to obtain RREK-F60. Continue to add anhydrous ethanol to the supernatant until the volume ratio is 80%, seal and place in a refrigerator for 6 h. Take the precipitate, concentrate and freeze-dry to obtain RREK-F80.

[0037] S3: Dissolve 5 g of crude polysaccharide RREK-F40 from S2 in water in 200 mL of pure water to prepare a 25 mg / mL solution. Centrifuge at 8000 r for 10 min to remove insoluble matter, then place in a 44 mm 3500 Da dialysis bag, leaving 2 cm of space to remove air. Dialyze in pure water for 48 h, until no obvious color is observed in pure water within 4 h. Freeze-dry, weigh, and dissolve to prepare a 25 mg / mL polysaccharide solution. Repeat the above operation, dialyze again for 2 days, and then freeze-dry.

[0038] S4 The polysaccharides after S3 dialysis were dynamically adsorbed and decolorized using D101 macroporous resin with a column size of 6*40 cm. The polysaccharide loading concentration was 5 mg / mL, and the loading volume was 200 mL. The eluent was eluted with pure water until the eluent was developed by the phenol-sulfuric acid method. The absorbance value measured by the microplate reader was lower than 0.1. The eluents were then combined, concentrated, and lyophilized.

[0039] S5. The decolorized polysaccharide obtained in S4 was filtered through a 0.45 μm aqueous filter membrane and loaded onto a G-200 gel column (3×100cm). Pure water was used as the eluent for elution, and 10 mL was collected per tube. The elution peak was developed by the phenol-sulfuric acid method and collected. The mixture was concentrated and lyophilized to obtain a homogeneous polysaccharide.

[0040] S6 The purity of the homogeneous polysaccharide obtained in S5 was identified by using the phenol-sulfuric acid method to determine the total sugar content, the Coomassie brilliant blue method to determine the protein content, and the high-performance gel permeation chromatography to determine the molecular weight. The specific procedures are as follows:

[0041] a. Neutral sugar determination: Prepare a 100 ug / mL glucose solution, and take 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 mL of each solution, and add water to make up to 2 mL. Add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid to each solution, allow to stand for 30 min, and measure at 490 nm. Prepare a 0.1 mg / mL solution of the homogeneous polysaccharide obtained in S5, take 1 mL of each solution, and perform the same procedure. The neutral sugar content of the homogeneous polysaccharide obtained in S5 was found to be 39.87%.

[0042] b. Determination of uronic acid: Prepare 100 μg / mL galacturonic acid solution. Take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of each solution, add water to make up to 1 mL, add 6 mL of 0.0238 mol / L sodium tetraborate-sulfuric acid solution, shake well, boil in a water bath for 5 min, cool in an ice bath, add 0.1 mL of 8.81 mmol / L m-hydroxybiphenyl solution, react for 5 min, and then measure at 527 nm. Prepare a 0.1 mg / mL solution of the homogeneous polysaccharide obtained in S5, take 1 mL, and perform the same procedure. The uronic acid content of the homogeneous polysaccharide obtained in S5 was determined to be 59.34%.

[0043] c. Protein content determination: Accurately weigh 100 mg of Coomassie Brilliant Blue into a 1 L volumetric flask, add 50 mL of 95% ethanol and 100 mL of phosphoric acid, dissolve, and then dilute to the mark with water. Filter through a 0.45 μm filter to obtain the Coomassie Brilliant Blue solution. Prepare a 0.1 mg / mL bovine serum albumin solution and the homogeneous polysaccharide solution obtained in S5. Take 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of bovine serum albumin solution, respectively, add water to a final volume of 2 mL, add 5 mL of Coomassie Brilliant Blue solution, let stand for 3 min, and measure the absorbance at 595 nm. Take 0.5 mL of the solution obtained in S5 and perform the same determination. The protein content of the homogeneous polysaccharide obtained in S5 was found to be below the detection limit.

[0044] d. Pulum blue polysaccharides of different molecular weights (P5, P10, P20, P50, P100, P200, P400, P800) and the homogeneous polysaccharide obtained from S5 were prepared into 1 mg / mL solutions and analyzed using Agilent high-performance liquid chromatography (HPLC). The detector was an evaporative light detector, the mobile phase was ultrapure water, the flow rate was 0.8 mL / min, the column temperature was 35℃, and the chromatographic column was an OHpak SB-804 HQ (8.0 × 300 mm). The molecular weight of the homogeneous polysaccharide obtained from S5 was determined to be 6.39 × 10⁻⁶. 5 Da, with a weight-average molecular weight of 7.04 × 10⁻⁶. 5 Da, with a number-average molecular weight of 5.58 × 10⁻⁶. 5 Da, the dispersion index is 1.26.

[0045] In this embodiment, the G-200 gel column elution curve of thornless prickly pear fruit polysaccharide is as follows: Figure 1 As shown in the image, a homogeneous polysaccharide is... Figure 2 As shown in the figure, the purity and molecular weight determination of homogeneous polysaccharides are as follows. Figure 3 As shown.

[0046] Comparative Example 1

[0047] S1 is the same as in Example 1 (S1).

[0048] S2 is the same as Example 1 (S2).

[0049] S3. Take 5 g of crude polysaccharide RREK-F40 from S2 for decolorization. The macroporous resin is D101 macroporous resin, the column size is 6*40cm, the polysaccharide loading concentration is 5 mg / mL, elute with pure water, combine and concentrate, and freeze dry.

[0050] S4: 2 g of the decolorized polysaccharide from S3 was dissolved in 100 mL of pure water, filtered through a 0.45 μm aqueous filter membrane, and loaded onto a G200 gel column (3 × 100 cm). Elution peaks were collected at 10 mL / tube, combined, and analyzed by HPLC. The results showed that homogeneous polysaccharide was not obtained. This indicates that dialysis to remove low molecular weight components before decolorization and column chromatography purification is crucial for separating homogeneous polysaccharides.

[0051] In this comparative example, the elution curve of thornless prickly pear fruit polysaccharide G-200 without dialysis pretreatment is as follows: Figure 4 As shown, the chromatograms of each elution peak are as follows: Figure 5 As shown.

[0052] Comparative Example 2

[0053] S1 is the same as in Example 1 (S1).

[0054] S2 is the same as Example 1 (S2).

[0055] S3. Take 5 g of crude polysaccharide RREK-F40 from S2 for decolorization. The macroporous resin is D101 macroporous resin, the column size is 6*40cm, the polysaccharide loading concentration is 5 mg / mL, elute with pure water, combine and concentrate, and freeze dry.

[0056] S4: 1 g of the decolorized polysaccharide from S3 was dissolved in 50 mL of pure water, filtered through a 0.45 μm aqueous filter membrane, and loaded onto a DEAE-52 cellulose column (6 × 40 cm). Elution was performed using pure water, 0.2 mol / L, and 0.4 mol / L NaCl, with 30 mL collected per tube. The phenol-sulfuric acid method was used for color development to establish an elution curve. Elution peaks were combined, dialyzed, lyophilized, and analyzed by HPLC. The results showed that a homogeneous polysaccharide was not obtained. In this comparative example, the RREK-F40 sample was initially separated by DEAE-52 cellulose column chromatography without dialysis pretreatment. Although the pure water elution stage effectively removed most small molecule impurities, and the remaining components mainly showed single chromatographic peaks, the homogeneity was still not ideal. Furthermore, this method resulted in significant material loss on the DEAE-52 column, and a secondary purification via gel column chromatography was required, leading to a long overall purification cycle.

[0057] In this comparative example, the elution curve of DEAE-52 polysaccharide from thornless prickly pear fruit without predialysis treatment is as follows: Figure 6 As shown, the chromatograms of the elution peaks of RREK-F40-DEAE-H2O, 0.2M, and 0.4M NaCl are as follows. Figure 7 As shown.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for isolating and purifying homogeneous polysaccharides from thornless prickly pear fruit, characterized in that, Includes the following steps: Step 1: Take thornless prickly pear fruit, remove seeds, dry, crush, add anhydrous ethanol, heat under reflux 2-3 times, filter, dry, and obtain pretreated material; Step 2: Take the pretreated material and extract it with pure water 2-3 times at a material-to-liquid ratio of 1:(18-22). Then combine the concentrated extracts and use a gradient alcohol precipitation method. Add anhydrous ethanol and make its volume percentages 38-42%, 58-62%, and 78-82% respectively. Precipitate at 3-5℃ for 5.5-6.5 hours. Collect the precipitate by centrifugation and freeze-dry to obtain thornless prickly pear crude polysaccharides RREK-F40, RREK-F60 and RREK-F80 respectively. Step 3: First, take 4-6 g of RREK-F40 and dissolve it in 180-220 mL of pure water. Centrifuge to remove insoluble matter, then dialyze in pure water for 45-50 h until there is no obvious color in the pure water for 3.5-4.5 h. Finally, freeze dry, weigh, and dissolve in a polysaccharide solution with a concentration of 25 mg / mL. Repeat the above operation and dialyze again for 1-3 days. Step 4: Dynamic adsorption and decolorization of the dialysis polysaccharide, elution with pure water, then combining the eluents, concentrating and lyophilizing. Step 5: Dissolve the decolorized sample in pure water, filter through a 0.4-0.5 μm filter membrane, load the sample onto a gel column, elute with pure water, collect in 10 ml / tube, develop color using the phenol-sulfuric acid method, combine the elution peaks, concentrate, and lyophilize to obtain the homogeneous polysaccharide.

2. The method according to claim 1, characterized in that, The thornless prickly pear fruit mentioned in step one is a mature, fresh thornless prickly pear fruit; the drying temperature is 35-45℃.

3. The method according to claim 2, characterized in that, The heating and reflux temperature in step one is 65-75℃, and the time is 2.5-3.5 h.

4. The method according to claim 3, characterized in that, The material-liquid ratio mentioned in step two is a weight-to-volume ratio; the extraction time for each pure water extraction is 2.5-3.5 h; and the concentration standard is to concentrate to one-fifth to one-third of the original volume.

5. The method according to claim 4, characterized in that, The specific steps of the gradient alcohol precipitation method described in step two include: first, adding anhydrous ethanol to a volume ratio of 38-42%, precipitating at 3-5℃ for 5.5-6.5 hours, centrifuging and freeze-drying the precipitate to obtain RREK-F40; then, adding anhydrous ethanol to the supernatant concentrate to a volume ratio of 58-62%, refrigerating, separating the precipitate, concentrating and freeze-drying to obtain RREK-F60; finally, continuing to add anhydrous ethanol to the supernatant to a volume ratio of 78-82%, sealing and storing for 5.5-6.5 hours, then taking the precipitate, concentrating and freeze-drying to obtain RREK-F80.

6. The method according to claim 5, characterized in that, The centrifugation conditions described in step three are 7500-8500 r for 8-12 min.

7. The method according to claim 6, characterized in that, The dialysis bags used in step three have a molecular weight cutoff of 3000-4000 Da and a flat width of 40-50 mm.

8. The method according to claim 7, characterized in that, The decolorization in step four utilizes D101 macroporous resin with a column size of 6*40 cm. The polysaccharide loading concentration is 4-6 mg / mL, and the loading volume is 180-220 mL. The standard for elution with pure water is that the absorbance value of the eluent measured by the phenol-sulfuric acid method is less than 0.1 when it is subjected to color development by an enzyme-linked immunosorbent assay (ELISA) reader.

9. The method according to claim 8, characterized in that, The gel column mentioned in step five is a G-200 gel column with a size of 3×100 cm.

10. The method for isolating and purifying homogeneous polysaccharides from thornless prickly pear fruit according to any one of claims 1-9, and its application in the development and utilization of thornless prickly pear resources and the preparation of related products.

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