Method for extracting and purifying Dendrobium officinale polysaccharides

Abstract

A method for extracting and purifying Dendrobium officinale polysaccharides comprises following steps: (1) fully disperse Dendrobium officinale powder in pure water to obtain crude liquid; (2) removing insoluble impurities from the crude liquid through a microfiltration membrane to obtain permeate 1 and retentate 1; (3) performing macroporous ultrafiltration treatment of the permeate 1 and collect permeate 2 and retentate 2; (4) adding an aqueous solution of edible alkali metal inorganic salt to the retentate 2, fully stirring and dissolving to obtain polysaccharide crude liquid, performing macroporous ultrafiltration treatment and collecting permeate 3 and retentate 3; (5) combining the permeate 2 and permeate 3, adding the combined permeate into an electrodialysis device for desalination, and collecting dilute solution and concentrated solution; (6) performing microporous ultrafiltration treatment of the dilute solution and collect retentate 4 and permeate 4; (7) carrying out freeze-drying of the retentate 4 to obtain Dendrobium officinale polysaccharides.

Claims

1. A method for extracting and purifying Dendrobium officinale polysaccharides, comprising following steps: (1) taking Dendrobium officinale powder and fully dispersing it in pure water to obtain crude liquid; (2) removing insoluble impurities from the crude liquid obtained in the step (1) through a microfiltration membrane to obtain permeate 1 and retentate 1; (3) performing ultrafiltration treatment of the permeate 1 collected in the step (2) through a macroporous ultrafiltration membrane with a molecular weight cut-off of 100-800 kDa, and collecting permeate 2 and retentate 2; (4) adding 0.5-1.5 mol/L of aqueous solution of edible alkali metal inorganic salt to the retentate 2 collected in the step (3), stirring and fully dissolving to obtain polysaccharide crude liquid, performing ultrafiltration treatment through a macroporous ultrafiltration membrane with a molecular weight cut-off of 100-800 kDa, and collecting permeate 3 and retentate 3; (5) combining the permeate 2 and permeate 3 obtained in the step (3) and step (4), adding the combined permeate into an electrodialysis device for desalination, and collecting dilute solution and concentrated solution; (6) performing ultrafiltration treatment of the dilute solution obtained in the step (5) through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa-8 kDa and collecting retentate 4 and permeate 4; (7) carrying out freeze-drying of the retentate 4 obtained in the step (6) to obtain Dendrobium officinale polysaccharides.

2. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein the concentration of Dendrobium officinale in the crude liquid is 10-30 wt %.

3. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein conditions of the microfiltration membrane treatment are as follows: operating temperature is 25-50° C., transmembrane pressure is 0.3-0.6 MPa, flow rate of feed liquid is 0.2-0.5 L/min.

4. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein in the steps (3) and (4), the molecular weight cut-off of the macroporous ultrafiltration membrane is 300-800 kDa.

5. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 4, wherein in the steps (3) and (4), the molecular weight cut-off of the macroporous ultrafiltration membrane is 500 kDa.

6. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein conditions of the ultrafiltration treatment are as follows: operating temperature is 25-50° C., transmembrane pressure is 0.3-0.6 MPa, flow rate of feed liquid is 0.2-0.5 L/min.

7. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein in the step (4), the edible alkali metal inorganic salt is one of NaCl, Na.sub.2SO.sub.4, KCl, K.sub.2SO.sub.4 or a mixture thereof.

8. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein the aqueous solution of the edible alkali metal inorganic salt is 0.5-1.5 M NaCl aqueous solution or 0.5-1M Na.sub.2SO.sub.4 aqueous solution.

9. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 8, wherein the aqueous solution of the edible alkali metal inorganic salt is 0.5M NaCl aqueous solution or 0.5M Na.sub.2SO.sub.4 aqueous solution.

10. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein conditions of the ultrafiltration treatment are as follows: pH of feed liquid is 4-10, operating temperature is 25-50° C., transmembrane pressure is 0.3-0.6 MPa, and flow rate of feed liquid is 0.2-0.5 L/min.

11. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 10, wherein in the step (4), the conditions of the ultrafiltration treatment are as follows: pH of the feed liquid is 6-7, operating temperature is 30-35° C., and flow rate of feed liquid is 0.2-0.5 L/min.

12. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein in the step (6), the molecular weight cut-off of the ultrafiltration membrane is 3-5 kDa.

13. The method for extracting and purifying Dendrobium officinale polysaccharides according to claim 1, wherein in the step (6), the molecular weight cut-off of the ultrafiltration membrane is 5 kDa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a conventional process route for preparing dendrobium polysaccharides.

(2) FIG. 2 is a process route for preparing dendrobium polysaccharides adopted in the present invention.

(3) FIG. 3 is a schematic diagram of the electrodialysis device used in the embodiments of the present invention, in which the dilute liquid tank, concentrated liquid tank, electrode liquid tank, circulating pump, etc. are not shown.

(4) FIG. 4 is a curve showing the change in the metal ion content in the extract over time during the electrodialysis process of Example 1.

(5) FIG. 5 is a curve showing the change in the conductivity of the liquid in the concentrated chamber and dilute chamber over time during the electrodialysis process of Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The technical solutions of the present invention are further described below with specific embodiments, but the scope of protection of the present invention is not limited thereto.

Example 1

(7) {circle around (1)} 200 g of Dendrobium officinale powder was fully dissolve in 1000 mL of pure water to obtain crude liquid of Dendrobium officinale;

(8) {circle around (2)} Insoluble impurities were removed from the Dendrobium officinale crude liquid through a microfiltration membrane with a pore size of 0.1 μm (microfiltration conditions: temperature of 25° C., transmembrane pressure of 0.3 MPa and solution flow rate of 0.4 L/min). The microfiltration was carried out five times in total. The retentate after each microfiltration was diluted with 1000 mL of pure water for the next microfiltration. The permeate of five times of microfiltration was combined to get a total of 5000 mL;

(9) {circle around (3)} 5000 mL of the permeate obtained in the step {circle around (2)} was subjected to dead-end ultrafiltration through a 500 kDa ultrafiltration membrane to extract polysaccharides (ultrafiltration conditions: temperature of 25° C., transmembrane pressure of 0.3 MPa, solution flow rate of 0.4 L/min), the obtained retentate was added with 1000 mL of pure water for the next ultrafiltration, the ultrafiltration was repeated for 3 times, and 300 mL of retentate and 7700 mL of permeate were collected;

(10) {circle around (4)} 300 mL of the retentate collected through the ultrafiltration membrane was added into 1000 mL of 0.5 mol/L NaCl aqueous solution (mixed solution at pH 7), fully stirred and then subjected to dead-end ultrafiltration through a 500 kDa ultrafiltration membrane, the obtained retentate was added with 1000 mL of the above saline solution for the next ultrafiltration, the ultrafiltration was repeated for 3 times, and the retentate and permeate were collected.

(11) {circle around (5)} The permeate obtained in step {circle around (4)} and step {circle around (3)} was combined. The obtained permeate contained polysaccharides, oligosaccharides, monosaccharides, heavy metal salts and added sodium chloride. The permeate was added to the dilute liquid tank of the electrodialysis device, pure water was added to the concentrated liquid tank, and 3% anhydrous sodium sulfate aqueous solution was added to the electrode liquid tank (the schematic diagram of the device is shown in FIG. 3). The membrane stack of the electrodialysis device was composed of 5 electrodialysis units. The cation exchange membranes with model TRJCM were purchased from Beijing Tingrun Membrane Technology Development Co., Ltd., and the anion exchange membranes with model TRJAM were purchased from Beijing Tingrun Membrane Technology Development Co., Ltd., with an effective membrane area of 6.4×10.sup.−3 m.sup.2. Both the anode and the cathode were electrodes coated with ruthenium. The circulating pump was started to make the feed liquid start circulating and the device was powered on for electrodialysis. The operating conditions were: voltage of 20V, and feed liquid flow rate of 40 L/h. When the conductivity tended to stable, stopped the operation and the operation lasted 45 min, and collected the dilute solution. In the process of electrodialysis, took the samples of dilute solution for freeze-drying every 5 minutes, and determined the content of heavy metals in the freeze-dried samples by ICP-MS. The results were shown in FIG. 4. In the process of electrodialysis, took the samples of dilute solution and concentrated solution every 5 min to determine the conductivity. The results were shown in FIG. 5. At the same time, after measurement, the concentration of NaCl in the dilute solution after electrodialysis was 24.3 mg/L, which was far lower than the sodium salt content in the human blood.

(12) {circle around (6)} The dilute solution obtained in the step {circle around (5)} was subjected to ultrafiltration treatment through an ultrafiltration membrane with a MWCO of 5 kDa (ultrafiltration condition: temperature 25° C., transmembrane pressure 0.3 MPa, solution flow rate 0.4 L/min), and the ultrafiltration treatment was carried out for 3 times to remove the substances such as oligosaccharides, monosaccharides, polyphenols, flavonoids, etc. The retentate of each ultrafiltration was diluted with 1000 mL of pure water before the next ultrafiltration.

(13) {circle around (7)} Freeze-drying of the retentate obtained in the step {circle around (6)} was carried out with a freeze dryer to obtain 83.9 g of Dendrobium officinale polysaccharides product (DOPs) with a content of heavy metal ions (lead content+copper content+cadmium content) of 0.296 mg.Math.Kg.sup.−1 and a purity of polysaccharides of 87.13%. The lead content in the polysaccharides was decreased from 2.260 mg/Kg before electrodialysis to 0.031 mg/Kg, the copper content was decreased from 0.857 mg/Kg to 0.063 mg/kg, and the cadmium content was decreased from 0.491 mg/Kg to 0.202 mg/Kg.

(14) Test methods were as follows:

(15) 1. Determination of Polysaccharide Content

(16) The polysaccharide content is the total carbohydrate in DOPs aqueous solution (obtained by dissolving the DOPs in water) minus the reducing sugar content.

(17) The total carbohydrate content was determined according to the phenol-sulfuric acid method and some modifications were made (Xie, J. H., Shen, M. Y., Nie, S. P., Zhao, Q., Li, C., Xie, M. Y., 2014. Separation of water-soluble polysaccharides from Cyclocarya paliurus by ultrafiltration process. Carbohydr Polym 101, 479-483). Approximately 2 mL of the DOPs aqueous solution was placed in a test tube. After adding 5% (w/w) phenol (1 mL) and H.sub.2SO.sub.4 (5 mL), the mixture was kept in a boiling water bath for 15 minutes and then cooled under a water stream. Absorbance was measured at 490 nm using a UV-visible spectrophotometer.

(18) The reducing sugar measurement was carried out according to the literature method (Zhu, J., Chen, Z., Chen, L., Yu, C., Wang, H., Wei, X., Wang, Y., 2019. Comparison and structural characterization of polysaccharides from natural and artificial Se-enriched green tea. Int J Biol Macromol 130, 388-398) and some modifications were made. Approximately 1 mL or more of the DOPs aqueous solution was absorbed into the tubes and mixed with 3 mL of DNS. The solution was kept in a boiling water bath for 5 minutes and then cooled under a stream of water. The solution was thoroughly mixed and its absorbance was measured at 540 nm in a UV-visible spectrophotometer.

(19) 2. Polysaccharide Purity

(20) The purity of polysaccharides (P) is the ratio of mass (W.sub.retentate) of polysaccharides in the DOPs aqueous solution to mass of DOPs (W.sub.feed). The definition is as follows:

(21) $p\%=\frac{W_{\mathrm{retentate}}}{W_{\mathrm{feed}}}\times 100\%$

(22) 3. Heavy Metal Content Determination

(23) The content of lead, copper, and chromium in DOPs was determined by inductively coupled plasma mass spectrometry (ICP-MS).

Comparative Example 1

(24) The steps {circle around (1)} and {circle around (2)} were the same as those in Example 1;

(25) {circle around (3)} 5000 mL of the permeate obtained in five times of microfiltration was subjected to dead-end ultrafiltration through a 500 kDa PES flat membrane to extract polysaccharides (ultrafiltration conditions: temperature of 25° C., transmembrane pressure of 0.3 MPa, solution flow rate of 0.4 L/min), the obtained retentate was added with 1000 mL of pure water for the next ultrafiltration treatment, the ultrafiltration treatment was repeated for 6 times, and the permeate was collected and combined.

(26) {circle around (4)} The permeate collected through the ultrafiltration membrane was added into a dilute liquid tank of the electrodialysis device (the electrodialysis device was the same as that in Example 1). The membrane stack of the electrodialysis device was composed of 5 electrodialysis units. The cation exchange membranes with model TRJCM were purchased from Beijing Tingrun Membrane Technology Development Co., Ltd., and the anion exchange membranes with model TRJAM were purchased from Beijing Tingrun Membrane Technology Development Co., Ltd., with an effective membrane area of 6.4×10.sup.−3 m.sup.2. Both the anode and the cathode were electrodes coated with ruthenium. Electrode liquid tank: 3% anhydrous sodium sulfate aqueous solution, concentrated liquid tank: pure water. The circulating pump was started to make the feed liquid start circulating and the device was powered on for electrodialysis. Operating conditions were as follows: voltage of 20V, feed liquid flow rate of 40 L/h. Stopped the operation when the conductivity tended to stable. The operation lasted 15 min, and the dilute solution was collected.

(27) {circle around (5)} The obtained dilute solution was subjected to ultrafiltration treatment through a 5 kDa PES flat membrane and the ultrafiltration treatment was carried out for three times (ultrafiltration condition: temperature 25° C., transmembrane pressure 0.3 MPa, solution flow rate 0.4 L/min) to remove the substances such as oligosaccharides, monosaccharides, polyphenols, flavonoids, etc. The retentate of each ultrafiltration was diluted with 1000 mL of pure water for the next ultrafiltration. The retentate and permeate were collected.

(28) {circle around (6)} Freeze-drying of the retentate obtained in the step {circle around (5)} was carried out with a freeze dryer to obtain 78.12 g of Dendrobium officinale polysaccharides with a content of heavy metal ions of 0.510 mg.Math.Kg.sup.−1 and a purity of polysaccharide of 78.11%. The lead content was 0.113 mg/Kg, the copper content was 0.170 mg/kg and the cadmium content was 0.227 mg/Kg in the polysaccharides.

Examples 2-4

(29) The concentration of the NaCl aqueous solution in step {circle around (4)} was changed to 1 mol/L (Example 2), 1.5 mol/L (Example 3), 2 mol/L (Example 4). Other procedures were the same as those in Example 1. The mass, purity and heavy metal content of obtained Dendrobium officinale polysaccharides were shown in the following table 1.

(30) TABLE-US-00001 TABLE 1 Comparative Exam- Exam- Exam- Exam- Example 1 ple 1 ple 2 ple 3 ple 4 .sup.aNaCl 0 0.5 1 1.5 2 concentration/M Mass of 78.12 83.9 81.21 82.13 79.1 polysaccharides/g Purity of 78.11 87.13 79.23 78.7 75.0 polysaccharides/% Content of heavy 0.510 0.296 0.291 0.311 0.316 metal ions/mg .Math. Kg.sup.−1 Lead content/ 0.113 0.031 0.028 0.032 0.029 mg .Math. Kg.sup.−1 Copper content/ 0.170 0.063 0.055 0.049 0.058 mg .Math. Kg.sup.−1 Cadmium content/ 0.227 0.202 0.208 0.23 0.229 mg .Math. Kg.sup.−1 .sup.bNaCl — 24.3 27.2 25.4 28.1 concentration/mg/L .sup.aThe concentration of the sodium chloride aqueous solution added in step {circle around (4)} .sup.bThe concentration of NaCl in the dilute solution after the electrodialysis was completed.

Example 5

(31) The steps {circle around (1)} to {circle around (3)} were the same as those in Example 1;

(32) {circle around (4)} 300 mL of the retentate collected through ultrafiltration membrane was added into 1000 mL of 0.5 mol/L Na.sub.2SO.sub.4 aqueous solution, fully stirred and subjected to dead-end ultrafiltration through a 500 kDa ultrafiltration membrane, the resulting retentate was added to 1000 mL of the above saline solution for the next ultrafiltration treatment, the ultrafiltration treatment was repeated for 3 times, and the retentate and permeate were collected.

(33) {circle around (5)} The permeate obtained in step {circle around (4)} and step {circle around (3)} was combined, and the obtained permeate contained polysaccharides, oligosaccharides, monosaccharides, heavy metal salts and added sodium chloride. The solution was added to the dilute liquid tank of the electrodialysis device (see FIG. 3 for a schematic diagram of the device), pure water was added to the concentrated liquid tank, and 3% sodium sulfate aqueous solution was added to the electrode liquid tank. The membrane stack of the electrodialysis device was composed of 5 electrodialysis units. The CEM-Type II cation exchange membranes and AEM-Type II anion exchange membranes were used, both of which were purchased from Fujifilm Corporation of Japan. The effective membrane area of each membrane was 6.4×10.sup.−3 m.sup.2. Both the anode and the cathode were electrodes coated with ruthenium. The circulating pump was started to make the feed liquid start circulating and the device was powered on for electrodialysis. Operating conditions were as follows: voltage of 20V, feed liquid flow rate of 40 L/h. The operation lasted 45 min, and the dilute solution was collected.

(34) {circle around (6)} The dilute solution obtained in the step {circle around (5)} was subjected to ultrafiltration treatment through an ultrafiltration membrane with a MWCO of 5 kDa (ultrafiltration condition: temperature 25° C., transmembrane pressure 0.3 MPa, solution flow rate 0.4 L/min) and the ultrafiltration treatment was carried out for 3 times to remove the substances such as oligosaccharides, monosaccharides, polyphenols, flavonoids, etc. The retentate of each ultrafiltration was diluted with 1000 mL of pure water for the next ultrafiltration.

(35) {circle around (7)} Freeze-drying of the retentate obtained in the step {circle around (6)} was carried out with a freeze dryer to obtain 82.4 g of Dendrobium officinale polysaccharides with a content of heavy metal ions of 0.219 mg.Math.Kg.sup.−1 and a purity of polysaccharide of 87.13%. The lead content was 0.028 mg/Kg, the copper content was 0.068 mg/kg and the cadmium content was 0.132 mg/Kg in the polysaccharides.

Example 6

(36) In step {circle around (4)}, 1000 mL of 1 mol/L Na.sub.2SO.sub.4 aqueous solution was added, and the other procedures were the same as Example 5, finally 76.7 g of Dendrobium officinale polysaccharides with a content of heavy metal ions of 0.241 mg.Math.Kg.sup.−1 and a purity of polysaccharide of 81.6% was obtained. The lead content was 0.036 mg/Kg, the copper content was 0.077 mg/kg and the cadmium content was 0.128 mg/Kg in the polysaccharides.

Examples 7-11

(37) In the step {circle around (4)}, the ultrafiltration operating temperature was changed to 30° C. (Example 7), 35° C. (Example 8), 40° C. (Example 9), 45° C. (Example 10), 50° C. (Example 11), respectively, other procedures were the same as those in Example 1. The mass, purity and content of heavy metals of the obtained Dendrobium officinale polysaccharides were shown in the following Table 2.

(38) TABLE-US-00002 TABLE 2 Example 1 Example 7 Example 8 Example 9 Example 10 Example 11 T/° C. 25 30 35 40 45 50 Mass of 83.9 89.3 92.4 90.3 87.5 85.3 polysaccharides/g Purity of 87.13 90.1 88.6 85.3 83.9 81.0 polysaccharides/% Content of heavy 0.296 0.303 0.34 0.302 0.298 0.32 metal ions/mg .Math. Kg.sup.-1

Examples 12-17

(39) In the step {circle around (4)}, the pH value of the ultrafiltration feed liquid was adjusted to 4 (Example 12), 5 (Example 13), 6 (Example 14), 8 (Example 15), 9 (Example 16), 10 (Example 17) using hydrochloric acid or NaOH, respectively, and the other procedures were the same as those in Example 1. The mass, purity and content of heavy metals of the obtained Dendrobium officinale polysaccharides were shown in the following Table 3.

(40) TABLE-US-00003 TABLE 3 Content Mass of Purity of of heavy polysac- polysac- metals/mg .Math. pH charides/g charides/% Kg.sup.−1 Example 12 4 78.6 79.6 0.314 Example 13 5 82.11 86.9 0.303 Example 14 6 84.1 88.3 0.299 Example 1 7 83.9 87.13 0.296 Example 15 8 82.6 80.9 0.45 Example 16 9 81.9 74.7 0.498 Example 17 10 74.6 64.3 0.78

Examples 18-19

(41) In the steps {circle around (3)} and {circle around (4)}, the MWCO of ultrafiltration membrane was changed to 300 kDa (Example 18) or 800 kDa (Example 19), and the other procedures were the same as those in Example 1. The mass, purity and content of heavy metals of the obtained Dendrobium officinale polysaccharides were shown in the following Table 4.

(42) TABLE-US-00004 TABLE 4 Example 1 Example 18 Example 19 MWCO/kDa 500 300 800 Mass of 83.9 78.4 96.8 polysaccharides/g Purity of 87.13 90.5 73.5 polysaccharides/% Content of heavy 0.296 0.363 0.387 metal ions/mg .Math. Kg.sup.−1

Examples 20-21

(43) In the step {circle around (6)}, the MWCO of ultrafiltration membrane was changed to 3 kDa (Example 20) or 8 kDa (Example 21), and the other procedures were the same as those in Example 1. The mass, purity and content of heavy metals of the obtained Dendrobium officinale polysaccharides were shown in the following Table 5.

(44) TABLE-US-00005 TABLE 5 Example 1 Example 20 Example 21 MWCO/kDa 5 3 8 Mass of polysaccharides/g 83.9 86.7 60.9 Purity of polysaccharides/% 87.13 85.9 88.6 Content of heavy metal 0.296 0.298 0.309 ions/mg .Math. Kg.sup.−1