NL2030622B1

NL2030622B1 - A Method for Extraction of Polygonatum Sibiricum Polysaccharide Using Deep Eutectic Solvent

Info

Publication number: NL2030622B1
Application number: NL2030622A
Authority: NL
Inventors: Aniya; Xia Qile; Cao Yan
Original assignee: Zhejiang Acad Agricultural Sci
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-11-03
Also published as: NL2030622A

Abstract

A method of extracting polygonatum sibiricum polysaccharide using deep eutectic solvent belongs to the field of natural product extraction technology. The method 5 includes: Extracting the lyophilized powder of polygonatum sibiricum in a deep eutectic solvent by heating, then separating it by alcohol precipitation, and the precipitate obtained is polygonatum sibiricum polysaccharide. The yield of the extracted polygonatum sibiricum polysaccharide obtained using the low eutectic solvent-ultrasonic assisted method of the present invention is 32.98i2.90%‚ which is 10 about 13 times higher than the extraction yield of polygonatum sibiricum polysaccharide (2.531048%) using the traditional hot water extraction method.

Description

NL 2030622 1 001902P-NL April 12, 2022 A Method for Extraction of Polygonatum Sibiricum Polysaccharide Using Deep Eutectic Solvent Technical field The present invention belongs to the field of natural product extraction technology, and specifically relates to a method of extracting polygonatum sibiricum polysaccharide using a deep eutectic solvent.

Background Technology Polygonatum sibiricum is a medicinal perennial plant, which is a dried tuber of the genus Polygonatum in the lily family. It is sweet in taste and flat in nature, and is useful for strengthening the spleen, moistening the lungs and tonifying the kidneys.

Studies have shown that polygonatum sibiricum has a variety of effects such as delaying aging, lowering blood sugar, anti-inflammatory, and regulating immunity. The content of polysaccharide, oligofructose and fructose in the rhizomes of polygonatum sibiricum is about 50%, and the proportion of protein is more than

11.16%, and contains 7 kinds of essential amino acids required by human body, among which the taste amino acids are rich, and the taste is good. There are various chemical components in polygonatum sibiricum, and the most functional component is polygonatum sibiricum polysaccharide, which is considered to be the main substance that provides various biological activities of polygonatum sibiricum. At present, the extraction of polysaccharides from polygonatum sibiricum is mainly done by using the solubility difference of polysaccharides in water and organic solvents. After extraction by hot water, alkaline water and biological enzymes, ethanol is added to the extraction solution to precipitate the polysaccharides. These methods are not only time-consuming, but also have low extraction rates. Deep eutectic solvent is a kind of deep eutectic mixture composed of two different components, hydrogen acceptor and hydrogen donor, whose freezing point is significantly lower than the melting point of the pure substance of each component, and is classified as a new class of ionic liquid, and the large amount of hydrogen bonds contained in it helps to extract the polysaccharide from polygonatum sibiricum.

NL 2030622 2 001902P-NL April 12, 2022 In the present invention, in order to solve the problems of time-consuming extraction and low extraction rate of polygonatum sibiricum polysaccharide, a deep eutectic solvent- sonication-assisted method is used to extract the polysaccharide from polygonatum sibiricum, and optimal extraction conditions were provided to investigate the yield of the polygonatum sibiricum polysaccharide as well as the monosaccharide composition of the polygonatum sibiricum polysaccharide. Content of the invention In response to the problems of the prior art, it is an object of the present invention to design a technical solution for providing a method of extracting polysaccharides from polygonatum sibiricum with a deep eutectic solvent. The present invention is specifically realized by the following technical solution.

A method of extracting polygonatum sibiricum polysaccharide by a deep eutectic solvent as described, the method comprises: extracting the lyophilized powder of polygonatum sibiricum by heating in a deep eutectic solvent and separating it by alcohol precipitation, and the precipitate obtained is polygonatum sibiricum polysaccharide. Further, the method specifically includes the steps of. 1) Preparation of lyophilized powder of Cortex luteum: Cortex luteum is cleaned and sliced, crushed after freeze-drying, sieved and stored in -4°C refrigerator for spare.

2) Preparation of deep eutectic solvent: mixing the selected hydrogen acceptor and hydrogen donor, heating them under stirring to melt them until they are transparent and clear, and the said ratio of hydrogen acceptor and hydrogen donor is 19:1-2g.

3) Extraction of crude polygonatum sibiricum polysaccharide: the lyophilized powder of polygonatum sibiricum from step 1) was mixed with the deep eutectic solvent from step 2) and then heated to extract, centrifuged and separated, ethanol was added to

NL 2030622 3 001902P-NL April 12, 2022 the supernatant and left overnight, and the precipitate obtained after centrifugation was polygonatum sibiricum polysaccharide. Further, the hydrogen acceptors in said step 2) are choline chloride, betaine, L- proline, and the hydrogen donors are urea, citric acid, propanetriol, ethylene glycol, L-lactic acid, DL-malic acid. Further, said step 2) in which the hydrogen acceptor is betaine and the hydrogen donor is propanetriol.

Further, said step 2) in which the heating temperature is 60-80°C and the time is 20- 60min. Further, said step 3) in which the ratio of lyophilized powder of polygonatum sibiricum mixed with deep eutectic solvent is 1g9:10-40g. Further, the extraction temperature in said step 3) is 40-80°C and the extraction time is 20-60min.

Further, the water content of the deep eutectic solvent in step 3) is 0.3-0.4 g/g. Further, the centrifugation condition in said step 3) is 4000-5000r/min for 10-25min. Further, said step 3) in the final concentration of ethanol is 60% to 90%. Further, said step 3) in the precipitation conditions is a temperature of 0 to 4°C and a time of 12 to 24h. Compared with the prior art, the beneficial effects of the present invention are: The yield of the extracted polygonatum sibiricum polysaccharide obtained by the present invention using deep eutectic solvent- sonication-assisted method was

32.9812.90%, which was about 13 times higher than the extraction yield of

NL 2030622 4 001902P-NL April 12, 2022 polygonatum sibiricum polysaccharide (2.53£0.48%) using the traditional hot water extraction method.

Description of the accompanying figures FIG. 1 shows the high performance liquid chromatogram in Example 3 (where A is the standard, B is the crude polygonatum sibiricum polysaccharide, C is the chromatographic column elution fraction 1, and D is the chromatographic column elution fraction 2).

FIG. 2 shows the infrared spectrum in Example 3 (where A is the crude polygonatum sibiricum polysaccharide, B is the chromatographic column elution fraction 1, and C is the chromatographic column elution fraction 2).

FIG. 3 is a scanning electron microscopy diagram in Example 3 (A:PP 100x, B:PP 1000x, C:PP-1 100x, D:PP-1 1000x, E:PP-2 100%, F:PP-3 1000x).

Specific implementation In order to make the present invention more obvious and understandable, the preferred embodiments, together with the accompanying drawings, are described in detail as follows.

Example 1 The hydrogen acceptors were set up as choline chloride, betaine, L-proline, and hydrogen donors as urea, citric acid, propanetriol, ethylene glycol, L-lactic acid, DL- malic acid, respectively, and 10 kinds of deep eutectic solvents were combined in the selected way, and mixed with the same mass of polygonatum sibiricum powder, using water as a comparison. Three parallel cases were set for each solvent mixture, the mixing ratio was 1g:10mL, the mixing temperature was 70°C, the mixing time was 50min, the solvent water content was 30%, the supernatant was obtained, four times the volume of ethanol was added to precipitate overnight, the precipitate was

NL 2030622 5 001902P-NL April 12, 2022 separated by centrifugation and dried to obtain the polygonatum sibiricum polysaccharide.

The above obtained yield of polygonatum sibiricum polysaccharide is calculated according to the following formula.

Polysaccharide yield (%)=Mass of Rhizome Polysaccharide (g)/Powder of Rhizome (9)x100% The extraction rates of polysaccharides obtained from different solvents mixed with polygonatum sibiricum are shown in Table 1. From Table 1, it can be seen that all the deep eutectic solvents extracted the of polygonatum sibiricum polysaccharide with higher yield than water, and among all the deep eutectic solvents, betaine - propanetriol had the highest yield of polygonatum sibiricum polysaccharide, at this time, the yield of polysaccharide of polygonatum sibiricum polysaccharide reached

18.43 £ 0.36 %.

Table 1 Composition of different extraction solvents and extraction rate of polysaccharide from polygonatum sibiricum DES Hydrogen Relative Hydrogen Relative Molar | Extraction rate/% acceptor molecular mass donor molecular mass ratio (average of 3 times) DES 1 Choline 139.62 Urea 120.08 12 8.80 chloride DES 2 Choline 139.62 Citric acid 105.07 11 398 chloride DES 3 Choline 139.62 Glycerol 92.09 1:2 4.04 chloride DES 4 Choline 139.62 Ethylene 62.07 1:2 3.90 chloride glycol DES 7 Betaine 117.15 Ethylene 62.07 12 14.76 glvcol DES 8 L-Proline 115.13 L-Lactic 90.08 11 435 acid DES9 L-Proline 115.13 DL-Malic 134.09 I 6.50 acid

NL 2030622 6 001902P-NL April 12, 2022 Example 2 By designing a four-factor three-level orthogonal test, with mixing temperature of 70°C, mixing material-liquid ratio of 19:20mL, mixing time of 40min, and solvent water content of 0.3g/g as the optimization center, and using polysaccharide yield as the evaluation index, the conditions of the extracted rate of polygonatum sibiricum polysaccharide were optimized and verified, and the effects of mixing temperature, material-liquid ratio, mixing time, and solvent water content on the extraction rate of polysaccharide were also analyzed. The results are shown in Table 2. The results are shown in Table 2. As can be seen from Table 2, the best extraction process was selected by combining the K values of the factors, the mixing temperature was 70°C, the mixing ratio was 1g:10mL, the mixing time was 50min, and the solvent water content was 0.3g/g. The extraction rate of polysaccharide from Rhizoma coptidis was

32.98% under this condition. Table 2 Orthogonal test design and result analysis B Material- A Temper oo C.Water ‚| Polysaccharid Test No. liquid D.Time/min ature/°C content/g/g e yield/% ratio/(g/mL) ae me

73.569 82.671 77.631 72.789 |

91.989 81.669 84.699 87.630 |

78.579 79.800 81.810 83.721 |

NL 2030622 7 001902P-NL April 12, 2022 [Www [mew [wer [ww | The significance of the effects of mixing temperature, mixing ratio, mixing time, and solvent water content on the extraction rate of polygonatum sibiricum polysaccharide were obtained by ANOVA of the orthogonal test, and the results are shown in Table

3. From Table 3, it can be seen that the effects of mixing temperature and mixing time on the extraction rate of polygonatum sibiricum polysaccharide reached significance. Table 3 Analysis of variance of orthogonal test Factor Sum of Degrees of F ratio F critical | Significance squared freedom value deviations

0.469 Material to 1.415 2 0.288 6.940 liquid ratio

5.423 72 | Gow | | Ewer | os | 4 | | | Note: * indicates a significant difference in this factor (p<0.05) Example 3 The extract was extracted by mixing the lyophilized powder of polygonatum sibiricum with deep eutectic solvent containing 0.3 g/g of water at a material-liquid ratio of 1 g:10 mL with ultrasonic power of 200 w, mixing temperature of 70°C and mixing time of 50 min to obtain the extract of polygonatum sibiricum polysaccharide, adding 4 times the volume of ethanol slowly to the extract and stirring well, then precipitating overnight at 4°C and collecting the precipitate. The precipitate was dissolved again with appropriate amount of water, and the protein was removed by Sevege method, ie. 1/4 volume of Sevage reagent (trichloromethane: n-butanol = 5:1 mixed well) was added to the polysaccharide solution, stirred for 20 min and then centrifuged to extract the uppermost layer of the solution, and the operation was repeated until no

NL 2030622 8 001902P-NL April 12, 2022 precipitate appeared after centrifugation and the upper layer of the solution had no absorbance value at 280 nm. After that, the supernatant will be alcoholic sediment again, and after centrifugation, the sediment will be dialyzed through a dialysis bag with a cut-off molecular weight of 3000 Da for 72 h. The water will be changed at least 8 times. After dialysis, the solution in the dialysis bag was pre-freezed at -80°C and freeze-dried to obtain the crude polygonatum sibiricum polysaccharide. The above obtained crude polygonatum sibiricum polysaccharide was tested separately as follows.

(1) The above crude polygonatum sibiricum polysaccharide (300-500 mg) was dissolved by adding 10 mL of distilled water, and then slowly added to a DEAE-52 chromatographic column (©3.5 cmx50 cm), eluted with distilled water, sodium chloride (0.1-0.5 mol/L) and sodium hydroxide (0.1-0.5 mol/L) in turn at a flow rate of 1 mL/min, and the eluate was measured by phenol-sulfuric acid method. The sugar content of each eluate was measured separately in each tube, and the eluates of the same components were combined, dialyzed and freeze-dried. The mass of each elution fraction was weighed, and the proportion of each elution fraction in all elution fractions was calculated according to the following formula.

The proportion of each elution component (%) = mf/mTx100 % Where, mf: mass of each elution component (mg), mT: total mass of elution component (mg).

The masses of each elution component are shown in Table 4. From Table 4, it can be seen that the obtained polygonatum sibiricum polysaccharides eluted five fractions in DEAE-52 chromatographic column, among which the masses of PP-3, PP-4 and PP-5 after lyophilization were beyond the minimum weighable range. the mass of PP-1 accounted for 56.02% of the upper sample mass, and the mass of PP- 2 accounted for 6.99% of the upper sample mass. Table 4 Table of polysaccharide fractions of DEAE-52 chromatographic column eluate

NL 2030622 9 001902P-NL April 12, 2022 Polysaccharide Elution solution Mass/g Content/% fraction

0.1 MNaCl PP-2 0.0220

0.2 MNaOH PP-3

0.4 MNaOH PP-4

0.5 MNaOH PP-5 (2) The monosaccharide composition of the obtained polygonatum sibiricum polysaccharide and the two main elution components PP-1 and PP-2 were analyzed by HPLC method. The sample was accurately weighed to 10 mg of polygonatum sibiricum polysaccharide, added to an ampoule, added trifluoroacetic acid (4M, 3mL), sealed, hydrolyzed at 110°C for 6h, cooled to room temperature, nitrogen blowing to remove trifluoroacetic acid, added appropriate amount of methanol and continued nitrogen blowing, repeated 3 times until the trifluoroacetic acid was completely removed, added 500uL of distilled water to dissolve. Take 100pL of the standard solution in the tube, add sodium hydroxide solution (0.30M, 100uL) and PMP- methanol solution (0.50M, 100uL), mix well, seal and react in a constant temperature water bath at 70°C for 1h, cool, add 100 u L of 0.30M hydrochloric acid to neutralize. Add 500 4 L trichloromethane, centrifuge, take the upper aqueous phase, repeat 3 times and then aspirate the aqueous phase and pass through 0.22 u m filter membrane to obtain the derivatized sample solution. The HPLC chromatographic detection conditions were as follows: LC-2030C 3D Plus high performance liquid chromatograph; Inertsil ODS-3 C18 column; mobility: phosphate buffer (0.1 M, pH 7.0): acetonitrile (V1:V2=83:17); column temperature: 30°C; flow rate: 1 mL/min; detection wavelength. 245 nm; injection volume: 10 pL.

NL 2030622 10 001902P-NL April 12, 2022 The HPLC chromatograms are shown in Figure 1, where A is the chromatogram of the standard sample, B is the chromatogram of the obtained polygonatum sibiricum polysaccharide sample, C is the chromatogram of the PP-1 fraction sample, and D is the chromatogram of the PP-2 fraction sample. The results of monosaccharide composition measurements are shown in Table 5. From Fig. 1 and Table 5, it can be seen that the two eluted fractions differed significantly in monosaccharide composition, with PP-1 mainly consisting of mannose and glucose and PP-2 mainly consisting of galactose and arabinose.

Table 5 Monosaccharide composition of different elution fractions Samples | ST Ki [| Go Cokin a oe tps pase pet we | o- = pT bo (3) Structural analyses of the obtained polygonatum sibiricum polysaccharide extract and two eluted fractions were performed by infrared spectroscopy.

The infrared spectra were measured by lyophilizing the obtained polygonatum sibiricum polysaccharide and the different fractions obtained by chromatography, fully grinding them and then pressing them to form transparent flakes before measurement, the measured results are shown in Figure 2, where A is the infrared spectra of crude polygonatum sibiricum polysaccharide, B is the infrared spectra of chromatographic fraction 1, and C is the infrared spectra of fraction 2.

From Figure 2, it can be seen that PP, PP-1 and PP-2 have polysaccharide characteristic absorption peaks with O-H stretching vibration at 3412 cm-1, 3390 cm- 1 and 3396 cm-1, respectively, and the peaks of PP at 2935 cm-1, PP-1 at 2935 cm- 1 and 2893 cm-1 and PP-2 at 2962 cm-1, 2933 cm-1 and 2877 cm-1 are due to C-H stretching vibrations, and the peaks at 1415 cm-1, 1425 cm-1, and 1406 cm-1 are C- H variable angle vibrational peaks. Differently, the C=0 stretching vibrations of COO- contained in PP-1 and PP-2 at 1641 cm-1 were different, which might be related to

NL 2030622 11 001902P-NL April 12, 2022 their neutral and acidic sugar contents, and the absorption peaks of PP and PP-2 at 875 cm-1 indicated that they contained B-glycosidic bonds.

(3) Scanning electron microscope analysis Appropriate amount of polysaccharide samples were weighed, gold sprayed in an ion injector to make them conductive, and observed under scanning electron microscope with magnification of 100 and 1000, respectively, and photographed for analysis.

Figure 3 shows the morphology of PP, PP-1, and PP-2 at 100x and 1000x under scanning electron microscopy. Figure 3A and B show the overall rough and bumpy surface of the crude polygonatum sibiricum polysaccharide, and the specific morphology is a smooth and tight long reticular structure, showing round tubes, intertwined and closely arranged, and also some spherical particles are distributed next to the reticular structure, and the tight structure indicates the existence of strong intermolecular interaction forces. Figure 3C and D show that the overall aggregation plane of PP-1 is rough and not smooth, in which spherical particles of different sizes are scattered, and the surface of the particles is uneven and has small holes. Figure 3E and F show that the overall aggregation plane of PP-2 is rough, and both are long strips of mesh with dense arrangement with PP, but the strips of PP-2 are angular and irregular. The different morphologies of PP, PP-1, and PP-2 shown by SEM images may have effects on the properties of polysaccharides, such as solubility, water retention, etc.

Claims

12 001902P-NL Conclusions

1. A method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent, characterized in that the method comprises: extracting the polygonatum sibiricum lyophilized powder by heating it in low eutectic solvent, and then separating it by alcohol precipitation, and the precipitate obtained is polygonatum sibiricum polysaccharide.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 1, which is characterized in that the method specifically comprises the steps of: (1) Preparation of lyophilized powder of polygonatum sibiricum: washing and cutting of polygonatum sibiricum, freeze dry then crush, strain and store in -4°C refrigerator for reserve.

(2) Preparation of low eutectic solvent: Mix selected hydrogen acceptor and hydrogen donor, heat with stirring to melt them until transparent and clear, said ratio of hydrogen acceptor and hydrogen donor is 1g:1-2g.

(3) Extraction of polygonatum sibiricum raw polysaccharide: The lyophilized powder of polygonatum sibiricum of step 1) is mixed with the low eutectic solvent of step 2) and then heated and extracted, ethanol is added to the supernatant after centrifugation separation and left overnight, and the precipitate obtained after centrifuge separation is polygonatum sibiricum polysaccharide.

13 001902P-EN

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the hydrogen acceptors in the above step 2) are choline chloride, betaine, L-proline, and the hydrogen donors are urea, citric acid, propanetriol, ethylene glycol , L-lactic acid, DL-lactic acid.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the hydrogen acceptor in said step 2) is betaine and the hydrogen donor is propanetriol.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the heating temperature in the above step 2) is 60-80°C and the time is 20-60min.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the ratio of freeze-dried powder of polygonatum sibiricum mixed with low eutectic solvent in the above step 3) is 1g:10-40mL.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the extraction temperature in said step 3) is 40-80°C and the extraction time is 20-60min.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the water content of low eutectic solvent in said step 3) is 0.3-0.4 g/g.

14 001902P-EN

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the centrifugation condition in the above step 3) is 4000-5000r/min and time is 10-25min.

The method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the final concentration of ethanol in the above step 3) is 60% to 90%.

A method of extracting polygonatum sibiricum polysaccharide by low eutectic solvent as claimed in claim 2, characterized in that the precipitation conditions in the above step 3) are temperature 0-4°C and time 12-24h.