CN113521188A - Method for purifying rhizoma polygonati total flavonoids - Google Patents
Method for purifying rhizoma polygonati total flavonoids Download PDFInfo
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- 229930003935 flavonoid Natural products 0.000 title claims abstract description 33
- 235000017173 flavonoids Nutrition 0.000 title claims abstract description 33
- 150000002215 flavonoids Chemical class 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000001179 sorption measurement Methods 0.000 claims abstract description 77
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 claims abstract description 40
- 229930003944 flavone Natural products 0.000 claims abstract description 40
- 235000011949 flavones Nutrition 0.000 claims abstract description 40
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims abstract description 40
- 150000002212 flavone derivatives Chemical class 0.000 claims abstract description 39
- 239000011347 resin Substances 0.000 claims abstract description 32
- 229920005989 resin Polymers 0.000 claims abstract description 32
- 238000000605 extraction Methods 0.000 claims abstract description 26
- 238000000746 purification Methods 0.000 claims abstract description 22
- 239000000287 crude extract Substances 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 19
- 239000002904 solvent Substances 0.000 claims abstract description 15
- 239000003480 eluent Substances 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 239000000284 extract Substances 0.000 claims abstract description 10
- 238000005303 weighing Methods 0.000 claims abstract description 9
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 8
- 238000007873 sieving Methods 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- 241000037831 Polygonatum sibiricum Species 0.000 claims description 8
- 238000000643 oven drying Methods 0.000 claims description 7
- 238000010298 pulverizing process Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000001035 drying Methods 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 36
- 230000000694 effects Effects 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 12
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 8
- BNGXYYYYKUGPPF-UHFFFAOYSA-M (3-methylphenyl)methyl-triphenylphosphanium;chloride Chemical compound [Cl-].CC1=CC=CC(C[P+](C=2C=CC=CC=2)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1 BNGXYYYYKUGPPF-UHFFFAOYSA-M 0.000 description 5
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- 235000010288 sodium nitrite Nutrition 0.000 description 5
- 238000011835 investigation Methods 0.000 description 4
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- 241000756042 Polygonatum Species 0.000 description 3
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8969—Polygonatum (Solomon's seal)
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Abstract
The invention discloses a process for purifying rhizoma polygonati total flavonoids, which comprises the steps of slicing rhizoma polygonati, drying, crushing and sieving with a 60-mesh sieve; weighing a proper amount of crushed rhizoma polygonati, adding an extraction solvent according to a material-liquid ratio of 1:18, performing water bath extraction, centrifuging to obtain a supernatant, performing rotary evaporation concentration on the supernatant, performing constant volume to a certain volume by using the extraction solvent to obtain a rhizoma polygonati total flavone crude extract, and adding the crude extract into a pretreated resin column; washing off water-soluble impurities after the effluent completely flows out, adding an extraction solvent for elution, collecting the eluent, and concentrating to obtain a rhizoma polygonati total flavone purified solution; the method has the advantages that the rhizoma polygonati total flavonoids are treated by macroporous adsorption resin, so that the purity of the flavonoids in the extract is greatly improved, the operation time is short, the subsequent research and development of the flavonoid extract are facilitated, and the problems of relatively original purification process, complicated operation steps, long operation time and very low production efficiency of the existing rhizoma polygonati total flavonoids are solved.
Description
Technical Field
The invention relates to the technical field of flavone purification, in particular to a method for purifying rhizoma polygonati total flavone.
Background
In China, polygonatum has a long history of medication, is an important medicinal and edible plant in China, has unique natural biological activity, and is one of natural biological active ingredients. Modern pharmacological research finds that the rhizoma polygonati total flavonoids have obvious pharmacological effects of resisting oxidation, fatigue, aging, tumors, bacteria, blood sugar and blood fat, and the like. The existing sealwort total flavone purification process is relatively original, the operation steps are very complicated, the operation time is long, and the production efficiency is very low, so that an efficient and rapid sealwort total flavone purification process is urgently needed. The method for extracting and separating multiple active ingredients in polygonatum sibiricum disclosed in the Chinese CN 104940609 patent adopts a process route of flash extraction, degreasing, repeated extraction, column loading and drying, and polygonatum sibiricum flavone is obtained through repeated separation and purification.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a purification process of rhizoma polygonati total flavonoids, which solves the problems of relatively original process means, very complicated operation steps, long operation time and low production efficiency when the conventional rhizoma polygonati total flavonoids are purified.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for purifying rhizoma polygonati total flavonoids comprises the following steps:
(1) slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with a pulverizer, and sieving with 60 mesh sieve to obtain rhizoma Polygonati powder;
(2) weighing a proper amount of the sealwort powder obtained in the step (1), adding an extraction solvent according to a material-liquid ratio of 1:18, performing water bath extraction, centrifuging, and taking a supernatant to obtain a supernatant;
(3) performing rotary evaporation concentration on the supernatant obtained in the step (2), and fixing the volume to a certain volume by using an extraction solvent to obtain a crude polygonatum sibiricum flavone extract;
(4) slowly adding the crude extract of the rhizoma polygonati general flavone obtained in the step (3) with the pH value of 3-7 into a pretreated macroporous adsorption resin column, wherein the adsorption time is 0-24 hours, and the adsorption temperature is 15-35 ℃; washing off water-soluble impurities after the effluent completely flows out, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified solution;
(5) and (3) adsorbing and purifying the crude extract of the rhizoma polygonati total flavonoids by using adsorption resin under an optimized condition, and calculating the purity before and after purification.
In the step (4), the macroporous adsorption resin is one or more of D101 type, NKA-9 type, AB-8 type and S-8 type macroporous adsorption resin, preferably NKA-9 type macroporous adsorption resin.
Preferably, in the step (4), the pH of the crude rhizoma polygonati extract is 5.
Preferably, in the step (4), the adsorption time is 8 h.
Preferably, in the step (4), the adsorption temperature is 25 ℃.
Preferably, in step (1), step (2) and step (5), the extraction solvent has an ethanol concentration of 70%.
Compared with the prior art, the method has the following beneficial effects:
the purification method of polygonatum sibiricum flavone disclosed by the invention has the advantages that the purity of flavone in the extract is greatly improved, the operation time is short, the subsequent further research and development of the flavone extract are facilitated, and the problems of original technological means, complicated operation steps, long operation time and low production efficiency in the purification of the existing polygonatum sibiricum total flavone are solved.
Drawings
FIG. 1 is a 24h adsorption curve of four macroporous adsorption resins;
FIG. 2 shows the effect of pH value of crude extract of rhizoma Polygonati on adsorption effect;
FIG. 3 is a graph showing the effect of adsorption time on adsorption effect;
FIG. 4 shows the effect of adsorption temperature on adsorption effect.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
(1) Slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve;
(2) weighing 50g of crushed rhizoma polygonati, adding 900mL of 70% ethanol solution, extracting in water bath, centrifuging, and taking supernatant;
(3) concentrating the supernatant by rotary evaporation, and diluting to a constant volume of 200mL with 70% ethanol solution to obtain rhizoma Polygonati total flavone crude extract;
(4) precisely weighing 1.00g of pretreated D101, NKA-9, AB-8 and S-8 macroporous resin respectively, placing the macroporous resin into four triangular flasks with the volume of 100mL, and adding 20mL of crude extract of rhizoma polygonati total flavonoids;
(5) placing in a shaking table at constant temperature of 25 deg.C and 110r/min, shaking for timing, and placing 2mL of supernatant at 0h, 0.5h, 1h, 2h, 4h, 8h, and 24h in a 25mL volumetric flask. Adding 1mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 10mL of 4% sodium hydroxide solution, fixing the volume to the scale with 70% ethanol, shaking up, standing for 15min, and determining the absorbance value at the wavelength of 510nm of a spectrophotometer by taking the ethanol concentration solution with the same concentration during extraction as a reference solution.
Drawing the static adsorption curve of rhizoma Polygonati total flavone on D101, NKA-9, AB-8, and S-8 macroporous resin, and referring to figure 1. It can be seen from the figure that the absorbance value of the rhizoma polygonati total flavone solution shows a descending trend along with the prolonging of the adsorption time and is stable after 8 hours. Meanwhile, the NKA-9 type macroporous adsorption resin has the adsorption effect obviously better than that of the other three types of macroporous adsorption resin, so that NKA-9 is selected as a resin material for subsequent experiments.
Examples 2 to 6
(1) Slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve;
(2) weighing 50g of crushed rhizoma polygonati, adding 900mL of 70% ethanol solution, extracting in water bath, centrifuging, and taking supernatant;
(3) concentrating the supernatant by rotary evaporation, and diluting to a constant volume of 200mL with 70% ethanol solution to obtain rhizoma Polygonati total flavone crude extract;
(4) slowly adding the crude extract of the rhizoma polygonati total flavonoids with the pH of 5 into a pretreated NKA-9 macroporous adsorption resin column; in the purification process, the adsorption time of the purification is 8h, and the adsorption temperature is 25 ℃.
(5) And after all the effluent liquid flows out, washing off water-soluble impurities, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified liquid.
(6) 2mL of the purified solution of the total flavonoids from the sealwort is put into a 25mL volumetric flask. Adding 1mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 10mL of 4% sodium hydroxide solution, fixing the volume to the scale with 70% ethanol, shaking up, standing for 15min, and determining the absorbance value at the wavelength of 510nm of a spectrophotometer by taking the ethanol concentration solution with the same concentration during extraction as a reference solution.
(7) The steps and parameters of the sealwort total flavone purification method implemented in 2-6 are basically the same, except that the pH values of the crude sealwort extraction used in the step (4) of the embodiment 2-6 are 3, 4, 5, 6 and 7 respectively. The influence of pH value of crude extract of rhizoma Polygonati on adsorption effect is analyzed by using light absorption value as investigation index, and is shown in figure 2. The graph shows that when the pH value of the sample solution is 3-4, the absorbance value of the rhizoma polygonati total flavonoids is gradually reduced, and the adsorption rate is increased; when the pH value is 4, the absorbance value of the total flavone is lowest, and the adsorption effect is best; when the pH value exceeds 4, the absorbance value gradually decreases as the pH value increases. This is because flavone is a polyhydroxyphenol compound, is acidic, and is adsorbed to resin by van der waals force, whereas under alkaline conditions, the hydroxyl group of the flavone molecule is deionized, and the flavone compound forms an ionic structure, and is not easily adsorbed. Therefore, when the pH value of the sample solution is 4, the NKA-9 macroporous adsorption resin has the best adsorption effect on the rhizoma polygonati total flavonoids.
Examples 7 to 11
(1) Slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve;
(2) weighing 50g of crushed rhizoma polygonati, adding 900mL of 70% ethanol solution, extracting in water bath, centrifuging, and taking supernatant;
(3) concentrating the supernatant by rotary evaporation, and diluting to a constant volume of 200mL with 70% ethanol solution to obtain rhizoma Polygonati total flavone crude extract;
(4) slowly adding the crude extract of the rhizoma polygonati general flavone into a pretreated NKA-9 macroporous adsorption resin column; in the purification process, the pH value of the purified crude rhizoma polygonati extract is 3, and the adsorption temperature is 15 ℃.
(5) And after all the effluent liquid flows out, washing off water-soluble impurities, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified liquid.
(6) 2mL of the purified solution of the total flavonoids from the sealwort is put into a 25mL volumetric flask. Adding 1mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 10mL of 4% sodium hydroxide solution, fixing the volume to the scale with 70% ethanol, shaking up, standing for 15min, and determining the absorbance value at the wavelength of 510nm of a spectrophotometer by taking the ethanol concentration solution with the same concentration during extraction as a reference solution.
(7) The steps and parameters of the sealwort total flavone purification method implemented in 7-11 are basically the same, except that the adsorption time used in the step (4) of the embodiment 7-11 is 4, 8, 12, 16 and 24 hours respectively. The absorption value is taken as an investigation index, and the influence of the adsorption time on the adsorption effect is analyzed, which is shown in figure 3 in detail. As can be seen, the absorbance values gradually decreased with time. Therefore, the flavone is gradually reduced after being adsorbed by NKA-9 macroporous adsorption resin, and the adsorption rate of the resin is gradually increased; after 8h, the curve gradually tends to be stable, and the macroporous resin adsorption tends to be saturated. Therefore, the optimal time of NKA-9 macroporous adsorption resin rhizoma polygonati total flavonoids should be controlled to be 7-8 hours when the NKA-9 macroporous adsorption resin rhizoma polygonati total flavonoids are stable.
Examples 12 to 16
(1) Slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve;
(2) weighing 50g of crushed rhizoma polygonati, adding 900mL of 70% ethanol solution, extracting in water bath, centrifuging, and taking supernatant;
(3) concentrating the supernatant by rotary evaporation, and diluting to a constant volume of 200mL with 70% ethanol solution to obtain rhizoma Polygonati total flavone crude extract;
(4) slowly adding the crude extract of the rhizoma polygonati general flavone into a pretreated NKA-9 macroporous adsorption resin column; in the purification process, the pH value of the purified crude rhizoma polygonati extract is 7, the adsorption temperature is 35 ℃, and the adsorption time is 24 hours.
(5) And after all the effluent liquid flows out, washing off water-soluble impurities, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified liquid.
(6) 2mL of the purified solution of the total flavonoids from the sealwort is put into a 25mL volumetric flask. Adding 1mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 10mL of 4% sodium hydroxide solution, fixing the volume to the scale with 70% ethanol, shaking up, standing for 15min, and determining the absorbance value at the wavelength of 510nm of a spectrophotometer by taking the ethanol concentration solution with the same concentration during extraction as a reference solution.
(7) The steps and parameters of the sealwort total flavone purification method implemented in 12-16 are basically the same, except that the adsorption temperatures used in the step (4) of the embodiment 12-16 are respectively 15, 20, 25, 30 and 35 ℃. The influence of the adsorption temperature on the adsorption effect is analyzed by taking the light absorption value as an investigation index, and the details are shown in an attached figure 4. As shown in the figure, when the temperature is 15-25 ℃, the absorbance value of the rhizoma polygonati total flavonoids is gradually reduced, and the adsorption rate is increased; when the temperature is 25 ℃, the absorbance value of the rhizoma polygonati total flavonoids is lower than that of other temperatures, the adsorption effect is optimal, and the temperature is close to room temperature. Therefore, 25 ℃ was chosen as the optimum adsorption temperature.
Example 17
According to the experimental result of a single factor, three factors of sample solution values, adsorption temperature and adsorption time influencing the adsorption effect of the flavonoid compound are selected for investigation, and the pH value, the adsorption temperature and the adsorption time of the sample solution are selected for orthogonal experiment (table 1).
TABLE 1 orthogonal experiment factor horizon
The results are shown in table 2, and it can be seen from the results that the adsorption temperature has the greatest influence on the adsorption effect of the macroporous adsorption resin for flavonoid compounds in polygonatum sibiricum, the pH value is the second place, and the adsorption time is the smallest influence. Therefore, when the pH value is 5, the adsorption temperature is 25 ℃, the adsorption time is 8h, and the adsorption rate of the rhizoma polygonati total flavonoids adsorbed by the NKA-9 macroporous adsorption resin is the highest. In the single-factor experiment process, the adsorption temperature is 25 ℃ and the adsorption time is 7-8 h, so that the adsorption effect is good, and the orthogonal experiment result is also met. Therefore, the optimal process conditions of the macroporous adsorption resin for purifying the polygonatum flavone are determined by a single-factor experiment and an orthogonal experiment as follows: the pH value is 5, the adsorption temperature is 25 ℃, and the adsorption time is 8 h.
Table 2 results of orthogonal experiments with adsorption conditions
Example 18
(1) Slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with pulverizer, and sieving with 60 mesh sieve;
(2) weighing 50g of crushed rhizoma polygonati, adding 900mL of 70% ethanol solution, extracting in water bath, centrifuging, and taking supernatant;
(3) concentrating the supernatant by rotary evaporation, and diluting to a constant volume of 200mL with 70% ethanol solution to obtain rhizoma Polygonati total flavone crude extract;
(4) slowly adding the crude extract of the rhizoma polygonati general flavone into a pretreated NKA-9 macroporous adsorption resin column; in the purification process, the pH value of the purified crude rhizoma polygonati extract is 5, the adsorption temperature is 25 ℃, and the adsorption time is 8 hours.
(5) And after all the effluent liquid flows out, washing off water-soluble impurities, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified liquid.
(6) 2mL of the crude extract and the purified solution of the total flavonoids of polygonatum are put into a 25mL volumetric flask. Adding 1mL of 5% sodium nitrite solution, shaking up, standing for 6min, adding 1mL of 10% aluminum nitrate solution, shaking up, standing for 6min, adding 10mL of 4% sodium hydroxide solution, fixing the volume to the scale with 70% ethanol, shaking up, standing for 15min, and determining the absorbance value at the wavelength of 510nm of a spectrophotometer by taking the ethanol concentration solution with the same concentration during extraction as a reference solution.
(7) Calculating the purity before and after purification by the following formula: the results show in table 3, the purity of the polygonatum sibiricum total flavonoids is increased from 33.5% to 78.34%, the purity is improved by nearly two times, and the purification effect is good.
TABLE 3 comparison of the purity of total flavonoids before and after purification
The foregoing lists merely illustrate specific embodiments of the invention. The present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (3)
1. A purification process of rhizoma polygonati total flavonoids is characterized by comprising the following steps:
(1) slicing rhizoma Polygonati, oven drying at 45 deg.C to constant weight, pulverizing with a pulverizer, and sieving with 60 mesh sieve to obtain rhizoma Polygonati powder;
(2) weighing a proper amount of the sealwort powder obtained in the step (1), adding an extraction solvent according to a material-liquid ratio of 1:18, performing water bath extraction, centrifuging, and taking a supernatant to obtain a supernatant;
(3) performing rotary evaporation concentration on the supernatant obtained in the step (2), and fixing the volume to a certain volume by using an extraction solvent to obtain a crude polygonatum sibiricum flavone extract;
(4) slowly adding the crude extract of the rhizoma polygonati general flavone obtained in the step (3) with the pH value of 3-7 into a pretreated macroporous adsorption resin column, wherein the adsorption time is 0-24 hours, and the adsorption temperature is 15-35 ℃; washing off water-soluble impurities after the effluent completely flows out, slowly adding an extraction solvent for elution, collecting the eluent, and concentrating the eluent to 100mL to obtain the rhizoma polygonati total flavone purified solution;
(5) and (3) adsorbing and purifying the crude extract of the rhizoma polygonati total flavonoids by using adsorption resin under an optimized condition, and calculating the purity before and after purification.
2. The method for purifying rhizoma polygonati total flavonoids according to claim 1, wherein the macroporous adsorption resin in the step (4) is one of macroporous resins of D101 type, NKA-9 type, AB-8 type and S-8 type.
3. The method for purifying rhizoma polygonati total flavonoids according to claim 1, wherein the extraction solvents in the steps (1), (2) and (5) are all 70% ethanol concentrated solutions.
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