CN115813967A - Preparation method of total saponins of adventitious roots of ginseng - Google Patents
Preparation method of total saponins of adventitious roots of ginseng Download PDFInfo
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- CN115813967A CN115813967A CN202211583245.2A CN202211583245A CN115813967A CN 115813967 A CN115813967 A CN 115813967A CN 202211583245 A CN202211583245 A CN 202211583245A CN 115813967 A CN115813967 A CN 115813967A
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- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 87
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method of total saponins of adventitious roots of ginseng, which comprises the following steps: (1) wall breaking: pulverizing dried tissue-cultured adventitious root of Ginseng radix, soaking in water, and breaking cell wall with ultrahigh pressure homogenizing equipment; (2) extracting: reflux-extracting the wall-broken material liquid, and concentrating; and (3) purification: adsorbing the concentrated solution by macroporous adsorption resin, sequentially washing the macroporous resin with water and impurity-removing solution, eluting with eluent, and collecting the eluent containing total saponins; and (4) drying: evaporating and concentrating the eluent, and drying to obtain the high-purity total saponins of adventitious roots of ginseng. The ginseng adventitious root total saponins prepared by the method have high purity and high extraction rate, and the method is simple and easy to implement, mild in reaction condition, less in used material consumption, environment-friendly, low in requirement on reagent equipment and suitable for large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of preparation of total saponins of ginseng, and relates to a preparation method of total saponins of adventitious roots of ginseng.
Background
Ginseng is one of the famous and precious Chinese medicinal materials, has the effects of reinforcing primordial qi, restoring pulse, relieving depletion, tonifying spleen, benefiting lung, promoting the production of body fluid, soothing nerves, benefiting intelligence and the like, is listed as the top grade in Shen nong Ben Cao Jing, and is a commonly used tonic. Modern researches show that ginseng contains various effective components such as saponin, polysaccharide, flavone, protein, amino acid and the like, wherein the most main active component is the ginsenoside, and pharmacological researches show that the ginsenoside has various pharmacological effects such as oxidation resistance, aging resistance, blood sugar reduction, tumor resistance and the like, can act on a plurality of systems such as a circulatory system, an immune system, a cardiovascular system, an endocrine system and the like, and further how to efficiently and scientifically extract the ginsenoside becomes a research subject at present widely.
The ginseng is divided into overground part and underground part, and the overground part comprises stems, leaves, ginseng flowers and fruits. The ginseng stem is the stem connecting the reed head with the ginseng leaves and the ginseng seeds, and generally, the higher the age of the ginseng, the longer the ginseng stem. The underground part of ginseng can be divided into a reed head, a body and an adventitious root. The ginseng adventitious root can be induced from ginseng seed source, and can be differentiated and cultured to form adventitious root, and is made up by using three-stage culture, cleaning and drying steps, etc. it has no limitation of land, season and environment, and its growth is quick, period is short, and these are incomparable with traditional culture method, so that it opens up a new way for producing plant medicine. The existing method for extracting and preparing total saponin from the adventitious roots of ginseng has the problem of low extraction rate.
The Chinese patent application with the application number of 202010867377.2 discloses an extraction process of total saponins in ginseng stems and leaves, which comprises the following steps: the method comprises the following steps: removing residues of the ginseng stem and leaf raw materials by using a low-carbon alcohol solution with the water content of 0-20wt% to obtain a pretreated ginseng stem and leaf stock solution; step two: introducing nitrogen into the stock solution of the pretreated ginseng stems and leaves in the first step, stirring simultaneously to obtain foams and residual solution containing the total saponins of ginseng, and separating the foams from the residual solution; step three: dissolving the foam obtained in the second step to obtain a ginseng total saponin solution, adjusting the pH value, passing the ginseng total saponin solution through a macroporous resin column, eluting with ethanol or an ethanol water solution, collecting the eluent, and evaporating to obtain the ginseng total saponin. In the scheme, pesticide residue is reduced through residue removal treatment, nitrogen and the like are required to be used, and the extraction rate needs to be improved.
Chinese patent application No. 200410011078.X discloses a method for extracting ginsenoside under ultrahigh pressure, which comprises the steps of firstly crushing dried ginseng, sieving with a 40-mesh sieve, adding chloroform or ether solvent into the dried ginseng powder according to a solid-to-liquid ratio of 1g to 40mL, refluxing and degreasing in a Soxhlet extractor for 3h, volatilizing filter residue at normal temperature after filtering, adding water or ethanol, methanol and n-butyl alcohol organic solvent into the degreased material according to a solid-to-liquid ratio of 1g to 10-100mL, mixing and sealing, soaking for 0-24h at normal temperature, placing the mixed material into a high-pressure container, applying 100-6000MPa pressure at normal temperature for leaching, keeping the pressure for 1-5min, filtering to remove material residue after pressure relief, and collecting filtrate to obtain an extracting solution containing ginsenoside. The scheme adopts ultrahigh pressure for extraction, but the purity is not high.
Therefore, it is desirable to provide a method for preparing ginsenoside with high extraction rate, high purity and relatively simple process.
The present invention has been made in view of this point.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of total adventitious root saponins of ginseng. In the preparation method, water is used as an extraction solvent, the ultrahigh-pressure homogenizing equipment is used for breaking cell walls, reflux extraction, macroporous resin adsorption purification and drying are carried out, and the optimized conditions are comprehensively acted, so that the extract containing the ginseng adventitious root total saponins with the purity of more than 60 percent is obtained, and the purity and the extraction rate are high. In addition, the method is simple and easy to implement, mild in reaction conditions, less in consumed materials, environment-friendly, low in requirements for reagent equipment and suitable for large-scale industrial production.
In order to solve the technical problems, the invention adopts the technical scheme that:
the invention aims to provide a preparation method of total saponins of adventitious roots of ginseng, which comprises the following steps:
(1) Wall breaking: pulverizing dried tissue-cultured adventitious root of Ginseng radix, soaking in water, and breaking cell wall with ultrahigh pressure homogenizing equipment;
(2) Extraction: reflux-extracting the wall-broken material liquid, and concentrating;
(3) And (3) purification: adsorbing the concentrated solution by macroporous adsorption resin, sequentially washing the macroporous resin with water and impurity-removing solution, eluting with eluent, and collecting the eluent containing total saponins;
(4) And (3) drying: evaporating and concentrating the eluent, and drying to obtain the high-purity total saponins of adventitious roots of ginseng.
According to the invention, firstly, water is adopted to soak the adventitious roots of the ginseng tissue culture, the ultrahigh pressure homogenization equipment is utilized to carry out cell wall breaking, then reflux extraction, macroporous resin adsorption purification and drying are carried out, and the optimized comprehensive effects of various conditions are realized, so that the extract containing the ginseng adventitious root total saponins with the purity of more than 60% is obtained, and the purity and the extraction rate are high.
In the step (1), when water is added for soaking, the material-water ratio of the ginseng adventitious roots to the water is 1 to 10-30, and the unit of the material-water ratio is g/mL, so that the dried ginseng tissue culture adventitious roots are rehydrated;
preferably, the feed-water ratio is 1;
preferably, the soaking time is 8-16h, and more preferably, the soaking time is 12h.
In the step (1), when the cell wall breaking treatment is carried out by using ultrahigh pressure homogenizing equipment, the wall breaking pressure is 100-150MPa, the flow rate is 100-200mL/min, and the treatment times are 2-4 times;
preferably, the wall breaking pressure is 130-140MPa, the flow rate is 120-160mL/min, and the treatment times are 3-4 times.
In the step (2), reflux extraction is carried out on the feed liquid after wall breaking for 2-4 times, filtrate is collected after each extraction, water is added into filter residue for subsequent extraction, the amount of the added water is the same as that of the primary liquid, the reflux extraction time is 1-3h each time, and the extraction temperature is 70-100 ℃; after extraction, combining the extracting solutions and concentrating;
preferably, reflux extraction is carried out for 2-3 times, each time is 1.5-2.5h, and the extraction temperature is 80-100 ℃;
preferably, the concentration of total ginsenosides in the concentrated extractive solution is 1-5mg/mL.
In the step (3), the type of the macroporous adsorption resin is selected from one of HPD-100, HPD-400, DM-130, D101, X-5 and AB-8;
preferably, the types of the macroporous adsorption resin are as follows: d101 or AB-8.
In the step (3), when the concentrated solution is loaded on the macroporous adsorption resin, the loading concentration is 2-6mg/mL, the loading flow rate is 2-5mL/min, and the loading volume is 40-60mL;
preferably, the feeding concentration is 3-5mg/mL, the feeding flow rate is 3-4mL/min, and the feeding volume is 50-60mL.
In the step (3), the impurity removal solution is an ethanol water solution with the volume percentage of 30-50%; the eluent is ethanol water solution with the volume percentage of 50-90%;
preferably, the impurity removing liquid is 35-45% by volume of ethanol water solution; the eluent is 60-80% ethanol water solution by volume percentage.
In the further scheme, in the step (3), elution is carried out by adopting eluent at the flow rate of 1.0-3.0 mL/min;
preferably, elution is performed at a flow rate of 1.5-2.5mL/min.
In the step (4), the eluent is subjected to rotary evaporation to recover ethanol to obtain a concentrated solution, and then the concentrated solution is dried at 50-100 ℃ to obtain high-purity ginseng tissue culture adventitious root total saponins with the water content of less than 5%;
preferably, the drying equipment is selected from one of a hot air circulation oven, a vacuum oven and an electric heating forced air drying oven.
According to a further scheme, the purity of the obtained ginseng tissue culture adventitious root total saponin is 30-80%;
preferably, the purity of the obtained total saponins of the tissue culture adventitious roots of the ginseng is 65-80%.
The ginseng adventitious roots in the present invention may be naturally-grown ginseng adventitious roots, or may be artificially tissue-cultured ginseng adventitious roots, and are not limited thereto. As a specific embodiment, the cultivation of the ginseng adventitious roots may be carried out with reference to the related patent of the ginseng adventitious roots cultivation method previously filed by the applicant. For example, see patent application No. 2021100618659 for adventitious root culture protocol.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. in the preparation method, water is used as an extraction solvent, the ultrahigh-pressure homogenizing equipment is used for breaking cell walls, reflux extraction, macroporous resin adsorption purification and drying are carried out, and the optimized conditions are comprehensively acted, so that the extract containing the ginseng adventitious root total saponins with the purity of more than 60 percent is obtained, and the purity and the extraction rate are high.
2. The method is simple and easy to implement, mild in reaction conditions, less in used material consumption, environment-friendly, low in requirement on reagent equipment and suitable for large-scale industrial production.
Detailed Description
The following embodiments are further described to help further understand the advantages and effects of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The ultrahigh pressure homogenizing device adopted in the invention is SCIENTZ-207A produced by Ningbo Xinzhi Biotechnology GmbH.
Example 1
S110, wall breaking treatment:
s111, taking 160g of dried ginseng adventitious roots, crushing the ginseng adventitious roots, sieving the fine powder with a 80-mesh standard sieve, adding purified water according to a material-water ratio (g/mL) of 1.
And S112, performing cell wall breaking treatment on the material treated in the step S111 by using ultrahigh-pressure homogenizing equipment, wherein the wall breaking pressure is 110MPa, the flow rate is 120mL/min, the treatment times are 2 times, and the material yield is 4800mL.
S120, extracting;
s121, carrying out reflux extraction on the feed liquid treated in the step S112 for 4 times, wherein each time lasts for 1.5 hours, filtering and collecting filtrate after each extraction, adding water into filter residues for subsequent extraction, wherein the amount of the added water is the same as that of the primary liquid, the extraction temperature is 80 ℃, merging the extracting solutions after the extraction is finished, and concentrating, wherein the material receiving amount is 1200mL, and each 1mL of the concentrated extracting solution contains 3mg of total ginsenoside;
s130, purifying;
s131, adsorbing the product obtained in the step S121 by macroporous adsorption resin, wherein the type of the macroporous adsorption resin is as follows: HPD-100; the feeding concentration is 3mg/mL, the feeding flow rate is 2mL/min, and the feeding volume is 40mL.
S132, after the purified materials are loaded, respectively washing the macroporous resin with 80mL of purified water and 80mL of 35% ethanol water solution (impurity removal solution) to remove non-saponin components in the ginseng adventitious root extract, eluting with 60% ethanol water solution (eluent) at the flow rate of 2.5mL/min, and collecting the eluent containing ginseng adventitious root total saponins;
s140, drying:
s141, performing rotary evaporation on the eluent containing the total saponins of adventitious roots of ginseng in the S132 to recover ethanol to obtain a concentrated solution containing the total saponins of adventitious roots of ginseng; the volume of the concentrated solution is 15mL;
and S142, recovering the solvent from the concentrated solution containing the total adventitious root saponins in the S141, and drying in a hot air circulation oven at the drying temperature of 50 ℃ to obtain the high-purity total adventitious root saponins with the water content of less than 5%.
Examples 2 to 6
Examples 2 to 6 each provide a method for preparing total saponins of ginseng adventitious roots, the procedure is the same as in example 1, and the parameters of each example are shown in table 1.
Table 1: parameters of examples 2 to 6
The contents of total saponins of adventitious roots of ginseng prepared in examples 1 to 6 were measured, and the results are shown in table 2 below.
Table 2 shows the amount and content of total saponins of adventitious roots of ginseng
Test example 1: optimization of wall breaking process
(I) Experimental method
The test optimizes the wall breaking process, and the parameters of each test group are shown in table 3.
Table 3: test groups 1-6 wall breaking parameters
The content of total saponins of Ginseng radix in the broken wall material is determined according to the determination method of total saponins of Ginseng radix in 2020 edition of Chinese pharmacopoeia.
1. Preparation of control solutions
Ginsenoside Re control solution: taking a proper amount of ginsenoside Re reference substance, precisely weighing, and adding methanol to prepare a solution containing 1.0mg of Re per 1 mL.
2. Preparation of test solution
Filtering the test materials to obtain a material liquid containing ginsenoside after wall breaking, taking 20mL of the material liquid to carry out reduced pressure concentration, recovering the solvent, drying the concentrated solution to obtain a crude saponin product, and weighing the crude saponin product.
Taking 20mL of the feed liquid after wall breaking, passing through a D101 type macroporous adsorption resin column, eluting with water until the feed liquid is colorless, eluting with 60% ethanol until the eluent is colorless, collecting the eluent of 60% ethanol, recovering the solvent until the eluent is nearly dry, evaporating to dryness, dissolving the residue with methanol, transferring the dissolved residue into a 10mL volumetric flask, adding methanol to dilute the dissolved residue to a scale, and shaking up. The mixture was filtered through a 0.45 μm needle filter and examined.
3. Preparation of the Standard Curve
Precisely sucking ginsenoside Re reference substance solutions of 40 mu L, 60 mu L, 80 mu L, 100 mu L, 120 mu L, 140 mu L and 160 mu L, respectively placing in a test tube with a plug, adding distilled water to complement the volume to 200 mu L, precisely adding 1% vanillin perchloric acid test solution of 0.5mL, placing in a constant-temperature water bath at 60 ℃, fully mixing uniformly, heating for 15min, immediately cooling for 2min by using an ice water bath, adding 77% sulfuric acid solution of 5.0mL, and shaking uniformly; the absorbance at a wavelength of 540nm was measured by ultraviolet-visible spectrophotometry (2020 th edition, fourth general rule 0401) using the corresponding reagent as a blank, and the results of absorbance are shown in Table 3.1. And drawing a standard curve by taking the absorbance (A) as the ordinate and the mass (m) of the ginsenoside Re as the abscissa.
4. Determination of sample content
Precisely measuring 50 mu L of test solution, measuring the absorbance by the method starting from 'placing in a test tube with a plug', reading the amount of the ginsenoside Re in the test solution from the standard curve, and multiplying the calculation result by 0.84 to obtain the content of the total ginsenoside in the sample.
(II) results of the experiment
The results of the crude saponin product weight and total saponin content for each test group are shown in table 4:
table 4: test groups 1-6 wall breaking treatment results
As can be seen from the test data in the table above, the test groups 1 and 6 have unsatisfactory effects and poor economy in the content of crude saponin and total saponin. For analysis reasons, the experiment group 1 may cause insufficient wall breaking of materials and poor wall breaking effect due to small wall breaking pressure and small wall breaking times; in the experimental group 6, the loss of functional components in the material and the poor wall-breaking effect may be caused by the large wall-breaking pressure and the large number of wall-breaking times; therefore, the wall breaking process parameters determined by the invention are that the material-water ratio (g: mL) is 1; preferably, the material-water ratio (g: mL) is 1.
Test example 2: optimization of extraction Process
(I) Experimental method
The extraction process is optimized in the experiment, the extraction treatment is carried out on the materials after the wall breaking treatment according to the feeding amount of 10g, the material-liquid ratio (g: mL) of 1 to 25, the wall breaking pressure of 140MPa, the wall breaking flow rate of 160mL/min and the wall breaking times of 4 times, and the parameters of each experimental group of the extraction treatment are shown in Table 5.
Table 5: test groups 7-12 extraction processing parameters
The extracted material is determined according to the content determination method of the total ginsenoside under the item of 'total ginsenoside' in 'Chinese pharmacopoeia' 2020 edition.
1. Preparation of control solutions
Ginsenoside Re control solution: taking a proper amount of ginsenoside Re reference substance, precisely weighing, and adding methanol to prepare 1mL solution containing 1.0mg of ginsenoside Re.
2. Preparation of test solution
Filtering the test materials to obtain a material liquid containing ginsenoside after extraction, taking 100mL of the material liquid to carry out reduced pressure concentration, recovering the solvent, drying the concentrated solution to obtain a crude saponin product, and weighing the crude saponin product.
Taking 100mL of the extracted feed liquid, passing through a D101 type macroporous adsorption resin column, eluting with water until the material liquid is colorless, eluting with 60% ethanol until the eluent is colorless, collecting the eluent of 60% ethanol, recovering the solvent until the solvent is nearly dry, evaporating to dryness, dissolving the residue with methanol, transferring the dissolved residue into a 10mL volumetric flask, adding methanol to dilute the dissolved residue to a scale, and shaking up. The mixture was filtered through a 0.45 μm needle filter and examined.
3. Preparation of the Standard Curve
Precisely sucking ginsenoside Re reference substance solutions of 40 mu L, 60 mu L, 80 mu L, 100 mu L, 120 mu L, 140 mu L and 160 mu L, respectively placing in a test tube with a plug, adding distilled water to complement the volume to 200 mu L, precisely adding 1% vanillin perchloric acid test solution of 0.5mL, placing in a constant-temperature water bath at 60 ℃, fully mixing uniformly, heating for 15min, immediately cooling for 2min by using an ice water bath, adding 77% sulfuric acid solution of 5.0mL, and shaking uniformly; the absorbance was measured at a wavelength of 540nm using the corresponding reagent as a blank in an ultraviolet-visible spectrophotometry (2020 th edition, fourth general rule 0401 in the Chinese pharmacopoeia) and the results of the absorbance are shown in Table 3.1. And drawing a standard curve by taking the absorbance (A) as the ordinate and the mass (m) of the ginsenoside Re as the abscissa.
4. Sample content determination
Precisely measuring 50 mu L of sample solution, measuring absorbance according to the method under the standard curve preparation item from 'placing in a test tube with a plug', reading the amount of ginsenoside Re in the sample solution from the standard curve, and multiplying the calculation result by 0.84 to obtain the content of the total ginsenoside in the sample.
(II) results of the experiment
The results of the crude saponin product weight and total saponin content for each test group are shown in table 6:
table 6: results of extraction treatment in test groups 7-12
From the test data in the table above, it can be seen that test group 7 and test group 12 have unsatisfactory effects and poor economy in both the crude saponin product and the total saponin content. For analysis reasons, the experimental group 7 may cause insufficient extraction of functional components in the material and poor extraction effect due to short extraction time, few extraction times and low extraction temperature; the experiment group 12 may cause loss of heat-sensitive functional components in the material and poor wall breaking effect due to long extraction time, more extraction times and long total high-temperature treatment time; therefore, the extraction process parameters determined by the invention are that the feed liquid after the wall breaking treatment is extracted by refluxing for 2-4 times, each time lasts for 1-3h, the ratio of the feed water extracted each time is the same as the ratio of the feed water extracted for the first time, the extraction temperature is 70-100 ℃, preferably, the reflux extraction is performed for 2-3 times, each time lasts for 1.5-2.5h, the ratio of the feed water extracted each time is the same as the ratio of the feed water extracted for the first time, and the extraction temperature is 80-100 ℃.
Test example 3: purification process optimization
Model verification of macroporous adsorption resin
1. Pretreatment of macroporous resin
Respectively placing the 6 selected macroporous adsorption resins in absolute ethyl alcohol for sealed soaking, placing in a water bath constant temperature oscillator (at 45 ℃ and 110 r/min) for 24h, washing with absolute ethyl alcohol until the washing liquid is clear and white turbid liquid, and then washing with distilled water until no alcohol smell exists. The resin was filtered off and excess water was blotted dry for use.
2. Adsorption test on different macroporous resins
Weighing 2.0g of each of 6 pretreated macroporous adsorption resins in a conical flask with a plug, adding 20mL of the crude extract of the total adventitious root saponins of ginseng with the concentration of 3.0mg/mL, oscillating in a shaking table at 45 ℃ and 110r/min in a dark place for 24h, taking the supernatant, measuring the concentration of the total saponins, and calculating the adsorption rate and the adsorption capacity according to the following formulas respectively.
Resin adsorption amount: q (mg/g) = (C) 0 -C 1 )V/m
Resin adsorption rate: e (%) = [ (C) 0 -C 1 )/C 0 ]×100％
In the formula: q is the resin adsorption capacity (mg/g);
e is resin adsorption (%);
C 0 the total saponin concentration (mg/mL) of the sample before adsorption;
C 1 the concentration (mg/mL) of the total saponins in the supernatant liquid during adsorption equilibrium;
v is the volume of the extract (mL);
m is the resin mass (g).
3. Desorption test of different macroporous resins
Filtering 6 kinds of macroporous resin completely adsorbed, washing with distilled water, filtering out resin, sucking excessive water, adding 30mL of 70% ethanol solution as eluent into a conical flask with a plug, oscillating in a shaking table at 45 ℃ and 110r/min in a dark place for 24 hours, taking supernate to measure the concentration of total saponin, and calculating the desorption rate according to the following formula:
resin desorption rate D (%) = [ C 2 *V 2 /C 0 *V 0 ]×100％
In the formula: d is the resin desorption ratio (%)
C 0 The concentration (mg/mL) of total saponins in the sample solution before adsorption;
C 2 the total saponin concentration (mg/mL) in the eluate was used.
V 0 Volume of sample solution before adsorption (mL)
V 2 Volume of eluent (mL)
The adsorption rate and desorption rate of the macroporous resin to the total saponins of adventitious roots of ginseng are taken as investigation indexes, and the appropriate macroporous resin is comprehensively screened out.
4. Test results
The results of screening 6 types of macroporous adsorbent resins using the adsorption and desorption rates of macroporous adsorbent resins as the index are shown in table 7.
TABLE 7 comparison of adsorption and desorption effects of macroporous resins of different types
| Type of resin | Adsorption Capacity (mg/g) | Adsorption Rate (%) | Desorption ratio (%) |
| HPD-100 | 65.94 | 54.95 | 82.22 |
| HPD-400 | 94.99 | 79.16 | 83.49 |
| DM-130 | 90.53 | 75.44 | 79.98 |
| D-101 | 97.96 | 81.63 | 85.53 |
| X-5 | 83.34 | 69.45 | 75.41 |
| AB-8 | 96.46 | 80.38 | 89.13 |
The table shows that the adsorption rate of the D-101 type adsorption resin is the highest and reaches 81.63%, and the desorption rate of the AB-8 type adsorption resin is the highest and reaches 89.13%, which indicates that under the same conditions, the different types of macroporous adsorption resins have different adsorption degrees and have selectivity on adsorption of total saponins of adventitious roots of ginseng. The higher the adsorption rate, the better the adsorption degree of the resin, and the more suitable the resin is selected as the adsorbent. The macroporous resin favorable for separating and purifying the total ginsenoside is selected, so that the macroporous resin not only needs to have stronger adsorption performance on the saponin, but also needs to consider the desorption capacity on the saponin, and therefore, the macroporous resin is the best choice for separating and purifying the total ginsenoside when the adsorption and desorption performance on the total saponin is stronger. As can be seen from the screening results, the D-101 type and AB-8 type resins have higher adsorption and desorption performances, so the invention determines the better macroporous resin types to be the D-101 type and AB-8 type macroporous adsorption resins.
(II) influence of feeding concentration on adsorption effect of macroporous adsorption resin
1. Test method
Accurately weighing 15g of each pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding crude extract of total ginsenosides with concentrations of 1, 2, 3, 4, 5, 6 and 7mg/mL, controlling the sample loading flow rate to be 3mL/min and the material loading volume to be 50mL, collecting sample liquid after passing the column, measuring the absorbance values before and after the sample liquid flows out, calculating the adsorption capacity and the adsorption rate of the D-101 type macroporous adsorption resin on the total ginsenosides with different sample loading mass concentrations according to an adsorption capacity and adsorption rate formula, and determining the optimal sample loading liquid mass concentration.
2. Test results
The influence of the feed concentration on the adsorption effect of the macroporous adsorbent resin is shown in Table 8
TABLE 8 influence of the feed concentration on the adsorption effect of macroporous adsorbent resins
As shown in the table above, when the mass concentration of the total saponins in the material loading is increased from 1mg/mL to 4mg/mL, the adsorption amount of the D-101 resin is gradually increased, the adsorption rate is gradually reduced, but the reduction is not obvious, and the adsorption capacity of the resin at this stage is not fully applied. After the feeding concentration exceeds 4mg/mL, the adsorption quantity and the adsorption rate are reduced along with the increase of the feeding concentration, and the analysis reason is probably that a large amount of saponin compounds are lost and wasted due to the fact that resin adsorption is saturated. Therefore, the feeding concentration determined by the invention is 2-6mg/mL, and the more preferable feeding concentration is 3-5mg/mL.
(III) influence of feeding flow velocity on adsorption effect of macroporous adsorption resin
1. Test method
Accurately weighing 15g of each pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding crude extract of total ginsenosides with the concentration of 4mg/mL, controlling the sample loading flow rate to be 1, 2, 3, 4, 5 and 6mL/min respectively, and the loading volume to be 50mL, collecting the sample liquid after passing the column, measuring the absorbance values before and after the sample liquid flows out, calculating the adsorption rate of the D-101 type macroporous adsorption resin on the total ginsenosides at different loading flow rates according to an adsorption rate formula, and determining the optimal sample loading flow rate.
2. Test results
The influence of the feed flow rate on the adsorption effect of the macroporous adsorbent resin is shown in Table 9
TABLE 9 influence of the feed concentration on the adsorption effect of macroporous adsorbent resins
As shown in the above table, the adsorption rate of the resin decreased with the increase of the flow rate, and the analysis reason may be that the saponin compounds in the sample liquid could not contact with the resin sufficiently due to the excessive flow rate, and could flow out of the resin column without being adsorbed. When the flow rate is low, the adsorption rate of the total saponin is high, but the working time is long and the efficiency is low. Therefore, the feeding flow rate determined by the invention is 2-5mL/min, and the more preferable feeding flow rate is 3-4mL/min.
Influence of volume of the upper part on adsorption effect of macroporous adsorption resin
1. Test method
Accurately weighing 15g of pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding crude extract of total adventitious root saponins of ginseng with the concentration of 4.0mg/mL, controlling the sample loading flow rate to be 3mL/min, collecting sample liquid after the column is passed, collecting the sample liquid after the column is passed, measuring the absorbance values before and after the sample liquid flows out, calculating the adsorption rate of the D-101 type macroporous resin on the total adventitious root saponins of ginseng at different loading flow rates according to an adsorption rate formula, and stopping sample injection when the adsorption rate is lower than 70%. The maximum sample loading volume was determined.
2. Test results
The influence of the upper volume on the adsorption effect of the macroporous adsorbent resin is shown in Table 10
TABLE 10 influence of the feed concentration on the adsorption effect of macroporous adsorbent resins
As shown in the table, the adsorption rate of the resin decreases with the increase of the loading volume, and the analysis reason is probably that the adsorption of the resin to the total saponins of the ginseng adventitious roots gradually approaches to saturation. Therefore, the feeding volume determined by the invention is 40-60mL, and the more preferable feeding volume is 50-60mL.
(V) influence of ethanol concentration of impurity removal liquid on elution effect
1. Test method
Accurately weighing 15g of each pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding the crude extract of the total saponins of adventitious roots of ginseng with the concentration of 4mg/mL, and controlling the flow rate of sample loading to be 3mL/min and the volume of the sample loading to be 50mL. And then washing the macroporous resin column after adsorption saturation with 80mL of purified water, eluting with 80mL of 20%, 30%, 35%, 45%, 50% and 60% ethanol aqueous solution (impurity-removed solution), collecting the sample solution after passing through the column, measuring the absorbance values before and after the sample solution flows out, calculating the desorption rate of the D-101 type macroporous adsorption resin on the total saponins of adventitious roots of ginseng under different concentrations of the impurity-removed ethanol solution according to a desorption rate formula, and determining the optimal concentration of the impurity-removed ethanol solution.
2. Test results
The influence of the ethanol concentration of the impurity-removed solution on the elution effect is shown in Table 11
TABLE 11 influence of ethanol concentration of impurity-removed solution on elution Effect
The purpose of this step is to remove non-total saponin components adsorbed on the chromatographic column. As shown in the table, the desorption rate of the resin is increased along with the increase of the concentration of the impurity-removed ethanol, namely, the saponin components are eluted in the impurity-removing process, so that the loss is caused. Therefore, the concentration of the impurity-removed ethanol solution is determined to be 30-50%, and the concentration of the more preferable impurity-removed ethanol solution is 35-45%.
(VI) Effect of elution flow Rate on elution Effect
1. Test method
Accurately weighing 15g of each pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding a crude extract of the total saponins of adventitious roots of ginseng with the concentration of 4mg/mL, and controlling the flow rate of sample loading to be 3mL/min and the volume of the sample loading to be 50mL. Then washing the macroporous resin column after adsorption saturation with 80mL of purified water, eluting with 80mL of 40% ethanol water solution, collecting none of the above solutions, eluting with 70% ethanol solution (eluent) at flow rates of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5mL/min, collecting the sample solution after passing through the column, measuring absorbance value of the sample solution, calculating the desorption rate of D-101 type macroporous adsorption resin on total ginsenosides with different elution flow rates according to a desorption rate formula, and determining the optimal elution flow rate.
2. Test results
The effect of elution flow rate on elution effect is shown in Table 12
TABLE 12 Effect of elution flow Rate on elution Effect
As shown in the above table, the desorption rate of total saponins gradually decreased as the flow rate of the eluent was increased. When the flow rate is 0.5-2.5mL/min, the desorption rate is slowly reduced, but the desorption rate is still kept above 85%. When the flow rate is 2.5-3.5mL/min, the desorption rate is obviously reduced; when the flow rate of the sample liquid is 3.0mL/min, the desorption rate can still be kept above 80%. When the flow rate of the eluent is too large, the eluent cannot fully react with the saponin compounds on the resin, so that the displacement efficiency of the saponin compounds from the adsorption sites of the macroporous resin is low. When the flow rate is low, the total saponin desorption rate is high, but the working time is long and the efficiency is low. Therefore, the invention determines the elution flow rate to be 1.0-3.0mL/min, and more preferably the elution flow rate to be 1.5-2.5mL/min.
(VII) influence of ethanol concentration of eluent on elution effect
1. Test method
Accurately weighing 15g of each pretreated D-101 type macroporous resin, filling the resin into a column by a wet method, adding a crude extract of the total saponins of adventitious roots of ginseng with the concentration of 4mg/mL, and controlling the flow rate of sample loading to be 3mL/min and the volume of the sample loading to be 50mL. Then washing the macroporous resin column after adsorption saturation with 80mL of purified water, eluting with 80mL of 40% ethanol water, collecting all the solutions, eluting with 40%, 50%, 60%, 70%, 80%, 90%, 100% ethanol solution (eluent) at the flow rate of 2.0mL/min, collecting sample solution after passing through the column, measuring the absorbance value of the sample solution, calculating the desorption rate of D-101 type macroporous adsorption resin on the ginseng adventitious root total saponins at different elution flow rates according to a desorption rate formula, and determining the optimal concentration of the eluted ethanol solution.
2. Test results
The influence of the ethanol concentration of the eluent on the elution effect is shown in Table 13
TABLE 13 influence of ethanol concentration of eluent on elution Effect
After the ginseng adventitious root total saponins are adsorbed by macroporous adsorption resin, the saponins compounds are washed off the resin by adopting eluent, thus achieving the purpose of separation and purification. Saponins compounds are generally applied to food and medicine health care industries, and the safety problem is considered. In the experiment, ethanol is selected as a desorbent, the influence of the concentration of the ethanol on the desorption rate of the total saponin is examined, and the result is shown in the table.
When the concentration of the ethanol solution is 40%, the desorption rate of the total saponin is 67.28%. As the concentration of the ethanol solution increases, the desorption rate increases significantly. The saponin compounds can generate hydrogen bond action with the macroporous resin to be adsorbed on the macroporous resin, so the saponin compounds are not easy to be washed off by water. When the concentration of the ethanol solution is low, only a small amount of saponin compounds can be eluted. The total saponin desorption rate is obviously increased along with the increase of the concentration of the ethanol solution. When the concentration of the ethanol is 70%, the elution effect of the total saponin is the best, and the desorption rate is 90.62%. When the concentration of the ethanol solution exceeds 70%, the desorption rate of the total saponin is reduced, and when the concentration of the ethanol is 100%, the desorption rate is reduced to 79.57%. Possibly due to excessive concentrations of ethanol solution desorbing some of the other substances tightly bound to the column. Therefore, the concentration of the ethanol eluting solution is determined to be 50% -90%, and the concentration of the more preferable ethanol eluting solution is determined to be 60% -80%.
Test example 4
In this test example, the contents of different monomeric ginsenosides in the ginseng adventitious root powder and the artificially planted ginseng powder were measured with slight modification according to the method in "determination of ginsenoside NY/T1842".
1 analytical procedure
1.1 sample extraction
Accurately weighing 2g (accurate to 0.001 g) of each of the pulverized powder of adventitious root of Ginseng radix and the artificially planted Ginseng radix powder, weighing 3 parts of each sample as parallel sample, adding 100mL of diethyl ether into a Soxhlet extractor, extracting for 1h, discarding diethyl ether, volatilizing diethyl ether in the residue, and adding methanol for reflux distillation for 8h.
1.2 sample purification
1.2.1SPE C 18 Pretreatment of columns
First, 20mL deionized water was used to elute SPE C 18 The column was then activated with 20mL of methanol and equilibrated with 20mL of deionized water. And (5) loading the sample when the water is close to the column sieve plate.
1.2.2 treatment of the extract
Concentrating the extract at 60 ℃ in a water bath by a rotary evaporator under reduced pressure to be nearly dry, blowing dry by nitrogen, and adding 4mL of deionized water to fully rock. 2mL of the solution is injected into the activated SPE C 18 In the column, when the liquid level is close to the sieve plate of the column, pouring 10mL of deionized water to drip SPE C 18 Column, discarding effluent liquid, adding 25mL70% ethanol solution to elute SPE C when the liquid level of eluate is near the sieve plate of column 18 And (4) performing column chromatography, collecting eluent in a 50mL graduated test tube, blowing nitrogen to below 25mL, metering the volume to 25mL by using methanol, uniformly mixing, filtering by using a 0.2um filter membrane, and detecting.
1.3 determination of
1.3.1 Instrument reference conditions
1.3.1.1 column: c 18 (4.6 mm X300mm X0.5 μm) or equivalent.
1.3.1.2 mobile phase: acetonitrile (a) ten 0.05% phosphoric acid aqueous solution (B).
1.3.1.3 column temperature: at 30 ℃.
1.3.1.4 flow rate: 1.3mL/min.
1.3.1.5 detection wavelength: 202nm.
1.3.1.6 sample size: 10 μ L.
1.3.1.7 gradient elution procedure see table 14.
TABLE 14 gradient elution procedure
1.3.2 drawing of Standard Curve
Weighing 0.2g of ginsenoside Rg1, rg3, re, rb1, rb2 and Rc (accurate to 0.001g and content of 98%) standard substances one by one, placing in a 100mL volumetric flask, adding methanol to constant volume, preparing into a mixed standard solution with mass concentration of 2.0g/L, storing in a refrigerator below-18 ℃, and keeping the effective period for 6 months. The mixed saponin standard working solution is used for analysis according to a gradient elution program. Accurately sucking 0mL, 2mL, 4mL, 6mL, 8mL and 10mL of mixed saponin standard solutions, respectively placing the mixed saponin standard solutions in 10mL volumetric flasks, diluting the mixed saponin standard solutions to a scale with methanol, accurately sucking 10 mu L of the standard solutions in the volumetric flasks, respectively injecting the standard solutions into a liquid chromatograph, and recording peak areas. And drawing a standard curve according to the mass concentration of each saponin sample injection to the peak area of each saponin sample injection.
1.3.3 sample determination
Accurately sucking 10 mu L of sample solution to be tested, injecting the sample solution into a liquid chromatograph, determining the nature of the sample solution by retention time, and comparing the peak area of the solution to be tested with the peak area of the standard solution for quantification.
1.3.4 blank experiment
The parallel procedure was performed using exactly the same assay procedure as the sample except that the sample was not weighed.
2 calculation of results
The content of ginsenoside in the sample is expressed by mass fraction omega, and the unit is milligram/gram (mg/g), and is calculated according to the formula (1):
in the formula: m is 1 -the mass of a certain ginsenoside in a sample is in milligrams (mg);
m 2 -mass of the sample in grams (g);
V 1 sample volume in milliliters (mL);
V 2 volumetric volume of the sample in milliliters (mL).
3 precision
The absolute difference between two independent test results obtained under repetitive conditions for each ginsenoside is not more than 5% of the arithmetic mean of the two measurements.
4 results of the test
The results of the correlation measurements are shown in the following table.
TABLE 15 ginsenoside content test results
The data in the table show that the content of each ginsenoside monomer in the ginseng adventitious root freeze-dried powder is superior to the corresponding content index in the artificially planted ginseng powder, wherein the content of ginsenoside Rg3 which is a rare saponin in the ginseng adventitious root freeze-dried powder is far higher than that of the artificially planted ginseng powder, and the difference is very obvious.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for preparing total saponins of adventitious roots of ginseng is characterized by comprising the following steps:
(1) Wall breaking: pulverizing dried adventitious root of Ginseng radix, soaking in water, and breaking cell wall with ultrahigh pressure homogenizing equipment;
(2) Extraction: reflux-extracting the wall-broken material liquid, and concentrating;
(3) And (3) purification: adsorbing the concentrated solution by macroporous adsorption resin, sequentially washing the macroporous resin with water and impurity-removing solution, eluting with eluent, and collecting the eluent containing total saponins;
(4) And (3) drying: evaporating and concentrating the eluent, and drying to obtain the high-purity total ginsenosides.
2. The preparation method according to claim 1, wherein in the step (1), when the ginseng tissue culture adventitious roots are soaked in water, the ratio of the ginseng adventitious roots to the water is 1;
preferably, the feed-water ratio is 1;
preferably, the soaking time is 8-16h, and more preferably, the soaking time is 12h.
3. The preparation method according to claim 1 or 2, wherein in the step (1), when the cell wall breaking treatment is performed by using an ultrahigh pressure homogenizing device, the wall breaking pressure is 100-150MPa, the flow rate is 100-200mL/min, and the treatment times are 2-4 times;
preferably, the wall breaking pressure is 130-140MPa, the flow rate is 120-160mL/min, and the treatment times are 3-4 times.
4. The preparation method according to any one of claims 1 to 3, wherein in the step (2), the wall-broken feed liquid is subjected to reflux extraction for 2 to 4 times, the filtrate is collected by filtration after each extraction, the filter residue is subjected to subsequent extraction by adding water, the amount of the added water is the same as that of the primary liquid, the reflux extraction time is 1 to 3 hours each time, and the extraction temperature is 70 to 100 ℃; after extraction, combining the extracting solutions and concentrating;
preferably, reflux extracting for 2-3 times, each time for 1.5-2.5h, at 80-100 deg.C;
preferably, the concentration of total ginsenoside in the concentrated extractive solution is 1-5mg/mL.
5. The preparation method according to any one of claims 1 to 4, wherein in the step (3), the model of the macroporous adsorption resin is selected from one of HPD-100, HPD-400, DM-130, D101, X-5 and AB-8;
preferably, the types of the macroporous adsorption resin are as follows: d101 or AB-8.
6. The preparation method according to any one of claims 1 to 5, wherein in the step (3), when the concentrate is loaded on the macroporous adsorbent resin, the loading concentration is 2 to 6mg/mL, the loading flow rate is 2 to 5mL/min, and the loading volume is 40 to 60mL;
preferably, the feeding concentration is 3-5mg/mL, the feeding flow rate is 3-4mL/min, and the feeding volume is 50-60mL.
7. The preparation method according to any one of claims 1 to 6, wherein in the step (3), the impurity removal solution is an ethanol water solution with a volume percentage of 30 to 50 percent; the eluent is ethanol water solution with the volume percentage of 50-90%;
preferably, the impurity removing liquid is 35-45% by volume of ethanol water solution; the eluent is 60-80% ethanol water solution by volume percentage.
8. The production method according to any one of claims 1 to 7, wherein in the step (3), elution is performed with an eluent at a flow rate of 1.0 to 3.0 mL/min;
preferably, elution is carried out at a flow rate of 1.5 to 2.5mL/min.
9. The preparation method according to any one of claims 1 to 8, wherein in step (4), the eluate is subjected to rotary evaporation to recover ethanol to obtain a concentrated solution, and then the concentrated solution is dried at 50 to 100 ℃ to obtain high-purity ginseng tissue culture adventitious root total saponins with a water content of less than 5%;
preferably, the drying equipment is selected from one of a hot air circulation oven, a vacuum oven and an electric heating forced air drying oven.
10. The preparation method according to any one of claims 1 to 9, wherein the purity of the obtained total saponins of adventitious roots in tissue culture of ginseng is 30 to 80%;
preferably, the purity of the obtained total saponins of the adventitious roots of the ginseng tissue culture is 65-80%.
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Citations (2)
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| CN1869054A (en) * | 2006-06-21 | 2006-11-29 | 海南亚洲制药有限公司 | Preparation method of ginseng group saponine |
| CN112876528A (en) * | 2021-01-15 | 2021-06-01 | 吉林农业大学 | Method for obtaining ginsenoside |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1869054A (en) * | 2006-06-21 | 2006-11-29 | 海南亚洲制药有限公司 | Preparation method of ginseng group saponine |
| CN112876528A (en) * | 2021-01-15 | 2021-06-01 | 吉林农业大学 | Method for obtaining ginsenoside |
Non-Patent Citations (5)
| Title |
|---|
| 张儒等: "响应面法优化人参不定根中总皂苷的提取工艺", 天然产物研究与开发, vol. 27, no. 4, pages 726 - 731 * |
| 王中立;欧阳柳凤;张蔷;王昕;赵玉男;: "人参皂苷的提取及总皂苷纯化工艺的研究进展", 世界科学技术-中医药现代化, no. 09, pages 1596 - 1601 * |
| 王辉;何伟;任军;: "大孔吸附树脂纯化人参皂苷类成分的工艺研究", 中国药房, no. 24 * |
| 袁媛;: "黑参中人参皂苷的大孔吸附树脂纯化工艺研究", 亚太传统医药, no. 05, pages 41 - 43 * |
| 魏永春;张卫;李冲;: "皂苷的提取与纯化工艺研究进展", 广东化工, no. 11 * |
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