CN111606899B - Method for preparing dehydrated safflower yellow B in safflower by high-speed counter-current chromatography - Google Patents
Method for preparing dehydrated safflower yellow B in safflower by high-speed counter-current chromatography Download PDFInfo
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Abstract
A method for preparing dehydrated safflower yellow B in safflower by high-speed countercurrent chromatography belongs to the technical field of compound separation and preparation. The method comprises the following steps: (1) preparing a high-speed counter-current two-phase solvent system; (2) preparing a sample solution; (3) Separating dehydrated safflower yellow B in the safflower by high-speed counter-current chromatography, and simultaneously carrying out single factor investigation and response surface optimization to obtain optimal process conditions; (4) And (4) collecting fractions of the target peak, and concentrating by adopting a freeze-drying method to obtain target product powder. The invention adopts HSCCC method to separate and prepare target product, combines response surface analysis method to optimize process, and establishes a high-efficiency separation and preparation method of dehydrated safflower yellow B. The final result of the method is that the stationary phase retention rate is 54%, the stationary phase loss volume after sample injection is 25mL, the total time is 225min, the purity is 98.32%, the yield is 66044.6, and the comprehensive score of AHSYB is finally converted to 0.687 by an entropy weight method.
Description
Technical Field
The invention belongs to the technical field of compound separation and preparation, and particularly relates to a method for preparing dehydrated safflower yellow B in safflower by high-speed counter-current chromatography.
Background
Carthami flos (Carthamus tinctorius L.) of CompositaeCarthamustinctoriusL. the dried tubular flower is a good medicine for promoting blood circulation to remove blood stasis, removing blood stasis and relieving pain. The safflower contains many compounds, and the water-soluble quinoid chalcone compound safflower yellow is the main active component in safflower, and mainly comprises hydroxysafflower yellow A (HSYA) and dehydrated safflower yellow B (AHSYB). HSYA is an important drug substance in safflower, and is clinically used for treating cardiovascular diseases. The content and the antioxidant activity of AHSYB in safflower are only second to HSYA, the pharmacological action is not small but the related AHSYB separation and preparation method and the pharmacological activityVery few studies have been made. In the earlier stage of the subject group, AHSYB is prepared by separation by preparative high performance liquid chromatography, the product purity is high, but the preparation amount at one time is small, a chromatographic column is easy to block, and the time consumption is long. If the pharmacological effect of AHSYB is further researched, an efficient and rapid separation preparation method is also needed to be established. The high-speed countercurrent chromatography (HSCCC) method is a liquid-liquid distribution chromatographic technique, can effectively avoid the conditions of irreversible adsorption, denaturation and inactivation and the like of a sample due to no adoption of a solid as a stationary phase, is simple and convenient to operate and large in preparation amount, can sample a sample of 10 mL at one time, and is a gradually developed and mature separation means. At present, no report of AHSYB separation and purification by HSCCC method exists.
Disclosure of Invention
In view of the above problems in the prior art, it is an object of the present invention to provide a method for preparing dehydrated safflower yellow B from safflower by high-speed countercurrent chromatography. The method takes a safflower extract purified by macroporous adsorption resin as a sample solution, then uses an HSCCC method to separate and prepare a target product, and combines a response surface analysis method to carry out process optimization, thereby establishing an efficient AHSYB separation and preparation method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing dehydrated safflower yellow B in safflower by high-speed countercurrent chromatography is characterized by comprising the following steps:
(1) Preparing a high-speed counter-current two-phase solvent system: selecting a two-phase solvent system consisting of water, n-butyl alcohol, methyl tert-butyl ether, acetonitrile and trifluoroacetic acid, sequentially adding into a separating funnel, fully shaking, standing for 12h for layering, separating an upper phase and a lower phase, wherein the upper phase is used as a stationary phase, the lower phase is used as a mobile phase, and respectively performing ultrasonic degassing for 20 min for later use;
(2) Preparation of sample solution: accurately weighing 50-250mg of freeze-dried powder of the safflower water extract, and mixing and dissolving 5mL of each of the upper phase and the lower phase obtained in the step (1) for later use;
(3) Separating dehydrated safflower yellow B in safflower by high-speed counter-current chromatography: adopting a head-tail washing mold release mode with the speed of 20 mL. Min -1 The stationary phase obtained in the step (1) is pumped by the flow velocityEntering the main machine, after the liquid flows out from the outlet, starting the main machine to rotate positively at the main machine rotating speed of 750-950 r. Min -1 The temperature of the constant temperature circulating water pump is 5-45 ℃, and the flow rate of the mobile phase is 1.5-6 mL. Min after the chromatographic baseline is stable -1 Pumping, starting a detector with fixed wavelength of 405nm to collect data when the instrument reaches dynamic balance, injecting the sample solution obtained in the step (2) through an injection valve after the baseline is balanced, and simultaneously carrying out single-factor and response surface optimization to obtain the optimal process condition;
(4) And (4) collecting fractions of the target peak, and concentrating by adopting a freeze-drying method to obtain target product powder.
The method for preparing the dehydrated safflower yellow B in the safflower by the high-speed countercurrent chromatography is characterized in that the volume ratio of water-n-butyl alcohol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid in the step (1) is 6:3:3:0.5:0.005.
the method for preparing the dehydrated safflower yellow B in the safflower by the high-speed counter-current chromatography is characterized in that the preparation method of the freeze-dried powder of the safflower water extract in the step (2) comprises the following steps: pulverizing Carthami flos, precisely weighing 50g, placing into a conical flask with a plug, adding ultrapure water according to a material-liquid ratio of 1 -1 Passing through macroporous adsorbent resin, and loading the sample with volume flow rate of 2.5 BV. H -1 Immediately after the sample is loaded, the sample is eluted by 75% ethanol, and the volume flow rate is 2.0 BV h -1 Collecting eluate, removing ethanol under reduced pressure, extracting with ethyl acetate at a volume ratio of 1:1 for 4 times, mixing water phases, freeze drying to obtain lyophilized powder of Carthami flos water extract, and storing at 4 deg.C in dark place.
The method for preparing the dehydrated safflower yellow B in the safflower by the high-speed countercurrent chromatography is characterized in that 100mg of safflower water extract freeze-dried powder is weighed in the step (2), namely the sample solution is 10mg/mL.
The method for preparing the dehydrated safflower yellow B in the safflower by the high-speed countercurrent chromatography is characterized in thatThe optimal process conditions in the step (3) are as follows: the rotating speed of the main engine is 853 r. Min -1 Inflow rate of the mobile phase pump is 2.3 mL. Min -1 And the temperature of the constant-temperature circulating water pump is 36 ℃.
The invention takes safflower extract purified by macroporous absorbent resin as sample solution, then uses HSCCC method to separate and prepare target product, combines response surface analysis method to carry out process optimization, and establishes a high-efficiency separation and preparation method of dehydrated safflower yellow B. According to the final result of the method, the stationary phase retention rate j1=54%, the stationary phase loss volume j2=25mL after sample injection, the total duration j3=225min, the purity j4=98.32%, the yield j5=66044.6, and the comprehensive score W =0.687 of AHSYB separated by the HSCCC method is finally converted by the entropy weight method.
Drawings
FIG. 1 shows the results of a solvent system screening for the isolation of AHSYB (i.e., dehydrated safflower yellow B from safflower), wherein (1) water: n-butanol: methyl tert-butyl ether: acetonitrile: trifluoroacetic acid (6; (2) water: n-butanol: methyl tert-butyl ether: acetonitrile: trifluoroacetic acid (6; (3) water: n-butanol: methyl tert-butyl ether: acetonitrile: trifluoroacetic acid (6; (4) water: n-butanol: methyl tert-butyl ether: acetonitrile: trifluoroacetic acid (6; (5) water: n-butanol: methyl tert-butyl ether: acetonitrile: trifluoroacetic acid (6;
fig. 2 is a chromatogram of the HSCCC method for isolating AHSYB, wherein the solvent system water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (6;
FIG. 3 is an HPLC chromatogram of AHSYB;
FIG. 4 is a full wavelength scanning absorption curve;
FIG. 5 is a graph of the effect of the rotational speed of a high speed countercurrent chromatograph on the separation process;
FIG. 6 is a graph of the effect of mobile phase flow rate on the separation process;
FIG. 7 is a graph of the effect of constant temperature water pump temperature on the separation process;
FIG. 8 is a graph of the effect of sample size on the separation process;
FIG. 9 is a graph of the effect of rotational speed and flow rate on the AHSYB composite score W;
FIG. 10 is a graph of the effect of speed and temperature on the AHSYB composite score W;
figure 11 is a graph of the effect of flow rate and temperature on the AHSYB composite score W.
Detailed Description
The invention will be further illustrated by the following examples and figures.
The embodiment is as follows:
1. establishment of method for separating AHSYB from safflower by high-speed counter-current chromatography
1.1 preparation of safflower Water extract powder
Pulverizing Carthami flos, and sieving with 20 mesh sieve. Precisely weighing 50g safflower coarse powder in a conical bottle with a plug, and mixing the raw materials according to a material-liquid ratio of 1:22 (w/v) adding ultrapure water, warm-bathing at 73 deg.C for 40min, filtering, and extracting the residue with the same method for 2 times. Mixing the filtrates, concentrating under reduced pressure to 20mL, adding ethanol to 80% volume, precipitating with ethanol to remove impurities such as polysaccharide, collecting supernatant, distilling under reduced pressure until no ethanol remains, adding ultrapure water to adjust the mass concentration to 0.06 g. ML -1 . Passing through HPD-300 macroporous adsorbent resin (resin: crude drug = 3:1), and the sample loading volume flow is 2.5 BV. H -1 Immediately after the sample loading, the sample is eluted by 75% ethanol, and the volume flow rate is 2.0 BV h -1 Then collecting the eluent, removing ethanol under reduced pressure, and mixing the concentrated solution with a mixed solution of 1:1, extracting for 4 times by adding ethyl acetate, combining water phases, freeze-drying the water phases into powder, and storing the powder at 4 ℃ in the dark for later use.
1.2 screening of high-speed countercurrent solvent systems
The selection of a suitable solvent system was carried out according to rule 3 in combination with the following method, with reference to the gold rule selected by doctor Ito high-speed countercurrent chromatography conditions.
And (3) preparing the determined solvent system into a separating funnel according to a determined proportion, adding a small amount of safflower water extract powder after the solvent system is balanced, and standing for a moment to ensure that two phases are layered. Precisely sucking the upper phase and the lower phase of 1 mL respectively, transferring the upper phase and the lower phase into a small beaker, and freeze-drying the small beaker in a freeze dryer. Dissolving the freeze-dried powder with 50% methanol in an equivalent amount respectively, filtering with a 0.22 mu m filter membrane, performing High Performance Liquid Chromatography (HPLC) sample injection analysis, and calculating the ratio of the corresponding peak areas of the target compound in the two phases, namelyDistribution coefficient K value: k = A Upper phase /A The lower phase.
After a proper solvent system is screened out, the stationary phase retention rate of the solvent system needs to be measured, and the measurement method is as follows: the high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase. First, the temperature is controlled to be 20 mL. Min -1 Pumping the stationary phase into a separation column at the flow rate, and calculating the volume of the pumped stationary phase to obtain the total volume V of the pipeline General assembly . When the outlet of the main machine flows out of the stationary phase, the stationary phase is indicated to be filled in the separation column. The rotating speed of the chromatographic instrument is adjusted to 900 r. Min -1 And simultaneously, a constant-temperature circulating water pump is started to adjust the temperature to 25 ℃. After the temperature is stabilized, the temperature is controlled to be 1.5 mL. Min -1 Is pumped into the mobile phase, the volume of the stationary phase pushed out by the mobile phase is V Go out . When the mobile phase flows out of the outlet of the main machine and the volume of the fixed phase does not change any more, the two-phase solvent reaches the dynamic equilibrium. The stationary phase retention rate, i.e., = ((V) stationary phase retention rate was calculated General assembly -V Go out ）/V General assembly ) X 100%. If the stationary phase retention value is too low, the solvent system needs to be screened again.
1.3 high-speed countercurrent two-phase solvent System and preparation of sample solutions
(1) Preparing a high-speed countercurrent two-phase solvent system: selecting an optimal solvent system, namely a two-phase solvent system consisting of water, n-butyl alcohol, methyl tert-butyl ether, acetonitrile and trifluoroacetic acid, sequentially adding the optimal solvent system into a separating funnel according to a determined proportion, fully shaking, and standing for 12h to separate layers. Separating upper and lower phases, the upper phase as stationary phase and the lower phase as mobile phase, and ultrasonic degassing the two phases for 20 min.
(2) Preparation of sample solution: accurately weighing lyophilized powder of Carthami flos water extract 100mg, and dissolving with equal amount of upper and lower phase liquid 5mL respectively for use.
1.4 high-speed countercurrent chromatography for separating AHSYB from safflower
The high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase. At 20 mL. Min -1 The stationary phase is pumped into the main machine at the flow rate, and after the liquid flows out from the outlet, the stationary phase is shown to be alreadyFilling the entire pipeline. Starting the main machine to rotate forward, and adjusting the rotating speed of the main machine to 900 r-min -1 And simultaneously adjusting the temperature of the constant-temperature circulating water pump to be 25 ℃. After the temperature is stabilized, the temperature is controlled to be 1.5 mL. Min -1 The flow rate of the sample is pumped into a mobile phase, when the instrument reaches dynamic balance, a detector with fixed wavelength of 405nm is started to collect data, and after a base line is balanced, sample injection is carried out through a sample injection valve.
1.5 Collection and treatment of the target product
Collecting the fraction of the target peak, and concentrating the target product by freeze drying.
1.6 sample analysis
(1) HPLC method for determining sample purity: the lyophilized target product powder was dissolved in 50% chromatographic methanol, passed through a 0.22 μm organic filter for HPLC analysis, and the purity of the compound was determined by area normalization.
(2) HPLC-MS analysis of samples: the lyophilized target product powder was dissolved in 50% chromatographic methanol and subjected to HPLC-MS analysis through a 0.22 μm organic filter.
2. Process optimization for separating AHSYB in safflower by high-speed countercurrent chromatography
2.1 Single factor experiment of separation Process
2.1.1 Determination of evaluation index
The evaluation indexes of the single-factor experiment in the experiment are the stationary phase retention rate (j 1), the stationary phase loss volume (j 2) after sample injection, the total duration (j 3), the purity (j 4) and the yield (j 5), and the comprehensive evaluation value W is obtained as the evaluation basis by endowing corresponding weight coefficients to all indexes by using an entropy weight method.
Since the HPLC peak area and yield of the AHSYB component in safflower are directly proportional, the separation process was optimized in the following experiment using the peak area as an index j5 instead of the yield.
2.1.2 calculating index weights and composite scores
Endowing each index with corresponding weight coefficient (w) by an entropy weight method j ) Where j =1,2,3. The entropy weight method calculation formula comprises the following steps:
(1) the negative indicators are processed by reciprocal, and then are standardized:
for convenience of expression, this indicator data x 'after normalization' ij Is still marked as x ij 。
(2) Calculating the proportion p of the ith scheme index value under the jth index ij ：
(3) Calculating the entropy e of the jth index j ：
Where the constant k is related to the number of samples m in the system, in this case
Then, then
。
(4) Calculating utility value of index
，d j The larger the index value is, the larger the weight of the index value is, and then the weight of the j-th index is:
(5) calculating a composite score W for each sample, where x ij For normalized data:
2.1.3 Investigation of high speed counter current chromatograph rotation speed
The high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase of a solvent system is used as a stationary phase, and the lower phase is used as a mobile phase. First, the temperature is controlled to be 20 mL. Min -1 Pumping the stationary phase into an empty separation column at the flow rate, and recording the pumping volume when the stationary phase flows out of an outlet, namely the total volume V of the pipeline General assembly . The rotating speeds of the chromatographic instruments are respectively adjusted to 750, 800, 850, 900, 950 r. Min -1 The temperature of the constant-temperature circulating water pump is adjusted to 25 ℃, and the value of 1.5 mL-min is calculated when the chromatographic baseline is stable -1 The mobile phase is pumped into the separation column at the flow speed, when the outlet liquid is layered and the stationary phase volume is stable, the two-phase solvent in the separation column reaches dynamic balance, and the stationary phase volume V pushed out by the mobile phase is recorded Go out . The stationary phase retention ratio (j 1) was calculated, i.e.
。
Accurately weighing Carthami flos sample powder 100mg when the chromatographic baseline is stable, dissolving with upper and lower phase liquids 5mL, and introducing sample through sample injection valve (the sample introduction amount is 10 mL, and the sample concentration is 10 mg. ML) -1 ). And (5) after sample injection, re-connecting and taking liquid at the outlet, stopping when the yellow sample solution flows out of the outlet, and recording the loss volume (j 2) of the stationary phase after sample injection. From the chromatographic peak, the components of the target peak were manually picked up and the total duration (j 3) was recorded at the end. The purity (j 4) and peak area (j 5) of AHSYB were measured by HPLC, and a comprehensive score W was calculated from the formulas 2 to 5.
2.1.4 investigation of flow velocity of mobile phase
The high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase of a solvent system is used as a stationary phase, and the lower phase is used as a mobile phase. First, the temperature is controlled to be 20 mL. Min -1 Pumping the stationary phase into an empty separation column at a flow rate of (1), and adjusting the rotation speed of the chromatographic instrument to 900 r. Min -1 The temperature of the constant-temperature circulating water pump is adjusted to 25 ℃, and when the chromatographic baseline is stable, the mobile phase is respectively controlled to be 2,3, 4, 5, 6 mL. Min -1 Is pumped into the separation column and j1 is calculated. And (5) injecting a sample when the chromatographic baseline is stable, and recording j2 and j3. AHSYB was measured by HPLC for j4, j5, and the composite score W was calculated from equations 2-5.
2.1.5 temperature investigation of constant temperature Water Pump
The high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase of a solvent system is used as a stationary phase, and the lower phase of the solvent system is used as a mobile phase. First, the temperature is controlled to be 20 mL. Min -1 Pumping the stationary phase into an empty separation column at a flow rate of (1), and adjusting the rotation speed of the chromatographic instrument to 900 r. Min -1 The temperature of the constant temperature circulating water pump is respectively adjusted to 5 ℃, 15 ℃, 25 ℃, 35 ℃ and 45 ℃, and when the chromatographic baseline is stable, the mobile phase is 1.5 mL-min -1 Is pumped into the separation column and j1 is calculated. And (5) injecting sample when the chromatographic baseline is stable, and recording j2 and j3. AHSYB was measured by HPLC for j4, j5, and the composite score W was calculated from equations 2-5.
2.1.6 investigation of sample size
The high-speed counter-current chromatograph adopts a head-tail washing demoulding mode, the upper phase of a solvent system is used as a stationary phase, and the lower phase is used as a mobile phase. First, the temperature is controlled to be 20 mL. Min -1 Pumping the stationary phase into an empty separation column at a flow rate of (1), and adjusting the rotation speed of the chromatographic instrument to 900 r. Min -1 The temperature of the constant-temperature circulating water pump is respectively adjusted to 25 ℃, and when the chromatographic baseline is stable, the mobile phase is adjusted to 3 mL-min -1 Is pumped into the separation column and j1 is calculated. Accurately weighing Carthami flos sample powder 50mg, 100mg, 150 mg, 200 mg, 250mg when the chromatographic baseline is stable, dissolving with upper and lower phase liquids 5mL, injecting through injection valve (i.e. the injection amount is 10 mL, the sample concentration is 5, 10, 15, 20, 25 mg. ML) -1 ) J2 and j3 are recorded. AHSYB was measured by HPLC for j4 and j5, and the composite score W was calculated from equations 2-5.
2.2 Response surface analysis
2.2.1 establishment and Experimental design of response surface analytical model
After single-factor experiment investigation, according to the center combination experiment principle of Box-Behnken, three factors of rotation speed (Q1), flow rate (Q2) and temperature (Q3) influencing AHSYB comprehensive score W are selected, the optimal value of the single-factor experiment result is taken as the middle level, 1 horizontal value is taken in each of the upper area and the lower area as the experimental design level of the response surface, and a three-factor three-level response surface method mathematical model is established, and is shown in Table 1.
TABLE 1 Box-Behnken Experimental factor levels
According to Design-Expert 10 software Design, factor levels are encoded as-1 (low level), 0 (medium level), 1 (high level). And (3) taking Q1, Q2 and Q3 as independent variables, taking the comprehensive score W of AHSYB as a response value, designing a scheme of 17 experimental points by using response surface software, and establishing a table, wherein a zero-point experiment is repeated for 5 times to estimate errors.
2.2.2 Establishment of regression model and significance analysis thereof
And (3) analyzing the experimental data by using Design-Expert 10 software, establishing a regression equation, and performing significance analysis.
2. Results
(one) screening of solvent systems
In the initial stage of the experiment, HPLC is adopted to measure the distribution coefficients of AHSYB and a substance HSYA with similar polarity in the safflower water extract, and the latter is measured to avoid the influence of substances with similar polarity on the separation of AHSYB. When the K value of the target component is between 0.5 and 2 and the separation degree is more than 1.5, the solvent system has high possibility of successfully separating the component. The results (tables 1-2) show that the solvent system water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid ratio is in the range of 5:2:2.5:2.5: at 0.01, AHSYB has proper K value and HSYA has high separation degree from it, so that the solution with the ratio is prepared in the initial stage of experiment for pre-experiment.
TABLE 2K values and degrees of separation in different solvents
(II) determination of solvent system proportion by high-speed counter-current chromatography
However, the partition coefficient can only theoretically assist in the screening of solvent systems, and the actual results may be far from the theoretical values due to various errors in the operation. Experimental results show that the solvent system water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (5. Therefore, a solvent system with a proper K value and proper separation degree in the table 1-2 is selected again for experiment, then the proportion of the n-butyl alcohol and the methyl tert-butyl ether is continuously adjusted, the proportion of the acetonitrile is reduced (figure 1), and the proportion of the water to the n-butyl alcohol is found to be close to 2:1 and n-butanol-methyl tert-butyl ether is 1:1, the obtained AHSYB has higher purity and better separation degree. At this time, AHSYB with higher purity cannot be separated by continuously adjusting the proportion of the solvent, and the possibility that the components in the safflower aqueous extract are complex and other substances are difficult to separate from the AHSYB is considered. As AHSYB is a hydrophilic substance, the safflower aqueous extract is extracted by petroleum ether and ethyl acetate in sequence according to the size of hydrophobicity, and the interference of hydrophobic impurities is successfully reduced. At this time, according to the solvent system water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid, the ratio is 6:3:3:0.5:0.01, AHSYB with a purity of 92% was isolated, see FIG. 2.
(III) separation result of high-speed countercurrent chromatography
Due to the poor stability of AHSYB, although HSCCC can avoid sample adsorption and denaturation, strong acid in a solvent system, namely trifluoroacetic acid, can also cause decomposition, and when the proportion of the solvent system is determined, water: trifluoroacetic acid is 6: a ratio of 0.01 adjusts the acidity, but stronger acidity was found to cause denaturation of AHSYB in the subsequent treatments, and by continuously decreasing the trifluoroacetic acid content, the water addition was finally determined: trifluoroacetic acid (6.
The isolation of the compound was performed using the solvent system water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (6. Injecting 100mg lyophilized powder of flos Carthami water extract at one time, separating within 320 min, and collecting target peak fraction by hand according to FIG. 2. By HPLC analysis, the retention time is 47.813 min (FIG. 3), the maximum absorption peak is 410 nm (FIG. 4), both retention time and spectrogram are consistent with literature reports, and AHSYB is preliminarily determined. Purity was 92% as calculated by HPLC peak area normalization.
(IV) liquid chromatography-mass spectrometry (HPLC-MS) analysis results of the sample
The sample is sent to HPLC-MS for detection, and [ M-H ] exists in a negative ion mode] - Ion peak m/z 1044.2. The relative molecular mass is 1044.2; structural formula (xvi): c 48 H 52 O 26 All consistent with literature reports, thus identifying it as AHSYB.
(V) Single factor experiment of separation Process
1. Inspection of rotation speed of high-speed countercurrent chromatograph
The results of the effect of the rotation speed of the high-speed countercurrent chromatography on the separation process are shown in FIG. 5. The comprehensive score W of AHSYB increases along with the increase of the rotating speed of the instrument, and when the rotating speed is 900 r. Min -1 When the value of W is maximum, then it is in descending trend, so the optimum rotating speed is selected to be 900 r. Min -1 。
2. Investigation of mobile phase flow velocity
The results of the effect of the mobile phase flow rate on the separation process are shown in fig. 6.W increases with the flow rate of the mobile phase, and when the flow rate of the mobile phase is 3 mL. Min -1 When W is the largest, then the flow rate decreases as the flow rate increases, so the optimum flow rate is selected to be 3 mL. Min -1 。
3. Temperature investigation of constant temperature water pump
The results of the effect of the thermostatic water pump temperature on the separation process are shown in fig. 7.W is increased along with the increase of the temperature of the constant temperature water pump, when the temperature of the constant temperature water pump is 35 ℃, the value of W is maximum, and then the value of W is in a descending trend, so that the optimal temperature is selected to be 35 ℃.
4. Investigation of sample size
The results of the effect of the sample size on the separation process are shown in FIG. 8.W can be increased along with the increase of the sample volume, and does not have a peak, but the sample volume concentration is too high, so that the problems of difficult sample introduction, pipeline blockage and the like are caused, and the sample volume investigation is not a factor for subsequent investigation.
(VI) analysis of the response surface
1. Establishment and experimental design of response surface analysis model
The 17 experimental points (see table 3) of the response surface analysis were divided into zero and factorial points, and the zero experiment was repeated five times to estimate the error, including experimental groups 1, 3, 5, 6, 17; the factorial experiment comprises experiment groups 2, 4 and 7 to 16.
TABLE 3 response surface analysis protocol and results
2. Establishment and significance analysis of regression model
According to Design-Expert software Design, performing polynomial regression analysis on experimental data of the table 3 to obtain a quadratic multiple regression equation of the W pair coding independent variables Q1, Q2 and Q3:
Z=0.58 - 0.023 Q1 - 0.091 Q2 + 0.029 Q3 + 0.014 Q1Q2 - 0.052 Q1Q3- 0.022 Q2Q3 + 0.031 Q1 2 - 0.047 Q2 2 - 0.14 Q3 2 . The model was analyzed for variance and the significance of the coefficients was examined and the results are shown in table 4. Analysis of model varianceP<0.0001, the model is proved to have good significance, so that the regression equation can be used for replacing an experimental true point to analyze and predict an experimental result. Correction decision coefficient R 2 Is 0.9775>0.80, which shows that the model can explain the change of 97.75% response value and can be used for the experimental theoretical numerical prediction of AHSYB comprehensive score.
According to the coefficient significance test result, the influence of the three factors on the AHSYB comprehensive score W is interactive and not a single linear relation, and the influence of the interactive item on the W can be analyzed by using a response surface generated by software.
TABLE 4 regression equation coefficients and their significance difference test
Note:P<0.05 indicates that there is a significant difference.
3. Influence of interactive item on response value
FIG. 9 is a graph showing the effect of rotation speed and flow rate on W, where W decreases with increasing flow rate when the rotation speed is constant; when the flow speed is constant, W is in a decreasing trend along with the increase of the rotating speed, which shows that the rotating speed and the flow speed have certain influence on W.
FIG. 10 is a graph showing the effect of rotation speed and temperature on W, and it can be seen from the graph that W increases with increasing temperature and decreases after reaching a peak when the rotation speed is constant; when the temperature is unchanged, W is reduced along with the increase of the rotating speed and is increased after reaching a peak, which shows that the rotating speed and the temperature have certain influence on W.
FIG. 11 is a graph showing the effect of flow rate and temperature on W, where W increases with increasing temperature and decreases after reaching a peak, when the flow rate is constant; when the temperature is unchanged, W is increased along with the increase of the flow velocity and is reduced after reaching a peak, which shows that the flow velocity and the temperature have certain influence on W.
(VII) response surface analytical method verification experiment
According to the design principle of a Box-Benhnken central combined experiment, taking the comprehensive score W of AHSYB as a response value, and performing a three-factor three-level response surface analysis experiment to obtain the optimal process: rotating speed of 853.317 r. Min -1 Flow rate 2.279 mL. Min -1 Temperature 36.346 ℃, under which W =0.690 is predicted.
Considering the feasibility of the experimental operation, the optimal separation process was determined as: rotating speed 853 r. Min -1 The flow rate was 2.3 mL. Min -1 A confirmatory experiment was performed at 36 ℃ (n = 3). The results were determined as stationary phase retention j1=54%, stationary phase loss volume after injection j2=25mL, total duration j3=225min, purity j4=98.32%, yield j5=66044.6. And finally converting the comprehensive score W =0.687 of AHSYB by an entropy weight method to reach 99.56% of a theoretical predicted value, and having no significant difference. Therefore, the model can reflect the condition of separating AHSYB in safflower to a certain extent by high-speed countercurrent chromatography, and has practical value.
Claims (3)
1. A method for preparing dehydrated safflower yellow B in safflower by high-speed countercurrent chromatography is characterized by comprising the following steps:
(1) Preparing a high-speed countercurrent two-phase solvent system: selecting a two-phase solvent system consisting of water-n-butyl alcohol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid, wherein the volume ratio of the water-n-butyl alcohol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid is 6:3:3:0.5:0.005, adding into a separating funnel in sequence, fully shaking, standing for 12h for layering, separating an upper phase and a lower phase, taking the upper phase as a stationary phase and the lower phase as a mobile phase, and respectively performing ultrasonic degassing for 20 min for later use;
(2) Preparation of sample solution: accurately weighing 50-250mg of freeze-dried powder of the safflower water extract, and mixing and dissolving 5mL of the upper phase and the lower phase obtained in the step (1) for later use;
the preparation method of the freeze-dried powder of the safflower water extract comprises the following steps: pulverizing Carthami flos, precisely weighing 50g, placing into a conical flask with a plug, adding ultrapure water according to a material-liquid ratio of 1 -1 Passing through macroporous adsorbent resin, and loading the sample with volume flow rate of 2.5 BV. H -1 Immediately after loading, the sample was eluted with 75% ethanol at a volume flow of 2.0 BV. H -1 Collecting eluate, removing ethanol under reduced pressure, extracting with ethyl acetate at a volume ratio of 1:1 for 4 times, mixing water phases, freeze drying to obtain lyophilized powder of Carthami flos water extract, and storing at 4 deg.C in dark place;
(3) Separating dehydrated safflower yellow B in safflower by high-speed countercurrent chromatography: adopting a head-tail washing mold release mode with the speed of 20 mL. Min -1 Pumping the stationary phase obtained in the step (1) into a host machine at the flow speed of 750-950 r. Min, and starting the host machine to rotate positively after liquid flows out of an outlet -1 The temperature of the constant temperature circulating water pump is 5-45 ℃, and the flow rate of the mobile phase is 1.5-6 mL. Min after the chromatographic baseline is stable -1 Pumping, starting a detector with a fixed wavelength of 405nm to collect data when the instrument reaches dynamic balance, injecting the sample solution obtained in the step (2) through an injection valve after baseline balance, and simultaneously carrying out single-factor and response surface optimization to obtain optimal process conditions, wherein single-factor evaluation indexes are a stationary phase retention rate, a stationary phase loss volume after injection, total duration, purity and yield;
(4) And (4) collecting fractions of the target peak, and concentrating by adopting a freeze-drying method to obtain target product powder.
2. The method for preparing dehydrated safflower yellow B in safflower according to claim 1, wherein in the step (2), 100mg of freeze-dried powder of an aqueous extract of safflower is weighed, i.e., 10mg/mL of a sample solution.
3. The process for preparing dehydrated safflower yellow B contained in safflower according to claim 1, wherein said optimum process conditions in the step (3) are: rotating speed of a main engine 853 r-min -1 Inflow rate of the mobile phase pump is 2.3 mL. Min -1 And the temperature of the constant-temperature circulating water pump is 36 ℃.
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