Method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals

Abstract

The invention relates to a method for efficiently preparing kaempferol-3-O-sophoroside from stigma croci petals, which comprises the following steps: 1) macroporous resin separation: taking a stigma croci petal extract as a raw material, loading the stigma croci petal extract on macroporous adsorption resin, sequentially eluting with distilled water and ethanol in a certain proportion, collecting an ethanol solution elution part after loading, and concentrating to obtain a concentrate A; and 2) high-speed countercurrent chromatography separation: taking an ethyl acetate-n-butyl alcohol-water two-phase solvent system, and carrying out separation and purification treatment on the concentrate A in an HSCCC (High Speed Counter Current Chromatography) instrument to obtain the kaempferol-3-O-sophoroside monomer. According to the method, the kaempferol-3-O-sophoroside compound can be simply, conveniently and efficiently obtained from the saffron petal extract, and the method is suitable for small-scale or industrial application and is beneficial to improving the additional value of saffron planting and processing.

Description

Technical Field

[0001] The invention belongs to the technical field of active ingredient extraction, and particularly relates to a method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals. Background Art

[0002] The dried red stigma of saffron is called "plant gold" because of its low yield and high price. It is mainly used as a spice, colorant and medicine. However, harvesting each kilogram of saffron stigma will produce more than 50 kilograms of petal byproducts. Therefore, making full use of the byproducts of saffron petals to create new added value for the saffron industry will also help promote the sustainable development of saffron cultivation. In recent years, phytochemical studies have shown that saffron petals are rich in flavonoids, anthocyanins, carotenoids, phenolic acids and other compounds. Among them, kaempferol-3-O-sophoroside (KS) is a typical kaempferol flavonoid, with a content of about 5% in saffron petals. It has multiple biological activities such as antioxidant, anthelmintic, myocardial endothelial cell protection, wound healing, gastritis prevention, anti-depression, etc. Therefore, obtaining KS from saffron red petals is of great significance for its further research and development in the fields of medicine, food, cosmetics, etc.

[0003] Previously, KS compounds were mainly obtained through gel column chromatography sephadex LH-20, silica gel column chromatography, and combined preparative high performance liquid chromatography. However, these processes are not suitable for large-scale application due to the use of a large amount of environmentally unfriendly solvents and cumbersome pre- and post-treatment. Macroporous resin is a low-cost material with the advantages of being eco-friendly, easy to operate, and suitable for large-scale enrichment of bioactive natural products. High-speed countercurrent chromatography (HSCCC) is a new chromatographic separation and purification technology based on liquid-liquid distribution mechanism. Since it does not use any solid support or carrier, it has the advantages of high sample recovery rate and few operating steps, and is particularly suitable for the separation of natural active monomer components. Therefore, the use of macroporous resin combined with HSCCC technology to develop a method for the efficient preparation of KS has great application potential. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for efficiently preparing high-purity kaempferol-3-O-sophoroside (KS) from saffron petal ethanol extract. The method first optimizes the separation process of a preferred macroporous resin to obtain a KS enrichment with a higher purity; then uses an ethyl acetate-n-butanol-water solvent system for HSCCC further separation and purification to obtain medium-to-high purity KS from the KS enrichment, which is specifically achieved by the following technical scheme:

[0005] A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals, comprising the following process steps:

[0006] 1) Macroporous resin separation: a KS solution of appropriate concentration is prepared with saffron petal ethanol extract as raw material, loaded on a macroporous adsorption resin with appropriate concentration and volume, eluted with distilled water and ethanol of appropriate concentration in sequence, collecting the ethanol elution portion of appropriate concentration, and concentrating under reduced pressure to obtain a concentrate A. The concentrate contains KS components with a purity greater than 50% after analysis;

[0007] 2) High-speed countercurrent chromatography separation: A two-phase solvent system of ethyl acetate-n-butanol-water solvent system with an appropriate volume ratio is used to separate and purify the concentrate A in a HSCCC chromatograph, and the effluent at the outlet is automatically collected according to the running time of the HSCCC chromatograph, and the effluent is tested by HPLC, and the collected liquids with KS purity greater than 98% are combined, and the solvent is recovered by a rotary evaporator to obtain a KS monomer compound.

[0008] Furthermore, the ethanol extract of saffron petals in step 1) is: an extract obtained by ultrasonically extracting saffron petals with 60% ethanol, filtering, concentrating the filtrate and then freeze-drying it to obtain the final saffron petal extract powder.

[0009] Furthermore, in step 1), the macroporous adsorption resin model is XAD-1600N.

[0010] Furthermore, in step 1), the KS concentration of the saffron petal extract solution loaded is 1.5 mg / mL, the loading flow rate is 2.3-3.7 BV / h, and the loading volume is 30 BV; the elution volume and flow rate of water are 5 BV and 2 BV / h respectively; the elution volume of 49-69% ethanol is 5 BV, and the elution flow rate is 1-3 BV / h.

[0011] Furthermore, the high-speed countercurrent chromatography separation in step 2) is specifically as follows: taking an ethyl acetate-n-butanol-water two-phase solvent system to prepare a mixed solution in a separatory funnel, taking the upper layer as the stationary phase and the lower layer as the mobile phase; pumping the stationary phase into the HSCCC chromatograph at a speed of 10 to 20 mL / min, rotating the main unit at 600 to 1000 rpm after the pipeline is completely filled with the stationary phase, and pumping the mobile phase at a speed of 1 to 3 mL / min until the mobile phase flows out and the ultraviolet detector monitors and displays a stable baseline, taking an appropriate amount of concentrate A dissolved in the mobile phase and injecting it into the HSCCC chromatograph with an injector, automatically collecting the effluent at the outlet according to the running time, performing HPLC detection on the effluent, combining the collected liquids with a KS purity greater than 98%, and recovering the solvent with a rotary evaporator to obtain a KS monomer compound.

[0012] Furthermore, the two-phase solvent system is an ethyl acetate-n-butanol-water two-phase solvent system with a volume percentage of 10-40:40-10:50.

[0013] The method of the present invention can efficiently obtain KS compounds from saffron petal extracts. After macroporous resin enrichment, the purity of KS in the extract is increased from 8% to about 50%, and the purity is greater than 98% after further HSCCC purification. Compared with the existing extraction method, the operation process is simpler, suitable for small-scale or industrial applications, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The HPLC spectrum of saffron petal extract solution (A) and KS standard solution (B);

[0015] Figure 2 Comparison of the adsorption and desorption capacity of KS by different macroporous resins in Example 1;

[0016] Figure 3 The effect of sample flow rate on dynamic adsorption in Example 1;

[0017] Figure 4 The effect of sample concentration on dynamic adsorption in Example 1;

[0018] Figure 5 The effect of water elution volume on dynamic desorption in Example 1;

[0019] Figure 6 The effect of different ethanol concentrations on dynamic desorption in Example 1;

[0020] Figure 7 The dynamic desorption curves of different elution flow rates of Example 1;

[0021] Figure 8 The effect of different elution flow rates on dynamic desorption in Example 1;

[0022] Fig. 9 is the effect of the interaction between the two factors of the response surface on the comprehensive score Z;

[0023] Fig.10 The optimized parameter operating ranges of the purification process parameters at different angles in Example 1;

[0024] Fig.11 The high-speed countercurrent chromatogram of the sample purified by macroporous resin of Osmanthus fragrans petals in Example 2;

[0025] Fig.12 HPLC chart of the sample of the effluent (130-143 min) collected by high-speed countercurrent chromatography separation in Example 2. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0027] The saffron petal extract of the present invention is an extract obtained by ultrasonically extracting saffron petals with 60% ethanol, and the filtrate is concentrated after filtration and then freeze-dried to obtain saffron petal extract powder.

[0028] The experiment used liquid chromatography (HPLC) conditions for analyzing KS in saffron petal extract solution and sample solution separated by macroporous resin and HSCCC: using InertSustain C18 column (4.6mm×150mm, 5μm) as filler, methanol A as mobile phase, 0.1% formic acid solution as mobile phase B, gradient elution time program: 0-5min, 27-40% A; 5-10min, 40-55% A; 10-15min, 55-80% A. The detection wavelength was 266nm, the flow rate was 1.0mL / min, the column temperature was 35℃, and the injection volume was 10μl. Figure 1 (A) is the HPLC spectrum of saffron petal extract solution, Figure 1 (B) HPLC spectrum of KS standard solution.

[0029] Example 1: Macroporous resin separation

[0030] Macroporous resin screening: Weigh 0.2g of pretreated macroporous resin and put it into a 50mL conical flask containing 25mL SPE solution (1.0mg / mLKS). After mixing, place it in a shaker at 25℃, 150r / min for 12h, and collect the supernatant. During desorption, wash the above resin with deionized water three times, add 25mL60% ethanol solution, shake it again under the above experimental conditions until desorption equilibrium, and collect the supernatant. Determine the KS concentration and calculate the equilibrium adsorption amount Q respectively. e , desorption amount Q d And desorption rate D. The experimental related formula is as follows:

[0031] Equilibrium adsorption capacity:

[0032] Desorption amount:

[0033] Resolution:

[0034] In the formula, C 0 , C e and C d (mg / mL) are the concentrations of KS at the initial, adsorption equilibrium and desorption equilibrium times, respectively. s and V d(mL) are the volume of sample solution and desorption solution, respectively, and M is the mass of resin (g).

[0035] The experimental results are as follows Figure 2 As shown in the figure, XAD-1600N and XAD-16 have higher adsorption capacity for KS in SPE solution than the other six resins, and the relatively large surface area may be the key factor. Generally speaking, resins with higher specific surface area tend to provide more adsorption sites and thus show excellent adsorption performance; relatively speaking, the desorption rates of the eight resins are not significantly different, which indicates that our desorption conditions can completely release KS from the resin. Taking all factors into consideration, XAD-1600N resin has obvious advantages in the adsorption and desorption of KS among the eight resins, so it was selected for further separation process research.

[0036] Effect of sample flow rate on dynamic adsorption of macroporous resin: Saffron petal extract solution (1 mg / mL KS) was injected into a chromatographic column (1.0×20 cm) filled with 1 g XAD-1600N resin at different sample flow rates (2, 3, 4 BV / h) through a peristaltic pump, and the effluent of each column volume BV was collected in a test tube, and the KS concentration was determined by HPLC. Figure 3 As shown in the figure, when the sample flow rate is 2, 3 and 4 BV / h, the breakthrough points appear at 41, 34 and 27 BV respectively. Because a higher sample flow rate may cause the target analyte to bind insufficiently to the site, resulting in unstable adsorption, which is manifested as excessive loss of effective components in the resin; on the contrary, a lower flow rate has better adsorption performance, thereby extending the adsorption residence time of the target compound in the resin. Therefore, 2 BV / h was selected as the sample flow rate condition for subsequent experiments.

[0037] Effect of sample concentration on dynamic adsorption of macroporous resin: Saffron petal extract solutions containing different KS concentrations (0.5, 1, 1.5, 2, 2.5 mg / mL) were prepared, and the optimal sample loading flow rate of 2 BV / h obtained by the above optimization was applied to the resin column. The effluent of each column volume BV was collected in a test tube, and the KS concentration was determined by HPLC. Figure 4 As shown in the figure, at a loading flow rate of 2BV / h, the breakthrough points are 83, 41, 30, 19 and 16BV, respectively, and the calculated KS adsorption amount is not significantly different between 160.61 and 168.35 mg / g. However, the loading amount of high-concentration KS solution can significantly shorten the time, but the high-concentration KS solution is unstable, has many impurities and is prone to resin clogging. 1.5 mg / mL was selected as the initial solution concentration for subsequent experiments. At the preferred flow rate and solution concentration, the loading volume was 30BV and the KS adsorption amount was 166.96 mg / g.

[0038] Effect of water elution volume on dynamic desorption: 5 different volumes (3-7 BV) of deionized water were used to elute impurities at an elution flow rate of 2 BV / h, and then 60% ethanol (elution volume and flow rate were 5 BV and 2 BV / h, respectively) was used for elution. Each column volume BV of effluent was collected in a test tube, and the KS concentration was determined by HPLC, and the dynamic desorption amount (D, mg / g) and recovery rate (R, %) were calculated. Figure 5 As shown, the desorption capacity and recovery rate increased significantly as the water flushing volume increased from 3BV to 5BV, and then decreased as the water flushing volume decreased from 5BV to 7BV. We speculate that a small amount of water elution can completely remove impurities, thereby affecting the desorption of KS from the resin; however, too much water elution will cause the loss of KS from the resin. Therefore, a water flushing volume of 5BV was selected for further experiments.

[0039] Effect of ethanol concentration on dynamic desorption: After reaching adsorption equilibrium under the above optimal sample conditions and washing with an appropriate volume of distilled water, different ethanol concentrations (20-100%) were used as elution solvents, and the elution volume and flow rate were 5BV and 2BV / h, respectively. Each column volume BV of effluent was collected in a test tube, and the KS concentration was determined by HPLC, and the dynamic desorption amount (D, mg / g) and recovery rate (R, %) were calculated. Figure 6 As shown in the figure, in the range of ethanol concentration of 20%-60%, the desorption capacity and recovery rate of KS increased rapidly; then, as the ethanol concentration increased to 100%, the desorption capacity and recovery rate of KS gradually decreased. This is because as the ethanol concentration increased to 60%, the polarity of the elution solvent decreased, which was conducive to the efficient desorption and dissolution of KS; but when the ethanol concentration exceeded 60%, non-polar impurities would be dissolved, resulting in a decrease in the desorption amount and recovery rate. Therefore, 60% ethanol was used as the elution solvent in subsequent experiments.

[0040] Effect of ethanol elution flow rate on dynamic desorption: Under the above optimal conditions, dynamic desorption experiments were performed at four optimal ethanol concentrations at four flow rates (1-4 BV / h). The effluent of each half column volume (BV) was collected in a test tube, and the KS concentration was determined by HPLC, and the dynamic desorption amount (D, mg / g) and recovery rate (R, %) were calculated. Figure 7 As shown in the figure, at flow rates of 1 and 4 BV / h, the elution curve is significantly broadened; on the contrary, clear and narrow elution curves can be seen at 2 and 3 BV / h, which means that the elution is more efficient. The relatively low flow rate of 1 BV / h may cause the resin to re-adsorb the eluted KS, thereby reducing the desorption efficiency. However, the excessively high flow rate of 4 BV / h leads to a shortened contact time, which may result in incomplete elution. Figure 8 As shown, the flow rate of 2 BV / h has a higher desorption capacity and recovery rate for KS on XAD-1600N resin than that of 3 BV / h.

[0041] Optimization of dynamic adsorption / desorption process of macroporous resin based on AHP-EWM-RSM method

[0042] The weight coefficient is determined by the analytic hierarchy process (AHP): AHP is a weight calculation method for the evaluation index system that combines qualitative and quantitative analysis. The importance of the pairing factors is graded according to the 1-9 scale method in the AHP theory, and the score results are converted into a matrix N (Table 1). The priority weight (R i ), according to the maximum eigenvalue (λ max ), consistency ratio (CR), consistency index (CI) and normalized eigenvector for comprehensive ranking. However, this method is subjective and requires consistency test of data, with CR value as evaluation index. If CR≤0.1, the evaluation is "credible"; conversely, if CR>0.1, the evaluation is meaningless. The relevant formula is as follows:

[0043]

[0044] Table 1: AHP indicator pairwise comparison matrix

[0045]

[0046] The weight coefficients of desorption rate, recovery rate and purity determined by AHP are 0.069, 0.155 and 0.777 respectively. From the table of n=3, RI=0.58 is obtained, that is, CR=0.07<0.1, and the evaluation is "credible".

[0047] Entropy Weight Method (EWM) determines the weight coefficient: The entropy weight method calculates the weight according to the different importance of each factor to obtain the final score of each factor, and then ranks the importance of risk factors according to the score results.

[0048] Normalization of indicators:

[0049]

[0050] Where: x i ' j is the normalized value of the jth option of the ith influencing factor; x ij is the number of choices for the jth option of the ith influencing factor; min(x j ) and max(x j ) are the minimum and maximum number of the j-th option respectively.

[0051] The proportion y of the jth option of the i-th factor ij for:

[0052] In the formula: m is the number of influencing factors considered;

[0053] The information entropy of the jth option is:

[0054] The weight of the jth option is:

[0055] Where: n is the number of samples.

[0056] AHP-EWM method determines composite weight coefficient: AHP method determines the weight of indicators based on subjective ranking. Because it relies too much on personal experience and intuition, it is often subjective and arbitrary. The EWM method is highly theoretical, its calculation process is complex, and the weight coefficient obtained is affected by many factors, so it may deviate from the actual situation. In order to overcome the fact that a single weighting method is not enough to reasonably and comprehensively evaluate the purification process, Formula 12 is used to integrate the scoring values ​​obtained by the two methods to obtain a comprehensive score. The weight coefficient results of different evaluation methods are shown in Table 2.

[0057]

[0058] Table 2: Weight coefficients of different evaluation methods

[0059]

[0060] Response surface (RSM) experimental design and analysis: Based on the previous experimental results of dynamic adsorption and desorption of macroporous resin, the Box-Behnken experimental design (BBD) in RSM was used to select three important factors: sample flow rate (2, 3, 4 BV / h), elution ethanol concentration (40%, 60%, 80%) and elution flow rate (1, 2, 3 BV / h) to further optimize the dynamic enrichment process of KS in saffron petal solution by XAD-1600N resin; the AHP-EWM method was used to calculate the comprehensive index Z for the weight coefficients assigned to the three response variables of desorption rate, recovery rate and purity, and used as a comprehensive evaluation index for column chromatography purification process. The specific experimental design and experimental results performed by DesignExpert software are shown in Tables 3 and 4. The variance analysis of the response surface model is shown in Table 5. The high F value (91.27) and low p value (p<0.0001) of the model illustrate that the regression model is extremely significant; the p value of the lack of fit term is 0.1909>0.05, showing a good fit. A quantitative mathematical model between process parameters and comprehensive evaluation indicators was established:

[0061] Z=57.94-1.69×B+7.50×AB+3.67×AC+2.65×BC-9.02×A 2 -8.04×B 2 -1.58×C 2 ;

[0062] The order of influence of each factor on the comprehensive evaluation index is: ethanol concentration > loading flow rate > elution flow rate. The response surface trend diagram of the interaction of the three factors on the comprehensive evaluation index is shown in the figure below. Fig. 9 .

[0063] Table 3: Factors and levels of RSM experiment

[0064]

[0065] Table 4: AHP-EWM-RSM experimental design and results

[0066]

[0067] A: loading flow rate (BV / h); B: ethanol concentration (%); C: elution flow rate (BV / h); Z: comprehensive index.

[0068] Table 5: ANOVA analysis of AHP-EWM-RSM experimental results

[0069]

[0070] A: loading flow rate (BV / h); B: ethanol concentration (%); C: elution flow rate (BV / h).

[0071] Significant level: p<0.05(*), p<0.01(**).

[0072] The median of the comprehensive score (50.72) was used as the lower limit of the constraint condition, and the 90% confidence interval was used to optimize it, and the operating parameter optimization range of the purification process parameters of saffron petal flavonoids was established. Fig.10 As shown in the figure, the bright yellow area indicates the operating parameter optimization range that meets the expected process evaluation index target; the dark yellow area indicates that there is a 10% probability of not meeting the process evaluation index target in the optimized parameter operating range; the gray area indicates the parameter range that cannot meet the evaluation index target. The shape of the calculated operating parameter optimization range is irregular. For ease of operation, the recommended operating parameter optimization range is: sample flow rate 2.3-3.7BV / h, elution ethanol concentration 49-69% and elution flow rate 1-3BV / h. In order to verify the reliability of the established optimized parameter operating range, 3 points within the operating parameter optimization range were selected for verification. The verification experimental parameter settings and results are presented in Table 6. The verification results show that the actual value is greater than the target lower limit and is close to the predicted value, indicating that the product quality can be guaranteed within the recommended operating parameter range.

[0073] Table 6: Verification test results

[0074]

[0075] Example 2: Separation and purification by high-speed countercurrent chromatography

[0076] Screening of solvent system: According to the distribution coefficient (K) of the target compound, HPLC was used to evaluate the composition of the two-phase solvent system. Starting from the classic system proposed by Dr. Ito, a variety of solution systems with different ratios were prepared in test tubes, and the test tubes were shaken vigorously and then allowed to stand for stratification. After the two-phase solution reached equilibrium, 2mL of the upper and lower phase solvents were taken respectively and placed in a 5mL test tube. At the same time, a certain amount of the sample purified by resin was added, and then the test tube was continuously shaken until the sample was completely dissolved. When equilibrium stratification was reached, the same volume of the upper and lower phase solutions were taken, and after passing through a 0.45μm filter membrane, the upper and lower phases of the target component were analyzed by 266nm HPLC, and the distribution coefficient was calculated.

[0077]

[0078] Among them, A U and A L Represent the peak areas of KS in the upper and lower phases, respectively.

[0079] The experimental results are shown in Table 7. For the ethyl acetate-n-butanol-water two-phase solvent system, the distribution coefficient K for KS in the sample should be in the range of 0.5≤K≤2.0 when the volume percentage is 10-40:40-10:50.

[0080] Table 7 K value of solvent system

[0081]

[0082] High-speed countercurrent chromatography separation KS: According to the results of the solvent system screening, ethyl acetate-n-butanol-water (4:1:5, v / v) was selected as the solvent system, added to the separatory funnel in proportion and mixed evenly, and allowed to stand and separate at room temperature. The upper phase is the stationary phase and the lower phase is the mobile phase. The layered upper and lower phases were placed in an ultrasonic cleaner for ultrasonic degassing for 30 minutes for later use. Weigh 391.4 mg of the sample purified by saffron petals macroporous resin and dissolved in the selected upper and lower phases, ultrasonically dissolved it fully, and passed through a 0.45 μm microporous filter membrane. At 30°C, the upper phase was pumped in at 20 mL / min, the speed was set to 900 rpm forward, and the lower phase was pumped into the instrument at 2 mL / min. The retention rate of the stationary phase was 50%. After the baseline is balanced, observe the spectrum at the detection wavelength of 266 nm as shown Fig.11 The effluent from the HSCCC outlet at 130-143 min was collected, and the purity of KS was 98.72% ( Fig.12 ), after concentration and drying, the residue was 35.10 mg.

Claims

1. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals, characterized in that The following steps are involved: 1) Preparation of ethanol extract of saffron petals: ultrasonically extracting saffron petals with 60% ethanol, filtering the extract, concentrating the filtrate and freeze-drying it to obtain saffron petal extract powder; 2) Macroporous resin separation: The saffron petal extract powder is prepared into a kaempferol-3-O-sophoroside solution of appropriate concentration, loaded on a macroporous adsorption resin, eluted with distilled water and ethanol in sequence, and the ethanol elution portion of appropriate concentration is collected, and concentrated under reduced pressure to obtain a concentrate A; 3) High-speed countercurrent chromatography separation: A two-phase solvent system of ethyl acetate-n-butanol-water solvent system with an appropriate volume ratio is used to separate and purify the concentrate A in a HSCCC chromatograph, and the effluent at the outlet is automatically collected according to the running time of the HSCCC chromatograph, and the effluent is tested by HPLC, and the collected liquids with KS purity greater than 98% are combined, and the solvent is recovered by a rotary evaporator to obtain a KS monomer compound.

2. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals as claimed in claim 1, characterized in that In step 2), the model of the macroporous adsorption resin is XAD-1600N.

3. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals as claimed in claim 1, characterized in that In step 2), the concentration of KS solution is 1.5 mg / mL, the loading flow rate is 2.3-3.7 BV / h, and the loading volume is 30 BV; the elution volume and flow rate of water are 5 BV and 2 BV / h respectively; the concentration of ethanol elution is 49-69%, the volume is 5 BV, and the elution flow rate is 1-3 BV / h.

4. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals as claimed in claim 1, characterized in that In step 3), high-speed countercurrent chromatography separation is specifically as follows: a two-phase solvent system of ethyl acetate-n-butanol-water is prepared into a mixed solution in a separatory funnel, the upper layer is taken as the stationary phase, and the lower layer is taken as the mobile phase; the stationary phase is pumped into the HSCCC chromatograph, and after the pipeline is completely filled with the stationary phase, the main engine is rotated, and the mobile phase is pumped in at the same time, until the mobile phase flows out and the ultraviolet detector monitors and displays a stable baseline, and then an appropriate amount of concentrate A dissolved in the mobile phase is taken and injected into the HSCCC chromatograph with an injector, and the effluent at the outlet is automatically collected according to the running time, and the effluent is subjected to HPLC detection, and the effluents with a KS purity greater than 98% are collected and combined, and the solvent is recovered by a rotary evaporator to obtain a KS monomer compound.

5. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals as claimed in claim 4, characterized in that The volume percentage of the ethyl acetate-n-butanol-water two-phase solvent system is 10-40:40-10:

50.

6. A method for efficiently preparing kaempferol-3-O-sophoroside from saffron petals as claimed in claim 4, characterized in that The stationary phase is pumped in at a speed of 10 to 20 mL / min; the main engine speed is 600 to 1000 rpm; the mobile phase is pumped in at a speed of 1 to 3 mL / min.

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