A method for efficient enrichment and separation of isoflavones from kudzu root

Abstract

This invention discloses a highly efficient method for the enrichment and separation of isoflavones from kudzu root, belonging to the field of traditional Chinese medicine extraction and separation technology. The method includes: adsorbing and enriching kudzu root juice using HP-20 macroporous adsorption resin, optimizing the sample loading and elution conditions, and eluting with 50% ethanol to obtain a crude isoflavone extract; then separating the crude extract using high-speed countercurrent chromatography, employing a solvent system of ethyl acetate-n-butanol-water with a volume ratio of 2:1:3 to achieve efficient and simultaneous separation of four isoflavone components: puerarin, 3′-methoxypuerarin, puerarin apigenin, and daidzein. This invention features a simple process, low solvent consumption, and high separation efficiency, simultaneously obtaining multiple high-purity isoflavone monomers, making it suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction and separation technology, and in particular to a method for the efficient enrichment and separation of isoflavone components from kudzu root. Background Technology

[0002] Kudzu root, the dried root of the legume *Pueraria lobata*, possesses properties such as relieving muscle tension and fever, and promoting the production of body fluids and quenching thirst. Its main active components are isoflavones, including puerarin, daidzein, and 3′-methoxypuerarin. These isoflavones exhibit various pharmacological activities, including vasodilating, improving microcirculation, antioxidation, and anti-inflammation, and are widely used in the treatment of cardiovascular and cerebrovascular diseases, health foods, and cosmetics. Therefore, the efficient, green, and large-scale enrichment and separation of high-purity kudzu root isoflavone monomers is of great significance for enhancing the added value of kudzu root medicinal materials and ensuring the consistency of quality and efficacy of related products.

[0003] Currently, the extraction of kudzu isoflavones mainly employs traditional methods such as reflux extraction and maceration extraction, which suffer from low extraction efficiency, high consumption of organic solvents, and complex processes. Furthermore, it is difficult to achieve simultaneous high-purity separation of multiple components. Traditional solvent extraction methods exhibit poor selectivity for isoflavones, resulting in unstable extraction rates and leaving a large amount of active ingredients in the residue. Since kudzu isoflavone components have similar polarities, conventional chromatography techniques struggle to achieve efficient simultaneous separation of multiple structural analogs, often requiring multi-step purification, resulting in lengthy processes. These processes frequently utilize large amounts of organic solvents (such as methanol and acetone), which are difficult to recover, increasing production costs and creating safety and environmental concerns. Existing processes cannot simultaneously obtain multiple high-purity (>95%) isoflavone monomers in a single batch, hindering the development of high-end formulations and standardized products. In summary, the overall extraction rate of kudzu isoflavones is low (often below 80%), the separation process cannot simultaneously obtain multiple high-purity (>95%) monomers, and it heavily relies on high-consumption, difficult-to-recover organic solvent systems. Summary of the Invention

[0004] This application provides a highly efficient method for enriching and separating isoflavones from kudzu root, which solves the problems of low extraction rate, low purity, and high consumption in the prior art.

[0005] It achieves efficient separation of four isoflavone components with high purity in a single countercurrent chromatography run.

[0006] This application provides a method for the efficient enrichment and separation of isoflavones from kudzu root, comprising the following steps: (1) The juice of kudzu root was enriched by macroporous adsorption resin, and then eluted with an ethanol aqueous solution as an eluent. The eluent was collected to obtain crude isoflavone extract. The concentration of ethanol as an eluent ranged from 10% to 90%, preferably 50%. Macroporous adsorption resin and kudzu root juice were mixed at a ratio of 1:6 (g / mL). The macroporous adsorption resin is selected from any one of HP-20, AB-8, HPD100, and HPD450, with HP-20 being preferred.

[0007] (2) The crude isoflavone extract is separated by high-speed countercurrent chromatography and purified by high-speed countercurrent chromatography. The solvent system used in the high-speed countercurrent chromatography is a mixed solvent system of ethyl acetate, n-butanol and water. The volume ratio of ethyl acetate, n-butanol and water is (0-10):(5-15):15, preferably 2:1:3.

[0008] Furthermore, the HP-20 macroporous adsorption resin described in step (1) is pretreated before use. The pretreatment includes soaking in 95% ethanol for 24 hours and washing until the washing liquid is colorless and transparent.

[0009] Furthermore, the amount of sample loaded for enrichment in step (1) is 1-2 ml of kudzu root juice per 1 gram of resin.

[0010] Furthermore, the volume of the 50% ethanol aqueous solution used for elution in step (1) is 6-8 column volumes.

[0011] Furthermore, the elution flow rate described in step (1) is 1 ml / min.

[0012] Furthermore, in step (2), the stationary phase of the high-speed countercurrent chromatography is the upper organic phase of the solvent system, and the mobile phase is the lower aqueous phase of the solvent system.

[0013] Furthermore, in step (2), the spiral tube rotation speed of the high-speed countercurrent chromatography is 800-1000 rpm, the column temperature is 20-30 degrees Celsius, and the mobile phase flow rate is 1.0-10.0 mL / min.

[0014] Furthermore, the isoflavone components include puerarin, 3′-methoxypuerarin, puerarin apigenin, and daidzein.

[0015] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: By using HP-20 macroporous adsorption resin and a 50% ethanol elution system, the enrichment efficiency and selectivity of isoflavones were significantly improved, with the extraction rate remaining stable at over 85% and the desorption rate exceeding 80%, effectively reducing the consumption of organic solvents.

[0016] By optimizing the sample loading amount and elution volume, the resin adsorption capacity was maximized, reducing raw material waste and process costs.

[0017] By using a high-speed countercurrent chromatography solvent system of ethyl acetate-n-butanol-water in a volume ratio of 2:1:3, four high-purity isoflavone monomers, namely puerarin, 3′-methoxypuerarin, puerarin apigenin and daidzein, can be obtained simultaneously in a single separation process, with a purity of over 95%.

[0018] The overall process is green and efficient, the solvent is recyclable, and it is suitable for industrial production, providing reliable technical support for the high-value-added development of kudzu isoflavones. Attached Figure Description

[0019] Figure 1 The adsorption kinetics curves of the four macroporous adsorption resins of this invention are shown. Figure 2 This is a comparison chart of the elution effects of different concentrations of ethanol eluent in this invention; Figure 3 This is a leakage curve diagram of HP-20 resin sample loading according to the present invention; Figure 4 This is a high-performance liquid chromatogram of kudzu root juice according to the present invention; Figure 5 This is a chromatogram of the high-efficiency countercurrent chromatography separation of the present invention; Figure 6 HPLC purity analysis of the four isoflavone components of this invention. Detailed Implementation

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] All kudzu roots used in the embodiments of this invention were collected and prepared in accordance with the Chinese Pharmacopoeia (2020).

[0022] Example 1: Effect of resin type (screening by static adsorption rate and desorption rate) on the enrichment of isoflavones; Accurately weigh 1.00 g each of HPD100, HPD450, HPD750, AB-8, D101 and HP-20 resins into 10 mL centrifuge tubes, pre-treat (soak in 95% ethanol for 24 h, filter and wash until the washing liquid is pure and transparent, and all subsequent numerical pre-treatment methods are the same) and set aside. Mix the kudzu root juice thoroughly, accurately measure 1 mL into a centrifuge tube containing resin, mix using a vortex mixer to remove air bubbles, and perform static adsorption by intermittent shaking for 24 h. Collect all the adsorbed liquid for later use. Then, add 7 times the volume of 95% ethanol to the resin, mix thoroughly and remove air bubbles, and perform static desorption by intermittent shaking for 24 h. Collect all the desorbed liquid for later use. Dilute the kudzu root juice, adsorbed liquid, and desorbed liquid 20 times and analyze them using high performance liquid chromatography (HPLC). Calculate the adsorption rate and desorption rate based on the HPLC chromatograms of the three. The adsorption and desorption rates of the above resins are shown in Tables 1 and 2. Overall, HP-20, HPD100, HPD450, and AB-8 resins have comparable effects. D101 and HPD750 have low desorption rates and are not suitable for enriching kudzu juice, so they will not be considered in the following steps.

[0023] High-performance liquid chromatography (HPLC) was performed under the following conditions: an AgiLent HPLC 1100 (USA) was used. The column selected was a YMC-C18 (4.6 mm id × 150 mm, 5 μm). A gradient elution was performed using methanol (A) and 0.03% TFA aqueous solution (B) as the mobile phase: 0-5 min, A from 10% to 20%; 5-25 min, A from 20% to 40%; 25-30 min, A from 40% to 100%; 30-35 min, A maintained at 100%. The mobile phase flow rate was 0.8 mL / min, the column temperature was controlled at 30 ℃, and the injection volume was 5 μL. The eluent was monitored using a 254 nm diode array detector. The resulting HPLC chromatogram of kudzu root juice is shown below. Figure 4 .

[0024] Adsorption rate (%) = [(Total concentration of isoflavones in the original kudzu root juice solution - Total concentration of isoflavones in the residue after resin adsorption (i.e., adsorbent solution) / Total concentration of isoflavones in the original kudzu root juice solution] × 100%; Desorption rate (%) = [Total concentration of isoflavones eluted in the desorption solution / (Total concentration of isoflavones in the original kudzu root juice solution - Total concentration of isoflavones in the residue after resin adsorption (i.e., adsorption solution))] × 100%; Table 1 Adsorption rate of various resin types Table 2 Desorption Rate of Resins of Various Types Example 2: Effect of resin type (adsorption kinetics screening) on ​​the enrichment of isoflavones; Accurately weigh 10.00 g each of HPD100, HPD450, AB-8, and HP-20 resins into 50 mL centrifuge tubes and pretreat them for later use. Mix kudzu root juice thoroughly, accurately measure 20 mL into a centrifuge tube containing the resin, and mix using a vortex mixer to remove air bubbles. For the first 60 min, take samples every 10 min for measurement, then every 30 min thereafter, continuously measuring until 240 min. Calculate the total adsorption capacity of isoflavones at each time point and plot the dynamic adsorption curve. The results show that there are significant differences in the enrichment capacity of different resin types for isoflavones. Specific results are shown in […]. Figure 1 As shown in Table 3, the data indicates that the second-order kinetics are more consistent with the adsorption process of each resin, and the reaction rate constant of HP-20 is better than that of other resins. Therefore, HP-20 is considered to have the best effect.

[0025] Table 3 Adsorption kinetic data for each type of resin Example 3: Effect of eluent type on the elution of isoflavones; In the dynamic adsorption experiment, the effect of the eluent on the desorption rate of isoflavones must be considered. A series of different volume fractions of ethanol (10%, 20%, 30%, 50%, 70%, and 95%, respectively) were set as desorbents to investigate the desorption effect of different volume fractions of ethanol solutions on puerarin and other main components in kudzu juice.

[0026] Measure 5 mL of HP-20 resin and load it with 3 mL of kudzu juice for gradient elution (elution rate set at 1 mL / min). First, elute with distilled water (eluting two column volumes) to remove impurities, then elute with the selected eluent. Collect one tube of eluent after every two column volumes, and determine the content of puerarin and other main components. Calculate the recovery rate. The results are shown in Table 4. Figure 2 The elution effect of 95% ethanol is slightly better than that of 50% ethanol. However, considering the increase in cost, the actual benefit is actually lower. Therefore, it is determined that 50% ethanol is more effective.

[0027] Table 4 Elution results of isoflavones by eluent type Example 4: Effect of sample loading amount on the enrichment of isoflavones; Take an appropriate amount of HP-20 resin and load it onto the column using the wet method, recording this as one column volume. Load the column with kudzu root juice at a flow rate of 1 mL / min. Collect one tube for every 1 mL of kudzu root juice and measure the content of puerarin and other main components in the effluent after column loading. Plot a leakage curve with the sample volume (based on column volume) as the x-axis and the content of puerarin and other main components in the effluent as the y-axis. The leakage point is recorded when the puerarin content in the effluent is 1 / 10 of the content of puerarin and other main components in the original waste liquid, indicating that the separation column resin is saturated. The results are shown in Table 5. Figure 3 The results showed that the optimal loading volume was 6 column volumes.

[0028] Table 5. Enrichment results of kudzu root samples based on loading amount Example 5: Effect of eluent volume on the enrichment of isoflavones; Measure 5 mL of HP-20 resin and load it with 3 mL of kudzu juice for gradient elution (elution rate set to 1 mL / min). First, elute with distilled water (two column volumes) to remove impurities, then elute with 50% ethanol. Collect one tube of eluent after every two column volumes and determine the content of puerarin and other main components. The results show that elution is complete and efficient when the eluent volume is 8 column volumes.

[0029] Example 6: Screening of solvent systems for high-speed countercurrent chromatography; The selection principles for the solvent system are as follows: it should not cause decomposition or denaturation of the sample; each component in the sample should have a suitable partition coefficient in the solvent system, generally considered to be within the range of 0.25-4.0; the partition coefficients of each component should have sufficient differences, and the separation factor should preferably be greater than or equal to 1.5; the solvent system should not interfere with the detection of the sample; to ensure that the retention rate of the stationary phase is not less than 50%, the separation time of the solvent system should not exceed 30 seconds; the volume ratio of the upper and lower phases should be appropriate to avoid wasting solvent; volatile solvents should be used as much as possible, and highly toxic solvents should be avoided to facilitate subsequent processing. Based on the above principles and the characteristics of kudzu root, three systems were initially selected for subsequent testing. The systems and related K values ​​are shown in Table 6. It was found that the solvent system of ethyl acetate: n-butanol: water = 2:1:3 had a good separation effect on each substance, and the K values ​​of each substance basically met the experimental requirements. Therefore, the 2:1:3 solvent system was selected for countercurrent separation.

[0030] The enriched kudzu root sample was separated by countercurrent chromatography under the pre-determined experimental conditions, including: Take approximately 200 mg of crude puerarin isoflavone extract obtained by enrichment and elution with HP-20 macroporous adsorption resin, dissolve it in the upper and lower phases of the selected solvent system using equal volume high-speed countercurrent chromatography, prepare a sample solution with a concentration of 20 mg / mL, filter it through a 0.45 μm microporous membrane, and set it aside for later use. Prepare a solvent system of ethyl acetate-n-butanol-water (volume ratio 2:1:3). Add each solvent to a separatory funnel in the specified proportions, shake thoroughly to mix, and allow to stand for separation. Separate the upper phase (organic phase) and the lower phase (aqueous phase), and degas each phase by sonication for 20 min for later use. A high-speed countercurrent chromatograph was used, equipped with polytetrafluoroethylene spiral tubing (column volume approximately 340 mL); the tubing was flushed with methanol-water and dried before use. Stationary phase: The upper phase (organic phase) is pumped into the spiral tube at a flow rate of 10 mL / min to serve as the stationary phase.

[0031] Start rotation: Turn on the main unit, set the spiral tube speed to 1000 rpm and the column temperature to 25 ℃; Establish the mobile phase: After the stationary phase is filled, switch to pumping in the lower phase (water phase) as the mobile phase at a flow rate of 4.0 mL / min.

[0032] Sample injection: When the mobile phase begins to flow out steadily, inject the sample solution (10 mL, about 200 mg) through the injection valve.

[0033] Collect flow fractions: Collect the corresponding flow fractions according to the effluent time of each peak; Stop and wash: After separation, stop rotating, discharge the stationary phase, and wash the column with methanol and water in sequence; The collected fractions were concentrated to dryness under reduced pressure, weighed, and the yield was calculated (Table 7). The purity of each fraction was determined by high-performance liquid chromatography (HPLC). Figure 6 The structures of the compounds were identified by nuclear magnetic resonance spectroscopy (NMR), and were confirmed to be puerarin, 3′-methoxypuerarin, puerarin apigenin, and daidzein, respectively.

[0034] Table 6. K-values ​​for each system and related information Table 7 Separation results of kudzu root samples after enrichment In actual long-term production processes, the combined separation of macroporous resin and countercurrent chromatography is prone to clogging, reducing purification efficiency and ultimately increasing costs. After 10 purification cycles, the purification effect is less than 80% of the first purification effect. To address this, the adsorbed resin was washed with 20% ethanol at a flow rate of 1.5 BV / h for 0.5 h, followed by a second wash with 5% acetic acid aqueous solution at a flow rate of 1.5 BV / h for 0.5 h. Then, 7 times the volume of 50% ethanol was added, mixed thoroughly, and air bubbles were removed. The mixture was then intermittently shaken for 24 h for static desorption. The eluent was collected for later use. Washing removes moderately polar impurities to prevent interfacial aggregation and removes metal ions and basic impurities to prevent precipitation. Reduced impurities significantly reduce the risk of column clogging, decrease the washing frequency, ensure more stable sample composition, and maintain consistent separation conditions over a long period. More than 65% of the key impurities in the resin concentrate that cause countercurrent chromatography performance degradation are removed beforehand. Even after more than 10 consecutive purification cycles, more than 95% of the initial purification effect can still be maintained.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the efficient enrichment and separation of isoflavones from kudzu root, characterized in that, Includes the following steps: (1) The juice of kudzu root was enriched by macroporous adsorption resin, and then eluted with an ethanol aqueous solution as an eluent. The eluent was collected to obtain crude isoflavone extract. The concentration of ethanol as an eluent ranged from 10% to 90%, preferably 50%. Macroporous adsorption resin and kudzu root juice were mixed at a ratio of 1:6 (g / mL). The macroporous adsorption resin is selected from any one of HP-20, AB-8, HPD100, and HPD450, with HP-20 being preferred; (2) The crude isoflavone extract is separated by high-speed countercurrent chromatography and purified by high-speed countercurrent chromatography. The solvent system used in the high-speed countercurrent chromatography is a mixed solvent system of ethyl acetate, n-butanol and water. The volume ratio of ethyl acetate, n-butanol and water is (0-10):(5-15):15, preferably 2:1:

3.

2. The method as described in claim 1, characterized in that, The HP-20 macroporous adsorption resin described in step (1) is pretreated before use. The pretreatment includes soaking in 95% ethanol for 24 hours and washing until the washing liquid is colorless and transparent.

3. The method as described in claim 1, characterized in that, The amount of sample loaded for enrichment in step (1) is 1-2 ml of kudzu root juice per 1 gram of resin.

4. The method as described in claim 1, characterized in that, The volume of the 50% ethanol aqueous solution used for elution in step (1) is 6-8 column volumes.

5. The method as described in claim 1, characterized in that, The elution flow rate described in step (1) is 1 ml / min.

6. The method as described in claim 1, characterized in that, In step (2), the stationary phase of the high-speed countercurrent chromatography is the upper organic phase of the solvent system, and the mobile phase is the lower aqueous phase of the solvent system.

7. The method as described in claim 1, characterized in that, In step (2), the spiral tube rotation speed of the high-speed countercurrent chromatography is 800-1000 rpm, the column temperature is 20-30 degrees Celsius, and the mobile phase flow rate is 1.0-10.0 mL / min.

8. The method as described in claim 1, characterized in that, The isoflavone components include puerarin, 3′-methoxypuerarin, puerarin apigenin, and daidzein.

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