Method for preparing high-purity isoquercitrin from polygonum viviparum

Abstract

The invention provides a method for preparing high-purity isoquercitrin from polygonum viviparum, which comprises the following steps: sequentially carrying out water extraction impurity removal and alcohol extraction on a polygonum viviparum raw material to obtain an alcohol extract; decolorizing the alcohol extract, then adjusting the pH value to 9-11 to precipitate and remove tannin, and then carrying out vacuum concentration to obtain a general flavone crude product; performing acid hydrolysis on the crude product, performing separation and purification by adopting high-pressure preparative liquid chromatography, performing online monitoring by utilizing an ultraviolet detector, and obtaining a fraction rich in isoquercitrin by accurately controlling starting and ending points of fraction collection; and concentrating under reduced pressure, washing with water and drying to obtain the product. By optimizing the pretreatment and chromatographic separation conditions, the purity and yield of the product are synchronously improved, the HPLC (High Performance Liquid Chromatography) purity of the obtained isoquercitrin reaches 98% or above, and the method is low in process operation cost, environment-friendly and suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and separation technology, and more specifically, to a method for preparing high-purity isoquercitrin using Polygonum villosa. Background Technology

[0002] Isoquercetin is a flavonoid compound with important medicinal value, widely found in various plants, with Polygonum bulbiferum being one of its main natural sources. Currently, conventional methods for extracting isoquercetin from plants generally suffer from problems such as complex processes, low separation efficiency, and difficulty in simultaneously achieving product purity and yield, making it challenging to achieve large-scale, low-cost production of high-purity isoquercetin. Therefore, developing an efficient, stable, and industrially applicable extraction and purification process is of significant practical importance and application demand.

[0003] While various chromatographic separation methods exist for purifying natural products, their application in the large-scale preparation of high-purity isoquercitrin from Polygonum bulbiferum often faces common technical bottlenecks, including lengthy pretreatment steps, easy column contamination, high separation costs, and difficulty in simultaneously ensuring high purity and high yield. In particular, there is a lack of a complete process scheme that can systematically integrate raw material pretreatment, selective impurity removal, and efficient chromatographic separation, while also enabling precise control and continuous operation. Summary of the Invention

[0004] The main objective of this invention is to provide a method for preparing high-purity isoquercitrin using Polygonum bulbiferum, so as to at least solve the problem that the existing technology cannot achieve low-cost, high-efficiency, large-scale production of high-purity isoquercitrin.

[0005] To achieve the above objectives, the present invention provides a method for preparing high-purity isoquercitrin from Polygonum bulbiferum, characterized by comprising the following steps: Raw material pretreatment and extraction: The Polygonum bulbifera raw material was subjected to water extraction and impurity removal and alcohol extraction in sequence, and the alcohol extract was collected; Impurity removal and total flavonoid enrichment: After decolorization of the alcohol extract, the pH was adjusted to 9-11, the precipitate was allowed to stand and the precipitate was removed. Then the filtrate was concentrated under reduced pressure to precipitate and collect the crude total flavonoids. Acid hydrolysis: The crude total flavonoids were hydrolyzed with an acidified alcohol reagent to obtain a hydrolysate; Chromatographic separation and purification: The hydrolysate was used as the loading solution and separated and purified by high-performance liquid chromatography (HPLC). An octadecylsilane-bonded silica gel column was used as the stationary phase, and a methanol-phosphoric acid aqueous solution was used as the mobile phase for elution. The elution process was monitored online using an ultraviolet detector. Based on the detected isoquercitrin peak signal, the fraction collector was activated when the peak began to rise and stopped collecting when the peak decreased to one-fifth to one-sixth of its height, thus obtaining an elution fraction rich in isoquercitrin. Post-processing: The eluted fraction rich in isoquercitrin was concentrated, washed, and dried to obtain high-purity isoquercitrin.

[0006] Furthermore, in the raw material pretreatment and extraction steps, the conditions for water extraction to remove impurities are as follows: using water as the solvent, the liquid-to-solid ratio is 10-15 mL / g, and extraction is performed 1-3 times at 90-100℃, each time for 1-3 hours; the conditions for alcohol extraction are as follows: using an ethanol solution with a mass fraction of 75% or higher as the solvent, the liquid-to-solid ratio is 6-12 mL / g, and extraction is performed 2-3 times at 60-85℃, each time for 1.5-2 hours. The finished medicinal material processed from the roots and / or stems (stems with bark) of Polygonum viviparum is washed clean with water and dried beforehand to obtain the raw material. Water extraction of the raw material from Polygonum viviparum can remove water-soluble impurities such as sugars and some tannins.

[0007] Further, in the impurity removal and total flavonoid enrichment steps, the decolorization treatment involves adding 0.3% to 1.0% (by weight) of activated carbon to the alcohol extract and stirring for adsorption at 40 to 80°C for 15 to 60 minutes. The pH adjustment involves using a 0.5 to 1.0 mol / L sodium hydroxide solution to adjust the pH of the decolorized liquid to 9 to 11 and allowing it to stand for 8 to 12 hours. The extract is treated with activated carbon to remove fat-soluble impurities, and then the impurities dissolved in the extract are precipitated by the added sodium hydroxide solution. After removing the precipitate formed during standing by filtration, the filtrate is concentrated under reduced pressure (at 40 to 60°C, with a vacuum degree of 0.09 to 0.15 MPa) to 1 / 5 to 1 / 3 of its original volume (i.e., the volume of the filtrate before concentration). The precipitate (i.e., crude total flavonoids) is then separated by filtration.

[0008] Furthermore, prior to the acid hydrolysis step, the method further includes washing the crude total flavonoids with water to a pH of 6-8.

[0009] Furthermore, in the acid hydrolysis step, the acidified alcohol reagent is a solution of methanol and hydrochloric acid solution with a mass fraction of 3%~15% mixed at a volume ratio of (30~60):(40~70); the hydrolysis conditions are: the ratio of crude total flavonoids to acidified alcohol reagent is 1:20~40 g / mL, and hydrolysis is carried out at 50~80℃ for 0.5~2.0 hours.

[0010] Further, in the chromatographic separation and purification step, the methanol-phosphoric acid aqueous solution is prepared by mixing methanol and a 0.2%~0.8% (w / w) phosphoric acid aqueous solution at a volume ratio of (55~65):(35~45); the elution flow rate is 10~200 mL / min. The elution time is 30~40 minutes. After elution, the fraction is collected. In the chromatographic separation, the peak shape of isoquercitrin is relatively symmetrical, but the peak does not reach baseline separation. Therefore, it is collected when the peak begins to rise and the collection ends when the peak drops to one-fifth to one-sixth of its total height.

[0011] Furthermore, in the chromatographic separation and purification step, the octadecylsilane-bonded silica gel packing material of the chromatographic column has a particle size of 5~20μm and a pore size of 40~70Å; the column length of the chromatographic column is 10~30cm and the diameter is 2~15cm.

[0012] Furthermore, in the chromatographic separation and purification step, the sample is loaded in multiple batches, with each batch containing 10-20 mL of the hydrolysate.

[0013] Furthermore, in the post-processing step, the washing temperature is 80-100℃, and the drying temperature is 50-80℃. The collected eluent fraction is concentrated under reduced pressure at 40-60℃ and a vacuum degree of 0.09-0.18 MPa, and the precipitate is then washed with water and dried sequentially. Hot water at 80-100℃ allows water-soluble substances to dissolve better, thus making it easier to remove these residual impurities, while also removing residual components of the mobile phase; isoquercitrin is poorly soluble in hot water, so there is no loss of isoquercitrin.

[0014] Furthermore, following the post-processing steps, a column regeneration step is included: when the column efficiency decreases, the column is activated and regenerated using a 3%–7% hydrochloric acid solution, followed by rinsing with a 2%–5% acetonitrile aqueous solution. This activation and regeneration step removes residual chloride ions from the column, preventing corrosion of the stationary phase and flow path system by acid residues.

[0015] The beneficial effects of the technical solution of this invention are as follows: 1. This invention utilizes extraction and separation methods such as leaching, adsorption, and silica gel column chromatography, and optimizes the solvent and extraction / separation route, achieving the extraction of isoquercitrin with a purity of over 98% from Polygonum viviparum. This invention enables rapid separation of isoquercitrin with minimal loss and a significantly improved yield. Furthermore, this invention uses fewer types of organic solvents and generates less waste during the extraction and separation of isoquercitrin, making it environmentally friendly and suitable for industrial production.

[0016] 2. This invention addresses impurities such as tannins in Polygonum villosa extract, which easily cause column clogging and contamination. After decolorization, an alkaline adjustment step is introduced, allowing these impurities to precipitate and be effectively removed in the alcohol system through static precipitation and filtration. This not only significantly improves the efficiency and stability of subsequent chromatographic separations but also substantially extends the lifespan of the chromatographic column, laying the foundation for continuous industrial production.

[0017] 3. This invention experimentally determined that methanol acidified with a certain concentration of dilute hydrochloric acid should be used as the hydrolysis reagent. At the same time, by controlling the ratio of methanol to dilute hydrochloric acid and the amount of total flavonoids, it is ensured that all the added total flavonoids can be dissolved and all the glycosides are hydrolyzed into aglycones (isoquercetin is the aglycone), thereby improving the separation efficiency.

[0018] 4. This invention applies high-pressure preparative liquid chromatography (HPLC) for the first time to the extraction and separation of isoquercitrin from Polygonum villosa. A silica gel column (e.g., octadecylsilane-bonded silica gel) is used, and the separation process employs only a single mobile phase—methanol-phosphoric acid solution—for constant-flow, constant-rate, and gradient elution. This not only results in high column efficiency but also allows for precise product collection through online monitoring, simplifying and accelerating the separation process. High-purity isoquercitrin can be separated after a single elution. Furthermore, the required mobile phase volume is small, and the solvent in the mobile phase can be recycled after elution, reducing the overall process cost. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is an HPLC chromatogram of isoquercitrin obtained by means of separation according to an embodiment of the present invention (the rightmost peak is isoquercitrin). Detailed Implementation

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] (a) Dried Polygonum bulbiferum, reagents and laboratory equipment 1. Dried Bulbus Polygonum Purchased from the medicinal herb market, with a moisture content of less than 10%.

[0022] 2. Reagents Sodium hydroxide, ethanol, methanol, hydrochloric acid, and phosphoric acid used in the extraction and separation were all of analytical grade, and the pure water was double-distilled water; the acetonitrile used in the HPLC quantitative analysis was of chromatographic grade.

[0023] 3. Experimental Apparatus The large-diameter (8cm) preparative column was an Agilent high-pressure column, the column packing material was octadecylsilane-bonded silica gel (particle size 5μm, pore size 40Å), the high-pressure preparative liquid chromatography system was an Agilent product, the ultraviolet detector was an Agilent product, the automatic fraction collector was an Agilent product, and the high-performance liquid chromatograph was a Waters product.

[0024] (II) Method for extracting and isolating high-purity isoquercitrin from Polygonum bulbiferum Example

[0025] 1) Take 1 kg of dried Polygonum bulbiferum, wash it with water, dry it and add it to 10 L of pure water. Heat and boil for 1.5 hours. Pour off the water and filter out the residue. Heat the residue with 90% ethanol solution in 3 portions, each time using 6 L of 90% ethanol solution, heating at 65℃ for 1.5 hours. After reflux, filter and combine the filtrates to obtain the ethanol extract. 2) Add 0.3% activated carbon (by weight of the extract) to the ethanol extract and stir at 50°C for 1 hour to decolorize. Then filter out the activated carbon. Adjust the pH of the decolorized ethanol extract to about 11 with 1.0 mol / L sodium hydroxide solution. After standing for 12 hours, remove the precipitate (containing tannins and other impurities) by filtration. Concentrate the filtrate after filtration under reduced pressure (at 60°C and a vacuum of 0.09 MPa) to about 1 / 3 of the original volume (5 L). Filter out a large amount of precipitated insoluble matter. The filtered insoluble matter is the crude total flavonoids (8.6 g, yield 0.86%). 3) Wash 8.6g of crude total flavonoids with pure water until pH 6-8 (after adding crude total flavonoids to pure water, stir and filter out insoluble matter; repeat this washing process several times). Then dissolve it in 100mL of methanol-5% hydrochloric acid solution (methanol:5% hydrochloric acid solution volume ratio is 50:50), heat (80℃) and reflux for 1 hour. Filter while hot to remove insoluble matter (including hydrolysis residue, impurities insoluble in methanol, etc.). Use the resulting filtrate (i.e., hydrolysate) as the loading solution. 4) Load the sample solution into a high-pressure preparative liquid phase for separation in multiple portions, with 10 mL of sample loaded each time. The column length is 10 cm. Use methanol-0.5% phosphoric acid solution as the mobile phase and elute at a constant flow rate of 20 mL / min. The volume ratio of methanol to 0.5% phosphoric acid solution in the mobile phase is 55:45. 5) Under UV detection (detection wavelength 360 nm), fractional collection was performed based on the chromatographic peak elution time. The chromatographic peaks were observed using software. The isoquercitrin product peak rose at 8.4 min. Collection was performed using an automatic fraction collector when the corresponding peak of isoquercitrin began to rise. The collection time started at 8.45 min during the rising phase of the isoquercitrin peak and ended when the peak dropped to one-sixth of its total height. The elution was completed after 30 min, and the sample loading and collection were repeated until the sample solution was completely injected. The collected eluent was used to recover the solvent in a rotary evaporator. After the eluent was concentrated under reduced pressure (at 60°C and a vacuum of 0.09 MPa) until no methanol odor was detected, the precipitated insoluble matter was filtered out, washed with boiling distilled water, and then dried (at 80°C) to obtain 0.63 g of powder. Quantitative analysis by high performance liquid chromatography (HPLC) showed that the content of isoquercitrin in the powder was 98.9%, and the yield of isoquercitrin was calculated to be 0.063% (relative to the dried Polygonum bulbiferum and the total flavonoids yield of 7.3%). Example

[0026] 1) Take 1 kg of dried Polygonum bulbiferum rhizomes, wash them with water, dry them, add them to 12 L of pure water, heat and boil for 1 hour, pour off the boiling liquid and filter out the residue; heat the residue with 95% ethanol solution in 3 portions, each time using 8 L of 95% ethanol solution, heating at 75℃, and refluxing for 2 hours. After reflux, filter and combine the filtrates obtained from each reflux to obtain the ethanol extract. 2) Add 0.6% activated carbon (by weight of the extract) to the ethanol extract and stir at 60℃ for 0.8 hours to decolorize. Then filter out the activated carbon. Adjust the pH of the decolorized ethanol extract to about 11 with 0.6 mol / L sodium hydroxide solution. After standing for 10 hours, remove the precipitate by filtration. Concentrate the filtrate (impurities) under reduced pressure (at 60℃ and a vacuum of 0.10 MPa) to about 1 / 4 of the original volume (6L). Filter out a large amount of precipitated insoluble matter. The filtered insoluble matter is the crude total flavonoids (8.1g). 3) Wash 8.1g of crude total flavonoids with pure water until pH 6-8 (after adding crude total flavonoids to pure water, stir and filter out insoluble matter; repeat this washing process several times). Then dissolve it in 120mL of methanol-5% hydrochloric acid solution (methanol:5% hydrochloric acid solution volume ratio is 50:50), heat (80℃) and reflux for 1 hour. Filter while hot to remove insoluble matter, and use the resulting filtrate as the loading solution. 4) The sample solution was loaded into a high-pressure preparative liquid phase for separation in multiple portions, with 15 mL of sample loaded each time. The column length was 20 cm. Methanol-0.5% phosphoric acid solution was used as the mobile phase, and constant flow elution was performed at a rate of 40 mL / min. The volume ratio of methanol to 0.5% phosphoric acid solution in the mobile phase was 57:43. 5) Under UV detection (detection wavelength 360 nm), the chromatographic peaks were collected in segments according to their elution times. The peaks were observed using software. The isoquercitrin peak rose at 8.0 min. Collection was initiated using an automatic fraction collector when the corresponding isoquercitrin peak began to rise, specifically starting at 8.05 min during the peak's rise phase. Collection ended when the peak decreased to one-sixth of its total height. Elution was completed after 40 min, and the process of loading and collecting was repeated until all sample was injected. The collected eluent was then used to recover the solvent in a rotary evaporator. The eluent was concentrated under reduced pressure (at 60℃ and a vacuum of 0.10 MPa) until no methanol odor remained. The precipitated insoluble matter was filtered out, washed with boiling distilled water, and then dried (at 80℃) to obtain 0.78 g of powder. Quantitative analysis by high-performance liquid chromatography showed that the isoquercitrin content in the powder was 98.6%. Figure 1 Meanwhile, calculations showed that the yield of isoquercitrin was 0.078%. Example

[0027] 1) Take 1 kg of dried Polygonum bulbiferum, wash it with water, dry it and add it to 15 L of pure water. Heat and boil for 2.0 hours. Pour off the water and filter out the residue. Heat the residue with 85% ethanol solution in 3 portions, each time using 12 L of 85% ethanol solution, heating at 80℃ for 2 hours. After reflux, filter and combine the filtrates to obtain the ethanol extract. 2) Add 1.0% activated carbon (by weight of the extract) to the ethanol extract and stir at 55℃ for 0.6 hours to decolorize. Then filter out the activated carbon. Adjust the pH of the decolorized ethanol extract to about 11 with 0.8 mol / L sodium hydroxide solution. After standing for 8 hours, remove the precipitate by filtration. Concentrate the filtrate (impurities) under reduced pressure (at 55℃ and a vacuum of 0.13 MPa) to about 1 / 5 of the original volume (7L). Filter out a large amount of precipitated insoluble matter. The filtered insoluble matter is the crude total flavonoids (7.5g). 3) Wash 7.5g of crude total flavonoids with pure water until pH 6-8 (after adding crude total flavonoids to pure water, stir and filter out insoluble matter; repeat this washing several times). Then dissolve it in 80mL of methanol-5% hydrochloric acid solution (methanol:5% hydrochloric acid solution volume ratio is 50:50), heat (70℃) and reflux for 1 hour. Filter while hot to remove insoluble matter, and use the resulting filtrate as the loading solution. 4) The sample solution was loaded into a high-pressure preparative liquid phase for separation in multiple portions, with 20 mL of sample loaded each time. The column length was 15 cm. Methanol-0.5% phosphoric acid solution was used as the mobile phase, and constant flow elution was performed at a rate of 10 mL / min. The volume ratio of methanol to 0.5% phosphoric acid solution in the mobile phase was 60:40. 5) Under UV detection (detection wavelength 360 nm), fractional collection was performed based on the chromatographic peak elution time. The peaks were observed using software. The isoquercitrin product peak rose at 7.4 min. Collection was initiated using an automatic fraction collector when the corresponding isoquercitrin peak began to rise, specifically starting at 7.45 min during the peak's ascent phase. Collection ended when the peak decreased to one-fifth of its total height. Elution was completed after 30 min, and the process was repeated. Sample loading and collection were carried out until the sample solution was completely injected. The collected eluent was used to recover the solvent in a rotary evaporator. After the eluent was concentrated under reduced pressure (at 50°C and a vacuum of 0.15 MPa) until there was no methanol odor, the precipitated insoluble matter was filtered out, washed with boiling distilled water, and then dried (at 80°C) to obtain 0.75 g of powder. Quantitative analysis by high performance liquid chromatography showed that the content of isoquercitrin in the powder was 98.3%, and the yield of isoquercitrin was calculated to be 0.075%.

[0028] After performing multiple chromatographic separations according to the different examples above, the separation efficiency of the high-pressure preparative liquid chromatography column decreases. In this case, the following measures can be taken to restore the column efficiency: backwash the column with a 5% hydrochloric acid solution at a flow rate of 10% to 20% of the separation flow rate, and then wash the column with a 3% acetonitrile aqueous solution until there are no chloride ions in the eluent.

[0029] (III) Comparative Experiment 1. The effect of water extraction for impurity removal on extraction and separation results The step of removing impurities by "water extraction" is omitted in the following examples, and is compared with Example 2.

[0030] 1) Take 1 kg of dried Polygonum bulbiferum, wash it with water, dry it, and then heat it under reflux for 3 times with 95% ethanol solution. Each time, the amount of 95% ethanol solution used is 8 L, the heating temperature is 75℃, and the reflux time is 2 hours. After the reflux is completed, filter it and combine the filtrates obtained from each time to obtain the ethanol extract. 2) Add 0.6% activated carbon (by weight of the extract) to the ethanol extract and stir at 60℃ for 0.8 hours to decolorize. Then filter out the activated carbon. Adjust the pH of the decolorized ethanol extract to about 11 with 0.6 mol / L sodium hydroxide solution. After standing for 10 hours, remove the precipitate by filtration. Concentrate the filtrate (impurities) under reduced pressure (at 60℃ and a vacuum of 0.10 MPa) to about 1 / 4 of the original volume (6L). Filter out a large amount of precipitated insoluble matter. The filtered insoluble matter is the crude total flavonoids (26.7g). 3) Wash 26.7g of crude total flavonoids with pure water until pH 6-8 (after adding crude total flavonoids to pure water, stir and filter out insoluble matter; repeat this washing several times). Then dissolve it in 350mL of methanol-5% hydrochloric acid solution (methanol:5% hydrochloric acid solution volume ratio is 50:50), heat (80℃) and reflux for 1 hour. Filter while hot to remove insoluble matter, and use the resulting filtrate as the loading solution. 4) The sample solution was loaded into a high-pressure preparative liquid phase for separation in multiple portions, with 15 mL of sample loaded each time. The column length was 20 cm. Methanol-0.5% phosphoric acid solution was used as the mobile phase, and constant flow elution was performed at a rate of 40 mL / min. The volume ratio of methanol to 0.5% phosphoric acid solution in the mobile phase was 57:43. 5) Under ultraviolet detection (detection wavelength 360 nm), the chromatographic peaks were collected in segments according to their elution times. The peaks were observed using software. When the peak corresponding to isoquercitrin began to rise, it was collected using an automatic fraction collector. The collection ended when the peak dropped to one-sixth of its total height. The sample loading and collection were repeated until the sample solution was completely injected. The collected eluent was used to recover the solvent in a rotary evaporator. After the eluent was concentrated under reduced pressure (at 60 °C and a vacuum of 0.10 MPa) until there was no methanol odor, the precipitated insoluble matter was filtered out, washed with boiling distilled water, and then dried (at 80 °C) to obtain 0.56 g of powder. Quantitative analysis by high performance liquid chromatography showed that the isoquercitrin content in the powder was 98.2%, and the isoquercitrin yield was calculated to be 0.056%.

[0031] Comparative analysis revealed that removing the water extraction and impurity removal step increased the total amount of crude flavonoids separated (due to factors including the separation of some impurities that are soluble in both water and alcohol, which could not be removed), and also resulted in a darker color of the crude flavonoids. The number of loading attempts and the loading time increased (approximately three times), which also significantly increased the solvent recovery time. The final product yield decreased (due to increased losses resulting from the increased number of loading attempts), and the resulting product was darker in color, with a yield reduction of 28.2%, although the isoquercitrin content remained relatively unchanged.

[0032] 2. The effect of alkali adjustment on extraction and separation results Preliminary experiments revealed that if sodium hydroxide is not used to adjust the pH of the filtrate to alkaline after decolorization, the most significant impact of the crude total flavonoids obtained from vacuum concentration on downstream processes is high-pressure preparative liquid chromatography (HPLC) separation: as the column pressure increases, residual tannins cause column blockage, reducing column life. Furthermore, the obtained crude total flavonoids are prone to moisture absorption and clumping, hindering subsequent separation operations and ultimately preventing the completion of the entire extraction and separation process.

[0033] 3. The effect of the composition of the acidified methanol reagent (used as a hydrolysis reagent) on the extraction and separation results. Previous experiments have shown that if the methanol-hydrochloric acid solution is prepared using 2% dilute hydrochloric acid, the hydrolysis will be incomplete, resulting in a low yield (approximately 0.046%); however, the concentration of dilute hydrochloric acid above 7% has little impact on the final result.

[0034] 4. The effect of mobile phase composition on extraction and separation results Previous experiments have shown that an excessively high proportion of phosphoric acid solution in the mobile phase will directly affect the column life or even damage it. The above examples demonstrate that slightly increasing or decreasing the proportion of phosphoric acid solution in the mobile phase does not affect the quantity and purity of the product, but directly affects the collection time of the eluted sample.

[0035] The method for extracting and separating isoquercitrin from Polygonum villosa provided by this invention is the first to introduce high-performance liquid chromatography (HPLC) into the extraction and separation process of isoquercitrin. This allows for product separation and precise collection under online monitoring with an ultraviolet detector. It not only obtains the target product (isoquercitrin) in a single separation, but also features a simple and rapid separation process with minimal isoquercitrin loss, short separation time, long-term recyclability of the packing material, and recyclable and reusable solvent. The method provided by this invention can produce isoquercitrin with a purity of over 98%, significantly improves the isoquercitrin yield, and has low operating costs and is environmentally friendly, making it suitable for industrial production.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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 preparing high-purity isoquercitrin from Polygonum bulbiferum, characterized in that, Includes the following steps: Raw material pretreatment and extraction: The Polygonum bulbifera raw material was subjected to water extraction to remove impurities and alcohol extraction in sequence, and the alcohol extract was collected; Impurity removal and total flavonoid enrichment: After decolorization, the pH of the alcohol extract was adjusted to 9-11, the precipitate was allowed to stand and the precipitate was removed. Then the filtrate was concentrated under reduced pressure to precipitate and collect the crude total flavonoids. Acid hydrolysis: The crude total flavonoids were hydrolyzed with an acidified alcohol reagent to obtain a hydrolysate; Chromatographic separation and purification: The hydrolysate was used as the loading solution and separated and purified by high-performance liquid chromatography (HPLC). An octadecylsilane-bonded silica gel column was used as the stationary phase, and a methanol-phosphoric acid aqueous solution was used as the mobile phase for elution. The elution process was monitored online using an ultraviolet detector. Based on the detected isoquercitrin peak signal, the fraction collector was activated when the peak began to rise and stopped collecting when the peak decreased to one-fifth to one-sixth of its height, thus obtaining an elution fraction rich in isoquercitrin. Post-processing: The eluted fraction rich in isoquercitrin was concentrated, washed, and dried to obtain high-purity isoquercitrin.

2. The method according to claim 1, characterized in that, In the raw material pretreatment and extraction steps, the conditions for water extraction to remove impurities are as follows: water is used as the solvent, the liquid-to-solid ratio is 10-15 mL / g, and extraction is performed 1-3 times at 90-100℃, each time for 1-3 hours; the conditions for alcohol extraction are as follows: ethanol solution with a mass fraction of 75% or higher is used as the solvent, the liquid-to-solid ratio is 6-12 mL / g, and extraction is performed 2-3 times at 60-85℃, each time for 1.5-2 hours.

3. The method according to claim 1, characterized in that, In the steps of impurity removal and total flavonoid enrichment, the decolorization treatment involves adding 0.3% to 1.0% of activated carbon by mass to the alcohol extract and stirring and adsorbing at 40 to 80°C for 15 to 60 minutes; the pH adjustment involves adjusting the pH of the decolorized liquid to 9 to 11 using a 0.5 to 1.0 mol / L sodium hydroxide solution and allowing it to stand for 8 to 12 hours.

4. The method according to claim 1, characterized in that, Prior to the acid hydrolysis step, the method further includes washing the crude total flavonoids with water to a pH of 6-8.

5. The method according to claim 1, characterized in that, In the acid hydrolysis step, the acidified alcohol reagent is a solution of methanol and hydrochloric acid solution with a mass fraction of 3%~15% mixed at a volume ratio of (30~60):(40~70); the hydrolysis conditions are: the ratio of crude total flavonoids to acidified alcohol reagent is 1:20~40 g / mL, and the hydrolysis is carried out at 50~80℃ for 0.5~2.0 hours.

6. The method according to claim 1, characterized in that, In the chromatographic separation and purification step, the methanol-phosphoric acid aqueous solution is prepared by mixing methanol and a phosphoric acid aqueous solution with a mass fraction of 0.2%~0.8% at a volume ratio of (55~65):(35~45); the elution flow rate is 10~200mL / min.

7. The method according to claim 1, characterized in that, In the chromatographic separation and purification step, the octadecylsilane-bonded silica gel packing material of the chromatographic column has a particle size of 5~20μm and a pore size of 40~70Å; the column length is 10~30cm and the diameter is 2~15cm.

8. The method according to claim 1, characterized in that, In the chromatographic separation and purification step, the sample is loaded in multiple batches, with each batch containing 10-20 mL of the hydrolysate.

9. The method according to claim 1, characterized in that, In the post-processing step, the washing temperature is 80~100℃, and the drying temperature is 50~80℃.

10. The method according to claim 1, characterized in that, Following the post-processing step, a column regeneration step is also included: when the column efficiency of the chromatographic column decreases, the column is activated and regenerated using a 3% to 7% hydrochloric acid solution, and then the column is rinsed with a 2% to 5% acetonitrile aqueous solution.

Images