Method for extracting and separating isoheteroside in ginkgo biloba
By using surfactant-assisted ultrasonic extraction and anion exchange column chromatography, the extraction and separation conditions were optimized, solving the problems of high energy consumption and low separation efficiency in traditional methods. This achieved efficient extraction and high-purity separation of isopropanol, reducing costs and environmental pollution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGZHOU UNIV
- Filing Date
- 2026-03-02
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, traditional hot solvent extraction methods result in high energy consumption, long production cycles, and difficulty in effectively separating isopropanol and isomers from Lophatherum gracile, posing risks of degradation and solvent residue. Traditional macroporous resin purification technology lacks specificity and is difficult to achieve efficient separation and purification.
Surfactant-assisted ultrasonic extraction combined with anion exchange column chromatography was employed. The extraction system and conditions were optimized through orthogonal experiments. Total flavonoids were extracted using SDS aqueous solution combined with ultrasound, followed by gradient elution on a Q Beads 6FF anion exchange column to achieve efficient separation of isopropanol.
It improved the yield of total flavonoids, significantly enhanced the purity and separation efficiency of isopropanol, simplified the process, reduced costs, and decreased the risk of environmental pollution.
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Figure CN122103107A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product chemistry and traditional Chinese medicine extraction and separation technology. Specifically, it relates to a method for extracting total flavonoids from the leaves of the grass plant Lophatherum gracile and separating the active ingredient isoharmonin using surfactant-assisted ultrasonic extraction technology optimized by orthogonal experimental design. Background Technology
[0002] Isohexin, a core C-glycoside flavonoid found in the leaves of the grass Lophatherum gracile, exhibits remarkable pharmacological activities such as antioxidant, anti-inflammatory, antiviral, and cardiovascular protection due to its unique chemical structure. These properties establish its key position in modern medicine, functional foods, and high-end daily chemical industries. Furthermore, the total flavonoid components associated with Lophatherum gracile can have a synergistic effect with it, further enhancing its development value. Therefore, constructing a highly efficient preparation process for enriching isohexin while also recovering total flavonoids is a prerequisite for realizing the high-value utilization of this resource.
[0003] However, current industrial preparation mainly relies on hot solvent extraction, which commonly uses high-concentration organic solvents. Although this traditional process is mature, it faces serious challenges: prolonged high-temperature operation not only leads to huge energy consumption and lengthy production cycles, but also easily induces the degradation or isomerization of thermosensitive C-glycosides, significantly reducing the yield of the target product; at the same time, dependence on organic solvents also brings safety hazards such as high costs, environmental pollution, and solvent residues.
[0004] Furthermore, the complex impurities in the crude extract are also a key factor restricting product quality. Traditional macroporous resin purification technology is mainly based on physical adsorption, but due to its lack of specificity, it is difficult to achieve effective separation of isopropanol and its isomers. Summary of the Invention
[0005] The present invention aims to overcome the limitations of the prior art and provide a method for the extraction and separation of isoharmonin from Lophatherum gracile. Specifically, it is a method for efficiently extracting total flavonoids from Lophatherum gracile and separating and purifying isoharmonin.
[0006] This invention is achieved through the following technical solution: A method for extracting and separating isoharmonin from Lophatherum gracile, comprising the following steps: (1) Total flavonoids were extracted from Lophatherum gracile leaves using surfactant-assisted ultrasonic extraction technology; (2) The total flavonoids were purified by anion exchange column chromatography to obtain isopropanol.
[0007] Step (1) includes the following steps: S1 single-factor experiments determined the extraction system and the range of conditions for ultrasonic extraction; S2 orthogonal experiments were used to determine the most relevant influencing factors of the extraction system and ultrasonic extraction. S3 was used to extract total flavonoids under the extraction system optimized by orthogonal experimental design and ultrasonic extraction conditions.
[0008] Specifically, in step S1, a single-factor experiment was conducted to investigate the composition of the extraction system, which included an organic solution and raw materials, an aqueous surfactant solution and raw materials; the aqueous surfactant solution was an aqueous solution of sodium dodecyl sulfonate (hereinafter referred to as SDS); the experimental factors of the single-factor experiment included the extraction solution (70% methanol, 70% ethanol, 70% methanol + SDS, 70% ethanol + SDS, SDS aqueous solution), the concentration of the extraction solution (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%), and the liquid-to-solid ratio (20 mL / g, 30 mL / g, 40 mL / g, 50 mL / g, 60 mL / g, 70 mL / g).
[0009] In the extraction system obtained by single-factor screening, the extraction solution was an SDS aqueous solution with a concentration of 0.5-3.0% and a liquid-to-solid ratio of 20-70 mL / g.
[0010] Furthermore, in the extraction system obtained by single-factor screening, the extraction solution was an SDS aqueous solution with a concentration of 1.5%; the liquid-to-solid ratio was 50 mL / g.
[0011] Based on the optimal extraction system obtained through single-factor experiments, further single-factor experiments were conducted on ultrasonic extraction conditions. The experimental factors included: ultrasonic time (20 min, 40 min, 60 min, 80 min, 100 min); ultrasonic temperature (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃).
[0012] The ultrasonic extraction conditions obtained through single-factor experiments were: ultrasonic time of 80 min and ultrasonic temperature of 50 ℃.
[0013] In step S2, a four-factor, three-level orthogonal experimental design was adopted to select the following experimental factors for the extraction system and ultrasonic extraction: SDS aqueous solution concentration (1.0%, 1.5%, 2.0%), liquid-to-solid ratio (40 mL / g, 50 mL / g, 60 mL / g), ultrasonic time (60 min, 80 min, 100 min), and ultrasonic temperature (40 ℃, 50 ℃, 60 ℃). The most relevant factors affecting the extraction system and ultrasonic extraction yield were analyzed.
[0014] In step S3, total flavonoids were extracted using the extraction system optimized by orthogonal experimental design and under ultrasonic extraction conditions. Experiments confirmed that the extraction system and ultrasonic extraction of the present invention yielded more than twice the total flavonoids compared to the initial extraction conditions (1.0 g of 40-mesh bamboo leaf powder, pure water as the extraction solution, a liquid-to-solid ratio of 50 mL / g, ultrasonic heating at 40 ℃ for 1 h, followed by three extractions with 3 times the amount of petroleum ether after filtrate to remove the upper petroleum ether phase and collect the lower solution).
[0015] In step (2), the extracted total flavonoids are filtered through a filter membrane and then loaded onto an anion exchange column for chromatography, followed by gradient elution to prepare isopropanol.
[0016] The stationary packing material for the anion exchange column chromatography described in this invention is Q Beads 6FF.
[0017] The gradient elution described in this invention uses an acidic methanol solution. The acidic methanol solution is a mixture of glacial acetic acid and methanol, with an acetic acid molar concentration of 0.1-1.0 M. Approximately 5-10 column volumes are collected for each gradient, with one tube collected per column volume. The content of isoharmonin is then determined using high-performance liquid chromatography (HPLC). The separation of isoharmonin is optimal when the acidic methanol solution concentration is 0.2 M.
[0018] The total flavonoids extracted according to the present invention should be filtered through a 0.45 μm filter membrane before loading onto anion exchange column chromatography to reduce impurities and prevent column clogging.
[0019] It should be noted that the raw material in this invention is prepared by grinding the Lophatherum gracile medicinal material into 40-mesh Lophatherum gracile powder, and then soaking it in the extraction system.
[0020] The isopropanol and total flavonoids produced by the method of this invention have broad application prospects and promotional value.
[0021] The beneficial effects of this invention are: This invention proposes a method for the efficient extraction of isopropargyl glycosides and total flavonoids from Lophatherum gracile leaves. This method uses an SDS-water solution combined with ultrasonic technology as the extraction method, replacing traditional organic solvent processes, and has the advantages of low cost, high safety, and environmental friendliness.
[0022] After systematically screening the optimal ranges of each key variable through single-factor experiments, this method employed a four-factor, three-level orthogonal experiment to optimize the SDS aqueous solution concentration, solid-liquid ratio, ultrasonic time, and temperature. The optimal process combination was determined to be: SDS concentration of 2.0%, solid-liquid ratio of 40 mL / g, ultrasonic time of 100 min, and ultrasonic temperature of 40℃. The results showed that, compared with the unoptimized process, this method increased the yield of total flavonoids by more than 100%.
[0023] Building upon this foundation, the present invention further proposes a highly efficient purification strategy based on strong anion exchange chromatography. The extracted total flavonoids are dissolved in 50 mM borate buffer (pH 8.5), adsorbed onto a Q Beads 6FF column, and eluted using an acidic methanol gradient. The optimal elution conditions were determined to be 0.2 M acidic methanol solution. After repeated rotary evaporation with methanol to remove boron, a high-purity, pale yellow isoharmonin product was obtained. This opens up a new technical pathway for the efficient and high-purity purification of isoharmonin.
[0024] In summary, the process of this invention is simple, the conditions are mild, the cycle is short, and the reproducibility is good, providing a reliable technical reference for the large-scale preparation of isopropanol and total flavonoids. Attached Figure Description
[0025] Figure 1 The effect of the extraction solution on the yield of total flavonoids extracted from Lophatherum gracile leaves; Figure 2 The effect of SDS aqueous solution concentration on the yield of total flavonoids extracted from Lophatherum gracile leaves; Figure 3 The effect of liquid-to-solid ratio on the yield of total flavonoids extracted from Lophatherum gracile leaves; Figure 4 The effect of ultrasound time on the yield of total flavonoids extracted from Lophatherum gracile leaves; Figure 5 The effect of ultrasonic temperature on the yield of total flavonoids extracted from Lophatherum gracile leaves; Figure 6 The standard curve for isopropanol; Figure 7 HPLC analysis of isopropargyl glycoside obtained by separation and preparation. Detailed Implementation
[0026] The present invention will be further described in detail below with reference to specific embodiments.
[0027] The raw material used in the following specific embodiments of the present invention is light bamboo leaf medicinal material prepared into light bamboo leaf powder with a mesh size of 40.
[0028] The method for extracting total flavonoids in this invention is as follows: Take about 1 g of 40-mesh light bamboo leaf powder, accurately weigh it, place it in a 50 mL stoppered Erlenmeyer flask, add the extraction solution, weigh it, sonicate it for a certain time, cool it, weigh it again, replenish the weight loss with the extraction solution, shake it well, filter it with filter paper, collect the filtrate, extract it 3 times with 3 times the amount of petroleum ether, remove the upper petroleum ether phase, collect the lower layer solution and calculate the total flavonoid yield.
[0029] The method for testing the total flavonoid yield in this invention is as follows: Preparation of rutin standard solution: Accurately weigh 12.5 mg of dried rutin into a 25 mL volumetric flask, add 7.5 mL of ethanol to dissolve, add water to make up to the mark, and shake well to obtain a 0.5 mg / mL rutin standard solution.
[0030] Preparation of the total flavonoid standard curve: Accurately pipette 0.0 mL, 0.5 mL, 0.8 mL, 1.0 mL, 1.3 mL, 1.5 mL, 1.8 mL, and 2.0 mL of rutin standard solution into 10 mL colorimetric tubes. Add 80% ethanol solution to a total volume of 4 mL, add 0.5 mL of sodium nitrite solution, shake well, and let stand for 8 min. Then add 0.5 mL of aluminum chloride solution, shake well, and let stand for 10 min. Add 4.0 mL of sodium hydroxide solution, and dilute to the mark with 80% ethanol solution, shake well, and let stand for 10 min. Prepare a series of standard solutions with concentrations of 0 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 1 mg / mL, 1.3 mg / mL, 1.5 mg / mL, 1.8 mg / mL, and 2.0 mg / mL. Using a reagent blank as a blank, measure the absorbance at 510 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis.
[0031] The reaction system for determining total flavonoids was as follows: 1.0 mL of the test solution was pipetted into a 10 mL colorimetric tube, and 80% ethanol solution was added to a total volume of 4 mL. 0.5 mL of sodium nitrite solution was added, shaken well, and allowed to stand for 8 min. Then, 0.5 mL of aluminum chloride solution was added, shaken well, and allowed to stand for 10 min. 4.0 mL of sodium hydroxide solution was added, and the mixture was brought to the mark with 80% ethanol solution, shaken well, and allowed to stand for 10 min. The absorbance was measured at 510 nm, and the total flavonoid yield was calculated based on the standard curve.
[0032] Preparation of the isoharmonic acid standard curve: Accurately pipette a certain amount of isoharmonic acid standard solution to prepare a series of standard solutions with concentrations of 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL. Inject 10 μL of each solution and perform chromatographic analysis under the specified chromatographic conditions. Plot the standard curve with the peak area on the ordinate and the isoharmonic acid concentration on the abscissa.
[0033] The reaction system for determining isoharmonic acid was as follows: the purified pale yellow isoharmonic acid powder was dissolved in methanol solution and diluted to 100 mL, then filtered through a 0.45 µm filter membrane. 10 μL of the solution was injected, and the concentration of isoharmonic acid in the test solution was calculated based on the standard curve, in mg / mL.
[0034] The purity of isoharonidine is calculated using the following formula:
[0035] C represents the concentration of isopropargyl glycoside in the test solution calculated based on the standard curve, in mg / mL; V represents the volume of the sample to be brought to a final volume, in mL; and M represents the mass of the sample taken, in mg.
[0036] Example 1
[0037] Single-factor experiments were conducted on the extraction system and ultrasonic extraction conditions to extract total flavonoids from Lophatherum gracile leaves, and the effects of the extraction system and ultrasonic extraction on the yield of total flavonoids were investigated.
[0038] (1) Effect of extraction solution on the yield of total flavonoids extracted from Lophatherum gracile leaves
[0039] The extraction solvents were selected from 70% methanol, 70% ethanol, pure water, 70% methanol + 1% SDS, 70% ethanol + 1% SDS, and 1% SDS aqueous solution. Other initial extraction conditions were as follows: 1.0 g of 40-mesh Lophatherum gracile powder, a liquid-to-solid ratio of 50 mL / g, ultrasonic heating at 40℃ for 1 h, and extraction three times with three times the volume of petroleum ether after filtrate extraction. The upper petroleum ether phase was removed, and the lower layer solution was collected. The total flavonoid yield was determined, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the total flavonoid yield reaches its maximum when the extraction solvent is an aqueous SDS solution. Therefore, an aqueous SDS solution was chosen as the extraction solution for subsequent experiments.
[0040] (2) Effect of SDS aqueous solution concentration on the yield of total flavonoids extracted from Lophatherum gracile leaves
[0041] Based on the optimal extraction solution, to further determine the effect of SDS aqueous solution concentration on the yield of total flavonoids extracted from Lophatherum gracile, six concentration levels of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0% were selected for the experiment. Other initial extraction conditions were as follows: 1 g of 40-mesh Lophatherum gracile powder, a liquid-to-solid ratio of 50 mL / g, ultrasonic heating at 40 ℃ for 1 h, followed by filtration and extraction three times with 3 times the amount of petroleum ether. The upper petroleum ether phase was removed, and the lower layer solution was collected. The yield of total flavonoids was determined, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the total flavonoid yield is highest when the SDS aqueous solution concentration is 1.5%, therefore, the SDS aqueous solution concentration of 1.5% was selected.
[0042] (3) Effect of liquid-to-solid ratio on the yield of total flavonoids extracted from Lophatherum gracile leaves
[0043] Based on the optimal extraction solution and its concentration, to further determine the effect of the liquid-to-solid ratio on the yield of total flavonoids extracted from Lophatherum gracile leaves, six levels of liquid-to-solid ratios were selected for experiments: 20 mL / g, 30 mL / g, 40 mL / g, 50 mL / g, 60 mL / g, and 70 mL / g. Other initial extraction conditions were as follows: 1 g of Lophatherum gracile leaf powder, ultrasonic heating at 40 ℃ for 1 h, followed by filtration and extraction three times with 3 times the volume of petroleum ether. The upper petroleum ether phase was removed, and the lower layer solution was collected. The yield of total flavonoids was then determined. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the total flavonoid yield has the maximum value when the liquid-to-solid ratio is 50 mL / g, so the liquid-to-solid ratio of 50 mL / g is selected.
[0044] (4) Effect of ultrasound time on the yield of total flavonoids extracted from Lophatherum gracile leaves
[0045] Based on the optimal extraction solution and its concentration, and the optimal liquid-to-solid ratio, to further determine the effect of ultrasonic time on the yield of total flavonoids extracted from Lophatherum gracile leaves, experiments were conducted at five levels: ultrasonic time of 20 min, 40 min, 60 min, 80 min, and 100 min. Other initial extraction conditions were as follows: 1 g of Lophatherum gracile leaf powder, ultrasonic heating at 40 ℃, and extraction three times with 3 times the volume of petroleum ether after filtrate extraction. The upper petroleum ether phase was removed, and the lower layer solution was collected. The yield of total flavonoids was then determined. The results are as follows: Figure 4 As shown. By Figure 4 As can be seen, the total flavonoid yield reaches its maximum when the ultrasound time is 80 min. Therefore, an ultrasound time of 80 min was chosen.
[0046] (5) Effect of ultrasonic temperature on the yield of total flavonoids extracted from Lophatherum gracile leaves
[0047] Based on the optimal extraction solution and its concentration, optimal liquid-to-solid ratio, and optimal ultrasonic time, to further determine the effect of ultrasonic temperature on the yield of total flavonoids extracted from Lophatherum gracile leaves, experiments were conducted at six ultrasonic temperature levels: 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, and 80 ℃. Other initial extraction conditions were used: 1 g of Lophatherum gracile leaf powder was used; the filtrate was extracted three times with three times the volume of petroleum ether; the upper petroleum ether phase was removed; the lower layer solution was collected; and the yield of total flavonoids was determined. The results are as follows: Figure 5 As shown. By Figure 5 As can be seen, the total flavonoid yield reaches its maximum when the ultrasonic temperature is 50 ℃. Therefore, an ultrasonic temperature of 50 ℃ was chosen.
[0048] The optimal extraction system and ultrasonic extraction conditions obtained above are: extraction solution of 1.5% SDS aqueous solution, optimal liquid-to-solid ratio of 50 mL / g, optimal ultrasonic time of 80 min, and optimal ultrasonic temperature of 50 ℃. Based on these conditions, total flavonoids were extracted from Lophatherum gracile leaves.
[0049] Example 2
[0050] To improve the extraction of total flavonoids from Lophatherum gracile using the single-factor experiments in Example 1, an orthogonal experiment was designed to determine the extraction system and ultrasonic extraction conditions, considering four factors: SDS aqueous solution concentration, liquid-to-solid ratio, ultrasonic time, and ultrasonic temperature. The specific factor levels for the orthogonal experiment are shown in Table 1. An orthogonal array was designed using orthogonal design software, and the experimental results are shown in Table 2.
[0051] Table 1. Factor Level Table for Orthogonal Experiment
[0052] Table 2. Results of Orthogonal Experiments
[0053] This embodiment illustrates that the factors affecting the total flavonoid yield, from most significant to least significant, are: liquid-to-solid ratio > ultrasonic time > SDS aqueous solution concentration > ultrasonic temperature. Therefore, the optimal extraction system and ultrasonic extraction conditions for total flavonoids from *Lophatherum gracile* leaves are: 2.0% SDS aqueous solution, optimal liquid-to-solid ratio of 40 mL / g, optimal ultrasonic time of 100 min, and optimal ultrasonic temperature of 40 °C. To further investigate the effect of the optimized extraction system and ultrasonic extraction conditions on the total flavonoid yield, this embodiment extracts total flavonoids from *Lophatherum gracile* leaf powder under the optimal extraction system and ultrasonic extraction conditions: 1.0 g of 40-mesh *Lophatherum gracile* leaf powder, 2.0% SDS aqueous solution, liquid-to-solid ratio of 40 mL / g, ultrasonic heating at 40 °C for 100 min, filtration, and extraction three times with 3 times the amount of petroleum ether. The upper petroleum ether phase is removed, and the lower layer solution is collected. Using the initial extraction conditions as a control (1.0 g of 40-mesh Lophatherum gracile powder, pure water as the extraction solution, a liquid-to-solid ratio of 50 mL / g, ultrasonic heating at 40 ℃ for 1 h, followed by filtration and extraction three times with 3 times the amount of petroleum ether to remove the upper petroleum ether phase and collect the lower layer solution), total flavonoids were extracted from Lophatherum gracile under the optimized extraction conditions. The total flavonoid yield was 2.71%, which is 131.6% higher than that under the unoptimized conditions (1.17%). This significantly unlocks the production potential of Lophatherum gracile, achieving the desired goal and contributing to the industrial application of total flavonoids.
[0054] Example 3
[0055] The total flavonoid extract from *Lophatherum gracile* leaves was separated and purified using a Q Beads 6FF anion exchange column. The extracted total flavonoid solution was added to 50 mM borate buffer (pH 8.5) as the loading stock solution. A suitable gravity chromatography column was used, the lower shim was installed, and the column and shim were thoroughly rinsed with deionized water. The Q Beads 6FF was mixed thoroughly, and an appropriate amount of the slurry was added to the gravity column (the actual volume of the medium should be half the volume of the suspension). The lower outlet was opened to allow the 20% ethanol protective solution to drain. The packing material was rinsed with deionized water until no ethanol odor remained. After the liquid in the column had drained by gravity, the lower outlet was closed. The column was then equilibrated with 50 mM borate buffer, with a rinsing volume of approximately 5 times the column volume, until the pH of the effluent stabilized at around 8.5.
[0056] The prepared sample stock solution was filtered through a 0.45 μm filter membrane before loading. The sample solution was slowly added to the column wall at a flow rate of approximately 1 mL / min. When the sample level dropped to the surface of the packing material, the column wall was washed with a small amount of 50 mM borate buffer and retained for 2 min to allow for sufficient adsorption. The eluent was collected and analyzed. Loading could be repeated as needed to improve the binding rate. After loading, the column was eluted with 50 mM borate buffer until the eluent was clear and colorless to remove unadsorbed impurities. Gradient elution was then performed using acidic methanol solutions (0.1 M-1.0 M acetic acid) of different concentrations. Approximately 5-10 column volumes were collected for each gradient, with one tube collected per column volume. The content of isopropanol was determined using high-performance liquid chromatography (HPLC). After purification, the remaining retained components were first washed with 1.0 M acidic methanol solution, followed by washing with at least 5 column volumes of 2.0 M NaCl solution, then washing with deionized water for approximately 5 column volumes, and finally washing with 2-3 column volumes of 20% ethanol solution. The solution was then stored at 4°C. A 0.2 M acidic methanol solution concentration yielded ideally separated isoharmonin. The isoharmonin-rich fraction obtained from elution with 0.2 M acidic methanol solution was collected, combined, and transferred to a rotary evaporator flask. The flask was then evaporated under vacuum at 50°C to remove most of the methanol and acetic acid. A suitable amount of methanol solution was added to the concentrate, and the flask was evaporated to dryness. This process was repeated 3-4 times to completely remove residual boric acid. Finally, a pale yellow isoharmonin product with a purity of 87.5% was obtained at the bottom of the flask.
[0057] Example 4
[0058] The total flavonoid extract from *Lophatherum gracile* leaves was separated and purified using an AB-8 macroporous adsorption resin column. The AB-8 macroporous resin was activated by soaking in 95% ethanol for 24 h, and then packed into a chromatography column. The resin column was washed with 5 column volumes of 95% ethanol and an equal volume of deionized water until no ethanol odor was detected in the eluent. The extracted total flavonoid solution was added to the equilibrated AB-8 resin column and washed with 5 column volumes of deionized water, followed by stepwise gradient elution with an ethanol-water solution. The eluents from different concentrations of ethanol were collected, and the isoharonidine content was determined by high-performance liquid chromatography (HPLC). The isoharonidine component was mainly concentrated in the fraction eluted with 70% ethanol. All 70% ethanol eluents rich in the target product were combined, and the ethanol was removed by rotary evaporation at 50 °C. Finally, the product was vacuum dried to obtain a pale yellow isoharonidine product with a purity of 68.8%.
[0059] This invention achieves efficient separation and purification of isoharmonin from total flavonoids in Lophatherum gracile. Based on optimized extraction process, a Q Beads 6FF strong anion exchange column is used for purification. Compared with the traditional AB-8 macroporous resin method (68.8%), the purity of the target product is significantly improved (87.5%), achieving the ideal separation target and providing an efficient technical route for the large-scale preparation of isoharmonin.
[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. For those skilled in the art, any obvious modifications made without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. A method for extracting and separating isoharmonin from Lophatherum gracile, characterized in that, Includes the following steps: (1) Total flavonoids were extracted from bamboo leaves using surfactant-assisted ultrasonic extraction technology; wherein the extraction solvent was an SDS aqueous solution with a mass fraction of 1-3%; (2) The total flavonoids were purified by anion exchange column chromatography to obtain isopropanol.
2. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 1, characterized in that, The specific method of step (1) is as follows: add the powder of light bamboo leaves to the SDS aqueous solution, with a liquid-to-solid ratio of 30-70 mL / g, and extract by ultrasonication at 40-80℃ for 10-100 min; collect the filtrate by solid-liquid separation, and collect the aqueous phase by extraction separation to obtain total flavonoids.
3. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 2, characterized in that, The mass fraction of SDS aqueous solution is 1.0-2.0%.
4. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 2, characterized in that, The material-to-liquid ratio is 40-60 mL / g.
5. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 2, characterized in that, The ultrasound time is 60-100 minutes.
6. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 2, characterized in that, The ultrasonic temperature is 40-60℃.
7. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 2, characterized in that, The specific method of step (1) is as follows: the mass fraction of the SDS aqueous solution is 2.0%, the liquid-to-solid ratio is 40 mL / g, and the solution is ultrasonically heated at 40 °C for 100 min.
8. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 1, characterized in that, The stationary packing material for the anion exchange column chromatography in step (2) is Q Beads 6FF.
9. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 1, characterized in that, Chromatographic purification was performed by gradient elution using acidic methanol solution as the eluent.
10. The method for extracting and separating isoharmonin from Lophatherum gracile according to claim 9, characterized in that, The acidic methanol solution is a mixture of glacial acetic acid and methanol, wherein the molar concentration of acetic acid is 0.1-1.0 M.