Preparation method of baccatin from a medicine source of selaginella

Abstract

The application relates to a preparation method of a bifendate from a medicine material source of Taxodium, which comprises the following steps of medicine material extraction, macroporous adsorption resin column chromatography and solid-liquid extraction. According to the acid property of the phenolic hydroxyl group of the bifendate, the extraction method of alkali extraction and acid precipitation is used to obtain an extract rich in the bifendate, then the macroporous adsorption resin process is combined with the solid-liquid extraction process to purify the extract, and finally the bifendate with the purity of more than 98% is obtained, and the yield can reach 1.319%. The application reduces the use of organic solvents, greatly simplifies the extraction and separation and purification process, is easy to scale up, and has the advantages of simple operation, low cost and high yield.

Description

Technical Field

[0001] This invention relates to a method for preparing flavonoids from Selaginella tamariscina, belonging to the field of traditional Chinese medicine extraction, separation and purification technology. Background Technology

[0002] Taxodium biflavonoids are widely found in plants of the genus *Selaginella*. Literature reports that taxodium biflavonoids possess various biological activities, including anti-inflammatory, antioxidant, antiviral, antitumor, hypoglycemic, neuroprotective, and vasodilatory effects. The inventors' team discovered that taxodium biflavonoids have significant wound-healing activity and can be used to develop drugs that promote wound healing. Taxodium biflavonoids have broad application prospects in the pharmaceutical and health product industries. With increasing emphasis on healthy lifestyles and growing demand for natural medicines and health products, the market demand for taxodium biflavonoids will continue to grow. Since *Selaginella* plants are rich in taxodium biflavonoids, researching and developing a preparation process for high-purity taxodium biflavonoids from *Selaginella* plants has high application value.

[0003] Currently, most methods for extracting cyperine flavonoids from Selaginella tamariscina involve ethanol reflux extraction followed by purification using various chromatography columns. However, these methods require a large amount of organic reagents, high extraction temperatures, multiple extraction cycles, and low extraction purity.

[0004] A search revealed that patent number 201811219566.8 and authorization announcement number CN109400566B discloses a method for extracting and separating high-purity paclitaxel flavonoids from Selaginella species. The method involves extracting the herb using ethanol-water reflux or warm soaking, enriching the paclitaxel flavonoids with macroporous adsorption resin, and further separating them through alkaline methanol dissolution, acid precipitation, and ethanol recrystallization to obtain paclitaxel flavonoids with a purity of over 98%, achieving a process transfer rate of over 50%. While this technical solution represents a significant improvement over previous methods, the poor solubility of paclitaxel flavonoids in organic solvents or pure water results in a low extraction rate with this reflux or warm soaking method. Furthermore, this method requires high-concentration ethanol and heating, leading to high energy consumption and demanding requirements for production equipment and conditions, resulting in high production costs. Additionally, the subsequent recrystallization step in this technical solution results in high losses, yielding only 3.6g of paclitaxel flavonoids from 1kg of herb, indicating a low yield.

[0005] Kang Wenyi et al. reported in their paper "Extraction Process and Activity Study of Acidic Components of Selaginella tamariscina" (Modern Technology of Traditional Chinese Medicine - Fine Chemicals, 2008, 12(25):1201-1205) that they used an alkali-dissolution and acid-precipitation method to extract acidic fractions rich in cypermethrin. The mass fraction of cypermethrin was stable at 10% to 12%, and the process was simple and stable, avoiding the use of large amounts of organic solvents. However, this paper did not further purify the cypermethrin, and did not obtain high-purity cypermethrin monomer compounds, nor could it solve the problem of high recrystallization loss. Summary of the Invention

[0006] The main objective of this invention is to overcome the problems existing in the prior art and provide a method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials. This method can produce high-purity cyperus flavonoids with less loss, higher yield, and is easy to scale up for industrial production.

[0007] The technical solution of this invention to solve its technical problem is as follows:

[0008] A method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials includes the following steps:

[0009] Step 1: Extraction of medicinal materials: The dried Selaginella tamariscina medicinal materials are made into Selaginella tamariscina medicinal material powder; a predetermined amount of Selaginella tamariscina medicinal material powder is soaked in NaOH solution with pH 9.0-13.0 overnight, and then placed in a water bath at 30℃-60℃ for 1-3 hours for warm extraction. The extract is then filtered to obtain the extract; the pH of the extract is adjusted to 1-4 using an acidic solution, and the mixture is allowed to stand at room temperature to settle. The solids are then filtered and dried to obtain the medicinal material extract.

[0010] Step 2: Macroporous adsorption resin column chromatography: Macroporous adsorption resin columns were prepared using macroporous adsorption resin; the herbal extract was dissolved in methanol by ultrasonication and mixed with macroporous adsorption resin of the same type; after dry loading, the sample was eluted with 30-55% ethanol aqueous solution for 5-10 column volumes, and then eluted with 60-80% ethanol aqueous solution for 12-25 column volumes; the elution fractions of 60-80% ethanol aqueous solution were combined and dried under reduced pressure to obtain crude paclitaxel biflavonoids.

[0011] Step 3, solid-liquid extraction: The crude cypermethrin was soaked in an acidic solution with a pH of 1.0 to 3.0 for 1 to 3 hours, wherein the weight of the crude cypermethrin was 20 ± 5 mg and the volume of the acidic solution was 1 mL. The suspension was then filtered through a nylon organic microporous membrane to obtain the solids. The solids were washed with pure water until neutral. The obtained solids were dissolved in methanol and filtered. The obtained solution was concentrated to remove the solvent and obtain the cypermethrin product.

[0012] This method utilizes the acidic nature of the phenolic hydroxyl groups in paclitaxel flavonoids. An extract rich in paclitaxel flavonoids is obtained through alkaline extraction and acid precipitation. The extract is then purified using a combination of macroporous adsorption resin and solid-liquid extraction, ultimately yielding paclitaxel flavonoids with a purity exceeding 98%. 13.19g of paclitaxel flavonoids can be obtained from 1kg of Selaginella tamariscina medicinal materials, achieving a yield of 1.319%. This method reduces the use of organic solvents and greatly simplifies the extraction and purification process, making it easy to scale up for production. It boasts advantages such as simple operation, low cost, and high yield.

[0013] The further improved technical solution of this invention is as follows:

[0014] Preferably, in the first step, the dried Selaginella tamariscina medicinal material is pulverized using a pulverizer to obtain Selaginella tamariscina medicinal material powder; the Selaginella tamariscina medicinal material is Selaginella tamariscina.

[0015] Preferably, in the first step, during soaking, the ratio of Selaginella tamariscina powder to NaOH solution is 1g:10-50mL; the acidic solution is hydrochloric acid solution.

[0016] More preferably, in the first step, during soaking, the ratio of Selaginella tamariscina powder to NaOH solution is 1g: 30-50mL; during warm extraction, the water bath temperature is 50℃-60℃, and the extraction time is 2hr; the pH of the extract is adjusted to 2-3 with an acidic solution.

[0017] By adopting the above preferred scheme, the specific details of the first step can be further optimized, thereby achieving better medicinal material extraction results.

[0018] Preferably, in the second step, the macroporous adsorption resin is one of AB-8, D101, HPD-100, NKA, or X-5.

[0019] More preferably, in the second step, during ultrasonic dissolution, the ratio of medicinal extract to methanol is 1g:8-10mL; during sample mixing, the ratio of medicinal extract weight to macroporous adsorption resin weight is 1:5-7; and the ratio of sample volume to column volume is 1:10-12.

[0020] More preferably, in the second step, after loading the sample, 5 column volumes are eluted with 54% ethanol aqueous solution, and then 12 column volumes are eluted with 60% ethanol aqueous solution.

[0021] By adopting the above preferred scheme, the specific details of the second step can be further optimized, thereby achieving better purification effect by macroporous adsorption resin column chromatography.

[0022] Preferably, in the third step, the pore size of the nylon organic microporous filter membrane is less than 0.22 μm; during the filtration process, it is rinsed several times with the same acidic solution.

[0023] Preferably, in the third step, an acidic solution with a pH of 2.0 to 3.0 is used; the soaking time is 2 hours.

[0024] Preferably, in the third step, the acidic solution is a hydrochloric acid solution.

[0025] By adopting the above preferred scheme, the specific details of the third step can be further optimized, thereby achieving better purification results from solid-liquid extraction.

[0026] Compared with existing technologies, this invention utilizes the acidic phenolic hydroxyl group of paclitaxel flavonoids to obtain an extract rich in paclitaxel flavonoids through an alkaline extraction and acid precipitation method. The extract is then purified using a macroporous adsorption resin process combined with a solid-liquid extraction process, ultimately yielding high-purity paclitaxel flavonoids with a yield far exceeding that of currently reported methods.

[0027] Compared with ethanol reflux extraction, the method of this invention requires fewer extractions, lower extraction temperature, and less energy consumption, reducing the use of organic reagents. It is highly targeted, reducing the extraction rate of other structural components, and yields higher content and purity of Taxodium biflavonoids, thus reducing the workload of subsequent separation and purification. The method innovatively uses solid-liquid extraction to purify crude Taxodium biflavonoids, cleverly utilizing the difference in solubility between the target compound and impurities to achieve separation.

[0028] The method of this invention is easy to scale up for production, avoiding the drawbacks of chromatographic preparation methods which require instruments and equipment and consume a large amount of reagents, and crystallization methods which are time-consuming and leave residual samples in the mother liquor. Attached Figure Description

[0029] Figure 1 This is a standard curve of cypress biflavonoids from Example 1 of the present invention.

[0030] Figure 2 Figure A shows the HPLC detection results of the content of cypermethrin in Example 3 of this invention. Figure B shows the HPLC detection results of a cypermethrin sample and the HPLC detection results of a cypermethrin standard.

[0031] Figure 3 This is an HPLC stacked chromatogram of the paclitaxel biflavonoid sample and the paclitaxel biflavonoid standard in Example 3 of the present invention.

[0032] Figure 4 The sample of Taxodium biflavonoids in Example 3 of this invention 1 H-NMR spectrum.

[0033] Figure 5 The stacking of the paclitaxel flavonoid standard (A) and the paclitaxel flavonoid sample (B) in Example 3 of this invention. 1 H-NMR spectrum.

[0034] Figure 6 This is the mass spectrum of the cypress biflavonoid sample from Example 3 of the present invention. Detailed Implementation

[0035] In specific implementation, the method for preparing cypress biflavonoids from Selaginella tamariscina medicinal materials of the present invention includes the following steps:

[0036] Step 1, Extraction of medicinal materials: Prepare dried Selaginella tamariscina (preferably Selaginella tamariscina) into Selaginella tamariscina powder; take a predetermined amount of Selaginella tamariscina powder, soak it overnight in NaOH solution with pH 9.0-13.0, and then place it in a water bath at 30℃-60℃ (preferably 50℃-60℃) for 1-3 hours (preferably 2 hours) for warm extraction, filter to obtain the extract; adjust the pH of the extract to 1-4 (preferably pH 2-3) with an acidic solution, allow it to settle at room temperature, filter to obtain the solids, and dry to obtain the medicinal extract.

[0037] In this process, dried Selaginella tamariscina medicinal materials are pulverized using a pulverizer to obtain Selaginella tamariscina medicinal material powder; during soaking, the ratio of Selaginella tamariscina medicinal material powder to NaOH solution is 1g: 10-50mL (preferably 30-50mL); the acidic solution is hydrochloric acid solution.

[0038] Step 2: Macroporous adsorption resin column chromatography: Macroporous adsorption resin columns were prepared using macroporous adsorption resin; the herbal extract was dissolved in methanol by ultrasonication and mixed with macroporous adsorption resin of the same type; after dry loading, the sample was first eluted with 30-55% ethanol aqueous solution for 5-10 column volumes, and then eluted with 60-80% ethanol aqueous solution for 12-25 column volumes (preferably eluted with 54% ethanol aqueous solution for 5 column volumes, and then eluted with 60% ethanol aqueous solution for 12 column volumes); the eluted fractions of 60-80% ethanol aqueous solution were combined and dried under reduced pressure to obtain crude cypermethrin.

[0039] Among them, the macroporous adsorption resin is one of AB-8, D101, HPD-100, NKA, or X-5; during ultrasonic dissolution, the ratio of medicinal extract to methanol is 1g: 8-10mL; during sample mixing, the ratio of the weight of medicinal extract to the weight of macroporous adsorption resin is 1: 5-7; and the ratio of sample volume to column volume is 1: 10-12.

[0040] Step 3, solid-liquid extraction: The crude cypermethrin flavonoids were soaked in an acidic solution with pH 1.0-3.0 (preferably pH 2.0-3.0) for 1-3 hours (preferably 2 hours), wherein the weight of the crude cypermethrin flavonoids and the volume of the acidic solution were 20±5 mg: 1 mL. The suspension was then filtered through a nylon organic microporous membrane to obtain the solids. The solids were washed with pure water until neutral. The obtained solids were dissolved in methanol and filtered. The obtained solution was concentrated to remove the solvent to obtain the cypermethrin flavonoids product.

[0041] The nylon organic microporous filter membrane has a pore size of less than 0.22 μm; it is rinsed several times with the same acidic solution during the filtration process; the acidic solution is hydrochloric acid solution.

[0042] Note: The molecular formula of paclitaxel flavonoids is C1 30 H 18 O 10 The chemical structural formula is shown below:

[0043]

[0044] The present invention provides a method for preparing cypermethrin from Selaginella tamariscina medicinal materials. Compared with ethanol reflux extraction, this method requires fewer extraction cycles, lower extraction temperature, and lower energy consumption, reducing the use of organic reagents. It is highly targeted, reducing the extraction rate of other structural components, and yields cypermethrin with higher content and purity, thus reducing the workload of subsequent separation and purification. The method innovatively uses solid-liquid extraction to purify the crude cypermethrin, cleverly utilizing the difference in solubility between the target compound and impurities to achieve separation. This method is easy to scale up for production, avoiding the drawbacks of chromatographic preparation methods which require equipment and consume large amounts of reagents, and crystallization methods which are time-consuming and leave residual samples in the mother liquor.

[0045] Using the method of this invention, 13.19g of paclitaxel flavonoids can be extracted from 1kg of dried Selaginella tamariscina medicinal material, with a yield of 1.319%, which is much higher than that of currently reported methods.

[0046] The present invention will be further illustrated by the following examples, but the invention is not limited to these examples. All reagents involved in the present invention are commercially available. Unless otherwise specified, the experimental methods in the following examples are conventional methods.

[0047] Example 1

[0048] This embodiment describes a method for detecting the content of flavonoids in Taxus chinensis using HPLC.

[0049] The specific details of this embodiment are as follows:

[0050] Accurately weigh 1.00 mg of Taxodium biflavonoid standard (HPLC ≥ 98.0%, Shanghai Yuanye Biotechnology Co., Ltd.) into a 2 mL EP tube, accurately add 1000 μL of methanol, and sonicate for 10 min to obtain a 1.000 mg / mL Taxodium biflavonoid standard solution.

[0051] A 10 μL solution was analyzed by HPLC using an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA). The chromatographic conditions used were as follows:

[0052] Column: Agilent, ZORBAX SB-C18 (4.6×250mm, 5μm).

[0053] Mobile phases: Phase A: phosphoric acid-water (pH = 5.00); Phase B: methanol

[0054] Flow rate: 1.0 mL / min, injection volume: 10 μL, column temperature: 35℃, detection wavelength: 337 nm.

[0055] Gradient elution was used, and the gradient elution procedure is shown in Table 1 below.

[0056] Table 1. HPLC elution gradient

[0057]

[0058] Establishment of the standard curve for cypermethrin:

[0059] Accurately pipette 1.000 mL of a 1.000 mg / mL solution of paclitaxel flavonoids standard, and dilute it to 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 of the original concentration to prepare a series of standard solutions. Inject 10 μL of each solution into the liquid chromatograph under the chromatographic conditions described above and record the chromatograms.

[0060] A standard curve was plotted with peak area on the ordinate and standard solution concentration (mg / mL) on the abscissa. Linear regression was then performed to obtain the regression equation. The results are shown in Table 2. Figure 1 As shown. The linear range of this standard curve is 0.0078 mg / mL to 1.0000 mg / mL, and the linear equation is: Y = 28308X + 244.15, R0 2 =0.9997.

[0061] Table 2. HPLC peak areas of different concentrations of paclitaxel flavonoid standards

[0062]

[0063] Example 2

[0064] This embodiment describes the extraction process of medicinal materials and the macroporous adsorption resin column chromatography process.

[0065] The specific details of this embodiment are as follows:

[0066] (1) Extraction of medicinal materials:

[0067] Accurately weigh 16.000g of NaOH powder (Xilong Scientific Co., Ltd., Shanghai, China) into a 5L beaker, add 4L of pure water to prepare a NaOH solution with pH=13, and set aside. Weigh 100.0g of Selaginella tamariscina powder (obtained by pulverizing dried Selaginella tamariscina using a pulverizer) and soak it in 4L of NaOH solution (pH=13) overnight. Then, place the powder suspension in a 50℃ constant temperature water bath (Tester Instruments Co., Ltd., Tianjin, China) for extraction for 120min. After extraction, filter the residue through gauze, adjust the pH of the filtrate to 3 using concentrated hydrochloric acid solution, let it stand for 2 hours to precipitate, filter it through a Buchner funnel using filter paper as the medium, and dry the filtered solid to obtain Selaginella tamariscina extract. HPLC analysis (i.e., the method in Example 1, the same below) showed that the content of paclitaxel flavonoids in the Selaginella tamariscina extract was 12.08%.

[0068] (2) Macroporous adsorption resin column chromatography:

[0069] Selaginella extract was prepared according to method (1) above. 10.00 g of Selaginella extract was weighed, dissolved in methanol by ultrasonication, and mixed with 60.0 g of D101 macroporous adsorption resin (Samsung Resin Technology Co., Ltd., Anhui, China) before dry loading. The same D101 macroporous adsorption resin was used in the column bed, with a column volume to sample volume ratio of 11:1 and a column volume of approximately 1000 mL. Elution was performed according to the elution gradient shown in Table 3.

[0070] Table 3. Elution gradient of macroporous adsorption resin

[0071]

[0072] Each 0.5 L eluent fraction was collected as a single fraction. HPLC analysis revealed that the content of paclitaxel flavonoids in the 54% ethanol-water eluent was extremely low, below the concentration range of the standard curve. The content and purity of paclitaxel flavonoids were higher in the 1.50 L–12.00 L 60% ethanol-water eluent fractions. A small amount of paclitaxel flavonoids was found in the 100% ethanol eluent fractions.

[0073] The 54% ethanol-water eluent was combined and dried under reduced pressure to obtain 0.931 g of brownish-yellow powder; the 3rd to 24th eluents of the 60% ethanol-water eluent gradient were combined and dried under reduced pressure to obtain 1.473 g of pale yellow powder; the 100% ethanol eluent was combined and dried under reduced pressure to obtain 1.230 g of brown powder.

[0074] Three 10.00 mg aliquots of sample powder were accurately weighed into 10 mL volumetric flasks, and the volume was adjusted to 10 mL with methanol. 10 μL of each sample solution was injected for HPLC analysis. The sample detection results are shown in Table 4: the content of paclitaxel flavonoids in the 54% ethanol-water eluent was low, below the standard curve range (i.e., paclitaxel flavonoids content was below 0.78%); the content of paclitaxel flavonoids in the 60% ethanol-water eluent was 71.89%; and the content of paclitaxel flavonoids in the 100% ethanol eluent was 5.26%.

[0075] Table 4. Sample Information for Macroporous Adsorption Resin Purification

[0076]

[0077] Based on the above experimental results, when purifying with macroporous adsorption resin, 5 BV of impurities can be removed by elution with 54% ethanol-water solvent, followed by collection of fractions 3-24 of 60% ethanol-water solvent, and drying under reduced pressure to obtain crude paclitaxel flavonoids (the paclitaxel flavonoid content was determined to be 71.89%). Finally, regeneration with 100% ethanol solvent allows for the recovery and reuse of the macroporous adsorption resin.

[0078] Example 3

[0079] This embodiment describes the solid-liquid extraction process and the testing of the product.

[0080] The specific details of this embodiment are as follows:

[0081] The crude product of Taxodium biflavonoids (content 71.89%) was prepared according to the method in Example 1.

[0082] Twenty-seven portions of crude flavonoids from Taxus chinensis were accurately weighed and an orthogonal experiment was conducted using hydrochloric acid solution pH (A), hydrochloric acid solution soaking time (B), and the volume of hydrochloric acid solution used (C) as factors (Table 5).

[0083] Table 5. Factor Levels

[0084]

[0085] The experimental procedure was as follows: The crude flavonoids of *Taxus chinensis* were soaked in hydrochloric acid solution, where the pH of the hydrochloric acid solution was factor A, the soaking time was factor B, and the volume of hydrochloric acid solution used was factor C. Afterwards, the sample was filtered using a 0.22 μm nylon organic microporous membrane, and rinsed multiple times with hydrochloric acid solution during filtration. Then, it was rinsed 10 times with purified water (conductivity ≤ 5.0 μS / cm) (Wahaha Group Co., Ltd., Hangzhou, China). The resulting solid was dissolved in methanol in portions and filtered again to obtain the sample solution. The solution was then dried by rotary evaporation to obtain the purified sample, accurately weighed, and its purity was determined by HPLC. The orthogonal experimental results are shown in Table 6.

[0086] Table 6. Results of the Three-Factor Orthogonal Experiment

[0087]

[0088] Based on the comprehensive analysis of the above results, the optimal conditions are A3B3C1, namely: hydrochloric acid solution pH = 3.0, hydrochloric acid solution soaking time = 120 min, and the volume of hydrochloric acid solution used = 1 mL per 20 mg sample. Scale-up verification was performed under these conditions, as detailed below.

[0089] 50.05 mg of crude paclitaxel flavonoids (content 71.89%) was soaked in 2.5 mL of pH 3.0 hydrochloric acid solution for 120 min. Afterwards, the solids were collected by vacuum filtration through a 0.22 μm nylon organic microporous membrane. During filtration, the sample was rinsed 10 times with pH 3.0 hydrochloric acid solution, with each rinse volume being 2 mL. This procedure utilizes the effectiveness of hydrochloric acid solution in dissolving various water-soluble trace impurities adhering to the sample, allowing them to dissolve in the hydrochloric acid solution and pass through the microporous membrane. Simultaneously, the acidic environment minimizes the loss of paclitaxel flavonoids and converts small amounts of paclitaxel flavonoids in salt form into molecular form, thus removing water-soluble impurities while ensuring sample yield.

[0090] The solids were then rinsed 10 times with purified water (conductivity ≤ 5.0 μS / cm), with each rinse consisting of 2 mL of water. This procedure effectively removes any residual hydrochloric acid solution from the sample, preventing it from affecting long-term storage.

[0091] The obtained solid was then dissolved in 2.5 mL of methanol in several portions, and the filtrates were filtered off. This process was repeated 10 times, and the filtrates were combined to obtain the sample solution. The sample solution was then dried by rotary evaporation to obtain 37.31 mg of paclitaxel flavonoids. This procedure removes methanol-insoluble impurities.

[0092] Two more samples of the same crude paclitaxel flavonoids (content 71.89%) were taken and purified using the above method, resulting in a total of three paclitaxel flavonoid samples.

[0093] The following tests will be performed next:

[0094] (1) Accurately weigh 1.00 mg of paclitaxel flavonoid standard (HPLC ≥ 98.0%, Shanghai Yuanye Biotechnology Co., Ltd.) into a 2 mL EP tube, accurately add 1000 μL of methanol, and sonicate for 10 min to obtain a 1.000 mg / mL paclitaxel flavonoid standard solution. Accurately weigh 10.00 mg of the above paclitaxel flavonoid samples into 10 mL volumetric flasks, and dilute to 10 mL with methanol to obtain a 1.000 mg / mL paclitaxel flavonoid sample solution. Take 10 μL of each sample and perform HPLC detection according to the method in Example 1.

[0095] The test results are shown in Table 7 and Figure 2 As shown, the results indicate that the purity of paclitaxel flavonoids in the three samples, determined by the external standard one-point method, was 98.1%, 99.1%, and 99.4%, respectively, with an average purity of 98.9%.

[0096] Table 7. Results of purity test for cypress biflavonoids

[0097]

[0098] (2) Inject 10 μL of the above-mentioned Taxodium biflavonoid standard solution and Taxodium biflavonoid sample solution respectively for HPLC detection according to the method in Example 1, and detect whether the retention times of the two are the same. The results are as follows: Figure 3 As shown, the retention time of the standard of paclitaxel flavonoids was 11.899 min, and the retention time of the paclitaxel flavonoid sample was 11.904 min, which were the same.

[0099] (3) The standard and sample of Taxodium tumefaciens were respectively tested. 1 ¹H NMR detection showed that: the sample of Taxodium biflavonoids... 1 The 1H NMR spectrum (300MHz, DMSO-d6) showed 12 olefin hydrogen signals in the aromatic region and 6 hydrogen signals in the low-field region. Figure 4 (and Table 8). The compounds in this sample 1 The 1H NMR data are consistent with the literature reference values ​​for paclitaxel flavonoids (Table 8). Stacking of paclitaxel flavonoid standards and paclitaxel flavonoid samples. 1 H-NMR spectrum ( Figure 5 It can be seen that both 1 The characteristic peaks of the HNMR spectrum are consistent.

[0100] Table 8. Taxodium flavonoids 1 1H NMR data (300MHz, DMSO-d6)

[0101]

[0102]

[0103] (4) The LC-MS analysis of the flavonoid sample from *Taxus chinensis* (Agilent 6530Q-TOF LC-MS / HDMS, Agilent Technologies, USA) was performed, and the results are as follows: Figure 6 As shown, the quasi-molecular ion peak of Taxodium biflavonoids can be observed at m / z = 539.0997 [M+H]. + The result is consistent with the theoretical calculation value of 539.0973.

[0104] The inventors' team summarized and calculated the specific process production yield based on the actual situation, as shown in Table 9.

[0105] Table 9. Total Yield of Extraction Process of Taxodium flavonoids from Selaginella Species Medicinal Materials

[0106]

[0107] In addition to the above embodiments, the inventors' team, through extensive scalability experiments, ultimately determined the following steps:

[0108] (1) Extraction of medicinal materials:

[0109] Dried Selaginella tamariscina (preferably Selaginella) is pulverized using a pulverizer to obtain Selaginella tamariscina powder. A predetermined amount of Selaginella tamariscina powder is soaked in a NaOH solution with pH 9.0–13.0 (Selaginella tamariscina powder: NaOH solution = 1g: 10–50mL, preferably 30–50mL) overnight, and then warm-extracted in a water bath at 30°C–60°C (preferably 50°C–60°C) for 1–3 hours (preferably 2 hours). The extract is then filtered to obtain the extract. The pH of the extract is adjusted to 1–4 (preferably pH 2–3) using an acidic solution (such as concentrated hydrochloric acid solution), and the extract is allowed to settle at room temperature. The solids are then filtered and dried to obtain the herbal extract.

[0110] (2) Macroporous adsorption resin column chromatography:

[0111] Macroporous adsorption resin columns were prepared using one of the following models: AB-8, D101, HPD-100, NKA, or X-5. The herbal extract was ultrasonically dissolved in methanol (herbal extract: methanol = 1 g: 8–10 mL), and mixed with the same model of macroporous adsorption resin (herbal extract weight: macroporous adsorption resin weight = 1: 5–7). After dry loading (sample volume: column volume = 1: 10–12), the sample was eluted with 30–55% ethanol aqueous solution for 5–10 column volumes, followed by elution with 60–80% ethanol aqueous solution for 12–25 column volumes (preferably eluted with 54% ethanol aqueous solution for 5 column volumes, followed by 60% ethanol aqueous solution for 12 column volumes). The 60–80% ethanol aqueous solution eluates were combined and dried under reduced pressure to obtain crude cypermethrin.

[0112] (3) Solid-liquid extraction:

[0113] The crude cypermethrin was soaked in an acidic solution (such as hydrochloric acid solution) with a pH of 1.0–3.0 (preferably pH 2.0–3.0) for 1–3 hours (preferably 2 hours), wherein the weight of the crude cypermethrin to the volume of the acidic solution was 20 ± 5 mg: 1 mL. The suspension was then filtered through a nylon organic microporous membrane (pore size less than 0.22 μm) to obtain the solids (rinsing several times with the same acidic solution during filtration). The solids were washed with pure water until neutral. The obtained solids were dissolved in methanol and filtered. The obtained solution was concentrated to remove the solvent to obtain the cypermethrin product.

[0114] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials, characterized in that, Includes the following steps: Step 1, Extraction of medicinal materials: The dried Selaginella tamariscina medicinal materials are made into Selaginella tamariscina medicinal material powder; a predetermined amount of Selaginella tamariscina medicinal material powder is soaked in NaOH solution with pH 9.0~13.0 overnight, and then placed in a water bath at 50℃~60℃ for 2 hours for warm extraction, and the extract is obtained by filtration. The pH of the extract was adjusted to 2-3 using an acidic solution, and the mixture was allowed to settle at room temperature. The solids were then filtered and dried to obtain the herbal extract. During soaking, the ratio of Selaginella tamariscina powder to NaOH solution is 1g: 30~50mL; the acidic solution is hydrochloric acid solution; the Selaginella tamariscina medicinal material is Selaginella tamariscina. Step 2: Macroporous adsorption resin column chromatography: Macroporous adsorption resin columns were prepared using macroporous adsorption resin; the herbal extract was ultrasonically dissolved in methanol and mixed with macroporous adsorption resin of the same type; during ultrasonic dissolution, the ratio of herbal extract to methanol was 1 g: 8~10 mL; during mixing, the weight ratio of herbal extract to macroporous adsorption resin was 1: 5~7; after dry loading, the sample was eluted with 30~55% ethanol aqueous solution for 5~10 column volumes, and then eluted with 60~80% ethanol aqueous solution for 12~25 column volumes; the eluted fractions of 60~80% ethanol aqueous solution were combined and dried under reduced pressure to obtain crude cypermethrin biflavonoids, wherein the ratio of sample volume to column volume was 1: 10~12. Step 3, solid-liquid extraction: The crude cypermethrin was soaked in an acidic solution with a pH of 2.0-3.0 for 2 hours, wherein the weight of the crude cypermethrin was 20 ± 5 mg and the volume of the acidic solution was 1 mL; the acidic solution was hydrochloric acid solution; then the suspension was filtered through a nylon organic microporous membrane to obtain the solids, the solids were washed with pure water until neutral, the obtained solids were dissolved in methanol and filtered, and the obtained solution was concentrated to remove the solvent to obtain the cypermethrin product.

2. The method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials according to claim 1, characterized in that, in the first step, dried Selaginella tamariscina medicinal materials are pulverized using a pulverizer to obtain Selaginella tamariscina medicinal material powder.

3. The method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials according to claim 1, characterized in that, In the second step, the macroporous adsorption resin is one of AB-8, D101, HPD-100, NKA, or X-5.

4. The method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials according to claim 1, characterized in that, In the second step, after loading the sample, 5 column volumes were eluted with 54% ethanol aqueous solution, and then 12 column volumes were eluted with 60% ethanol aqueous solution.

5. The method for preparing cyperus flavonoids from Selaginella tamariscina medicinal materials according to claim 1, characterized in that, In the third step, the pore size of the nylon organic microporous filter membrane is less than 0.22 μm; during the filtration process, it is rinsed several times with the same acidic solution.

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