Preparation method of amentoflavone from selaginella medicinal material

Abstract

The invention relates to a preparation method of amentoflavone from a selaginella medicinal material. The preparation method comprises the following steps: medicinal material extraction, macroporous adsorption resin column chromatography and solid-liquid extraction. According to the characteristic that the amentotaxus biflavone has an acidic phenolic hydroxyl group, the extract rich in the amentotaxus biflavone is obtained by utilizing an alkali extraction and acid precipitation extraction method, then the extract is purified by combining a macroporous adsorption resin process with a solid-liquid extraction process, finally the amentotaxus biflavone with the purity of 98% or above is obtained, and the yield can reach 1.319%. The method reduces the use of an organic solvent, greatly simplifies the extraction, separation and purification process, is easy to amplify production, and has the advantages of simplicity in operation, low cost and high yield.

Description

Technical Field

[0001] The invention relates to a method for preparing aconitum biflavonoids from Selaginella medicinal materials, and belongs to the technical field of traditional Chinese medicine extraction, separation and purification. Background Art

[0002] Selaginella flavonoids are widely present in Selaginella plants. Literature reports that Selaginella flavonoids have multiple biological activities such as anti-inflammatory, antioxidant, antiviral, antitumor, hypoglycemic, neuroprotective and vasodilation. The inventor's team found that Selaginella flavonoids has significant activity in promoting wound healing and can be used to develop drugs that promote wound healing. Selaginella flavonoids have broad application prospects in the pharmaceutical and health care products industries. As people pay more attention to healthy lifestyles and the demand for natural medicines and health care products continues to increase, the market demand for Selaginella flavonoids will continue to grow. Selaginella plants are rich in Selaginella flavonoids, and research and development of a preparation process for preparing high-purity Selaginella flavonoids from Selaginella plants has high application value.

[0003] At present, most methods for extracting flavonoids from Selaginella are through ethanol reflux extraction and subsequent purification through various chromatography columns. However, these methods require the use of a large amount of organic reagents, and the extraction temperature is high, the number of extractions is large, and the extraction purity is low.

[0004] After searching, it was found that the invention patent with patent number 201811219566.8 and authorization announcement number CN109400566B disclosed a method for extracting and separating high-purity sedum biflavonoids from Selaginella plants, extracting the medicinal materials by ethanol aqueous solution reflux or warm soaking, enriching sedum biflavonoids by macroporous adsorption resin, and further separating sedum biflavonoids with a purity of more than 98% by alkaline methanol dissolution acid precipitation and ethanol recrystallization process, and the process transfer rate was more than 50%. This technical solution has been greatly improved over the previous method, but due to the poor solubility of sedum biflavonoids in organic solvents or pure water, the extraction rate of this reflux or warm soaking extraction method is not high. Moreover, this method requires the use of high-concentration ethanol and heating, high energy consumption, high requirements for production equipment and production conditions, and high production costs. In addition, the technical solution has a high loss in the subsequent recrystallization step, and 3.6g of sedum biflavonoids product is extracted from 1kg of medicinal materials, with a low yield.

[0005] Kang Wenyi et al. reported in their paper "Study on the Extraction Process and Activity of Acidic Components of Selaginella" (Modernization Technology of Traditional Chinese Medicine - Fine Chemicals, 2008, 12(25): 1201-1205) that the acidic part rich in selaginella biflavonoids was extracted by alkali dissolution and acid precipitation. The mass fraction of selaginella biflavonoids was stable at 10% to 12%. The process was simple and stable, and the use of a large amount of organic solvents was avoided. However, the document did not further purify selaginella biflavonoids, and did not obtain high-purity selaginella biflavonoid monomer compounds, nor could it solve the problem of high recrystallization loss. Summary of the invention

[0006] The main purpose of the present invention is to overcome the problems existing in the prior art and provide a method for preparing sedum biflavonoids from Selaginella medicinal materials, which can prepare high-purity sedum biflavonoids with low loss, high yield and easy industrial scale-up production.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] A method for preparing aconitum biflavonoids from Selaginella medicinal materials, comprising the following steps:

[0009] The first step is to extract medicinal materials: prepare dried Selaginella medicinal materials into Selaginella medicinal material powder; take a predetermined amount of Selaginella medicinal material powder, soak it in a pH 9.0-13.0 NaOH solution overnight, place it in a 30°C-60°C water bath for warm extraction for 1-3 hours, and filter to obtain an extract; use an acidic solution to adjust the pH of the extract to 1-4, let it stand at room temperature for sedimentation, filter to obtain a solid, and dry it to obtain a medicinal material extract.

[0010] The second step is macroporous adsorption resin column chromatography: a macroporous adsorption resin column is prepared by using a macroporous adsorption resin; the medicinal material extract is ultrasonically dissolved with methanol, and the sample is mixed with a macroporous adsorption resin of the same type; after dry loading, 5 to 10 column volumes are first eluted with a 30 to 55% ethanol aqueous solution, and then 12 to 25 column volumes are eluted with a 60 to 80% ethanol aqueous solution; the eluted fractions with 60 to 80% ethanol aqueous solution are combined, and dried under reduced pressure to obtain a crude product of sedum biflavonoids.

[0011] The third step is solid-liquid extraction: soak the crude product of tachyphylla biflavonoids in an acidic solution of pH 1.0-3.0 for 1-3 hours, wherein the weight of the crude product of tachyphylla biflavonoids: the volume of the acidic solution = 20±5 mg: 1 mL; then filter the suspension with a nylon organic microporous filter membrane to obtain the solid, wash the solid with pure water until it is neutral, dissolve the obtained solid with methanol and filter, and concentrate the obtained solution to remove the solvent to obtain the tachyphylla biflavonoids product.

[0012] According to the characteristic that the phenolic hydroxyl group of sedum sedum biflavonoids is acidic, an extract rich in sedum sedum biflavonoids is obtained by an alkali extraction and acid precipitation extraction method, and then the extract is purified by a macroporous adsorption resin process combined with a solid-liquid extraction process, and finally sedum sedum biflavonoids with a purity of more than 98% are obtained. 13.19g of sedum sedum biflavonoids can be obtained from 1kg of Selaginella medicinal materials, and the yield can reach 1.319%. The method reduces the use of organic solvents and greatly simplifies the extraction and separation and purification process, is easy to scale up production, and has the advantages of simple operation, low cost and high yield.

[0013] The technical solution further improved by the present invention is as follows:

[0014] Preferably, in the first step, the dried Selaginella medicinal material is crushed by a powder grinder to obtain Selaginella medicinal material powder; the Selaginella medicinal material is Selaginella.

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

[0016] More preferably, in the first step, during soaking, Selaginella medicinal material powder: NaOH solution = 1g: 30-50mL; during warm soaking extraction, the water bath temperature is 50°C-60°C, and the extraction time is 2hr; the acidic solution is used to adjust the pH of the extract to 2-3.

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

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

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

[0020] More preferably, in the second step, after the sample is loaded, 5 column volumes of 54% ethanol aqueous solution are used for elution, and then 12 column volumes of 60% ethanol aqueous solution are used for elution.

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

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

[0023] Preferably, in the third step, an acidic solution with a pH of 2.0 to 3.0 is used; and 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 detailed features of the third step can be further optimized, thereby achieving a better solid-liquid extraction treatment and purification effect.

[0026] Compared with the prior art, the present invention uses an alkali-extraction and acid-precipitation extraction method to obtain an extract rich in tachyphylla flavonoids based on the property that the phenolic hydroxyl group of tachyphylla flavonoids is acidic, and then uses a macroporous adsorption resin process combined with a solid-liquid extraction process to purify the extract, ultimately obtaining high-purity tachyphylla flavonoids, with a yield far higher than that of currently reported methods.

[0027] Compared with the ethanol reflux extraction method, the method of the present invention has fewer extraction times, lower extraction temperature, less energy consumption, reduces the use of organic reagents, has strong specificity, reduces the extraction rate of other structural types of components, and the extracted tachycarpus flavonoids have higher content and purity, reducing the workload of subsequent separation and purification; the innovative use of solid-liquid extraction method is used to purify the crude tachycarpus flavonoids, and the difference in solubility between the target compound and the impurity is cleverly used to achieve separation.

[0028] The method of the invention is easy to scale up for production, and avoids the drawbacks of the chromatography preparation method requiring instruments and equipment and consuming a large amount of reagents, and the crystallization method being time-consuming and having residual samples in the mother liquor. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 The HPLC test results of the content of tachyphylla biflavonoids in Example 3 of the present invention are shown in Figure A. Figure A is a HPLC test result of a tachyphylla biflavonoids sample, and Figure B is a HPLC test result of a tachyphylla biflavonoids standard.

[0031] Figure 3 This is the HPLC stacked spectrum of the aconitum flavonoids sample and the aconitum flavonoids standard in Example 3 of the present invention.

[0032] Figure 4 The sample of the biflavonoids of Achyranthes bidentata in Example 3 of the present invention is 1 H-NMR spectrum.

[0033] Figure 5 The stacking of the standard product of sedge flavonoids (A) and the sample of sedge flavonoids (B) in Example 3 of the present invention is 1 H-NMR spectrum.

[0034] Figure 6 This is the mass spectrum of the aconitum biflavonoids sample in Example 3 of the present invention. DETAILED DESCRIPTION

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

[0036] The first step is to extract the medicinal material: prepare the dried Selaginella medicinal material (preferably Selaginella) into Selaginella medicinal material powder; take a predetermined amount of Selaginella medicinal material powder, soak it in a pH 9.0-13.0 NaOH solution overnight, place it in a 30°C-60°C (preferably 50°C-60°C) water bath for warm immersion extraction for 1-3 hours (preferably 2 hours), and filter to obtain an extract; use an acidic solution to adjust the pH of the extract to 1-4 (preferably pH 2-3), let it stand at room temperature for sedimentation, filter out the solid, and dry it to obtain a medicinal material extract.

[0037] The dried Selaginella medicinal material is crushed by a powder grinder to obtain Selaginella medicinal material powder; during soaking, Selaginella medicinal material powder: NaOH solution = 1g: 10-50mL (preferably 30-50mL); the acidic solution uses hydrochloric acid solution.

[0038] The second step is macroporous adsorption resin column chromatography: a macroporous adsorption resin column is prepared by using a macroporous adsorption resin; the medicinal material extract is ultrasonically dissolved with methanol, and the sample is mixed with a macroporous adsorption resin of the same type; after dry loading, 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 with 60-80% ethanol aqueous solution are combined and dried under reduced pressure to obtain a crude product of sedum biflavonoids.

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

[0040] The third step is solid-liquid extraction: soak the crude product of tachyphylla biflavonoids in an acidic solution of pH 1.0-3.0 (preferably pH 2.0-3.0) for 1-3 hr (preferably 2 hr), wherein the weight of the crude product of tachyphylla biflavonoids: the volume of the acidic solution = 20±5 mg: 1 mL; then filter the suspension with a nylon organic microporous filter membrane to obtain the solid, wash the solid with pure water until it is neutral, dissolve the obtained solid with methanol and filter, and concentrate the obtained solution to remove the solvent to obtain the tachyphylla biflavonoids product.

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

[0042] Note: The molecular formula of tachyphylla biflavonoids is C 30 H 18 O 10 , the chemical structure is as follows:

[0043]

[0044] The method for preparing sedum biflavonoids from Selaginella medicinal materials of the present invention has fewer extraction times, lower extraction temperature, less energy consumption, reduced use of organic reagents, strong pertinence, reduced extraction rates of components of other structural types, and higher content and purity of the extracted sedum biflavonoids, reducing the workload of subsequent separation and purification; the crude sedum biflavonoids are purified by innovative solid-liquid extraction method, and the difference in solubility of the target compound and impurities is cleverly used to achieve separation. The method of the present invention is easy to scale up production, avoiding the disadvantages of the chromatographic preparation method requiring instruments and equipment and consuming a large amount of reagents, and the crystallization method being time-consuming and having residual samples in the mother liquor.

[0045] By using the method of the present invention, 13.19 g of aconitum biflavonoids can be extracted from 1 kg of dried Selaginella medicinal materials, and the yield can reach 1.319%, which is much higher than the currently reported methods.

[0046] The present invention is further described below by way of examples, but the present invention is not limited to these examples. The reagents involved in the present invention are all commercially available. The experimental methods of the following examples are conventional methods unless otherwise specified.

[0047] Example 1

[0048] This example is a method for detecting the content of aconitum biflavonoids by HPLC.

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

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

[0051] 10 μL of the solution was taken and HPLC was performed using an Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc., USA). The chromatographic conditions used were as follows:

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

[0053] Mobile phase: 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 °C, detection wavelength: 337 nm.

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

[0056] Table 1. HPLC elution gradient

[0057]

[0058] Establishment of standard curve of aconitum biflavonoids:

[0059] Accurately pipette 1.000 mL of 1.000 mg / mL standard solution of Metasequoia gmelinii, and dilute it to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32 of the original concentration to prepare a series of standard solutions. According to the above chromatographic conditions, pipette 10 μL of each solution and inject it into the liquid chromatograph to record the chromatogram.

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

[0061] Table 2. HPLC peak area of ​​different concentrations of sedum biflavonoids standard

[0062]

[0063] Example 2

[0064] This embodiment is a process of extracting medicinal materials and a process of macroporous adsorption resin column chromatography.

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

[0066] (1) Extraction of medicinal materials:

[0067] 16.000g NaOH powder (Xilong Science Co., Ltd., Shanghai, China) was accurately weighed into a 5L beaker, and 4L pure water was added to prepare a NaOH solution with a pH of 13 for use. 100.0g Selaginella medicinal material powder (obtained by crushing dried Selaginella medicinal materials with a powder grinder) was weighed and soaked in 4L NaOH solution (pH = 13) overnight, and then the medicinal material powder suspension was placed in a 50°C constant temperature water bath (Test Instrument Co., Ltd., Tianjin, China) for heating and extraction for 120min. After the extraction was completed, the drug residue was removed by gauze filtration, and the filtrate was adjusted to pH = 3 with concentrated hydrochloric acid solution, and the precipitation was allowed to stand for 2hr. The solid obtained by filtration was dried and filtered using a Buchner funnel with filter paper as the medium to obtain a Selaginella extract. The content of sedum biflavonoids in the Selaginella extract was 12.08% as detected by HPLC (i.e., the detection method of Example 1, the same below).

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

[0069] The Selaginella extract was prepared according to the method (1) above. 10.00 g of Selaginella extract was weighed and dissolved by ultrasonication with methanol. The sample was mixed with 60.0 g of D101 macroporous adsorption resin (Samsung Resin Technology Co., Ltd., Anhui, China) and then dry-loaded. The column bed used the same D101 macroporous adsorption resin, the column volume to sample volume ratio was 11:1, and the column volume was about 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 0.5L eluate was collected as a fraction, and HPLC detection revealed that the content of sedum biflavonoids in the 54% ethanol-water eluate was very low, lower than the concentration range of the standard curve. The content and purity of sedum biflavonoids in the 60% ethanol-water eluate 1.50L to 12.00L were relatively high; a small amount of sedum biflavonoids was present in the 100% ethanol elution fraction.

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

[0074] Weigh three samples of powder (10.00 mg) into a 10 ml volumetric flask, dilute to 10 ml with methanol, and inject 10 μL of each sample solution for HPLC detection. The sample detection results are shown in Table 4: the content of 54% ethanol-water elution fraction is low, lower than the standard curve range (i.e., the content of 54% ethanol-water elution fraction is lower than 0.78%); the content of 54% ethanol-water elution fraction is 71.89%; the content of 54% ethanol-water elution fraction is 5.26%.

[0075] Table 4. Macroporous adsorption resin purification sample information

[0076]

[0077] Based on the above experimental results, it can be seen that when using macroporous adsorption resin for purification, 54% ethanol-water solvent can be used to elute 5BV to remove impurities, and then the 3rd to 24th fractions of 60% ethanol-water solvent are collected and dried under reduced pressure to obtain a crude product of sedum biflavonoids (the content of sedum biflavonoids is 71.89% after testing). Finally, 100% ethanol solvent is used for regeneration, and the macroporous adsorption resin can be recovered for repeated use.

[0078] Example 3

[0079] This embodiment is a solid-liquid extraction process and the detection of the product.

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

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

[0082] 27 portions of crude flavonoids from Metasequoia gmelinii were accurately weighed and an orthogonal experiment was conducted with the pH of the hydrochloric acid solution (A), the soaking time of the hydrochloric acid solution (B), and the volume of the hydrochloric acid solution used (C) as factors (Table 5).

[0083] Table 5. Factor levels

[0084]

[0085] The experimental steps are as follows: soak the crude product of schizonepeta biflavonoids in hydrochloric acid solution, wherein the pH of the hydrochloric acid solution is factor A, the soaking time is factor B, and the volume of the hydrochloric acid solution used is factor C. Afterwards, use a 0.22 μm nylon organic microporous filter membrane for suction filtration, and rinse with hydrochloric acid solution several times during the suction filtration process; then use pure water (conductivity ≤ 5.0 μS / cm) (Wahaha Group Co., Ltd., Hangzhou, China) to rinse 10 times, and add methanol to the solid obtained after filtration to dissolve and filter in batches to obtain a sample solution, and rotary evaporate to obtain a dried post-processing purified sample, accurately weigh and perform HPLC purity detection. The orthogonal test results are shown in Table 6.

[0086] Table 6. Results of three-factor orthogonal test

[0087]

[0088] According to the comprehensive analysis of the above results, the best condition is A 3 B 3 C 1 , that is, the pH of the hydrochloric acid solution is 3.0, the soaking time of the hydrochloric acid solution is 120 minutes, and the volume of the hydrochloric acid solution used is 1 mL of hydrochloric acid solution for every 20 mg of sample. The scale-up verification is carried out under this condition, as follows.

[0089] Take 50.05 mg of crude flavonoids of Metasequoia gracile (content 71.89%) and soak them in 2.5 mL of pH = 3.0 hydrochloric acid solution for 120 minutes. Afterwards, use a 0.22 μm nylon organic microporous filter membrane to filter out the solids. During the filtration process, use a pH = 3.0 hydrochloric acid solution to rinse 10 times, and the volume of each rinse is 2 mL. This operation step uses the hydrochloric acid solution to effectively dissolve various water-soluble trace impurities attached to the sample, dissolving them in the hydrochloric acid solution and passing through the microporous filter membrane; at the same time, the acidic environment can minimize the loss of flavonoids of Metasequoia gracile, and convert a small amount of salt form of flavonoids of Metasequoia gracile into a molecular form, thereby removing water-soluble impurities while ensuring the sample yield.

[0090] Afterwards, the solids were rinsed 10 times with pure water (conductivity ≤ 5.0 μS / cm), with a volume of 2 mL each time. This operation step can rinse away a small amount of residual hydrochloric acid solution in the sample, thus preventing the hydrochloric acid from affecting the long-term storage of the sample.

[0091] Afterwards, 2.5 mL of methanol was added to the solid to dissolve it and the filtrate was filtered out, and the addition was performed 10 times in total. The filtrate was combined to obtain a sample solution, and the sample solution was rotary evaporated and dried to obtain 37.31 mg of aconitum biflavonoids sample. This operation step can remove methanol-insoluble impurities.

[0092] Two more identical crude products of aconitum flavonoids (with a content of 71.89%) were taken and purified using the above method to obtain three aconitum flavonoids samples in total.

[0093] Next, perform the following tests:

[0094] (1) Accurately weigh 1.00 mg of the standard product of schizophyllum biflavonoids (HPLC ≥ 98.0%, Shanghai Yuanye Biotechnology Co., Ltd.) into a 2 mL EP tube, accurately add 1000 μL of methanol, and ultrasonically dissolve for 10 min to obtain a 1.000 mg / mL schizophyllum biflavonoids standard solution. Accurately weigh 10.00 mg of the above schizophyllum biflavonoids samples into a 10 mL volumetric flask, dilute to 10 mL with methanol, and obtain a 1.000 mg / mL schizophyllum biflavonoids 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 showed that the purity of aconitum biflavonoids in three samples detected by external standard one-point method was 98.1%, 99.1%, and 99.4%, respectively, with an average purity of 98.9%.

[0096] Table 7. Purity test results of sedum biflavonoids

[0097]

[0098] (2) 10 μL of the above-mentioned standard solution of sedum biflavonoids and the sample solution of sedum biflavonoids were injected respectively and subjected to HPLC detection according to the method of Example 1 to detect whether the retention times of the two were the same. The results are as follows Figure 3 As shown, the retention time of the standard substance of the flavonoids from Metasequoia gracile is 11.899 min, and the retention time of the flavonoids from Metasequoia gracile sample is 11.904 min, and the retention times of the two substances are the same.

[0099] (3) The standard product of tachyphylla biflavonoids and the sample of tachyphylla biflavonoids were respectively 1 H NMR detection results show that: 1 H NMR spectrum (300 MHz, DMSO-d 6 ) The aromatic region shows 12 olefinic hydrogen signals and the low-field region has 6 hydrogen signals ( Figure 4 and Table 8). 1 The H NMR spectrum data are consistent with the literature reference values ​​of sedum biflavonoids (Table 8). 1 H-NMR spectrum ( Figure 5 ) It can be seen that both 1 The characteristic peaks of HNMR spectrum are consistent.

[0100] Table 8, Aconitifolia biflavonoids 1 H NMR data (300 MHz, DMSO-d 6 )

[0101]

[0102]

[0103] (4) The samples of flavonoids from Aquilegia sutchuenensis were analyzed by LC-MS (Agilent 6530Q-TOF high-resolution mass spectrometer, Agilent Technologies, USA). Figure 6 As shown in the figure, the quasi-molecular ion peak of aconitum biflavonoids m / z = 539.0997 [M+H + ], which is consistent with the theoretical calculated value of 539.0973.

[0104] The inventor team calculated the specific process production yield based on the actual situation as shown in Table 9.

[0105] Table 9. Total yield of the process for extracting sedum biflavonoids from Selaginella medicinal materials

[0106]

[0107] In addition to the above embodiments, the inventor team finally determined the steps as follows through a large number of expansive experiments:

[0108] (1) Extraction of medicinal materials:

[0109] The dried Selaginella medicinal material (preferably Selaginella) is crushed with a powder grinder to obtain Selaginella medicinal material powder; a predetermined amount of Selaginella medicinal material powder is taken, soaked in a pH 9.0-13.0 NaOH solution (Selaginella medicinal material powder: NaOH solution = 1g: 10-50mL, preferably 30-50mL) overnight, placed in a 30°C-60°C (preferably 50°C-60°C) water bath for warm immersion extraction for 1-3hr (preferably 2hr), and filtered to obtain an extract; an acidic solution (such as a concentrated hydrochloric acid solution) is used to adjust the extract to pH 1-4 (preferably pH 2-3), the extract is allowed to stand at room temperature for sedimentation, the solid is filtered to obtain, and the extract is dried to obtain a medicinal material extract.

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

[0111] A macroporous adsorption resin column is prepared by using a macroporous adsorption resin (one of the models AB-8, D101, HPD-100, NKA and X-5); the medicinal material extract is ultrasonically dissolved with methanol (medicinal material extract: methanol = 1 g: 8-10 mL), and the sample is mixed with a macroporous adsorption resin of the same model (the weight of the medicinal material extract: the weight of the macroporous adsorption resin = 1: 5-7); after dry loading (sample volume: column volume = 1: 10-12), 5-10 column volumes are firstly eluted with a 30-55% ethanol aqueous solution, and then 12-25 column volumes are eluted with a 60-80% ethanol aqueous solution (preferably, 5 column volumes are firstly eluted with a 54% ethanol aqueous solution, and then 12 column volumes are eluted with a 60% ethanol aqueous solution); the eluted fractions with the 60-80% ethanol aqueous solution are combined, and dried under reduced pressure to obtain a crude product of scutellaria aviculare biflavonoids.

[0112] (3) Solid-liquid extraction:

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

[0114] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A method for preparing aconitum biflavonoids from Selaginella medicinal materials, characterized in that: The following steps are involved: Step 1, extracting medicinal materials: preparing dried Selaginella medicinal materials into Selaginella medicinal materials powder; taking a predetermined amount of Selaginella medicinal materials powder, soaking it in a pH 9.0-13.0 NaOH solution overnight, placing it in a 30°C-60°C water bath for warm soaking extraction for 1-3 hours, and filtering to obtain an extract; The pH of the extract is adjusted to 1-4 using an acidic solution, and the extract is allowed to settle at room temperature, and the solid matter is filtered out and dried to obtain the medicinal material extract; Step 2, macroporous adsorption resin column chromatography: a macroporous adsorption resin is used to prepare a macroporous adsorption resin column; the medicinal material extract is ultrasonically dissolved with methanol, and the sample is mixed with a macroporous adsorption resin of the same type; after dry loading, 5 to 10 column volumes are first eluted with a 30 to 55% ethanol aqueous solution, and then 12 to 25 column volumes are eluted with a 60 to 80% ethanol aqueous solution; the eluted fractions with 60 to 80% ethanol aqueous solution are combined, and dried under reduced pressure to obtain a crude product of sedum biflavonoids; The third step is solid-liquid extraction: soak the crude product of tachyphylla biflavonoids in an acidic solution of pH 1.0-3.0 for 1-3 hours, wherein the weight of the crude product of tachyphylla biflavonoids: the volume of the acidic solution = 20±5 mg: 1 mL; then filter the suspension with a nylon organic microporous filter membrane to obtain the solid, wash the solid with pure water until it is neutral, dissolve the obtained solid with methanol and filter, and concentrate the obtained solution to remove the solvent to obtain the tachyphylla biflavonoids product.

2. The method for preparing aconitum biflavonoids from a Selaginella medicinal material according to claim 1, characterized in that, in the first step, the dried Selaginella medicinal material is crushed by a powder grinder to obtain Selaginella medicinal material powder; the Selaginella medicinal material is Selaginella.

3. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 1, characterized in that, in the first step, during soaking, the Selaginella medicinal material powder: NaOH solution = 1g: 10-50mL; the acidic solution is a hydrochloric acid solution.

4. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 3, characterized in that: In the first step, during soaking, the Selaginella medicinal material powder: NaOH solution = 1g: 30-50mL; during warm soaking extraction, the water bath temperature is 50°C-60°C, and the extraction time is 2hr; the acidic solution adjusts the extract pH to 2-3.

5. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 1, characterized in that: In the second step, the model of the macroporous adsorption resin is one of AB-8, D101, HPD-100, NKA, and X-5.

6. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 5, characterized in that: In the second step, during ultrasonic dissolution, the medicinal material extract: methanol = 1g: 8-10mL; during sample mixing, the weight of the medicinal material extract: the weight of the macroporous adsorption resin = 1: 5-7; the sample volume loaded: the column volume = 1: 10-12.

7. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 6, characterized in that: In the second step, after loading, the sample was eluted with 54% ethanol aqueous solution for 5 column volumes and then with 60% ethanol aqueous solution for 12 column volumes.

8. The method for preparing aconitum biflavonoids from Selaginella 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; and the membrane is rinsed several times with the same acidic solution during the filtration process.

9. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 1, characterized in that: In the third step, an acidic solution with a pH of 2.0 to 3.0 is used; the soaking time is 2 hours.

10. The method for preparing aconitum biflavonoids from Selaginella medicinal materials according to claim 1, characterized in that: In the third step, the acidic solution is a hydrochloric acid solution.

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