A method for preparing a monkey ear ring

Abstract

This invention provides a method for preparing simian cyclophosphamide, comprising the following steps: loading simian cyclophosphamide extract into a macroporous adsorption resin column, performing first column chromatography using a first eluent to obtain simian cyclophosphamide component; loading the simian cyclophosphamide component into a reversed-phase silica gel column, performing second column chromatography using a second eluent to obtain crude simian cyclophosphamide; and loading the crude simian cyclophosphamide into a gel chromatography column, performing third column chromatography using a third eluent to obtain purified product. This preparation method utilizes a combination of macroporous adsorption resin column, reversed-phase silica gel column, and gel chromatography for purification, along with a specific elution procedure and eluent. The resulting simian cyclophosphamide I has a purity of over 99%, meeting the purity requirements for detection reference standards (>98%) and content determination reference standards (>99%), with an extraction yield of over 50%.

Description

Technical Field

[0001] This invention belongs to the field of compound extraction technology, specifically relating to a method for preparing monkey ear cyclosporine. Background Technology

[0002] Monkey earrings ( Archidendron clypearia (Jack) IC Nielsen is a plant belonging to the genus *Cyclocarya* in the legume family, mainly distributed in southern China, such as Guangdong, Guangxi, and Yunnan. Its branches, leaves, seeds, and bark are used medicinally, making it a distinctive and authentic southern Chinese medicinal herb. According to the Guangdong Provincial Standard for Traditional Chinese Medicine, *Cyclocarya* can be used to treat mastitis, stomach pain, and damp-heat diarrhea. Clinically, it is mainly used to treat upper respiratory tract infections, acute pharyngitis, acute tonsillitis, and acute gastroenteritis. Modern pharmacological studies have shown that *Cyclocarya* extract has the effects of clearing heat and detoxifying, cooling blood and reducing swelling, stopping diarrhea, and removing dampness and astringing sores. Clinically, it is mainly used to treat upper respiratory tract infections, acute pharyngitis, acute tonsillitis, and acute gastroenteritis.

[0003] Monkey earring extract contains a large number of structurally similar flavonoids and flavans, among which 7 are galloyl-substituted flavans. Galloyl terlicinone (abbreviated as 7-caryocycline I) GTDF (Gross Taeniae Flavescentis) is a unique component of plants in the genus *Taeniae*. Currently, the extraction technology for GTDF is patented in CN1305870C. This patent describes adsorption of crude extracts from *Taeniae* onto a polyamide column or macroporous resin column, followed by silica gel column chromatography, and finally recrystallization to obtain GTDF with a purity of over 95%. However, the GTDF obtained by the above method does not meet the purity requirements for detection standards (>98%) and content determination standards (>99%). To conduct research on GTDF, such as identification, testing, and content determination, there is an urgent need to develop a rapid and efficient method for separating and purifying GTDF to achieve a purity of over 98%. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide a method for preparing monkey ear cyclodextrin.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a method for preparing monocyclonine, wherein the monocyclonine is 7-galloylterlicinonane; The preparation method of the monkey ear cyclophosphamide includes the following steps: S1. Extract the pulverized monkey earring medicinal material by soaking it in a polar solvent to obtain the extract; S2. The extract is packed into a macroporous adsorption resin column and separated by first column chromatography using the first eluent to obtain the monkey ear cyclosporine component. The first eluent includes an ethanol-containing solution with a volume percentage of 30-100%. S3. The monkey ear cyclophosphine component is packed into a reversed-phase silica gel chromatographic column and separated by second column chromatography using a second eluent to obtain crude monkey ear cyclophosphine. The second eluent includes a methanol-containing solution with a volume percentage of 20-50%. S4. The crude monkey ear cyclophosphine is loaded into a gel chromatography column and separated by third column chromatography using a third eluent to obtain the purified product. The third eluent includes a methanol-containing solution with a volume percentage of 90-100%. The purified product was concentrated to obtain pure monkey ear cyclosporine.

[0007] The structural formula of the monkey ear cyclophosphine is shown in Formula A: Formula A.

[0008] Preferably, in step S1, the polar solvent includes at least one of water, methanol, ethanol, and ethyl acetate.

[0009] More preferably, in step S1, the polar solvent is an aqueous methanol solution or an aqueous ethanol solution. Even more preferably, the volume percentage of ethanol in the aqueous ethanol solution is 90%-100%.

[0010] Preferably, in step S1, the extraction time is 5-10 days.

[0011] Preferably, in step S1, the extraction temperature is 15-40℃.

[0012] Preferably, in step S1, the extraction is performed 1-5 times.

[0013] Preferably, in step S1, the mass-to-volume ratio of the monkey earring medicinal material to the polar solvent is 1 kg:(3-6)L.

[0014] Preferably, in step S1, the particle size of the pulverized monkey earring medicinal material is less than 1 mm.

[0015] More preferably, in step S1, the particle size of the pulverized monkey earring medicinal material is less than 0.8 mm.

[0016] Preferably, step S2 specifically includes the following steps: preparing a loading solution with ethanol, wherein the concentration of the loading solution is 3-15 mg / mL, and loading the loading solution into a macroporous adsorption resin column.

[0017] More preferably, the concentration of the loading solution is 6, 7, 8, 9, 10, 11, 12, 13 or 14 mg / mL.

[0018] More preferably, the sample loading flow rate of the sample loading solution is 0.5-2 mL / min.

[0019] More preferably, the loading volume of the loading solution is 90-110 mL. Alternatively, the loading volume of the loading solution is 3 BV (1 BV is one bed volume of eluent).

[0020] Preferably, the diameter-to-height ratio of the macroporous adsorption resin column is 1:(3-10).

[0021] Preferably, in step S2, the macroporous resin of the macroporous adsorption resin column is macroporous polymethyl methacrylate.

[0022] More preferably, the macroporous resin has a particle size range of 0.56-0.71 mm.

[0023] More preferably, the surface area of ​​the macroporous resin is ≥380m². 2 / g.

[0024] Preferably, in step S2, the first column chromatography separation includes: performing staged gradient elution using a first eluent, wherein the staged gradient elution includes a first stage elution and a second stage elution; the eluent for the first stage elution is an aqueous ethanol solution with a volume percentage of 30% to 40%; and the eluent for the second stage elution is an aqueous ethanol solution with a volume percentage of 60% to 80%. The elution products containing monocyclonol obtained from the first and second elution stages were combined to obtain the monocyclonol component.

[0025] More preferably, the eluent for the first stage of elution is an aqueous ethanol solution with a volume percentage of 40%.

[0026] More preferably, the eluent for the second stage of elution is an aqueous ethanol solution with a volume percentage of 70%.

[0027] More preferably, the amount of eluent used in the first stage of elution is 1-3 BV.

[0028] More preferably, the amount of eluent used in the second stage of elution is 3-8 BV.

[0029] More preferably, the flow rate of the eluent in the second stage of elution is 0.5-2 mL / min.

[0030] Preferably, in step S3, the reversed-phase silica gel column is a C18 reversed-phase silica gel column.

[0031] Preferably, step S3 specifically includes the following steps: preparing a loading solution by dispersing the monocyclocycline component with methanol, wherein the concentration of the loading solution is 2-10 mg / mL, and loading the loading solution into a reversed-phase silica gel column.

[0032] More preferably, the loading volume of the loading solution is 120-150 mL. Alternatively, the loading volume of the loading solution is 4 BV.

[0033] Preferably, the diameter-to-height ratio of the reversed-phase silica gel column is 1:(3-6).

[0034] Preferably, in step S3, the second column chromatography separation includes: performing staged gradient elution using a second eluent, wherein the staged gradient elution includes a first stage elution and a second stage elution; the eluent for the first stage elution is a methanol aqueous solution with a volume percentage of 20% to 30%; and the eluent for the second stage elution is a methanol aqueous solution with a volume percentage of 30% to 40%. The elution products containing simian cyclophosphine obtained from the first and second elution stages were combined to obtain crude simian cyclophosphine.

[0035] More preferably, the eluent for the first stage of elution is a methanol aqueous solution with a volume percentage of 20%.

[0036] More preferably, the eluent for the second stage of elution is a methanol aqueous solution with a volume percentage of 30%.

[0037] More preferably, the amount of eluent used in the first stage of elution is 3 BV.

[0038] More preferably, the amount of eluent used in the second stage of elution is 3-7 BV.

[0039] Preferably, the gel chromatography column is a dextran gel chromatography column.

[0040] Preferably, the third column chromatography separation includes isocratic elution using a third eluent, wherein the third eluent is methanol.

[0041] The beneficial effects of this invention are: This invention provides a method for preparing monocyclonine, which employs a combination of macroporous adsorption resin column, reversed-phase silica gel column and gel chromatography column for purification, and uses a specific elution procedure and eluent. The resulting monocyclonine I has a purity of over 99%, meeting the purity requirements for reference standards for detection (>98.5%) and content determination (>99%), and the extraction yield is over 50%. Attached Figure Description

[0042] Figure 1This is a chromatogram of the sample after purification with macroporous resin; Figure 2 The chromatogram of the sample after purification using a C18 silica gel column is shown. Figure 3 The image shows the chromatogram of the sample after purification using dextran gel LH-20. Detailed Implementation

[0043] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0044] The monkey ear cyclosporine I used in the following examples is 7-galloylterylsulforane.

[0045] The following information is provided regarding the instruments and reagents used in the experiment: AB-8, XAD-7HP, XAD-16N, XAD-2, HPD-600, HPD-722 macroporous adsorption resins (Zhengzhou Hecheng New Material Technology Co., Ltd.); C18 reverse silica gel (12nm, S-50nm, YMC Corporation, Japan); dextran gel LH-20 (Shanghai Yuanye Biotechnology Co., Ltd.).

[0046] The sample of *Hylocereus undatus* (monkey earring) medicinal material was collected from the Guangzhou Laitai Pharmaceutical Monkey Earring Base in Maogang Village, Zhongluotan Town, Baiyun District, Guangzhou. Professor Yang Depo of the School of Pharmaceutical Sciences, Sun Yat-sen University, identified it as *Hylocereus undatus*, a plant belonging to the genus *Hylocereus* of the Fabaceae family. Its Latin name is... Archidendron clypearia (Jack) IC Nielsen.

[0047] Example 1 This embodiment provides a preparation process for monkey ear cyclophosphamide I, and the specific preparation process is as follows: (1) Extraction: Crush an appropriate amount of dried monkey ear ring leaves and branches (<2cm) with a pulverizer and pass them through a 24-mesh sieve. Weigh 5kg of the herbal powder and soak it in 95% ethanol aqueous solution at room temperature (7 days / time, 20L each time, 3 extractions). Combine the obtained extracts and evaporate the extracts under reduced pressure to obtain 900g of total extract.

[0048] (2) Column chromatography enrichment process of monkey ear cyclosporin I: The extract obtained in step (1) was dissolved in 30% ethanol to prepare a 10 mg / mL solution. The 3 BV solution was loaded into an XAD-7HP macroporous adsorption resin column at a loading rate of 1 mL / min for adsorption. After elution with 2 BV 40% ethanol, 4 BV 70% ethanol was used as the eluent and eluted at a rate of 1 mL / min. The eluent was collected, concentrated under reduced pressure, and dried to obtain monkey ear cyclosporin I fraction.

[0049] (3) Preparation process of crude monocytin I: For the monocytin I component obtained in step (2), dissolve it in 20% methanol to prepare a 5 mg / mL solution, adsorb it onto a reverse silica gel column with 4 BV solution, elute with 3 BV 20% methanol, elute with 3 BV 30% methanol as the eluent, collect the eluent, concentrate under reduced pressure, and dry to obtain crude monocytin I.

[0050] (4) Purification process of monocytin I: The crude monocytin I obtained in step (3) was dissolved in methanol and filtered through a 0.22 μm filter membrane. It was then adsorbed onto a dextran gel LH-20 chromatography column with a top diameter to height ratio of 1:20 using a wet loading method. The loading mass: gel mass = 1:100. The elution solvent was 4 BV methanol solution, and elution was carried out at a volumetric flow rate of 0.5 mL / min. The solution was concentrated under reduced pressure and dried to obtain pure monocytin I.

[0051] HPLC analysis showed that the purity of the prepared monkey cloxacin I was 99.21%, and the yield was 60.69%.

[0052] Example 2 This embodiment provides a preparation process for monkey ear cyclophosphamide I, and the specific preparation process is as follows: (1) Crush an appropriate amount of dried monkey ear ring leaves, branches (<2cm) and flowers with a pulverizer and pass them through a 24-mesh sieve. Weigh 5kg of the medicinal powder and extract it by soaking in 70% methanol aqueous solution at room temperature (7 days / time, 20L each time, 3 extractions). Combine the obtained extracts and evaporate the extracts under reduced pressure to obtain a total extract of 940g.

[0053] (2) Column chromatography enrichment process of monkey ear cyclosporin I: The extract obtained in step (1) was dissolved in 30% ethanol to prepare a 5 mg / mL solution. The 4 BV solution was loaded into an XAD-7HP macroporous adsorption resin column at a loading rate of 1 mL / min for adsorption. After elution with 3 BV 40% ethanol, 4 BV 70% ethanol was used as the eluent and eluted at a rate of 1 mL / min. The eluent was collected, concentrated under reduced pressure, and dried to obtain monkey ear cyclosporin I fraction.

[0054] (3) Preparation process of crude monocytin I: For the monocytin I component obtained in step (2), dissolve it in 20% methanol to prepare a 5 mg / mL solution, adsorb it onto a reverse silica gel column with 3 BV solution, elute with 3 BV 20% methanol, elute with 4 BV 30% methanol as the eluent, collect the eluent, concentrate under reduced pressure, and dry to obtain crude monocytin I.

[0055] (4) Purification process of monocytin I: The crude monocytin I obtained in step (3) was dissolved in methanol and filtered through a 0.22 μm filter membrane. It was then adsorbed onto a dextran gel LH-20 chromatography column with a top diameter to height ratio of 1:17 using the wet loading method. The sample mass: gel mass = 1:50. The elution solvent was 4 BV methanol solution, and elution was performed at a volumetric flow rate of 0.5 mL / min. The elution was detected by high performance liquid chromatography, concentrated under reduced pressure, and dried to obtain pure monocytin I.

[0056] The purity of the prepared monkey cyclophosphine I was 99.01%, and the yield was 50.69%.

[0057] Screening experiment of macroporous adsorption resin (1) Pretreatment According to the literature, appropriate amounts of AB-8, XAD-7HP, XAD-16N, XAD-2, HPD-600, and HPD-722 resins were soaked in distilled water for 3 hours to remove impurities and broken resins. The resins were then soaked in 95% ethanol for 24 hours to allow them to fully swell. Finally, the resins were rinsed with distilled water until there was no alcohol odor.

[0058] (2) Static adsorption test Weigh 2g of each of the seven resins, make three parallel portions, and place them in 50mL Erlenmeyer flasks. Add 30mL of 0.05 / 0.2 / 0.5g monkey ear extract, shake at 37℃ and 100r / min for 24h, then filter. Determine the content of monkey ear cyclin I, and calculate the adsorption rate and specific adsorption capacity. Wash the saturated resin with an appropriate amount of distilled water, filter dry, place in an Erlenmeyer flask, add 30mL of anhydrous ethanol, and elute at a constant temperature for 24h. Collect the eluent, determine the content of monkey ear cyclin I, and calculate the desorption rate and specific desorption capacity.

[0059] The formulas are as follows: ① Adsorption rate = [(C1-C2) / C1]×100%, ② Specific adsorption capacity = [C1V1-C2V2) / M, ③ Desorption rate = [(C3V3) / (C1V1-C2V2)]×100%, ④ Specific desorption capacity = C3V3 / M; where C1 is the mass concentration of cyclophosphamide I before resin adsorption, C2 is the mass concentration of cyclophosphamide I after resin adsorption, C3 is the mass concentration of cyclophosphamide I after resin desorption, V1 is the volume of sample liquid before resin adsorption, V2 is the volume of sample liquid after resin adsorption, V3 is the volume of eluent, and M is the mass of resin.

[0060] The results are shown in Table 1. It can be seen that the XAD-7HP resin had the highest adsorption rate, while the desorption rate showed no significant difference; therefore, it was chosen for the separation and purification of monkey cloxacin I.

[0061] Table 1. Results of static adsorption tests on seven macroporous adsorption resins

[0062] Optimization of macroporous adsorption resin column chromatography process (1) Sample concentration Five portions (20g each) of pretreated XAD-7HP macroporous adsorption resin were weighed and slowly added to the adsorption column using a wet method. 200mL of sample solutions with concentrations of 1, 2, 5, 10, and 15 mg / mL were measured and loaded at a flow rate of 1 mL / min. Dynamic adsorption occurred within the resin column, and unadsorbed liquid was collected. The samples were then analyzed by high-performance liquid chromatography (HPLC), and the specific adsorption capacity and adsorption rate were calculated. The results are shown in Table 2. It can be seen that as the mass concentration of the sample solution increases, the specific adsorption capacity increases, but the leakage also increases, and the adsorption rate decreases, thus reducing adsorption efficiency and increasing production costs. Therefore, a sample solution mass concentration of 10 mg / mL was ultimately selected.

[0063] Table 2 Screening results of macroporous resin loading mass concentration

[0064] (2) Diameter-to-height ratio screening Four portions (20g each) of pretreated XAD-7HP macroporous adsorption resin were weighed and slowly added to glass columns with diameter-to-height ratios of 1:3, 1:4, 1:6, and 1:8 using a wet method. 200mL of a 10mg / mL sample solution was loaded at a flow rate of 1mL / min, allowing for dynamic adsorption within the resin column. Unadsorbed liquid was collected. The adsorption capacity and rate were determined under high-performance liquid chromatography (HPLC), and the results are shown in Table 3. It can be seen that a larger diameter-to-height ratio results in a higher adsorption rate, but the overall change is not significant. The optimal diameter-to-height ratio was ultimately determined to be 1:8.

[0065] Table 3 Screening results of macroporous resin diameter-to-height ratio

[0066] (3) Sample loading volume flow rate Four portions (20g each) of pretreated XAD-7HP macroporous adsorption resin were weighed and slowly added to a glass column with a diameter-to-height ratio of 1:8 using a wet method. 200mL of a 10mg / mL sample solution was added, and the samples were loaded at flow rates of 0.5, 1, 2, and 5mL / min, respectively. Dynamic adsorption was observed within the resin column, and unadsorbed liquid was collected. The adsorption rate was calculated. The results are shown in Table 4. It can be seen that the adsorption rate decreases significantly when the mass flow rate exceeds 1mL / min. For efficiency considerations, a flow rate of 1mL / min was chosen.

[0067] Table 4 Screening Results of Macroporous Resin Loading Volumetric Flow Rate

[0068] (4) Sample loading volume Weigh 20g of pretreated XAD-7HP macroporous adsorption resin (diameter-to-height ratio 1:8), and measure 200mL of a 10mg / mL loading solution. Load the resin at a flow rate of 1mL / min for dynamic adsorption within the resin column. Collect 10mL of eluent from each tube and perform high-performance liquid chromatography (HPLC) analysis. The results are shown in Table 5. It can be seen that when the loading volume is 100mL, the leakage reaches 10% of the original mass concentration of cyclophosphamide I. When the mass concentration of this component in the eluent is 10% of the loading solution, the leakage point can be considered reached. Therefore, 100mL (3BV) was ultimately selected as the loading volume.

[0069] Table 5. Mass concentration of cyclodextrin I collected from each tube of monkey.

[0070] (5) Screening of eluent volume fraction Weigh 20g of pretreated XAD-7HP macroporous adsorption resin (diameter-to-height ratio 1:8), and measure 100mL of 10mg / mL loading solution. Load the sample at a flow rate of 1mL / min for dynamic adsorption within the resin column. Elute with 30%, 40%, 50%, 60%, 70%, 80%, and 90% 3BV anhydrous ethanol at a flow rate of 1mL / min, collect the eluent, and calculate the desorption rate.

[0071] Therefore, it can be seen that 50% and 60% ethanol can elute most of this component, while 80%, 90%, and anhydrous ethanol can hardly elute it. Taking into account the maximum enrichment effect of moniliformin I and the green and economical use of solvent, it was finally determined that after removing impurities with 40% (volume fraction) ethanol, elution should be carried out with 70% (volume fraction) ethanol.

[0072] Table 6 Screening results of macroporous resin eluent volume fraction

[0073] (6) Volume of impurity removal liquid The sample was loaded and placed according to the method described in section (5). Elution was performed with 40% anhydrous ethanol at a flow rate of 1 mL / min. The eluent was collected for each column volume, and the desorption rate was calculated. It was found that significant leakage of the compound occurred when the eluent volume reached 3 BV. Therefore, a final selection of 2 BV was made for the amount of purging solution (40% ethanol).

[0074] Table 7 Results of volume screening for macroporous resin impurity removal solution

[0075] (7) Eluent volume The column was packed, sample loaded, and impurities removed according to the method in section (5). Elution was performed with 70% ethanol at a volumetric flow rate of 1 mL / min. The eluent was collected for each column volume, and the desorption rate was calculated. The results are shown in Table 8. It can be seen that the desorption rate gradually stabilizes when the amount used is greater than 4 BV. Considering the green and economical use of solvent, 4 BV was finally selected.

[0076] Table 8 Results of volume screening of macroporous resin eluent

[0077] (8) Eluent volumetric flow rate The column was packed, sample loaded, and impurities removed according to the method described in section (5). Elution was performed with 4 BV 70% ethanol at flow rates of 0.5, 1, 2, and 5 mL / min, respectively. The desorption rates were calculated, and the results are shown in Table 9. It can be seen that the elution rate decreases significantly when the mass flow rate is higher than 1 mL / min. For efficiency considerations, 1 mL / min was chosen.

[0078] Table 9 Screening Results of Macroporous Resin Eluent Volumetric Flow Rate

[0079] (9) Verification test Based on the results of the single-factor experiments, the optimal process was determined to be a diameter-to-height ratio of 1:8, a sample loading volume of 3 BV, a flow rate of 1 mL / min, a mass concentration of 10 mg / mL, a purification liquid fraction of 40% and a volume of 2 BV, and an elution liquid fraction of 70% and a volume of 4 BV and a flow rate of 1 mL / min. Three batches of validation experiments were conducted using the optimized process, and the results are shown in Table 10, indicating that the process is stable and feasible.

[0080] Table 10 Results of macroporous resin validation tests (n=3)

[0081] Optimization of reverse silica gel column chromatography process (1) Silicone pretreatment Take an appropriate amount of C18 reverse silicone and soak it in methanol.

[0082] (2) Sample concentration Weigh out four portions of pretreated C18 silica gel, each 20g, and slowly add them to the adsorption column using a wet method. Take 200mL of sample solution at concentrations of 1, 2, 5, and 10 mg / mL respectively, and allow dynamic adsorption within the resin column. Collect the unadsorbed liquid, and perform high-performance liquid chromatography (HPLC) to determine the specific adsorption capacity and adsorption rate. The results are shown in Table 11. It can be seen that as the mass concentration of the sample solution increases, the specific adsorption capacity increases, but the leakage also increases, and the adsorption rate decreases, thus reducing the adsorption efficiency. Considering both the specific adsorption rate and the adsorption capacity, a sample solution mass concentration of 5 mg / mL was ultimately selected.

[0083] Table 11 Screening Results of C18 Sample Mass Concentration

[0084] (3) Diameter-to-height ratio Four portions (20g each) of pretreated C18 resin were weighed and slowly added to glass columns with diameter-to-height ratios of 1:3, 1:4, and 1:6 using a wet method. 200mL of a 5mg / mL sample solution was measured and allowed to dynamically adsorb within the resin column. Unadsorbed solution was collected. The adsorption capacity and adsorption rate were determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 12. This indicates that the adsorption rate does not change significantly with increasing diameter-to-height ratio.

[0085] Table 12 Screening Results of C18 Diameter-to-Height Ratio

[0086] (4) Sample loading volume screening Weigh 20g of pretreated C18 resin and measure 200mL of 5mg / mL loading solution for dynamic adsorption within the resin column. Collect 10mL of eluent from each test tube, and determine the leakage using high-performance liquid chromatography (HPLC). Plot the leakage curve, and the results are shown in Table 13. It can be seen that when the loading volume is 140mL, the leakage is 10% of the original mass concentration of cyclophosphamide I. The leakage point can be considered reached when the mass concentration of this component in the eluent is 10% of the loading solution. Finally, 130mL (4BV) was selected as the loading volume.

[0087] Table 13. Mass concentrations of cyclodextrin I collected from each tube of monkey.

[0088] (5) Eluent volume fraction Weigh 20g of pretreated C18 resin and measure 130mL of 5mg / mL loading solution for dynamic adsorption in the resin column. Elute with 3BV 20%, 30%, 40%, and 50% methanol, respectively, collect the eluent, and calculate the desorption rate.

[0089] Therefore, it can be seen that 30% methanol can elute most of this component, while 40% and 50% methanol can hardly elute it. Taking into account the maximum enrichment effect of moniliformin I and the green and economical use of solvent, it was finally determined that after removing impurities with 20% (methanol) volume fraction, elution should be carried out with 30% (methanol) volume fraction.

[0090] Table 14 Screening Results of C18 Eluent Volume Fraction

[0091] (6) Amount of impurity removal solution Load the sample according to the method described in section (5). Elute with 20% methanol, and collect the eluent for each column volume. When the eluent volume reaches 3 BV, the eluent is almost colorless. Finally, select 3 BV of impurity removal solution (40% ethanol).

[0092] (7) Eluent dosage The column was packed, sample loaded, and impurities removed according to the method in section (5). Elution was performed with 30% methanol. The eluent was collected for each column volume, and the desorption rate was calculated. The results are shown in Table 15. It can be seen that the desorption rate gradually stabilizes when the amount used is greater than 3 BV. Considering the green and economical use of solvent, 3 BV was finally selected.

[0093] Table 15 Screening Results of C18 Eluent Dosage

[0094] (8) Verification test Based on the results of the single-factor experiments, the optimal process was determined to be: sample loading volume 4 BV, mass concentration 5 mg / mL, impurity removal liquid fraction 20% (volume 3 BV), and elution liquid fraction 30% (volume 3 BV). Three batches of validation experiments were conducted using the optimized process, and the results are shown in Table 16. This demonstrates that the process is stable and feasible.

[0095] Table 16 C18 Verification Test Results

[0096] Dextran gel column chromatography process optimization (1) Resin pretreatment Take an appropriate amount of dextran gel LH-20, soak it in methanol for 24 hours to allow it to swell fully, and set it aside for later use.

[0097] (2) Sample loading quality 100g of pretreated dextran gel was weighed and slowly added to the adsorption column using a wet method. 0.6, 1, and 1.5g of crude moniliformin I were weighed, dissolved in methanol, filtered through a 0.22μm filter membrane, and loaded onto the column using a wet method. Elution was performed with 4 BV methanol, collecting one tube per 10mL, and combining the eluents without impurity peaks. The results are shown in Table 17. When the sample loading amount was not higher than 1g, the yield did not change significantly. When the sample loading amount reached 1.5g, the yield decreased significantly. To maximize sample collection efficiency, a sample loading mass of 1g was ultimately chosen.

[0098] Table 17 Screening of Dextran Gel Loading Quality

[0099] (3) Diameter-to-height ratio 100g of pretreated dextran gel resin was weighed and slowly added to glass columns with diameter-to-height ratios of 1:10, 1:15, 1:17, and 1:20 using a wet method. 1g of crude moniliformin I was weighed, dissolved in methanol, filtered through a 0.22μm filter membrane, and loaded onto the column using a wet method. Elution was performed with 4 BV methanol, collecting one tube per 10 mL, and combining the eluents without impurity peaks. The results are shown in Table 18; the yield gradually increased with increasing diameter-to-height ratio. Considering space limitations and ease of operation, a diameter-to-height ratio of 1:20 was ultimately chosen.

[0100] Table 18 Screening results of diameter-to-height ratio of dextran gel LH-20

[0101] (4) Eluent volume 100g of pretreated dextran gel resin was weighed and slowly added to a glass column with a diameter-to-height ratio of 1:20 using a wet method. 1g of crude moniliformin I was weighed, dissolved in methanol, filtered through a 0.22μm filter membrane, and loaded onto the column using a wet method. Elution was performed with 8 BV methanol, collecting one tube per 10mL, and combining the eluents without impurity peaks. The results are shown in Table 19. When the eluent volume was less than 4 BV, the yield gradually increased with increasing eluent volume; however, when the eluent volume was greater than 4 BV, the yield no longer changed. Considering the environmental friendliness and economy of the solvent, 4 BV was ultimately chosen.

[0102] Table 19 Results of volume screening for LH-20 eluent for dextran gel

[0103] (5) Eluent volumetric flow rate Following step (4), the column was packed and sample loaded, and eluted with 4 BV methanol at 0.5, 1, and 2 mL / min, respectively. One tube was collected for every 10 mL, and the eluents without impurity peaks were combined. The results are shown in Table 20. As the elution rate decreased, the yield gradually increased, and gradually stabilized at 1 mL / min. Considering efficiency, a final elution rate of 0.5 mL / min was selected.

[0104] Table 20 Screening Results of Macroporous Resin Sample Loading Volumetric Flow Rate

[0105] (6) Verification test Based on the results of single-factor experiments, the optimal process was determined to be a diameter-to-height ratio of 1:20, a sample mass to gel mass ratio of 1:100, an eluent volume of 4 BV, and an eluent flow rate of 0.5 mL / min. Validation experiments conducted according to the optimized process showed that the process is stable and feasible.

[0106] Table 21 Validation test results of dextran gel LH-20

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing monkey cloxacin, characterized in that, The monkey ear cyclophenone is 7-galloylterylsulforhanane; The preparation method of the monkey ear cyclophosphamide includes the following steps: S1. Extract the pulverized monkey earring medicinal material by soaking it in a polar solvent to obtain the extract; S2. The extract is packed into a macroporous adsorption resin column and separated by first column chromatography using the first eluent to obtain the monkey ear cyclosporine component. The first eluent includes an ethanol-containing solution with a volume percentage of 30-100%. S3. The monkey ear cyclophosphine component is packed into a reversed-phase silica gel chromatographic column and separated by second column chromatography using a second eluent to obtain crude monkey ear cyclophosphine. The second eluent includes a methanol-containing solution with a volume percentage of 20-50%. S4. The crude monkey ear cyclophosphine is loaded into a gel chromatography column and separated by third column chromatography using a third eluent to obtain the purified product. The third eluent includes a methanol-containing solution with a volume percentage of 90-100%. The purified product was concentrated to obtain pure monkey ear cyclosporine.

2. The method for preparing monkey cloxacin according to claim 1, characterized in that, The macroporous resin in the macroporous adsorption resin column is macroporous polymethyl methacrylate.

3. The method for preparing monkey cloxacin according to claim 1, characterized in that, The first column chromatography separation includes: performing staged gradient elution using a first eluent, wherein the staged gradient elution includes a first stage elution and a second stage elution; the eluent for the first stage elution is an aqueous ethanol solution with a volume percentage of 30% to 40%; and the eluent for the second stage elution is an aqueous ethanol solution with a volume percentage of 60% to 80%. The elution products containing monocyclonol obtained from the first and second elution stages were combined to obtain the monocyclonol component.

4. The method for preparing monkey cloxacin according to claim 1, characterized in that, The reversed-phase silica gel column is a C18 reversed-phase silica gel column.

5. The method for preparing monkey cloxacin according to claim 1, characterized in that, The second column chromatography separation includes: staged gradient elution using a second eluent, wherein the staged gradient elution includes a first stage elution and a second stage elution; the eluent for the first stage elution is a methanol-water solution with a volume percentage of 20% to 30%; and the eluent for the second stage elution is a methanol-water solution with a volume percentage of 30% to 40%. The elution products containing simian cyclophosphine obtained from the first and second elution stages were combined to obtain crude simian cyclophosphine.

6. The method for preparing monkey cloxacin according to claim 1, characterized in that, The gel chromatography column is a dextran gel chromatography column.

7. The method for preparing monkey cloxacin according to claim 1, characterized in that, The third column chromatography separation includes isocratic elution using a third eluent, wherein the third eluent is methanol.

8. The method for preparing monkey cloxacin according to claim 1, characterized in that, The polar solvent includes at least one of water, methanol, ethanol, and ethyl acetate.

9. The method for preparing monkey cloxacin according to claim 1, characterized in that, The pulverized monkey earring medicinal material has a particle size of less than 1 mm.

10. The method for preparing monkey cloxacin according to claim 1, characterized in that, Step S2 specifically includes the following steps: preparing a loading solution with ethanol, wherein the concentration of the loading solution is 3-15 mg / mL, and loading the loading solution into a macroporous adsorption resin column.

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