Preparation method and quality control method of high-purity cagayanone A
High-purity cagayanone A was prepared by ethanol extraction, silica gel column chromatography, and recrystallization techniques. Combined with thin-layer chromatography and liquid chromatography detection, this method solved the problem of insufficient purity of traditional Chinese medicine chemical reference standards, achieving efficient quality control and enhancing international market competitiveness.
Patent Information
- Application Number
- CN202610190254.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies make it difficult to prepare high-purity cagayanone A chemical reference standard, and there is a lack of effective quality control methods, which fails to meet the purity requirements of chemical reference standards for traditional Chinese medicine, thus limiting the development of traditional Chinese medicine in the international market.
The roots, stems and leaves of Schisandra chinensis were extracted by reflux with 95% ethanol. Combined with dichloromethane extraction, silica gel column chromatography and recrystallization, an efficient extraction process was established. The purity and content were detected by thin-layer chromatography and high-performance liquid chromatography to ensure product quality.
The preparation and quality control of high-purity (over 98%) cagayanone A were achieved, providing a scientific analytical testing method, laying the foundation for the research and quality standards of chemical reference standards for traditional Chinese medicine, and enhancing the international competitiveness of traditional Chinese medicine.
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Figure CN122103077A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of compound preparation technology, and more specifically to a method for preparing high-purity cagayanone A and its quality control method. Background Technology
[0002] Chemical reference standards, also known as standards, are physical comparisons used in the research, testing, and control of traditional Chinese medicine (TCM) quality standards. The research of TCM chemical reference standards is a crucial part of TCM standardization research. They play an extremely important role in product quality evaluation, especially in the quality control of pharmaceutical production, and are the foundation and core of TCM quality control. Cagayanone A, as a chemical reference standard for plants, medicinal materials, and their products, is a key technical element in quality control. Many enterprises, research institutions, and testing departments require high-purity reference standards, resulting in a large market demand. Cagayanone A is a lignin-based chemical component and one of the active ingredients in plants such as Schisandra chinensis. It is also an indicator component for the quality control of many plant and pharmaceutical standards. However, the content of cagayanone A in medicinal materials is low, and the extraction and separation techniques are highly demanding, making the preparation of high-purity cagayanone A very difficult. Currently, there is no corresponding national pharmaceutical standard substance, and no systematic research on cagayanone A as a TCM chemical reference standard has been reported domestically or internationally.
[0003] Cagayanone A is an active substance isolated from *Schisandra henrgi* CBClarke subsp. Marginalis (AC Smith) RMK Saund., a plant belonging to the Schisandraceae family. According to publicly available literature, the extraction, separation, and content determination methods for cagayanone A mainly include the following:
[0004] 1. Dinorcassane diterpenoid from Boesenbergia rotunda rhizomes collected in Lower Myanmar: Dried rhizomes of Boesenbergia rotunda (200 g) were extracted with chloroform (1 L) for 90 min (3 times) under ultrasonic conditions at 30 °C to obtain chloroform extract (7.00 g). Separation was performed by silica gel column chromatography with ethyl acetate-n-hexane gradient elution (10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 50:50) to obtain 7 fractions (1: 372 mg; 2: 950 mg; 3: 200 mg; 4: 730 mg; 5: 567 mg; 6: 1.97 g; 7: 1.50 g). Fraction 4 (730 mg) was subjected to Cosmosil 75C18-OPN column chromatography (methanol-water, 5:1) to yield two subfractions [4-1: 300 mg; 4-2: 100 mg]. Subfraction 4-2 (100 mg) was subjected to Sephadex LH-20 gel column chromatography (methanol elution) to yield kaempferol-7,4'-dimethyl ether (15.8 mg) (Nwet Nwet Win, May Mon Kyaw, Prema, et al. Dinorcassanediterpenoid from Boesenbergia rotunda rhizomes collected in Lower Myanmar. Chemistry & Biodiversity, 2019, 16(4): 1-6.).
[0005] The methods described above, from different perspectives, illustrate the extraction and separation of cagayanone A. While the separation purity is relatively high, most of these methods do not meet the requirements for chemical reference standards of traditional Chinese medicine (i.e., purity greater than 98%), thus failing to meet the need for high-purity cagayanone A chemical reference standards. Furthermore, no relevant reports have been found in the existing technology regarding the determination of cagayanone A or the quality control and evaluation of cagayanone A chemical reference standards.
[0006] In the future, all kinds of pharmaceuticals, whether produced domestically or entering the international market, will need to rely on high-level quality standards and advanced analytical testing technologies to achieve development and improvement; otherwise, they will easily lose market share. Product quality standards and testing methods are becoming increasingly important. The standard of "safe, effective, and quality-controllable" medication has become an international consensus, and pharmaceutical production should revolve around this central theme, with the core being the level of quality standard control, while chemical reference standards play a crucial role.
[0007] However, most Chinese medicinal herbs and their preparations currently lack clear chemical composition or chemical reference standards, making it impossible to elucidate the chemical basis of their effects and to conduct quality control. This hinders their acceptance by modern society and becomes a key constraint preventing Chinese herbal medicines and natural drugs from entering the international pharmaceutical market. These technical barriers have created difficulties for the development of the Chinese medicine industry. Therefore, research on the chemical composition of Chinese herbal medicines and the standardization of quality standards are essential for the modernization of Chinese medicine. This research is crucial for elucidating the material basis of the effects of Chinese herbal medicines, formulating production and processing technologies for Chinese herbal preparations, and identifying counterfeit and substandard products.
[0008] Therefore, conducting quality standard research on cagayanone A, establishing standardized analytical testing methods, and developing high-tech detection indicators and analytical methods to control the quality of cagayanone A, making it more scientific and standardized, enhancing its international competitiveness, and creating conditions for traditional Chinese medicine to enter the international market, has significant practical and academic value. Summary of the Invention
[0009] In view of this, the present invention provides a method for preparing high-purity cagayanone A and a method for quality control thereof, thereby solving the problem of high-purity cagayanone A chemical reference standard.
[0010] This invention, referencing the technical requirements of chemical reference standards for traditional Chinese medicine (for content determination), studies the chemical reference standard cagayanone A, establishes a batch extraction process for cagayanone A, and develops analytical methods for purity, content, and impurity testing. This establishes a technical standard for cagayanone A chemical reference standard, providing a scientific basis and guarantee for its use as a chemical reference standard for traditional Chinese medicine and for quality standard research on medicinal materials and preparations.
[0011] The cagayanone A of this invention is obtained from the roots, stems, and leaves of *Schisandrahenrgi CB Clarke subsp. Marginalis (AC Smith) RMK Saund.*, a plant belonging to the Schisandraceae family, through extraction, separation, purification, and refining. Its chemical name, molecular formula, and structural formula are as follows: Chinese name: cagayanone A; Chemical name: 3,5-dihydroxy-7,4'-dimethoxy-flavone.
[0012] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing high-purity cagayanone A includes the following steps: (1) Take the dried roots, stems and leaves of Schisandra chinensis, crush them, and extract them by reflux with 95% ethanol. Filter the extract, combine the filtrates, recover the ethanol, and obtain the extract: (2) The extract was then extracted with dichloromethane, allowed to stand for more than 2 hours, the dichloromethane layers were combined, and concentrated by rotary evaporation to obtain the dichloromethane extract: (3) The dichloromethane extract was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate system, and the fraction containing cagayanone A was collected: (4) The fractions were detected by thin-layer chromatography, combined, concentrated, and recrystallized with petroleum ether-ethyl acetate to obtain cagayanone A crystals with a purity of 98% by weight.
[0013] Preferably, in step (1), the amount of 95% ethanol added is 5 to 10 times the weight of the dried roots, stems and leaves of Schisandra chinensis; the number of reflux extractions is 2 to 6.
[0014] Preferably, the gradient elution in step (3) is: 0~A minutes, with petroleum ether as the eluent; B~C minutes, with petroleum ether-ethyl acetate as the eluent, wherein A is 10~30, B is 11~50, and C is 30~70. The volume ratio of petroleum ether to ethyl acetate is (10~100):1.
[0015] Preferably, the parameters for the thin-layer chromatography method in step (4) are: Thin-layer plate: Silicone G; Three developing solvent systems were used: System 1, with a volume ratio of petroleum ether-acetone-glacial acetic acid of 6:4:0.1; System 2, with a volume ratio of petroleum ether-ethyl acetate-glacial acetic acid of 7:3:0.1; and System 3, with a volume ratio of cyclohexane-dichloromethane-glacial acetic acid of 3:4:0.3. Spotting: Prepare a 100 μg / mL solution with methanol and spot it onto the same silica gel G thin-layer plate at different sample volume gradients: 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg. Develop the plate in a developing tank with a development distance of 10 cm. Positioning: Spray with 10% sulfuric acid ethanol solution, air dry, and heat at 105℃ until the spots are clearly visible. Examine under a 365nm UV lamp. The results show that under the three developing solvent systems in thin-layer chromatography, five different concentration gradient spots were all single spots, and no impurity spots were observed.
[0016] Preferably, the recrystallization conditions in step (4) are: a volume ratio of petroleum ether to ethyl acetate of 1:1, a temperature of 25°C, and a standing time of 72h.
[0017] Another objective of this invention is to provide a quality control method for high-purity cagayanone A, which involves treating the cagayanone A to be tested with a 50% methanol solution to obtain an extract, and then using high-performance liquid chromatography to detect the content of cagayanone A in the extract.
[0018] Preferably, the chromatographic conditions are as follows: column C-18, 4.6X250mm, 5~10 m; flow rate 0.8~1.2ml / min; injection volume: 10~20uL; quantification is performed using the area normalization method.
[0019] Preferably, the system conditions are one of the following two conditions: Condition 1: The mobile phase is methanol-water solution with a volume ratio of 70~80:20~30, and the detection wavelength is 260~365nm; Condition 2: The fluid is an acetonitrile-water solution with a volume ratio of 60~70:30~40, and the detection wavelength is 260~365nm.
[0020] Preferably, it also includes: peak purity detection, specifically: taking an appropriate amount of reference standard, according to the mobile phase system, and checking the peak purity using a diode array (DAD) detector on a high-performance liquid chromatograph.
[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: 1. The present invention is reasonably designed and the process is simple. It uses alcohol-water solvent extraction, and after one silica gel column chromatography and recrystallization, a chemical reference standard with a purity of 98% can be obtained. The method is simple and easy to implement.
[0022] 2. The compound obtained by this invention was confirmed as cagayanone A by spectral analysis. In this invention, the dried roots, stems, and leaves of Schisandra chinensis were first pulverized and extracted with 95% ethanol under reflux. The extracts were combined, the ethanol was recovered, and then extracted with dichloromethane to obtain a dichloromethane extract. This extract was then subjected to silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate system. Thin-layer chromatography was used for detection, and the eluent containing cagayanone A was collected, combined, concentrated under reduced pressure, and recrystallized to obtain cagayanone A with a purity of 90%–98% and a fraction with a purity greater than 98%.
[0023] 3. This invention, through research on the chemical reference standard of cagayanone A, establishes a batch extraction process, purity and content analysis method, and impurity testing method for cagayanone A chemical reference standard. This establishes a technical standard for cagayanone A chemical reference standard, providing a scientific basis and guarantee for its use as a chemical reference standard for traditional Chinese medicine and for quality standard research of medicinal materials and preparations. This invention can provide a more complete basic chemical basis for cagayanone A chemical reference standard, mastering its chemical information and analytical testing technology, which is beneficial to the further development and utilization of related products. Moreover, it has potential and immeasurable social and economic benefits for developing products unique to my country, developing high-tech, high-value-added products, and improving market competitiveness.
[0024] 4. This invention employs thin-layer chromatography and high-performance liquid chromatography for purity testing, content determination, and quality control to ensure product quality. The process design is reasonable, the process is simple, the separation speed is fast, the production cycle is short, and the resulting product has high purity and controllable quality, making it suitable for industrial production and possessing excellent application prospects.
[0025] 5. This invention prepares cagayanone A from the roots, stems and leaves of Schisandra chinensis with a purity meeting the requirements of chemical reference standards and a content of over 98%, solving the supply problem of cagayanone A chemical reference standard and providing a scientific basis and guarantee for the quality control of Schisandra chinensis and other medicines containing cagayanone A. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0027] Figure 1 The thin-layer chromatograms are shown in the figure. A is System 1, B is System 2, and C is System 3. Figure 2 For the use of system test conditions - liquid chromatography Figure 1 ; Figure 3 For the use of system testing conditions, two-phase liquid chromatography Figure 1 ; Figure 4 Three-phase liquid chromatography was used to perform system testing. Figure 1 ; Figure 5 The liquid chromatogram was used to check peak purity. Figure 6A three-dimensional spectrum; Figure 7 A 5-point spectral plot; Figure 8 For the use of system test conditions - liquid chromatography Figure 2 ; Figure 9 For the use of system testing conditions, two-phase liquid chromatography Figure 2 ; Figure 10 Three-phase liquid chromatography for system testing conditions Figure 2 . Detailed Implementation
[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0029] In this invention, the percentage of methanol is a volume percentage, and v / v represents the volume ratio of the solution.
[0030] Example 1 A method for preparing high-purity cagayanone A includes the following steps: (1) Take 20 kg of dried rhizomes and leaves of Schisandra chinensis, crush them, and extract them by reflux with 95% ethanol. Filter the extract, combine the filtrates, recover the ethanol, and obtain the extract: (2) The extract was then extracted with dichloromethane, allowed to stand, the dichloromethane layers were combined, and concentrated to obtain the dichloromethane extract: (3) The dichloromethane extract was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate system, and the fraction containing cagayanone A was collected: (4) The fractions were analyzed by thin-layer chromatography, combined, concentrated, and recrystallized with petroleum ether-ethyl acetate to obtain cagayanone A crystals with a purity of 98% by weight. In this embodiment, the amount of 95% ethanol added in step (1) is 6 times the weight of the medicinal material, and the number of reflux extractions is 4.
[0031] In step (3), the gradient elution parameters of the petroleum ether-ethyl acetate system are: From 0 to 20 minutes, the eluent is petroleum ether; from 21 to 50 minutes, the eluent is petroleum ether-ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 10:1.
[0032] The thin-layer chromatography parameters in step (4) are as follows: Thin-layer plate: Silicone G; Three developing solvent systems were used: System 1, with a volume ratio of petroleum ether-acetone-glacial acetic acid of 6:4:0.1; System 2, with a volume ratio of petroleum ether-ethyl acetate-glacial acetic acid of 7:3:0.1; and System 3, with a volume ratio of cyclohexane-dichloromethane-glacial acetic acid of 3:4:0.3. Spotting: Prepare a 100 μg / mL solution with methanol and spot it onto the same silica gel G thin-layer plate at different sample volume gradients: 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg. Develop the plate in a developing tank with a development distance of 10 cm. Positioning: Spray with 10% sulfuric acid ethanol solution, air dry, heat at 105℃ until the spots are clearly visible, and examine under a 365nm ultraviolet lamp; Results: In thin-layer chromatography, for three developing solvent systems and five different concentration gradients, all spots were single spots, and no impurity spots were observed. The recrystallization conditions were: a volume ratio of petroleum ether to ethyl acetate of 1:1, a temperature of 25°C, and a standing time of 72 h.
[0033] Example 2 A method for preparing high-purity cagayanone A includes the following steps: (1) Take 20 kg of dried rhizomes and leaves of Schisandra chinensis, crush them, and extract them by reflux with 95% ethanol. Filter the extract, combine the filtrates, recover the ethanol, and obtain the extract: (2) The extract was then extracted with dichloromethane, allowed to stand, the dichloromethane layers were combined, and concentrated to obtain the dichloromethane extract: (3) The dichloromethane extract was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate system, and the fraction containing cagayanone A was collected: (4) The fractions were analyzed by thin-layer chromatography, combined, concentrated, and recrystallized with petroleum ether-ethyl acetate to obtain cagayanone A crystals with a purity of 98% by weight. In this embodiment, the amount of 95% ethanol added in step (1) is 6 times the weight of the medicinal material, and the number of reflux extractions is 4.
[0034] In step (3), the gradient elution parameters of the petroleum ether-ethyl acetate system are: From 0 to 20 min, the eluent is petroleum ether; from 21 to 50 min, the elution system is petroleum ether-ethyl acetate in a ratio of 10:1.
[0035] The thin-layer chromatography parameters in step (4) are as follows: Thin-layer plate: Silicone G; Three developing solvent systems were used: System 1, with a volume ratio of petroleum ether-acetone-glacial acetic acid of 6:4:0.1; System 2, with a volume ratio of petroleum ether-ethyl acetate-glacial acetic acid of 7:3:0.1; and System 3, with a volume ratio of cyclohexane-dichloromethane-glacial acetic acid of 3:4:0.3. Spotting: Prepare a 100 μg / mL solution with methanol and spot it on the same silica gel G plate at different sample volume gradients: 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg. Develop the plate in a developing tank with a development distance of 10 cm. Positioning: Spray with 10% sulfuric acid ethanol solution, air dry, heat at 105℃ until the spots are clearly visible, and examine under a 365nm UV lamp; Results: In thin-layer chromatography, for three developing solvent systems and five different concentration gradient spots, all were single spots, and no impurity spots were observed.
[0036] The recrystallization conditions were: a volume ratio of petroleum ether to ethyl acetate of 1:1, a temperature of 25°C, and a standing time of 72 h.
[0037] Example 3 The quality control method for product cagayanone A in this invention is as follows: Accurately weigh an appropriate amount of the product from Example 1, which has been dried to constant weight at 105°C, and set aside. Purity testing: Thin-layer chromatography purity test: Take an appropriate amount of cagayanone A candidate chemical reference standard, weigh it accurately, and prepare a 100 μg·mL solution with methanol. -1 The solution was spotted onto the same silica gel G plate at different sample concentration gradients: 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg. Developed using three different solvent systems (System 1, System 2, and System 3), the plates were removed, dried, and sprayed with 10% sulfuric acid ethanol solution for color development. Observed under a UV lamp (365 nm). In the thin-layer chromatograms of five different sample concentrations in the three different solvent systems, all samples showed a single yellow-green fluorescent spot, without any other impurity spots. The thin-layer chromatograms are shown below. Figure 1 .
[0038] High performance liquid chromatography (HPLC) examination: Weigh an appropriate amount of cagayanone A candidate chemical reference standard accurately, dissolve it in methanol, and prepare a solution of 1 mg / mL. -1 The test solution was filtered through a 0.22 μm microporous membrane, and the filtrate was collected. An Agilent 1260 high-performance liquid chromatograph was used with an Elite Supersil ODS2-C18 column (250 mm × 4.6 mm, 5 μm), an injection volume of 20 μL, and a flow rate of 1.0 mL / min. -1 The values were measured under three different system test conditions.
[0039] System test conditions 1: The mobile phase is methanol-water solution (70:30), and the detection wavelength is 250 nm; System test condition 2: The mobile phase is methanol-water solution (70:30), and the detection wavelength is 300 nm; System test conditions 3: The mobile phase is acetonitrile-water solution (55:45), and the detection wavelength is 300 nm.
[0040] The selected chemical reference standard, cagayanone A, produced single peaks in the liquid chromatograms under three chromatographic conditions, indicating good peak purity. The relative percentage content, calculated using the peak area normalization method, was above 98%. (See results below.) Figure 2-4 .
[0041] Peak purity check: Take an appropriate amount of cagayanone A product, mix with mobile phase two, and perform peak purity checks on a high-performance liquid chromatograph using a diode array (DAD) detector. The HPLC chromatographic peak of cagayanone A is >98%, and its chromatogram ( Figure 5 ) is a single peak, three-dimensional spectrum ( Figure 6 ) and 5-point spectral diagram ( Figure 7 The peaks completely overlap, indicating that they are a single pure substance peak.
[0042] Area normalization purity check: Under the specified conditions, 20 μL of the test solution was injected into the liquid chromatograph. Chromatograms were recorded under system test conditions one, two, and three until the retention time of the main component peak was at least 2.5 times the normalization time. The content was calculated using the area normalization method. The results showed that the content of the reference standard was consistently above 98%, and the total area of all impurity peaks (excluding the solvent peak) was <2.0%. The results are shown in Table 1. Figure 8-10 .
[0043] Table 1. Quantitative analysis results of HPLC (System 1, System 2, System 3) using normalization method
[0044] The present invention examines the HPLC chromatographic analysis methodology as follows: Chromatographic conditions: Elite Supersil ODS2-C18 column (250 mm × 4.6 mm, 5 μm), methanol-water (70:30) as mobile phase, detection wavelength 300 nm, injection volume 20 μl; flow rate 1 mL / min.
[0045] Examining linear relationships: Take the above-mentioned cagayanone A candidate chemical reference standard, dry it to constant weight at 105℃, accurately weigh about 5 mg, place it in a 100 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare a reference standard stock solution with a concentration of 50.0 μg / mL. Pipette 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 6 ml of the reference standard stock solution into 10 mL volumetric flasks, dilute to the mark with methanol, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain a series of reference standard solutions of different concentrations. Inject these solutions into the liquid chromatograph under the above chromatographic conditions, injecting each concentration twice, and determine the peak area. Plot a standard curve with the injection volume (μg) on the x-axis and the peak area integral on the y-axis. The regression equation is calculated as Y = 13.086X - 0.6267, R = 0.9991. The cagayanone A candidate chemical reference standard is used in injections of 5.0–30.0 μg·mL. -1 It exhibits a good linear relationship within the range.
[0046] Reproducibility, stability, and precision studies: Precision: The same test solution was injected six times consecutively according to the above chromatographic conditions. The chromatogram and peak area integral of cagayanone A were recorded, and the content was calculated. The RSD was 0.07%, indicating that the instrument has good precision.
[0047] Reproducibility: Six samples of the same test sample were taken and measured using the above method and chromatographic system (mobile phase two). The chromatograms and peak area integral values of cagayanone A were recorded, and the content was calculated. The average content was 98.96%, and the RSD was 0.09%, indicating that the method has good reproducibility.
[0048] Stability: The same test solution was placed at room temperature for 2, 4, 6, 8, 10, 12 and 24 hours, and the sample was injected and determined according to the method. The chromatogram of cagayanone A and the peak area integral value were recorded. The results showed that RSD=0.53%, indicating that the test solution had good stability within 24 hours.
[0049] The durability of the method was examined. Three chromatographic columns from different manufacturers and brands were used to determine retention time, theoretical plate number, resolution, and impurity separation effect. Under the above analytical conditions, the cagayanone A peak achieved baseline separation from other impurity peaks, with a resolution greater than 1.5. When the theoretical plate number calculated based on the cagayanone A peak was not less than 4000, the determination requirements were met, as shown in Table 2.
[0050] Table 2
[0051] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.
[0052] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for preparing high-purity cagayanone A, characterized in that, Includes the following steps: (1) Take the dried roots, stems and leaves of Schisandra chinensis, crush them, and extract them by reflux with 95% ethanol. Filter the extract, combine the filtrates, recover the ethanol, and obtain the extract: (2) The extract was then extracted with dichloromethane, allowed to stand, and the dichloromethane layers were combined and concentrated by rotary evaporation to obtain the dichloromethane extract: (3) The dichloromethane extract was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate system, and the fraction containing cagayanone A was collected: (4) The fractions were detected by thin-layer chromatography, combined, concentrated, and recrystallized with petroleum ether-ethyl acetate to obtain cagayanone A crystals.
2. The method for preparing high-purity cagayanone A according to claim 1, characterized in that, In step (1), the amount of 95% ethanol added is 5 to 10 times the weight of the dried roots, stems and leaves of Schisandra chinensis; the number of reflux extractions is 2 to 6. The settling time in step (2) is >2h.
3. The method for preparing high-purity cagayanone A according to claim 1, characterized in that, The gradient elution in step (3) is as follows: 0~A minutes, with petroleum ether as the eluent; B~C minutes, with petroleum ether-ethyl acetate as the eluent, where A is 10~30, B is 11~50, and C is 30~70. The volume ratio of petroleum ether to ethyl acetate is (10~100):
1.
4. The method for preparing high-purity cagayanone A according to claim 1, characterized in that, The parameters for the thin-layer chromatography method described in step (4) are: Thin-layer plate: Silicone G; Three developing solvent systems were used: System 1, with a volume ratio of petroleum ether-acetone-glacial acetic acid of 6:4:0.1; System 2, with a volume ratio of petroleum ether-ethyl acetate-glacial acetic acid of 7:3:0.1; and System 3, with a volume ratio of cyclohexane-dichloromethane-glacial acetic acid of 3:4:0.
3. Spotting: Prepare a 100 μg / mL solution with methanol and spot it onto the same silica gel G thin-layer plate at different sample volume gradients: 2 μg, 4 μg, 6 μg, 8 μg, and 10 μg. Develop the plate in a developing tank with a development distance of 10 cm. Positioning: Spray with 10% sulfuric acid ethanol solution, air dry, and heat at 105℃ until the spots are clearly visible. Examine under a 365nm UV lamp. The results show that under the three developing solvent systems in thin-layer chromatography, five different concentration gradient spots were all single spots, and no impurity spots were observed.
5. The method for preparing high-purity cagayanone A according to claim 1, characterized in that, The recrystallization conditions described in step (4) are: a volume ratio of petroleum ether to ethyl acetate of 1:1, a temperature of 25°C, and a standing time of 72 h.
6. A quality control method for high-purity cagayanone A, characterized in that, The cagayanone A sample was obtained by treating the sample with a 50% methanol solution, and the content of cagayanone A in the extract was detected by high performance liquid chromatography.
7. The quality control method for high-purity cagayanone A according to claim 6, characterized in that, Chromatographic conditions were as follows: column C-18, 4.6 x 250 mm, 5–10 m; flow rate 0.8–1.2 ml / min; injection volume 10–20 μL; quantification was performed using the area normalization method.
8. The quality control method for high-purity cagayanone A according to claim 7, characterized in that, The mobile phase system meets one of the following two conditions: Condition 1: Methanol-water solution, with a volume ratio of 70~80:20~30, and a detection wavelength of 260~365 nm; Condition 2: Acetonitrile-water solution, with a volume ratio of 60~70:30~40, and a detection wavelength of 260~365nm.
9. The quality control method for high-purity cagayanone A according to claim 8, characterized in that, Also includes: Peak purity detection is performed as follows: Take an appropriate amount of reference standard, and according to the mobile phase system, check the peak purity using a diode array (DAD) detector on a high-performance liquid chromatograph.