Method for separating and purifying ganoderic acid reference substance

Abstract

The invention discloses a method for separating and purifying a ganoderic acid reference substance, which comprises the following steps: S1, taking a lucid ganoderma extract, adding absolute methanol, carrying out ultrasonic heating extraction twice, combining extracting solutions, carrying out suction filtration, and concentrating filtrate; s2, uniformly stirring the sample solution with silica gel, drying, loading the sample with a dry method for normal phase chromatography preparation, eluting with a petroleum ether-ethyl acetate system, concentrating to remove a normal phase reagent, and dissolving with methanol to obtain a three-section ganoderic acid component; s3, performing membrane-passing clarification, performing reversed-phase chromatography preparation by using an upper-medium pressure preparative column, performing isocratic elution, performing segmentation by using an acetonitrile-acid water system, and performing high-performance liquid chromatography detection to obtain N sections of ganoderic acid samples; s4, respectively performing reversed phase chromatography preparation, isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system preparation, high performance liquid chromatography detection, concentration to remove an organic solvent, and drying under reduced pressure to obtain 22 ganoderic acid series reference substances; the method has the advantages of high ganoderic acid separation efficiency, high yield, short time and the like, and a large amount of gt can be obtained; the purity of the ganoderic acid reference substance is 98%.

Description

Technical Field

[0001] The invention relates to the technical field of ganoderic acid preparation, in particular to a method for separating and purifying a ganoderic acid reference substance. Background Art

[0002] Lingzhi (scientific name: Ganoderma lucidum (Curtis) P. Karst.) is a rare edible and medicinal fungus belonging to the Ganodermaceae family and the genus Ganoderma. Most of the fruiting bodies of Lingzhi are annual, and a few are perennial, with stalks and lateral stalks. As early as in the "Shennong's Herbal Classic", there were relatively detailed records of the medicinal effects of Lingzhi. Lingzhi has the effects of clearing away heat and detoxifying, benefiting the kidney and protecting the liver, nourishing and strengthening the body, strengthening the body, beautifying and nourishing the skin, calming the mind and benefiting the essence.

[0003] The chemical composition of Ganoderma lucidum is complex. The natural ingredients of Ganoderma lucidum contain effective ingredients such as Ganoderma polysaccharides, Ganoderma terpenes, Ganoderma polyphenols, fatty acids, organic germanium, amino acids, etc. Ganoderma triterpenoid compounds are divided into tetracyclic triterpenes and pentacyclic triterpenes, mainly including ganoderic acid, ganoderin, and ganoderin ketone.

[0004] Among the main components of Ganoderma lucidum, Ganoderma lucidum polysaccharide and Ganoderma acid are considered to be the main effective components. Ganoderma acid is mostly tetracyclic triterpenes, which are highly oxidized lanostanes. Ganoderma acid can inhibit the release of cellular histamine, enhance the functions of various organs of the digestive system, and has the effects of lowering blood lipids, lowering blood pressure, protecting the liver, and regulating liver function. It is a natural organic compound with multiple functions such as analgesia, sedation, anti-cancer, and detoxification. The reason why Ganoderma acid can lower blood lipids and blood pressure is that Ganoderma acid blocks the ability of lanosterol or dioxylanosterol to synthesize cholesterol, thereby slowing down the process of atherosclerosis and slowing down the increase in blood pressure. Since Ganoderma acid has the above-mentioned positive effects, the content of Ganoderma acid has become one of the important indicators of Ganoderma lucidum and its products.

[0005] Triterpenoid compounds, one of the main components of Ganoderma lucidum, are of various types. Since Kubota et al. first isolated ganoderic acid A and ganoderic acid B from the fruiting body of Ganoderma lucidum in 1982, nearly 100 ganoderic acid compounds have been isolated. The research on ganoderic acid in China started late. Tang Wen et al.'s research showed that the separation degree and capacity factor of ganoderic acid were the best under the condition of elution temperature of 40°C, and the addition of 1% acetic acid in C18 chromatographic column could achieve better separation. Zhu Zhongmin et al.'s research showed that HZ-816 macroporous resin combined with high-speed countercurrent chromatography can separate ganoderic acid compound monomers with a purity of more than 85%. Li Chengfei et al.'s authorized patent CN201410223365.0 discloses that ganoderic acid A with a purity of 97.5% was separated by normal phase silica gel purification, LH-20 gel chromatography column separation, and finally recrystallization. Feng Na et al.'s authorized patent CN201510134329.1 discloses that ganoderic acid T with a purity of 92% was separated by high-speed countercurrent chromatography. The authorized patent CN201110443487.7 of Zhang Shufang et al. discloses the separation of 95% pure Ganoderic A using a supercritical carbon dioxide extractor and high-speed countercurrent chromatography.

[0006] Ganoderic acid has many types and similar structures, so there are great difficulties in quality control research. Ganoderma products with high content of ganoderic acid have extremely high commercial value, so scientific and strict quality control must be carried out on the production of high-content ganoderic acid products, and ganoderic acid reference substances are indispensable for quality control. Therefore, in-depth research on the separation and purification technology of ganoderic acid to obtain high-purity ganoderic acid reference substances is necessary for the systematic research on enriching ganoderic acid, effectively developing medicinal ingredients of ganoderic acid, producing high-value-added ganoderic products, forming the industrialization and product standardization of ganoderic products, giving full play to my country's advantages in ganoderic resources, and ensuring the stable and sustainable development of the ganoderic industry, which has important economic benefits and social significance.

[0007] Existing research on separation and purification is only aimed at the separation of a very small amount of triterpenoids from Ganoderma lucidum, and the methods mainly use high-speed countercurrent chromatography, silica gel column chromatography, macroporous resin, etc. High-speed countercurrent chromatography is currently the most important method for the separation and purification of Ganoderma lucidum acid, but this separation method is not efficient and the purity is difficult to reach 98%. Summary of the invention

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for separating and purifying ganoderic acid reference substances, which can efficiently and quickly separate a large number of ganoderic acid series reference substances with a purity of 98%.

[0009] To achieve the above object, the present invention provides the following technical solutions: A method for separating and purifying a ganoderic acid reference substance comprises the following steps: S1 Ganoderma lucidum raw material pretreatment: Ganoderma lucidum extract was extracted twice with anhydrous methanol and ultrasonic heating, the extracts were combined, filtered, and the filtrate was concentrated to obtain a sample solution; S2 silica gel column chromatography for decolorization and impurity removal: the sample solution is mixed with silica gel, dried, and prepared for normal phase chromatography by dry loading, eluted with petroleum ether-ethyl acetate system, concentrated to remove the normal phase reagent, and dissolved with methanol to obtain three sections of ganoderma lucidum acid components; S3 reverse phase chromatography column split preparation: The three sections of Ganoderma lucidum components were clarified by membrane, and then respectively applied to medium pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, acetonitrile-acid water system for splitting, and high performance liquid chromatography detection to obtain N sections of Ganoderma lucidum acid samples; Preparation of S4 ganoderic acid reference substance: The N-segment ganoderic acid samples were separately loaded onto medium-pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system for preparation, and detected by high performance liquid chromatography. The organic solvent was concentrated to remove the organic solvent, and the samples were dried under reduced pressure to obtain a total of 22 ganoderic acid series reference substances.

[0010] Preferably, in S2, 60-100 mesh silica gel is used for sample mixing, and the silica gel column is filled with 200-300 mesh silica gel, and the mass ratio of sample mixing silica gel to column filling silica gel is 1:1.1.

[0011] Preferably, the elution using petroleum ether-ethyl acetate system in S2 is as follows: first, 3 BV of petroleum ether is eluted, then 4 BV of petroleum ether: ethyl acetate is 10:1, then 10 BV of petroleum ether: ethyl acetate is 2:1 to obtain ganoderic acid A sample, then 5 BV of ethyl acetate is eluted to obtain ganoderic acid B sample, and finally 6 BV of ethyl acetate is eluted to obtain ganoderic acid C sample.

[0012] Preferably, in the acetonitrile-acid water system in S3, the acid water is 1% acetic acid, the acetonitrile elution ratio is 35%-75%, the flow rate is 300 ml / min, the elution time is 60-80 min, and the wavelength is 252 nm.

[0013] Preferably, S3 uses a C18 filler with a particle size of 10 μm and a medium pressure preparative column with a sample loading of 8-15%.

[0014] Preferably, S4 is prepared using an ethanol-acid water system, an acetonitrile-acid water system, or a methanol-acid water system, wherein the acid water is 1% acetic acid, the elution ratio of ethanol is 45%-65%, the elution ratio of acetonitrile is 40%-60%, the elution ratio of methanol is 55%-80%, the flow rate is 300 ml / min, the elution time is 15-65 min, and the wavelength is 252 nm.

[0015] Preferably, the N-segment ganoderic acid sample obtained in S3 is prepared according to S4, and when the purity of the obtained ganoderic acid sample is less than 98%, a second preparation is performed according to S4.

[0016] Preferably, the filler used in S4 is C18WAT filler, which is an octadecylsilane bonded silica filler with a particle size of 10 μm.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. Compared with the traditional purification method, the present invention has the advantages of high efficiency, high yield, short time and the like in the separation of ganoderic acid, and can obtain a large amount of ganoderic acid reference substance with a purity of >98%.

[0018] 2. During the preparation process, since the preparation is carried out in an isocratic manner, the organic reagents used in the preparation process can be recycled by a concentration method of reagent rotary evaporation recovery, which is more environmentally friendly and can significantly reduce production costs, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Process flow chart of the preparation method of ganoderic acid reference substance; Figure 2 HPLC analysis of ganoderic acid reference substance a; Figure 3 Figure b: HPLC analysis of ganoderic acid reference substance; Figure 4 Comparison of the separation effects of ganoderic acid on conventional C18 filler and reversed phase chromatography filler C18WAT. DETAILED DESCRIPTION

[0020] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0021] The present invention provides the following technical solutions: A method for separating and purifying a ganoderic acid reference substance comprises the following steps: S1 Ganoderma lucidum raw material pretreatment: Ganoderma lucidum extract was extracted twice with anhydrous methanol and ultrasonic heating, the extracts were combined, filtered, and the filtrate was concentrated to obtain a sample solution; S2 silica gel column chromatography for decolorization and impurity removal: the sample solution is mixed with silica gel, dried, and prepared for normal phase chromatography by dry loading, eluted with petroleum ether-ethyl acetate system, concentrated to remove the normal phase reagent, and dissolved with methanol to obtain three sections of ganoderma lucidum acid components; S3 reverse phase chromatography column split preparation: The three sections of Ganoderma lucidum components were clarified by membrane, and then respectively applied to medium pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, acetonitrile-acid water system for splitting, and high performance liquid chromatography detection to obtain N sections of Ganoderma lucidum acid samples; Preparation of S4 ganoderic acid reference substance: The N-segment ganoderic acid samples were separately loaded onto medium-pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system for preparation, and detected by high performance liquid chromatography. The organic solvent was concentrated to remove the organic solvent, and the samples were dried under reduced pressure to obtain a total of 22 ganoderic acid series reference substances.

[0022] Preferably, in S2, 60-100 mesh silica gel is used for sample mixing, and the silica gel column is filled with 200-300 mesh silica gel, and the mass ratio of sample mixing silica gel to column filling silica gel is 1:1.1.

[0023] Preferably, the elution using petroleum ether-ethyl acetate system in S2 is as follows: first, 3 BV of petroleum ether is eluted, then 4 BV of petroleum ether: ethyl acetate is 10:1, then 10 BV of petroleum ether: ethyl acetate is 2:1 to obtain ganoderic acid A sample, then 5 BV of ethyl acetate is eluted to obtain ganoderic acid B sample, and finally 6 BV of ethyl acetate is eluted to obtain ganoderic acid C sample.

[0024] Preferably, in the acetonitrile-acid water system in S3, the acid water is 1% acetic acid, the acetonitrile elution ratio is 35%-75%, the flow rate is 300 ml / min, the elution time is 60-80 min, and the wavelength is 252 nm.

[0025] Preferably, S3 uses a C18 filler with a particle size of 10 μm and a medium pressure preparative column with a sample loading of 8-15%.

[0026] Preferably, S4 is prepared using an ethanol-acid water system, an acetonitrile-acid water system, or a methanol-acid water system, wherein the acid water is 1% acetic acid, the elution ratio of ethanol is 45%-65%, the elution ratio of acetonitrile is 40%-60%, the elution ratio of methanol is 55%-80%, the flow rate is 300 ml / min, the elution time is 15-65 min, and the wavelength is 252 nm.

[0027] Preferably, the N-segment ganoderic acid sample obtained in S3 is prepared according to S4, and when the purity of the obtained ganoderic acid sample is less than 98%, a second preparation is performed according to S4.

[0028] Preferably, the filler used in S4 is C18WAT filler, which is an octadecylsilane bonded silica filler with a particle size of 10 μm.

[0029] The addition of an appropriate amount of acetic acid during the preparation of ganoderic acid is because the molecules of ganoderic acid compounds contain both hydrophobic lanostane parent rings and carboxyl and hydroxyl groups, which can be protonated. Therefore, the addition of acetic acid in the eluent can provide sufficient protons to make the ganoderic acid molecules neutral molecules, so that they can be more fully distributed with the stationary phase C18.

[0030] Example 1 like Figures 1 to 3 , A method for separating and purifying a ganoderic acid reference substance comprises the following steps: S1 Pretreatment of Ganoderma lucidum raw materials: Take 5 kg of Ganoderma lucidum extract and add 30 L of anhydrous methanol, extract twice by ultrasonic heating at 40 °C, combine the extracts, filter, and concentrate the filtrate to obtain the sample solution.

[0031] S2 silica gel column chromatography decolorization and impurity removal: use 60-100 mesh silica gel to mix the sample solution evenly (the silica gel name: column chromatography silica gel, manufacturer: Qingdao Ocean Chemical Co., Ltd., specification: 60-100 mesh), dry, use dry loading method for normal phase chromatography preparation, the silica gel column is filled with 200-300 mesh silica gel (name: column chromatography silica gel, manufacturer: Qingdao Ocean Chemical Co., Ltd., specification: 200-300 mesh), the mass ratio of sample mixing silica gel to column silica gel is 1:1.1, use petroleum ether-ethyl acetate system for elution, concentrate to remove the normal phase reagent, dissolve with methanol, and obtain 3 sections of ganoderma lucidum acid components; The petroleum ether-ethyl acetate system was used for elution as follows: first, 3 BV of petroleum ether was used to elute, then 4 BV of petroleum ether: ethyl acetate was used in a ratio of 10:1, then 10 BV of petroleum ether: ethyl acetate was used in a ratio of 2:1 to elute to obtain ganoderic acid A sample, then 5 BV of ethyl acetate was used to elute to obtain ganoderic acid B sample, and finally 6 BV of ethyl acetate was used to elute to obtain ganoderic acid C sample, which were named LZ-A, LZ-B, and LZ-C, respectively.

[0032] S3 reverse phase chromatography column split preparation: The three sections of Ganoderma lucidum components were clarified by membrane, and were loaded on medium pressure preparative columns with a sample volume of 8-15% for reverse phase chromatography preparation. The model was 100×650mm, using C18 filler, particle size 10μm, isocratic elution, acetonitrile-acid water system for splitting, acid water was 1% acetic acid, acetonitrile elution ratio was 35%-75%, flow rate was 300ml / min, elution time was 60-80min, HPLC detection was performed, and the wavelength was 252nm. A total of 9 sections of Ganoderma lucidum acid samples were obtained, as follows: LZ-A was loaded on a medium-pressure preparative column with a model of 100×650mm, filled with C18 filler, particle size of 10μm, and a flow rate of 300ml / min at a sample loading of 15%. After loading, it was eluted with 35% acetonitrile-1% acetic acid water for 60min. High performance liquid chromatography was used for analysis and detection at a wavelength of 252nm. The target segment samples were collected, and a total of 3 segments of ganoderic acid components were obtained, which were named LZS-1D, LZS-2D, and LZS-3D respectively.

[0033] LZ-B was continuously injected at a sample loading volume of 15% and a flow rate of 300 ml / min. After loading, it was eluted with 50% acetonitrile-1% acetic acid water for 80 min. High performance liquid chromatography was used for detection at a wavelength of 252 nm. The target segment samples were collected and a total of 4 segments of ganoderic acid components were obtained, which were named LZS-4D, LZS-5D, LZS-6D, and LZS-7D respectively.

[0034] LZ-C was injected at a sample loading of 15% and a flow rate of 300 ml / min. After loading, it was eluted with 75% acetonitrile-1% acetic acid water for 80 min. High performance liquid chromatography was used for analysis and detection at a wavelength of 252 nm. The target segment samples were collected and 2 segments of ganoderic acid components were obtained, named LZS-8D and LZS-9D respectively.

[0035] S4 Preparation of Ganoderic Acid Reference Substance: 9 sections of Ganoderic Acid samples were separately loaded onto medium pressure preparative columns for reverse phase chromatography preparation, model 100×650mm, using C18WAT filler, which is octadecylsilane bonded silica filler, particle size 10μm, isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system for preparation, wherein the acid water is 1% acetic acid, the elution ratio of ethanol is 45%-65%, the elution ratio of acetonitrile is 40%-60%, the elution ratio of methanol is 55%-80%, the flow rate is 300ml / min, the elution time is 15-65min, and the HPLC detection is performed at a wavelength of 252nm. The organic solvent is concentrated and dried under reduced pressure to obtain a total of 22 Ganoderic Acid series reference substances. The specific steps are as follows: (1) LZS-1D, LZS-2D, and LZS-3D were prepared in one dimension using a C18WAT medium-pressure preparative column, using a 55% isocratic methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 35 min for LZS-1D, 50 min for LZS-2, and 55 min for LZS-3D, and the target components were collected at a wavelength of 252 nm: LZS-1D was used to prepare ganoderic acid C6 with a HPLC purity of 91%, ganoderic acid G with a purity of 96%, and ganoderic acid A with a purity of 89%.

[0036] LZS-2D was used to prepare ganoderic acid C2 with an HPLC purity of 94.3% and ganoderic acid G with a purity of 95%.

[0037] LZS-3D was used to prepare ganoderic acid A with a purity of 93.7% by HPLC, ganoderic acid B with a purity of 92.2% and component LZ-3D-1.

[0038] Ganoderic acid C6, ganoderic acid G, ganoderic acid A, ganoderic acid G, ganoderic acid C2, ganoderic acid A, ganoderic acid B, and LZ-3D-1 were prepared in two dimensions using C18WAT medium-pressure preparative column, and ganoderic acid C6, ganoderic acid G, ganoderic acid A, ganoderic acid G, ganoderic acid C2, ganoderic acid A, and ganoderic acid B were prepared using 40% acetonitrile-1% acetic acid water system with a flow rate of 300 mL / min and an elution time of 16 min for ganoderic acid C6. , elution time of ganoderic acid G is 18min, elution time of ganoderic acid A is 23min, elution time of ganoderic acid G is 27min, elution time of ganoderic acid C2 is 30min, elution time of ganoderic acid B is 37min, elution time of ganoderic acid A is 38min, LZ-3D-1 is prepared by 45% ethanol-1% acetic acid water system, flow rate is 300mL / min, elution time is 28min, and target components are collected at 252nm wavelength: LZS-1D prepared ganoderic acid C6 with an HPLC purity of 99.7% and a mass of 13.8 g; ganoderic acid G with an HPLC purity of 98.9% and a mass of 18.7 g; and ganoderic acid A with an HPLC purity of 99.3% and a mass of 15.4 g.

[0039] LZS-2D prepared ganoderic acid C2 with an HPLC purity of 99.5% and a mass of 21.3 g; and ganoderic acid G with an HPLC purity of 99.2% and a mass of 19.7 g.

[0040] LZS-3D prepared ganoderic acid A with an HPLC purity of 98.7% and a mass of 45.8 g; ganoderic acid B with an HPLC purity of 99.2% and a mass of 29.5 g; ganoderic acid D with an HPLC purity of 99.9% and a mass of 21.5 g; and ganoderic acid B with an HPLC purity of 98.5% and a mass of 19.1 g.

[0041] (2) LZS-5D was prepared in one dimension using a C18WAT medium-pressure preparative column in a 50% ethanol-1% acetic acid water system, with a flow rate of 300 mL / min, an elution time of 30 min, and a wavelength of 252 nm to collect the target components, LZ-5-1 and LZ-5-2.

[0042] The LZ-5-1 and LZ-5-2 components were prepared in two dimensions using a C18WAT medium-pressure preparative column, using a 65% methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 42 min for LZ-5-1, an elution time of 40 min for LZ-5-2, and a wavelength of 252 nm to collect the target components: LZS-5D was used to prepare ganoderic acid D with an HPLC purity of 98.9%, with a mass of 26.7 g; ganoderic acid A2 with an HPLC purity of 99.8%, with a mass of 15.3 g; and ganoderic acid DM with an HPLC purity of 98.6%, with a mass of 23.8 g.

[0043] (3) LZS-4D, LZS-6D, and LZS-7D were prepared in one dimension using a C18WAT medium-pressure preparative column, using a 65% methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 45 min for LZS-4D, 50 min for LZS-6D, and 50 min for LZS-7D, and the target components were collected at a wavelength of 252 nm: LZS-4D was used to prepare ganoderic acid F with an HPLC purity of 96.7%. The ganoderic acid F was concentrated to a small volume to obtain solid precipitation. The solid was filtered and washed with water to obtain ganoderic acid F with an HPLC purity of 98.3% and a mass of 17.1 g.

[0044] LZS-6D was used to prepare ganoderic acid E with an HPLC purity of 96.8% and ganoderic acid L with an HPLC purity of 99.5%, with a mass of 21.8 g.

[0045] LZS-7D was used to prepare ganoderic acid SZ with an HPLC purity of 81.4%, ganoderic acid H with an HPLC purity of 94.5%, and ganoderic acid TR with an HPLC purity of 91.7%.

[0046] Ganoderic acid E, Ganoderic acid SZ, Ganoderic acid H, and Ganoderic acid TR were prepared in two dimensions using a C18WAT medium-pressure preparative column, using a 45% isocratic acetonitrile-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 18 min for Ganoderic acid E, 27 min for Ganoderic acid SZ, 50 min for Ganoderic acid H, and 39 min for Ganoderic acid TR, and the target components were collected at a wavelength of 252 nm: Ganoderic acid E with HPLC purity of 99.5% was prepared by LZS-6D.

[0047] LZS-7D prepared ganoderic acid SZ with an HPLC purity of 98.2% and a mass of 8.9 g; ganoderic acid H with an HPLC purity of 99.5% and a mass of 31 g; and ganoderic acid TR with an HPLC purity of 98.6% and a mass of 12.8 g.

[0048] (4) LZS-8D and LZS-9D were prepared in one dimension using a C18WAT medium-pressure preparative column, using an 80% isocratic methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 50 min for LZS-8D, and an elution time of 65 min for LZS-9D, and the target components were collected at a wavelength of 252 nm: LZS-8D was used to prepare ganoderic acid T with an HPLC purity of 98.9% and a mass of 29.5 g.

[0049] LZS-9D was used to prepare ganoderic acid I with an HPLC purity of 94.2%, ganoderic acid S with an HPLC purity of 95.3%, and ganoderic acid C with an HPLC purity of 92.3%.

[0050] Ganoderic acid I, Ganoderic acid C and Ganoderic acid S were prepared in two dimensions using C18WAT medium-pressure preparation column. Ganoderic acid I and Ganoderic acid C were prepared using 60% isocratic acetonitrile-1% acetic acid water system at a flow rate of 300 mL / min. The elution time of Ganoderic acid I and Ganoderic acid C was 44 min and 47 min, respectively. Ganoderic acid S was prepared using 65% isocratic ethanol-1% acetic acid water system at a flow rate of 300 mL / min. The elution time of Ganoderic acid S was 24 min. The target components were collected at a wavelength of 252 nm. LZS-9D prepared ganoderic acid C with an HPLC purity of 99.2% and a mass of 31 g; ganoderic acid I with an HPLC purity of 99.6% and a mass of 17.3 g; and ganoderic acid S with an HPLC purity of 99.5% and a mass of 15.9 g.

[0051] Example 2 like Figure 2-Figure 3 , a method for separating and purifying a ganoderic acid reference substance, comprising the following steps: S1 Ganoderma lucidum raw material pretreatment: take 5 kg of Ganoderma lucidum extract and add 30 L of anhydrous methanol, extract twice by ultrasonic heating at 40 ° C, combine the extracts, filter, and concentrate the filtrate to obtain the sample solution; S2 silica gel column chromatography decolorization and impurity removal: 60-100 mesh silica gel (name: column chromatography silica gel, manufacturer: Qingdao Ocean Chemical Co., Ltd., specification: 60-100 mesh) was used to mix the sample solution evenly, dry it, and prepare it for normal phase chromatography by dry loading. The silica gel column was filled with 200-300 mesh silica gel (name: column chromatography silica gel, manufacturer: Qingdao Ocean Chemical Co., Ltd., specification: 200-300 mesh). The mass ratio of the sample mixing silica gel to the column silica gel was 1:1.1. The petroleum ether-ethyl acetate system was used for elution, the normal phase reagent was removed by concentration, and the mixture was dissolved with methanol to obtain 3 sections of ganoderma lucidum acid components; The specific elution process using the petroleum ether-ethyl acetate system is as follows: first, 3 BV of petroleum ether is used to elute, then 4 BV of petroleum ether: ethyl acetate is used in a ratio of 10:1, then 10 BV of petroleum ether: ethyl acetate is used in a ratio of 2:1 to elute to obtain a ganoderic acid sample A, then 5 BV of ethyl acetate is used to elute to obtain a ganoderic acid sample B, and finally 6 BV of ethyl acetate is used to elute to obtain a ganoderic acid sample C, which are named LZ-A, LZ-B, and LZ-C, respectively.

[0052] S3 reverse phase chromatography column split preparation: The three sections of Ganoderma lucidum components were clarified by membrane, and then respectively loaded on medium pressure preparative columns for reverse phase chromatography preparation, with a sample loading of 8-15%, using C18 filler, particle size of 10 μm, isocratic elution, acetonitrile-acid water system for splitting, acid water was 1% acetic acid, acetonitrile elution ratio was 35%-75%, flow rate was 300 ml / min, elution time was 60-80 min, HPLC detection was performed, wavelength was 252 nm, and a total of 16 sections of Ganoderma lucidum acid samples were obtained, as follows: LZ-A was loaded on a medium-pressure preparative column with a sample volume of 8%, model 100×650mm, filled with C18 filler, particle size 10μm, flow rate 300ml / min, and eluted with 35% acetonitrile-1% acetic acid water for 60min. High performance liquid chromatography analysis was performed at a wavelength of 252nm. The target segment samples were collected, and a total of 7 segments of ganoderic acid components were obtained, which were named LZS-1D, LZS-2D, LZS-3D, LZS-4D, LZS-5D, LZS-6D, and LZS-7D respectively.

[0053] LZ-B was continuously injected with a sample loading volume of 8% and a flow rate of 300 ml / min. After loading, it was eluted with 50% acetonitrile-1% acetic acid water for 80 min. High performance liquid chromatography was used for detection at a wavelength of 252 nm. The target segment samples were collected and a total of 6 segments of ganoderic acid components were obtained, which were named LZS-8D, LZS-9D, LZS-10D, LZS-11D, LZS-12D, and LZS-13D respectively.

[0054] LZ-C was continuously injected with a sample loading volume of 8% and a flow rate of 300 ml / min. After loading, it was eluted with 75% acetonitrile-1% acetic acid water for 80 min. High performance liquid chromatography was used for detection at a wavelength of 252 nm. The target segment samples were collected and a total of 3 segments of ganoderic acid components were obtained, namely LZS-14D, LZS-15D, and LZS-16D.

[0055] S4 Preparation of Ganoderic Acid Reference Substance: 16 sections of Ganoderic Acid samples were respectively loaded onto medium pressure preparative columns for reverse phase chromatography preparation. The filler used was C18WAT filler, which is an octadecylsilane bonded silica filler with a particle size of 10 μm. Isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system were used for preparation, wherein the acid water was 1% acetic acid, the elution ratio of ethanol was 45%-65%, the elution ratio of acetonitrile was 40%-60%, and the elution ratio of methanol was 55%-80%. The flow rate was 300 ml / min, and the elution time was 15-65 min. HPLC detection was performed at a wavelength of 252 nm. The organic solvent was concentrated and dried under reduced pressure. A total of 22 Ganoderic Acid series reference substances were obtained. The specific steps are as follows: (1) LZS-1D, LZS-2D, LZS-3D, LZS-4D, LZS-5D and LZS-6D were prepared in one dimension using a C18WAT medium pressure preparative column, using a 55% isocratic methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 25 min for LZS-1D and LZS-2D, 28 min for LZS-3D, 37 min for LZS-4D, and 50 min for LZS-5D and LZS-6D, and the target components were collected at a wavelength of 252 nm: LZS-1D prepared ganoderic acid C6 with an HPLC purity of 99.3% and a mass of 15.8 g; and ganoderic acid G with an HPLC purity of 99.8% and a mass of 16.3 g.

[0056] LZS-2D prepared ganoderic acid A with an HPLC purity of 99.9% and a mass of 20.1 g.

[0057] LZS-3D prepared ganoderic acid C2 with an HPLC purity of 98.6% and a mass of 25.6 g.

[0058] LZS-4D was used to prepare ganoderic acid G with an HPLC purity of 99.4% and a mass of 17.2 g.

[0059] LZS-5D was used to prepare ganoderic acid A with an HPLC purity of 99.6% and a mass of 37.3 g.

[0060] LZS-6D was used to prepare ganoderic acid B with an HPLC purity of 99.5% and a mass of 97.0 g.

[0061] (2) LZS-7D was prepared in one dimension using a C18WAT medium-pressure preparative column, using a 45% isocratic ethanol-1% acetic acid water system, a flow rate of 300 mL / min, an LZS-7D elution time of 45 min, and a wavelength of 252 nm: LZS-7D was used to prepare ganoderic acid D with an HPLC purity of 99.9% and a mass of 28.3 g; and ganoderic acid B with an HPLC purity of 98.6% and a mass of 25.3 g.

[0062] (3) LZS-8D, LZS-10D, LZS-11D, LZS-12D, and LZS-13D were prepared in one dimension using a C18WAT medium-pressure preparative column, using a 65% isocratic methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 30 min for LZS-8D, 38 min for LZS-10D and LZS-11D, 50 min for LZS-12D, and 45 min for LZS-13D, and the target components were collected at a wavelength of 252 nm: LZS-8D was used to prepare ganoderic acid D with an HPLC purity of 99.3% and a mass of 30.3 g.

[0063] LZS-10D was used to prepare ganoderic acid E with an HPLC purity of 98.8% and a mass of 11.2 g; and ganoderic acid DM with an HPLC purity of 99.6% and a mass of 20.8 g.

[0064] LZS-11D was used to prepare ganoderic acid L with an HPLC purity of 99.5% and a mass of 21.9 g; and ganoderic acid SZ with an HPLC purity of 99.2% and a mass of 7.9 g.

[0065] LZS-12D prepared ganoderic acid H with an HPLC purity of 99.1% and a mass of 23.3 g; and ganoderic acid TR with an HPLC purity of 98.9% and a mass of 13.4 g.

[0066] LZS-13D prepared ganoderic acid F with an HPLC purity of 99.5% and a mass of 15.7 g.

[0067] (4) LZS-9D was prepared in one dimension using a C18WAT medium-pressure preparative column, using a 50% isocratic ethanol-1% acetic acid water system, a flow rate of 300 mL / min, an LZS-9D elution time of 38 min, and a wavelength of 252 nm: LZS-9D prepared ganoderic acid A2 with an HPLC purity of 99.7% and a mass of 18.5 g.

[0068] (5) LZS-14D, LZS-15D, and LZS-16D were prepared in one dimension using a C18WAT medium-pressure preparative column, using an 80% isocratic methanol-1% acetic acid water system, a flow rate of 300 mL / min, an elution time of 28 min for LZS-14D, 35 min for LZS-15D, and 40 min for LZS-16D, and the target components were collected at a wavelength of 252 nm: LZS-14D was used to prepare ganoderic acid T with an HPLC purity of 99.8% and a mass of 22.6 g.

[0069] LZS-15D was used to prepare ganoderic acid C with an HPLC purity of 99.9% and a mass of 28.7 g; and ganoderic acid I with an HPLC purity of 99.1% and a mass of 25.6 g.

[0070] LZS-16D was used to prepare ganoderic acid S with an HPLC purity of 98.7% and a mass of 25.9 g.

[0071] Example 3 like Figure 4 , Comparison of separation effects between conventional C18 filler and C18WAT filler: In the preparation process of ganoderic acid, conventional C18 filler and C18WAT filler were used for simulation preparation respectively, and the same specification chromatographic column was used: 10um filler and 100dac thick column.

[0072] The ganoderic acid component was loaded on a C18 preparative column at a loading amount of 1%, and prepared using a 55% methanol-1% acetic acid water system, a flow rate of 300 mL / min, elution for 35 min, and the target components were collected at a wavelength of 252 nm to prepare ganoderic acid A with an HPLC purity of 72.3% and ganoderic acid B with an HPLC purity of 61.6%.

[0073] The ganoderic acid component was loaded on a C18WAT preparative column at a loading amount of 5%. The filler was acid-resistant and had a particle size of 10 um. It was prepared using a 55% methanol-1% acetic acid water system with a flow rate of 300 mL / min and an elution time of 30 min. The target components were collected at a wavelength of 252 nm to prepare ganoderic acid A with an HPLC purity of 99.3% and ganoderic acid B with an HPLC purity of 98.7%.

[0074] In the preparation process of ganoderic acid, C18WAT filler has higher selectivity than traditional C18 filler. C18WAT has better separation effect at high sample load than traditional C18 at low sample load, which is of great significance for the industrial production of ganoderic acid series reference substances.

[0075] Conclusion: 1. The isocratic preparation method can save reagents and time by continuous sampling, and different samples can be obtained by the same preparation method. Therefore, compared with the traditional purification method, the separation of ganoderic acid in this patent invention has the advantages of high efficiency and short time, and has a high yield, and can obtain a large amount of ganoderic acid reference substances with a purity of >98%.

[0076] 2. The preparation is carried out in an isocratic manner. The organic reagents such as methanol, acetonitrile, ethanol, etc. used in the preparation process can be recycled by a concentration method of reagent rotary evaporation recovery. Specifically, the waste liquid generated by the preparation is concentrated and recovered by rotary evaporation, which is more environmentally friendly and can significantly reduce production costs, and is conducive to industrial production.

[0077] It should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0078] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for separating and purifying a ganoderic acid reference substance, It is characterized in that The following steps are involved: S1 Ganoderma lucidum raw material pretreatment: Ganoderma lucidum extract was extracted twice with anhydrous methanol and ultrasonic heating, the extracts were combined, filtered, and the filtrate was concentrated to obtain a sample solution; S2 silica gel column chromatography for decolorization and impurity removal: the sample solution is mixed with silica gel, dried, and prepared for normal phase chromatography by dry loading, eluted with petroleum ether-ethyl acetate system, concentrated to remove the normal phase reagent, and dissolved with methanol to obtain three sections of ganoderma lucidum acid components; S3 reverse phase chromatography column split preparation: The three sections of Ganoderma lucidum components were clarified by membrane, and then respectively applied to medium pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, acetonitrile-acid water system for splitting, and high performance liquid chromatography detection to obtain N sections of Ganoderma lucidum acid samples; Preparation of S4 ganoderic acid reference substance: The N-segment ganoderic acid samples were separately loaded onto medium-pressure preparative columns for reverse phase chromatography preparation, using isocratic elution, ethanol-acid water system or acetonitrile-acid water system or methanol-acid water system for preparation, and detected by high performance liquid chromatography. The organic solvent was concentrated to remove the organic solvent, and the samples were dried under reduced pressure to obtain a total of 22 ganoderic acid series reference substances.

2. A method for separating and purifying a ganoderic acid reference substance according to claim 1, It is characterized in that In S2, 60-100 mesh silica gel was used for sample mixing, and 200-300 mesh silica gel was used to fill the silica gel column. The mass ratio of sample mixing silica gel to column filling silica gel was 1:1.

1.

3. A method for separating and purifying a ganoderic acid reference substance according to claim 1, It is characterized in that The petroleum ether-ethyl acetate system was used for elution in S2 as follows: first, 3 BV of petroleum ether was used to elute, then 4 BV of petroleum ether: ethyl acetate was used at a ratio of 10:1, then 10 BV of petroleum ether: ethyl acetate was used at a ratio of 2:1 to elute to obtain ganoderic acid sample A, then 5 BV of ethyl acetate was used to elute to obtain ganoderic acid sample B, and finally 6 BV of ethyl acetate was used to elute to obtain ganoderic acid sample C.

4. The method for separating and purifying a ganoderic acid reference substance according to claim 1, It is characterized in that In the acetonitrile-acid water system of S3, the acid water is 1% acetic acid, the acetonitrile elution ratio is 35%-75%, the flow rate is 300ml / min, the elution time is 60-80min, and the wavelength is 252nm.

5. The method for separating and purifying a ganoderic acid reference substance according to claim 1, It is characterized in that S3 uses C18 filler with a particle size of 10 μm and a medium pressure preparative column with a sample loading of 8-15%.

6. The method for preparing a series of ganoderic acid reference substances according to claim 1, It is characterized in that S4 is prepared using an ethanol-acid water system, an acetonitrile-acid water system, or a methanol-acid water system, wherein the acid water is 1% acetic acid, the elution ratio of ethanol is 45%-65%, the elution ratio of acetonitrile is 40%-60%, the elution ratio of methanol is 55%-80%, the flow rate is 300 ml / min, the elution time is 15-65 min, and the wavelength is 252 nm.

7. The method for preparing a series of ganoderic acid reference substances according to claim 6, It is characterized in that The N-segment ganoderic acid sample obtained in S3 is prepared according to S4. When the purity of the obtained ganoderic acid sample is less than 98%, a second preparation is performed according to S4.

8. The method for preparing a series of ganoderic acid reference substances according to claim 1, It is characterized in that The filler used in S4 is C18WAT filler, which is an octadecylsilane bonded silica filler with a particle size of 10 μm.

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