Preparation method of gambogic acid series reference substance

Abstract

The invention relates to a preparation method of gambogic acid series reference substances. With gamboge as a raw material, the method comprises the following steps: (1) pretreatment of gamboge; (2) a column chromatography enrichment process of gamboge; (3) a gamboge segmentation process; and (4) a preparation process of a gambogic acid reference substance. The method adopts a reversed-phase strong-acid-resistant C18STE and reversed-phase C18 alternate separation mode, has the advantages of high efficiency, easiness in operation and the like, and can be used for preparing various gambogic acid series reference substances with the purity of 98% or above on a large scale.

Description

Technical Field

[0001] The invention belongs to the technical field of gambogic acid preparation methods, and specifically relates to a method for preparing a gambogic acid series reference substance. Background Art

[0002] Garcinia is the colloid resin of the plant Garcinia of the family Garcinia. It was first recorded in the wood section of the "Haiyao Bencao Bencao". It tastes sour and moist. As a traditional Chinese medicine, it has the functions of reducing swelling, removing lumps, stopping bleeding and killing insects. It is mainly used in the treatment of carbuncle, swelling, stubborn ulcers and other diseases in clinical practice. In addition, it can also be used as a yellow dye. It is mainly distributed in India, Thailand and other places, and is also cultivated in Guangdong, Guangxi, Fujian, Hainan and other regions in my country.

[0003] In recent years, more than 20 compounds have been reported to be isolated and identified from the Chinese medicine Garcinia, most of which are xanthones containing bridge rings, mainly Garcinia acid, isogarcinia acid, neogarcinia acid, as well as morilin, garcinol and other substances of the Garcinia acid class. Among them, Garcinia acid and neogarcinia acid are their main active ingredients. Studies have shown that Garcinia acid and neogarcinia acid both have strong anti-tumor effects, a wide anti-tumor spectrum, low toxicity, can selectively act on tumor cells, and have little effect on the hematopoietic system and immune function of normal animals. They have good therapeutic effects on lung cancer, pancreatic cancer, gastric cancer, etc., and are natural anti-tumor drugs with great development prospects. There are many reports on the activity of the two, but there are relatively few reports on the extraction and preparation methods of the two.

[0004] The extraction process of new gambogic acid is mainly based on methanol reflux extraction. Liu Xiushu et al. used new gambogic acid as an indicator and determined the extraction process as 12 times methanol reflux extraction 3 times, each time for 3 hours. On this basis, Wang Ke et al. proposed that the optimal extraction process of new gambogic acid is to soak the medicinal materials in 12 times methanol and reflux extraction 2 times, each time for 5 hours. The separation process of gambogic acid and new gambogic acid mainly includes multi-silica gel column chromatography, medium-pressure preparative liquid phase method, pyridine salt crystallization method, etc. Liu Weihai et al. used a dry chromatography column with thin layer silica gel G as the stationary phase and eluted with trichloromethane: methanol: diethylamine (10:1:1) as the mobile phase. When the eluent reached the bottom of the column, the target color band was scraped out and loaded into the chromatography column, and then eluted with methanol and concentrated to an extract. Use a concentration of 0.01g / mL NaHCO 3The extract was dissolved in the solution, and then the pH was adjusted to 2.0 with HCl solution at pH = 1.0. A bright yellow precipitate was precipitated, filtered, and washed with distilled water until neutral to obtain new gambogic acid. Hu Zhong et al. used the mother liquor of gambogic acid pyridine salt as raw material, acidified it, and filled the column with 100-200 mesh silica gel. The new gambogic acid was obtained by column chromatography with a variety of eluents, and the yield was 20%. Li Xiang et al. used medium-pressure liquid phase preparation, medium- and high-pressure glass preparation chromatography tower 310×100mm, filler ODS (C18) (30-50μm), sample amount 50g, flow rate 180ml / min, and eluted with methanol-0.1% formic acid water (80:20), but the purity of the obtained new gambogic acid was about 95%. Liu Ju et al. mixed the garcinia resin powder and diatomaceous earth, extracted it by ethyl acetate percolation, and then obtained the orange-yellow garcinia acid monomer through pyridine salt formation, three recrystallizations, and displacement extraction, with a yield of 23.9%.

[0005] However, the above methods are either cumbersome to operate, or the purity cannot meet the requirements of the reference substance, or it is impossible to obtain multiple gram-level gambogic acid series reference substances. Therefore, it is particularly important to study and develop a new and efficient technology for separating and purifying gamboge and ultimately realize industrial production. The purpose of the present invention is to develop a method for preparing multiple gram-level gamboge acid series reference substances that can be separated and purified quickly and efficiently with a purity of more than 98%, and can realize industrial production. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a series of gambogic acid reference substances. The present invention provides a method for preparing a series of gambogic acid reference substances that is easy to operate, high in purity, and can be industrialized on a large scale, and comprises the following steps in sequence: The pretreatment process of Garcinia cambogia: crush the Garcinia cambogia raw material, extract with ethanol, filter, and concentrate the filtrate until there is no alcohol taste to obtain Garcinia cambogia concentrate.

[0007] Column chromatography enrichment process of Garcinia: The Garcinia concentrate was mixed with silica gel, eluted with 200-300 mesh silica gel as the stationary phase, petroleum ether and ethyl acetate as the eluent, and the Garcinia eluate was collected and concentrated into an extract, dissolved in methanol, and filtered to obtain the Garcinia clarified liquid.

[0008] Garcinia segmentation process: The Garcinia clarified liquid was cut into segments on a medium-pressure preparative column with a sample loading of 1% to 4%. The elution solvents were acetonitrile and acid water. A high organic phase isocratic elution method was adopted with an organic phase ratio of 80% to 90%. High performance liquid chromatography was used for detection to obtain a total of 11 Garcinia components.

[0009] Preparation process of garcinia acid reference substance: 11 sections of garcinia components are placed on a medium-pressure preparative column for column concentration and impurity removal, and then reverse phase preparation is performed. The elution solvents are methanol and water. HPLC detection is performed, and reduced pressure concentration is performed, filtration is performed, 40%~50% methanol is used for elution, and freeze-drying is performed to obtain 11 garcinia acid series reference substances.

[0010] In a preferred embodiment of the present invention, the pretreatment process of Garcinia adopts 70%~100% ethanol or methanol extraction, the solid-liquid ratio is 1:1~4:1, the extraction temperature is 45~50°C, the extraction time is 2~3h, and the number of extractions is 2~3 times.

[0011] In a preferred embodiment of the present invention, the filler in the medium-pressure preparation column of the Garcinia segmentation process is a reverse phase strong acid-resistant C18STE filler (a type of octadecylsilane bonded silica filler with low pH resistance and high temperature resistance) with a particle size of 5-10 μm.

[0012] In a preferred embodiment of the present invention, the column concentration in the garcinia segmentation process is to concentrate the garcinia component at 40°C and a vacuum degree of -0.09Mpa to an organic phase ratio of about 60% to 65%, and then prepare a column for concentration, wherein sections 1 to 5 are concentrated with C18 fillers; sections 6 to 11 are concentrated with C18STE fillers, the elution solvents are acetonitrile and water, and the organic phase ratio is 40% to 85%.

[0013] In a preferred embodiment of the present invention, the acid in the acetonitrile and acid water in the Garcinia segmentation process is 0.1% to 5.0% acetic acid.

[0014] In a preferred embodiment of the present invention, the reverse phase preparation of the 11-stage garcinia component in the garcinia reference substance preparation process adopts C18 and C18STE alternating separation and purification, the elution solvents are methanol and water, a high organic phase isocratic elution method is adopted, and the organic phase ratio is selected to be 80%~90%.

[0015] In a preferred embodiment of the present invention, the concentration in the preparation process of the garcinia reference substance is concentrated to an organic phase ratio of 30% to 50% under the conditions of 35° C. and a vacuum degree of -0.03 to 0.08 MPa.

[0016] Beneficial effects of the present invention: 1. This patent can quickly obtain a series of high-purity garcinia acid reference substances, and can provide high-quality samples for further research on the pharmacological activity of garcinia acid.

[0017] 2. In view of the particularity and strong retention of the series of garcinia acid samples, the present invention uses reverse phase strong acid resistant C18STE filler for segmentation.

[0018] 3. The present invention simplifies the process flow of separation and purification of Garcinia: Garcinia pretreatment process - Garcinia column chromatography enrichment process - Garcinia segmentation process - Garcinia acid reference substance preparation process, which has the advantages of high efficiency and high yield, and can quickly obtain a variety of gram-level Garcinia acid series reference substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Process flow chart of the preparation method of the series of garcinia acid reference substances; Figure 2 HPLC analysis of garcinia acid series reference substances; Figure 3 Comparison of the analysis results of Garcinia in C18 filler and C18STE filler; Figure 4 Comparison chart of the preparation method development of Garcinia on C18 filler and C18STE filler; Figure 5 Effect of temperature on the purity of garcinia acid components. DETAILED DESCRIPTION

[0020] The organic solvents such as methanol, ethanol and acetonitrile used in the preparation of the present invention are all reused in a concentrated recovery manner, thereby saving solvent costs.

[0021] There is no particular limitation on the concentration method in the present invention, which is mainly concentrated under reduced pressure. There is no particular limitation on the filtering method, either a conventional filtering device or a suction filtering device can be used. The water used is mainly distilled water or deionized water.

[0022] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are only exemplary to illustrate and explain the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0023] Example 1 According to the process flow chart (such as Figure 1 ) Steps are as follows: Step A: Extraction of medicinal materials: Purchase 5 kg of Garcinia cambogia and crush it into powder using a grinder. Add 10 L of 90% ethanol for heating and soaking extraction at a temperature of 45-50°C. Extract 2-3 times, filter twice with filter paper, and concentrate the filtrate at 45-50°C until there is no alcohol taste to obtain Garcinia cambogia concentrate.

[0024] Step B: Column chromatography enrichment: Add 2kg of 60-100 mesh silica gel to the concentrate of step A, stir evenly, and air dry. Add the sample to 2.5kg of 200-300 mesh silica gel column for normal phase preparation. Use seven mixing ratios of petroleum ether (5BV), petroleum ether: ethyl acetate = 50:1 (5BV), petroleum ether: ethyl acetate = 25:1 (8BV), petroleum ether: ethyl acetate = 10:1 (3BV), petroleum ether: ethyl acetate = 5:1 (3BV), petroleum ether: ethyl acetate = 1:1 (3BV), and ethyl acetate (3BV) for elution. Concentrate the eluate to extract.

[0025] Step C: Dissolving and clarifying: Dissolve the extract in step B with methanol and filter through a membrane to obtain a clarified solution. Step D: Garcinia segmentation: The clarified liquid in step C was loaded on a medium-pressure preparative column with a sample loading volume of 1% for preparation. The model was 100×650mm, the filler was a reversed-phase strong acid-resistant C18STE filler, the particle size was 10μm, the flow rate was 330ml / min, the mobile phase A was acetonitrile, and B was 0.1% acetic acid water. The mobile phases were eluted with 80% A for 55min and 90% A for 10min, respectively. HPLC analysis and detection were performed at a wavelength of 362nm. The target segment samples were collected, and a total of 11 segments of Garcinia components were obtained, which were named TH_1~TH_11 respectively.

[0026] Step E: Preparation of garcinia acid by reverse phase chromatography: TH_1~TH_5 in step D are purified using C18 filler; TH_6 is prepared and purified alternately using C18 and C18STE; TH_7~TH_11 are purified using C18STE filler, and the particle size of C18 and C18STE is 10μm. The components in the preparation process are stored in a -20℃ refrigerator, and crystals are precipitated after low-temperature concentration to a low organic phase, filtered, rinsed with 40%~50% methanol water, and freeze-dried to obtain a high-purity reference solid. The specific operation method for the preparation of 11 segments of garcinia components is as follows: TH_1~TH_5 were concentrated by C18 preparative column, mobile phase A was acetonitrile, B was pure water, flow rate was 300ml / min, wavelength was 362nm, gradient elution was 40~85% phase A, elution time was 0~50min, column concentrate was obtained and then concentrated under reduced pressure, concentrated into solid, dissolved in methanol, passed through membrane, and TH_1~TH_5 clarified liquid was obtained; The clarified liquids of TH_1~TH_5 were prepared by C18 preparative columns, using an isocratic preparation of 80% recovered methanol-water system, a flow rate of 330ml / min, a wavelength of 362nm, and continuous injection to collect the target components.

[0027] TH_1 was used to prepare Forbesione with a HPLC purity of 99.50% and a mass of 2.10 g; TH_2 was prepared to obtain Gaudichaudic acid with an HPLC purity of 99.86% and a mass of 2.20 g; TH_3 was used to prepare β-morellic acid with a HPLC purity of 99.65% and a mass of 7.20 g; TH_4 was used to prepare α-morellic acid with a HPLC purity of 99.31% and a mass of 48.00 g; TH_5 was used to prepare Isomorellinol with an HPLC purity of 99.56% and a mass of 5.70 g; TH_6 was concentrated using a C18STE preparative column, with mobile phase A being acetonitrile, mobile phase B being pure water, a flow rate of 300 ml / min, a wavelength of 362 nm, a gradient elution of 70-80% phase A, and an elution time of 0-50 min. The column concentrate was obtained and then concentrated under reduced pressure to form a solid, dissolved in methanol, and passed through a membrane to obtain a clear liquid.

[0028] The clarified liquid was prepared in one dimension using a C18 preparative column, and a 90% recovered methanol-water system was used for isocratic preparation, a flow rate of 330 ml / min, and a wavelength of 362 nm to prepare a sample with a HPLC purity of 97.88%. The clarified liquid was prepared in two dimensions using a reversed-phase C18STE filler, and an 80% recovered methanol-water system was used for isocratic preparation, a flow rate of 330 ml / min, and a wavelength of 362 nm, and continuous injection was used to prepare Isogambogenic acid with a HPLC purity of 99.80%, with a mass of 17.40 g. TH_7~TH_11 were concentrated by C18STE preparation column, mobile phase A was acetonitrile, B was pure water, flow rate was 300ml / min, wavelength was 362nm, gradient elution was 42~85% phase A, elution time was 0~60min, column concentrate was obtained and then concentrated under reduced pressure, concentrated into solid, dissolved in methanol, passed through membrane, and TH_7~TH_11 clarified solution was obtained; The clarified solutions of TH_7~TH_11 were prepared using C18STE preparative columns, using an isocratic preparation of 85% recovered methanol-water system, a flow rate of 330 ml / min, a wavelength of 362 nm, and continuous injection to collect the target components; TH_7 was used to prepare Neogambogic acid with an HPLC purity of 99.42% and a mass of 152.83 g; TH_8 was used to prepare R-isogarcinia acid with a HPLC purity of 99.48% and a mass of 51.90 g; After TH_9 was concentrated, crystals were precipitated, eluted with methanol, and tested by high performance liquid chromatography. The purity was 99.25%. The structural nuclear magnetic spectrum analysis confirmed that it was S-isogarcinia acid, with a mass of 49.53g. TH_10 was prepared to obtain Gambogic acid with an HPLC purity of 98.43% and a mass of 169.31 g.

[0029] Epigambogic acid with a HPLC purity of 99.37% and a mass of 156.44 g was obtained from TH_11.

[0030] Example 2 According to the process flow chart, the steps are as follows: Step A: Extraction of medicinal materials: 5 kg of Garcinia cambogia was crushed into powder using a pulverizer. 10 L of 90% ethanol was added for heating and soaking extraction. The extraction temperature was 45-50°C. The extraction was repeated 2-3 times, and the filtrate was filtered twice. The filtrate was concentrated at 45-50°C until there was no alcohol taste, and Garcinia cambogia concentrate was obtained.

[0031] Step B: Column chromatography enrichment: Add 1.1kg of 60-100 mesh silica gel to the concentrate of step A, stir evenly, and air dry. Add the sample to 2kg of 200-300 mesh silica gel column for normal phase preparation. Use six mixing ratios of petroleum ether (2BV), petroleum ether: ethyl acetate = 50:1 (6BV), petroleum ether: ethyl acetate = 25:1 (6BV), petroleum ether: ethyl acetate = 10:1 (2BV), petroleum ether: ethyl acetate = 1:1 (10BV), and ethyl acetate (4BV) for elution. The eluate is concentrated to an extract.

[0032] Step C: Dissolving and clarifying: Dissolve the extract in step B with methanol and filter through a membrane to obtain a clarified solution. Step D: Garcinia segmentation: The clarified liquid in step C was loaded on a medium-pressure preparative column with a sample loading volume of 4%, model 100×650mm, filler C18STE filler, particle size 10μm, flow rate 330ml / min, mobile phase A was acetonitrile, B was 0.1% acetic acid water, and 80% A was used for isocratic elution for 55min and 90% A for isocratic elution for 10min, respectively. HPLC analysis and detection was carried out at a wavelength of 362nm. The target segment samples were collected, and a total of 11 segments of Garcinia components were obtained, which were named TH_1~TH_11 respectively.

[0033] Step E: Preparation of garcinia acid by reverse phase chromatography: TH_1~TH_5 in step D are purified using C18 filler; TH_6 is prepared and purified alternately using C18 and C18STE; TH_7~TH_11 are purified using C18STE filler, and the particle size of C18 and C18STE is 10μm. The components in the preparation process are stored in a -20℃ refrigerator, and crystals are precipitated after low-temperature concentration to a low organic phase, filtered, rinsed with 40%~50% methanol water, and freeze-dried to obtain a high-purity reference solid. The specific operation method for the preparation of 11 segments of garcinia components is as follows: TH_1~TH_5 were concentrated by C18 preparative column, mobile phase A was acetonitrile, B was pure water, flow rate was 300ml / min, wavelength was 362nm, gradient elution was 40~85% phase A, elution time was 0~50min, column concentrate was obtained and then concentrated under reduced pressure, concentrated into solid, dissolved in methanol, passed through membrane, and TH_1~TH_5 clarified liquid was obtained; The clarified liquids of TH_1~TH_5 were prepared by C18 preparative columns, using an isocratic preparation of 80% recovered methanol-water system, a flow rate of 330ml / min, a wavelength of 362nm, and continuous injection to collect the target components.

[0034] TH_1 was used to prepare Forbesione with a HPLC purity of 99.35% and a mass of 1.95 g; TH_2 was prepared to obtain Gaudichaudic acid with an HPLC purity of 99.77% and a mass of 1.75 g; TH_3 was used to prepare β-morellic acid with an HPLC purity of 98.73% and a mass of 6.44 g; TH_4 was prepared to obtain α-morellic acid with an HPLC purity of 98.50% and a mass of 53.35 g; TH_5 was used to prepare Isomorellinol with an HPLC purity of 99.57% and a mass of 9.65 g; TH_6 was concentrated using a C18STE preparative column, with mobile phase A being acetonitrile, mobile phase B being pure water, a flow rate of 300 ml / min, a wavelength of 362 nm, a gradient elution of 70-80% phase A, and an elution time of 0-50 min. The column concentrate was obtained and then concentrated under reduced pressure to form a solid, dissolved in methanol, and passed through a membrane to obtain a clear liquid.

[0035] The clarified liquid was prepared in one dimension using a C18 preparative column, and a 90% recovered methanol-water system was used for isocratic preparation, a flow rate of 330 ml / min, and a wavelength of 362 nm to prepare a sample with a HPLC purity of 97.64%. The clarified liquid was prepared in two dimensions using a reversed-phase C18STE filler, and an 80% recovered methanol-water system was used for isocratic preparation, a flow rate of 330 ml / min, and a wavelength of 362 nm, and continuous injection was used to prepare Isogambogenic acid with a HPLC purity of 98.64%, with a mass of 22.18 g. TH_7~TH_11 were concentrated by C18STE preparation column, mobile phase A was acetonitrile, B was pure water, flow rate was 300ml / min, wavelength was 362nm, gradient elution was 40~85% phase A, elution time was 0~60min, column concentrate was obtained and then concentrated under reduced pressure, concentrated into solid, dissolved in methanol, passed through membrane, and TH_7~TH_11 clarified solution was obtained; After TH_9 was concentrated, crystals were precipitated, eluted with methanol, and detected by high performance liquid chromatography. The purity was 98.86%. Structural nuclear magnetic resonance spectrum analysis confirmed that it was S-isogarcinia acid with a mass of 52.20 g.

[0036] The clarified solutions of TH_7, TH_8 and TH_11 were prepared by C18STE preparative columns, using an isocratic preparation with 85% recovered methanol-water system, a flow rate of 330 ml / min, a wavelength of 362 nm, and continuous injection to collect the target components.

[0037] TH_7 was used to prepare Neogambogic acid with an HPLC purity of 99.71% and a mass of 148.70 g; TH_8 was prepared to obtain R-isogarcinia acid with an HPLC purity of 98.77% and a mass of 49.38 g; Epigambogic acid with a HPLC purity of 99.31% and a mass of 162 g was obtained from TH_11; The TH_10 clarified liquid was prepared using a C18STE preparative column, using an 83% recovered methanol-water system for isocratic preparation, a flow rate of 330 ml / min, a wavelength of 362 nm, continuous injection, and collection of the target components to obtain gambogic acid with an HPLC purity of 98.51% and a mass of 164 g.

[0038] Comparative Example 1: Comparison of the separation effects of C18 and C18STE on garcinia acid In the process of segmentation of gambogic acid, C18 and C18STE were used for liquid phase analysis (e.g. Figure 3), C18 has no separation effect on R-isogarcinia acid and S-isogarcinia acid, and poor separation effect on Epigambogic acid and Gambogi acid; while C18STE has higher selectivity for R-isogarcinia acid, S-isogarcinia acid, Epigambogic acid and Gambogi acid, good separation effect and high repeatability.

[0039] C18 detection conditions: Liquid phase conditions: Chromatographic column Tnature C18, 4.6×250mm; Column temperature 25°C; Flow rate: 1.0mL / min; Mobile phase: acetonitrile (A), 0.1% acetic acid water (B); Elution conditions: Isocratic elution; 0~60min, 75% isocratic phase A. Detector: UV detector C18STE detection conditions: Liquid phase conditions: Unitary C18STE, 4.6×250mm; column temperature 25°C; flow rate: 1.0mL / min; mobile phase: acetonitrile (A), 0.1% acetic acid water (B); elution conditions: gradient elution; 0~20min, 70~70% A phase; 20~50min, 70~80% A phase; 50-60min, 80-80% A phase. Detector: UV detector Method development for the preparation of C18 and C18STE simultaneously (e.g. Figure 4 ), under the same column specifications and preparation methods, it was found that Garcinia acid was strongly retained on C18 and could not be completely eluted in a short time, and had a relatively short retention time on C18STE.

[0040] During the segmentation process of Garcinia acid, the sample has strong retention. Compared with C18 filler, C18STE filler has higher selectivity and less reagent usage. At the same time, the preparation conditions are simple, the stability is high, and the repeatability is good, which can achieve large-scale production of Garcinia acid series reference substances.

[0041] Comparative Example 2: Effect of Temperature on the Purity of Garcinia Acid Components During the preparation of the Garcinia acid reference substance, the series of Garcinia acid reference substances were unstable to heat. For example, after the preparation components of Epigambogic acid with an HPLC purity of 99.81% were stored in the dark at room temperature (18°C) for 10 hours, the purity of the components decreased to 97.82%. However, after being stored in a -20°C refrigerator in the dark for 10 hours, the purity of the components was 99.68%, which was relatively stable.

[0042] In the preparation process of the garcinia acid reference substance, in order to avoid degradation of sample components, the prepared components were stored in a -20℃ refrigerator away from light. The components were post-processed by low-temperature concentration, concentrated to a low organic phase, and crystals were precipitated, filtered, rinsed with 40%~50% methanol water, and freeze-dried to obtain a freeze-dried powder with a purity of >98%, and the garcinia acid reference substance solid was vacuum-packed and stored in a -40℃ refrigerator.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

Claims

1. A method for preparing a series of gambogic acid reference substances, It is characterized in that The following steps are included: (1) Pretreatment process of garcinia: crushing the garcinia raw material, extracting with ethanol or methanol, filtering, and concentrating the filtrate until there is no alcohol taste to obtain garcinia concentrate; (2) Column chromatography enrichment process of garcinia: the garcinia concentrate was mixed with silica gel, eluted with 200-300 mesh silica gel as stationary phase, petroleum ether and ethyl acetate as eluent, and the garcinia eluate was collected and concentrated into a solid, dissolved in methanol, and filtered to obtain a garcinia clarified liquid; (3) Garcinia segmentation process: The Garcinia clarified liquid was cut into segments on a medium-pressure preparative column with a sample loading of 1% to 4%. The elution solvents were acetonitrile and acid water. A high organic phase isocratic elution method was used with an organic phase ratio of 80% to 90%. HPLC detection was performed to obtain a total of 11 Garcinia segments. (4) Preparation process of garcinia acid reference standard: 11 sections of garcinia components were concentrated and impurities were removed by medium pressure preparative column, and then reverse phase preparation was performed. The elution solvents were methanol and water. HPLC detection was performed, vacuum concentration was performed, filtration was performed, 40%~50% methanol was used for elution, and lyophilization was performed to obtain 11 garcinia acid series reference standards.

2. A method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that The pretreatment process of garcinia adopts 70%~100% ethanol or methanol extraction, the solid-liquid ratio is 1:1~4:1, the extraction temperature is 45~50℃, the extraction time is 2~3h, and the extraction times are 2~3 times.

3. A method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that In the column chromatography enrichment process of Garcinia, seven mixing ratios of petroleum ether (5BV), petroleum ether: ethyl acetate = 50:1 (5BV), petroleum ether: ethyl acetate = 25:1 (8BV), petroleum ether: ethyl acetate = 10:1 (3BV), petroleum ether: ethyl acetate = 5:1 (3BV), petroleum ether: ethyl acetate = 1:1 (3BV), and ethyl acetate (3BV) are used for elution.

4. A method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that The filler in the medium-pressure preparation column of the Garcinia segmentation process is a reverse phase strong acid resistant C18STE filler; C18STE filler is a type of octadecylsilane bonded silica filler, has the characteristics of low pH resistance and high temperature resistance, and has a particle size of 5-10 μm.

5. A method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that The acid in the acetonitrile and acid water in the garcinia segmentation process is 0.1% to 5.0% acetic acid.

6. A method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that The reverse phase preparation of 11 sections of garcinia components in the preparation process of garcinia acid reference substance adopts C18 and C18STE alternating separation, the elution solvents are methanol and water, a high organic phase isocratic elution method is adopted, and the organic phase ratio is selected to be 80%~90%.

7. The method for preparing a series of gambogic acid reference substances according to claim 1, It is characterized in that The concentration described in the preparation process of the garcinia reference substance is carried out at 35° C. and a vacuum degree of -0.03 to 0.08 MPa, and the concentration is performed until the organic phase ratio is 30% to 50%.

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