CN104817569B - It is a kind of at the same separate gamboge in four kinds of gambogic acid compositions method - Google Patents

Abstract

The invention discloses a method for simultaneously separating four gambogic acid components in garcinia cambogia, which comprises the following steps: the gamboge medicinal material is crushed and then extracted with acetone to obtain an extract, the extract is concentrated under reduced pressure, then added with water, fully stirred and dissolved, Filtrate to obtain the insoluble matter; dissolve the insoluble matter with 40% acetone solution until completely dissolved, centrifuge to obtain the supernatant; inject the supernatant into a treated BS‑16 macroporous resin chromatography column for elution, Obtain the eluent, and after the eluent is concentrated to an extract, dissolve with ethanol; use preparative liquid chromatography to separate four gambogic acid components 2α-hydroxy-3β-acetoxy betulinic acid, neogambogic acid , R‑gambogic acid and S‑gambogic acid. The process of the invention is simple, four kinds of gambogic acid components can be obtained simultaneously, and the utilization value of the gamboge is improved.

Description

A method for simultaneously separating four gambogic acid components in gamboge

technical field

The invention relates to the technical field of biomedicine, in particular to a method for simultaneously separating four gambogic acid components in gamboge.

Background technique

The gelatinous resin secreted by Garcinia hanburyi Hook.f., a plant of Garcinia genus, is in the form of cylindrical or irregular lumps after drying, containing acid components such as gambogic acid, neogambogic acid, and allogambogic acid. In recent years, Chinese scholars have done a lot of research on the active ingredients in the traditional Chinese medicine Garcinia cambogia. It has been found through research that gambogic acid has strong anti-tumor activity against various tumors, and its toxic and side effects are relatively small within the effective dose range. It has a very high selectivity for tumor cell inhibition and can selectively kill cancer cells. , but has no effect on normal animal hematopoietic system and immune function.

Contents of the invention

The purpose of the present invention is to provide a method for simultaneously separating four gambogic acid components in gamboge, which improves the utilization value of gamboge.

In order to achieve the above object, the technical scheme that the present invention takes is:

A method for simultaneously separating four gambogic acid components in gamboge, comprising the steps of:

S1, pulverize the original medicinal material of Garcinia Cambogia, weigh 1Kg quantitatively, place it in a 20L extraction tank, extract twice with 10L acetone, each time for 3 hours, filter, and combine the filtrate twice to obtain the extract;

S2. Concentrate the extract obtained in step S1 to 800mL by rotary evaporation to obtain the garcinia acetone concentrate, add 1600mL of water, fully stir and dissolve, leave at room temperature for 24h, and filter to obtain insoluble matter;

S3. Add 40% acetone solution to the insoluble matter obtained in step S2 until the insoluble matter is completely dissolved, then centrifuge in a high-speed centrifuge to obtain a supernatant;

S4. Inject the supernatant obtained in step S3 into the treated BS-16 macroporous resin chromatography column, and then use 3 times the column volume for distilled water to elute, and 3 times the column volume for elution with 40% acetone , eluted with 100% acetone of 3 times the column volume to obtain the eluate of gambogic acid macroporous resin, which was concentrated to extract, and dissolved in ethanol (20g/100mL);

S5. The solution obtained in step S4 is separated by preparative liquid chromatography. Equilibrate the chromatographic column with the initial mobile phase for 20 minutes, each injection volume is 5mL, and elute according to the set gradient. The elution gradient is shown in Table 1, and the ultraviolet detector is monitored in real time. min and 17.9 min of the mixed sample); after 20 min, equilibrate the chromatographic column according to the initial ratio of the gradient, inject the sample again, and inject the sample 6 times in total, and combine the collected eluted components according to the retention time. Concentrate, recover under reduced pressure, stand at room temperature for 8 hours, precipitate a large amount of powder, filter, and dry at 50°C for 12 hours to obtain the product;

S6, with the sample collected when the retention time obtained in step S5 is 17min, dissolve it into a sample with a concentration of 5mg/mL with ethanol, repeat the sample injection several times under the conditions described in step S5, each injection volume is 1mL, collect and retain The time is 17.5min and 17.9min for the effluent, the effluent is concentrated under reduced pressure, left at room temperature for 8 hours, the powder is precipitated, filtered, and dried at 50°C for 12 hours to obtain R-gambogic acid and S-gambogic acid;

S7, each component product of gained is 98.5% through HPLC purity analysis; Analysis condition is: c18 (5 μ m, 250mm * 4.6mm), mobile phase is acetonitrile: water: phosphoric acid=85: 14.8: 0.2, detection wavelength is 360nm, flow velocity The temperature is 1 mL/min, and the column temperature is 25°C.

Wherein, the frequency of the high-speed centrifuge in the step S3 is 25000r/min.

Wherein, the chromatographic column in the step S5 is c18 (10 μm, 250mm×50mm); the ultraviolet detection wavelength is 360nm; the elution flow rate is 60mL/min; the elution gradient is: the eluent adopts acetonitrile-water-phosphoric acid system, First eluted with 70% acetonitrile by volume and 0.2% phosphoric acid by volume; then eluted with 80% acetonitrile by volume and 0.2% phosphoric acid by volume; finally eluted with 95% acetonitrile by volume The percentage is 0.2% phosphoric acid elution, and a separation cycle is completed in 20 minutes.

Wherein, the product in the step S5 is: retention time 8.0min is 2α-hydroxyl-3β-acetoxy betulinic acid, 10.3min is neogambogic acid, 17min is R-gambogic acid and S-gambogic acid A mixture of isomers.

After testing: the components of the obtained gambogic acids, the retention time of 8.0min is 2α-hydroxy-3β-acetoxy betulinic acid, 10.3min is neogambogic acid, 17.5min and 17.9min are R-gambogic acid and S-gambogic acid.

The present invention has the following beneficial effects:

The invention has simple process, can obtain four kinds of gambogic acid components, and improves the utilization value of gamboge.

Description of drawings

Fig. 1 is the HPLC collection of illustrative plates of garcinia macroporous resin acetone elution part in the embodiment of the present invention.

Fig. 2 is the preparative HPLC spectrum of the separation of various gambogic acid monomer components in gamboge in the embodiment of the present invention.

detailed description

In order to make the objects and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The embodiment of the present invention provides a method for simultaneously separating four gambogic acid components in gamboge, comprising the steps of:

S1, pulverize the original medicinal material of Garcinia Cambogia, weigh 1Kg quantitatively, place it in a 20L extraction tank, extract twice with 10L acetone, each time for 3 hours, filter, and combine the filtrate twice to obtain the extract;

S2. Concentrate the extract obtained in step S1 to 800mL by rotary evaporation to obtain the garcinia acetone concentrate, add 1600mL of water, fully stir and dissolve, leave at room temperature for 24h, and filter to obtain insoluble matter;

S3. Add 40% acetone solution to the insoluble matter obtained in step S2 until the insoluble matter is completely dissolved, then centrifuge in a high-speed centrifuge with a frequency of 25000r/min to obtain a supernatant;

S4. Inject the supernatant obtained in step S3 into the treated BS-16 macroporous resin chromatography column, and then use 3 times the column volume for distilled water to elute, and 3 times the column volume for elution with 40% acetone , 3 times column volume 100% acetone elution, obtain gambogic acid macroporous resin eluate, concentrate to extract, dissolve with ethanol (20g/100mL);

S5. The solution obtained in step S4 is separated by preparative liquid chromatography. Equilibrate the chromatographic column with the initial mobile phase for 20min, and the chromatographic column is c18 (10μm, 250mm×50mm); the ultraviolet detection wavelength is 360nm; the elution flow rate is 60mL/min; For the water-phosphoric acid system, first elute with 70% acetonitrile by volume and 0.2% phosphoric acid by volume; then elute with 80% acetonitrile by volume and 0.2% phosphoric acid by volume; finally use 95% acetonitrile, 0.2% by volume phosphoric acid elution, 20min to complete a separation cycle.

Table 1 The elution gradient of preparative liquid chromatography

time Acetonitrile water phosphoric acid 0 70 29.8 0.2 10 80 19.8 0.2 20 95 4.8 0.2

Each injection volume is 5mL, eluted according to the set gradient, monitored by the ultraviolet detector in real time, and the three components with the retention time of 8.0min, 10.3min and 17min (the mixed sample of 17.5min and 17.9min) are collected; after 20min , equilibrate the chromatographic column according to the initial ratio of the gradient, inject the sample again, and inject the sample 6 times in total, combine the collected eluted components according to the retention time, concentrate, recover under reduced pressure, let stand at room temperature for 8 hours, precipitate a large amount of powder, filter , dried at 50°C for 12 hours to obtain the product (2α-hydroxy-3β-acetoxy betulinic acid with retention time of 8.0min, neogambogic acid at 10.3min, two isotropic acids of R-gambogic acid and S-gambogic acid at 17min) mixture of conformers);

S6, the collected sample (the mixture of two isomers of R-gambogic acid and S-gambogic acid) when the retention time obtained in step S5 is 17min is dissolved into a sample with a concentration of 5mg/mL with ethanol, and in step S5 Under the conditions described above, the injection was repeated several times, each injection volume was 1mL, the effluent with retention time of 17.5min and 17.9min was collected, the effluent was concentrated under reduced pressure, left standing at room temperature for 8h, precipitated powder, filtered, and dried at 50°C for 12 Hours, get R-gambogic acid and S-gambogic acid;

S7, each component product of gained is 98.5% through HPLC purity analysis; Analysis condition is: c18 (5 μ m, 250mm * 4.6mm), mobile phase is acetonitrile: water: phosphoric acid=85: 14.8: 0.2, detection wavelength is 360nm, flow velocity The temperature is 1 mL/min, and the column temperature is 25°C.

After testing, the components of the obtained gambogic acids, the retention time of 8.0min is 2α-hydroxy-3β-acetoxy betulinic acid, 10.3min is neogambogic acid, 17.5min and 17.9min are R-gambogic acid and S-gambogic acid.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be It is regarded as the protection scope of the present invention.

Claims (4)

1. it is a kind of while separating the method for four kinds of gambogic acid compositions in gamboge, it is characterised in that to comprise the following steps：

S1, gamboge crude drug crushed, quantitatively weighs 1Kg, be placed in 20L extractors, 10L acetone extractions twice, each 3h, mistake Filter, merges filtrate twice, obtains extract solution；

S2, the extract solution obtained by step S1 is concentrated under reduced pressure into 800mL, obtains gamboge acetone concentrate, add water 1600mL, fully Stirring and dissolving, is stored at room temperature after 24h, filtering, obtains insoluble matter；

S3, toward the acetone soln that 40% is added in the insoluble matter obtained by step S2, until after insoluble matter is completely dissolved, with high speed from Scheming is centrifuged, and obtains supernatant；

S4, by the supernatant sample introduction obtained by step S3 into processed good BS-16 type macroreticular resin chromatographic columns, successively with 3 times The distillation water elution of column volume, the 3 times of acetone of column volume 40% elutions, the 3 times of acetone of column volume 100% elutions obtain gambogic acid big Hole resin eluent, is concentrated into medicinal extract shape, is dissolved with ethanol；

S5, solution obtained by step S4 is taken to be separated using preparative liquid chromatography, balanced each other chromatographic column with initial flow first 20min, each sampling volume 5mL, by setting gradient elution, UV-detector monitors in real time, collections retention time be 8.0min, 10.3min, 17min 3 components, wherein, the sample that retention time is 17min is that retention time is 17.5min and 17.9min Biased sample；After 20min, chromatographic column is balanced according to gradient initial proportion, again sample introduction, sample introduction 6 times, collection is obtained altogether Elution fraction merge same section by retention time, concentration is recovered under reduced pressure, is stored at room temperature 8h, separates out a large amount of powder, filtering, 50 DEG C of dryings 12 hours, obtain product, wherein, retention time 8.0min is 2-3 β of Alpha-hydroxy-acetoxyl group betulic acid, 10.3min for Neo-garcinolic acid；

S6, by the retention time obtained by step S5 be 17min when collected sample, the sample that concentration is 5mg/mL is dissolved into ethanol Product, under the conditions of described in step S5 repeatedly sample introduction repeatedly, each sampling volume 1mL, collect retention time be 17.5min and 17.9min efflux, efflux is concentrated under reduced pressure, and is stored at room temperature 8h, separates out powder, filtering, 50 DEG C of dryings 12 hours, obtains R- rattans Yellow acid and S- gambogicacids；

S7, gained each component product are 98.5% through HPLC purity analysis；Analysis condition is：C18,5 μm, 250mm × 4.6mm, Mobile phase is acetonitrile: water: phosphoric acid=85: 14.8: 0.2, Detection wavelength is 360nm, and flow velocity is 1mL/min, and column temperature is 25 DEG C.

2. it is according to claim 1 a kind of while separating the method for four kinds of gambogic acid compositions in gamboge, it is characterised in that The frequency of the step S3 high speed centrifuges is 25000r/min.

3. it is according to claim 1 a kind of while separating the method for four kinds of gambogic acid compositions in gamboge, it is characterised in that Chromatographic column in the step S5 is c18,10 μm, 250mm × 50mm；Ultraviolet detection wavelength is 360nm；Elution flow rate is 60mL/min；Gradient is：Eluant, eluent uses acetonitrile-water-Phosphoric Acid, first with the acetonitrile that percent by volume is 70%, Percent by volume elutes for 0.2% phosphoric acid；Again with the acetonitrile that percent by volume is 80%, percent by volume is 0.2% phosphorus Pickling takes off；It is finally 95% acetonitrile with percent by volume, percent by volume elutes for 0.2% phosphoric acid, and 20min completes one point From the cycle.

4. it is according to claim 1 a kind of while separating the method for four kinds of gambogic acid compositions in gamboge, it is characterised in that Product in the step S5 is：Retention time 8.0min is 2-3 β of Alpha-hydroxy-acetoxyl group betulic acid, and 10.3min is new Gambogicacid, 17min is the mixture of two kinds of isomers of R- gambogicacids and S- gambogicacids.