CN104817569B - It is a kind of at the same separate gamboge in four kinds of gambogic acid compositions method - Google Patents
It is a kind of at the same separate gamboge in four kinds of gambogic acid compositions method Download PDFInfo
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- CN104817569B CN104817569B CN201510111176.9A CN201510111176A CN104817569B CN 104817569 B CN104817569 B CN 104817569B CN 201510111176 A CN201510111176 A CN 201510111176A CN 104817569 B CN104817569 B CN 104817569B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
- C07D493/20—Spiro-condensed systems
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- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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Abstract
The invention discloses it is a kind of and meanwhile separate gamboge in four kinds of gambogic acid compositions method, comprise the following steps：Extract solution is obtained after gamboge pulverizing medicinal materials with acetone extraction, extract solution adds water after being concentrated under reduced pressure, be sufficiently stirred for dissolving, filtering obtains insoluble matter；Not tolerant, to being completely dissolved, is centrifuged with 40% acetone soln, obtains supernatant；Eluted in the type macroreticular resin chromatographic columns of BS 16 that supernatant sample introduction is handled well to oneself, obtain eluent, eluent is concentrated into after medicinal extract shape, dissolved with ethanol；Four kinds of β acetoxyl groups betulic acids of 2 α hydroxyls of gambogic acid component 3, Neo-garcinolic acid, R gambogicacids and S gambogicacids are separated to obtain using preparative liquid chromatography.Present invention process is simple, and four kinds of gambogic acid compositions can be obtained simultaneously, the value to gamboge is improved.
Description
Technical field
The present invention relates to biomedicine technical field, and in particular to a kind of to separate four kinds of gambogic acid compositions in gamboge simultaneously
Method.
Background technology
Colloidal resin secreted by Garcinia maingayii gamboge Garcinia hanburyi Hook.f., after drying, in cylinder
Shape or irregular block, containing acrylic components such as gambogicacid, Neo-garcinolic acid, allogambogic acids.In recent years, Chinese scholar is to Chinese medicine rattan
Active component in Huang has done substantial amounts of research.It has been investigated that, gambogicacid shows stronger antitumor activity to kinds of tumors,
And toxic side effect is smaller in effective dosage ranges, there is very high selectivity to the suppression of tumour cell, can be selective
Cancer cell is killed, and intact animal hemopoietic system and immunologic function are not influenceed.
The content of the invention
Object of the present invention is to provide it is a kind of and meanwhile separate gamboge in four kinds of gambogic acid compositions method, improve
To the value of gamboge.
To achieve the above object, the technical scheme taken of the present invention is：
It is a kind of at the same separate gamboge in four kinds of gambogic acid compositions method, comprise the following steps：
S1, gamboge crude drug crushed, quantitatively weighs 1Kg, be placed in 20L extractors, 10L acetone extractions twice, every time
3h, filtering merges filtrate twice, obtains extract solution；
S2, the extract solution rotary evaporation obtained by step S1 is concentrated into 800mL, obtains gamboge acetone concentrate, add
1600mL water, is sufficiently stirred for dissolving, is stored at room temperature after 24h, filters, obtains insoluble matter；
S3, the acetone soln toward addition 40% in the insoluble matter obtained by step S2, until after insoluble matter is completely dissolved, it is too high
Fast centrifuge, obtains supernatant；
S4, by the supernatant sample introduction obtained by step S3 into processed good BS-16 type macroreticular resin chromatographic columns, use successively
The distillation water elution of 3 times of column volumes, the 40% acetone elution of 3 times of column volumes, the 100% acetone elution of 3 times of column volumes obtains gamboge
Acids macroreticular resin eluent, is concentrated into medicinal extract shape, is dissolved (20g/100mL) 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
20min, each sampling volume 5mL, by setting gradient elution, gradient is shown in Table 1, and UV-detector is monitored in real time, collects and protects
Stay 3 components that the time is 8.0min, 10.3min, 17min (being 17.5min and 17.9min biased sample)；20min with
Afterwards, chromatographic column is balanced according to gradient initial proportion, again sample introduction, altogether sample introduction 6 times, during by collecting obtained elution fraction by retaining
Between merge same section, concentrate, be recovered under reduced pressure, be stored at room temperature 8h, separate out a large amount of powder, filtering, 50 DEG C of dryings 12 hours must be produced
Product；
S6, by the retention time obtained by step S5 be 17min when collected sample, it is 5mg/mL that concentration is dissolved into ethanol
Sample, 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℃。
Wherein, the frequency of the step S3 high speed centrifuges is 25000r/min.
Wherein, the 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, is with percent by volume first
70% acetonitrile, percent by volume elutes for 0.2% phosphoric acid；Again with the acetonitrile that percent by volume is 80%, percent by volume
Eluted for 0.2% phosphoric acid；It is finally 95% acetonitrile with percent by volume, percent by volume elutes for 0.2% phosphoric acid,
20min completes a separation cycle.
Wherein, the product in the step S5 is：Retention time 8.0min is 2-3 β of Alpha-hydroxy-acetoxyl group white birch fat
Acid, 10.3min is Neo-garcinolic acid, and 17min is the mixture of two kinds of isomers of R- gambogicacids and S- gambogicacids.
After testing：Gained gambogic acid each component, retention time 8.0min is 2-3 β of Alpha-hydroxy-acetoxyl group white birch fat
Acid, 10.3min is Neo-garcinolic acid, and 17.5min and 17.9min are respectively R- gambogicacids and S- gambogicacids.
The invention has the advantages that：
Present invention process is simple, can obtain four kinds of gambogic acid compositions, improve the value to gamboge.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of gamboge macroreticular resin acetone elution fraction in the embodiment of the present invention.
The preparation HPLC collection of illustrative plates that Fig. 2 separates for various gambogic acid monomer components in gamboge in the embodiment of the present invention.
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
The embodiments of the invention provide a kind of while separate the method for four kinds of gambogic acid compositions in gamboge, including following step
Suddenly：
S1, gamboge crude drug crushed, quantitatively weighs 1Kg, be placed in 20L extractors, 10L acetone extractions twice, every time
3h, filtering merges filtrate twice, obtains extract solution；
S2, the extract solution rotary evaporation obtained by step S1 is concentrated into 800mL, obtains gamboge acetone concentrate, add
1600mL water, is sufficiently stirred for dissolving, is stored at room temperature after 24h, filters, obtains insoluble matter；
S3, the acetone soln toward addition 40% in the insoluble matter obtained by step S2, until after insoluble matter is completely dissolved, it is too high
Fast centrifuge, the frequency of centrifuge is 25000r/min, obtains supernatant；
S4, by the supernatant sample introduction obtained by step S3 into processed good BS-16 type macroreticular resin chromatographic columns, use successively
The distillation water elution of 3 times of column volumes, the 40% acetone elution of 3 times of column volumes, the 3 times of acetone of column volume 100% elutions obtain gambogicacid
Class macroreticular resin eluent, is concentrated into medicinal extract shape, is dissolved (20g/100mL) 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
20min, chromatographic column is c18 (10 μm, 250mm × 50mm)；Ultraviolet detection wavelength is 360nm；Elution flow rate is 60mL/min；Wash
De- gradient is as shown in table 1：Eluant, eluent uses acetonitrile-water-Phosphoric Acid, first with the acetonitrile that percent by volume is 70%, volume
Percentage elutes for 0.2% phosphoric acid；Again with the acetonitrile that percent by volume is 80%, percent by volume is washed for 0.2% phosphoric acid
It is de-；It is finally 95% acetonitrile with percent by volume, percent by volume elutes for 0.2% phosphoric acid, 20min completes a separation week
Phase.
The gradient of the preparative liquid chromatography of table 1
| Time | Acetonitrile | Water | Phosphoric acid |
| 0 | 70 | 29.8 | 0.2 |
| 10 | 80 | 19.8 | 0.2 |
| 20 | 95 | 4.8 | 0.2 |
Each sampling volume 5mL, by setting gradient elution, UV-detector is monitored in real time, is collected retention time and is
8.0min, 10.3min, 17min (being 17.5min and 17.9min biased sample) 3 components；After 20min, according to ladder
Initial proportion balance chromatographic column is spent, again sample introduction, altogether sample introduction 6 times, obtained elution fraction will be collected by retention time and merge phase
Same part, concentration, is recovered under reduced pressure, and is stored at room temperature 8h, separates out a large amount of powder, and filtering, 50 DEG C of dryings obtain product for 12 hours (during reservation
Between 8.0min be 2-3 β of Alpha-hydroxy-acetoxyl group betulic acid, 10.3min is Neo-garcinolic acid, and 17min is R- gambogicacids and S- rattans
The mixture of two kinds of isomers of yellow acid)；
S6, by the retention time obtained by step S5 be 17min when collected sample (two kinds of R- gambogicacids and S- gambogicacids are different
The mixture of structure body) sample that concentration is 5mg/mL is dissolved into ethanol, sample introduction is multiple repeatedly under the conditions of described in step S5, often
Secondary sampling volume 1mL, collects the efflux that retention time is 17.5min and 17.9min, and efflux is concentrated under reduced pressure, is stored at room temperature
8h, separates out powder, and filtering, 50 DEG C of dryings 12 hours obtain R- gambogicacids 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℃。
After testing, gained gambogic acid each component, retention time 8.0min is 2-3 β of Alpha-hydroxy-acetoxyl group white birch fat
Acid, 10.3min is Neo-garcinolic acid, and 17.5min and 17.9min are respectively R- gambogicacids and S- gambogicacids.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as 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.
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| CN109053761A (en) * | 2018-09-16 | 2018-12-21 | 湖州展舒生物科技有限公司 | The preparation method of gambogicacid |
| CN110437246B (en) * | 2019-08-19 | 2021-05-11 | 济宁医学院 | Method for separating neogambogic acid, gambogic acid and N-aryl gambogic amide by medium-low pressure gradient silica gel dry column chromatography |
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| US7592367B2 (en) * | 2005-12-23 | 2009-09-22 | Hong Kong Jockey Club Institute Of Chinese Medicine Ltd. | Compounds from Garcinia hanburyi, their use in treating cancer and method of separating epimers thereof |
| CN101607978A (en) * | 2008-06-20 | 2009-12-23 | 秦继红 | Two new compounds, its preparation method and pharmaceutical uses in the gamboge |
| TWI432191B (en) * | 2010-06-11 | 2014-04-01 | Taiwan Sunpan Biotechnology Dev Co Ltd | Compounds isolated from gamboge resin , and pharmaceutical compositions comprising the same |
| CN102372725A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Purification method for high-content gambogic acid |
| CN102617590A (en) * | 2012-03-07 | 2012-08-01 | 广州牌牌生物科技有限公司 | Preparation process for neogambogic acid |
| CN103664997A (en) * | 2013-11-28 | 2014-03-26 | 江苏康缘药业股份有限公司 | Antitumor compound extracted from gamboge and preparation method and application thereof |
| CN103816147B (en) * | 2014-03-14 | 2016-01-20 | 吉林农业大学 | The medical usage of gamlogic acid, neogambogic acid and compositions thereof |
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Inventor after: Wang Xiaoling Inventor after: Tang Shaoqi Inventor after: Zhang Renrui Inventor after: Yang Desuo Inventor after: Xiao Jian Inventor before: Wang Xiaoling Inventor before: Tang Shaoqi Inventor before: Yang Desuo Inventor before: Xiao Jian |
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Effective date of registration: 20190408 Address after: 721016 No. 1 Gaoxin Avenue, Baoji City, Shaanxi Province Co-patentee after: BAOJI CHENGUANG BIO-TECH Co.,Ltd. Patentee after: BAOJI University OF ARTS AND SCIENCES Address before: 721016 No. 1 Gaoxin Avenue, Baoji City, Shaanxi Province Patentee before: BAOJI UNIVERSITY OF ARTS AND SCIENCES |
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