CN101456803A - Method for purifying hypericin - Google Patents
Method for purifying hypericin Download PDFInfo
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- CN101456803A CN101456803A CNA2007101790549A CN200710179054A CN101456803A CN 101456803 A CN101456803 A CN 101456803A CN A2007101790549 A CNA2007101790549 A CN A2007101790549A CN 200710179054 A CN200710179054 A CN 200710179054A CN 101456803 A CN101456803 A CN 101456803A
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Abstract
The invention discloses technology for purifying hypericin. The technology is to take hypericum perforatum, overground parts provided with flowers of other plants containing the hypericin, and a crude extract or flower of the plants as raw materials, and to prepare hypericin purified products of which the content is higher than 98 percent after solvent extraction, separation and purification. The purified products can be used as chemical standard substances, reference substances or raw materials of preparation.
Description
Technical field
The present invention relates to a kind of purifying process of hypericin, particularly a kind of from the hypericum Herba Hyperici perforati and contain other plant of hypericin or the purifying process of the hypericin lyophilized powder that obtains by purifying the crude extract of above-mentioned plant.
Background technology
Hypericin (Hypericin) is one of antidepressant effective constituent in hypericum Herba Hyperici perforati (Hypericum PerforatumL.) and other plant that contains hypericin.
Hypericin is a benzo dianthrone constituents, promptly 4,4 ', 5,5 ', 7, and 7 '-hexahydroxy--2,2 '-dimethyl-meta benzo dianthrone.Molecular formula is C
30H
16O
8, molecular weight is 504.43.Black-and-blue needle crystal is soluble in pyridine and other organic amine and is orange red and fluorescence redly, is dissolved in alkali aqueous solution, is dissolved in other organic solvent hardly.The solution that takes on a red color when being lower than pH11.5 is green solution and fluorescence redly when being higher than pH11.5.Hypericin content in plant is very low by 0.01～0.05%, and poor stability, and heating is easily decomposed, and does not have the fixed fusing point.60 ℃ surpassed for six weeks, and hypericin all significantly descends in powder extract, tablet, the aqueous extract.Hypericin is unstable under illumination, acid, alkali, hot conditions, extraction separation and when preserving need lucifuge, minimizing contact, avoid heat with soda acid.
The derivative of hypericin comprises pseudohypericin (pseudohypericin) and some precursor substances, as former hypericin (protohypericin), former pseudohypericin (protopseudohypericin).
1) R=CH
3Hypericin hypericin 3) R=CH
3Former hypericin protohypericin
2) R=CH
2OH pseudohypericin pseudohypericin 4) R=CH
2The former pseudohypericin protopseudohypericin of OH
The purification process of bibliographical information has four classes at present, we find during by the above method of lot of experiment validation: what the method for pyridine-20% hydrochloric acid/recrystallizing methanol obtained is the mixture of hypericin and pseudohypericin, rather than hypericin monomer, if use this method recrystallization repeatedly, can cause sample seriously to decompose, yield is more and more lower; The product that silica gel column chromatography method obtains contains aliphatic chain compositions such as big weight polyalcohol, and a large amount of assorted peaks are arranged on the NMR spectrogram, and hypericin and silica filler generation absorption more by force, causes the sample elution peak seriously to take off tail, has reduced content and yield; Gel method Sephadex LH-20 can not separate hypericin and pseudohypericin well, always has pseudohypericin to exist in the effluent liquid that is received; The macroporous resin partition method
[8,9]Can enrichment benzo dianthrone constituents, but can't separate hypericin and pseudohypericin, can not get monomer equally.Hypericin and pseudohypericin are closely similar in structure, and only the group that connects on a carbon has difference, hypericin connection-CH3, pseudohypericin connection-CH2OH.Existing method fails effectively to separate this two kinds of compositions.
Summary of the invention
The object of the present invention is to provide a kind of purifying process of hypericin.
The present invention seeks to be achieved through the following technical solutions:
The extraction of hypericin of the present invention, separation, purifying process are as follows:
A, extraction and enrichment: get the Herba Hyperici perforati medicinal material of grinding, extract by backflow, diacolation, dipping, supersound extraction or with continuous extraction with solvent; United extraction liquid at 20～60 ℃ of following decompression and solvent recoveries, adds 3～10 times to medicinal material weight of water and alcohol dilution, and transfers to that to contain alcohol amount be 10%～55%, and mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid; Distilled water is washed till does not have the alcohol flavor; Supernatant liquor is crossed above macroporous adsorbent resin, and amount of resin is 1/8～1/3 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1～2 times of resin volume discards water lotion; Be lower than 55% ethanol elution with concentration, elutriant discards; Use 60%～90% ethanol elution again, collecting the part by weight that elutriant is concentrated into raw medicinal material is 1: 0.5～3;
B, crystallization: concentrated hydrochloric acid added to make content of hydrochloric acid reach 1%～5% in the concentrated solution, in 4～5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out; Dry;
C, gel filtration chromatography: the rough hypericin precipitation of above exsiccant removes by filter insolubles with the solution dissolving of methyl alcohol or pyridine: methyl alcohol=4-6:94-96, crosses gel column and separates; Solution or ethyl acetate with methyl alcohol or methyl alcohol: water=5-10:1: the solution of methyl alcohol=1: 3-7 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=50～75:10～15:5～10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography: C
18The used filler of column chromatography is unsetting or spheric octadecyl bonding phase silica gel, and particle diameter is 50 μ m or 100 μ m; With ultrasonic method, decompress filter method or feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method; The isolating component of above-mentioned gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; Solution with methyl alcohol: water=1-19:1 is eluting solvent, feeds N in the solvent elution process
2, collect wherein hypericin flow point; Flow point is known with the TLC inspection: thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=50～75:10～15:5～10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical flow point;
E, lyophilize: in 20 ℃～60 ℃ following decompression and solvent recoveries, surplus solution is put in the Ultralow Temperature Freezer more than the freezing 10h with the effluent liquid of above collection, lyophilize, the hypericin lyophilized powder.
The extraction of hypericin of the present invention, separation, purifying process are preferably:
A, extraction and enrichment: get the Herba Hyperici perforati medicinal material of grinding, extract by backflow, diacolation, dipping, supersound extraction or with continuous extraction with solvent; United extraction liquid, 40 ℃ of decompression and solvent recoveries add 4～5 times to medicinal material weight of water and alcohol dilution, and transfer to that to contain alcohol amount be 30%, mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid; Distilled water is washed till does not have the alcohol flavor; The macroporous adsorbent resin that supernatant liquor is handled well more than excessively, amount of resin is 1/6～1/4 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1～2 times of resin volume discards water lotion; Be lower than 55% ethanol elution with concentration, elutriant discards; Use 80% ethanol elution hypericin again, collecting the part by weight that elutriant is concentrated into raw medicinal material is 1:1;
B, crystallization: concentrated hydrochloric acid added to make content of hydrochloric acid reach 3% in the concentrated solution, in 4～5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out;
C, gel filtration chromatography: the rough hypericin of above exsiccant precipitation is with methyl alcohol or pyridine: the solution dissolving of methyl alcohol=5: 95, remove by filter insolubles, and the row gel column separates; Solution or ethyl acetate with methyl alcohol or methyl alcohol: water=95:5: the solution of methyl alcohol=1: 5 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography: C
18The used filler of column chromatography is a spheric octadecyl bonding phase silica gel, and particle diameter is 50 μ m; With ultrasonic method, decompress filter method, feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method, and the isolating component of above-mentioned gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; With methyl alcohol: water=70: 30-80: 20 mixing solutionss are eluting solvent, feed N in the solvent elution process
2, collect wherein hypericin flow point; Used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
E, lyophilize: with 40 ℃ of decompression and solvent recoveries of preparation effluent liquid of above collection, surplus solution is put in the Ultralow Temperature Freezer more than the freezing 10h, with the vacuum freeze drier lyophilize, the hypericin lyophilized powder.
Herba Hyperici perforati medicinal material in the wherein above-mentioned purifying process replaces with the flower of natural hypericum Herba Hyperici perforati, the crude extract that contains the wounded in the battle over-ground part of other plant of hypericin, above-mentioned plant or above-mentioned plant.Wherein extract used solvent in the steps A and be the mixture of arbitrary proportion of alcohols, ketone, alcohol-water mixture, ketone water mixture or the alcohol ketone water of 1～4 carbon atom, wherein alcohols is methyl alcohol, ethanol, Virahol, n-propyl alcohol or butanols., be preferably 75% ethanol, methyl alcohol or acetone.Wherein the macroporous resin in the steps A is the non-polar resin of styrene type, ethyl styrene type, vinyl toluene type.When wherein extracting the Herba Hyperici perforati medicinal material and replacing, collect elutriant in the steps A and be concentrated into 3～7 times of crude extract weight, preferred 5 times with the crude extract of above-mentioned plant.Wherein the used gel model of gel column is SephadexLH-20 gel or HW-40C gel among the step C.
Description of drawings:
Fig. 1: the HPLC figure of silica gel column chromatography sample separation;
Fig. 2: the HPLC figure of gel filtration chromatography sample separation;
Fig. 3: C
18The HPLC figure of column chromatography for separation sample;
Fig. 4: C
18The hydrogen spectrum of column chromatography for separation sample;
Fig. 5: C
18The carbon spectrum of column chromatography for separation sample.
The present invention is by solvent extraction, macroreticular resin separation, crystallization, gel filtration chromatography, C18The column chromatography branch Obtain sterling from purifying. The content of hypericin in plant only is ten thousand/several, by solvent extraction, The intermediate Determination of Hypericin from Extraction that macroreticular resin separates and crystallization obtains can reach 14%, the gel column branch From content being increased to more than 50% C18Column chromatography is further purified content 〉=98%. C18The post layer Step before analysing is enrichment, deimpurity process; C18Column chromatography is the method for effectively separating hypericin and pseudohypericin than existing method for separating the committed step of hypericin and pseudohypericin, a step C18Column chromatography can separate hypericin with pseudohypericin fully. Prepare by method of the present invention Hypericin is through a series of Structural Identification and assay, and content 〉=98% can be used as the chemistry mark Accurate product, reference substance or use as preparation raw material.
Following experimental example is used for further specifying but is not limited to the present invention.
Experimental example 1 is investigated different solvents to the impact of hypericin recovery rate
The hypericum perforatum pulverizing medicinal materials is crossed 20 mesh sieves, mixing. Get respectively powder 10g, each adds 75% Ethanol, 95% ethanol, methyl alcohol, acetone 50mL weigh, and heating and refluxing extraction 1h adds respectively phase The solvent of answering is supplied weight; Filter, get filtrate, measure the content of hypericin in the filtrate with the HPLC method. The results are shown in Table 1:
Table 1: investigate the different solvents extraction to the impact (n=3) of Determination of Hypericin from Extraction
Learn from upper table: P＜0.05, Determination of Hypericin from Extraction difference in each extract is in the acetone extract Determination of Hypericin from Extraction is the highest, secondly is 75% ethanol.
The addition of hydrochloric acid when experimental example 2 investigation salt adding acid outs go out the hypericin precipitation.
Get respectively the concentrate 1000ml of hypericum perforatum after with macroporous resin enrichment, each adds concentrated hydrochloric acid to it Middle content of hydrochloric acid is respectively 1%, 2%, 3%, 4%, 5%. Mixing, refrigerator and cooled are hidden and are placed; Wait to sink Precipitation goes out rear filtration, weighs, and drying, the HPLC method is measured wherein Determination of Hypericin from Extraction; The results are shown in Table 2
Table 2: investigate the hydrochloric acid addition to the impact of precipitation hypericin
The result: along with the increase of content of hydrochloric acid, the content of hypericin all increases in precipitation capacity and the precipitation, But content of hydrochloric acid is increased to 5% from 3%, and the content of hypericin in precipitation capacity and the precipitation is improved impact not Greatly; Because therefore the too high stability that may destroy hypericin of acidity, get content of hydrochloric acid 3% for suitable Suitable condition.
Experimental example 3 is investigated impact (contrast silica gel, polyamide, gel, the C that different fillers separate hypericin18The result who separates)
Get the wounded in the battle position, ground of 2kg hypericum perforatum, grind 80 ℃ of heating and refluxing extraction 3 of 75% ethanol Time, quantity of solvent is respectively 5000,4000,3000ml, and the time is respectively 2h, 1h, 1h, and merging is carried Get liquid, 40 ℃ of decompression and solvent recoveries add water and ethanol and are diluted to 5 times of medicinal material weight, and transfer to and contain alcohol Amount is 30%, and mixing is centrifugal. Supernatant is crossed through the good D-101 type macroporous absorbent resin of preliminary treatment, Amount of resin is 1/6 of supernatant volume, stops upper sample after resin adsorption is saturated; With 1 times of resin volume Water elution discards water lotion; Continue with 40% ethanol elution, eluent discards; Wash with 80% ethanol again Take off, the collection eluent is concentrated into extract and the medicinal material ratio is 1: 1. Concentrated hydrochloric acid added in the concentrate make Content of hydrochloric acid reaches 3%, and refrigerator and cooled is hidden after 4～5 ℃ of placements the i.e. toner powder darkly of rough hypericin Precipitation. Above precipitation is divided into 4 parts.
(1), silica gel column chromatography
Get 100～200 order silica gel for chromatography or aluminium oxide, with ethyl acetate wet method dress post. Sample is with few The amount acetone solution, the silica gel that adds 2 times is mixed thoroughly, reclaims solvent to doing. Sample on the silica gel mixed sample dry method, earlier Go out yellow and green impurity with ethyl acetate, change again ethyl acetate: the acetone gradient elution, use thin layer Analyse the TLC inspection and know, used lamellae is the silica G plate, and solvent is carrene: acetone: the methyl alcohol volume Ratio is 75: 15: 10, takes advantage of wet the observation under the 365nm uviol lamp, merges identical component. Decompression is steamed Dried solvent.
Result: 1. check, contain hypericin and pseudohypericin in the sample, illustrate that silica filler is bad to the hypericin selectivity through HPLC; 2. strong adsorption is arranged between sample and the silica filler during silica gel column chromatography, cause the sample elution peak seriously to take off tail, reduced content and yield; 3. sample need just can obtain the higher hypericin of content through column chromatography repeatedly, but because post absorption causes productive rate to reduce greatly; 4. product HPLC figure has only hypericin and pseudohypericin absorption peak, separate through preparation HPLC again, remove pseudohypericin, on HPLC figure, have only single hypericin peak, but a large amount of assorted peaks are arranged on the NMR spectrogram, illustrate that containing the impurity that does not have absorption under a large amount of 200～800nm exists.See accompanying drawing 1.The method of proof silica gel column chromatography is undesirable to separating hypericin.
(2), polyamide column chromatography
Sample dissolves with small amount of methanol, admixes polymeric amide dry powder, with the solvent evaporated under reduced pressure, and the dress post.Use by rare to dense ethanolic soln 10%～95%, methyl alcohol, 3.5% ammoniacal liquor wash-out successively.
The result: hypericin and polymeric amide filling adsorption are too strong, can't elute.As seen polyamide column chromatography and be not suitable for SEPARATION OF GOLD silk peach element.
(3), gel filtration chromatography
The dissolve with methanol sample filters, the row gel filtration chromatography.With methanol-eluted fractions, collect hypericin effluent liquid wherein.Effluent liquid is checked with HPLC.The evaporated under reduced pressure solvent is observed the NMR collection of illustrative plates.See accompanying drawing 2
The result: separate through gel column, can't separate hypericin and pseudohypericin, HPLC figure shows pseudohypericin and exists.Also need separate through preparation HPLC.But gel column can be removed aliphatic chain compositions such as big weight polyalcohol.
(4), C
18Column chromatography
The dissolve with methanol sample filters, row C
18Column chromatography.With methyl alcohol-water ratio is 80: 20 wash-outs.Collect hypericin effluent liquid wherein.Effluent liquid is checked with HPLC.The evaporated under reduced pressure solvent is observed the NMR collection of illustrative plates.See accompanying drawing 3, accompanying drawing 4, accompanying drawing 5.
Result: through a C
18Column chromatography can separate hypericin with pseudohypericin fully, and HPLC checks that the peak area per-cent of hypericin is about 100%.The NMR collection of illustrative plates does not have impurity peaks.
Following embodiment all can realize the described effect of above-mentioned experimental example.
Embodiment
Embodiment 1:
A, extraction and enrichment:
Get the wounded in the battle position, ground of 500g Herba Hyperici perforati, grind, 80 ℃ of heating and refluxing extraction of 75% ethanol 3 times, quantity of solvent is respectively 5000ml, 4000ml, 3000ml, time is respectively 2h, 1h, 1h, united extraction liquid, 50 ℃ of decompression and solvent recoveries add 4 times to medicinal material weight of water and alcohol dilution, and transfer to that to contain alcohol amount be 50%, mixing, centrifugal, get supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid; Distilled water is washed till does not have the alcohol flavor; Supernatant liquor is crossed through the good D-101 type macroporous adsorbent resin of above pre-treatment, and amount of resin is 1/5 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 2 times of resin volumes discards water lotion; Continue with 40% ethanol elution, elutriant discards; Use 85% ethanol elution again, the collection elutriant is concentrated into the volume of extracting solution and the part by weight of raw medicinal material is 1: 1;
B, crystallization:
Concentrated hydrochloric acid added to make content of hydrochloric acid reach 4% in the concentrated solution, refrigerator and cooled hide after 5 ℃ of placements rough hypericin promptly darkly toner powder precipitation separate out;
C, gel filtration chromatography:
More than precipitation is with pyridine: the solution dissolving of methyl alcohol=5: 95, remove by filter insolubles, and the row gel column separates; With ethyl acetate: the solution of methyl alcohol=1: 5 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography:
The used filler of C18 column chromatography is unsetting octadecyl bonding phase silica gel, and particle diameter is 50 μ m; With ultrasonic method, decompress filter method, feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method, and the isolating component of above gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; With methanol-water=3: 1 solution is eluting solvent, feeds N in the solvent elution process
2, collect wherein hypericin flow point; Used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
E, lyophilize:
40 ℃ of decompression and solvent recoveries of preparation effluent liquid with above collection, the surplus solution branch is filled in the brown cillin bottle, cillin bottle froth butyl rubber plug suitable for reading, put in the Ultralow Temperature Freezer more than the freezing 10h, with the vacuum freeze drier lyophilize, gland seal is taken out, at plug external pressure aluminium lid, the hypericin lyophilized powder that score installs.
Embodiment 2:
A, extraction and enrichment:
Get 1000g Herba Hyperici perforati crude extract, add the acetone diacolation and be extracted into colourlessly, 55 ℃ of decompression and solvent recoveries add dissolve with ethanol, the elimination precipitation, supernatant liquor is with to medicinal material weight 8 times of water and alcohol dilution, and transfers to that to contain the alcohol amount be 15%, mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid.Distilled water is washed till does not have the alcohol flavor; Supernatant liquor is crossed through the good HZ-816 type macroporous adsorbent resin of above pre-treatment, and amount of resin is 1/4 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1 times of resin volume discards water lotion; Continue with 50% ethanol elution, elutriant discards; Use 80% ethanol elution again, the collection elutriant is concentrated into 5 times of crude extract weight;
B, crystallization:
Concentrated hydrochloric acid added to make content of hydrochloric acid reach 2% in the concentrated solution, in 5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out;
C, gel filtration chromatography:
The rough hypericin of above exsiccant precipitates with pyridine: methyl alcohol=solution dissolving in 5: 95, remove by filter insolubles, and the row gel column separates; With methyl alcohol is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=(75: 15: 10), under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography:
The used filler of C18 column chromatography is a spheric octadecyl bonding phase silica gel, and particle diameter is 100 μ m; With ultrasonic method, decompress filter method, feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method, and the isolating component of above gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; With methanol-water=2: 1 solution is eluting solvent, feeds N in the solvent elution process
2, collect wherein hypericin flow point; Used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
E, lyophilize:
35 ℃ of decompression and solvent recoveries of preparation effluent liquid with above collection, the surplus solution branch is filled in the brown cillin bottle, cillin bottle froth butyl rubber plug suitable for reading, put in the Ultralow Temperature Freezer more than the freezing 10h, with the vacuum freeze drier lyophilize, gland seal is taken out, at plug external pressure aluminium lid, the hypericin lyophilized powder that score installs.
Embodiment 3:
A, extraction and enrichment:
Get 200g Herba Hyperici perforati medicinal material, add the methyl alcohol supersound extraction to colourless, 40 ℃ of decompression and solvent recoveries add dissolve with ethanol, the elimination precipitation, supernatant liquor is with to medicinal material weight 5 times of water and alcohol dilution, and transfers to that to contain the alcohol amount be 30%, mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid.Distilled water is washed till does not have the alcohol flavor; Supernatant liquor is crossed through the good LSI-106 type macroporous adsorbent resin of above pre-treatment, and amount of resin is 1/5 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1.5 times of resin volumes discards water lotion; Continue with 30% ethanol elution, elutriant discards; Use 80% ethanol elution again, the collection elutriant is concentrated into the volume of extracting solution and the part by weight of raw medicinal material is 1: 1;
B, crystallization:
Concentrated hydrochloric acid added to make content of hydrochloric acid reach 3% in the concentrated solution, in 5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out;
C, gel filtration chromatography:
The rough hypericin precipitation of above exsiccant removes by filter insolubles with dissolve with methanol, and the row gel column separates; With methyl alcohol: the solution of water=95: 5 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography:
The used filler of C18 column chromatography is a spheric octadecyl bonding phase silica gel, and particle diameter is 50 μ m, with ultrasonic method, decompress filter method, feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method, and the isolating component of above gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; Solution with methanol-water=6: 2 is eluting solvent, feeds N in the solvent elution process
2, collect wherein hypericin flow point; Used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
E, lyophilize:
40 ℃ of decompression and solvent recoveries of preparation effluent liquid with above collection, the surplus solution branch is filled in the brown cillin bottle, cillin bottle froth butyl rubber plug suitable for reading, put in the Ultralow Temperature Freezer more than the freezing 10h, with the vacuum freeze drier lyophilize, gland seal is taken out, at plug external pressure aluminium lid, the hypericin lyophilized powder that score installs.
Claims (9)
1, a kind of purifying process of hypericin is characterized in that this technology comprises the steps:
A, extraction and enrichment: get the Herba Hyperici perforati medicinal material of grinding, extract by backflow, diacolation, dipping, supersound extraction or with continuous extraction with solvent; United extraction liquid at 20～60 ℃ of following decompression and solvent recoveries, adds 3～10 times to medicinal material weight of water and alcohol dilution, and transfers to that to contain alcohol amount be 10%～55%, and mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid; Distilled water is washed till does not have the alcohol flavor; Supernatant liquor is crossed above macroporous adsorbent resin, and amount of resin is 1/8～1/3 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1～2 times of resin volume discards water lotion; Be lower than 55% ethanol elution with concentration, elutriant discards; Use 60%～90% ethanol elution again, collecting the part by weight that elutriant is concentrated into raw medicinal material is 1: 0.5～3;
B, crystallization: concentrated hydrochloric acid added to make content of hydrochloric acid reach 1%～5% in the concentrated solution, in 4～5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out; Dry;
C, gel filtration chromatography: the rough hypericin precipitation of above exsiccant removes by filter insolubles with the solution dissolving of methyl alcohol or pyridine: methyl alcohol=4-6:94-96, crosses gel column and separates; Solution or ethyl acetate with methyl alcohol or methyl alcohol: water=5-10:1: the solution of methyl alcohol=1: 3-7 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=50～75:10～15:5～10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography: C
18The used filler of column chromatography is unsetting or spheric octadecyl bonding phase silica gel, and particle diameter is 50 μ m or 100 μ m; With ultrasonic method, decompress filter method or feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method; The isolating component of above-mentioned gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; Solution with methyl alcohol: water=1-19:1 is eluting solvent, feeds N in the solvent elution process
2, collect wherein hypericin flow point; Flow point is known with the TLC inspection: thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=50～75:10～15:5～10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical flow point;
E, lyophilize: in 20 ℃～60 ℃ following decompression and solvent recoveries, surplus solution is put in the Ultralow Temperature Freezer more than the freezing 10h with the effluent liquid of above collection, lyophilize, the hypericin lyophilized powder.
2, purifying process as claimed in claim 1 is characterized in that this technology comprises the steps:
A, extraction and enrichment: get the Herba Hyperici perforati medicinal material of grinding, extract by backflow, diacolation, dipping, supersound extraction or with continuous extraction with solvent; United extraction liquid, 40 ℃ of decompression and solvent recoveries add 4～5 times to medicinal material weight of water and alcohol dilution, and transfer to that to contain alcohol amount be 30%, mixing is centrifugal, supernatant liquor; With adorning post behind the 95% alcohol immersion macroporous adsorbent resin 24h, wash post with 95% ethanol, add water no longer till the muddiness to effluent liquid; Distilled water is washed till does not have the alcohol flavor; The macroporous adsorbent resin that supernatant liquor is handled well more than excessively, amount of resin is 1/6～1/4 of a supernatant liquor volume, stops to go up sample after resin absorption is saturated; Water elution with 1～2 times of resin volume discards water lotion; Be lower than 55% ethanol elution with concentration, elutriant discards; Use 80% ethanol elution hypericin again, collecting the part by weight that elutriant is concentrated into raw medicinal material is 1: 1;
B, crystallization: concentrated hydrochloric acid added to make content of hydrochloric acid reach 3% in the concentrated solution, in 4～5 ℃ of refrigerator and cooled hide place the rough hypericins in back promptly darkly toner powder precipitation separate out;
C, gel filtration chromatography: the rough hypericin of above exsiccant precipitation is with methyl alcohol or pyridine: the solution dissolving of methyl alcohol=5: 95, remove by filter insolubles, and the row gel column separates; Solution or ethyl acetate with methyl alcohol or methyl alcohol: water=95:5: the solution of methyl alcohol=1: 5 is eluting solvent, collects wherein hypericin flow point; Flow point is known with the TLC inspection: used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
D, C
18Column chromatography: C
18The used filler of column chromatography is a spheric octadecyl bonding phase silica gel, and particle diameter is 50 μ m; With ultrasonic method, decompress filter method, feeding N
2The oxygen in the eluting solvent is removed in the combination degassing of method or this several method, and the isolating component of above-mentioned gel column is with the dissolve with methanol of 3～5 times of amount volumes, last C
18Post; With methyl alcohol: water=70: 30-80: 20 mixing solutionss are eluting solvent, feed N in the solvent elution process
2, collect wherein hypericin flow point; Used thin layer plate is the silica gel G plate, and developping agent is a methylene dichloride: acetone: methyl alcohol=75: 15: 10, under the 365nm ultraviolet lamp, take advantage of wet the observation, and merge identical component;
E, lyophilize: with 40 ℃ of decompression and solvent recoveries of preparation effluent liquid of above collection, surplus solution is put in the Ultralow Temperature Freezer more than the freezing 10h, with the vacuum freeze drier lyophilize, the hypericin lyophilized powder.
3, purifying process as claimed in claim 1 or 2 is characterized in that the Herba Hyperici perforati medicinal material replaces with the flower of natural hypericum Herba Hyperici perforati, the crude extract that contains the wounded in the battle over-ground part of other plant of hypericin, above-mentioned plant or above-mentioned plant.
4, purifying process as claimed in claim 1 or 2, it is characterized in that in the steps A extracting used solvent and be the mixture of arbitrary proportion of alcohols, ketone, alcohol-water mixture, ketone water mixture or the alcohol ketone water of 1～4 carbon atom, wherein alcohols is methyl alcohol, ethanol, Virahol, n-propyl alcohol or butanols.
5, purifying process as claimed in claim 4 is characterized in that extracting used solvent in the steps A is 75% ethanol, methyl alcohol or acetone.
6, purifying process as claimed in claim 1 or 2 is characterized in that the macroporous resin in the steps A is styrene type, ethyl styrene type or vinyl toluene type resin.
7, purifying process as claimed in claim 3 when it is characterized in that the Herba Hyperici perforati medicinal material replaces with the crude extract of above-mentioned plant, is collected elutriant and is concentrated into 3～7 times of crude extract weight in the steps A.
8, purifying process as claimed in claim 7 when it is characterized in that the Herba Hyperici perforati medicinal material replaces with the crude extract of above-mentioned plant, is collected elutriant and is concentrated into 5 times of crude extract weight in the steps A.
9, purifying process as claimed in claim 1 or 2 is characterized in that gel column is SephadexLH-20 gel column or HW-40C gel column among the step C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102132920A (en) * | 2011-02-21 | 2011-07-27 | 华南师范大学 | Potassium enriched rhodomyrtus tomentosa hassk beverage and preparation method thereof |
| CN102204941A (en) * | 2011-05-19 | 2011-10-05 | 南京工业大学 | Antidepressant effect and application of effective parts of hypericum wightianum |
| CN103450000A (en) * | 2013-10-08 | 2013-12-18 | 白心亮 | Method for extracting hypericin from hyperforin perforatum |
| CN105777522A (en) * | 2014-12-18 | 2016-07-20 | 六安裕发农业科技有限公司 | Method for extracting hypericin from Hypericum perforatum |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1176893C (en) * | 2002-07-18 | 2004-11-24 | 中山大学 | Extracting and purifying method for hypericum perforatum component in plant |
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2007
- 2007-12-10 CN CN2007101790549A patent/CN101456803B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102132920A (en) * | 2011-02-21 | 2011-07-27 | 华南师范大学 | Potassium enriched rhodomyrtus tomentosa hassk beverage and preparation method thereof |
| CN102204941A (en) * | 2011-05-19 | 2011-10-05 | 南京工业大学 | Antidepressant effect and application of effective parts of hypericum wightianum |
| CN103450000A (en) * | 2013-10-08 | 2013-12-18 | 白心亮 | Method for extracting hypericin from hyperforin perforatum |
| CN103450000B (en) * | 2013-10-08 | 2015-03-11 | 白心亮 | Method for extracting hypericin from hyperforin perforatum |
| CN105777522A (en) * | 2014-12-18 | 2016-07-20 | 六安裕发农业科技有限公司 | Method for extracting hypericin from Hypericum perforatum |
| CN105777522B (en) * | 2014-12-18 | 2017-12-26 | 安徽农达生物技术有限公司 | A kind of method that hypericin is extracted from hypericum perforatum |
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| CN101456803B (en) | 2011-10-26 |
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