CN101088519B - Tripterygium glycosides extract and its extraction process - Google Patents
Tripterygium glycosides extract and its extraction process Download PDFInfo
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Abstract
The process of extracting tripterygium glycosides includes the following steps: 1. extracting coarse tripterygium glycosides product through water extracting tripterygium powder, extracting with alcohol or alcohol aqua, merging the extracted solution, concentrating to volume content of alcohol not more than 20 %, organic solvent extracting and concentrating to obtain dry coarse tripterygium glycosides product; and 2. refining through dissolving the coarse product in organic solvent, adsorbing with adsorbent, filtering, eluting the filter cake, concentrating the eluted liquid, column chromatographic separation, eluting with organic solvent, and concentrating the eluted liquid to obtain dry product. The process has simple operation, low cost, short production period, less environmental pollution, and easy application in industrial production, and the product has high effective component content and high pharmaceutical effect.
Description
Technical field
The present invention relates to a kind of plant extract and extracting method thereof, be specifically related to a kind of tripterygium glycosides extract and extracting method thereof.
Background technology
The Thunder God Calamus mainly is distributed in the Yangtze river basin with Nanshan District and northeast Changbaishan area in China, and widespread among the people is used as the medical herbs of treatment of arthritis, traumatic injury, dermatosis etc., also is used for pesticide.The domestic clinically research to Radix Tripterygii Wilfordii has obtained significant effect and breakthrough, multiple commonly encountered diseases and difficult miscellaneous diseases such as treatment lupus erythematosus, psoriasis, nephritis and tumor research have been developed into from treatment rheumatic arthritis, because its wide application, evident in efficacy, and to difficult pertinacious disease have unique therapeutical effect and become gradually both at home and abroad, focus that the traditional Chinese medical science, doctor trained in Western medicine, each expert of Chinese and western medicine are attracted attention and studied.
In the Chinese medicine, the traditional extraction process of Radix Tripterygii Wilfordii adopts water boiling concentration.The method inventory is limited, the temperature requirement height, and operation has higher requirements, and yields poorly, and is not suitable for suitability for industrialized production.Existing application mainly contains following several in the extracting method of the Radix Tripterygii Wilfordii extract of suitability for industrialized production:
1. extraction method: with the tripterygium wilfordii water boiling and extraction.This method is polluted, and water consumption is big, and energy consumption is big.
2. ethanol extraction method: Radix Tripterygii Wilfordii is used ethanol extraction, afterwards with chloroform extraction and be concentrated into dried.Production efficiency is extremely low, ethanol feed intake big and loss big, be not easy to large-scale industrial production.
3. ethyl acetate extraction method: Radix Tripterygii Wilfordii is used ethanol extraction, use ethyl acetate extraction afterwards, the reuse chloroform extraction also is concentrated into dried.
The product quality that above-mentioned 3 kinds of methods are extracted is bad, and wherein the extraction ratio of terpenoid such as active ingredient triptolide is low.
4. column chromatography: this method product purity is higher, but inventory is little, and the large-scale production operation requires high, is not easy to large-scale industrial application.
After the twentieth century initial stage eighties was found the Radix Tripterygii Wilfordii curative effect, many pharmaceutical factories produced the Radix Tripterygii Wilfordii extract product one after another.But, because at present seldom to the pharmacological research of Radix Tripterygii Wilfordii preparation, lack regular pharmacology data, and there is not unified quality control standard for active ingredient, the contained component of Radix Tripterygii Wilfordii preparation and the content of drug effect components of each manufacturer production are not quite similar, therefore, the pharmacological effect of Radix Tripterygii Wilfordii preparation on the market and toxic and side effects have very big difference.Wherein, a lot of product pharmacological effects are bad, and toxic and side effects is big.
Summary of the invention
Technical problem to be solved by this invention is that to overcome in the tripterygium glycosides that the extracting method of prior art extracts content of drug effect components not high, product quality is bad, perhaps technology is not easy to problems such as large-scale industrial production, and a kind of product efficacy component height is provided, and pharmacological effect is good, and it is easy and simple to handle, cost is low, and is with short production cycle, and environmental pollution is few, easily be applied to large-scale industrial tripterygium glycosides extracting method, with and the tripterygium glycosides product.
Method of the present invention adopts water to carry and alcohol extraction, and the purifying technique that absorption is parallel with column chromatography specifically comprises the steps:
(1) extract crude product: Common Threewingnut Root is extracted with decocting in water, and reuse alcohol or pure extraction with aqueous solution merge extracting solution, be concentrated into pure content and be less than or equal to percent by volume 20%, use organic solvent extraction afterwards, make the extract that contains crude product, be concentrated into dried crude product.Method of the present invention is extracted respectively at the aqueous solution of slightly carrying part selection decocting in water and alcohol, effective ingredient is more extracted preserved, and reduced water consumption.
Wherein, Radix Tripterygii Wilfordii can be selected leaf, flower, root, each one of stem for use, and the preferable xylem of selecting for use makes powder after selected grinding.
What wherein, the number of times of described decocting in water extraction was preferable is 1～3 time; The consumption of water is preferable is 3～10 times of Common Threewingnut Root weight; What the temperature of decocting in water was preferable is 50～100 ℃; What the time of decocting in water was preferable is 30～60 minutes.
What wherein, the number of times of the extraction with aqueous solution of described alcohol or alcohol was preferable is 1～3 time; Described alcohol can be selected from lower alcohol, and preferable is methanol, ethanol, isopropyl alcohol or n-butyl alcohol; What the concentration of alcohol was preferable in the aqueous solution of alcohol is that percent by volume is more than 40%; The consumption of pure or pure aqueous solution is preferable is 6～10 times of Common Threewingnut Root weight; What the temperature of the extraction with aqueous solution of alcohol was preferable is 20～50 ℃; What the time of the extraction with aqueous solution of alcohol was preferable is 8～24 hours.
Wherein, the number of times of described extraction be preferable be 1～3 time; The organic solvent that uses is preferable is selected from petroleum ether, lower halogenated alkane, cycloalkane, alkane, lower aliphatic ketone or ester; The consumption of organic solvent is preferable 1～3 times of concentrating the back volume for extracting solution; What the time of each extraction was preferable is 30～60 minutes.
The tripterygium glycosides crude product is loose rufous powder, and yield is about 5～18wt ‰.It is as shown in table 1 to adopt different organic solvents to extract the yield of crude product of gained.
Table 1 adopts different organic solvents to extract the crude product yield of gained
| Organic solvent | Petroleum ether | The lower halogenated alkanes | Ring, alkanes | Lower aliphatic ketone | The lower aliphatic ester |
| Yield | 5～11wt‰ | 7～15wt‰ | 5～10wt‰ | 10～18wt‰ | 9～17wt‰ |
(2) crude product refining: use the organic solvent dissolution crude product, adding adsorbent adsorbs, filter, filter cake is carried out eluting, the eluent of collection live part with the solution of gradient organic solvent, concentrate, carry out column chromatography again, carry out eluting, collect the eluent that contains effective composition with the solution of gradient organic solvent, be concentrated into driedly, promptly get and make with extra care tripterygium glycosides.Adsorption process in the inventive method can effectively improve product purity, and is simple to operate, is convenient to the heavy industrialization operation, can effectively save labor cost, shortens the production cycle, and cost saves time; Gradient elution has improved the separating effect of column chromatography, and end product quality effectively improves.
Wherein, dissolving tripterygium glycosides crude product used with organic solvent preferable be petroleum ether, halogenated alkane, cycloalkane, alkane, lower aliphatic ketone or ester.
What wherein, described adsorbent was preferable is silica gel, neutral alumina, cellulose or kieselguhr; The consumption of adsorbent is preferable is 10～20 times of crude product quality. The time of absorption be preferable be 30～60 minutes.
The absorption after-filtration carries out eluting with filter cake with the solution of gradient organic solvent, the eluent of collection live part.Wherein, described organic solvent is preferable is selected from petroleum ether, halogenated alkane, cycloalkane, alkane, lower aliphatic ketone or ester, what the solution of described gradient organic solvent was preferable is that gradient is at the halogenated alkane of 1～20% of percent by volume or the alcoholic solution of alkane, or gradient maybe can reach other gradient organic solvent of same expansion effect at the acetone soln of the cycloalkane of percent by volume 1～20%; What described halogenated alkane was preferable is dichloromethane, dichloroethanes, chloroform or carbon tetrachloride; What described cycloalkane was preferable is cyclohexane extraction or Pentamethylene.; What described alkane was preferable is hexane or pentane; What described lower aliphatic ketone was preferable is acetone or butanone; What described ester was preferable is ethyl acetate.The solution of the organic solvent of preferable gradient elution and consumption thereof are: with every gram adsorbent is reference, 1% gradient, and consumption is 3～4 milliliters; 3% gradient, consumption are 1.5～2 milliliters; 5% gradient, consumption are 2～3 milliliters; 7%, 10% and 12% gradient, consumption are 3～5 milliliters; 15% gradient, consumption are 4～6 milliliters; Percentage ratio is percent by volume.
Wherein, the eluent of described live part detects down to adopt thin layer chromatography and ultraviolet 364nm, confirms with standard control.After eluent concentrated, make dry extract, can add organic solvent again, carry out the second adsorption eluting as stated above.Concentrate behind the absorb-elute, preferably be concentrated into driedly, add organic solvent dissolution again, carry out follow-up column chromatography.
The method of column chromatography is operated routinely and is carried out, and uses the solution of gradient organic solvent to carry out eluting, collects the eluent that contains effective composition.Wherein, the consumption of the solution of the organic solvent of each gradient and time, the kind of the solution of gradient organic solvent and proportioning, the solution of the organic solvent of preferable gradient elution and consumption thereof are with aforementioned.The eluent of the live part behind the eluting is admitted aforementioned really.
Eluent behind the column chromatography is concentrated into dried, promptly makes refining tripterygium glycosides product.It is as shown in table 2 to adopt different organic solvents to carry out the yield of refining tripterygium glycosides product of gradient elution gained.
Table 2 adopts different organic solvents to carry out the yield of the refining tripterygium glycosides product of gradient elution gained
| Organic solvent | The lower halogenated alkanes | Ring, alkanes | Lower aliphatic ketone | The lower aliphatic ester |
| Yield | 3-8wt‰ | 1-5wt‰ | 4-8wt‰ | 4-9wt‰ |
In the method for the present invention, used all recycling uses of pure and mild other organic solvents, thus reduced cost, environmentally friendly.
The invention still further relates to the tripterygium glycosides extract that makes by said method, and the pharmaceutical composition that contains this tripterygium glycosides extract.This pharmaceutical composition comprises the tripterygium glycosides of the present invention and the pharmaceutically acceptable carrier for the treatment of effective dose.Among the present invention, described pharmaceutically acceptable carrier is meant the pharmaceutical carrier of pharmaceutical field routine, as diluent, excipient (as water etc.), binding agent (as cellulose derivative, gelatin, polyvinylpyrrolidone etc.), filler (as starch etc.), the agent of bursting apart (as calcium carbonate, sodium bicarbonate).In addition, other adjuvant can also be added, as flavouring agent and sweeting agent etc. in compositions.
Pharmaceutical composition of the present invention can put on the patient who needs treatment by intravenous injection, subcutaneous injection or oral form.Be used for when oral, it can be prepared into conventional solid preparation such as tablet, powder or capsule etc.; When being used to inject, it can be prepared into injection.The various dosage forms of pharmaceutical composition of the present invention can adopt the method for medical domain routine to be prepared.Wherein the content of wilforlide A can be and is no less than percentage by weight 0.01%.The general dosage that aforementioned pharmaceutical compositions puts on the patient who needs treatment can be 1～1.5mg tripterygium glycosides composition/kg body weight/sky, specifically can be according to variations such as the patient's age and the state of an illness.
Agents useful for same of the present invention and raw material are all commercially available to be got.
Positive progressive effect of the present invention is: method of the present invention adopts water to carry and alcohol extraction, the purifying technique that absorption is parallel with column chromatography, the tripterygium glycosides content of drug effect components height that makes, and easy and simple to handle, cost is low, and is with short production cycle, the all recycling uses of used organic reagent, environmentally friendly, easily be applied to large-scale commercial production, have higher industrial application value.The tripterygium glycosides content of drug effect components height that makes by method of the present invention, and content ratio appropriateness, pharmacological effect is good, and toxic and side effects is little.
Description of drawings
Fig. 1 is a triptolide standard curve among the effect embodiment.
Fig. 2 is a wilforlide A standard curve among the effect embodiment.
The specific embodiment
Mode below by embodiment further specifies the present invention, but does not therefore limit the present invention among the described scope of embodiments.
The Radix Tripterygii Wilfordii xylem is through choosing, be treated to fritter, and being pulverized by pulverizer is powder.Take by weighing the 150Kg powder, place manyly with extracting pot, stir, directly logically in extracting pot be steam heated to 80 ℃-100 ℃ with the water of 5 times of weight, steaming and decocting 1h, to be cooled to room temperature, collection filtrate.The ethanol that adds 6 times of weight again soaks 8h, and 30 ℃ of temperature are emitted ethanol extract, add ethanol once more with same method, soaks once merge extractive liquid.
Extracting solution is evacuated to boiler, at first open the inlet valve of condenser and cooler, slowly the cooling water in reaction pot passageway is emitted again, controls the flow of steam and about kettle temperature 70-100 ℃ well, in medicinal liquid, contain concentration of alcohol less than percent by volume 20% till.
Add and the isopyknic chloroform reflux, extract, of concentrated solution three times, extract 60min at every turn after, it is two-layer up and down that chloroform extraction liquid and former ethanol extract are told, and emits chloroform extraction liquid, three extracts are merged, dewatering and filtering, chloroform is reclaimed in air-distillation.Control kettle temperature and pressure well, decompression vacuum pumping below vacuum-0.07MPa, concentrates to such an extent that loose rufous powder is tripterygium glycosides crude product extract.
With the tripterygium glycosides coarse powder chloroform in last step, be dissolved into solution earlier, slowly adding is mixed with in the silica gel (100200 order) of chloroform then, and the consumption of silica gel is 15 times of crude product quality, stirs to adsorb 1h, and filtration is drained, and filtrate is invalid element.
Filter cake is with the chloroform alcohol solution gradient eluting of 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume), and solution usage is reference with every gram adsorbent, 1% gradient, and consumption is 3.5 milliliters; 3% gradient, consumption are 1.8 milliliters; 5% gradient, consumption are 2.5 milliliters; 7%, 10% and 12% gradient, consumption are 4 milliliters; 15% gradient, consumption are 5 milliliters.Adopt thin layer chromatography and ultraviolet 364nm to detect down, with standard control efficiency confirmed gradient.Filtration is drained, and effective filtrate is concentrated, and reclaims chloroform, gets dry extract after concentrating.Dry extract is added chloroform again be diluted to solution, repeat the second adsorption eluting, get dry extract after decompression vacuum pumping concentrates, treat that column chromatography is standby.
In the chromatographic column of the diameter 9cm left and right sides, pack into earlier chloroform a little, push absorbent cotton (doing glomeration) then and place the column bottom to pave.Wet method dress silica gel drains bubble in the post, leaves standstill the above compacting of 24h, opens piston and emits chloroform unnecessary above the silica gel agent, and plastochondria begins to shrink, and compresses cylinder naturally.
To add the diluent of dry extract in the post, the chloroform alcohol liquid that adds 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume) respectively carries out gradient elution, and solution usage is reference with every gram adsorbent, 1% gradient, and consumption is 3.5 milliliters; 3% gradient, consumption are 1.8 milliliters; 5% gradient, consumption are 2.5 milliliters; 7%, 10% and 12% gradient, consumption are 4 milliliters; 15% gradient, consumption are 5 milliliters.Collect eluent with minute bottle.With the bottle eluent collected respectively point sample after carrying out thin layer chromatography on the silica gel G plate, with differentiating under the uv analyzer respective wavelength whether eluent contains effective composition, collects effective bottle of sample, with the eluent of effective bottle of sample concentrated drain active ingredient.With active ingredient drying, chemical examination, the proportioning that branch is got, mixed 100 mesh sieves, packing is the former powder of finished product tripterygium glycosides.Wherein, the content of triptolide is 0.15wt%, and the content of wilforlide A is 0.32wt%.With used all organic reagent recycling uses.
The method of inspection of tripterygium glycosides powder adopts thin layer chromatography, and consistent with standard control, composition is identical.Many glycosides powder adopts LD50 acute toxicity test: LD50=155 ± 15mg/Kg, physicochemical property: tripterygium glycosides is yellow or pale brown toner end, bitter in the mouth, 100～120 ℃ of fusing points.Tripterygium glycosides is carried out by No. 76 literary composition of the Su Wei of Jiangsu Province Department of Public Health medicine duckweed word [84].
The Radix Tripterygii Wilfordii xylem is through choosing, be treated to fritter, and being pulverized by pulverizer is powder.Take by weighing the 150Kg powder, place manyly with extracting pot, stir, directly logically in extracting pot be steam heated to 50 ℃-60 ℃ with the water of 3 times of weight, steaming and decocting 40 minutes, to be cooled to room temperature, collection filtrate.Repeat to use water extraction 2 times.Percent by volume 90% methanol aqueous solution (percent by volume) that adds 10 times of weight again soaks 24h, and 20 ℃ of temperature are emitted methanol extract liquid.Merge extractive liquid.
Extracting solution is evacuated to boiler, at first open the inlet valve of condenser and cooler, slowly the cooling water in reaction pot passageway is emitted again, controls the flow of steam and about kettle temperature 70-100 ℃ well, in medicinal liquid, contain methanol concentration less than percent by volume 10% till.
The petroleum ether reflux, extract, that adds 3 times of concentrated solution volumes once, extract 40min after, it is two-layer up and down that petroleum ether extraction liquid and former extracting solution are told, and emits petroleum ether extraction liquid, dewatering and filtering, petroleum ether is reclaimed in air-distillation.Control kettle temperature and pressure well, decompression vacuum pumping below vacuum-0.07MPa, concentrates to such an extent that loose rufous powder is tripterygium glycosides crude product extract.
The tripterygium glycosides coarse powder in last step is dissolved into solution earlier with petroleum ether, slowly adding is mixed with in the neutral alumina (100-200 order) of petroleum ether then, and the consumption of neutral alumina is 10 times of crude product quality, stirs to adsorb 1h, filtration is drained, and filtrate is invalid element.
The filter cake petroleum ether alcoholic solution gradient elution of %, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume), solution usage is: with every gram adsorbent is reference, 1% gradient, consumption is 3 milliliters; 3% gradient, consumption are 1.5 milliliters; 5% gradient, consumption are 2 milliliters; 7%, 10% and 12% gradient, consumption are 3 milliliters; 15% gradient, consumption are 4 milliliters.Adopt thin layer chromatography and ultraviolet 364nm to detect down, with standard control efficiency confirmed gradient.Filtration is drained, and effective filtrate is concentrated, and reclaims petroleum ether, gets dry extract after concentrating, and treats that column chromatography is standby.
In the chromatographic column of the diameter 10cm left and right sides, pack into earlier petroleum ether a little, push absorbent cotton (doing glomeration) then and place the column bottom to pave.Wet method dress silica gel drains bubble in the post, leaves standstill the above compacting of 24h, opens piston and emits petroleum ether unnecessary above the silica gel agent, and plastochondria begins to shrink, and compresses cylinder naturally.
The diluent of dry extract will be added in the post, the petroleum ether ethanol liquid that adds 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume) respectively carries out gradient elution, solution usage is: with every gram adsorbent is reference, 1% gradient, and consumption is 3 milliliters; 3% gradient, consumption are 1.5 milliliters; 5% gradient, consumption are 2 milliliters; 7%, 10% and 12% gradient, consumption are 3 milliliters; 15% gradient, consumption are 4 milliliters.Collect eluent with minute bottle.With the bottle eluent collected respectively point sample after carrying out thin layer chromatography on the silica gel G plate, with differentiating under the uv analyzer respective wavelength whether eluent contains effective composition, collects effective bottle of sample, with the eluent of effective bottle of sample concentrated drain active ingredient.With active ingredient drying, chemical examination, the proportioning that branch is got, mixed 100 mesh sieves, packing is the former powder of finished product tripterygium glycosides.Wherein, the content of triptolide is 0.12wt%, and the content of wilforlide A is 0.30wt%.With used all organic reagent recycling uses.
Embodiment 3
The Radix Tripterygii Wilfordii xylem is through choosing, be treated to fritter, and being pulverized by pulverizer is powder.Take by weighing the 150Kg powder, place manyly with extracting pot, stir, directly logically in extracting pot be steam heated to 95 ℃-100 ℃ with the water of 10 times of weight, steaming and decocting 30 minutes, to be cooled to room temperature, collection filtrate.Repeat to use water extraction 1 time.Percent by volume 40% isopropanol water solution (percent by volume) that adds 6 times of weight again soaks 15h, and 50 ℃ of temperature are emitted isopropanol extraction liquid, repeat to extract twice, merge extractive liquid, again.
Extracting solution is evacuated to boiler, at first open the inlet valve of condenser and cooler, slowly the cooling water in reaction pot passageway is emitted again, controls the flow of steam and about kettle temperature 70-100 ℃ well, in medicinal liquid, contain isopropyl alcohol concentration less than percent by volume 5% till.
The cyclohexane extraction reflux, extract, twice that adds 2 times of concentrated solution volumes, extract 50min at every turn after, it is two-layer up and down that cyclohexane extraction extract and former extracting solution are told, and emits the cyclohexane extraction extract, three extracts are merged, dewatering and filtering, cyclohexane extraction is reclaimed in air-distillation.Control kettle temperature and pressure well, decompression vacuum pumping below vacuum-0.07MPa, concentrates to such an extent that loose rufous powder is tripterygium glycosides crude product extract.
With the tripterygium glycosides coarse powder cyclohexane extraction in last step, be dissolved into solution earlier, slowly adding is mixed with in the cellulose (100-200 order) of cyclohexane extraction then, cellulosic consumption is 18 times of crude product quality, stirring was adsorbed 50 fens, and filtration is drained, and filtrate is invalid element.
The cyclohexane extraction acetone soln gradient elution of filter cake 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume), solution usage is: with every gram adsorbent is reference, 1% gradient, consumption is 4 milliliters; 3% gradient, consumption are 2 milliliters; 5% gradient, consumption are 3 milliliters; 7%, 10% and 12% gradient, consumption are 5 milliliters; 15% gradient, consumption are 6 milliliters.Adopt thin layer chromatography and ultraviolet 364nm to detect down, with standard control efficiency confirmed gradient.Filtration is drained, and effective filtrate is concentrated, and reclaims cyclohexane extraction, gets dry extract after concentrating, and carries out the second adsorption eluting again, and the dry extract after concentrating treats that column chromatography is standby.
In the chromatographic column of the diameter 12cm left and right sides, pack into earlier cyclohexane extraction a little, push absorbent cotton (doing glomeration) then and place the column bottom to pave.Wet method dress silica gel drains bubble in the post, leaves standstill the above compacting of 24h, opens piston and emits cyclohexane extraction unnecessary above the silica gel agent, and plastochondria begins to shrink, and compresses cylinder naturally.
The diluent of dry extract will be added in the post, the cyclohexane extraction acetone soln that adds 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume) respectively carries out gradient elution, solution usage is: with every gram adsorbent is reference, 1% gradient, and consumption is 4 milliliters; 3% gradient, consumption are 2 milliliters; 5% gradient, consumption are 3 milliliters; 7%, 10% and 12% gradient, consumption are 5 milliliters; 15% gradient, consumption are 6 milliliters.Collect eluent with minute bottle.With the bottle eluent collected respectively point sample after carrying out thin layer chromatography on the silica gel G plate, with differentiating under the uv analyzer respective wavelength whether eluent contains effective composition, collects effective bottle of sample, with the eluent of effective bottle of sample concentrated drain active ingredient.With active ingredient drying, chemical examination, the proportioning that branch is got, mixed 100 mesh sieves, packing is the former powder of finished product tripterygium glycosides.Wherein, the content of triptolide is 0.10wt%, and the content of wilforlide A is 0.25wt%.With used all organic reagent recycling uses.
Embodiment 4
The Radix Tripterygii Wilfordii xylem is through choosing, be treated to fritter, and being pulverized by pulverizer is powder.Take by weighing the 150Kg powder, place manyly with extracting pot, stir, directly logically in extracting pot be steam heated to 70 ℃-80 ℃ with the water of 8 times of weight, steaming and decocting 50 minutes, to be cooled to room temperature, collection filtrate.Add 8 times percent by volume 60% n-butanol aqueous solution again, soak 20h, 25 ℃ of temperature are emitted n-butanol extracting liquid, add percent by volume 60% n-butanol aqueous solution once more with same method, soak once merge extractive liquid.
Extracting solution is evacuated to boiler, at first open the inlet valve of condenser and cooler, slowly the cooling water in reaction pot passageway is emitted again, controls the flow of steam and about kettle temperature 70-100 ℃ well, in medicinal liquid, contain n-butyl alcohol concentration less than percent by volume 10% till.
Adding is with the isopyknic hexane reflux, extract, of concentrated solution three times, extract 30min at every turn after, it is two-layer up and down that hexane extract and former extracting solution are told, and emits hexane extract, three extracts are merged, dewatering and filtering, hexane is reclaimed in air-distillation.Control kettle temperature and pressure well, decompression vacuum pumping below vacuum-0.07MPa, concentrates to such an extent that loose rufous powder is tripterygium glycosides crude product extract.
With the tripterygium glycosides coarse powder hexane in last step, be dissolved into solution earlier, slowly adding is mixed with in the kieselguhr (100-200 order) of hexane then, and diatomaceous consumption is 18 times of crude product quality, stirs to adsorb 30 fens, and filtration is drained, and filtrate is invalid element.
The methanol solution gradient elution of the hexane of filter cake 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume), solution usage is: with every gram adsorbent is reference, 1% gradient, consumption is 3.5 milliliters; 3% gradient, consumption are 1.6 milliliters; 5% gradient, consumption are 2.8 milliliters; 7%, 10% and 12% gradient, consumption are 4.5 milliliters; 15% gradient, consumption are 5.5 milliliters.Adopt thin layer chromatography and ultraviolet 364nm to detect down, with standard control efficiency confirmed gradient.Filtration is drained, and effective filtrate is concentrated, and reclaims hexane, after concentrating dry extract, carry out the second adsorption eluting again, be concentrated into driedly, treat that column chromatography is standby.
In the chromatographic column of the diameter 8cm left and right sides, pack into earlier hexane a little, push absorbent cotton (doing glomeration) then and place the column bottom to pave.Wet method dress silica gel drains bubble in the post, leaves standstill the above compacting of 24h, opens piston and emits hexane unnecessary above the silica gel agent, and plastochondria begins to shrink, and compresses cylinder naturally.
With spissated dry powder behind a small amount of hexane dissolving eluting, add in the post, the methanol solution liquid that adds the hexane of 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume) respectively carries out gradient elution, solution usage is: with every gram adsorbent is reference, 1% gradient, consumption are 3.5 milliliters; 3% gradient, consumption are 1.6 milliliters; 5% gradient, consumption are 2.8 milliliters; 7%, 10% and 12% gradient, consumption are 4.5 milliliters; 15% gradient, consumption are 5.5 milliliters.Collect eluent with minute bottle.With the bottle eluent collected respectively point sample after carrying out thin layer chromatography on the silica gel G plate, with differentiating under the uv analyzer respective wavelength whether eluent contains effective composition, collects effective bottle of sample, with the eluent of effective bottle of sample concentrated drain active ingredient.With active ingredient drying, chemical examination, the proportioning that branch is got, mixed 100 mesh sieves, packing is the former powder of finished product tripterygium glycosides.Wherein, the content of triptolide is 0.11wt%, and the content of wilforlide A is 0.25wt%.With used all organic reagent recycling uses.
Embodiment 5
The Radix Tripterygii Wilfordii xylem is through choosing, be treated to fritter, and being pulverized by pulverizer is powder.Take by weighing the 150Kg powder, place manyly with extracting pot, stir, directly logically in extracting pot be steam heated to 60 ℃-70 ℃ with the water of 8 times of weight, steaming and decocting 40 minutes, to be cooled to room temperature, collection filtrate.Add 8 times percent by volume 80% ethanol water again, soak 10h, 20 ℃ of temperature are emitted n-butanol extracting liquid, add percent by volume 80% ethanol water once more with same method, soak once merge extractive liquid.
Extracting solution is evacuated to boiler, at first open the inlet valve of condenser and cooler, slowly the cooling water in reaction pot passageway is emitted again, controls the flow of steam and about kettle temperature 70-100 ℃ well, in medicinal liquid, contain concentration of alcohol less than percent by volume 10% till.
Adding is extracted three times with the isopyknic ethyl acetate backflow of concentrated solution, extract 60min at every turn after, it is two-layer up and down that acetic acid ethyl acetate extract and former extracting solution are told, emit acetic acid ethyl acetate extract, three extracts are merged, dewatering and filtering, ethyl acetate is reclaimed in air-distillation.Control kettle temperature and pressure well, decompression vacuum pumping below vacuum-0.07MPa, concentrates to such an extent that loose rufous powder is tripterygium glycosides crude product extract.
With the tripterygium glycosides coarse powder acetone in last step, be dissolved into solution earlier, slowly adding is mixed with in the kieselguhr (100-200 order) of acetone then, and diatomaceous consumption is 20 times of crude product quality, stirs to adsorb 30 fens, and filtration is drained, and filtrate is invalid element.
The alcoholic solution gradient elution of the carbon tetrachloride of filter cake 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume), solution usage is: with every gram adsorbent is reference, 1% gradient, consumption is 3 milliliters; 3% gradient, consumption are 1.7 milliliters; 5% gradient, consumption are 2.2 milliliters; 7%, 10% and 12% gradient, consumption are 4.2 milliliters; 15% gradient, consumption are 4.5 milliliters.Adopt thin layer chromatography and ultraviolet 364nm to detect down, with standard control efficiency confirmed gradient.Filtration is drained, and effective filtrate is concentrated, and reclaims carbon tetrachloride, after concentrating dry extract, carry out the second adsorption eluting again, be concentrated into driedly, treat that column chromatography is standby.
In the chromatographic column of the diameter 8cm left and right sides, pack into earlier hexane a little, push absorbent cotton (doing glomeration) then and place the column bottom to pave.Wet method dress silica gel drains bubble in the post, leaves standstill the above compacting of 24h, opens piston and emits hexane unnecessary above the silica gel agent, and plastochondria begins to shrink, and compresses cylinder naturally.
With spissated dry powder behind a small amount of carbon tetrachloride dissolving eluting, add in the post, the alcoholic solution that adds the carbon tetrachloride of 1%, 3%, 5%, 7%, 10%, 12%, 15% (percent by volume) respectively carries out gradient elution, solution usage is: with every gram adsorbent is reference, 1% gradient, consumption are 3 milliliters; 3% gradient, consumption are 1.7 milliliters; 5% gradient, consumption are 2.2 milliliters; 7%, 10% and 12% gradient, consumption are 4.2 milliliters; 15% gradient, consumption are 4.5 milliliters.Collect eluent with minute bottle.With the bottle eluent collected respectively point sample after carrying out thin layer chromatography on the silica gel G plate, with differentiating under the uv analyzer respective wavelength whether eluent contains effective composition, collects effective bottle of sample, with the eluent of effective bottle of sample concentrated drain active ingredient.With active ingredient drying, chemical examination, the proportioning that branch is got, mixed 100 mesh sieves, packing is the former powder of finished product tripterygium glycosides.Wherein, the content of triptolide is 0.10wt%, and the content of wilforlide A is 0.25wt%.With used all organic reagent recycling uses.
Effect embodiment
High-performance liquid chromatogram determination triptolide and wilforlide A content (instrument: the Agilent.1100Series high performance liquid chromatograph)
(1) triptolide is measured:
1, the preparation of reference substance solution: it is an amount of to get the triptolide reference substance, and accurate the title decides, and adds dissolve with methanol and makes every milliliter of solution that contains 50ug approximately.
2, the preparation of need testing solution: get each 50 of each producer's tripterygium glycosides sheets, get about 1.5 grams, accurate claim surely, add ethyl acetate 10mL dissolving, weigh, supersound extraction, weighing adds ethyl acetate and supplies loss amount again, shakes up centrifugal (3000r/min).The accurate supernatant 5mL that draws, add to neutral alumina (the 3g internal diameter 1cm that installs with the ethyl acetate wet method, the 70-350 order) on the post (under the room temperature condition), uses eluent ethyl acetate, collect eluent, Rotary Evaporators steams and removes ethyl acetate, residue is transferred in the 5mL measuring bottle with methanol, adds methanol constant volume to scale, shakes up, filtering with microporous membrane is as need testing solution.
3, measure: the accurate need testing solution 10 μ L that draw, according to HPLC method (Chinese Pharmacopoeia 2005 version one one) operation.Chromatographic condition: 30 ℃ of column temperatures, mobile phase: methanol-0.01mol/L potassium dihydrogen phosphate (35:65), flow velocity 1.00mL/min.Measure the peak area of test sample and reference substance at 225nm wavelength place, calculate promptly.
(2) wilforlide A is measured:
1, the preparation of reference substance solution: it is an amount of to get the wilforlide A reference substance, and accurate the title decides, and contains the solution of 0.1mg in adding dissolve with methanol and making every milliliter approximately.
2, the preparation of test sample solution: get each 50 of each producer's tripterygium glycosides sheets, get about 1.5 grams, the accurate title, decide, and adds ethyl acetate 10mL dissolving, weighs, and supersound extraction is weighed and added ethyl acetate and supplies loss amount, centrifugal (3000r/min).The accurate supernatant 5mL that draws is to the neutral alumina (3g that has installed with the ethyl acetate wet method, internal diameter 1cm, the 70-350 order) on the post (under the room temperature condition), uses eluent ethyl acetate, collect eluent, rotary evaporation is removed ethyl acetate, residue is transferred in the 5mL measuring bottle with methanol, adds methanol constant volume to scale, shakes up, filtering with microporous membrane is as need testing solution.
3, measure: the accurate need testing solution 20 μ L that draw, according to HPLC method (appendix of Chinese Pharmacopoeia version in 2005) operation.Chromatographic condition: 30 ℃ of column temperatures, mobile phase: methanol-second is fine-water (60: 25: 15), flow velocity 1.0mL/min.Measure the peak area of test sample and reference substance at 210nm wavelength place, calculate promptly.
[preparation of standard curve]
Precision takes by weighing triptolide and wilforlide A reference substance 8.15mg, 6.04mg respectively, with dissolve with methanol and be settled to 25mL as storing solution.Accurate respectively absorption storing solution 0.25,0.5,1.0,1.5,2.0,2.5mL and 0.5,1.0,2.0,3.0,4.0,5.0 put in the 5mL measuring bottle, press the test of chromatographic condition and system suitability.The difference curve plotting, and calculate regression equation: Y
1=1714.2X+0.7818, Y
2=507.02X 1; R
1=1.0000, R
2=0.9998.Show that both have better linearity relation (seeing Fig. 1 and Fig. 2) between 0.163～1.630 μ g, 0.484～4.840 μ g.
Table 1 triptolide standard curve determination
Table 2 Radix Tripterygii Wilfordii ester first standard curve determination
Table 3 has provided the content contrast of triptolide and wilforlide A in the tripterygium glycosides sheet that tripterygium glycosides sheet that 5 batches of former powder of the tripterygium glycosides that makes of method by embodiment 1 make and other manufacturing enterprises produce.
The different manufacturing enterprise of table 3 Glucosidorum Tripterygll Totorum first element, lactone first content contrast table
| Manufacturing enterprise | Lot number | Triptolide (ug/ sheet) | Wilforlide A (ug/ sheet) |
| Huangshi Feiyun Pharmaceutical Co., Ltd. | 050601 | 0.85 | 19.2 |
| Huangshi Feiyun Pharmaceutical Co., Ltd. | 050301 | 0.88 | 19.5 |
| ShangHai Fudan Fuhua Pharmaceutical Co., Ltd | 050801 | 11.8 | 168.3 |
| ShangHai Fudan Fuhua Pharmaceutical Co., Ltd | 050301 | 15.5 | 20.5 |
| Xieli Pharmaceutical Co., Ltd., Hunan | 050301 | 2.6 | 9.1 |
| Xieli Pharmaceutical Co., Ltd., Hunan | 051002 | 2.2 | 11.5 |
| Manufacturing enterprise | Lot number | Triptolide (ug/ sheet) | Wilforlide A (ug/ sheet) |
| Guizhou hanfang Pharmaceutical Co., Ltd | 040603 | 2.2 | 28.3 |
| Zhejiang De'ende Pharmaceutical Co., Ltd. | 0601103 | 0.53 | 24.0 |
| Jiangsu Mei Tong pharmaceutical Co. Ltd (the present invention) | 050802 | 10.9 | 27.2 |
| Jiangsu Mei Tong pharmaceutical Co. Ltd (the present invention) | 050905 | 10.4 | 31.0 |
| Jiangsu Mei Tong pharmaceutical Co. Ltd (the present invention) | 051102 | 11.2 | 30.1 |
| Jiangsu Mei Tong pharmaceutical Co. Ltd (the present invention) | 051220 | 10.8 | 36.6 |
| Jiangsu Mei Tong pharmaceutical Co. Ltd (the present invention) | 060211 | 11.7 | 37.5 |
Find out by table 3 data, the tripterygium glycosides sheet that tripterygium glycosides extract of the present invention makes is compared with the tripterygium glycosides sheet that other manufacturing enterprises produce, the content of wherein contained triptolide and wilforlide A and ratio have more different, are in moderate scope.Can be detected as to be divided into by above two kinds and show that content of drug effect components and ratio are moderate in the tripterygium glycosides sheet that tripterygium glycosides extract of the present invention makes.
Experiment by following doubling dosage tripterygium glycosides treatment nephrotic syndrome shows: the tripterygium glycosides pharmacological effect that is made by method of the present invention is good, and toxic and side effects is little.
1 object and method
1.1 case is selected
18 routine patients all meet following condition: 1. clinical manifestation is simple NS, does not have or only has and seldom measure microscopic hematuria, and renal function is normal, does not have complication such as infection and thrombosis.Wherein a part of patient once used hormone therapy; 2. Histological change is the mesentery proliferative lesion; 3. do not have abnormal liver function, the peripheral blood leucocyte counting is normal; 4. get rid of secondary glomerulonephritis.Male's 13 examples wherein, women's 5 examples, 24.4 ± 8.6 years old mean age, course of disease two weeks～12 year.Clinical setting before the treatment sees Table 1.
18 routine patients' kidney biopsy is learned diagnosis: 1. IgA nephropathy (IgAN) 8 examples, and there are 5 examples once to accept hormone therapy in the past, 2 examples are responsive, and 3 examples are invalid; 2. IgM nephropathy (IgMN) 4 examples, 2 example hormone-sensitives; 3. mesangial proliferative glomerulonephritis (MsPGN) 6 examples, 3 example hormones in the past, 1 example is responsive, and 2 examples are invalid.
1.2 Therapeutic Method
1.2.1T II Taizhou pharmaceutical factory produces Glucosidorum Tripterygll Totorum (tripterygium glycosides sheet of the present invention)
1.2.2 dosage regimen
Initial dose 2mg/ (kg.d) divides three times oral after the meal (be generally the 10mg/ sheet, 4, every day three times), changes 1.5mg/ after continuing for 4 weeks into and reduces to 1mg/ (kg.d) after (kg.d) * 4 week and keep.
1.3 observation item
1. clinical observation: edema, urine quantitative changeization, digestive tract reaction has or not erythra etc.; 2. lab testing: urine amount, urine protein quantitation; Peripheral hemogram; Hepatic and renal function and plasma protein etc. are checked once weekly, carry out urine protein SDS-PAGE disk electrophoresis before the part patient treatment, analyze urine protein and form.
1.4 curative effect is judged
1. alleviate: urine protein disappears or reduces to trace, quantitatively below 0.4g/24h; 2. improve: the urine protein ratio is controlled
Reduce more than 50% before treating; 3. invalid: as not reach above-mentioned standard.
2 results
2.1 doubling dosage T II is to the curative effect of NS
This organizes 18 routine primary glomerulonephritises, and the clinical setting after the treatment sees Table 4.Have 15 examples and alleviated fully, account for 83.3% (seeing Table 5).7 examples are alleviated (accounting for 87.5%) among the 8 routine IgAN, and 1 example is improved.5 examples are alleviated (83.3%) among the 6 routine MsPGN, and 4 routine IgMN 3 examples are alleviated (75.0%).There are 5 examples once invalid but obtain alleviation in the past in 18 examples through heavy dose of T II treatment back with the capacity hormone therapy.There are 1 routine IgMN and 1 routine MsPGN invalid in this group to T II.In 15 examples of alleviating, treat 1 week promptly alleviation person 4 examples are arranged, treating 2 all alleviation persons has 8 examples, therefore has 12 examples (account for alleviation group 80%) urine protein disappearance (seeing Table 6) in 2 weeks of treatment.The patient of original edema, oliguria after taking T II, visible significantly diuretic reaction, the very fast disappearance of edema.At the IgMN that 1 routine T II fails to respond to any medical treatment, although albuminuria does not obviously reduce, the urine amount obviously increases, and edema also disappears rapidly.
General clinical setting of table 4 and lab testing result
| Project | Before the treatment | Treated for 4 weeks |
| Edema | 14 | 0 |
| Nephrotic syndrome | 16 | 2 |
| |
1 | 0 |
| |
0 | 0 |
| Scr＞133μmol/ |
0 | 0 |
| Hb(g/L) | 134±17 | 134±19 |
| WBC(×10 9/L) | 7.15±2.93 | 6.32±2.52 |
| Urine protein (g/24h) | 2.34±1.14 | 0.50±0.59 |
| Serum albumin (g/L) | 18.6±7.9 | 30.4±9.4 |
Table 5T II is to the curative effect of different pathological types NS
| Histological type | n | Alleviate (%) | Improve (%) | Invalid (%) |
| IgAN | 8 | 7(87.5) | 1(12.5) | 0 |
| MsPGN | 6 | 5(83.3) | 0 | 1(16.7) |
| Histological type | n | Alleviate (%) | Improve (%) | Invalid (%) |
| IgMN | 4 | 3(75.0) | 0 | 1(25.0) |
| Sum | 18 | 15(83.3) | 1(5.5) | 2(11.1) |
Table 6 patient remission time of occurrence
2.2T the relation of II curative effect and urine protein composition
Carried out the inspection of urine SDS2PAGE disk electrophoresis before the 11 routine patient treatments, the urine protein of finding alleviation group patient is mainly albumin, no macromolecule and small protein, but not alleviation group urine protein contains the protein (accounting for 8.5 ± 4.6%) of more molecular weight＜20,000 in forming, and has more macromole urine protein (accounting for 21.0 ± 14.7%) simultaneously.
2.3 the side reaction of doubling dosage T II treatment
Only have in the treatment indivedual cases have nauseating, vomit or degradation gastrointestinal upset under the appetite arranged, this organizes 18 routine patients all can tolerate doubling dosage T II treatment well.(only 1 example＞100U/L), recovery is normal again after continuing to treat, and the serum-free bilirubin raises in the of short duration slight rising of treatment initial stage SGPT for objective determination minority case.Leukopenia to 215 * 10 appear in none example
9Below/the L, only 1 routine WBC reduces to 3.1 * 10
9/ L, but not influence treatment.In the observation period, all case renal functioies keep normal (seeing Table 7).
The side reaction (n=18) of table 7 treatment
| Project | Example number (%) |
| Feel sick, vomit | 1(5.5) |
| Appetite descends | 2(1.1) |
| WBC＜2.5×10
9/ |
0 |
| SGPT raises | 4(22.2) |
| SGOT raises | 1(5.5) |
| Last sense | 2(11.1) |
2.4 the recurrence situation after the doubling dosage treatment
2 routine patients rapidly reduce to 1mg/ (kg.d) with T II dosage using 4 weeks of T II 2mg/ (kg.d) behind the clinical symptom remission, and 2 week of administration, back NS all recurred.In addition 2 routine patients after urine protein is alleviated, follow up a case by regular visits in the process because of on the sense albuminuria increase the weight of, continue to use the urine protein disappearance of doubling dosage treatment back.All the other cases are followed up a case by regular visits to, and do not see the recidivist as yet.
Claims (13)
1. the extracting method of a tripterygium glycosides is characterized in that comprising the steps:
(1) extract crude product: Common Threewingnut Root is extracted with decocting in water, and reuse alcohol or pure extraction with aqueous solution merge extracting solution, be concentrated into pure content and be less than or equal to percent by volume 20%, use organic solvent extraction afterwards, make the extract that contains crude product, be concentrated into dried crude product;
(2) crude product refining: use the organic solvent dissolution crude product, adding adsorbent adsorbs, filter, filter cake is carried out eluting, the eluent of collection live part with the solution of gradient organic solvent, concentrate, carry out column chromatography again, carry out eluting, collect the eluent that contains effective composition with the solution of gradient organic solvent, be concentrated into driedly, promptly get and make with extra care tripterygium glycosides;
In the step (1), the concentration of alcohol is that percent by volume is more than 40% in the aqueous solution of described alcohol;
In the step (2), described adsorbent is silica gel, neutral alumina, cellulose or kieselguhr;
In the step (2), the solution of described gradient organic solvent be gradient at the halogenated alkane of 1～20% of percent by volume or the alcoholic solution of alkane, or gradient is at the acetone soln of the cycloalkane of percent by volume 1～20%;
Wherein, described organic solvent is petroleum ether, halogenated alkane, cycloalkane, alkane, lower aliphatic ketone or ester; Described halogenated alkane is dichloromethane, dichloroethanes, chloroform or carbon tetrachloride; Described cycloalkane is cyclohexane extraction or Pentamethylene.; Described alkane is hexane or pentane; Described lower aliphatic ketone is acetone or butanone; Described ester is an ethyl acetate; Described alcohol is methanol, ethanol, isopropyl alcohol or n-butyl alcohol.
2. the method for claim 1 is characterized in that: in the step (1), the number of times that described decocting in water extracts is 1～3 time; The number of times of the extraction with aqueous solution of described alcohol or alcohol is 1～3 time; The number of times of described extraction is 1～3 time; In the step (2), the described organic solvent dissolution crude product of using adds adsorbent and adsorbs, and filters, and filter cake is carried out eluting with the gradient organic solvent, and the number of times of the step of the eluent of collection live part is 1～2 time.
3. the method for claim 1, it is characterized in that: in the step (1), the temperature of described decocting in water is 50～100 ℃; The time of decocting in water is 30～60 minutes.
4. the method for claim 1 is characterized in that: in the step (1), the temperature of the extraction with aqueous solution of described alcohol or alcohol is 20～50 ℃; The time of pure or pure extraction with aqueous solution is 8～24 hours.
5. the method for claim 1, it is characterized in that: in the step (1), the consumption of described water is 3～10 times of Common Threewingnut Root weight; The consumption of the aqueous solution of described alcohol or alcohol is 6～10 times of Common Threewingnut Root weight; The consumption of the organic solvent of described extraction usefulness is 1～3 times that extracting solution concentrates the back volume.
6. the method for claim 1, it is characterized in that: in the step (1), each time of described extraction is 30～60 minutes.
7. the method for claim 1, it is characterized in that: in the step (2), the consumption of described adsorbent is 10～20 times of crude product quality.
8. the method for claim 1, it is characterized in that: in the step (2), the time of described absorption is 30～60 minutes.
9. the method for claim 1, it is characterized in that: in the step (2), the consumption of the solution of described gradient organic solvent and the solution of each gradient is: with every gram adsorbent is reference, 1% gradient, consumption is 3～4 milliliters; 3% gradient, consumption are 1.5～2 milliliters; 5% gradient, consumption are 2～3 milliliters; 7%, 10% and 12% gradient, consumption are 3～5 milliliters; 15% gradient, consumption are 4～6 milliliters; Percentage ratio is percent by volume.
10. the method for claim 1 is characterized in that: in the step (2), the eluent of described live part detects down to adopt thin layer chromatography and ultraviolet 364nm, confirms with standard control.
11. the method for claim 1 is characterized in that: described alcohol and/or organic solvent are reclaimed, recycle.
12. the tripterygium glycosides that the method for claim 1 makes.
13. contain the pharmaceutical composition of tripterygium glycosides as claimed in claim 12.
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| CN101991633B (en) * | 2009-10-10 | 2012-11-07 | 上海复旦复华药业有限公司 | Method for extracting tripterygium glycosides, and product and inclusion compound and medicinal composition thereof |
| CN102008536B (en) * | 2010-12-02 | 2012-05-23 | 吴江迪星科技有限公司 | Tripterygium wilfordii hook extract cataplasm and preparation method thereof |
| CN102793737B (en) * | 2011-05-26 | 2014-01-08 | 澳门科技大学 | Method for preparing powder of effective part of thunder god vine |
| CN106418124A (en) * | 2016-09-07 | 2017-02-22 | 华南师范大学 | Yeast extract taste removing method and yeast taste component identification method |
| CN111297934A (en) * | 2019-12-18 | 2020-06-19 | 湖南千金协力药业有限公司 | Tripterygium glycosides extraction method |
| CN115073278B (en) * | 2022-07-20 | 2023-07-04 | 江苏知原药业股份有限公司 | Method for extracting tripterygium wilfordii p-quinone B from tripterygium wilfordii |
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