CN104739955A - Preparation method of purified short-pedicel aconite root alkaloid by employing cation exchange resin method - Google Patents
Preparation method of purified short-pedicel aconite root alkaloid by employing cation exchange resin method Download PDFInfo
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- CN104739955A CN104739955A CN201510058109.5A CN201510058109A CN104739955A CN 104739955 A CN104739955 A CN 104739955A CN 201510058109 A CN201510058109 A CN 201510058109A CN 104739955 A CN104739955 A CN 104739955A
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Abstract
The invention discloses a preparation method of purified short-pedicel aconite root alkaloid by employing a cation exchange resin method. The method comprises the following steps: extracting a short-pedicel aconite root liquid medicine by employing a water decoction method; concentrating an extract liquid into 0.25 crude drug per ml; carrying out refrigeration and centrifugal treatment, and taking a supernatant liquid; carrying out pretreatment of cation exchange resin, adsorbing and purifying the supernatant liquid by employing the pretreated cation exchange resin and eluting to obtain an eluent; collecting the eluent, and drying to obtain a dried product; refluxing the dried product with ethanol to remove salt; recovering ethanol, concentrating and drying to obtain a crude extract containing salt impurities; and further removing salt with chloroform, distilling and recovering chloroform, and drying concentrated liquor to obtain a short-pedicel aconite root purified product. The preparation method for preparing a short-pedicel aconite root alkaloid purified product has the beneficial effects of being simple and low in cost.
Description
Technical field
The invention belongs to biological technical field, relate to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani).
Background technology
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) derives from the dried root of ranunculaceae plant aconitum brachypodum (Aconitum brachypodum Diels), Radix aconiti szechenyiani (Aconitumpendulum Busch) and Aconitum nagarum var.lasiandrum W.T.Wang (Aconitum subrosullatum H-M.).Warm in nature, bitter in the mouth, pungent, very toxic, return liver, kidney channel, there is expelling wind and removing dampness, dissipating blood stasis cures the wound and the effect such as promoting blood circulation and stopping pain, be used for the treatment of muscles and bones caused by anemofrigid-damp arthralgia and arthralgia, the disease such as pain due to blood stasis and worm venom caused by traumatic injury clinically.By consulting literatures finds, existing pharmacological evaluation proves that it has significant analgesia, antiinflammatory action, and its effective ingredient is Diterpenoid Alkaloids class material.But the short supply of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid extract in the market; have one, Kunming Pharma Inc. only also at production Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid; but its technique is more complicated, be applied to a large amount of chloroforms, this solvent is harmful to operator, environmental conservation and product safety aspect.Therefore need study further the alkaloidal purification technique of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani).
Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid often adopts the methods such as dipping, decoction, solvent refluxing, percolation to extract from medical material, also there are other fat-soluble or water-solubility impurities a large amount of in the alkaloid solution after solvent extraction except alkaloid and salt, need to be further purified process, alkaloid component is therefrom separated.Its purification treating method is normally used is the methods such as organic solvent extraction, chromatograph and resin absorption.Resin absorption is a kind of method of effective separating effective ingredient.Be characterized in that treatment effeciency is high, flux large, the resin according to adopting can be divided into Flavonoids by Macroporous Adsorption Resin and ion-exchange-resin process two kinds.
Existing Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified price is high on the market at present, and cost of manufacture is high, and manufacture method is complicated.This seminar once carried out purification research with macroporous adsorbent resin to Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid, found that, the adsorbance of macroporous adsorbent resin is less, and when washing impurity with water, alkaloid component leakage rate is larger.Therefore last cation exchange resin processes carries out purifying process investigation to Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid, result: Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid component adsorption rate on cation exchange resin is higher, can reach more than 98%.Investigate eluent, final selection 0.02g/ml NaCl-50% ethanol is eluent, and eluting rate can reach more than 90%.Sodium chloride as impurity residual in eluent can remove major part with ethanol, then uses the further remove impurity of a small amount of chloroform, and in gained purified, total alkaloids purity reaches more than 60%, and the alkaloidal dissolubility of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) that its dissolubility ratio is bought improves greatly.
Summary of the invention
The object of the present invention is to provide the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani), solve existing Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified cost of manufacture high, the problem of manufacture method complexity.
The technical solution adopted in the present invention is carried out according to following steps:
Step 1: extract Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid by decocting cooking method, extracting solution is concentrated into 0.25g crude drug/ml, gets clear liquid medicine after cold preservation centrifugal treating;
Step 2: the pretreatment of cation exchange resin;
Step 3: adopt the good cation exchange resin of pretreatment to carry out adsorption and purification to upper clear liquid medicine, and eluting, obtain eluent, and collection eluent is dry obtains dry thing;
Step 4: dry thing alcohol reflux desalination, after reclaiming ethanol, concentrate drying must contain the thick pure thing of salt impurity;
Step 5: the further desalination of chloroform, after Distillation recovery chloroform, concentrated solution is dry obtains Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) purified.
Further, in described step 1, decocting cooking method extraction Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid process is: first that medical material is broken, add the water boiling and extraction 1h of 12 times amount at every turn, extract 3 times, each filtration, merges 3 extracting solution, is concentrated into 0.25g crude drug/mL with slow fire, centrifugal treating to 4 DEG C of refrigerator cold-storages, gets clear liquid medicine and namely obtains Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid.
Further, the pretreatment of described step 2 cationic exchanger resin: select sodium form 732 cation exchange resin, be placed in 2 ~ 3 times of resin volumes, 10% NaCl and aqueous solution, soak 2 ~ 4 hours, remove saline solution, with distilled water rinsing to close to colourless; With the alcohol solution dipping 2 ~ 4 hours of 2 ~ 3 times of resin volumes, be washed till without alcohol taste with distilled water; With the 1molL of 2 ~ 3 times of resin volumes
-1hCl and aqueous solution soaking resin 2 ~ 4 hours, remove acid solution, and distilled water flushing is to close to neutral; With the 1molL of 2 ~ 3 times of volumes
-1naOH and aqueous solution soaking 2 ~ 4 hours, remove alkali liquor, with distilled water flushing to close to neutral; Use 2 ~ 3 times of 1molL again
-1hCl and aqueous solution soaking 2 ~ 4 hours, make resin transfer be H
+type, with distilled water flushing to close to neutral, for subsequent use.
Further, adsorption and purification process in described step 3: select 15mm*200mm chromatographic column, cation exchange resin, the medicinal liquid loading 4-6BV of 0.25g crude drug/ml, with 2ml/min flow velocity loading, presses 2ml/min flow velocity washing 8-10BV with distilled water.
Further, in described step 3, eluting adopts 0.02g/ml NaCl-50% ethanol elution.
Further, eluent dried in described step 3: be 6 ~ 7 with saturated sodium hydroxide solution adjust pH by the eluent of collection, reclaim ethanol with Rotary Evaporators, temperature is 70 DEG C, concentrated solution is put in vacuum drying oven dry further, must contain the thick pure thing of salt impurity.
Further, in described step 4, dry thing alcohol reflux desalination method is dry thing 10 times amount alcohol reflux 1 time.
Further, in described step 5, the further desalting process of chloroform is chloroform reflux, extract, 2 times, for the first time 8 times amount chloroforms, second time 6 times amount chloroforms, each 1.5h.
The invention has the beneficial effects as follows and make Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified manufacture method simply, cost is low.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in detail.
Specific embodiments of the present invention are as follows:
(1) preparation of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid: medical material is broken, decocting cooking method is adopted to extract, extract 3 times, add the water extraction 1h of 12 times amount at every turn, filter, merge 3 extracting solution, slow fire is concentrated into 0.25g crude drug/mL, to 4 DEG C of refrigerator cold-storages centrifugal treating after 2 days, the centrifugal 30min of 2400r/min revolution, gets clear liquid medicine and get final product.
(2) pretreatment of cation exchange resin: select sodium form 732 cation exchange resin, be placed in 2 ~ 3 times of resin volumes, 10% NaCl and aqueous solution, soak 2 ~ 4 hours, remove saline solution, with distilled water rinsing to close to colourless, remove partial impurities and mechanical debris in resin; By 95% ethanol of 2 ~ 3 times of resin volumes and aqueous solution soaking 2 ~ 4 hours, wash away the alcohol soluble organism in resin, be washed till without alcohol taste with distilled water; With the 1molL of 2 ~ 3 times of resin volumes
-1hCl and aqueous solution soaking resin 2 ~ 4 hours, remove acid solution, and distilled water flushing is to close to neutral; Then the 1molL of 2 ~ 3 times of volumes is used
-1naOH and aqueous solution soaking 2 ~ 4 hours, remove alkali liquor, with distilled water flushing to close to neutral; Use 2 ~ 3 times of 1molL again
-1hCl and aqueous solution soaking 2 ~ 4 hours, make resin transfer be H
+type, with distilled water flushing to close to neutral, for subsequent use.
(3) cationic exchange resin adsorption purification: select 15mm*200mm chromatographic column (diameter 15mm, high 200mm, blade diameter length ratio is that 1:9 (needs 24.5ml resin, it is wet method dress post herein, 24.5ml is the volume of resin), the medicinal liquid loading 4-6BV of 0.25g crude drug/ml, with 2ml/min flow velocity loading, 2ml/min flow velocity washing 8-10BV is pressed with distilled water, with 0.02g/ml sodium chloride-50% ethanol for eluent, by 0.5ml/min flow velocity eluting 20BV, collect eluent.
Amplification technique: each technological parameter is pressed chromatographic column diameter amplification and amplified.If select chromatographic column diameter to amplify n doubly than the diameter of 15mm*200mm, then its purifying process parameter is: blade diameter length ratio is the consumption that 1:9 determines cation exchange resin, concentration is the Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid loading 4-6BV of 0.25g crude drug/ml, with 2nml/min flow velocity loading, 2n ml/min flow velocity washing 8-10BV is pressed with distilled water, with 0.02g/ml sodium chloride-50% ethanol with 1.5n ml/min flow velocity eluting 20BV, collect eluent.
(4) eluent dried: be 6 ~ 7 with saturated sodium hydroxide solution adjust pH by the eluent of collection, ethanol (temperature is 70 DEG C) is reclaimed with Rotary Evaporators, concentrated solution vacuum drying oven is dry further, must contain the dry thing of salt impurity.
(5) except Ficus caricaL: by dry thing 10 times of quality alcohol reflux 1 time, extracting solution moderate snow A prime extraction rate reached more than 92%.With the further desalination of chloroform after extracting solution Rotary Evaporators recovery ethanol concentrate drying, chloroform reflux, extract, 2 times, for the first time 8 times amount chloroforms, second time 6 times amount chloroforms, each 1.5h.Filter to obtain chloroform solution, Distillation recovery chloroform, concentrated solution is dry obtains Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified.
The mensuration of total alkaloid content in gained Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) purified: take appropriate purified, bullatine A reference substance and be configured to certain density solution with pH=3 dissolve with hydrochloric acid solution respectively.All the other measure its Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) total alkaloids content according to Guizhou Province's Chinese crude drug, Ethnic crude drugs quality standard method, count more than 60% with bullatine A.
The mensuration of bullatine A content in purified: take appropriate purified, bullatine A reference substance and be configured to certain density solution with dissolve with methanol respectively.Adopt high performance liquid chromatography (HPLC) method, by following chromatographic condition: chromatographic column: Kromasil-C18 (150mm × 4.6mm, 5 μm); Mobile phase: phosphate buffer (pH7.3): acetonitrile (80:20); Flow velocity: 1.0mLmin
-1; Determined wavelength: 199nm; Column temperature: 30 DEG C; Sample size: 10 μ L.The content recording bullatine A in purified is more than 19%.
Utilize the alkaloidal technique study of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani), first decocting cooking method is adopted to extract Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medical material, extracting solution is through concentrated, by cation exchange resin that pretreatment is good after cold preservation centrifugal treating, resin uses 0.02g/ml NaCl-50% ethanol elution with after distillation washing, eluent removes a large amount of NaCl with alcohol reflux after reclaiming ethanol drying under reduced pressure, ethanol extract reclaims ethanol to dry, dry thing adds the backflow of a small amount of chloroform again and removes residual impurity further, improve the purity of purified, obtain Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified.The method technique is simple, cost is low, greatly reduce the consumption of chloroform toxic reagent.Gained Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified has the pharmacological action of analgesia, antiinflammatory.
The present invention be advantageous in that:
(1) overcome the complicated difficult problem of Technology that pharmaceutical factory uses, reduce the use amount of toxic solvent chloroform.Lower-cost cation exchange resin is utilized to carry out purification to Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid, medicinal liquid has good adsorption on resin, adsorption rate can reach more than 98%, after washing impurity with water, direct 0.02g/ml NaCl-50% ethanol is that eluent carries out eluting to alkaloid component, and eluting rate reaches more than 90%.Remove most of sodium chloride as impurity with ethanol after eluent drying, then use the further remove impurity of a small amount of chloroform, in gained purified, total alkaloids purity reaches more than 60%.
(2) in the present invention, the preparation of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid adopts decocting cooking method to extract, and method is easy, cost-saving.
(3) easy, the reproducible utilization of cation exchange resin low price, pretreatment, is more satisfactory resin.The technological operation of cation exchange resin purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid is easy and adsorption rate is higher.
(4) elute soln adopts 0.02g/mlNaCl-50% alcoholic solution, and reduce the consumption of organic solvent, environmental protection, and elute effect is good, eluting rate reaches more than 90%.
(5) desalting process first removes most of salt with ethanol, then uses the further remove impurity of a small amount of chloroform, compared with the existing technique in pharmaceutical factory, greatly reduces the consumption of chloroform toxic solvent.
(6) compared with gained Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid purified is prepared with pharmaceutical factory, there is good solubility property, solve pharmaceutical factory and prepare the difficult problem that purified dissolving is difficult, preparation drug loading is little.
(7) pharmacological evaluation proves that Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid has the effect of analgesia, antiinflammatory, can be used for the diseases such as treatment arthralgia, pain due to blood stasis caused by traumatic injury and worm venom, has good medical value.And Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) is alkaloidal under-supply in the market, the present invention can solve the problem of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) alkaloid shortage of resources.
The above is only to better embodiment of the present invention, not any pro forma restriction is done to the present invention, every any simple modification done above embodiment according to technical spirit of the present invention, equivalent variations and modification, all belong in the scope of technical solution of the present invention.
Claims (8)
1. the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani), is characterized in that carrying out according to following steps:
Step 1: extract Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid by decocting cooking method, extracting solution is concentrated into 0.25g crude drug/ml, gets clear liquid medicine after cold preservation centrifugal treating;
Step 2: the pretreatment of cation exchange resin;
Step 3: adopt the good cation exchange resin of pretreatment to carry out adsorption and purification to upper clear liquid medicine, and eluting, obtain eluent, and collection eluent is dry obtains dry thing;
Step 4: dry thing alcohol reflux desalination, after reclaiming ethanol, concentrate drying must contain the thick pure thing of salt impurity;
Step 5: the further desalination of chloroform, after Distillation recovery chloroform, concentrated solution is dry obtains Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) purified.
2. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: in described step 1, decocting cooking method extraction Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid process is: first that medical material is broken, add the water boiling and extraction 1h of 12 times amount at every turn, extract 3 times, each filtration, merges 3 extracting solution, is concentrated into 0.25g crude drug/mL with slow fire, centrifugal treating to 4 DEG C of refrigerator cold-storages, gets clear liquid medicine and namely obtains Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) medicinal liquid.
3. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: the pretreatment of described step 2 cationic exchanger resin: select sodium form 732 cation exchange resin, be placed in 2 ~ 3 times of resin volumes, 10% NaCl and aqueous solution, soak 2 ~ 4 hours, remove saline solution, with distilled water rinsing extremely close to colourless; By 95% ethanol of 2 ~ 3 times of resin volumes and aqueous solution soaking 2 ~ 4 hours, be washed till without alcohol taste with distilled water; With the 1molL of 2 ~ 3 times of resin volumes
-1hCl and aqueous solution soaking resin 2 ~ 4 hours, remove acid solution, and distilled water flushing is to close to neutral; With the 1molL of 2 ~ 3 times of volumes
-1naOH and aqueous solution soaking 2 ~ 4 hours, remove alkali liquor, with distilled water flushing to close to neutral; Use 2 ~ 3 times of 1molL again
-1hCl and aqueous solution soaking 2 ~ 4 hours, make resin transfer be H
+type, with distilled water flushing to close to neutral, for subsequent use.
4. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: adsorption and purification process in described step 3: select 15mm*200mm chromatographic column, cation exchange resin, the medicinal liquid loading 4-6BV of 0.25g crude drug/ml, with 2ml/min flow velocity loading, press 2ml/min flow velocity washing 8-10BV with distilled water.
5. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: in described step 3, eluent adopts 0.02g/ml NaCl-50% ethanol.
6. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: eluent dried in described step 3: be 6 ~ 7 by the eluent of collection with saturated sodium hydroxide solution adjust pH, ethanol is reclaimed with Rotary Evaporators, temperature is 70 DEG C, concentrated solution vacuum drying oven is dry further, must contain the thick pure thing of salt impurity.
7. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: in described step 4, dry thing alcohol reflux desalination method is dry thing 10 times amount alcohol reflux 1 time.
8. according to the alkaloidal preparation method of cation exchange resin processes purification Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) described in claim 1, it is characterized in that: in described step 5, the further desalting process of chloroform is chloroform reflux, extract, 2 times, 8 times amount chloroforms for the first time, second time 6 times amount chloroforms, each 1.5h.
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Cited By (1)
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| CN108061764A (en) * | 2017-12-01 | 2018-05-22 | 云南枝蒿制药有限公司 | The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment |
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| CN102532023A (en) * | 2010-12-24 | 2012-07-04 | 苏州宝泽堂医药科技有限公司 | Purification method of yunaconitine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108061764A (en) * | 2017-12-01 | 2018-05-22 | 云南枝蒿制药有限公司 | The method of quality control of Radix Aconiti Brachypodi (Radix Aconiti Szechenyiani) quick-acting antalgesic liniment |
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