CN1704405A - Method for analyzing and separating preparation of Huperzine A and Huperzine B - Google Patents
Method for analyzing and separating preparation of Huperzine A and Huperzine B Download PDFInfo
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- CN1704405A CN1704405A CN 200410042760 CN200410042760A CN1704405A CN 1704405 A CN1704405 A CN 1704405A CN 200410042760 CN200410042760 CN 200410042760 CN 200410042760 A CN200410042760 A CN 200410042760A CN 1704405 A CN1704405 A CN 1704405A
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Abstract
The invention relates to a process for analyzing and separating preparation of Huperzine A and Huperzine B, which comprises the steps of disintegrating and immersing raw material, enriching and concentrating with macroporous absorption resin, reversed phase column chromatography, non-alkyl bonded phase silica gel medium column chromatography, concentrating and crystallizing.
Description
Technical field
The present invention relates to analytical chemistry field and a kind of traditional Chinese medicine extraction method, be specifically related to analyze and separate the method for preparing selagine and huperzine B.
Background technology
Selagine is an isolated new alkaloids in the remedies araucaria plant Herba Lycopodii serrati therefrom, it is a kind of potent, reversible and highly selective acetylcholinesterase (AChE) inhibitor, belong to s-generation AChE inhibitor, also be the most successful treatment alzheimer's disease of present domestic-developed (Alzheimer ' s Disease, AD, senile dementia is otherwise known as) medicine, the medicinal application of conduct treatment AD is used to improve memory by the FDA approval as foodstuff additive or dietary supplements in the U.S. in clinical at home.In addition, still contain the strong AChE of other tool in the Herba Lycopodii serrati and suppress active alkaloid, wherein the most representative with huperzine B, and its content is only second to selagine.Because huperzine B and selagine structural similitude, therefore become the most difficult isolating material in the selagine production, though it is 1/10th of selagine that the AChE of huperzine B suppresses activity,, therefore can show higher therapeutic index because it has longer active duration.
The correlation analysis method of having reported mainly concentrates on uses the high effective liquid chromatography for measuring selagine, the filler of chromatographic column all adopts octadecyl bonding phase silica gel, because therefore the absorption of not totally enclosed silicon hydroxyl in group in the selagine molecule and the stationary phase cause serious, the problems such as sensitivity is low, poor repeatability of peak hangover.Utilization is added organic amine, ion pair reagent and is adopted methods such as gradient elution, raising column temperature to address the above problem to a certain extent in moving phase, but has increased the complicacy of analyzing, and also makes analytical procedure be subjected to the restriction of instrument condition simultaneously.Present in addition HPLC (high performance liquid chromatography) seldom relates to the quantitative analysis of huperzine B.
Mainly be traditional solvent extraction crystallization process to separating the method for preparing selagine at present, perhaps combination is the normal phase column chromatography and the normal phase high performance liquid chromatography of filler with silica gel simultaneously, these methods need consume a large amount of as poisonous and harmful solvents such as chloroform, ether, acetone, and loaded down with trivial details time-consuming, yield is low, all ten thousand/below.Simultaneously, huperzine B is used as impurity and removes in the production, makes huperzine B can not get effective recycling, causes the waste of resource.
Summary of the invention
The purpose of this invention is to provide a kind of easy and analyze the method for selagine and huperzine B content accurately, and overcome the existing mass consumption poisonous and harmful organic solvent that exists in the preparation selagine technology that separates, process cycle is long, yield is low, effective constituent such as huperzine B defective such as be abandoned, thereby provide a kind of chromatography integrated technology that utilizes, use the organic solvent of low toxicity, the method of high efficiency separation purification selagine and huperzine B is to obtain the selagine and the pure product of huperzine B of high purity and high-recovery.
The objective of the invention is to realize by following technical scheme:
The method for preparing selagine and huperzine B provided by the invention is characterized in that comprising and adopts non-alkyl linked phase silica gel medium to carry out the step of column chromatography for separation selagine and huperzine B as chromatographic column filler.
Described non-alkyl linked phase silica gel medium refers to silica gel to be the medium of the non-alkyl group of surface bond of matrix; Described non-alkyl group comprises phenyl, pentafluorophenyl group, itrile group and phenolic group.
The method for preparing selagine and huperzine B provided by the invention, it is characterized in that: with non-alkyl linked phase silica gel medium dress post, the charging after immersion extraction, enrichment concentrate, purify of Herba Lycopodii serrati raw material, with certain density aqueous solutions of organic solvent wash-out, collect the elutriant of selagine and huperzine B place elution peak respectively, obtain selagine and huperzine B finished product after two kinds of elutriant crystallizations respectively, the drying.The aqueous solution of described organic solvent is the aqueous solution of methyl alcohol, ethanol or the acetone of concentration 20～100% (v/v).
The method of extracting selagine and huperzine B of soaking from raw material provided by the invention is: after the raw material Herba Lycopodii serrati is pulverized, extract with the aqueous solution soaking of 0.1%～2% (v/v) sulfuric acid or nitric acid, obtain the Herba Lycopodii serrati leach liquor.
The spissated method of the enrichment of selagine provided by the invention and huperzine B is: is that absorb-elute is carried out by macroporous adsorptive resins in 8～14 backs with the Herba Lycopodii serrati leach liquor with ammoniacal liquor, sodium hydroxide or potassium hydroxide adjust pH, and the elutriant concentrating under reduced pressure obtains separated product just.
The method of purification of selagine provided by the invention and huperzine B is: the method that adopts reversed phase chromatography, to be the inverted medium of matrix with silica gel or be the inverted medium dress post of matrix with the polymkeric substance, after first separated product charging, with the aqueous solution wash-out of 5%～80% (v/v) methyl alcohol, ethanol or acetone, collect the elutriant of selagine and huperzine B place elution peak.The inverted medium that wherein with silica gel is matrix is commercially available C8 and C18 inverted medium, is that the inverted medium of matrix comprises that with polystyrene etc. be the inverted medium of matrix with the polymkeric substance.
The method of analysis selagine provided by the invention and huperzine B is characterized in that comprising that adopting non-alkyl linked phase silica gel medium is the step that chromatographic column filler carries out stratographic analysis.
The method of analysis selagine provided by the invention and huperzine B is characterized in that it is the non-alkyl group of surface bond of matrix such as the medium of phenyl, pentafluorophenyl group, itrile group and phenolic group that described non-alkyl linked phase silica gel refers to silica gel.
The method of analysis selagine provided by the invention and huperzine B, it is characterized in that sample ligand makes the analytic sample solution that is complementary with separation system, use high performance liquid chromatography (HPLC method) to measure the concentration of selagine and huperzine B then, be converted into the content of selagine and huperzine B in the sample.
The method of analysis selagine provided by the invention and huperzine B, chromatographiccondition are 20～35 ℃ of column temperatures, and moving phase is the volume ratio=50: 50～90: 10 of first alcohol and water, and flow velocity is 0.5～2.0ml/min.After sample to be tested is separated, adopt the liquid chromatography UV-detector to measure the peak area of selagine and huperzine B, calculate the concentration of selagine and huperzine B, be converted into the content of selagine and huperzine B in the sample with the typical curve correction equation.
Compared with the prior art the present invention has following advantage:
1.CN1430059A disclose Xi Pulin and selagine Plasma Concentration analytical procedure, huperzine B joins in the sample as internal standard substance in this method, analyze with the LC/MS/MS system, selagine and huperzine B resolution are low as can be seen from patent accompanying drawing 3, peak spreading is serious, and the LC/MS/MS system of application of expensive is difficult for implementing in most of assay laboratories; The present invention analyzes selagine and has utilized the high performance liquid chromatography of non-alkyl linked silica gel mutually for chromatographic column filler with the method for huperzine B content, have simple quick, characteristics such as presenting property is good, resolving power is high, sensitivity height, can get rid of of the interference of other materials to selagine and huperzine B, linearity range is wide, and the minimum detectable activity of selagine and huperzine B is respectively 0.5 and 0.6ng.More than 95%, RSD is less than 1.00% to the rate of recovery of selagine in the sample and huperzine B in the present invention.
2. the separation preparation of existing selagine such as US5177082, CN1306960A, CN1448390A all adopt the enrichment from leach liquor of traditional alkaloid extracting method to concentrate selagine, the organic solvent consumption is big, process is loaded down with trivial details time-consuming, liquid-liquid extraction method is owing to easily produce emulsion simultaneously, the loss of product is very serious, thereby causes yield to descend; The present invention utilizes selagine and the huperzine B in the absorption with macroporous adsorbent resin leach liquor, and most of impurity is removed in absorption and water washing process owing to be not adsorbed, thereby a step reaches the purpose that enrichment concentrates selagine and huperzine B, effect is similar to solvent extraction, but weak point consuming time, solvent for use toxicity is little, consumption is few and product yield is high.
3.CN1448390A will obtain medicinal extract after the extraction liquid drying, admix silica gel and wet method dress post, carry out gradient elution with chloroform-methanol, the detection thin layer chromatography of process, this technology and CN1035503A, US5177082 chromatography resemble process, be the normal-phase chromatography method, have shortcomings such as the detection method level of automation is low, elutriant toxicity is big; This process using mesolow reversed phase chromatography, can detect with on-line ultraviolet, first separated product through macroporous adsorbent resin can be removed most impurity through this step chromatography, obtains the mixture of selagine and huperzine B, the aqueous solution of the solvent that toxicity such as elutriant methyl alcohol, ethanol are less.
4. because the structural similitude of huperzine B and selagine, adopt conventional method difficult with both separation, the present invention adopts preparative chromatography, the mixture of selagine and huperzine B is separated completely on non-alkyl linked phase silica gel medium and is reclaimed the product that makes separately, facility investment is low, and treatment capacity is big.
5. all renewable back of used chromatography media of the present invention such as macroporous adsorbent resin, reversed phase chromatography medium and non-alkyl linked phase silica gel medium is reused; Used organic solvent all can evaporate recovery, thereby reduces production costs.
6. existing production technique is not mentioned the selagine and the huperzine B rate of recovery, and represents with yield, and the raw material Herba Lycopodii serrati is different owing to the source, and the content difference of its selagine and huperzine B is bigger, therefore can't embody the suitability of technology with yield; The present invention is owing to the selagine and the huperzine B that have adopted in a kind of simple analysis of efficient liquid-phase chromatography method fast raw material, intermediate product and the product, thereby can the purity and the rate of recovery of middle product and end product be detected, the present invention simultaneously utilizes the chromatography integrated technology, can differ from content and extract selagine and huperzine B the very large different Herba Lycopodii serrati raw material, technology has higher suitability, the rate of recovery of selagine and huperzine B can reach more than 85% and 80% respectively, and purity is more than 98%.
7. technology of the present invention is simple, the efficient height, (less than 7 days) with short production cycle, cost is low and environmental pollution is little.
Description of drawings
The Herba Lycopodii serrati leach liquor of Fig. 1 for adopting method of the present invention to obtain, the HPLC collection of illustrative plates that utilizes analytical procedure provided by the invention to detect;
The first separated product of Fig. 2 for adopting method of the present invention to obtain, the HPLC collection of illustrative plates that utilizes analytical procedure provided by the invention to detect;
Fig. 3 with selagine and the huperzine B mixture that obtains behind the first separated product purifying, utilizes the HPLC collection of illustrative plates of analytical procedure detection provided by the invention for adopting method of the present invention;
Fig. 4 carries out the selagine solution that column chromatography obtains as chromatographic column filler, the HPLC collection of illustrative plates that utilizes analytical procedure provided by the invention to detect for adopting non-alkyl linked phase silica gel medium;
Fig. 5 carries out the huperzine B solution that column chromatography obtains as chromatographic column filler, the HPLC collection of illustrative plates that utilizes analytical procedure provided by the invention to detect for adopting non-alkyl linked phase silica gel medium.
Embodiment
The present invention is further described with the following Examples, but does not limit the present invention.
The technical process that the present invention separates preparation selagine and huperzine B is: the enrichment of raw material pulverizing immersion → macroporous adsorbent resin concentrates → reversed phase column chromatography → non-alkyl linked medium of silica gel mutually column chromatography → condensing crystal → finished product.Wherein the product that obtains of each step all detects the content of selagine and huperzine B with analytical procedure provided by the invention.
Embodiment 1, to contain selagine 0.053%, the Herba Lycopodii serrati of huperzine B 0.013% is a raw material, separate the to purify selagine of preparation 99.1% and 98.4% huperzine B, process adopts the content of high-efficient liquid phase chromatogram technique analysis selagine and huperzine B.
The HPLC analysis condition of sample is: Waters Alliance 2695 high performance liquid chromatographs, 2996 ultraviolet diode-array detectors, chromatographic column filler are phenyl bonding phase silica gel Inertsil Ph-3, mobile phase methanol: water=90: 10, sample size is 20 μ l, flow velocity 1ml/min.
1) 1 kilogram contains selagine 0.053%, after the dry Herba Lycopodii serrati of huperzine B 0.013% is pulverized, with 10 liter of 0.1% nitric acid dousing 24 hours; Incline supernatant liquid after, added 5 liter of 0.1% nitric acid dousing again 12 hours to resistates; Filter, for several times with same acid solution washing filter residue; Merging filtrate and washings obtain about 20 liters of leach liquor.The HPLC analytical results shows and contains 520mg selagine and 129mg huperzine B in the leach liquor that leaching yield is respectively 98.1% and 99.2%;
2) leach liquor is 8 with the sodium hydroxide adjust pH, filters, and filtrate is adsorbed by the X-5 resin that Chemical Plant of Nankai Univ. produces; After finishing, absorption uses distilled water washing resin, 1% hydrochloric acid wash-out, and elutriant that concentrating under reduced pressure is received obtains separated product 17.5g just.Just separated product is accurate claims surely, with sample introduction behind 80% dissolve with methanol, measures wherein that selagine content is 2.92%, and the rate of recovery is 98.3%; Huperzine B content is 0.72%, and the rate of recovery is 97.7%.
3) with C18 inverted medium dress post, with 20% aqueous ethanolic solution balance; With step 2) first separated product 20% aqueous ethanolic solution that obtains fully dissolves the back charging; With 25～40% aqueous ethanolic solution gradient elutions; On-line ultraviolet detects, and collects elution peak HPLC and detects; The elutriant at concentrating under reduced pressure selagine and huperzine B place also reclaims solvent, obtains selagine and huperzine B mixture 716mg, measures wherein through HPLC that selagine content is 68.2%, and the rate of recovery is 95.5%; Huperzine B content is 16.6%, the rate of recovery 94.3%.
4) with pentafluorophenyl group bonded silica gel medium dress post, methanol aqueous solution balance with 30%; The mixture that step 3) is obtained with 30% methanol aqueous solution fully dissolves the back charging, 30% methanol-eluted fractions, and on-line ultraviolet detects, and collects elution peak and obtains selagine solution; 70% methanol-eluted fractions is collected elution peak and is obtained huperzine B solution.Contain the 480mg selagine, the rate of recovery 98.2% in the HPLC analysis revealed selagine solution; Contain the 114mg huperzine B in the huperzine B solution, the rate of recovery 95.6%.
5) respectively the selagine solution that obtains of concentrating under reduced pressure step 4) and huperzine B solution to there being crystallization to separate out, lyophilize, 475mg purity is that 99.1% selagine and 110mg purity are 98.4% huperzine B, the rate of recovery is respectively 98.1% and 94.9%.
This technology total yield is respectively selagine 88.8%, huperzine B 83.3%.
The HPLC analytical procedure of sample is: Beckman FL750 high performance liquid chromatograph, ultraviolet multiwavelength detector, chromatographic column filler are pentafluorophenyl group bonding phase silica gel Curosil-PFP, and mobile phase methanol: water=70: 30, sample size are 10 μ l, flow velocity 2ml/min.
1) will contain selagine 0.016%, after the dry Herba Lycopodii serrati of huperzine B 0.0033% is pulverized, soak 24 hours with 1% sulfuric acid; Incline supernatant liquid after, add 1% sulfuric acid again to resistates and soaked 12 hours; Filter, for several times with same acid solution washing filter residue; Merging filtrate and washings obtain leach liquor.
2) leach liquor is 10 with the sodium hydroxide adjust pH, filters, and filtrate is adsorbed by the XAD-4 resin that Romn Hass produces; After finishing, absorption uses distilled water washing resin, 50% acetone wash-out, and elutriant that concentrating under reduced pressure is received obtains separated product just.
3) with C8 inverted medium dress post, with 10% aqueous ethanolic solution balance; With step 2) first separated product 10% aqueous ethanolic solution that obtains fully dissolves the back charging; With 50% aqueous ethanolic solution wash-out; On-line ultraviolet detects, and collects elution peak HPLC and detects; The elutriant at concentrating under reduced pressure selagine and huperzine B place also reclaims solvent, obtains selagine and huperzine B mixture.
4) with itrile group bonded silica gel medium dress post, methanol aqueous solution balance with 40%; The mixture that step 3) is obtained with 40% methanol aqueous solution fully dissolves the back charging, 40% methanol-eluted fractions, and on-line ultraviolet detects, and collects elution peak and obtains selagine solution; 100% methanol-eluted fractions is collected elution peak and is obtained huperzine B solution.
5) respectively the selagine solution that obtains of concentrating under reduced pressure step 4) and huperzine B solution to there being crystallization to separate out, lyophilize, purity is that 98.5% selagine and purity are 98.2% huperzine B.
This technology total yield is respectively selagine 86.9%, huperzine B 82.2%.
Embodiment 3, to contain selagine 0.0035%, the Herba Lycopodii serrati of huperzine B 0.0014% is a raw material, separate the purify selagine of preparation 98.5% and 98.1% huperzine B, process adopts the content of high-efficient liquid phase chromatogram technique analysis selagine and huperzine B.
The HPLC analytical procedure of sample is: Agilent 1100 high performance liquid chromatographs, ultraviolet multiwavelength detector, chromatographic column filler are itrile group bonding phase silica gel Lichrospher 100 CN, mobile phase methanol: water=60: 40, sample size is 20 μ l, flow velocity 0.5ml/min.
1) will contain selagine 0.053%, after the dry Herba Lycopodii serrati of huperzine B 0.013% is pulverized, soak 36 hours with 2% sulfuric acid; Incline supernatant liquid after, add 2% sulfuric acid again to resistates and soaked 12 hours; Filter, for several times with same acid solution washing filter residue; Merging filtrate and washings obtain leach liquor.
2) leach liquor is 14 with the sodium hydroxide adjust pH, filters, and the LSA-21 resin that filtrate is changed the production of sorbing material company limited by the blue deep friendship in Xi'an adsorbs; After finishing, absorption uses distilled water washing resin, 95% ethanol elution, and elutriant that concentrating under reduced pressure is received obtains separated product just.
3) with Polyspher PST 10 (MERCK, styrene diethylene benzene copoly mer) inverted medium dress post, with 30% methanol aqueous solution balance; With step 2) first separated product 30% aqueous ethanolic solution that obtains fully dissolves the back charging; With 80% methanol aqueous solution wash-out; On-line ultraviolet detects, and collects elution peak HPLC and detects; The elutriant at concentrating under reduced pressure selagine and huperzine B place also reclaims solvent, obtains selagine and huperzine B mixture.
4) with phenyl bonded silica gel medium dress post, aqueous ethanolic solution balance with 60%; The mixture that step 3) is obtained with 60% aqueous ethanolic solution fully dissolves the back charging, 60% ethanol elution, and on-line ultraviolet detects, and collects elution peak and obtains selagine solution; 80% ethanol elution is collected elution peak and is obtained huperzine B solution.
5) respectively the selagine solution that obtains of concentrating under reduced pressure step 4) and huperzine B solution to there being crystallization to separate out, lyophilize, purity is that 98.5% selagine and purity are 98.1% huperzine B.
This technology total yield is respectively selagine 85.2%, huperzine B 80.1%.
The organic solvent of using in the present embodiment recycles after can reclaiming once more.
Claims (12)
1. a method for preparing selagine and huperzine B is characterized in that comprising and adopts non-alkyl linked phase silica gel medium to carry out the step of column chromatography for separation selagine and huperzine B as chromatographic column filler.
2. the described method of claim 1, it is characterized in that: non-alkyl linked phase silica gel refers to silica gel to be the non-alkyl group of surface bond of matrix such as the medium of phenyl, pentafluorophenyl group, itrile group and phenolic group.
3. claim 1 or 2 described methods, it is characterized in that: with non-alkyl linked phase silica gel medium dress post, the charging after immersion extraction, enrichment concentrate, purify of Herba Lycopodii serrati raw material, with certain density aqueous solutions of organic solvent wash-out, collect the elutriant of selagine and huperzine B place elution peak respectively, obtain selagine and huperzine B finished product after two kinds of elutriant crystallizations respectively, the drying.
4. the described method of claim 3, it is characterized in that: the aqueous solution of described organic solvent is the aqueous solution of methyl alcohol, ethanol or the acetone of concentration 20～100% (v/v).
5. the described method of claim 3, it is characterized in that soaking from raw material the method for extracting selagine and huperzine B is: after the raw material Herba Lycopodii serrati is pulverized, with concentration is the aqueous solution soaking extraction of 0.1%～2% (v/v) sulfuric acid or nitric acid, obtains the Herba Lycopodii serrati leach liquor.
6. claim 3 or 5 described methods, the spissated method of enrichment that it is characterized in that selagine and huperzine B is: is that absorb-elute is carried out by macroporous adsorptive resins in 8～14 backs with the Herba Lycopodii serrati leach liquor with ammoniacal liquor, sodium hydroxide or potassium hydroxide adjust pH, and the elutriant concentrating under reduced pressure obtains separated product just.
7. claim 3 or 6 method, the method of purification that it is characterized in that selagine and huperzine B is: the method that adopts reversed phase chromatography, to be the inverted medium of matrix with silica gel or be the inverted medium dress post of matrix with the polymkeric substance, after first separated product charging, with the aqueous solution wash-out of 5%～80% (v/v) methyl alcohol, ethanol or acetone, collect the elutriant of selagine and huperzine B place elution peak.
8. claim 3 or 7 described methods is characterized in that: described is that the inverted medium of matrix is commercially available C8 and C18 inverted medium with silica gel, is that the inverted medium of matrix comprises that with polystyrene etc. be the inverted medium of matrix with the polymkeric substance.
9. a method of analyzing selagine and huperzine B is characterized in that comprising that adopting non-alkyl linked phase silica gel medium is the step that chromatographic column filler carries out stratographic analysis.
10. by the described method of claim 9, it is characterized in that: non-alkyl linked phase silica gel refers to silica gel to be the non-alkyl group of surface bond of matrix such as the medium of phenyl, pentafluorophenyl group, itrile group and phenolic group.
11. claim 9 or 10 described methods, it is characterized in that sample ligand makes the analytic sample solution that is complementary with separation system, use high performance liquid chromatography (HPLC method) to measure the concentration of selagine and huperzine B then, be converted into the content of selagine and huperzine B in the sample.
12. by claim 9,10 or 11 described methods, it is characterized in that: chromatographiccondition is 20～35 ℃ of column temperatures, moving phase is volume ratio=50: 50～90: 10 (v/v) of first alcohol and water, flow velocity is 0.5～2.0ml/min, after sample to be tested is separated, adopt the high performance liquid chromatography UV-detector to measure the peak area of selagine and huperzine B, calculate the concentration of selagine and huperzine B with the typical curve correction equation, be converted into the content of selagine and huperzine B in the sample.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100383122C (en) * | 2006-06-15 | 2008-04-23 | 河南太龙药业股份有限公司 | Process of extracting lycopdine A from plant |
| CN100445268C (en) * | 2006-12-01 | 2008-12-24 | 河南太龙药业股份有限公司豫中制药厂 | Process of extracting huperzine B from plant medicine material huperzine serrate |
| CN102070527A (en) * | 2011-01-25 | 2011-05-25 | 赵勇彪 | Method for extracting high-purity huperzine A and huperzine B from medicinal plant phlegmariurus crutomerianus |
| CN102432535A (en) * | 2011-12-29 | 2012-05-02 | 重庆市秀山红星中药材开发有限公司 | Method for extracting and separating huperzine A and huperzine B from huperzia serrata |
| CN112457251A (en) * | 2020-11-30 | 2021-03-09 | 湖南华诚生物资源股份有限公司 | Method for separating various high-content alkaloid monomers from huperzia serrata extract |
| CN113354642A (en) * | 2021-07-13 | 2021-09-07 | 上海天慈生物谷生物工程有限公司 | Huperzine B crystal and preparation and application thereof |
-
2004
- 2004-05-26 CN CN 200410042760 patent/CN1704405A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100383122C (en) * | 2006-06-15 | 2008-04-23 | 河南太龙药业股份有限公司 | Process of extracting lycopdine A from plant |
| CN100445268C (en) * | 2006-12-01 | 2008-12-24 | 河南太龙药业股份有限公司豫中制药厂 | Process of extracting huperzine B from plant medicine material huperzine serrate |
| CN102070527A (en) * | 2011-01-25 | 2011-05-25 | 赵勇彪 | Method for extracting high-purity huperzine A and huperzine B from medicinal plant phlegmariurus crutomerianus |
| CN102432535A (en) * | 2011-12-29 | 2012-05-02 | 重庆市秀山红星中药材开发有限公司 | Method for extracting and separating huperzine A and huperzine B from huperzia serrata |
| CN112457251A (en) * | 2020-11-30 | 2021-03-09 | 湖南华诚生物资源股份有限公司 | Method for separating various high-content alkaloid monomers from huperzia serrata extract |
| CN113354642A (en) * | 2021-07-13 | 2021-09-07 | 上海天慈生物谷生物工程有限公司 | Huperzine B crystal and preparation and application thereof |
| WO2023284804A1 (en) * | 2021-07-13 | 2023-01-19 | 上海天慈生物谷生物工程有限公司 | Huperzine b crystal and preparation and application thereof |
| CN113354642B (en) * | 2021-07-13 | 2023-11-28 | 上海天慈生物谷生物工程有限公司 | Huperzine B crystal and preparation and application thereof |
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