CN104435047A - Process for extracting, separating and purifying total flavonoids of herba epimedii - Google Patents
Process for extracting, separating and purifying total flavonoids of herba epimedii Download PDFInfo
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- CN104435047A CN104435047A CN201410739922.4A CN201410739922A CN104435047A CN 104435047 A CN104435047 A CN 104435047A CN 201410739922 A CN201410739922 A CN 201410739922A CN 104435047 A CN104435047 A CN 104435047A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
- A61K36/296—Epimedium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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Abstract
The invention discloses a process for extracting, separating and purifying total flavonoids of herba epimedii. An L9 orthogonal design method is adopted, the extract yield and the content of total flavonoids are taken as detection indexes, and a preferential extraction process is adopted. The process comprises the following steps: separating by using an AB-8 type macroporous resin column, extracting and purifying by using ethyl acetate and n-butyl alcohol. According to the research, the total flavonoids of herba epimedii are extracted by the methods, macroporous resin column separation is performed, the content difference of the total flavonoids of herba epimedii is small, a method for performing water extraction, performing macroporous resin column separation and extracting by using ethyl acetate and n-butyl alcohol is adopted during the experiment in order to improve the content of the total flavonoids of herba epimedii, and the extract is refined. The result proves that the content of the total flavonoids of herba epimedii obtained by extraction is improved to 61 percent. Therefore, a macroporous resin column separation method and an extraction method are combined, so that the aim of purifying the total flavonoids of herba epimedii can be achieved.
Description
Technical field
The present invention relates to a kind of Extraction and separation purifying process of Herba Epimedii total flavones, belong to biomedicine technical field.
Background technology
Herba Epimedii is Berberidaceae plant Herba Epimedii Epimediumbrevi-cornuMaxim, Epimedium sagittatum Epimedium.sagittatum(Seb.etZucc.) Maxim, the dried leaves of E. Pubescens Epimedium pu-bescens Maxim or Herba Epimedii Epimedium koreanum Nakai, , acrid-sweet flavor, warm in nature, there is kidney-replenishing, bone and muscle strengthening, efficacy of dispelling wind, modern pharmacology research shows, Herba Epimedii is in immunity, reproduction, nucleic acid metabolism, cardiovascular and defying age aspect have multiple drug effect.
Summary of the invention
The object of the invention is: the Extraction and separation purifying process that a kind of Herba Epimedii total flavones is provided, greatly can improve the obtaining ratio and content of total flavones, to overcome the deficiencies in the prior art.
Technical scheme of the present invention
An Extraction and separation purifying process for Herba Epimedii total flavones, adopts L9 orthogonal design, with yield of extract and general flavone content for Testing index Optimized extraction techniques; Be separated with AB-8 type macroporous adsorptive resins, ethyl acetate, n-butanol extraction purification.
In the Extraction and separation purifying process of aforesaid a kind of Herba Epimedii total flavones, optimum extraction process is add 27 times of water gagings in the coarse powder of Folium Epimedii, after soaking 2h, decocts 3 times, each 1h; On extraction ofHerba Epimedii, AB-8 type macroporous adsorptive resins is separated, then uses ethyl acetate, n-butanol extraction.
Owing to adopting technique scheme, compared with prior art, research report about Herba Epimedii total flavones extracting method in prior art is many, common method has decocting cooking method, ethanol refluxing process, soxhlet extraction, ultrasonic extraction, this research extracts Herba Epimedii total flavones by these methods, be separated through macroporous adsorptive resins again, the content obtaining Herba Epimedii total flavones is more or less the same, although with 60% ethanol for the Herba Epimedii total flavones content extracted during solvent is higher, but content is all lower than 50%, for improving the content of Herba Epimedii total flavones, consider that glassware for drinking water has the features such as cheap environmental protection, water extraction is adopted during experiment, after crossing macroporous adsorptive resins separation, use ethyl acetate again, n-butyl alcohol carries out the method extracted, extract is refined, found that, after extraction, the content of gained Herba Epimedii total flavones brings up to 61%, show, macroporous adsorptive resins partition method and extraction combine and can reach the object of purification Herba Epimedii total flavones.
Accompanying drawing explanation
Accompanying drawing 1 is extraction process factor level table;
Accompanying drawing 2 is epimedium total flavone extracting process orthogonal test and result schematic diagram;
Accompanying drawing 3 is extraction process analysis of variance table;
Accompanying drawing 4 is repeatability test schematic diagram;
Accompanying drawing 5 is that different eluant affects schematic diagram to flavone amount;
Accompanying drawing 6 is the result figure after extraction;
Accompanying drawing 7 is general flavone content schematic diagram after extraction.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail, but not as any limitation of the invention.
Embodiments of the invention:
1 instrument and reagent
UV-2401 type ultraviolet-uisible spectrophotometer (Prism Optical Technology Co), Japan Shimadzu AY-120 electronic analytical balance, Tokyo N1000DNA Rotary Evaporators, HHS-21-6 electric-heated thermostatic water bath (Hangzhou Ai Pu instrument and equipment company limited).Folium Epimedii is purchased from Guiyang three bridge Chinese Medicinal Materials Markets, icariin reference substance is Chengdu Man Site biological limited scientific & technical corporation product, lot number MUST-10070901, AB-8 type macroporous adsorbent resin (Tianjin sea light Chemical Co., Ltd.), 95% ethanol, ethyl acetate (AR, Shanghai Shen Bo Chemical Co., Ltd.), n-butyl alcohol (AR, Shanghai Shen Bo Chemical Co., Ltd.), methanol (AR, Tianjin great Mao chemical reagent factory), purified water (laboratory self-control).
2 methods and result
2.1 extraction processes preferred
2.1.1 the selection of factor level
For determining the optimum extraction process of Herba Epimedii total flavones, with amount of water, soak time, decocting time and decoction number of times for influence factor, by each factor 3 level, select L9(3
4) orthogonal table arrangement test, the results are shown in accompanying drawing 1, with yield of extract and general flavone content for inspection target, choose optimised process.
2.1.2 the drafting of standard curve
Precision takes icariin reference substance 10.0mg, is placed in 100ml measuring bottle, is settled to scale, shakes up, obtain the icariin reference substance solution that concentration is 100.0mg/L with dissolve with methanol.Accurate draw reference substance solution 0.50,1.00,2.00,3.00,4.00,5.00ml is placed in 25ml measuring bottle respectively, with methanol dilution to scale, take methanol as blank, absorbance is measured at 270nm place with ultraviolet spectrophotometer, with absorbance A, icariin concentration C is returned, obtaining regression equation is: A=0.0363C+0.0707, r=0.9998, result shows that, within the scope of 2.00 ~ 20.00mg/L, linear relationship is good.
2.1.3 the preparation of sample and measurement result
By L9(3
4) orthogonal table arrangement test, take Herba Epimedii coarse powder 50g, extract by setting scheme, filter, extracting solution is concentrated into 50ml, and adding 95% ethanol to alcohol precipitation concentration is 50%, 4 DEG C of standing 12h, elimination precipitates, and decompression recycling ethanol, to without alcohol taste, extracts with appropriate ethyl acetate gradation, combined ethyl acetate layer, reclaim under reduced pressure, vacuum drying, calculates extractum weight.Measure general flavone content by method under " 2.1.2 " item, and calculate total flavones yield, the results are shown in accompanying drawing 2 and accompanying drawing 3.
From extreme difference value R size in accompanying drawing 2, RD > RA > RB > RC, each influence factor's order is D > A > B > C, therefore D is the key factor affecting Herba Epimedii total flavones content, factor A, B take second place, and factor C impact is minimum; From accompanying drawing 3 variance analysis, factor D is to the extraction tool significance (P < 0.05) of Herba Epimedii total flavones, and comprehensively analyze in conjunction with extreme difference value R size, optimum extraction process is A
3b
3c
2d
3.
2.1.4 technique repeatability is investigated
Take 3 parts of medicinal material coarse powder, every part of 50g, add the water of 27 times amount by optimum extraction process, soak 2h, reflux 3 times, each 1h, merge extractive liquid, concentrated, precipitate with ethanol, add extraction into ethyl acetate, reclaim under reduced pressure, vacuum drying, measures yield of extract and total flavones yield, this technique repeatability is good, sees accompanying drawing 4.
The research of 2.2 separating technologies
2.2.1 macroporous adsorbent resin is preferred
AB-8 type macroporous adsorbent resin is the optimum resin of Herba Epimedii total flavones in separation and purification Herba Epimedii, and therefore this technique adopts AB-8 type macroporous adsorbent resin to be separated Herba Epimedii total flavones extract.
2.2.2 the pretreatment of macroporous adsorbent resin
Adopt ethanol wet method dress post (4.8cm × 25cm), with ethanol elution, detect ethanol stream fluid, after ethanol stream fluid to mix with water (1: 3) and does not present white milkiness phenomenon, use large-scale purification water eluting repeatedly again, until no longer decline without alcohol taste, resin height, the parallel pillar (4.8cm × 25cm) filling 4 same specifications.
2.2.3 the selection of eluting solvent
Take 200g Herba Epimedii coarse powder, extract by optimised process.Extracting solution is concentrated into 400ml, is divided into 4 parts, respectively loading, after absorption 1h, the Sufficient purified water elution of all pillars, when eluent is close to time colourless, use 20% respectively again, 60%, 70%, 90% ethanol elution pillar, flow velocity is 2ml/min, by ethanol elution respectively evaporate to dryness, weigh, record general flavone content, calculate total flavones resolution factor, the results are shown in accompanying drawing 5
2.2.4 the determination of eluting solvent consumption
Most of impurity can be removed with 20% ethanol of 3 column volumes, then use 60% ethanol elution of 5 column volumes, effective ingredient almost can be eluted completely.In 60% ethanol elution thing, general flavone content is the highest as shown in Table 5, resolution factor is best, so select 60% ethanol to be eluant, select purified water and 20% ethanol to be the solvent removing water-solubility impurity simultaneously, for improving general flavone content further, extraction process is carried out again to the eluent of 60% ethanol.
2.3 abstraction purification technical studies
2.3.1 the selection of extractant
According to optimum extraction process, extract 2 batches of Herba Epimedii coarse powder, often criticize 50g, extracting solution is concentrated into 50ml, adding 95% ethanol to alcohol precipitation concentration is 50%, 4 DEG C of standing 12h, filter, get supernatant, decompression recycling ethanol is extremely without alcohol taste, go up macroporous resin column to be respectively separated, the eluent of separate collection 60% ethanol, decompression recycling ethanol, every part is made to be concentrated into about 50ml, first part is extracted with ethyl acetate, second part with n-butanol extraction, decompression and solvent recovery, vacuum drying, measure general flavone content in extractum, the results are shown in accompanying drawing 6, more known with accompanying drawing 5, after extraction, the content of Herba Epimedii total flavones is not higher than before extracting, but the Herba Epimedii total flavones in extracting solution all can not thoroughly extract by these two kinds of solvents completely, for ensureing yield of extract and general flavone content, therefore study with two kinds of solvent extractions.
2.3.2 two kinds of solvent-extracted investigations take 5 parts of Herba Epimedii coarse powder, every part of 50g.Extract according to optimum, separating technology carries out, after obtaining the eluent (5 parts) of about 50ml60% ethanol, 9 volumes extraction into ethyl acetate is doubly added successively to every part of solution, and then add 10 volumes n-butanol extraction doubly, merge after decompression and solvent recovery to thick paste respectively, vacuum drying, obtain extractum, measure content, the results are shown in accompanying drawing 7, result shows, use ethyl acetate successively, the yield of extract that n-butanol extraction obtains exceeds about 1.5 times than being only extracted with ethyl acetate, the general flavone content obtained is than only exceeding about 6.2% with n-butanol extraction.As can be seen here when ensureing total flavones obtaining ratio and content, ethyl acetate, n-butanol extraction are more satisfactory successively.
Claims (2)
1. an Extraction and separation purifying process for Herba Epimedii total flavones, is characterized in that: adopt L9 orthogonal design, with yield of extract and general flavone content for Testing index Optimized extraction techniques; Be separated with AB-8 type macroporous adsorptive resins, ethyl acetate, n-butanol extraction purification.
2. the Extraction and separation purifying process of a kind of Herba Epimedii total flavones according to claim 1, is characterized in that: optimum extraction process is add 27 times of water gagings in the coarse powder of Folium Epimedii, after soaking 2h, decocts 3 times, each 1h; On extraction ofHerba Epimedii, AB-8 type macroporous adsorptive resins is separated, then uses ethyl acetate, n-butanol extraction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106261265A (en) * | 2016-08-16 | 2017-01-04 | 中州大学 | A kind of Herba Epimedii fatigue resistant sport drink and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106261265A (en) * | 2016-08-16 | 2017-01-04 | 中州大学 | A kind of Herba Epimedii fatigue resistant sport drink and preparation method thereof |
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