CN104557893A - Extraction and purification technology of four kinds of flavone C-glycosides in moso bamboo leaves - Google Patents
Extraction and purification technology of four kinds of flavone C-glycosides in moso bamboo leaves Download PDFInfo
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- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
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Abstract
The invention discloses an extraction and purification technology of four kinds of flavone C-glycosides in moso bamboo leaves. The active ingredients of moso bamboo leaves are extracted by a heat reflux extraction process, and the best extraction technology for obtaining four kinds of flavone C-glycosides through a single-factor experiment and an orthogonal experiment comprises the following steps: extracting with 75% ethanol for 2h at 70 DEG C according to a solid-liquid ratio of 1:20, and repeatedly extracting for 4 times; purifying the crude extract of flavone by use of macroporous resin, wherein D101 macroporous resin is the best choice according to comparison of static saturated adsorption capacity and desorption capacity; and further purifying the component by use of polyamide resin, wherein the particle size of the polyamide resin is 0.15-0.25mm according to the comparison of the dynamic adsorption screening results of three common kinds of polyamide resin, and a 40% ethanol solution is adopted for purifying the four kinds of flavone C-glycosides. Through the treatment, the final purity of the four kinds of flavone C-glycosides bamboo leaf flavone powder reaches 76.939mg.g<-1>.
Description
Technical field
The present invention relates to a kind of flavone c-glycoside Isolation and purification technique, be specifically related to four kinds of flavone c-glycoside Isolation and purification techniques in a kind of Moso Bamboo Leaves.
Background technology
Flavonoid compound because of its structure with source different, dissolution characteristics also has very big-difference, therefore suitable solvent can be selected to extract according to polarity.At present, extracting method mainly contains: water extraction, alkaline water or rare alcohol extracting are followed the example of, organic solvent water bath reflux method, using microwave-assisted, ultrasonic extraction, supercritical extraction etc.
Water extraction, extract for flavonoid glycoside substance, cost is low, is applicable to industrial production, but containing the impurity that protein, polysaccharide etc. are water-soluble in extracting solution, extraction efficiency is low, easily goes mouldy, and later separation bothers.Alkaline water or rare alcohol extracting are followed the example of, utilize phenolic hydroxy group in flavonoid compound to be dissolved in feature that buck meets Precipitation again after acid, for the extraction of flavonoid compound.In leaching process, the acid base concentration added wants appropriate, and alkali concn is too high can destroy flavones mother nucleus structure, and acid concentration is too high can generate with the hydroxyl of flavones salt of pretending, and again dissolves, reduction yield after causing throw out to be separated out.Organic solvent water bath reflux method, be current domestic and international most popular extracting method, conventional organic solvent has ethyl acetate, acetone, ethanol, methyl alcohol etc.; Some strong polarity phenolic acid (phenylformic acid, styracin etc.) can not use pure organic solvent extraction completely, adopts alcohol-water proportioning mixed solution extraction effect better; The solvent that some polarity such as methylene dichloride, chloroform, normal hexane is less is applicable to the extraction of non-polar compound, as wax, sterol, chlorophyll etc.Certain density ethanol or methanol aqueous solution are most commonly used to the extraction of flavonoid compound, and the alcoholic solution (more than 90%) of high density is suitable for extracting aglycon, and the alcoholic solution of lower concentration is then suitable for extracting glycoside.Using microwave-assisted, the difference utilizing different materials to absorb microwave ability in microwave field, in thing to be extracted, first some component is heated by selectivity, enter after being separated with system that specific inductivity is little, the extraction agent of microwave absorption capacity relative mistake, there is highly selective, convenient post-treatment, the environmentally safe feature such as harmless.Ultrasonic extraction utilizes ultrasonic cavitation effect, accelerates the process that effective ingredients in plant leaches, have the features such as efficient, quick, simple to operate.Supercritical fluid extraction mainly utilizes the characteristic of supercutical fluid to be separated, the existing feature large to solute solubility of supercutical fluid, has again gas to be easy to the feature of disperseing and moving; When subjected to the critical condition, the minor alteration of temperature and pressure, causes the noticeable change of supercritical fluid densities, thus makes its dissolving power also respective change occur, and generally often selects CO
2as supercutical fluid, the effective constituent in extraction plant.Supercritical fluid extraction extraction efficiency is high, and active ingredient and thermally labile component are not easily decomposed, and energy consumption is low, does not produce any " three wastes " material newly, favourable to environment protection.Researchist utilizes supercritical CO
2abstraction technique is extracting flavone class and aldehydes matter from vine tea stem, and is optimized extraction process; Also investigated vine tea stem extract to DPPH radical scavenging activity and and the sequestering action of ferrous ion, result shows, extract shows extremely strong Scavenging activity to DPPH free radical, but it is very weak with the sequestering action of ferrous ion, in addition, some flavonoid compounds utilizing HPLC technology to detect from extract not report in document, comprise apigenin, Vitexina, luteolin etc.
In actual production process, conventional main equipment carries out circumfluence distillation to the leaf of bamboo, though extraction efficiency is high not as good as the effect of ultrasonic extraction, microwave auxiliary extraction and supercritical fluid extraction, circumfluence distillation production cost is low, easy handling, is applicable to mass industrialized production.
The purification process related in prior art mainly contains macroreticular resin absorbing method, column chromatography, Paper Chromatography, membrane sepn, recrystallization etc.Macroporous resin is the high molecular polymer sorbent material that a class has macroporous structure, its principle is the absorption to flavonoid compound by Van der Waals force or hydrogen bond action realization, in addition, because residual cellular structure after the removing of inertia pore-creating agent also plays the effect of molecular sieve.Macroporous resin has that adsorption selectivity is good, desorption condition is gentle, life cycle is long, regenerate the advantages such as easy, and extraction yield is higher, cost is low, is adapted at applying in preparative separation technique, is widely used in the separation and purification of material.Kang Jiasheng etc. determine the adsorption and desorption process of 7 kinds of macroporous resins to Folium Bambusae total flavones, and wherein AB-8 macroporous resin shows good Adsorption and desorption characteristics.Polyamide resin produces hydrogen bond action by the phenolic hydroxyl group in molecule and the amide group in resin and realizes adsorption process, its adsorption strength depend primarily on hydroxy number in flavonoid compound molecule, position and eluent and flavonoid compound or and polymeric amide between form the size of hydrogen bond association ability.Polyamide resin has good inrichment to flavonoid compound, is suitable for a large amount of preparative separation techniques of flavonoid compound.Liu can utilize polyamide resin to carry out purifying to flavones ingredient in Herba Lophatheri, obtains optimised process, and collect 70% ethanol eluate and obtain Herba Lophatheri total flavones, its content reaches 36%.Silica gel column chromatography; mainly for separating of the flavonoid compound of low-pole; as isoflavones, flavanone (alcohol) and high methylation or acetylizad flavones and flavonol; in purge process, frequent mixed solvent carries out gradient elution, and chloroform-methanol, sherwood oil-acetone etc. are all conventional solvent elution combinations.Membrane separation technique is that when applying pressure in the external world, when extracting solution is by filter membrane, the isolation technique that polymer is trapped, effectively can remove macromolecular substance, as protein, polysaccharide, macromole pigment etc.In sepn process, without phase change, and improve liquid clarity, active constituent content is also greatly improved, and can realize selective separation.Tang Haoguo etc. are by comparative study to 5 kinds of ultra-filtration membranes, and select molecular weight cut off to be that the polysulfone membrane PSF-500 of 50000D carries out purification effect to sinocalamus latiflorus Folium Bambosae flavone better, product yield reaches 4.29%.High red peaceful grade is also contrasted by the separating effect of ceramic membrane and alcohol precipitation, result show micro-filtration in conjunction with macroporous resin purification technology to the adsorption rate of total flavones process in kuh-seng Aqueous extracts and impurity-eliminating effect better.Paper chromatography and recrystallizing technology also have in the literature and relate to, but the shortcoming all existed in various degree and limit its suitability for industrialized production.
Now, also have a lot of patent report to cross the purification process of Folium Bambosae flavone, Guo Xuefeng etc. obtain the total flavones of at least 50%, patent adopts extraction using alcohol, sherwood oil or the process of ether degrease, and macroporous resin column is separated, ultra-filtration membrane and nanofiltration membrane retain macromolecular substance, finally obtain Folium Bambosae flavone crude extract.Wang Chengzhang etc. adopt HPLC method from 30 kinds of leaf of bamboves, to screen the high bamboo kind of flavones content as raw materials for production, C
1~ C
3ethanol-extracted, concentrated, ceramic membrane, ultra-filtration membrane and nanofiltration membrane, then 40% ~ 65% bamboo leaf flavone must be reached by flavones content after macroporous resin purification.
Clear and definite along with Folium Bambosae flavone composition, its using value also obtains the concern of scholars, is mainly used in food, medicines and health protection, the industries such as makeup.By the search to national inventing patent, existing many patents are extracted flavones effective constituent and are utilized.Appropriate Folium Bambosae flavone adds in beer by Zhang Ying, produces bamboo beer beverage, not only remains nutritive value and the taste of beer, also has and promotes health microcirculation, reduces the multiple nourishing functions such as blood fat, simultaneously with typical bamboo delicate fragrance.Also useful Folium Bambosae flavone develops medicine or the healthcare products of thrombotic diseases prevention or treatment.In addition, Hu Linfu and Chen Yong is good for binding film treatment process, and Folium Bambosae flavone is used for fodder additives, healthy food material, foodstuff additive, the collection of medicine intermediate and cosmetic material, these products have that extraction yield is high, technical process is simple, product purity high.Liu Zhihe and Li Wei attempts Folium Bambosae flavone class material to add in oral cavity cleaning and nursing product, not only can remove peculiar smell, also have anti-inflammatory antibacterial, prevent and treat effect of oral disease, have certain repair in addition to the ulcer surface in oral cavity.Visible Folium Bambosae flavone has a very important role to human health.
The leaf of bamboo has long edible and medicinal history in China, also increasing year by year in recent years, but the research in Folium Bambosae extract still has weak point, need to further investigate further to the research of natural resources of bamboo leaf chemical utilization.
At present, the optimization of the extraction and purification process carried out for object with the leaf of bamboo four kinds of flavone c-glycosides (Orientin, Lutonaretin, Vitexina and Saponaretin) is also fewer, and purity is relatively low, does not also realize suitability for industrialized production.
Summary of the invention
For current Problems existing, the invention provides four kinds of flavone c-glycoside Isolation and purification techniques in a kind of Moso Bamboo Leaves, take mao bamboon as research object, with the yield of four kinds of flavone c-glycosides for index, be optimized Moso Bamboo Leaves four kinds of flavone c-glycoside Isolation and purification techniques, make with optimal conditions, extraction yield and the purity of four kinds of flavone c-glycosides are all the highest, the raw material of medicine intermediate or functional health-care food exploitation can be used as, for the suitability for industrialized production realizing flavone c-glycoside lays the first stone.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
Four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves, four kinds of described flavone c-glycosides are Orientin, Lutonaretin, Vitexina and Saponaretin, comprise the steps:
(1) accurately take Moso Bamboo Leaves powder, add in aqueous ethanolic solution and carry out circumfluence distillation, repeat extraction 4 times, filter and filtrate is merged to obtain extracting solution; The technique of described circumfluence distillation is: the ethanol of 70% ~ 80%, and solid-liquid ratio is that 1 ~ 3h is extracted in 1: 15 ~ 1: 25,60 ~ 80 DEG C of waters bath with thermostatic control;
(2) adopt the organic solvent in distillation under vacuum removing extracting solution, add isopyknic petroleum ether extraction with extracting solution, removing pigment and fat-soluble component, re-extract three times, obtains Moso Bamboo Leaves flavone gruff bring up substance, and sherwood oil reclaims, and recycles;
(3) again carry out purification process through polyamide resin: Moso Bamboo Leaves flavone gruff bring up substance is carried out purification process through macroporous resin: macroporous resin carries out pre-treatment, by macroporous resin column on Moso Bamboo Leaves flavone gruff bring up substance, wet method dress post, adds 10mgmL
-1folium Bambosae flavone crude extract, coutroi velocity 3BVh
-1, macroporous resin is 5: 1 with sample applied sample amount ratio, collects sample effluent liquid, after effluent liquid repeats upper prop 3 times, with 15% ethanolic soln with 4BVh
-1the preliminary wash-out removal of impurities of flow velocity, concentrate eluant, then uses the ethanolic soln of 60% with 4BVh
-1flow velocity wash-out, finally collects 60% ethanol eluate, and concentrated and lyophilize obtains Moso Bamboo Leaves flavones dry powder after purifying;
(4) by the Folium Bambosae flavone powder after above-mentioned macroporous resin purification, after dissolving, by polyamide resin wet method dress post in 2cm × 35cm chromatography column, dress post height is 12.5cm, adds 10mgmL
-1the solution of above-mentioned Folium Bambosae flavone dry composition, control 3BVh
-1flow velocity crosses post, collects effluent liquid, repeats upper prop 3 times, then uses distilled water wash-out, collects elutriant, concentrated, then uses equal-volume 40% ethanolic soln wash-out, finally collects 40% ethanol eluate, and concentrated and lyophilize obtains the Folium Bambosae flavone dry powder after purifying.
In above-mentioned Moso Bamboo Leaves in four kinds of flavone c-glycoside Isolation and purification techniques, through experiment sieving, the optimal process of described circumfluence distillation is: at 75% ethanol, solid-liquid ratio 1: 20,70 DEG C, 2h is extracted in water bath with thermostatic control.
In above-mentioned Moso Bamboo Leaves in four kinds of flavone c-glycoside Isolation and purification techniques, described macroporous resin is ADS-17, DM130 or D101, is preferably D101.
In above-mentioned Moso Bamboo Leaves in four kinds of flavone c-glycoside Isolation and purification techniques, the particle diameter of described polyamide resin is 0.15 ~ 0.25mm.
In above-mentioned Moso Bamboo Leaves in four kinds of flavone c-glycoside Isolation and purification techniques, before carrying out purification process through macroporous resin or polyamide resin, pre-treatment is carried out to macroporous resin or polyamide resin, specific as follows:
Tiny resin and impurity is washed away with dehydrated alcohol, then 12h is soaked, make macroporous resin or polyamide resin fully swelling, wet method dress post, elutant is washed till without till white opacity with dehydrated alcohol, be zero with distilled water flushing to alcohol concn again, then proceed to acid-alkali treatment, first use the 5%NaOH solution 3BVh of 3BV
-1flow velocity by resin layer, then distilled water with identical speed by being washed till pH in neutral; Use 3BV5%HCl solution with 3BVh again
-1flow velocity is by resin layer, and distilled water continues to be eluted to pH in neutral with same flow velocity, and last macroporous resin or polyamide resin are placed in dehydrated alcohol and preserve stand-by, and using distilled water to be washed till alcohol concn is before use zero, gets final product loading.
For design parameter of the present invention, applicant has carried out a series of research to it, is optimized, finally obtains optimum implementation by scientific and reasonable experiment to significant parameter of the present invention, specific as follows:
One, the research of four kinds of flavone c-glycoside extraction processes in Moso Bamboo Leaves
(1) single factor experiment
Quantitative Moso Bamboo Leaves is shattered process, adds certain density ethanolic soln, carry out circumfluence distillation, investigate alcohol concn, solid-liquid ratio, Extracting temperature, extraction time respectively to the impact of four kinds of flavone c-glycoside extracted amounts;
1, alcohol concn is on the impact of four kinds of flavone c-glycoside extracted amounts
Accurately take Moso Bamboo Leaves powder 5g, add the aqueous ethanolic solution of 50%, 60%, 70%, 80%, 90% respectively, solid-liquid ratio is 1: 20,2h is extracted in 70 DEG C of waters bath with thermostatic control, repeats extraction 4 times, filters and filtrate is merged, concentrate and be settled to 100mL, get 1mL constant volume in 25mL volumetric flask.Carry out detection by quantitative with HPLC, measure four kinds of flavone c-glycoside content.Result as shown in Figure 1.
As seen from Figure 1, along with the raising of alcohol concn, the extracted amount of four kinds of flavone c-glycosides increases gradually, and when alcohol concn is near 80%, extracted amount is up to 4.207mgg
-1.When alcohol concn is more than 80%, four kinds of flavone c-glycoside extracted amounts sharply decline, now because some composition such as alcohol dissolubility impurity, pigment stripping quantity increases, compete and aqueous ethanolic solution combining site with four kinds of flavone c-glycosides, cause the extracted amount of Orientin, Lutonaretin, Vitexina and Saponaretin to decline.So alcohol concn select 70% ~ 80% be optimized more reasonable.
2, solid-liquid ratio is on the impact of four kinds of flavone c-glycoside extracted amounts
Accurately take Moso Bamboo Leaves powder 5g, 80% aqueous ethanolic solution extracts 2h in 70 DEG C of waters bath with thermostatic control, and solid-liquid ratio is respectively 1: 10,1: 15,1: 20,1: 25,1: 30, repeat extraction 4 times, filter and filtrate is merged, concentrating and be settled to 100mL, get 1mL constant volume in 25mL volumetric flask.Carry out detection by quantitative with HPLC, measure four kinds of flavone c-glycoside content.Result as shown in Figure 2.Along with the increase of mao bamboon sample and aqueous ethanolic solution solid-liquid ratio, four kinds of flavone c-glycoside extracted amounts are slow rising tendency, but when solid-liquid ratio is more than 1: 20, extracted amount does not almost have noticeable change.This is because along with the increase of solid-liquid ratio, the contact area of material and solvent increases, make ethanolic soln be more prone to infiltrate Moso Bamboo Leaves cell, add the extracted amount of four kinds of flavone c-glycosides; But solid-liquid ratio is increased to a certain degree, material and solvent frictional belt concentration difference reduce, and diffusion reaches balance, and in solution, content tends towards stability.So solid-liquid ratio select 1: 15 ~ 1: 25 be optimized more reasonable.
3, Extracting temperature is on the impact of four kinds of flavone c-glycoside extracted amounts
Accurately take Moso Bamboo Leaves powder 5g, add 100mL80% aqueous ethanolic solution and extract, extract 4 times respectively at water bath with thermostatic control at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, each 2h, filter and filtrate is merged, concentrating and be settled to 100mL, get 1mL constant volume in 25mL volumetric flask.Carry out detection by quantitative with HPLC, measure four kinds of flavone c-glycoside content.Result as shown in Figure 3.Along with the rising of Extracting temperature, four kinds of flavone c-glycoside extracted amounts constantly increase, and when temperature is near 70 DEG C, extracted amount is up to 4.087mgg
-1, after 70 DEG C, extracted amount declines to some extent, this is because temperature is too high make four kinds of carbon glycosides generations destruction to a certain degree, the stripping quantity of impurity also can increase simultaneously, affects it and measures.Result shows, the Extracting temperature of certain limit is conducive to the stripping of four kinds of flavone c-glycosides in the leaf of bamboo, but exceedes certain temperature, then flavone c-glycoside structure can be caused to destroy, stripping quantity reduce, so Extracting temperature select 60 ~ 80 DEG C be optimized more reasonable.
4, extraction time is on the impact of four kinds of flavone c-glycoside extracted amounts
Accurately take Moso Bamboo Leaves powder 5g, add 100mL80% aqueous ethanolic solution, extract 1h, 2h, 3h, 4h, 5h respectively at water bath with thermostatic control heat at 70 DEG C, repeat extraction 4 times, filter and merging filtrate, be concentratedly settled to 100mL, get 1mL constant volume in 25mL volumetric flask.Carry out detection by quantitative with HPLC, measure four kinds of flavone c-glycoside content.The results are shown in Figure 4.Extraction time, the extracted amount to four kinds of flavone c-glycosides had a certain impact, along with the increase of extraction time, four kinds of flavone c-glycoside extracted amounts are also increasing gradually, this is because material and solvent contacts area more and more abundant, be conducive to the stripping of four kinds of flavone c-glycosides, but the loss when the extraction time is increased, in material transfer process is also in increase.So extraction time select 1 ~ 3h be optimized more reasonable.
(2) orthogonal test
1, orthogonal experiment
According to single factor experiment result, select alcohol concn, solid-liquid ratio, Extracting temperature, extraction time four factors three levels carry out orthogonal test, and range analysis and variance analysis are carried out to test-results, obtain four principal elements to the impact order of four kinds of flavone c-glycoside extracted amounts and suitable extraction process, result is as shown in following table 1 and 2.
As shown in Table 1, alcohol concn extreme difference: level 1 reduces gradually to level 3, and the extreme difference of solid-liquid ratio, Extracting temperature, extraction time: level 1 is increased to level 2, then is reduced to level 3.Each factor and the extracted amount of level to four kinds of flavone c-glycosides thereof show certain otherness, and the extreme difference of factor is larger, and the change of test-results that the change of its level causes is larger, then more obvious on the impact of test-results.Calculated by extreme difference, the order that factor affects size to four kinds of flavone c-glycoside yield is alcohol concn > extraction time > solid-liquid ratio > Extracting temperature.
Table 1 Moso Bamboo Leaves four kinds of flavone c-glycosides extract test orthogonal design and result
Table 1 is the variance analysis carried out the test-results of four kinds of flavone c-glycoside extracted amounts, compared by calculating F value and significance, its result and range analysis result are consistent, and alcohol concn is extremely remarkable factor, solid-liquid ratio and extraction time are remarkable factor, and the impact of Extracting temperature is minimum.According to above-mentioned analytical results, the suitable extraction process of four kinds of flavone c-glycosides is: extract 2h at 75% ethanol, solid-liquid ratio 1: 20,70 DEG C.
Table 2 Moso Bamboo Leaves four kinds of flavone c-glycosides extract the results of analysis of variance of test
Note: f
0.05 (2,18)=3.55; f
0.01 (2,18)=6.01
2, under being suitable for extraction process, extraction time is on the impact of four kinds of flavone c-glycoside extracted amounts
Accurately take Moso Bamboo Leaves powder 5g, add 100mL75% aqueous ethanolic solution, at 70 DEG C, water bath with thermostatic control heat extracts 2h, extracts 1,2,3,4,5 time continuously, filters and merging filtrate, is concentrated into respective volume.Carry out detection by quantitative with HPLC, measure four kinds of flavone c-glycoside content.Result as shown in Figure 5.Fig. 5 shows, increases the stripping that extraction time is conducive to four kinds of flavone c-glycosides in the leaf of bamboo, but when after extraction 4 times, extracted amount does not almost significantly increase, now in the leaf of bamboo, four kinds of flavone c-glycosides may extract completely, and extracted amount trend is stable, extraction time select 4 times more suitable.
3, extraction process checking
By suitable extraction process, 2kg leaf of bamboo powder obtains 579.809g bamboo leaf flavone after extracting concentrate drying, wherein, the content of Orientin, Lutonaretin, Vitexina and Saponaretin is respectively 1.223g, 5.496g, 0.395g, 1.799g, the total amount of four kinds of flavone c-glycosides is 8.913g.Thus, four kinds of flavone c-glycoside total amounts are 4.457mgg relative to the extracted amount of leaf of bamboo sample
-1, the purity of four kinds of flavone c-glycosides in bamboo leaf flavone reaches 15.372mgg
-1.
Two, the research of four kinds of flavone c-glycoside purifying process in Moso Bamboo Leaves
(1) macroporous resin purification technical study
(1) macroporous resin Static Adsorption-desorption experiment
(A) mensuration of saturated extent of adsorption and desorption amount
Take six kinds of pretreated (AB-8, ADS-17, D101, DM130, NKA-9 and X-5, all purchased from Zhengzhou Qin Shi Science and Technology Ltd.) macroporous resins (dry weight 1g) and in 100mL tool plug triangular flask, add 10mgmL
-1folium Bambosae flavone crude extract 55mL, is placed in constant-temperature table vibrates 12h (preliminary experiment shows in a basic balance), turn up 200rmin by triangular flask
-1.Suction filtration after macroporous resin fully adsorbs, filtrate crosses 0.45 μm of microporous membrane, and HPLC measures four kinds of flavone c-glycoside content in filtrate.The results are shown in Table 3.
Table 3 six kinds of macroporous resins are to the Static Adsorption of four kinds of flavone c-glycosides and desorption result
Adsorptive capacity Q
erepresenting the true adsorptive power of resin, is the important parameter selecting resin types and evaluating resin regeneration effect; And desorption quantity is the index of evaluating resin desorption ability and eluent ability; Adsorption rate and desorption efficiency are that resin absorption and desorption ability represent with the form of per-cent respectively, and as the important parameter that macroporous resin screens.As can be seen from Table 3, adsorption rate and the desorption efficiency of X-5 resin are minimum, and this is that the model ylid bloom action force rate of the functional group of four kinds of flavone glycosides and X-5 resin is more weak, can not fully be adsorbed on post bed; When adopting 70% ethanolic soln wash-out, can not fully desorb, four kinds of flavone c-glycosides losses are serious, can not be used for the purifying of these four kinds of flavone c-glycosides.NKA-9 resins act goes out high adsorption rate and desorption efficiency, but its market sale price is the highest, considers cost problems of operation during production in enormous quantities, also wouldn't select NKA-9.Adsorption rate and the desorption efficiency of AB-8, ADS-17, DM130 and D101 resin all respectively have strengths and weaknesses, then in conjunction with its polarity and constructional feature, select ADS-17, DM130 and D101 as the resin used being further purified four kinds of flavone c-glycosides.
(B) Static Adsorption isothermal curve measures
Select ADS-17, DM130 and D101 tri-kinds of macroporous resins to carry out pre-treatment, each six parts, every part of 1g, load in 100mL tool plug triangular flask, add 3mgmL respectively
-1, 5mgmL
-1, 8mgmL
-1, 10mgmL
-1, 12mgmL
-1with the Folium Bambosae flavone crude extract 55mL of 15mgmL-1, in shaking table vibration 12h under differing temps (15 DEG C, 25 DEG C, 35 DEG C).Filtrate is also crossed 0.45 μm of microporous membrane by suction filtration, and HPLC measures four kinds of flavone c-glycoside content in filtrate.
This test selects Langmuir and Freundlich absorption experimental formula to carry out matching to adsorption isotherm data, and obtain each regression equation and parameter thereof respectively, result is as shown in table 4 and table 5.As can be seen from relation conefficient, the adsorption process of four kinds of flavone c-glycosides on ADS-17, DM130 and D101 tri-kinds of macroporous resins meets Langmuir and Freundlich equation substantially, the relation conefficient of the two is all greater than 0.93, and at different temperatures, the relation conefficient of Langmuir equation closer to 0.99, then adopts Langmuir equation to describe this adsorption process better.According to the supposition of Langmuir isotherm adsorption model, can initial guess ADS-17, DM130 and D101 tri-kinds of macroporous resin absorption to four kinds of flavone c-glycosides be homogeneous based on solid adsorbent surfaces, the unimolecular layer isothermal adsorption that adsorptive power can be identical everywhere.Q
mfor saturated extent of adsorption, characterize the quality of the actual maximum adsorption of unit mass resin four kinds of flavone c-glycosides, when temperature rises to 35 DEG C, the Q of three kinds of macroporous resins
mvalue increases all to some extent, illustrates that temperature raises and is conducive to adsorption process.But consider that in macroporous resin purification technique, temperature arranges wayward, often select normal temperature to carry out purifying.The isothermal adsorption characteristics constant n of Freundlich equation is between 2 ~ 10, be called " preferential absorption ", illustrate that three kinds of macroporous resins easily realize the absorption to these four kinds of flavone c-glycosides of Orientin, Lutonaretin, Vitexina and Saponaretin, and n value is higher, then corresponding 1/n value is low, illustrate that resin adsorption rate is slow, adsorption time is long, shows that this resin has better selectivity to adsorbate; But adsorption rate can not be too low, otherwise adsorption time can be caused long, reduce production efficiency.
Under table 4 differing temps, the Langmuir adsorption equation of ADS-17, DM130, D101 macroporous resin and parameter
Under table 5 differing temps, the Freundlich adsorption equation of ADS-17, DM130, D101 macroporous resin and parameter
(C) Static Adsorption kinetic curve measures
Select ADS-17, DM130 and D101 tri-kinds of macroporous resins (each 1g of dry weight) to carry out pre-treatment, load in 100mL tool plug triangular flask, measure the adsorptive capacity of resin when t (t=0.5,1,2,4,8,12h) respectively.With Q
tto t mapping, carry out matching according to Lagergren pseudo-first-order kinetic equation.As shown in Figure 6, fit equation and adsorbing filament technique are in table 6 for result.
Table 6 Lagergren pseudo-first-order kinetic equation and parameter
As can be seen from Table 6, the relation conefficient of three kinds of resins, all more than 0.99, illustrates that Liquid film diffusion is the prevailing rate rate-determining steps of three kinds of resins, but can not illustrate that Liquid film diffusion is unique rate determining step, may be the coefficient result of multiple adsorption process, this test be no longer gone into seriously.Can be drawn by table 6, ADS-17 and D101 resin absorption speed K
fvalue, a little more than DM130, selects ADS-17 and D101 resin to carry out dynamic adsorption test.
(2) dynamic adsorption test and each factor are on the impact of purification effect
(A) macroporous resin the selection result
Take ADS-17 and D101 two kinds of macroporous resins (dry weight 20g), wet method is loaded in 2cm × 35cm chromatography column, and dress post height is 12.5cm, adds 10mgmL
-1folium Bambosae flavone crude extract 900mL, control 3BVh
-1flow velocity crosses post, collects effluent liquid, repeats upper prop 3 times, and concentrated also constant volume is in 50mL volumetric flask, and HPLC measures residue four kinds of flavone c-glycoside content in effluent liquid.Equal-volume distilled water wash-out, remove portion carbohydrate and soluble protein, collect elutriant, and concentrated, constant volume, in 50mL volumetric flask, measures four kinds of flavone c-glycoside content.Then use equal-volume 60% ethanolic soln wash-out, collect elutriant, constant volume, to 500mL volumetric flask, measures four kinds of flavone c-glycoside content in ethanol eluate.Finally remove impurity with ethanol elution, reclaim ethanol and continue to use, and manipulation of regeneration is carried out to macroporous resin.With macroporous resin adsorption rate and ethanol desorption efficiency for evaluation index, and consider target compound constructional feature and production cost, select suitable macroporous resin.Test-results is as shown in table 7 below.
As can be seen from Table 7, ADS-17 and D101 resin all has reasonable sorption and desorption ability to four kinds of flavone c-glycosides, but these two kinds of resins are each variant again to the adsorptive power of Orientin, Lutonaretin, Vitexina and Saponaretin, this is relevant with the polarity of four kinds of flavone c-glycosides with the structure of macroporous resin.The surface-area of D101 macroporous resin is higher than ADS-17 resin, thus add the contact area of adsorbate and solid adsorbent, reactive force between the two increases, so D101 macroporous resin can higher than ADS-17 resin to the adsorption rate of Orientin, Lutonaretin, Vitexina and Saponaretin.Organic solvent elution process is that elutriant and solid adsorbent are to the result of four kinds of flavone c-glycoside Competitions, the desorption efficiency of D101 macroporous resin is a little less than ADS-17 resin, synthesis resin cost and economic benefit, finally select D101 macroporous resin as the comparatively select tree fat of four kinds of flavone c-glycosides in purifying Moso Bamboo Leaves.
Table 7 ADS-17 and D101 two kinds of macroporous resin dynamic adsorption shaker test results
(B) D101 macroporous resin reveals curve and dynamic desorption curve
Take D101 macroporous resin (dry weight 20g) and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5cm).Add 10mgmL
-1folium Bambosae flavone crude extract, control 3BVh
-1flow velocity crosses post, and every 40mL collects 1 pipe, measures the content of each Guan Zhongsi kind flavone c-glycoside, obtains macroporous resin dynamic adsorption break-through curve, as Fig. 7.
As seen from Figure 7, when applied sample amount is lower, the binding site on resin bed is more sufficient, and now four kinds of flavone c-glycoside amount of leakage are little, almost nil; When loading volume reaches 400mL, in effluent liquid, four kinds of flavone c-glycoside content start to increase, this is because progressively increase along with applied sample amount, the content of impurity is also increasing gradually, impurity and four kinds of flavone c-glycosides compete the binding site on resin bed jointly, impurity replaces the combination of flavone c-glycoside and resin, causes in effluent liquid four kinds of flavone c-glycosides increase sharply and detected by HPLC; Increase applied sample amount if continue, when loading volume reaches 900mL, resin bed adsorptive power reaches capacity, and four kinds of flavone c-glycosides now on resin bed reach a kind of running balance, and the carbon glycosides content in effluent liquid also no longer includes obvious change.
After resin absorption is saturated, 60% ethanol carries out wash-out, and every 10mL collects 1 pipe, measures each Guan Zhongsi kind flavone c-glycoside content, obtains dynamic desorption kinetic curve, be illustrated in fig. 8 shown below.When collection the 1st pipe, four kinds of flavone c-glycoside summation 0.09mg just can be detected; Continue to collect, in the 3rd pipe and the 4th pipe elutriant, four kinds of flavone c-glycoside summations reach maximum, and this section belongs to the concentrated adsorption stage of flavone c-glycoside, and now in elutriant, four kinds of flavone c-glycoside summations reach 125.841mg; Continue wash-out, four kinds of flavone c-glycoside collecting amounts decline to some extent, flavone c-glycoside now in post bed is eluted gradually, when ethanol elution volume reaches 120mL (about 3BV), in elutriant, four kinds of flavone c-glycoside content are little, almost can ignore, so when ethanol elution volume is 3BV, post bed adsorbs four kinds of flavone c-glycosides fully can be eluted.
(C) sample starting point concentration is on the impact of resin absorption flavones performance
Take D101 macroporous resin (dry weight 20g) and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5cm).Respectively by 6mgmL
-1, 8mgmL
-1, 10mgmL
-1, 12mgmL
-1the each 400mL of Folium Bambosae flavone crude extract adds in 4 same resin columns, and flow velocity is 3BVh
-1, collect sample effluent liquid, concentrated also constant volume is in 50mL volumetric flask.After dynamic adsorption is saturated, with 3BV distilled water wash-out, concentrate eluant, constant volume is in 50mL volumetric flask.Then use 60% ethanol of 3BV with 3BVh
-1flow velocity wash-out, last constant volume is in 100mL volumetric flask, and get 1mL dilution and be settled in 10mL volumetric flask, cross 0.45 μm of microporous membrane, HPLC measures four kinds of flavone c-glycoside content in filtrate.Comparing adsorption rate and desorption efficiency by calculating, selecting the suitableeest sample concentration.Correlation parameter is in table 8.
Table 8 sample starting point concentration is on the impact of D101 resin absorption performance
As can be seen from Table 8, the Adsorption and desorption characteristics of sample starting point concentration to four kinds of flavone c-glycosides has a certain impact.When starting point concentration is lower than 10mgmL
-1time, adsorption rate is more or less the same, and all more than 95%, the Orientin now in Folium Bambosae flavone crude extract, Lutonaretin, Vitexina and Saponaretin compounds main have all been adsorbed on macroporous resin; And when concentration increases, resin absorption reaches capacity, adsorption rate comparatively speaking declines to some extent; When carrying out wash-out with elutriant, desorption efficiency also presents larger difference, the desorption efficiency that target compound on resin bed larger for applied sample amount elutes by ethanol is higher, now four kinds of flavone c-glycoside major parts are eluted, desorption efficiency reaches more than 97%, and the resin layer of low sample concentration presents lower desorption efficiency.Comprehensive the above results, selects 10mgmL
-1as sample starting point concentration.
(D) ethanol elution concentration is on the impact of resin absorption flavones performance
After Folium Bambosae flavone crude extract enters post bed, select certain density ethanolic soln to carry out wash-out as elutriant, per sample, the model ylid bloom action power size of component and resin, can get off the component of opposed polarity successively drip washing, realize the rough segmentation of Folium Bambosae flavone.For farthest removing impurity, retain target compound, lower concentration and high concentration ethanol solution in addition wash-out is selected in test respectively, improves four kinds of flavone c-glycoside content, reduction impurity ratio.
1) low-concentration ethanol wash-out
Take D101 macroporous resin (dry weight 20g) and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5 cm).Add 10mgmL
-1folium Bambosae flavone crude extract, coutroi velocity 3BVh
-1, collect sample effluent liquid, concentrated also constant volume is in 50mL volumetric flask.After absorption completely, with 3BV distilled water wash-out, concentrate eluant, constant volume is in 50mL volumetric flask.Then with 10%, 15%, 20%, 25%, 30% ethanolic soln, desorption is carried out to chromatography column respectively, elution volume is 3BV, collect elutriant, last constant volume is in 100mL volumetric flask, getting 1mL dilution is settled in 10mL volumetric flask, cross 0.45 μm of microporous membrane, HPLC measures four kinds of flavone c-glycoside content in filtrate.By the size of desorption efficiency, suitable low-concentration ethanol solution is selected to carry out wash-out.
Result as shown in Figure 9.When alcohol concn lower than 15% time, desorption efficiency is lower than 4%, and the target compound content now eluted is few, and foreign matter content is relatively large; Along with the continuation of alcohol concn increases, the desorption efficiency of four kinds of flavone c-glycosides increases rapidly, and when ethanol solution concentration is 30%, desorption efficiency reaches 46.66%, shows that the target compound of half nearly on resin layer is eluted.Although ethanol elution concentration increases, the foreign matter content of wash-out also can synchronously increase, and also increases the elution amount of target compound simultaneously, causes target compound to lose serious.So test and Selection 15% alcohol concn is as the eluting solvent of lower concentration, now the loss amount of four kinds of flavone c-glycosides is few, and impurity ratio is relatively large.
2) high concentration ethanol wash-out
Take D101 macroporous resin (dry weight 20g) and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5cm).Add 10mgmL
-1folium Bambosae flavone crude extract, coutroi velocity 3BVh
-1, collect sample effluent liquid, concentrated also constant volume is in 50mL volumetric flask.After absorption completely, with 3BV distilled water wash-out, concentrate eluant, constant volume is in 50mL volumetric flask.First use 15% ethanol elution of 3BV, use the ethanolic soln 3BV wash-out of 40%, 50%, 60%, 70%, 80% more respectively, collect elutriant, last constant volume is in 100mL volumetric flask, getting 1mL dilution is settled in 10mL volumetric flask, cross 0.45 μm of microporous membrane, HPLC measures four kinds of flavone c-glycoside content in filtrate.By the size of desorption efficiency, suitable high concentration ethanol solution is selected to carry out wash-out.
Result as shown in Figure 10.When alcohol concn is increased to 60% from 40%, the desorption efficiency of four kinds of flavone c-glycosides significantly increases, this is because ethanolic soln and macroporous resin competition, the solubleness of target compound in ethanolic soln is increased, and namely target compound gets off from desorb chromatography column gradually; But when ethanolic soln is greater than 60%, desorption efficiency change is not obvious, and now target compound may farthest elute from resin column by ethanolic soln, and the component that can not elute forms dead adsorption phenomena on resin bed.So test and Selection 60% alcohol concn is as the eluting solvent of high density, now the desorption efficiency of four kinds of flavone c-glycosides is higher, and decreases consumption of organic solvent.
(E) elution rate is on the impact of resin absorption flavones performance
Take D101 macroporous resin (dry weight 20g) and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5cm).Add 10mgmL
-1folium Bambosae flavone crude extract, respectively with 1BVh
-1, 2BVh
-1, 3BVh
-1, 4BVh
-1, 5BVh
-1flow velocity by chromatography column, collect sample effluent liquid, concentrated and constant volume is in 50mL volumetric flask.After absorption completely, with 3BV distilled water wash-out, concentrate eluant, constant volume is in 50mL volumetric flask.Then use 60% ethanol of 3BV with 3BVh
-1flow velocity wash-out, last constant volume is in 100mL volumetric flask, and get 1mL dilution and be settled in 10mL volumetric flask, cross 0.45 μm of microporous membrane, HPLC measures four kinds of flavone c-glycoside content in filtrate.Comparing adsorption rate and desorption efficiency by calculating, selecting the suitableeest elution rate.
As seen from Figure 11, when elution rate is 1BVh
-1time, the eluting rate of four kinds of flavone c-glycosides is the highest; When speed continues to increase, the change of desorption efficiency is not obvious.When elution rate reaches 1BVh
-1time, the desorption efficiency of four kinds of flavone c-glycosides is up to 97.47%, and now elutriant fully contacts with target compound, by the competition with resin layer, to greatest extent target compound is eluted, but simultaneously low elution rate extends again the production cycle of target compound, be unfavorable for the raising of economic benefit.Comprehensive desorption efficiency and production cycle are considered, select 4BVh
-1as suitable elution rate.
(3) macroporous resin purification process certification
By suitable macroporous resin purification technique, 150g bamboo leaf flavone is purified, concentrate drying obtains 25.7g Folium Bambosae flavone powder, wherein, the quality of Orientin, Lutonaretin, Vitexina and Saponaretin is respectively 487.147mg, 928.844mg, 88.4mg, 252.431mg, the total amount of four kinds of flavone c-glycosides is 1756.822mg.Thus, four kinds of flavone c-glycosides are 11.712mgg relative to the purifying amount of crude extract
-1, the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 68.359mgg
-1.
(2) polyamide purifying technical study
(1) polyamide resin is selected
Take the polyamide resin (dry weight 10g) that particle size range is 0.25 ~ 0.55mm, 0.15 ~ 0.25mm, 0.12 ~ 0.15mm, wet method dress post is in 2cm × 35cm chromatography column, and dress post height is 12.5cm, adds 10mgmL
-1the solution 200mL of above-mentioned Folium Bambosae flavone dry composition, control 3BVh
-1flow velocity crosses post, collects effluent liquid, repeats upper prop 3 times, and concentrated also constant volume is in 25mL volumetric flask, and HPLC measures residue four kinds of flavone c-glycoside content in effluent liquid.With equal-volume distilled water wash-out, collect elutriant, concentrated also constant volume, in 25mL volumetric flask, measures four kinds of flavone c-glycoside content.Then use equal-volume 40% ethanolic soln wash-out, collect elutriant, constant volume, to 100mL volumetric flask, is got 1mL solution and is settled to 10mL volumetric flask, measures four kinds of flavone c-glycoside content in ethanol eluate.With Adsorbent rate and ethanol desorption efficiency for evaluation index, select to be applicable to polyamide resin loading.Specifically in table 9.
Table 9 different-grain diameter polyamide resin shaker test result
Test-results is as shown in table 9.Particle size range is starkly lower than the less resin of all the other two kinds of particle diameters in the adsorption rate of the polyamide resin of 0.25 ~ 0.55mm and desorption efficiency, this is because polymeric amide particle diameter is larger, for the adsorbed material of same concentration, cause the target molecule of resin local ambient too much, some target compound is made not have enough time to be adsorbed just outflow, target compound loses seriously after introduction of the sample, and actual absorption target compound on the resin layer reduces, and the target compound eluted is corresponding minimizing also.When polyamide resin particle size range is at 0.15 ~ 0.25mm and 0.12 ~ 0.15mm, adsorption rate and desorption efficiency entirety do not have considerable change.Consider that particle diameter is larger, elution rate is faster simultaneously, and from enhancing productivity, angle is considered, selection particle size range is polyamide resin being further purified for four kinds of flavone c-glycosides of 0.15 ~ 0.25mm.
(2) ethanol elution concentration is on the impact of resin absorption flavones performance
According to reagent eluting characteristics and security, test adopts ethanolic soln to carry out desorb as elute soln, and research does not allow concentration ethanol solution on the impact of resin absorption flavones performance, with Adsorbent rate and elutriant desorption efficiency for evaluation index, select suitable ethanol solution concentration.
Take the above-mentioned polyamide resin (dry weight 10g) filtered out and carry out pre-treatment, wet method dress post (2cm × 35cm, dress post height 12.5cm).Add 10mgmL
-1above-mentioned Folium Bambosae flavone liquid, coutroi velocity 3BVh
-1, collect sample effluent liquid, concentrated also constant volume in 50mL volumetric flask, then uses 3BV distilled water wash-out, concentrate eluant, and constant volume is in 50mL volumetric flask.Then desorption is carried out with 10%, 20%, 30%, 40%, 50%, 60%, 70% ethanolic soln respectively, elution volume is 3BV, and collect elutriant, last constant volume is in 100mL volumetric flask, cross 0.45 μm of microporous membrane, HPLC measures four kinds of flavone c-glycoside content in filtrate.By desorption efficiency size, suitable ethanolic soln is selected to carry out wash-out.The results are shown in shown in Figure 12.
Test and Selection 7 different ethanol concentration carry out wash-out to resin bed, when alcohol concn is 10%, four kinds of flavone c-glycosides have had part to get off from desorb resin bed, and now desorption efficiency reaches 16.34%, show when alcohol concn is lower, four kinds of flavone c-glycosides are just easy to by wash-out; Along with the increase of ethanol elution concentration, the desorption efficiency of target compound also in corresponding increase, this is because target compound in the solution solubleness increase, followed by solution and get off from desorb resin layer; But when alcohol concn is greater than 40%, desorption efficiency change is not obvious, and now target compound may elute from resin bed by ethanolic soln as much as possible, but also have the target compound of part and impurity still to remain on the resin layer, cause dead adsorption phenomena.So test and Selection 40% alcohol concn is as elute soln, now the desorption efficiency of four kinds of flavone c-glycosides is higher, and ethanol consumption also reduces greatly simultaneously.
(3) polyamide resin purifying process checking
By above-mentioned purifying process, get 20g Folium Bambosae flavone powder after polyamide resin purifying, concentrated, drying 4.26g Folium Bambosae flavone powder, wherein, the quality of Orientin, Lutonaretin, Vitexina and Saponaretin is respectively 37.26mg, 182.556mg, 23.506mg, 79.243mg, the total amount of four kinds of flavone c-glycosides is 322.565mg.Thus, four kinds of flavone c-glycosides are 16.128mgg relative to the purifying amount of crude extract
-1, and the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 76.939mgg
-1.
The invention has the beneficial effects as follows:
(1) single factor experiment that four kinds of flavone c-glycoside extracted amounts affected by alcohol concn, solid-liquid ratio, Extracting temperature and extraction time of the present invention, suitable factor and level is selected to carry out orthogonal test, range analysis and variance analysis are carried out to result, obtains best extraction conditions.
(2) the present invention is through two-step purifying process, and has carried out systematic research to its actual conditions, has summed up best purification condition, and obtained Folium Bambosae flavone carbon glycosides is purer.
(3) by Static Adsorption, dynamic adsorption screening and optimizing macroporous resin type, select suitable macroporous resin to be further purified Folium Bambosae flavone crude extract, obtain suitable sample starting point concentration, loading volume, ethanol elution concentration and elution volume, elution rate.Four kinds of flavone c-glycosides are 11.712mgg relative to the purifying amount of crude extract as a result
-1, and the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 68.359mgg
-1.
(4) polyamide resin and ethanol elution concentration by selecting appropriate particle size scope are further purified the Folium Bambosae flavone dry powder after macroporous resin purification, and four kinds of flavone c-glycosides are 16.128mgg relative to the purifying amount of crude extract as a result
-1, and the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 76.939mgg
-1.
(5) the flavone c-glycoside purity that obtains of the present invention is high, and Stability Analysis of Structures, is easy to go deep into lesions position, and play drug effect, the exploitation that can be medicine intermediate provides source, and for instructing large-scale industrial production to provide basis.
Accompanying drawing explanation
Fig. 1 is the impact of different ethanol concentration of the present invention on four kinds of flavone c-glycoside extracted amounts.
Fig. 2 is the impact of different feed liquid comparison of the present invention four kinds of flavone c-glycoside extracted amounts.
Fig. 3 is the impact of Extracting temperature of the present invention on four kinds of flavone c-glycoside extracted amounts.
Fig. 4 is the impact of extraction time of the present invention on four kinds of flavone c-glycoside extracted amounts.
Fig. 5 is the impact of extraction time of the present invention on four kinds of flavone c-glycoside extracted amounts.
Fig. 6 is ADS-17, DM130 and D101 macroporous resin Static Adsorption kinetic curve of the present invention.
Fig. 7 is macroporous resin dynamic leakage curve of the present invention.
Fig. 8 is D101 macroporous resin dynamic desorption curve of the present invention.
Fig. 9 is that low-concentration ethanol elutriant of the present invention is on the impact of four kinds of flavone c-glycoside desorption efficiencies.
Figure 10 is that high concentration ethanol elutriant of the present invention is on the impact of four kinds of flavone c-glycoside desorption efficiencies.
Figure 11 is the impact of elution rate of the present invention on four kinds of flavone c-glycoside desorption efficiencies.
Figure 12 is that different concentration ethanol elutriant of the present invention is on the impact of four kinds of flavone c-glycoside desorption efficiencies.
Embodiment
Embodiment 1
Orientin, Lutonaretin, Vitexina and Saponaretin Isolation and purification technique in a kind of Moso Bamboo Leaves, comprise the steps:
(1) accurately take Moso Bamboo Leaves powder 2kg, add in aqueous ethanolic solution and carry out circumfluence distillation, repeat extraction 4 times, filter and filtrate is merged to obtain extracting solution; The technique of described circumfluence distillation is: at 75% ethanol, solid-liquid ratio 1: 20,70 DEG C, 2h is extracted in water bath with thermostatic control;
(2) adopt the organic solvent in distillation under vacuum removing extracting solution, add isopyknic petroleum ether extraction with extracting solution, removing pigment and fat-soluble component, re-extract three times, obtain 579.809g Moso Bamboo Leaves flavone gruff bring up substance, sherwood oil reclaims, and recycles;
(3) macroporous resin D101 pre-treatment: wash away tiny resin and impurity with dehydrated alcohol, then soak 12h, makes macroporous resin D101 fully swelling.Wet method dress post, is washed till elutant without till white opacity with dehydrated alcohol, then is zero with distilled water flushing to alcohol concn, then proceed to acid-alkali treatment.First use the 5%NaOH solution 3BVh of 3BV (BV is column volume, lower same)
-1flow velocity by resin layer, then distilled water with identical speed by being washed till pH in neutral; Use 3BV5%HCl solution with 3BVh again
-1flow velocity is by resin layer, and distilled water continues to be eluted to pH in neutral with same flow velocity.Last macroporous resin D101 is placed in dehydrated alcohol and preserves stand-by, and using distilled water to be washed till alcohol concn is before use zero, gets final product loading.
(4) Moso Bamboo Leaves flavone gruff bring up substance is carried out purification process through macroporous resin D101: by macroporous resin column on Moso Bamboo Leaves flavone gruff bring up substance, wet method dress post, adds 10mgmL
-1folium Bambosae flavone crude extract, coutroi velocity 3BVh
-1, macroporous resin D101 is 5: 1 with sample applied sample amount ratio, collects sample effluent liquid, after effluent liquid repeats upper prop 3 times, with 15% ethanolic soln with 4BVh
-1the preliminary wash-out removal of impurities of flow velocity, concentrate eluant, then uses the ethanolic soln of 60% with 4BVh
-1flow velocity wash-out, finally collects 60% ethanol eluate, and concentrated and lyophilize obtains Moso Bamboo Leaves flavones dry powder 99.3g after purifying;
HPLC detection method is utilized to measure its final purity: realizing the chromatographic condition that four kinds of flavone c-glycosides are effectively separated is: Waters XTerra MS C18 chromatographic column (250mm × 4.6mm, 5 μm); Adopt double pump system, mobile phase A is acetonitrile, and Mobile phase B is 0.5% formic acid/aqueous solution (v/v), 14%A Gradient elution 25min; Flow velocity is 1mLmin
-1, column temperature reaches 30 DEG C, and sample size is 10 μ L, and determined wavelength is set to 360nm;
Now, four kinds of flavone c-glycosides are 11.712mgg relative to the purifying amount of crude extract
-1, and the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 68.359mgg
-1.
(5) polyamide resin pre-treatment
Select particle diameter to be the polyamide resin of 0.15 ~ 0.25mm, use soaked in absolute ethyl alcohol 12h, stir removing bubble, wash away the fine powder of suspension simultaneously.Wet method dress post, is washed till effluent liquid with the ethanol of 3BV transparent, and without residue (or few residue) after evaporate to dryness.Be zero with distilled water flushing to alcohol concn again, then proceed to acid-alkali treatment.First use the 5%NaOH solution of 3BV with 3BVh
-1flow velocity through resin layer, then with distilled water with same flow velocity by being washed till pH in neutrality; Then use the 5%HCl solution of 3BV with 3BVh
-1flow velocity is by resin layer, and distilled water continues to be eluted to pH in neutral with same flow velocity.Last polyamide resin is placed in dehydrated alcohol and preserves stand-by, and using distilled water to be washed till alcohol concn is before use zero, gets final product loading;
(6) by the Folium Bambosae flavone powder after above-mentioned macroporous resin D101 purifying, after dissolving, by polyamide resin wet method dress post in 2cm × 35cm chromatography column, dress post height is 12.5cm, adds 10mgmL
-1the solution of above-mentioned Folium Bambosae flavone dry composition, control 3BVh
-1flow velocity crosses post, collects effluent liquid, repeats upper prop 3 times, then uses distilled water wash-out, collects elutriant, concentrated, then uses equal-volume 40% ethanolic soln wash-out, finally collects 40% ethanol eluate, and concentrated and lyophilize obtains the Folium Bambosae flavone dry powder 21g after purifying;
HPLC detection method is utilized to measure its final purity equally: chromatographic condition is the same.
Recording four kinds of flavone c-glycosides relative to the purifying amount of crude extract is 16.128mgg
-1, and the purity of four kinds of flavone c-glycosides in Folium Bambosae flavone powder reaches 76.939mgg
-1.
Claims (6)
1. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves, four kinds of described flavone c-glycosides are Orientin, Lutonaretin, Vitexina and Saponaretin, it is characterized in that, comprise the steps:
(1) accurately take Moso Bamboo Leaves powder, add in aqueous ethanolic solution and carry out circumfluence distillation, repeat extraction 4 times, filter and filtrate is merged to obtain extracting solution; The technique of described circumfluence distillation is: the ethanol of 70% ~ 80%, and solid-liquid ratio is that 1 ~ 3h is extracted in 1: 15 ~ 1: 25,60 ~ 80 DEG C of waters bath with thermostatic control;
(2) adopt the organic solvent in distillation under vacuum removing extracting solution, add isopyknic petroleum ether extraction with extracting solution, removing pigment and fat-soluble component, re-extract three times, obtains Moso Bamboo Leaves flavone gruff bring up substance, and sherwood oil reclaims, and recycles;
(3) Moso Bamboo Leaves flavone gruff bring up substance is carried out purification process through macroporous resin: macroporous resin carries out pre-treatment, by macroporous resin column on Moso Bamboo Leaves flavone gruff bring up substance, wet method dress post, adds 10mgmL
-1folium Bambosae flavone crude extract, coutroi velocity 3BVh
-1, macroporous resin is 5: 1 with sample applied sample amount ratio, collects sample effluent liquid, after effluent liquid repeats upper prop 3 times, with 15% ethanolic soln with 4BVh
-1the preliminary wash-out removal of impurities of flow velocity, concentrate eluant, then uses the ethanolic soln of 60% with 4BVh
-1flow velocity wash-out, finally collects 60% ethanol eluate, and concentrated and lyophilize obtains Moso Bamboo Leaves flavones dry powder after purifying;
(4) again carry out purification process through polyamide resin: by the Folium Bambosae flavone powder after above-mentioned macroporous resin purification, after dissolving, by polyamide resin wet method dress post in 2cm × 35cm chromatography column, dress post height is 12.5cm, adds 10mgmL
-1the solution of above-mentioned Folium Bambosae flavone dry composition, control 3BVh
-1flow velocity crosses post, collects effluent liquid, repeats upper prop 3 times, then uses distilled water wash-out, collects elutriant, concentrated, then uses equal-volume 40% ethanolic soln wash-out, finally collects 40% ethanol eluate, and concentrated and lyophilize obtains the Folium Bambosae flavone dry powder after purifying.
2. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves as claimed in claim 1, it is characterized in that, the technique of described circumfluence distillation is: at 75% ethanol, solid-liquid ratio 1: 20,70 DEG C, 2h is extracted in water bath with thermostatic control.
3. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves as claimed in claim 1 or 2, it is characterized in that, described macroporous resin is ADS-17, DM130 or D101.
4. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves as claimed in claim 3, it is characterized in that, described macroporous resin is D101.
5. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves as claimed in claim 1 or 2, it is characterized in that, the particle diameter of described polyamide resin is 0.15 ~ 0.25mm.
6. four kinds of flavone c-glycoside Isolation and purification techniques in Moso Bamboo Leaves as claimed in claim 1 or 2, is characterized in that, before macroporous resin or polyamide resin purification process, carry out pre-treatment to macroporous resin or polyamide resin, specific as follows:
Tiny resin and impurity is washed away with dehydrated alcohol, then 12h is soaked, make macroporous resin or polyamide resin fully swelling, wet method dress post, elutant is washed till without till white opacity with dehydrated alcohol, be zero with distilled water flushing to alcohol concn again, then proceed to acid-alkali treatment, first use the 5%NaOH solution 3BVh of 3BV
-1flow velocity by resin layer, then distilled water with identical speed by being washed till pH in neutral; Use 3BV 5%HCl solution with 3BVh again
-1flow velocity is by resin layer, and distilled water continues to be eluted to pH in neutral with same flow velocity, and last macroporous resin or polyamide resin are placed in dehydrated alcohol and preserve stand-by, and using distilled water to be washed till alcohol concn is before use zero, gets final product loading.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105859700A (en) * | 2016-04-26 | 2016-08-17 | 中国广州分析测试中心 | Method for extracting and preparing isovitexin from patrinia villosa juss |
| CN106667865A (en) * | 2016-11-15 | 2017-05-17 | 浙江汉篁生物技术有限公司 | Bamboo leaf extract for skin care product and preparation method thereof |
| CN107496727A (en) * | 2017-08-10 | 2017-12-22 | 合肥果成科技有限公司 | A kind of extracting method of flavones |
| CN107497409A (en) * | 2017-07-12 | 2017-12-22 | 浙江理工大学 | A kind of preparation method for being used for the macroreticular resin that total polyphenols purify in Bai le |
| CN108567773A (en) * | 2018-04-23 | 2018-09-25 | 山东大学 | It is a kind of to be used to remove mouth sprays of halitosis and preparation method thereof |
| CN109157465A (en) * | 2018-08-08 | 2019-01-08 | 浙江大学 | High-purity leaf of bamboo carbon glycosides flavones nanoparticle and its preparation method and application |
| CN110372655A (en) * | 2019-08-13 | 2019-10-25 | 武汉工程大学 | The method and application of antimycotic flavonoid component are extracted from indicalamus leaf |
| CN110420295A (en) * | 2019-09-03 | 2019-11-08 | 四川丰泰食品科技有限公司 | A kind of technique separating bamboo-leaves flavones |
| CN111388608A (en) * | 2020-03-24 | 2020-07-10 | 江西维莱营健高科有限公司 | Phoenix bambusoides extract and preparation method and application thereof |
| CN112321578A (en) * | 2020-11-18 | 2021-02-05 | 劲牌有限公司 | Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC |
| CN113813642A (en) * | 2021-10-14 | 2021-12-21 | 浙江新篁生物技术有限公司 | Bamboo leaf flavone extraction equipment for removing impurities by using absolute ethyl alcohol |
| CN115124601A (en) * | 2022-07-29 | 2022-09-30 | 浙江医药股份有限公司新昌制药厂 | Purification method of vancomycin hydrochloride |
| CN115403551A (en) * | 2022-10-18 | 2022-11-29 | 贵州汇腾科技有限公司 | Method for extracting and purifying high-quality bamboo leaf flavone from Sasa veitchii Rehd |
| CN116211970A (en) * | 2023-03-22 | 2023-06-06 | 安徽科技学院 | Method for extracting flavone from bamboo leaves and application thereof |
| CN117618514A (en) * | 2024-01-26 | 2024-03-01 | 广州绿徽新材料研究院有限公司 | Preparation method of ultra-high purity nanoscale bamboo leaf total flavone dispersoid |
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| CN105859700A (en) * | 2016-04-26 | 2016-08-17 | 中国广州分析测试中心 | Method for extracting and preparing isovitexin from patrinia villosa juss |
| CN106667865A (en) * | 2016-11-15 | 2017-05-17 | 浙江汉篁生物技术有限公司 | Bamboo leaf extract for skin care product and preparation method thereof |
| CN107497409A (en) * | 2017-07-12 | 2017-12-22 | 浙江理工大学 | A kind of preparation method for being used for the macroreticular resin that total polyphenols purify in Bai le |
| CN107497409B (en) * | 2017-07-12 | 2020-11-27 | 浙江理工大学 | Preparation method of macroporous resin for purifying total polyphenols in ilicifolius trifoliate |
| CN107496727A (en) * | 2017-08-10 | 2017-12-22 | 合肥果成科技有限公司 | A kind of extracting method of flavones |
| CN108567773B (en) * | 2018-04-23 | 2020-04-17 | 山东大学 | Oral spray for eliminating halitosis and preparation method thereof |
| CN108567773A (en) * | 2018-04-23 | 2018-09-25 | 山东大学 | It is a kind of to be used to remove mouth sprays of halitosis and preparation method thereof |
| CN109157465B (en) * | 2018-08-08 | 2020-11-13 | 浙江大学 | High-purity bamboo leaf carbon glycoside flavone nano particle, preparation method and application thereof |
| CN109157465A (en) * | 2018-08-08 | 2019-01-08 | 浙江大学 | High-purity leaf of bamboo carbon glycosides flavones nanoparticle and its preparation method and application |
| CN110372655A (en) * | 2019-08-13 | 2019-10-25 | 武汉工程大学 | The method and application of antimycotic flavonoid component are extracted from indicalamus leaf |
| CN110420295A (en) * | 2019-09-03 | 2019-11-08 | 四川丰泰食品科技有限公司 | A kind of technique separating bamboo-leaves flavones |
| CN111388608A (en) * | 2020-03-24 | 2020-07-10 | 江西维莱营健高科有限公司 | Phoenix bambusoides extract and preparation method and application thereof |
| CN112321578A (en) * | 2020-11-18 | 2021-02-05 | 劲牌有限公司 | Method for preparing four monomers in bamboo leaf flavone by Prep-HPLC |
| CN113813642A (en) * | 2021-10-14 | 2021-12-21 | 浙江新篁生物技术有限公司 | Bamboo leaf flavone extraction equipment for removing impurities by using absolute ethyl alcohol |
| CN113813642B (en) * | 2021-10-14 | 2022-09-27 | 浙江新篁生物技术有限公司 | Bamboo leaf flavone extraction equipment for removing impurities by using absolute ethyl alcohol |
| CN115124601A (en) * | 2022-07-29 | 2022-09-30 | 浙江医药股份有限公司新昌制药厂 | Purification method of vancomycin hydrochloride |
| CN115403551A (en) * | 2022-10-18 | 2022-11-29 | 贵州汇腾科技有限公司 | Method for extracting and purifying high-quality bamboo leaf flavone from Sasa veitchii Rehd |
| CN116211970A (en) * | 2023-03-22 | 2023-06-06 | 安徽科技学院 | Method for extracting flavone from bamboo leaves and application thereof |
| CN117618514A (en) * | 2024-01-26 | 2024-03-01 | 广州绿徽新材料研究院有限公司 | Preparation method of ultra-high purity nanoscale bamboo leaf total flavone dispersoid |
| CN117618514B (en) * | 2024-01-26 | 2024-04-12 | 广州绿徽新材料研究院有限公司 | Preparation method of ultra-high purity nanoscale bamboo leaf total flavone dispersoid |
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