CN102863485A - Process for purifying arctiin crude product - Google Patents
Process for purifying arctiin crude product Download PDFInfo
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Abstract
The invention discloses a process for purifying an arctiin crude product. The process comprises that the arctiin alcohol extract crude product is dynamically adsorbed and eluted by D4006 macroporous adsorbent resin to obtain a primary purifying product, and then high-purity arctiin is obtained through silica gel column chromatographic purification. According to the process, the D4006 macroporous adsorbent resin which is high in adsorption quantity ratio and desorption rate of the arctiin is screened out through static and dynamic adsorption and elution. The elution scheme is optimized, an arctiin sample solution is subjected to D4006 macroporous adsorbent resin adsorption-elution and silica gel column chromatographic purification, the recovery ratio of the arctiin in the purifying product obtained through high-performance liquid chromatography is 95.4%, the content can be within a range of 81.3-82.4%, and the purifying effect is significantly higher than that reported previously. The process has the advantages of being low in production cost, simple, practical, high in operability, good in purifying effect and the like, and the process is applicable to industrial production.
Description
Technical field
The invention belongs to the Chinese drug preparation technique field, relate to a kind of purification process of drug extract, be specifically related to a kind of purification process of arctinin crude product.
Background technology
Great Burdock Achene is the fruit of the drying and ripening of feverfew burdock (Arctium lappa L.), and original name is disliked real, begins to be stated from " Mingyi Bielu ", classifies middle product as.Great Burdock Achene all has distribution in China various places, records that " Chinese pharmacopoeia has dispelling wind and heat pathogens, a surname's lung promoting eruption, the effect of resolving toxin and disinhibiting the throat in going through version.Be used for the treatment of clinically common cold due to wind-heat, coughing with a lot of sputum, measles, rubella, swelling and pain in the throat, mumps erysipelas, carbuncle sore tumefacting virus.
Arctinin (Arctiin) content in Great Burdock Achene is the main biological activity precursor substance of its performance pharmacological action (Chinese Pharmacopoeia, 2005 editions) more than 5%.Arctinin is not participated in the regulation and control body physiological function directly, but after being metabolized to l-arctigenin (Arctigenin) under the effect of human or animal's intestinal microflora, body is brought into play effect (the Nose M such as clearing heat and detoxicating, antiviral and antitumor, et al.Planta Medica 1992,58 (6): 520-523; Kato T, et al.Anticancer Research 1998,18 (2A): 1053-1057).On chemical structure, arctinin lacks a part glucosyl group than l-arctigenin.
At present, the method for preparing arctinin mainly contains alcohol extracting method, ultrasonic auxiliary alcohol extracting method, microwave-assisted alcohol extracting method and super critical extraction, the purity high (about 40%-60%) of the arctinin that rear three kinds of methods are extracted, but needed plant and instrument is expensive, therefore difficult realization commercial running scale production; And the alcohol extracting rule is mainly used extraction using alcohol, and reagent is cheap, and method is easy, but the purity low (about 5%) of the arctinin product that extracts often contains and could use after the compounds such as more polysaccharide, protein all need to be further purified.At present, relate to the less of the sub-glycosides ethanol extraction of purified edible burdock purifying crude research, Wang Weidong etc. (" Food science ", 2009,20 (18): the purity that 187-191) adopts the HPD-700 type macroporous adsorbent resin than adsorptive capacity large (50.38mg/kg), desorption efficiency higher (88.50%) that the microwave-assisted alcohol extracting method is extracted is that 39.2% arctinin crude product carries out purifying, make the concentration of final sterling reach 63.8%, be 1.6 times of crude product, purification effect is better.And Sun Yu etc. (" Chinese experimental pharmacology of traditional Chinese medical formulae magazine ", 2011,17 (7): the desorption efficiency that 13-15) filters out is 33.78% D101 macroporous adsorbent resin to purity is that 5.7% arctinin extraction using alcohol crude product carries out purifying, the concentration of gained arctinin sterling only is 46.95%-60.53%, and is relatively low.
Summary of the invention
The objective of the invention is the above-mentioned deficiency for prior art, a kind of purification process of arctinin crude product is provided.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of purification process of arctinin crude product, the method that application D4006 type macroporous adsorbent resin and silica gel column chromatography combine is carried out purifying to Great Burdock Achene alcohol extracting thing crude product and is got highly purified arctinin, and the content of arctinin is 4.5 ~ 6.0% in the described Great Burdock Achene alcohol extracting thing crude product.Be that Great Burdock Achene alcohol extracting thing crude product obtains elementary purified product through D4006 type macroporous adsorbent resin dynamic adsorption-wash-out, obtain highly purified arctinin through purification by silica gel column chromatography again.
Wherein, described Great Burdock Achene alcohol extracting thing crude product prepares by the following method: get the Great Burdock Achene medicinal powder, behind 8 ~ 12 times of amount petroleum ether degreasings, add 70% ethanol of 7 ~ 9 times of amounts, refluxing extraction 2 ~ 3 times, each 1 ~ 1.5h, united extraction liquid.
Described Great Burdock Achene alcohol extracting thing crude product is by the following method preparation preferably: get the Great Burdock Achene medicinal powder, behind 10 times of amount petroleum ether degreasings, 70% ethanol that adds 8 times of amounts, refluxing extraction 3 times, each 1h, united extraction liquid, the content of measuring arctinin through high performance liquid chromatography (HPLC) is that the 5.4%(chromatographic condition is: Agilent ZorbaXSB-C18(5 μ m, the chromatographic column of 4.6mm * 250mm); Moving phase is methyl alcohol: water (1:1.1); Measure wavelength 280nm; Flow velocity 0.5mL/min; Sample size 10 μ L; Column temperature is room temperature).
Described extracting solution is through decompressed concentrate, add 20% ethanol, fully stir, supersound process 20min, it is fully dissolved, 0.45 (methyl alcohol: water=1:1) be settled to 1000mL, the mass concentration of arctinin is 4.90mg/mL in HPLC working sample solution, and is for subsequent use with moving phase behind the μ m membrane filtration.
Described D4006 type macroporous adsorbent resin is nonpolar macroporous adsorption resin, and outward appearance is the opaque particle of oyster white, and particle size range is 0.3～1.0mm, and specific surface area is 400～440m
2/ g, the aperture is 6.5～7.5nm, pore volume is 0.73～0.77mL/g, the D4006 type macroporous adsorbent resin that preferred Chemical Plant of Nankai Univ. produces.
The new resin pretreatment process of D4006 type is: 95% alcohol immersion 24h, behind the dress post with 95% ethanol be washed till effluent liquid mix with water do not produce white casse till, then wash most ethanol with deionized water, use again 5%(V/V) HCl solution soaking 3h, washing, use again 5%(V/V) NaOH soak 3h, deionized water is washed till neutrality, and is for subsequent use.
The macroporous adsorbent resin elution protocol is, the resin column volume is 130mL, the loading flow rate control be 1 ~ 2 column volume/hour, the post blade diameter length ratio is controlled to be 1:5 ~ 1:8; After washing is except desaccharification and protein, change again 45% ~ 55% ethanol elution with 4BV, collect 50% pure washing lotion, concentrate drying namely gets elementary purified product.
The macroporous adsorbent resin elution protocol is preferred: the resin column volume is 130mL(and is equivalent to dried resin 20g), the loading flow rate control is 1 column volume/hour (BV/h), the post blade diameter length ratio is controlled to be 1:5; After washing is except desaccharification and protein, change again the ethanol elution with 4BV50%, collect 50% pure washing lotion, concentrate drying namely gets elementary purified product.
The elementary purified product of gained arctinin is with chloroform: after methyl alcohol (9:1) dissolving, silica gel H column chromatography on the dry method, isocratic elution, with thin-layer chromatography chromatography monitoring stream fluid, collect and also to merge the arctinin effluent liquid, in the described arctinin effluent liquid of high effective liquid chromatography for measuring the purity of arctinin reach 81.3% or more than.
The invention has the beneficial effects as follows: the present invention filters out the ratio adsorptive capacity of arctinin and the high D4006 macroporous adsorbent resin of desorption efficiency by static, dynamic adsorption-elution test.By optimizing elution protocol, the arctinin sample liquid is through D4006 type macroporous adsorbent resin dynamic adsorption-elution process and purification by silica gel column chromatography, the rate of recovery of arctinin is 95.4% in efficient liquid phase chromatographic analysis gained sterling, content can reach 81.3-82.4%, arctinin purity behind this method purifying is more than 15 times of original crude product (5.4%), changes effect and is significantly higher than report before.Therefore, method of the present invention can be used in the purification process of arctinin crude product.
The method of the invention has the advantages such as production cost is low, simple and practical, strong operability, and purification effect is good, is fit to suitability for industrialized production.The present invention has successfully solved the problem that arctinin purifying crude technique falls behind.Use the high purity arctinin of gained of the present invention to can be used as in the medicament that bulk drug is applied to various relative diseases.
Description of drawings
Fig. 1 different in flow rate is to the dynamic adsorption curve of D-4006 type macroporous adsorbent resin.
Fig. 2 D-4006 macroporous resin is to the dynamic analysis curve of arctinin.
Embodiment
The preparation of embodiment 1 arctinin crude product
Take by weighing Great Burdock Achene medicinal powder 100g, after the degreasing of 10 times of amount sherwood oils (being the 1L sherwood oil), 70% ethanol (being 800ml70% ethanol) that adds 8 times of amounts, refluxing extraction 3 times, each 1h, united extraction liquid, the content of measuring arctinin through high performance liquid chromatography (HPLC) is that the 5.4%(chromatographic condition is: Agilent ZorbaXSB-C18 (5 μ m, the chromatographic column of 4.6mm * 250mm); Moving phase is methyl alcohol: water (1:1.1); Measure wavelength 280nm; Flow velocity 0.5mL/min; Sample size 10 μ L; Column temperature is room temperature.); Alcohol extract adds 20% ethanol through decompressed concentrate, fully stirs, supersound process 20min fully dissolves it, and (methyl alcohol: water=1:1) is settled to 1000mL with moving phase behind the 0.45 μ m membrane filtration, the mass concentration of arctinin is 4.90mg/mL in HPLC working sample solution, and is for subsequent use.
The screening of embodiment 2 macroporous adsorbent resins
1) pre-treatment of resin
Selected D4006, AB-8, NKA-9, NKA-II type macroporous adsorbent resin are available from Chemical Plant of Nankai Univ.; HZ807, HP100 type macroporous resin provide (physicals of each resin sees Table 1) by the Zheng Yonghua of food science and technology institute of Agricultural University Of Nanjing professor laboratory.The pretreatment process of new resin is: 95% alcohol immersion 24h, behind the dress post with 95% ethanol be washed till effluent liquid mix with water do not produce white casse till, then wash most ethanol with deionized water, use again 5%(V/V) HCl solution soaking 3h, washing, use again 5%(V/V) NaOH soak 3h, deionized water is washed till neutrality, and is for subsequent use.
The physicals of table 1 different model macroporous adsorbent resin
2) screening of resin
The pretreated resin of equivalent is dressed up the resin column of 6 equal-specifications, and each adds 20mL sample solution (embodiment 1 preparation, arctinin concentration is 4.90mg/mL) and carries out dynamic adsorption, collects effluent liquid and measures wherein total arctinin content, calculates the resin absorption amount.Adsorptive capacity (mg/g dried resin)=[(sample liquid concentration-outflow concentration) * adsorption liquid volume]/resin quality.The various resin filter that above-mentioned absorption is saturated again, water filling wash-out fully after (the Monish reaction is negative), 70% ethanol that adds again 20mL is eluted to terminal point with identical flow velocity, measures the content of arctinin in the stripping liquid, calculates the desorption efficiency of each resin.Desorption efficiency (%)=[(stripping liquid concentration * stripping liquid volume)/((sample liquids concentration * adsorption volume)-(outflow concentration * adsorption liquid volume))] * 100.Six kinds of absorption with macroporous adsorbent resin amounts and desorption efficiency the results are shown in Table 2.
Six kinds of absorption with macroporous adsorbent resin amounts of table 2 and desorption efficiency
As seen from table, D4006 type macroporous adsorbent resin is adsorptive capacity large (41.9mg/g) not only, and desorption efficiency high (96.9%), determines to select D4006 type resin.
The process parameter optimizing of embodiment 3 D4006 type anion exchange resin separation and concentration arctinins
1) the resin blade diameter length ratio is investigated
The D4006 type macroporous adsorbent resin that regeneration is good, wet method is loaded on 3 diameters and is in the post of 2cm, and resin column is respectively 6,10,16cm is high, and the resin column volume is about respectively 80,130,210mL.Get the sample solution that concentration is 4.9mg/mL, loading is opened switch by resin absorption respectively, and flow velocity is 1 column volume/hour (1BV/h), checks effluent liquid with thin layer chromatography, during to generation and the corresponding spot of reference substance chromatogram, stops loading.Resin column is with an amount of washing, and is negative to the Monish reaction, collects all effluent liquid, be concentrated into proper volume after, constant volume is drawn above-mentioned every duplicate samples solution 10 μ L, the HPLC method is measured the content of arctinin, calculates the ratio adsorptive capacity of each resin column, the results are shown in Table 3.
The different post blade diameter length ratios of table 3 are on the impact of D-4006 type macroporous resin adsorption arctinin
The result shows that the adsorption effect of resin is best during blade diameter length ratio 1:5, and along with the increase of blade diameter length ratio, the utilization ratio of resin is relatively low.Therefore, blade diameter length ratio is decided to be 1:5.
2) the absorption flow velocity is investigated
Get respectively the good D4006 type macroporous adsorbent resin of having regenerated, wet method is loaded on 3 diameters and is in the post of 2cm, and resin all fills the 10cm height, and the resin column volume is about 130mL(and is equivalent to dried resin 20g), for subsequent use.Get the sample solution that concentration is 4.9mg/mL.Loading is opened switch by resin absorption respectively, and the absorption flow rate control is 1BV/h, 2BV/h, 3BV/h, every 10mL collects an effluent liquid, has collected respectively 12,9 and 8 parts, checks effluent liquid with the thin-layer chromatography chromatography simultaneously, during to generation and the corresponding spot of reference substance chromatogram, stop loading.Every part of equal precision of effluent liquid pipettes 1.0mL, add moving phase after volatilizing and be settled to 10mL, draw above-mentioned every duplicate samples solution 10 μ L, by aforementioned chromatographic condition, content with HPLC external standard method arctinin, calculate adsorptive capacity, than adsorptive capacity, draw the dynamic adsorption curve of 3 kinds of flow velocitys, the results are shown in Table 4 and Fig. 1.
The different absorption of table 4 flow velocity is on the impact of D-4006 type macroporous resin adsorption arctinin
Table 4 shows that macroporous adsorbent resin was more to the adsorptive capacity of arctinin when flow velocity was 1BV/h, had higher practical value, and flow velocity is when being 2BV/h and 3BV/h, and macroporous resin is less to the adsorptive capacity of arctinin.
Fig. 1 shows that when flow velocity was 1BV/h, resin had leakage since the 13rd part; When flow velocity was 2BV/h, resin also had leakage since the 9th part; When flow velocity was 3BV/h, resin had leakage since the 8th part.Fig. 1 shows, take the flow velocity of 1BV/h as better, consistent with the result of table 4, it is comparatively moderate that adsorb flow velocity this moment, larger than adsorptive capacity, can satisfy the greatly needs of production to the absorption of arctinin for D4006 type macroporous adsorbent resin.
3) investigation of eluent
Pretreated D4006 resin dress post, the sample solution of getting 4.9mg/mL adsorbs according to upper method, use successively 30%, 50%, 75%, 95% ethanol, press the abundant wash-out of 1BV/h flow velocity, collect respectively rear every part of the suitable dilution of elutriant and get 10 μ L, by aforementioned chromatographic condition, with the content of HPLC external standard method arctinin, calculate the eluting rate of each wash-out post, the results are shown in Table 5.
Table 5 different concentration ethanol is to the wash-out situation of arctinin on the D-4006 type macroporous resin column
As shown in Table 5, during 50% ethanol elution, the D-4006 resin is to the elution amount of arctinin, all higher than elution amount and eluting rate, and therefore selecting 50% ethanol is eluent.
4) investigation of elution curve
Pretreated D4006 resin fills post, gets the sample solution of 4.9mg/mL, the difference loading, and the absorption flow rate control is 1BV/h, checks effluent liquid with the thin-layer chromatography chromatography, during to generation and the corresponding spot of reference substance chromatogram, stops loading.Wash through 3 times of water gagings, washing speed 1BV/h, negative to the Monish reaction.Again with 8 times of amounts of 50% ethanol resin volume wash-out, elution speed 2BV/h, Fractional Collections 50% ethanol eluate.Each resin volume (humid volume) is collected 1 part, collects altogether 8 parts.Measure respectively arctinin concentration, and take the wash-out umber as X-coordinate, arctinin concentration (mg/mL) is drawn elution curve (Fig. 2) for ordinate zou.
Fig. 2 shows that arctinin content obviously reduces in the 4th part of elutriant, and wash-out is complete can to think the arctinin that adsorbs on the resin column, thus determine that the eluent consumption is 4 times of resin volumes, this moment elution peak concentrate, symmetrical, without obvious conditions of streaking.
Determine that according to above experimental data elution protocol is: the loading flow rate control is 1BV/h, and the post blade diameter length ratio is controlled to be 1:5; After washing is except desaccharification and protein, change again 50% ethanol elution with 4BV, collect 50% pure washing lotion, concentrate drying namely gets elementary purified product.
The sub-glycosides of embodiment 4 silica gel column chromatography purified edible burdocks
The elementary purified product of arctinin of getting gained among 3 parts of embodiment 3 is with an amount of chloroform: after methyl alcohol (9:1) dissolving, and silica gel H column chromatography on the dry method, isocratic elution with thin-layer chromatography chromatography monitoring stream fluid, is collected the arctinin effluent liquid.Merge effluent liquid, detect its purity through the HPLC method, the results are shown in Table 6.
Table 6 silica gel column chromatography is on the impact of arctinin purity
As shown in Table 6, through macroporous resin and purification by silica gel column chromatography, the purity 81.3-82.4% of arctinin is more than 15 times of arctinin purity (5.4%) in the alcohol extracting crude product.
Present method simple possible, with low cost, purity is effective, is adapted at using in the enterprise scale production, has good prospects.
Claims (9)
1. the purification process of an arctinin crude product, it is characterized in that using method that D4006 type macroporous adsorbent resin and silica gel H column chromatography combine is carried out purifying to Great Burdock Achene alcohol extracting thing crude product and highly purified arctinin, the content of arctinin is 4.5 ~ 6.0% in the described Great Burdock Achene alcohol extracting thing crude product.
2. purification process according to claim 1, it is characterized in that, described Great Burdock Achene alcohol extracting thing crude product prepares by the following method: get the Great Burdock Achene medicinal powder, behind 8 ~ 12 times of amount petroleum ether degreasings, 70% ethanol that adds 7 ~ 9 times of amounts, refluxing extraction 2 ~ 3 times, each 1 ~ 1.5h, united extraction liquid.
3. purification process according to claim 2 is characterized in that, described Great Burdock Achene alcohol extracting thing crude product prepares by the following method: get the Great Burdock Achene medicinal powder, behind 10 times of amount petroleum ether degreasings, add 70% ethanol of 8 times of amounts, refluxing extraction 3 times, each 1h, united extraction liquid.
4. purification process according to claim 3 is characterized in that, described extracting solution adds 20% ethanol through decompressed concentrate, fully stirs, and supersound process 20min fully dissolves it, is used for upper purification with macroreticular resin.
5. purification process according to claim 1 is characterized in that, described D4006 type macroporous adsorbent resin is nonpolar macroporous adsorption resin, and particle size range is 0.3～1.0mm, and specific surface area is 400～440m
2/ g, the aperture is 6.5～7.5nm, pore volume is 0.73～0.77mL/g; The D4006 type macroporous adsorbent resin that the preferred Chemical Plant of Nankai Univ. of described D4006 type macroporous adsorbent resin produces.
6. purification process according to claim 1 is characterized in that, the macroporous adsorbent resin elution protocol is, the resin column volume is 130mL, the loading flow rate control be 1 ~ 2 column volume/hour, the post blade diameter length ratio is controlled to be 1:5 ~ 1:8; After washing is except desaccharification and protein, change again 45% ~ 55% ethanol elution with 4BV, collect 50% pure washing lotion, concentrate drying namely gets elementary purified product.
7. purification process according to claim 6 is characterized in that, the macroporous adsorbent resin elution protocol is, the resin column volume is 130mL, the loading flow rate control be 1 column volume/hour, the post blade diameter length ratio is controlled to be 1:5; After washing is except desaccharification and protein, change again 50% ethanol elution with 4BV, collect 50% pure washing lotion, concentrate drying namely gets elementary purified product.
8. purification process according to claim 1, it is characterized in that, claim 4 or the elementary purified product of 5 gained arctinins are with chloroform: after methyl alcohol=9:1 dissolving, silica gel H column chromatography on the dry method, isocratic elution, with thin-layer chromatography chromatography monitoring stream fluid, collect and merging arctinin effluent liquid.
9. purification process according to claim 8 is characterized in that, in the described arctinin effluent liquid of high effective liquid chromatography for measuring the purity of arctinin reach 81.3% or more than.
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