CN104829456A - Method for purifying caffeoyl guinic acid compounds in purple sweet potato stem and leaf through macro-porous resin - Google Patents
Method for purifying caffeoyl guinic acid compounds in purple sweet potato stem and leaf through macro-porous resin Download PDFInfo
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- CN104829456A CN104829456A CN201510158086.5A CN201510158086A CN104829456A CN 104829456 A CN104829456 A CN 104829456A CN 201510158086 A CN201510158086 A CN 201510158086A CN 104829456 A CN104829456 A CN 104829456A
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- rhizoma dioscoreae
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- C07—ORGANIC CHEMISTRY
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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Abstract
The invention discloses a method for purifying caffeoyl guinic acid compounds in purple sweet potato stem and leaf through macro-porous resin. The method comprises the following steps: drying purple sweet potato stem and leaf at 60DEG C, crushing, extracting the above obtained dried raw material at 80-100DEG C for 0.5-1.5h, filtering, extracting obtained raw material residues under same conditions through a same extraction process once, mixing the above obtained extraction liquids, carrying out vacuum concentration on the above obtained extraction liquid mixture at 55DEG C to recover ethanol in order to obtain a purple sweet potato stem and leaf extraction liquid, adjusting the extract liquid to the content of the caffeoyl guinic acid compounds of 1-3mg/ml and the pH value of 2-3, and adding the obtained sample to macro-porous resin according to an adding flow velocity of 2-3BV/h and a sample volume of 5BV or less; and eluting by adopting 5-6BV of water until colorlessness, sequentially eluting by using different concentrations of ethanol according to a flow velocity of 2-3BV/h, respectively concentrating the above obtained eluates, and freeze-drying the eluates to obtain the following two products with different specifications: a 20% ethanol elution product with the content of the caffeoyl guinic acid compounds of above 20%, and a 40-70% ethanol elution product with the content of the caffeoyl guinic acid compounds of above 60%. The method enlarges the natural source of the caffeoyl guinic acid compounds.
Description
Technical field
The invention belongs to caffeoylquinic acid research and extract field, particularly relate to the method for caffeoylquinic acid research in a kind of macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf.
Background technology
Caffeoylquinic acid research is plant class phenylpropanoids through the synthesis of phosphopentose pathway intermediate in aerobic repiration process, they are extensively present in plant, there is significant anti-oxidant activity and pharmacologically active, as anti-inflammatory, HIV (human immunodeficiency virus)-resistant activity, antibacterial, suppress multiple enzymic activity, protect the liver, suppress the multiple effects such as platelet aggregation, be the effective constituent of numerous medicinal material, and become the important indicator of some Chinese medicine preparation quality control.Except medicinal, also can be used as antioxidant and be applied to foodstuffs industry, also there is flavouring and color-protecting function simultaneously, can be used for food and fruit fresh-keeping.
Sweet potato be convolvulaceae sweet potato genus 1 year with raw or the perennial herbaceous plant that overgrows, cultivation many countries and regions all over the world of current sweet potato, annual production reaches 1.4 × 10
8ton is important grain, beverage and industrial raw material.China is sweet potato cultivation in the world first big country, accounts for 85.9% of Gross World Product, total product arrangement the 4th in China's grain effect is produced.The exploitation of current people to leaf potato leaf are less, and to act as vegetables edible outer and be processed into outside feed except a small amount of, are most ofly abandoned in field, cause the serious waste of resource.
Applicant's early-stage Study is isolation identification chlorogenic acid, 4 from sweet potato leaves and stems; the two caffeoyl quinic acid, 3 of 5-; the two caffeoyl quinic acid, 3 of 5-; the flavonoid compounds such as caffeoylquinic acid research and isoquercitrin such as the two caffeoyl quinic acids of 4-; further research shows to be mainly caffeoylquinic acid research in purple potato cauline leaf, and flavonoid content is extremely low.
Summary of the invention
The object of the embodiment of the present invention is the method providing caffeoylquinic acid research in a kind of macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf, be intended to solution less to the exploitation of leaf potato leaf at present, cause the problem of the wasting of resources, expand the natural origin of caffeoylquinic acid research simultaneously.
The present invention is achieved in that the method for caffeoylquinic acid research in a kind of macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf comprises:
Step one, Rhizoma Dioscoreae esculentae cauline leaf crushed after being dried at 60 DEG C, gets the Rhizoma Dioscoreae esculentae cauline leaf extraction using alcohol 0.5-1.5h of this drying and crushing, filters, and the raw material slag of gained extracts once by identical method, united extraction liquid;
Step 2,55 DEG C of vacuum concentration reclaim ethanol, obtain Rhizoma Dioscoreae esculentae stem leaf extract, adjust the pH of caffeoylquinic acid research content and extracting solution in this extract concentration;
Macroporous adsorbent resin crossed by step 3, extracting solution step 2 obtained, and washed away by the impurity in macroporous adsorbent resin, then uses eluent successively, by elutriant concentrated, the lyophilize respectively obtained.
Further, for extracting 5-15 times that the quality of the ethanol of the Rhizoma Dioscoreae esculentae cauline leaf of drying and crushing is the Rhizoma Dioscoreae esculentae cauline leaf quality of drying and crushing in step one, volume fraction is 50-70%.
Further, the Rhizoma Dioscoreae esculentae stem leaf extract obtained in step 2 is as sample solution, and adjusting caffeoylquinic acid research mass concentration in this sample solution is 1-3mg/ml, and the pH of sample solution is 2-3, and sample solution flow velocity 2-3BV/h, loading volume is less than or equal to 5BV.
Further, the resin selected is NKA-2 resin.
Further, macroporous adsorbent resin is contained in a vertical chromatography, and the sample solution obtained in step 2 flows through macroporous adsorbent resin, and adsorption flow rate is 2-3BV/h.
Further, elution flow rate is 2-3BV/h.
Further, eluent selects volume fraction to be the ethanol of 20% and 40%-70%.
Rhizoma Dioscoreae esculentae cauline leaf is crushed after being dried at 60 DEG C, the 50-70% ethanol getting a certain amount of dried feed 5-15 times quality extracts 0.5-1.5h at 80-100 DEG C, filter, raw material slag extracts once with method again, united extraction liquid, and 55 DEG C of vacuum concentration reclaim ethanol, obtain Rhizoma Dioscoreae esculentae stem leaf extract, adjust this extract concentration to caffeoylquinic acid research content at 1-3mg/ml and pH 2-3, sample solution flow velocity 2-3BV/h, sample Ti Ji≤5BV is advisable; Elution requirement: be washed to colourless with 5-6BV, use 6BV 20% ethanol, 4-6BV 40-70% ethanol with 2-3BV/h flow velocity wash-out more successively, elutriant is concentrated, lyophilize respectively, the product of two kinds of different sizes can be obtained: in 20% ethanol elution product, caffeoylquinic acid research content is greater than 20%, in 40-70% ethanol elution product, caffeoylquinic acid research content is greater than 60%, expands the natural origin of caffeoylquinic acid research.
Accompanying drawing explanation
Fig. 1 is the method flow diagram of caffeoylquinic acid research in the macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf that provides of the embodiment of the present invention.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, this present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain this present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, embodiments of the invention are achieved in that the method for caffeoylquinic acid research in a kind of macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf comprises:
S101, Rhizoma Dioscoreae esculentae cauline leaf crushed after being dried at 60 DEG C, gets the Rhizoma Dioscoreae esculentae cauline leaf extraction using alcohol 0.5-1.5h of this drying and crushing, filters, and the raw material slag of gained extracts once by identical method, united extraction liquid;
S102,55 DEG C of vacuum concentration reclaim ethanol, obtain Rhizoma Dioscoreae esculentae stem leaf extract, adjust the pH of caffeoylquinic acid research content and extracting solution in this extract concentration;
Macroporous adsorbent resin crossed by S103, the extracting solution obtained by step S102, washed away by the impurity in macroporous adsorbent resin, then uses eluent successively, by elutriant concentrated, the lyophilize respectively obtained.
Further, for extracting 5-15 times that the quality of the ethanol of the Rhizoma Dioscoreae esculentae cauline leaf of drying and crushing is the Rhizoma Dioscoreae esculentae cauline leaf quality of drying and crushing in step S101, volume fraction is 50-70%.
Further, the Rhizoma Dioscoreae esculentae stem leaf extract obtained in step S102 is as sample solution, and adjusting caffeoylquinic acid research mass concentration in this sample solution is 1-3mg/mL, and the pH of sample solution is 2-3, and sample solution flow velocity 2-3BV/h, loading volume is less than or equal to 5BV.
Further, the resin selected is NKA-2 resin.
Further, macroporous adsorbent resin is contained in a vertical chromatography, and the sample solution obtained in step S102 flows through macroporous adsorbent resin, and adsorption flow rate is 2-3BV/h.
Further, elution flow rate is 2-3BV/h.
Further, eluent selects volume fraction to be the ethanol of 20% and 40%-70%.
Embodiment one
1. caffeoylquinic acid research content detection in Rhizoma Dioscoreae esculentae cauline leaf
Precision take purity be 98% chlorogenic acid standard substance 6.17mg in 25mL volumetric flask, with 50% ethanol constant volume.Accurate draw above-mentioned reference substance solution 0,0.1,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6mL is in 10mL volumetric flask, add the distilled water of 5ml, shake up, add the Folin-C solution of 0.5mol/L, the sodium carbonate solution of 1.5ml 20% is added after shaking up, constant volume, 25 DEG C of reaction 2h, obtain the standard solution of different concns, with sample blank zeroing, measure absorbancy at 760nm wavelength place, with standard substance quality (mg) for X-coordinate, absorbancy is ordinate zou, equation of linear regression y=4.0976x+0.001, R2=0.993.
Take 1.0g different varieties Rhizoma Dioscoreae esculentae stem, leaf raw material respectively, add 60% ethanolic soln 100ml, put refluxing extraction 1h in the water-bath of 90 DEG C, filter, be settled to 100ml, get 0.2ml sample solution by the colour developing of standard specimen method, measure absorbancy at 760nm wavelength place, according to the caffeoylquinic acid research content in typical curve calculation sample, the results are shown in Table 1.As known from Table 1, between different varieties, caffeoylquinic acid research content has certain difference, and the different sites content of same kind has significant difference, and leaf content is significantly higher than stem and petiole.Be generally Rhizoma Dioscoreae esculentae leaf and tender stem thereof and petiole when considering actual use, subsequent experimental adopts 4 kinds of Rhizoma Dioscoreae esculentae cauline leaf mixtures as experimental raw, and wherein caffeoylquinic acid research content is 34.61mg/g.
The comparison (n=2) of table 1 different sites raw material chlorogenic acid compounds content
2, the screening of resin
After the various macroporous adsorbent resins that pre-treatment is good remove surface water, each precision takes 1.0g, be positioned in tool plug ground Erlenmeyer flask, add the stock liquid that 50mL concentration is 3.40mg/ml respectively, constant temperature 25 DEG C vibrates 24h in shaking table, after balance upon adsorption, filter, measure the concentration of caffeoylquinic acid research in filtrate.The desorb of 30mL70% ethanol respectively used again by the resin of adsorption equilibrium, after desorb completely, measure the concentration of stripping liquid caffeoylquinic acid research, calculate adsorptive capacity and the desorption efficiency of resin, determine the optimum resin type being separated Rhizoma Dioscoreae esculentae leaf caffeoylquinic acid research, the results are shown in Table 2, as seen from the table, the adsorptive capacity of NKA-2 resin and desorption efficiency are all best, therefore select as follow-up work resin.
The different macroporous resin adsorption amount of table 2 and desorption efficiency
3, the selection of sample solution pH
Get concentration 2.13mg/mL concentrated solution 5 parts, every part of 50ml, adjust pH is 2,3 respectively, 4,5,6, add in 1g NKA-2 resin, in 25 DEG C of shaking tables, vibration absorption 5h measures the content of caffeoylquinic acid research in solution before and after absorption, draws the variation relation figure of adsorptive capacity with pH.The results are shown in Table 3, when stock liquid pH is 2-3, the adsorptive capacity of NKA-2 resin is larger.Caffeoylquinic acid research generally exists in the feed in a salt form, and when pH≤3, caffeoylquinic acid research will become molecule free state, thus be conducive to the absorption of resin.So stock liquid adjusts pH to be that 2-3 is advisable before upper prop.
Table 3 stock liquid pH is on the impact of adsorptive capacity
4, the selection of sample solution concentration
Get 4 chromatography columns respectively, load 30ml resin, get mother liquor 25ml, add respectively water 6.5,12.5,25,75,125ml, and regulate pH3, with the flow velocity of 3BV/h by chromatography column, collect effluent liquid, sampling detects.The stock liquid of different concns on the impact of dynamic adsorption in table 4.Can find out from table, along with the increase of concentration, resin leakage also increases, Adsorbent rate reduces, but too low concentration needs longer loading time, is unfavorable for production efficiency, therefore, consider caffeoylquinic acid research mass concentration in sample solution to be advisable with 1-3mg/mL.
Table 4 upper prop concentration is on the impact of absorption
5, the selection of upper column flow rate
Get 4 chromatography columns, load 30ml resin, sample solution respectively with 2,3,4, the flow velocity of 5BV/h by chromatography column, in sample solution, caffetannic acid mass concentration is 2.13mg/mL, pH3, loading volume 50ml, collects effluent liquid sampling and detects, the results are shown in Table 5.As can be seen from the table, along with the increase of upper column flow rate, resin leakage increases, and adsorption rate reduces, but flow velocity is slow, and inefficiency, considers absorption property and the working efficiency of resin, determine that adsorption flow rate is advisable with 2-3BV/h.
On table 5, column flow rate is on the impact of adsorptive capacity
6, the selection of loading volume
By above-mentioned determined adsorption conditions, get pH=3, concentration be the Rhizoma Dioscoreae esculentae leaf caffeoylquinic acid research stock liquid of 2.12mg/mL with the flow velocity of 3BV/h by being equipped with the chromatography column of 50mL NKA-2 resin, collect effluent liquid, sampling detects, the results are shown in Table 6, from table, along with the increase of loading volume, in effluent liquid, caffetannic acid concentration increases, when loading volume is 6BV, adsorption rate, lower than 80%, therefore considers loading Ti Ji≤5BV and is advisable.
Table 6 upper prop volume is on the impact of adsorptive capacity
7, the selection of elution flow rate
Get 4 chromatography columns, fill resin 50ml respectively, get pH=3, concentration be 2.12mg/mL sample liquid 50ml with the above-mentioned condition determined by resin, 100ml washing after carries out wash-out with the flow velocity of 2BV/h, 3BV/h, 4BV/h, 5BV/h respectively with 150ml 70% ethanol, as shown in Table 7, during eluent by same volume, elution flow rate is slower, and eluting rate is higher, and 2-3BV/h is advisable.
Table 7 flow velocity is on the impact of eluting rate
8, the selection of desorption solvent
Get the chromatography column that 1 is equipped with 120ml resin, with pH=3, concentration is that the Rhizoma Dioscoreae esculentae leaf caffeoylquinic acid research stock liquid of 2.12mg/mL is with the flow velocity loading of 3BV/h, loading volume 400ml, leave standstill 0.5h after loading, use the ethanol desorb of 360ml different concns respectively, collect stripping liquid, sampling detects, and the results are shown in Table 8.As can be seen from the table, water, 10% ethanol, 20% ethanol are lower to caffeoylquinic acid research eluting rate, and in elutriant, impurity is many simultaneously.After 40% ethanol elution is complete, total eluting rate can be made to reach 91.58%, when use 70% ethanol elution complete after, total eluting rate can be made to reach 93.68%.After considering that determining alcohol is greater than 70%, the problem such as production cost and security, therefore, eluent 40%-70% ethanol.
The desorption efficiency effect of table 8 different concentration ethanol
9, the determination of elution volume
Get on 300ml load solution by stating the condition determined by being equipped with the chromatography column of 50ml resin, wash-out is carried out with 70% ethanol with 3BV/h flow velocity after being washed to very slight color with 5BV, every 1BV receives once, and the results are shown in Table 9, considering elutriant consumption is that 4-6BV is advisable.
Table 9 flow velocity is on the impact of eluting rate
The above-mentioned purifying process parameter determined is carried out twice amplification test.
Experiment one: chromatography column is amplified to
column length 80cm, resin dress column volume 500ml, feed concentration 1.96mg/mL, loading volume 2290ml, loading flow velocity 2BV/h, 0.5h is left standstill after loading, be washed to 5BV colourless, then use 6BV 20% ethanol, 5BV 70% ethanol with 3BV/h flow velocity wash-out successively, receive elutriant respectively, measure solid substance weight and content, the results are shown in Table 10.
Table 10 amplification technique product situation 1
Experiment two: chromatography column is amplified to
column length 80cm, resin dress column volume 500ml, feed concentration 2.98mg/mL, loading volume 2000ml, loading flow velocity 3BV/h, 0.5h is left standstill after loading, 6BV is washed to colourless, then uses the ethanol of 6BV 20%, the ethanol of 6BV 40% with 2BV/h flow velocity wash-out successively, receives elutriant respectively, measure solid substance weight and content, the results are shown in Table 10.
Table 11 amplification technique product situation 2
In 20% ethanol elution product, caffeoylquinic acid research content is greater than caffeoylquinic acid research content in 20%, 40-70% ethanol elution product and is greater than 60%.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (7)
1. the method for caffeoylquinic acid research in purification with macroreticular resin Rhizoma Dioscoreae esculentae cauline leaf, it is characterized in that, in described macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf, the method for caffeoylquinic acid research comprises:
Step one, Rhizoma Dioscoreae esculentae cauline leaf crushed after being dried at 60 DEG C, gets the Rhizoma Dioscoreae esculentae cauline leaf extraction using alcohol 0.5-1.5h of drying and crushing, filters, and the raw material slag of gained extracts once by identical method, united extraction liquid;
Step 2,55 DEG C of vacuum concentration reclaim ethanol, obtain Rhizoma Dioscoreae esculentae stem leaf extract, the pH of caffeoylquinic acid research content and extracting solution in adjustment extract concentration;
Macroporous adsorbent resin crossed by step 3, extracting solution step 2 obtained, and washed away by the impurity in macroporous adsorbent resin, then uses eluent successively, by elutriant concentrated, the lyophilize respectively obtained.
2. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, it is characterized in that, for extracting 5-15 times that the quality of the ethanol of the Rhizoma Dioscoreae esculentae cauline leaf of drying and crushing is the Rhizoma Dioscoreae esculentae cauline leaf quality of drying and crushing in step one, volume fraction is 50-70%.
3. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, it is characterized in that, the Rhizoma Dioscoreae esculentae stem leaf extract obtained in step 2 is as sample solution, adjusting caffeoylquinic acid research mass concentration in this sample solution is 1-3mg/ml, the pH of sample solution is 2-3, sample solution flow velocity 2-3BV/h, loading volume is less than or equal to 5BV.
4. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, it is characterized in that, the resin selected is NKA-2 resin.
5. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, it is characterized in that, macroporous adsorbent resin is contained in a vertical chromatography column, and the sample solution obtained in step 2 flows through macroporous adsorbent resin, and adsorption flow rate is 2-3BV/h.
6. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, it is characterized in that, elution flow rate is 2-3BV/h.
7. the method for caffeoylquinic acid research in macroporous resin purification Rhizoma Dioscoreae esculentae cauline leaf as claimed in claim 1, is characterized in that, eluent selects volume fraction to be the ethanol of 20% and 40%-70%.
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Cited By (1)
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| CN111067926A (en) * | 2020-01-14 | 2020-04-28 | 仲恺农业工程学院 | Method for extracting and enriching total flavonoids and quinic acid ester compounds in rhinacanthus nasutus |
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