CN103432193A - Microwave-assisted aqueous two-phase extraction and separation method of kudzu root total flavones - Google Patents
Microwave-assisted aqueous two-phase extraction and separation method of kudzu root total flavones Download PDFInfo
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Abstract
The invention relates to a microwave-assisted aqueous two-phase extraction method for separating kudzu root total flavone, which comprises the following steps: 1) preparation of an aqueous two-phase system: taking an inorganic salt, adding water to dissolve the inorganic salt, and adding an organic solvent to obtain a aqueous two-phase extractant; and 2) microwave-assisted aqueous two-phase extraction: adding kudzu root powder into the aqueous two-phase extractant obtained in the step 1), carrying out microwave-assisted extraction, and carrying out vacuum filtration to obtain an obvious phase-separated kudzu root flavone extracting solution. Compared with the prior art, the method provided by the invention has the following advantages: (1) the method is simple to operate and does not need to wait for phase separation; 2) the process is integrated; 3) the extraction process is quick and efficient; 4) the extraction solvent is low in toxicity; 5) the interfacial tension is small, thereby being beneficial to mass transfer between the two phases; 6) abundant impurities can be removed along with solid matters, thereby being beneficial to purifying the sample; and 7) the extraction method does not have obvious scale-up effect, can easily implement technique amplification and continuous operation, and can be directly connected with the subsequent purification procedure without special treatment.
Description
Technical field
The invention belongs to the extraction separation and purification field of active ingredient of natural product, be specifically related to a kind of microwave-assisted aqueous two-phase and extract the method for separating Radix Puerariae total flavones.
Background technology
The dry root that Radix Puerariae is legume pueraria lobata, be Chinese crude drug commonly used, and since the successive dynasties, book on Chinese herbal medicine is all on the books, and sweet in the mouth, suffering, property are put down, but expelling pathogenic factors from muscles for reducing heat, promoting the production of body fluid to quench thirst, rash, yang invigorating antidiarrheal, dredge the meridian passage, alcoholic intoxication.At present, Radix Puerariae is widely used in and improves cardiovascular and cerebrovascular disease, and in the important compound preparations such as treatment diarrhoea, diabetes, consumption is very big, is the important component of preparation.Include nearly more than 140 in the Pharmacopoeia of the People's Republic of China and " the Sanitation Ministry medicine standard " and plant Gegen compound, wherein mainly contain Radix Puerariae tablet, injection etc.The main effective ingredient of Radix Puerariae is Radix Puerariae total flavones, and especially Herba Gelsemii Elegantis is more containing Radix Puerariae total flavones, mainly as the raw material of kudzu vine root preparation, medicine, health product etc.The medicine aspect, Radix Puerariae flavone is mainly used in treating cardio-cerebralvascular diseases at present, as diseases such as hypertension, hyperlipidemia, migraine, coronary heart disease, myocardial infarctions, substitutes the Chinese medicament kudzu-vine root in Chinese patent medicine.Simultaneously because of the low and significantly unique biological activity of its toxicity, Radix Puerariae flavone is used as natural additive, is widely used in health food (complication relieve the effect of alcohol, delaying female climacteric, diabetes caused etc.) or the fields such as food, cosmetic industry (as food coloring, sweeting agent, antioxidant etc.).
The flavone compound contained in Radix Puerariae exists mainly with the form of glucosides, and while generally extracting, water, methanol, the alcoholic solution of adopting are made the extraction solvent more, with n-butyl alcohol, make extractant.But the extractant of selecting often toxicity is larger, extraction time is also longer, time consumption and energy consumption.
The microwave-assisted technology is to utilize the function of volume heating and the selectivity heating of microwave field, accelerates the carrying out of leaching process.Select microwave or translucent and have the medium of stronger solvability to make solvent to target component, in microwave field, the opaque material internal molecule of microwave is mutually collided, pushes under microwave action, be conducive to the Rapid Leaching of target component.Microwave method has high selectivity and high efficiency, when greatly having shortened the response time, can improve reaction yield again.
Aqueous two phase extraction technique (Aqueous two-phase extraction, ATPS) claims again aqueous solution biphase distribution technique, is a novel extraction isolation technics that has development prospect of studying in recent decades and growing up.Aqueous two phase extraction technique but, due to its mild condition, mass transport is fast, cost is low, easy to operate, scalable is easily amplified, but the characteristics such as continued operation with traditional organic solvent extraction system, compare, original superior part is arranged in the separation of biological substance.Traditional high polymer/high polymer or high polymer/inorganic salt double-aqueous phase system, success for separating of with extract range protein (polypeptide and enzyme), the fields such as active ingredient of the various antibiotic of separation and extraction, aminoacid, metal and Chinese medicine.
The double-aqueous phase system that high polymer forms, although extraction efficiency is high, cost is too high, and the system viscosity is large for Polyethylene Glycol used (being called for short PEG) and glucosan (being called for short Dextran), it is difficult to reclaim, thereby cost is high, is unsuitable for industrial mass production.And lower alcohol (as ethanol) is low with the double-aqueous phase system toxicity of the aqueous solution formation of salt, easy to the reclaimer operation of organic solvent, can recycle, cost is low.Its principle is to utilize the hydration of salt ion, thereby jointly compete hydrone with ethanol, to form the aqueous solution of ethanol water and salt biphase.When the effective ingredient in biological substance or natural product enters double-aqueous phase system, due to the existence of surface nature, charge effect and various power (hydrophobic key, hydrogen bond and ionic bond) and the impact of environmental factors (kind of concentration, temperature, pH value and ion and intensity etc.), the effective ingredient in biological substance or natural product easily optionally distributes in two alternate formation.
Summary of the invention
Technical problem to be solved by this invention is for above-mentioned prior art, to provide a kind of microwave-assisted aqueous two-phase to extract the method for separating Radix Puerariae total flavones, not only avoided the use of poisonous extractant, improved the extraction ratio of total flavones in the Radix Puerariae, and extraction and separating step one step are completed, greatly simplified operating procedure.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of microwave-assisted aqueous two-phase extracts the method for separating Radix Puerariae total flavones, it is characterized in that comprising the following steps:
1) preparation double-aqueous phase system: get inorganic salt, add water dissolution, add organic solvent, i.e. phase-splitting becomes the aqueous two-phase extractant;
2) the microwave-assisted aqueous two-phase extracts: add powder of Radix Puerariae in step 1) gained aqueous two-phase extractant, adopt microwave-assisted to extract, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.
Press such scheme, described inorganic salt is three water dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium carbonate or ammonium sulfate.
Press such scheme, described organic solvent is dehydrated alcohol, the 35%-55% that described dehydrated alcohol consumption is aqueous two-phase extractant gross mass.
Press such scheme, the 20%-40% that the consumption of described inorganic salt is aqueous two-phase extractant gross mass.
Press such scheme, described powder of Radix Puerariae and aqueous two-phase extractant solid-to-liquid ratio are 1: 5-1: 30, and unit is g: ml.
Press such scheme, the extraction temperature that described microwave-assisted extracts is 40 ℃-90 ℃, and extraction time is 5-20 minute, and microwave power is 400-700W.
Press such scheme, the pH of described aqueous two-phase extractant is 7-11.
The present invention uses double-aqueous phase system to make solvent in leaching process, and under operative temperature, biphase also phase-splitting is obvious, powder of Radix Puerariae just be suspended in biphase between, enter phase after the flavone that is soluble in the ethanol phase extracts, the polysaccharide that is soluble in water is dissolved in lower phase.Like this, extraction and separation process are carried out simultaneously.The Radix Puerariae flavone significant enrichment is in upper phase, lower almost do not have in mutually, the mass fraction of the salt added by change changes the volume of upper and lower phase, reduce upper phase volume, reach the concentrated purpose of enrichment, Radix Puerariae flavone is due to its construction features, be soluble in weakly alkaline solution, the aqueous two-phase extractant that dipotassium hydrogen phosphate and ethanol form is alkalescence, and the pH scope, at 7-11, is conducive to the stripping of Radix Puerariae flavone.Experiment confirms, from yield and Radix Puerariae flavone in the distribution condition of phase up and down, the effect of dipotassium hydrogen phosphate all is undoubtedly best, if use ammonium sulfate, is subject to temperature limiting, when room temperature is low, be difficult for to form aqueous two-phase, if use potassium carbonate, Radix Puerariae flavone has a distribution biphase up and down, and difference is little, and the purpose of this experiment is to wish that Radix Puerariae flavone is enriched in phase.
Compared with the prior art, the advantage of the method for aqueous two-phase extraction Radix Puerariae flavone provided by the present invention is:
1) simple to operate, without waiting for phase-splitting
Observe in experiment, after salt, water, ethanol are added successively, add Amylum Puerariae Radicis can enter extraction step, need not wait for that phase-splitting is obvious, yield is constant.
2) process integration
Microwave-assisted aqueous two-phase extraction method is integrated into a step leaching process by extraction, extract and separate and enriching step, simple to operation, completed and extracted enrichment in leaching process, Radix Puerariae flavone and the component of pueraria polysaccharide and other phases soluble in water are separated, and Radix Puerariae flavone is enriched in phase, cycles of concentration can reach more than 4 times.
3) leaching process is rapid, efficient
By microwave irradiation effect, will originally need the leaching process of some hours to foreshorten in 15 minutes, the yield of Radix Puerariae flavone is high.
4) extract the solvent low toxicity
Adopt ethanol/inorganic salt aqueous two-phase to make extractant, avoided the use of toxic solvent as n-butyl alcohol, the raw material environmental protection.
5) interfacial tension is little, contributes to the quality transmission between biphase
Described double-aqueous phase system, upper is organic solution ethanol mutually, lower is the solution of salt mutually, up and down mutually between surface tension little, free energy is little, contribute to biphase between the transmission of material.
6) a large amount of impurity can together be removed with solid matter, is conducive to purification of samples
After extract finishing, the solid medicinal residues be suspended in biphase between or lower phase, a large amount of impurity can enter lower phase with medicinal residues, with Radix Puerariae flavone, separates.
7) this extracting method enlarge-effect is not obvious, is easy to technique and amplifies and continued operation, with the subsequent purification operation, can directly be connected, without carrying out special handling.
The specific embodiment
Following examples are to further illustrate of the present invention, are not limitations of the present invention.
Embodiment 1:
(1) preparation double-aqueous phase system
Get 12gK
2hPO
43H
2o, add the 22mL water dissolution, adds 18mL ethanol, is formulated as the aqueous two-phase extractant;
(2) the microwave-assisted aqueous two-phase extracts
Gained double-aqueous phase system in step (1), add the powder of Radix Puerariae 0.2g of accurately weighed mistake 60 mesh sieves, and setting microwave condition is 60 ℃, 10min, 500W, carries out the extraction of microwave-assisted aqueous two-phase, and after extracting and finishing, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.
(3) utilize the concentration of Radix Puerariae total flavones in the determined by ultraviolet spectrophotometry two phase aqueous extraction system, press formula (3) calculate recovery rate:
R=V
on/ V
under(1)
K=C
on/ C
under(2)
Y=C
on* V
on/ M * 100% (3)
In formula: R is for comparing; V
onand V
underfor the volume of phase (ml) up and down; C
on, C
underit is respectively the upper and lower mass concentration (mg/mL) of middle target separator mutually; K is partition coefficient; The quality that M is the powder of Radix Puerariae that adds.
The assay of total flavones adopts spectrophotography.Concrete steps are as follows: take a certain amount of extract product of general flavone of kudzuvine root, and accurately weighed, do reference with blank solution, measure absorbance at the 248nm place, the substitution standard curve calculates the content of Radix Puerariae total flavones.
The yield of Radix Puerariae flavone is 12.10%.
Embodiment 2:
(1) preparation double-aqueous phase system
Get 16gK
2hPO
43H
2o, add the 22mL water dissolution, adds 18mL ethanol, is formulated as the aqueous two-phase extractant;
(2) the microwave-assisted aqueous two-phase extracts
Gained double-aqueous phase system in step (1), add the powder of Radix Puerariae 0.2g of accurately weighed mistake 60 mesh sieves, and setting microwave condition is 60 ℃, 10min, 600W, carries out the extraction of microwave-assisted aqueous two-phase, and after extracting and finishing, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.The yield of Radix Puerariae flavone is 12.92%.
Embodiment 3:
(1) preparation double-aqueous phase system
Get 16gK
2hPO
43H
2o, add the 22mL water dissolution, adds 18mL ethanol, is formulated as the aqueous two-phase extractant;
(2) the microwave-assisted aqueous two-phase extracts
Gained double-aqueous phase system in step (1), add the powder of Radix Puerariae 1g of accurately weighed mistake 60 mesh sieves, and setting microwave condition is 60 ℃, 10min, 600W, carries out the extraction of microwave-assisted aqueous two-phase, and after extracting and finishing, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.The yield of Radix Puerariae flavone is 12.72%.
Embodiment 4:
(1) preparation double-aqueous phase system
Get 16gK
2hPO
43H
2o, add the 22mL water dissolution, adds 18mL ethanol, is formulated as the aqueous two-phase extractant;
(2) the microwave-assisted aqueous two-phase extracts
Gained double-aqueous phase system in step (1), add the powder of Radix Puerariae 2g of accurately weighed mistake 60 mesh sieves, and setting microwave condition is 60 ℃, 10min, 700W, carries out the extraction of microwave-assisted aqueous two-phase, and after extracting and finishing, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.The yield of Radix Puerariae flavone is 11.48%.
Embodiment 5:
(1) preparation double-aqueous phase system
Get 14gK
2cO
3, add the 22mL water dissolution, add 18mL ethanol, be formulated as the aqueous two-phase extractant;
(2) the microwave-assisted aqueous two-phase extracts
Gained double-aqueous phase system in step (1), add the powder of Radix Puerariae 1.5g of accurately weighed mistake 60 mesh sieves, and setting microwave condition is 60 ℃, 10min, 700W, carries out the extraction of microwave-assisted aqueous two-phase, and after extracting and finishing, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.The yield of Radix Puerariae flavone is 10.23%.
Claims (8)
1. a microwave-assisted aqueous two-phase extracts the method for separating Radix Puerariae total flavones, it is characterized in that comprising the following steps:
1) preparation double-aqueous phase system: get inorganic salt, add water dissolution, add organic solvent, i.e. phase-splitting becomes the aqueous two-phase extractant;
2) the microwave-assisted aqueous two-phase extracts: add powder of Radix Puerariae in step 1) gained aqueous two-phase extractant, adopt microwave-assisted to extract, sucking filtration obtains the Radix Puerariae flavone extracting solution of obvious phase-splitting.
2. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, and it is characterized in that: described inorganic salt is three water dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium carbonate or ammonium sulfate.
3. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, it is characterized in that described organic solvent is dehydrated alcohol, the 35%-55% that described dehydrated alcohol consumption is aqueous two-phase extractant gross mass.
4. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, the 20%-40% that the consumption that it is characterized in that described inorganic salt is aqueous two-phase extractant gross mass.
5. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, it is characterized in that described powder of Radix Puerariae and aqueous two-phase extractant solid-to-liquid ratio are 1: 5-1: 30, and unit is g: ml.
6. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, it is characterized in that the extraction temperature that described microwave-assisted extracts is 40 ℃-90 ℃, and extraction time is 5-20 minute, and microwave power is 400-700W.
7. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, and the pH that it is characterized in that described aqueous two-phase extractant is 7-11.
8. microwave-assisted aqueous two-phase according to claim 1 extracts the method for separating Radix Puerariae total flavones, the 27%-34% that the consumption that it is characterized in that described inorganic salt is aqueous two-phase extractant gross mass.
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