CN105748570A - Ultrasonic-assisted method for extracting total flavonoid in alfalfa - Google Patents
Ultrasonic-assisted method for extracting total flavonoid in alfalfa Download PDFInfo
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
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- A—HUMAN NECESSITIES
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
The invention provides an ultrasonic-assisted method for extracting total flavonoid in alfalfa.The method comprises the following steps: (1) alfalfa is dried and smashed to obtain alfalfa powder; (2) an organic solvent (such as petroleum ether) is added to the alfalfa powder obtained in the step (1), heating reflux is performed for 4-24 hours, filtering is performed so as to remove the organic solvent, the alfalfa powder is obtained, and then the alfalfa powder is dried; (3) an ethanol solution with the volume fraction ranging from 20% to 70% and the alfalfa powder obtained in the step (1) are mixed according to the liquid-to-solid ratio of mL:g ranging from (20:1) to (70:1), extraction is performed at the temperature of 30 DEG C to 80 DEG C and under ultrasonic assistance, and an alfalfa total flavonoid extraction solution is obtained.The extraction rate of the total flavonoid can be improved with the method, the ultrasonic extraction is a physical process, the optimum temperature of the extraction process is about 60 DEG C, active ingredients of the flavonoid material in the alfalfa can be prevented from being changed by high temperature, and the extraction efficiency of the total flavonoid in the alfalfa is improved.
Description
Technical field
The present invention relates to active components of plants extractive technique field, extract the method for total flavones in Herba Medicaginis more particularly to a kind of ultrasonic assistant.
Background technology
Alfalfa is pulse family (Leguminosae) Papillionoideae (PapilionataeTaub) clover race (Trib.trifolieaeBronn) Medicago (MedicagaL) herbaceos perennial, it is distributed widely in China, yield is high, strong adaptability, vegetal pole horn of plenty, enjoying the good reputation of " King of Pasture ", it is because of the attention as good natural plant resource.Flavone compound (Flavonoids) is a class Secondary Metabolite Production in Plants, is distributed widely in plant.Herba Medicaginis flavone compound is one of main component of alfalfa plant extract, has many-sided biological effect, can effectively remove human free radical, have the effect of slight estrogen-like, increases reproductive capacity and the immunity of body within the specific limits.Meanwhile, Herba Medicaginis flavone compound can also as the additive of the additive of functional food, animal feed.Therefore, Herba Medicaginis flavone compound has broad application prospects in fields such as food additive, natural drug, functional foods.
Some are now had been disclosed and extracts the method for flavone compound and total flavones in Herba Medicaginis.Such as " Guangdong feedstuff " the 20th volume the 2nd phase " optimization of a Herba Medicaginis total flavones extraction process by water " literary composition, disclosed extraction conditions is: Extracting temperature is 90 DEG C, and extraction time is 4 hours, and solid-to-liquid ratio is 1: 40 (g: mL)；Under this condition the extraction yield of Herba Medicaginis total flavones be 5.862mg/g (the application in full in extractions yield be by every gram of dry material Herba Medicaginis raw material extraction obtain in the amount of the Herba Medicaginis total flavones of milligram).Main drawback is that of the method (1) uses water extracting directly, increases the dissolution of the impurity such as water-soluble saccharides and protein, causes that the purity of total flavones in extract reduces；(2) extraction time is long, increases the consumption of the energy, reduces the service efficiency of equipment.And for example " meadow journal " the 16th volume the 1st phase " research of a microwave radiation exaraction Herba Medicaginis Flavonoids Method " literary composition; disclosed extraction conditions is: concentration of alcohol 40%; solid-to-liquid ratio 1: 30 (g: mL); act on 60 seconds under microwave 500W power; under this extraction conditions, the extraction yield of flavone is only 4.92mg/g.
Summary of the invention
In the present context, term " extraction yield " refer to extracted by every gram of dry material raw material obtain in the amount of the product of milligram.Especially, in the present context, " the extraction yield of Herba Medicaginis total flavones " refer to extracted by every gram of dry material Herba Medicaginis obtain in the amount of the Herba Medicaginis total flavones of milligram.
Unless otherwise indicated, the concentration of alcohol in the present invention refers to the volume fraction of ethanol.
For problems of the prior art, it is an object of the invention to provide a kind of new method extracting total flavones from Herba Medicaginis.The method of the present invention overcomes defect and the deficiency of prior art.It is an object of the invention to by providing techniques below scheme to realize.
The invention provides a kind of ultrasonic assistant and extract the method for total flavones in Herba Medicaginis, the method comprises the following steps:
(1) Alfalfa be dry, pulverize, obtain alfalfa meal；
(2) in the alfalfa meal that step (1) obtains, organic solvent is added, wherein, the ratio of described organic solvent and alfalfa meal is calculated as 10: 1 to 15: 1 by mL: g, it is heated to reflux 4～24 hours, filter to remove organic solvent, obtain alfalfa meal, then the alfalfa meal of gained is dried；Wherein, described organic solvent is one or more the mixed solvent in petroleum ether, pentane, trimethylpentane, hexane, hexamethylene and isobutyltrimethylmethane.；
(3) alfalfa meal that the alcoholic solution that volume fraction is 20～70% is obtained with step (2) according in mL: g 20: 1 to 70: 1 liquid ratio mix, extract at the temperature of 30 DEG C～80 DEG C and ultrasonic assistant, obtain Herba Medicaginis total flavones extracting solution.
Preferably, in the step (1) of the inventive method, before pulverizing, Alfalfa is dried to moisture≤5wt%.
Preferably, in an embodiment of the inventive method, the method for the present invention is additionally included in step (1) by the step sieved of alfalfa meal after pulverizing；Preferably, employing 40～60 mesh sieve that sieves carries out.
Preferably, in the step (2) of the inventive method, described backflow is undertaken by soxhlet extraction.It is highly preferred that in soxhlet extraction, described extraction carries out 18～24 hours.
Preferably, in the step (2) of the inventive method, alfalfa meal is dried to moisture≤5wt%.
Preferably, in the step (2) of the inventive method, described organic solvent is petroleum ether.
Preferably, in the step (3) of the inventive method, the volume fraction of described alcoholic solution is 40～70%, more preferably 52%.
Preferably, in the step (3) of the inventive method, described liquid ratio is calculated as 50: 1 to 70: 1 by mL: g, it is preferred to 57: 1.
Preferably, in the step (3) of the inventive method, described in be extracted in 60～80 DEG C, more preferably carry out at the temperature of 62 DEG C.
Preferably, in the step (3) of the inventive method, described extraction carries out 20～120min；More preferably 60～100min, it is most preferred that 57min.
Preferably, in the step (3) of the inventive method, when extracting under ultrasonic assistant, hyperacoustic power is 100W.
In a preferred embodiment of the inventive method, in step (2), liquid ratio is 57: 1 (mL: g), and concentration of alcohol is 52%, and Extracting temperature is 62 DEG C, and extraction time is 57min.
In an embodiment according to the inventive method, described method also includes step (4): the Herba Medicaginis total flavones extracting solution obtained by step (3) is after vacuum filtration, collect filtrate, obtain general flavone ethanol-aqueous solution, remove the ethanol in Herba Medicaginis general flavone ethanol-aqueous solution, obtaining extractum, then drying obtains the powder containing Herba Medicaginis total flavones.Preferably, in step (4), ethanol is removed by rotary evaporation in vacuo.It is highly preferred that in step (4), described dry as vacuum lyophilization.
The present inventor is surprisingly found out that, in the method for the invention, chlorophyll in can removing the leaf that is present in Alfalfa with organic solvent (such as petroleum ether) and spending and lipid, avoid chlorophyll and lipid to affect the extraction of total flavones in follow-up operation, also improve the purity of extractive of general flavone simultaneously.
Compared with existing extractive technique, the ultrasonic assistant extractive technique that the present invention adopts is extracted the method for Herba Medicaginis total flavones from Herba Medicaginis and is had the following advantages and excellent effect:
(1) improve extraction yield: extraction yield respectively 5.862mg/g and the 4.92mg/g of two described in background of invention kind method, and the extraction yield of the Herba Medicaginis total flavones that the method for the present invention obtains is improved.Although according to the existing technical merit of the art, it is extremely difficult that extraction yield raising percentage ratio is greatly improved, but it is important that, when the general flavone content of Herba Medicaginis itself is limited, the method of the present invention ensure that the purity of extraction initial stage extractive of general flavone, and the subsequent treatment for extractive of general flavone provides solid foundation.The present inventor finds in the extraction process of Herba Medicaginis total flavones, and when not using organic solvent (such as petroleum ether) that alfalfa meal is carried out defat pretreatment, the extraction ratio of Herba Medicaginis total flavones can substantially reduce.The present inventor also surprisingly, it is found that, by the method for the present invention, after alfalfa meal being carried out pretreatment with the organic solvent of 10～15 times (mL/g), the wax coat of Herba Medicaginis material can be destroyed, remove the fat-soluble pigment such as chlorophyll, carotene and oils and fats etc., so be conducive to the infiltration of Extraction solvent and the dissolution of flavones ingredient, thus increasing the extraction ratio of total flavones.The present inventor has also attempted other organic solvents similar with petroleum ether polarity such as pentane, trimethylpentane, hexane, hexamethylene, isobutyltrimethylmethane. etc., have also been obtained desirable extraction effect, and when adopting petroleum ether, the extraction effect of total flavones is best, cost is relative moderate also.
(2) shortening extraction time: ultrasonic assistant extracts generally can obtain optimum extraction rate in the short period of time, the extracting method shortening 3 to 5 times that extraction time is more traditional, thus improve equipment treating capacity and treatment effeciency.
(3) pollution-free: it is a physical process that ultrasonic assistant extracts, and therebetween without chemical reaction, decreases the change of bioactive substance, be conducive to effective ingredient to separate and purification.
(4) operation is simple, and plant maintenance is convenient.
(5) not needing high temperature, energy consumption is low: the present inventor in the extraction process of Herba Medicaginis total flavones it was unexpectedly observed that Extracting temperature higher than 90 DEG C time, the heat-sensitive material being easily caused in Herba Medicaginis is degraded, thus reducing the extraction yield of total flavones.And in the method for the invention, case of thermal instability, oxidizable or hydrolysis active component, at about 60 DEG C, are had the effect of being effectively protected by the optimum temperature that ultrasonic assistant extracts, the physiologically active of thermal activity composition will not be destroyed.Meanwhile, ultrasonic assistant extracts and may also operate as energy-conservation effect.
The present invention passes through repeatedly parallel test, with Herba Medicaginis total flavones extraction ratio for index, utilizes single factor experiment to determine the liquid ratio when total flavones extraction ratio is maximum, Extracting temperature, extraction time and concentration of alcohol successively.It addition, optimize liquid ratio when extracting, Extracting temperature, extraction time and concentration of alcohol by response surface experiments (central composite design), so that it is determined that the optimum extraction condition of Herba Medicaginis total flavones, for the exploitation of Herba Medicaginis total flavones with produce and provide foundation.
Response surface optimization method (ResponseSurfaceMethodology, RSM) it is a kind of mathematical method and optimum experimental method that statistical method combines, it expresses inexplicit performance function by one multinomial with clear and definite expression-form of approximation, the response that multiple independent variables are affected is modeled and analyzes, and final purpose is to optimize response value.Owing to have employed more rational experimental design, in most economical mode, can effectively and rapidly determine the optimum condition of multiple-factor system, it is possible to by analyzing the 3-D graphic impact that must find out each factors on test result directly perceived obtained.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 is the standard curve adopting the standard substance rutin used in the inventive method to obtain；
Fig. 2 indicates that the liquid ratios different in the method for the present invention curve chart on the impact of the extraction yield of Herba Medicaginis total flavones；
Fig. 3 indicates that the extraction times different in the method for the present invention curve chart on the impact of the extraction yield of Herba Medicaginis total flavones；
Fig. 4 indicates that the Extracting temperature different in the method for the present invention curve chart on the impact of the extraction yield of Herba Medicaginis total flavones；
Fig. 5 indicates that the concentration of alcohol different in the method for the present invention curve chart on the impact of the extraction yield of Herba Medicaginis total flavones；
Fig. 6 indicates that liquid ratio and the Extracting temperature response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention；
Fig. 7 indicates that liquid ratio and the extraction time response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention；
Fig. 8 indicates that liquid ratio and the concentration of alcohol response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention；
Fig. 9 indicates that Extracting temperature and the extraction time response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention；
Figure 10 indicates that Extracting temperature and the concentration of alcohol response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention；
Figure 11 indicates that extraction time and the concentration of alcohol response surface solid scattergram on the impact of the extraction yield of Herba Medicaginis total flavones in the method for the present invention.
Detailed description of the invention
Embodiment 1
Ultrasonic assistant extracts the technique of total flavones in Herba Medicaginis and the optimization of conditional parameter
1. the preparation of alfalfa meal
Herba Medicaginis is dried in 65 DEG C of thermostatic drying chambers 12 hours (after drying, moisture is 4.74wt%), is then placed in pulverizer and pulverizes, cross 60 mesh sieves standby.Alfalfa meal filter paper after sieving is wrapped, it is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (in petroleum ether: alfalfa meal=mL: the g after sieving, lower same), surname extraction 24 hours, to remove chlorophyll and lipid.Then be positioned in air dry oven after alfalfa meal being volatilized petroleum ether 65 DEG C dry 2 hours (after drying, moisture is 4.7wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 52% (v/v) is handled well with step 1 is mixed according to liquid ratio 57: 1 (mL: g), it it is 62 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder of Herba Medicaginis total flavones.
3. the mensuration of Herba Medicaginis total flavones
(1) preparation of reference substance stock solution: precision weighs control substance of Rutin 0.005g, with the alcoholic solution constant volume that volume fraction is 70% and transfer to the volumetric flask of 25mL.
(2) drafting of standard curve: accurately measure the reference substance stock solution of 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, it is respectively placed in 25mL volumetric flask, the ethanol of each addition 70% is to 10mL, then addition concentration is the NaNO of 5% in each volumetric flask2Solution 1mL, shakes up, and adds the Al (NO that concentration is 10% after placing 6min3)3Solution 1mL, places 6min, is subsequently adding the NaOH solution 10mL of 4%, places 15min, and distilled water constant volume is to 25mL.Being mensuration absorbance under 510nm at wavelength, with the concentration of reference substance for abscissa (X) value, absorbance is vertical coordinate (Y) value, then carries out linear regression, and obtaining correlation coefficient is R2The standard curve Y=12.219X+0.0036 of=0.9998, standard curve is shown in Fig. 1.
(3) mensuration of general flavone content in sample: take the Herba Medicaginis total flavones powder 20mg after lyophilization, dissolve with the alcoholic solution of 70% (v/v) and constant volume is in 25mL volumetric flask, precision measures 5mL extracting solution and is placed in 25mL test tube, adds the ethanol 5mL that concentration is 70%.Then according to the method described in step (2), it is sequentially added into NaNO2、Al(NO3)3, NaOH solution, then with distilled water constant volume to 25mL, under 510nm wavelength, measure absorbance.Finally, measured absorbance is substituted in equation Y=12.219X+0.0036, calculate the purity of total flavones in testing sample.
4. below by single factor experiment method and response surface optimization method, the impact of the extraction yield of Herba Medicaginis total flavones has been tested by liquid ratio, extraction time, Extracting temperature, ethanol solution concentration, to choose the liquid ratio of optimization, extraction time, Extracting temperature, ethanol solution concentration.
(1) single factor experiment includes: the liquid ratio of varying level, extraction time, Extracting temperature, the concentration of alcohol impact on the extraction yield of Herba Medicaginis total flavones, concrete test method is in Table 1.
Table 1 single factor experiment investigates factor and level
(2) response surface optimization extraction conditions
Adopt response phase method research liquid ratio, extraction time, Extracting temperature and the concentration of alcohol impact on the extraction yield of Herba Medicaginis total flavones.Specific implementation method is in Table 2.
Table 2 response surface experiments factor and level
(3) result of the test:
A) liquid ratio impact on extracting yield
Being 60min in extraction time, Extracting temperature is 60 DEG C, when concentration of alcohol is 40%, investigates liquid ratio (mL: g), the impact such as 20: 1,30: 1,40: 1,50: 1,60: 1,70: 1, on the extraction yield of Herba Medicaginis total flavones.It is shown that the extraction yield the highest (see Fig. 2) of the Herba Medicaginis total flavones of gained when liquid ratio is 60: 1 (mL: g).
B) extraction time impact on extracting yield
In liquid ratio 50: 1 (mL: g), when Extracting temperature is 60 DEG C, concentration of alcohol is 40%, investigate extraction time, such as 20,40,60,80,100,120min, the impact on the extraction yield of Herba Medicaginis total flavones.It is shown that gained extracts yield the highest (see Fig. 3) when extraction time is 60min.
C) Extracting temperature impact on extracting yield
In liquid ratio 50: 1 (mL: g), extraction time is 60min, when concentration of alcohol is 40%, investigates Extracting temperature, such as 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, and the impact on the extraction yield of Herba Medicaginis total flavones.It is shown that gained extracts yield the highest (see Fig. 4) when Extracting temperature is 70 DEG C.
D) concentration of alcohol impact on extracting yield
In liquid ratio 50: 1 (mL: g), extraction time is 60min, when Extracting temperature is 60 DEG C, investigates concentration of alcohol, the impact such as 20%, 30%, 40%, 50%, 60%, on the extraction yield of Herba Medicaginis total flavones.It is shown that gained extracts yield the highest (see Fig. 5) when concentration of alcohol is 50%.
(4) response surface optimization extraction conditions
The condition that employing response phase method carries out studying is: liquid ratio ranges for 30: 1～70: 1 (mL: g), extraction time ranges for 20～100min, Extracting temperature ranges for 40～80 DEG C, and concentration of alcohol ranges for 20%～60%, and other conditions are ultrasonic power is 100W.By response surface experimental design, finally determine that ultrasonic assistant extracts the optimised process of Herba Medicaginis total flavones and is: liquid ratio is 57: 1 (mL: g), extraction time to be 57min, Extracting temperature be 62 DEG C, concentration of alcohol be 52%, now the extraction yield of Herba Medicaginis total flavones reaches 6.458mg/g, and purity is 3.915%.
Embodiment 2
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 12 hours (moisture is 4.25wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 60 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal after sieving puts into flask, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (mL: g), is heated to reflux 4 hours to remove chlorophyll and lipid.Mixture after backflow is filtered, use petroleum ether filter cake, then after the alfalfa meal obtained being volatilized petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.12wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 65% (v/v) is handled well with step 1 is mixed according to liquid ratio 60: 1 (mL: g), then it it is 50 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.10mg/g, and its purity is 3.421%.
Embodiment 3
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 12 hours (moisture is 4.18wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 60 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal after sieving puts into flask, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (mL: g), is heated to reflux 24 hours to remove chlorophyll and lipid.Mixture after backflow is filtered, use petroleum ether filter cake, then after the alfalfa meal obtained being volatilized petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.20wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 65% (v/v) is handled well with step 1 is mixed according to liquid ratio 60: 1 (mL: g), then it it is 50 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.15mg/g, and its purity is 3.510%.
Embodiment 4
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 12 hours (moisture is 4.25wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 60 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 12 times of volumes (mL: g), and surname extraction 18 hours is to remove chlorophyll and lipid.Then, after alfalfa meal volatilizes petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.17wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 50% (v/v) is handled well with step 1 is mixed according to liquid ratio 60: 1 (mL: g), then it it is 50 DEG C in temperature, ultrasonic extraction 40min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.07mg/g, and its purity is 3.514%.
Embodiment 5
It is as follows that the ultrasonic assistant of the present embodiment extracts the method step of total flavones in Herba Medicaginis:
1. the preparation of alfalfa meal
By alfalfa meal (aerial parts) 10 hours (moisture is 4.55wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 40 mesh sieves (meeting the R40/3 series of national standard) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (mL: g), and surname extraction 18 hours is to remove chlorophyll and lipid.Then, after alfalfa meal volatilizes petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.28wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 50% (v/v) is handled well with step 1 is mixed according to liquid ratio 60: 1 (mL: g), it it is 70 DEG C in temperature, ultrasonic extraction 40min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.19mg/g, and its purity is 3.192%.
Embodiment 6
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 8 hours (moisture is 4.88wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 60 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 10 times of volumes (mL: g), and surname extraction 24 hours is to remove chlorophyll and lipid.Then, after alfalfa meal volatilizes petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.59wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 60% (v/v) is handled well with step (1) is mixed according to liquid ratio 50: 1 (mL: g), then it it is 60 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution obtains extractum, and then vacuum lyophilization obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.23mg/g, and its purity is 3.847%.
Embodiment 7
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 8 hours (moisture is 4.88wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 60 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the hexamethylene of 10 times of volumes (mL: g), and surname extraction 24 hours is to remove chlorophyll and lipid.Then, after alfalfa meal volatilizes hexamethylene in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.59wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 60% (v/v) is handled well with step (1) is mixed according to liquid ratio 50: 1 (mL: g), then it it is 60 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution obtains extractum, then obtains the powder containing Herba Medicaginis total flavones through vacuum lyophilization, and now the extraction yield of Herba Medicaginis total flavones is 6.18mg/g, and its purity is 3.247%.
Embodiment 8
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 10 hours (moisture is 4.78wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 40 mesh sieves (meeting the R40/3 series of national standard) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (mL: g), and surname extraction 18 hours is to remove chlorophyll and lipid.Then, after volatilizing petroleum ether in alfalfa meal fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.57wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 40% (v/v) is handled well with step 1 is mixed according to liquid ratio 50: 1 (mL: g), it it is 60 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution obtains extractum, and then vacuum lyophilization obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 6.28mg/g, and its purity is 3.149%.
Embodiment 9
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 8 hours (moisture is 4.71wt% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 40 mesh sieves (meeting the R40/3 series of CNS) standby.Alfalfa meal filter paper after sieving is wrapped, and is put in apparatus,Soxhlet's, is subsequently adding the petroleum ether (boiling range 30～60 DEG C) of 15 times of volumes (mL: g), and surname extraction 24 hours is to remove chlorophyll and lipid.Then, after alfalfa meal volatilizes petroleum ether in fume hood, be positioned in air dry oven 65 DEG C dry 2 hours (after drying, moisture is 4.6wt%), obtain the alfalfa meal handled well.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 60% (v/v) is handled well with step (1) is mixed according to liquid ratio 50: 1 (mL: g), then it it is 90 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution, obtains extractum, then vacuum lyophilization, obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 5.48mg/g, and its purity is 2.839%.
Embodiment 10
Do not add the comparative example of organic removal of solvents chlorophyll and lipid
1. the preparation of alfalfa meal
By Alfalfa (aerial parts) 8 hours (moisture is 4.80% after drying) of 65 DEG C of drying in air dry oven, it is then placed in pulverizer and pulverizes, cross 40 mesh sieves (meeting the R40/3 series of national standard) standby.
2. the extraction of Herba Medicaginis total flavones
The alfalfa meal that the alcoholic solution that volume fraction is 40% (v/v) is handled well with step (1) is mixed according to liquid-solid ratio 50: 1 (mL: g), then it it is 60 DEG C in temperature, ultrasonic extraction 60min when ultrasonic power is 100W, obtains Herba Medicaginis total flavones extracting solution.Herba Medicaginis total flavones extracting solution, after vacuum filtration, is collected filtrate, is obtained general flavone ethanol-aqueous solution.The rotated evaporative removal ethanol of Herba Medicaginis general flavone ethanol-aqueous solution obtains extractum, and then vacuum lyophilization obtains the powder containing Herba Medicaginis total flavones, and now the extraction yield of Herba Medicaginis total flavones is 5.63mg/g, and its purity is 2.189%.
Listed above is only several specific embodiments of the present invention, and the present invention is not limited to above example, and in all deformation extracted in key element parameter area, being regarded as is protection scope of the present invention.
Claims (10)
1. ultrasonic assistant extracts a method for total flavones in Herba Medicaginis, and the method comprises the following steps:
(1) Alfalfa be dry, pulverize, obtain alfalfa meal；
(2) in the alfalfa meal that step (1) obtains, organic solvent is added, the ratio of described organic solvent and alfalfa meal is calculated as 10: 1 to 15: 1 by mL: g, is heated to reflux 4～24 hours, filters to remove organic solvent, obtain alfalfa meal, then alfalfa meal is dried；Wherein, described organic solvent is one or more the mixed solvent in petroleum ether, pentane, trimethylpentane, hexane, hexamethylene and isobutyltrimethylmethane.；
(3) alfalfa meal that the alcoholic solution that volume fraction is 20～70% is obtained with step (2) according in mL: g 20: 1～70: 1 liquid ratio mix, extract at the temperature of 30 DEG C～80 DEG C and ultrasonic assistant, obtain Herba Medicaginis total flavones extracting solution.
2. method according to claim 1, it is characterised in that in step (1), before pulverizing, is dried to moisture≤5wt% by Alfalfa.
3. method according to claim 1, it is characterised in that described method is additionally included in step (1), sieves the alfalfa meal after pulverizing；Preferably, employing 40～60 mesh sieve that sieves carries out.
4. method according to claim 1, it is characterised in that in step (2), described backflow is undertaken by soxhlet extraction.
5. method according to claim 4, it is characterised in that in soxhlet extraction, described extraction carries out 18～24 hours.
6. method according to claim 1, it is characterised in that in step (2), is dried to moisture≤5wt% by alfalfa meal.
7. method according to claim 1, it is characterised in that in step (2), described organic solvent is petroleum ether.
8. method according to claim 1, it is characterised in that in step (3), the volume fraction of described alcoholic solution is 40%～70%, more preferably 52%；
Preferably, in step (3), described liquid ratio is calculated as 50: 1～70: 1 by mL: g, more preferably 57: 1；
Preferably, in step (3), it is extracted in 60～80 DEG C, more preferably carries out at the temperature of 62 DEG C；
Preferably, in step (3), described extraction carries out 20～120min, more preferably 60～100min, it is most preferred that 57min.
9. method according to claim 1, it is characterised in that in step (3), hyperacoustic power is 100W.
10. method according to any one of claim 1 to 9, it is characterized in that, described method also includes step (4): the Herba Medicaginis total flavones extracting solution obtained by step (3) is after vacuum filtration, collect filtrate, obtain general flavone ethanol-aqueous solution, removing the ethanol in Herba Medicaginis general flavone ethanol-aqueous solution, obtain extractum, then drying obtains the powder containing Herba Medicaginis total flavones；
Preferably, in step (4), ethanol is removed by rotary evaporation in vacuo；
Preferably, in step (4), described dry as vacuum lyophilization.
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