CN108395459A - The method for extracting phloridzin, astragalin and afzclin from apple flower using ionic liquid - Google Patents
The method for extracting phloridzin, astragalin and afzclin from apple flower using ionic liquid Download PDFInfo
- Publication number
- CN108395459A CN108395459A CN201810160898.7A CN201810160898A CN108395459A CN 108395459 A CN108395459 A CN 108395459A CN 201810160898 A CN201810160898 A CN 201810160898A CN 108395459 A CN108395459 A CN 108395459A
- Authority
- CN
- China
- Prior art keywords
- ionic liquid
- astragalin
- afzclin
- phloridzin
- apple flower
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
Abstract
The present invention relates to a kind of methods for extracting phloridzin, astragalin and afzclin from apple flower using ionic liquid, and ultrasonic extraction is carried out in room temperature after dry apple flower powder is mixed with extractant, and after ultrasonic extraction, centrifugation takes supernatant to obtain the final product；The extractant, which is mixed by ionic liquid with methanol, to be formed.The total content of phloridzin, astragalin and afzclin is 145.4 mg/g in verification test.Compared with traditional extracting method, total recovery rate of phloridzin, astragalin and afzclin in apple flower after ionic liquid is added up to 25.4%.The result shows that the present invention screens optimum extraction process using ionic liquid combining response Surface Method, and apple flower methanol extract liquid has certain activation to tyrosinase, and methanol ionic liquid extract liquid has certain inhibiting effect to tyrosinase.Energy development and utilization to apple flower provide scientific basis.
Description
Technical field
The invention belongs to medicine or/and health product technology fields, and in particular to a kind of bent using ionic liquid combining response
The method that face method extracts phloridzin, astragalin and afzclin from apple flower.
Background technology
Apple flower is rose family Malus appleMalus pumilaThe dry flower of Mill.Apple flower chemistry at
Dividing mainly has dihydrochalcone-like such as phloridzin, phloretin and other flavones ingredients such as Quercetin, Kaempferide, rutin etc..
Apple flower tea, which has, enriches blood, and discongests neuralgia, chloasma hepaticum, and blackspot, pimples, acne are treated in improving eyesight, and skin maintenance helps digest, and are protected
Liver promoting blood circulation and other effects.Pharmaceutical research shows：Flavone compound have anti-oxidant, anti-inflammatory, analgesia, adjust immune, anti-aging,
The pharmacological actions such as reducing blood lipid, antitumor.Additionally it has been reported that some flavone compounds can inhibit the synthesis of melanin,
It can be used to treat the pigmentations relevant disease such as chloasma；Some flavone compounds can stimulation melanin synthesis, for treating
The depigmentations such as leucoderma disease.
Ionic liquid (ionic liquid, IL) is a kind of novel green organic solvent, also known as room temperature fused salt, by specific
, volume is relatively large, the asymmetric organic cation of structure and the relatively small inorganic anion composition of volume, have good
The features such as good thermal stability and chemical stability, range of viscosities is wide, controllability and dissolubility are good.It not only reduces to environment
Pollution, moreover it is possible to the structure for destroying cellulose then improves recovery rate to promote the dissolution of active ingredient.In recent years, have
Document report, ionic liquid are applied to the extraction of active Chinese drug component compound, have no ionic liquid to flavones ingredient in apple flower
Extract the report of research.
Therefore, the present invention uses ionic liquid-ultrasound-assisted extraction-high performance liquid chromatography, using Design-
Expert8.06 statistical analysis softwares, 5 factors of design Box-Benhnken, 3 horizontal center combination design, utilize HPLC methods
Assay is carried out to 3 kinds of phloridzin, astragalin and afzclin ingredients in apple flower simultaneously, sieve takes best extraction work
Skill, and the activity research of tyrosinase has been carried out to it.
Invention content
Present invention aims to overcome that prior art defect, provides and a kind of optimizing extraction from apple flower using ionic liquid
The method of phloridzin, astragalin and afzclin, the extracting method is easy to operate, and extraction efficiency is high.
To achieve the above object, the present invention adopts the following technical scheme that：
A method of optimizing extraction phloridzin, astragalin and afzclin from apple flower using ionic liquid, will do
Dry apple flower powder mixed with extractant after in room temperature（25-30 DEG C）Ultrasonic extraction is carried out, after ultrasonic extraction, centrifugation,
Take supernatant to obtain the final product.
Specifically, in said extracted method, the extractant, which is mixed by ionic liquid with methanol, to be formed, the ionic liquid
For ［ BMIM ］ BF4, ［ BMIM ］ Br, ［ BMIM ］ PF6Or ［ HMIM ］ PF6；Extractant intermediate ion strength of fluid is 0.1-1.0 mol/L
（Wherein, preferably 0.4-0.8 mol/L）.In text unless otherwise specified, methanol refers to volumetric concentration.
It further, can be by apple flower powder and extractant according to solid-to-liquid ratio 1g：20-100 mL（Wherein, preferably
1g：60-1g：100 mL）It is added, the suitable mesh number range of apple flower powder is in 10-90 mesh（Wherein, preferably 50-70
Mesh）.
In order to obtain preferable extraction effect, the ultrasonic extraction time is advisable with 10-60 min（Wherein, preferably 40-
60min）, centrifugal rotational speed is advisable with 2000-9000 r/min（Wherein, preferably 7000-9000 r/min）.
The methodology that the present invention extracts phloridzin, astragalin and afzclin using ionic liquid from apple flower walks
It is rapid as follows：
1）It selects suitable solvent to carry out dissolving ion liquid first and determines best extractant type；It is respectively compared methanol, second
Nitrile, ethyl acetate, 70% second alcohol and water carry out dissolving ion liquid and are extracted to apple flower, filter out methanol to target analysis
The recovery rate of object is best.Then it has been respectively compared ［ BMIM ］ BF4/ methanol, ［ BMIM ］ Br/ methanol, ［ BMIM ］ PF6/ methanol,
HMIM ］ PF6For four kinds of extractants of/methanol to the extraction yield of target analytes, final determining ［ BMIM ］ Br/ methanol is best extraction
Agent；
2）In step 1）On the basis of, the factor level of extraction conditions is designed, to extract the different control ginsengs in apple flower process
Number is the factor, and the factor level of response surface method is determined by single-factor analysis therapy；Screen the control of multiple extraction processes
After parameter, it is the factor that choosing, which influences maximum several control parameters,：With solid-to-liquid ratio（Times）, extractant concentration（mol/L）, ultrasound when
Between（min）, centrifugal rotational speed（r/min）, crush mesh number（Mesh）For five factors, tested；
3）Response surface design experiment is designed using Design-Expert 8.06, by variance analysis and determines phloridzin, Chinese milk vetch
The maximum extracted rate and its corresponding factor level of glycosides and afzclin；
4）According to the corresponding control parameter factor level of the maximum extracted rate of phloridzin, astragalin and afzclin, optimal
Under the conditions of extraction apple flower in phloridzin, astragalin and afzclin, Detection and Extraction rate（By test solution efficient liquid phase
Chromatograph detects peak area at 270 nm, compared with phloridzin, astragalin and afzclin standard curve, calculates root skin
Total extraction quantity of glycosides, astragalin and afzclin）.Actual extracting rate is compared with conventional solvent detected value, determines ion
The advance of liquid extraction；
5）Under same system, tyrosinase is carried out to the ionic liquid extract liquid, methanol extract liquid and ionic liquid of apple flower
Determination of activity.
Ionic liquid combining response curved surface experimental method optimization extraction phloridzin, astragalin are utilized the present invention provides a kind of
With the technique of afzclin 5 factors 3 of Box-Benhnken are designed using Design-Expert8.06 statistical analysis softwares
Horizontal center combination design, using HPLC methods simultaneously to 3 kinds of phloridzin, astragalin and afzclin ingredients in apple flower
Assay is carried out, sieve takes best extraction process, and the activity research of tyrosinase has been carried out to it.The present invention grinds for Chinese medicine
The persons of studying carefully provide new thinking and approach, and the optimum extraction condition that this experiment is established is that science is feasible, can be used for optimizing choosing
The extractive technique for taking suitable industry extension production, improves outmoded technique, improves efficiency, reality is provided for the comprehensive utilization of apple flower
Test foundation.In addition, extracting method of the present invention is easy to operate, extraction efficiency is high, is suitble to large-scale industrial production.
Description of the drawings
Fig. 1 apple flower test samples（a）With mix reference substance（b）HPLC figures, in figure, 1. phloridzin, 2. astragalin 3.
Afzclin；
Fig. 2 different solvents are to 3 kinds of phloridzin in apple flower, astragalin and afzclin flavone compound extraction yields
It influences；
Shadow of Fig. 3 extractants type to 3 kinds of phloridzin in apple flower, astragalin and afzclin chromocor compound extraction yields
It rings；
Fig. 4 crushes influence of the mesh number to 3 kinds of phloridzin, astragalin and afzclin chromocor compound extraction yields in apple flower；
Shadow of Fig. 5 extractant concentrations to 3 kinds of phloridzin in apple flower, astragalin and afzclin chromocor compound extraction yields
It rings；
Influence of Fig. 6 ultrasonic times to 3 kinds of phloridzin, astragalin and afzclin chromocor compound extraction yields in apple flower；
Influence of Fig. 7 solid-to-liquid ratios to 3 kinds of phloridzin, astragalin and afzclin chromocor compound extraction yields in apple flower；
Influence of Fig. 8 centrifugal rotational speeds to 3 kinds of phloridzin, astragalin and afzclin chromocor compound extraction yields in apple flower；
Influence of Fig. 9 reciprocations to 3 kinds of phloridzin, astragalin and afzclin chromocor compound extraction yields in apple flower.
Specific implementation mode
Technical scheme of the present invention is further discussed in detail with reference to embodiments, but protection scope of the present invention
It is not limited thereto.
Instrument and reagent.
Astragalin (lot number 17042804) is purchased from Chengdu Puffy moral Bioisystech Co., Ltd, and HPLC detections purity is more than
98%；Afzclin and phloridzin, HPLC detections purity are all higher than 98%.1- hexyl -3- methylimidazoles hexafluorophosphates [HMIM]
PF6 (Thermo Fischer Scient Inc., the U.S.), 1-butyl-3-methylimidazolium bromide [BMIM] Br (Moke Stock Limited Partnership,
Germany), 1- butyl -3- methyl imidazolium tetrafluoroborates [BMIM] BF4 (Moke Stock Limited Partnership, Germany), 1- butyl -3- first
Base limidazolium hexafluorophosphate [BMIM] PF6 (Moke Stock Limited Partnership, Germany).Acetonitrile, methanol (HPLC grades), phosphoric acid (Tianjin
Fu Yu Fine Chemical Co., Ltd of city), water is pure water (the sharp Food Co., Ltd of Hangzhou Wahaha hundred).
Shimadzu LC-20AT series of high efficiency liquid chromatograph (Japanese Shimadzu)：LC-20AT liquid chromatograies infusion pump, CTO-
10AS column ovens, SPD-20A UV detector, LC-Solution Chromatographic data systems；KQ-500DB type ultrasonic cleanings
Device (Kunshan Ultrasonic Instruments Co., Ltd., Jiangsu)；(plum Teller-support benefit instrument has 100,000 grades of electronic balances of AB135-S types
Limit company, Switzerland).
Experimental method and result.
The foundation of 2.1 phloridzins, astragalin and afzclin content assaying method.
2.1.1 the preparation of reference substance solution
Phloridzin, astragalin and appropriate afzclin are taken, it is accurately weighed, add methanol to dissolve, obtained concentration is respectively 0.204,
The mixed reference substance solution of 0.196 and 0.199 mg/mL.
2.1.2 the preparation of test solution
Precision weighs apple flower sample powder（70 mesh）Extractant is added in 10 mg（The extractant is by ionic liquid ［ BMIM ］ Br
Composition, a concentration of 0.52 mol/L of extractant intermediate ion liquid ［ BMIM ］ Br are mixed with methanol.）1mL, room temperature ultrasonic extraction
After 60min, 7000 r/min centrifuge 3 min, draw supernatant liquor and take filtrate to get confession through 0.22 μm of filtering with microporous membrane
Test sample solution（Crushing mesh number, extractant, solid-to-liquid ratio, ultrasonic time and centrifugal rotational speed described above are to screen most
Excellent condition）.
2.1.3 chromatographic condition and system suitability experiment.
The chromatographic condition of 1 apple flower assay of table
。
According to above-mentioned chromatographic condition, sample introduction reference substance solution and test solution, 10 μ L of sampling volume record color respectively
Spectrogram is shown in Fig. 1.Under this chromatographic condition, phloridzin, the chromatographic peak of astragalin and afzclin and reference substance appearance in sample
Time consistency, three kinds of compound separating degrees are good.
2.1.4 linear relationship is investigated.
Precision draws the mixed reference substance solution of various concentration, is measured by 2.1.3 lower chromatographic condition sample introductions, records each group
Swarming area.With peak area（Y）For ordinate, sample introduction quality（X, μ g）For abscissa, regression equation of mapping to obtain：Phloridzin：Y=
643736X+ 158998,r=0.9987；Astragalin：Y=518051X+ 145776,r=0.9979；AfzclinY=835408X-
359973,r=0.9942.The result shows that phloridzin, astragalin and afzclin sample size are respectively in 0.4080 ~ 14.28 μ
G, it is in good linear relationship with peak area response when 0.3920 ~ 13.72 μ g and 0.3980 ~ 13.93 μ g.
2.1.5 repeated experiment.
6 parts of accurate weighing apple flower sample prepares sample by above-mentioned 2.1.2 conditions, and according to 2.1.3 lower chromatographic conditions
10 μ L of sample introduction, as a result show：The RSD of phloridzin, astragalin and afzclin is respectively 1.16%, 1.8% and 2.1%, is shown
This test method is reproducible.
2.1.6 Precision Experiment.
Precision draws 10 μ L of mixed reference substance solution, according to chromatographic condition continuous sample introduction 6 times under 2.1.3, as a result shows
Show：The RSD of phloridzin, astragalin and afzclin is respectively 0.61%, 0.73% and 0.51%, shows that instrument precision is good,
The amount of substance can be accurately reflected.
2.1.7 stability experiment.
Precision weighs 1 part of apple flower, prepares sample by above-mentioned 2.1.2 conditions, and according to 2.1.3 lower chromatographic condition sample introductions
Respectively in 0,4,8,12,16,20,24 h sample introductions, 10 μ L, as a result show：The RSD of phloridzin, astragalin and afzclin points
Not Wei 2.58%, 0.97% and 1.42%, show that phloridzin in test liquid, astragalin and afzclin are basicly stable in 24 h.
2.1.8 it is loaded recovery test.
6 parts of apple flower sample for weighing known phloridzin, astragalin and afzclin content, by above-mentioned 2.1.2 conditions
Prepare test solution, contained phloridzin in determination sample, astragalin and afzclin content, then be separately added into and be equivalent to
80% standard items of phloridzin in sample, astragalin and afzclin quality, according to 2.1.3 lower chromatographic condition sample introductions 10
The mean sample recovery rate of μ L, phloridzin, astragalin and afzclin are respectively 97.70%, 101.32% and 106.0%,
RSD values are respectively 0.90%, 1.23% and 2.08%, illustrate that the accuracy of the test result of its reaction meets the requirements.
2.2 single factor experiment.
2.2.1 the selection of extractant.
Influence of the selection of extractant type to target analytes extraction yield is very big.[BMIM] BF is chosen in this experiment first4,
[BMIM] Br, [BMIM] PF6, [HMIM] PF6 For the ionic liquid of 4 types as extractant, this 4 kinds of ionic liquids are imidazoles
Class is stablized in air and solution, can make plant with lignocellulosic competitive binding to make cellulose high-efficiency dissolution
Intracellular ingredient more completely dissolves out, and increases extraction yield.But since ionic liquid is mostly sticky liquid, and [BMIM] Br
In solid crystal shape, so it is particularly important to select suitable solvent to carry out dissolving ion liquid.Experiment compare 70% ethyl alcohol, methanol,
5 kinds of acetonitrile, ethyl acetate and water solvents prepare sample according to above-mentioned 2.1.2 lower condition, and pass through above-mentioned 2.1.3 chromatography
Condition injects HPLC analyses, compares 3 kinds of target analytes phloridzins, astragalin and afzclin content.The results show that acetonitrile
Two kinds of flavones ingredients in apple flower can only be extracted, as shown in Fig. 2, methanol is best to the recovery rate of target analytes, therefore are selected
It is Extraction solvent to select methanol.
Fig. 3 compares [BMIM] BF4/ methanol, [BMIM] Br/ methanol, [BMIM] PF6/ methanol, [HMIM] PF64 kinds of/methanol
Extractant to the extraction results of target analytes, the results show that [BMIM] Br/ methanol to the extraction yields of target analytes most
Height, this may be related with itself composed structure of ionic liquid.Therefore this experiment selects [BMIM] Br/ methanol as extractant.
2.2.2 the selection of mesh number is crushed.
According to above-mentioned test method, other experimental conditions are identical, and it is 10,40,50,60,70 to have investigated crushing mesh number respectively
Influence with 90 mesh to target analytes extraction quantity.If Fig. 4 is shown, with the increase for crushing mesh number, extraction quantity has to be increased therewith
Trend maximum is reached to the extraction yield of target analytes and when it is 60 mesh to crush mesh number.This may be because of ionic liquid
With stickiness, with being gradually reduced for powder size, the chemical composition in sample is more easily extracted, if but when powder size is too small
When, and be easy, by ionic liquid conglomerate, to hinder the release of its chemical composition instead.
2.2.3 the selection of extractant concentration.
As shown in figure 5, when extractant concentration is 0.1 ~ 1.0 mol/L, with the increase of extractant concentration, target analysis
The extraction yield of object is in increased trend, and further increasing with extractant concentration, is in reduction trend instead.This may be because
To be reduced with the extension of the increase of ionic liquid concentration, solvent, hardly enters inside medicinal material, can not fully extract medicine
Ingredient in material, and extraction yield is caused to decline.Therefore, select 0.6 mol/L for best extractant concentration.
2.2.4 the selection of ultrasonic time.
It is ultrasonic time to select 10,20,30,40,50 and 60 min respectively, and extractant concentration is 0.6 mol/L, according to
Above-mentioned experimental condition, influence of the more different ultrasonic times to target analytes extraction yield, the results are shown in Figure 6.With ultrasound
The trend gradually increased is presented to the extraction yield of target analytes in the extension of time substantially；It is right when ultrasonic time is 50 min
The extraction yield of target analytes reaches maximum, and later as time increases, downward trend is presented in the extraction yield of target analytes.
Above-mentioned reason may be because of the long structure that can destroy ionic liquid and target analytes of ultrasonic time, and specific reason needs
Further research.
2.2.5 the selection of solid-to-liquid ratio.
It is 1 to investigate solid-to-liquid ratio:20、1:40、1:50、1:60、1:80 and 1:When 100, ionic liquid is to target analytes
Extraction yield.The results are shown in Figure 7, when solid-to-liquid ratio reaches 1:When 80, extraction yield reaches maximum value, when continuing growing ratio, extraction
Rate is taken to decline instead.The reason of this may be because of ionic liquid itself result and physical characteristic.
2.2.6 the selection of centrifugal rotational speed.
Under the optimum condition of above-mentioned optimization, 2000,4000,6000,7000,8000 and 9000 rmin are selected-1It investigates
Influence of the centrifugal speed to target analytes extraction yield, as shown in figure 8, extraction yield is in 8000 rmin-1When reach maximum, therefore
8000 rmin of experiment selection-1The central point " 0 " of face factor level is horizontal in response.
2.3 response phase method Optimum Experiments design.
According to the center combination experimental design principle of Box-Benhnken, with phloridzin in apple flower, astragalin and
The total content of afzclin is response（Y）, choose and crush mesh number（A）, extractant concentration（B）, ultrasonic time（C）Solid-to-liquid ratio
（D）, centrifugal rotational speed（E）5 factors, using the horizontal Response surface methodology of 5 factor 3.Experimental factor is shown in Table with level design
2。
2 response surface factor level of table designs table
。
To investigate the influence of each influence factor and its reciprocation to extraction process, Design Expert 8.06 are utilized
Software carries out multiple regression fitting to the data in table 2, obtains secondary multinomial regression model equation Y=125.10+4.05A-
0.95B-1.97C+23.88D+E-2.93AB-1.38AC+12.54AD+0.88AE-3.29BC-3.52BD+1.65BE+
2.43CD-6.81CE+0.47DE+1.42A2-0.89B2-2.68C2+24.76 D2-3.28E2.It is fitted quadratic polynomial model
Variance analysis is shown in Table 3, model P<0.0001 shows highly significant, can be utilized for the prediction of response；Model, which loses, intends Xiang Buxian
It writes, illustrates that degree of fitting of the regression equation in entirely recurrence space is preferable, multiple correlation coefficient R2=0.9572, R2adj=
0.9230, R2Pre=0.8472 illustrates that model is preferable to experiment actual conditions fitting.In Response Surface Method, model coefficient effect
The performance for indicating response, when positive-effect occurs in a coefficient, response modes increase as variable increases；It is when one
When number has negative effect, response modes are reduced with the increase with variable.The absolute value of coefficient is higher, the power of response variable
It is more important.Solid-to-liquid ratio is most significant factor in this experiment, therefore solid-to-liquid ratio is maximum to the influence entirely tested.
3 regression equation significance test of table
* * extremely significantly (P<0. 001), * * highly significants (P<0. 01), * significantly (P<0. 05) 。
2.3.1 response surface design map analysis.
Response surface design figure is as shown in figure 9, show the relationship between the effect of independent variable and reciprocation and response.
In Fig. 9, shown in a and c figures, one timing of ultrasonic time, with the increase for crushing mesh number, response linearly rises；Crush mesh number
When constant, the influence unobvious with the increase of ionic liquid concentration or the extension of ultrasonic time to extraction quantity.B, d, e, g scheme institute
Show, when another factor determines, solid-to-liquid ratio is bigger, and the extraction yield of target analytes is bigger, this may be when solid-liquid ratio becomes larger
When, the contact area and touch opportunity of apple flower and solvent are also opposite to become larger, and active ingredient can be made more to be contacted with solvent,
It preferably dissolves, while increasing for quantity of solvent makes the meltage of active ingredient in a solvent increase, and can make more targets point
Analyse object dissolving in a solvent.Shown in f figures, centrifugal rotational speed and ultrasonic time reciprocation on the extraction yield of target analytes influence compared with
It is small.
2.3.2 the optimization of Extraction technique and model verification.
Surface analysis according to response obtains the optimum extraction condition of apple flower：Crush 70 mesh of mesh number, extractant concentration 0.52
Mol/L, 59.52 min of ultrasonic time, solid-to-liquid ratio 1:99.90,7052.37 r/min of centrifugal rotational speed, due to the limit of practical operation
The best condition is changed to by system：Crush 70 mesh of mesh number, 0.52 mol/L of extractant concentration, 60 min of ultrasonic time, solid-to-liquid ratio 1:
100,7000 r/min of centrifugal rotational speed, the total content for measuring phloridzin in apple flower, astragalin and afzclin are 145.4
Mg/g is not much different with model calculation value, therefore accurate and reliable using the extracting parameter that Response Surface Method obtains, and has and actually answers
With value.
2.4 tyrosinase activities measure.
Take 45 μ L phosphate buffers (pH 6.8), 5 μ L samples solution (or methanol), 25 μ L, 0. 2 UmL-1Junket
Propylhomoserin enzyme aqueous solution, 37 DEG C incubate 0. 5 gL of addition after 10 min-125 μ L of levodopa solution, shaken well, 37
DEG C reaction 5 min, survey absorbance at 492 nm with microplate reader.Simultaneously be ［ BMIM ］ Br of ionic liquid under same system and
The parallel test of methanol mixed extract, pure methanol extract liquid and pure ionic liquid ［ BMIM ］ Br, and 8- methoxyl groups are added
The activator of psoralen (8-MOP) is as positive controls.
4 different material of table to the activity ratio of tyrosinase (X ± s,n =3)
Note:8-MOP is positive control；Compared with 8-MOP*** P≤0. 001
5 different solvents extracting solution of table to the activity ratio of tyrosinase (X ± s,n =3)
Note:Compared with ionic liquid*** P≤0. 001
Micromethod is aoxidized using external tyrosinase DOPA rate, it is Bu Tong dense eventually to measure phloridzin, astragalin and afzclin
To the influence of tyrosinase activity under degree.4 are the results are shown in Table, compared with positive control 8-MOP, under same final concentration, Chinese milk vetch
Glycosides and afzclin are above positive control to the activity ratio of tyrosinase；Phloridzin is in final concentration of 1 mmol/L and 0.5
When mmol/L to the activity ratio of tyrosinase pole be significantly higher than positive control (P≤ 0. 001), and in final concentration of 2 mmol/
When L, phloridzin to the activation of tyrosine not as good as positive control it is good (P≤ 0. 001), on the contrary, it has pole very to tyrosinase
Strong inhibiting effect.This illustrates that concentration is different to the active function direction of tyrosinase.Measure apple flower different solvents extracting solution
Influence to tyrosinase activity, the results are shown in Table 5, and under identical conditions, methanol extract liquid is extremely aobvious to the activity ratio of tyrosinase
Write higher than ionic liquid (P≤0. 001)；Methanol ionic liquid extract liquid to tyrosinase have extremely significant inhibiting effect (P≤
0. 001), this may be the substances of more activation tyrosinase activities to be extracted because of methanol, and ionic liquid extract goes out more
The substance inhibited tyrosinase activity.
Reference examples 1
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, precision weighs apple
Fruit spends sample powder（50 mesh）The methanol of 20 mL is added in 0.5 g, after 30 min of room temperature ultrasonic extraction, 3000 r/min centrifugations
3 min, draw supernatant liquor to get.
Known to after testing：Total content 82.77mg/g (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 1
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 0.5 g is done
Dry apple flower powder（50 mesh）With 20 mL extractants（The extractant is by ionic liquid ［ BMIM ］ BF4Group is mixed with methanol
At extractant intermediate ion liquid ［ BMIM ］ BF4A concentration of 0.7 mol/L.）After mixing, in 30 min of room temperature ultrasonic extraction, ultrasound
After extraction, 3000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：88.54 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 2
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 0.5 g is done
Dry apple flower powder（50 mesh）With 20 mL extractants（The extractant is mixed with methanol and is formed by ionic liquid ［ BMIM ］ Br,
A concentration of 0.7 mol/L of extractant intermediate ion liquid ［ BMIM ］ Br.）After mixing, in 30 min of room temperature ultrasonic extraction, ultrasonic extraction
After, 3000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：99.98 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 3
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 0.5 g is done
Dry apple flower powder（50 mesh）With 20 mL extractants（The extractant is by ionic liquid ［ BMIM ］ BF6Group is mixed with methanol
At extractant intermediate ion liquid ［ BMIM ］ BF6A concentration of 0.7 mol/L.）After mixing, in 30 min of room temperature ultrasonic extraction, ultrasound
After extraction, 3000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：92.66 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 4
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 0.5 g is done
Dry apple flower powder（50 mesh）With 20 mL extractants（The extractant is by ionic liquid ［ HMIM ］ BF6Group is mixed with methanol
At extractant intermediate ion liquid ［ HMIM ］ BF6A concentration of 0.7 mol/L.）After mixing, in 30 min of room temperature ultrasonic extraction, ultrasound
After extraction, 3000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：90.96 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Reference examples 2
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, precision weighs apple
Fruit spends sample powder（70 mesh）The methanol of 1 mL is added in 10 mg, after 60 min of room temperature ultrasonic extraction, 7000 r/min centrifugations 3
Min, draw supernatant liquor to get.
Known to after testing：Total content 115.7mg/g (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 5
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 10 mg are done
Dry apple flower powder（70 mesh）With 1 mL extractants（The extractant is mixed with methanol and is formed by ionic liquid ［ BMIM ］ Br,
A concentration of 0.52 mol/L of extractant intermediate ion liquid ［ BMIM ］ Br.）After mixing, in 60 min of room temperature ultrasonic extraction, ultrasound extraction
After taking, 7000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：145.4 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Embodiment 6
A method of phloridzin, astragalin and afzclin being extracted from apple flower using ionic liquid, and 10 mg are done
Dry apple flower powder（70 mesh）With 1 mL extractants（The extractant is by ionic liquid ［ BMIM ］ BF4Composition is mixed with methanol,
Extractant intermediate ion liquid ［ BMIM ］ BF4A concentration of 0.52 mol/L.）After mixing, in 60 min of room temperature ultrasonic extraction, ultrasound extraction
After taking, 7000 r/min centrifuge 3 min, take supernatant to obtain the final product.
Known to after testing：121.8 mg/g of total content (n=3) of phloridzin, astragalin and afzclin in supernatant.
Conclusion：The present invention uses ionic liquid-ultrasound-assisted extraction-high performance liquid chromatography for the first time, with [BMIM] Br/
Methanol solution is extractant, and crushing mesh number is 70 mesh, and extractant concentration is 0.52 mol/L, and ultrasonic time is 60 min, solid-liquid
Than being 1:100,7000 r/min of centrifugal rotational speed, the extraction yield for obtaining phloridzin in apple flower, astragalin and afzclin carries
It is high by 25.4%, can be that the utilization of apple flower be provided fundamental basis.But herein not yet find ionic liquid type from it is different
The regularity of active constituent needs further to further investigate.The present invention is that traditional Chinese medicine research persons provide new thinking and way
Diameter, the optimum extraction condition that the present invention establishes are that science is feasible, can be used for carrying for the suitable industry extension production of optimum option
Technology is taken, outmoded technique is improved, efficiency is improved, experimental basis is provided for the comprehensive utilization of apple flower.
Result of the present invention is shown：Astragalin and afzclin have an apparent activation to tyrosinase, and with
The increase of concentration, activity ratio are continuously increased, and are expected to the medicine that exploitation is leucoderma；Phloridzin is in final concentration of 2 mmol/L
When have inhibiting effect to tyrosinase；There is activation to tyrosinase in final concentration of 1 mmol/L, 0.5 mmol/L, but swashs
Effect living increases with the reduction of concentration, this may be because the phloridzin of various concentration, which influences tyrosinase activity, different machines
System, can be furtherd investigate again.
Claims (5)
1. a kind of method for being extracted phloridzin, astragalin and afzclin from apple flower using ionic liquid, feature are existed
In in room temperature progress ultrasonic extraction after mixing dry apple flower powder with extractant, after ultrasonic extraction, centrifugation takes
Supernatant to obtain the final product.
2. extracting the side of phloridzin, astragalin and afzclin from apple flower using ionic liquid as described in claim 1
Method, which is characterized in that the extractant, which is mixed by ionic liquid with methanol, to be formed, and the ionic liquid is ［ BMIM ］ BF4,
BMIM ］ Br, ［ BMIM ］ PF6Or ［ HMIM ］ PF6；Extractant intermediate ion strength of fluid is 0.1-1.0 mol/L.
3. extracting the side of phloridzin, astragalin and afzclin from apple flower using ionic liquid as claimed in claim 2
Method, which is characterized in that apple flower powder is with extractant according to solid-to-liquid ratio 1g：20-100 mL are added, apple flower powder mesh
Number is in 10-90 mesh.
4. extracting phloridzin, astragalin and A Fu beans from apple flower using ionic liquid as described in claims 1 to 3 is any
The method of glycosides, which is characterized in that the ultrasonic extraction time is 10-60 min.
5. extracting the side of phloridzin, astragalin and afzclin from apple flower using ionic liquid as claimed in claim 4
Method, which is characterized in that centrifugal rotational speed is 2000-9000 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810160898.7A CN108395459B (en) | 2018-02-26 | 2018-02-26 | Method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810160898.7A CN108395459B (en) | 2018-02-26 | 2018-02-26 | Method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108395459A true CN108395459A (en) | 2018-08-14 |
| CN108395459B CN108395459B (en) | 2020-11-13 |
Family
ID=63096628
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810160898.7A Active CN108395459B (en) | 2018-02-26 | 2018-02-26 | Method for extracting phlorizin, astragalin and afzerin from apple flowers by using ionic liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108395459B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975456A (en) * | 2019-04-02 | 2019-07-05 | 河南大学 | The method that 7 active constituents in psoralea corylifolia are extracted in ionic liquid combination PB design and the optimization of BBD response phase method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864864A (en) * | 2014-03-27 | 2014-06-18 | 江苏斯威森生物医药工程研究中心有限公司 | Method for efficiently extracting phlorizin from plants |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
-
2018
- 2018-02-26 CN CN201810160898.7A patent/CN108395459B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864864A (en) * | 2014-03-27 | 2014-06-18 | 江苏斯威森生物医药工程研究中心有限公司 | Method for efficiently extracting phlorizin from plants |
| CN107029686A (en) * | 2017-04-01 | 2017-08-11 | 大连理工大学 | Hydrophilic Interaction Chromatography stationary phase, preparation method and applications based on amine functionalized imidazole ion liquid |
Non-Patent Citations (2)
| Title |
|---|
| MATEJIA SENICA ET AL.: "Fruit Seeds of the Rosaceae Family: A Waste, New Life, or a Danger to Human Health?", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 李明英: "离子液体在天然活性物质提取中的应用研究", 《药学进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975456A (en) * | 2019-04-02 | 2019-07-05 | 河南大学 | The method that 7 active constituents in psoralea corylifolia are extracted in ionic liquid combination PB design and the optimization of BBD response phase method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108395459B (en) | 2020-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108830041B (en) | Virtual screening method of alpha-glucosidase inhibitor | |
| CN104306745B (en) | A kind of detection method of gastrodia tuber refreshment capsule | |
| CN103884811B (en) | A kind of biological chromatography compares screening system and application thereof | |
| CN104458990B (en) | Method for measuring content of vitamin K2 (MK-7) | |
| CN109503323A (en) | A kind of preprocess method extracting natural d-borneol from Borneolum leaf piece | |
| CN105439925B (en) | A kind of preparation of lipoic acid polymeric impurities and its detection method | |
| CN109134583A (en) | The method of triterpene compound is extracted from schefflera leaf using ionic liquid | |
| Sarvin et al. | LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design | |
| Dong et al. | Natural deep eutectic solvents as tailored and sustainable media for the extraction of five compounds from compound liquorice tablets and their comparison with conventional organic solvents | |
| Feng et al. | Tailor-made deep eutectic solvents extraction combined with UPLC-MS/MS determination of icarrin and icarisid II in rat plasma and its comparative pharmacokinetic application | |
| CN103494843B (en) | Yellow mushroom standardized component manufacturing method and application of components to treatment of lung cancer | |
| CN108395459A (en) | The method for extracting phloridzin, astragalin and afzclin from apple flower using ionic liquid | |
| Li et al. | Efficient determination of three flavonoids in Malus pumila flowers by ionic liquid-HPLC | |
| CN103808835A (en) | Method for simultaneously measuring contents of 10 chemical components in four-substance soup decoction by HPLC-DAD (high performance liquid chromatography-diode array detection) method | |
| CN106749114B (en) | A method of extracting taxol from Chinese yew | |
| CN104447892A (en) | Arginine-fructosyl-glucose detection method and medical application thereof | |
| Itoh et al. | Effects of unripe Citrus hassaku fruits extract and its flavanone glycosides on blood fluidity | |
| CN102384907A (en) | Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content | |
| AU2018102217A4 (en) | Use of high-speed countercurrent chromatograph in bioconversion of glycoside component | |
| CN107655984A (en) | Nitrofuran metabolites method for detecting residue in a kind of poultry | |
| Shi et al. | Optimization of microwave-assisted extraction of wedelolactone from Eclipta alba using response surface methodology | |
| CN103091408B (en) | Method for determining content of hesperidin in Citrus Bioflavonoids | |
| CN102998386A (en) | Method for measuring radix paeoniae alba diglucoside in radix paeoniae alba callus by utilizing high performance liquid chromatography | |
| CN103520243A (en) | Cyclic adenosine monophosphate snow chrysanthemum oil resin composite soft capsule and preparation method thereof | |
| CN108218935B (en) | Method for extracting myricitrin, quercitrin and afzelin from Chinese redbud leaves by using ionic liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |