CN109456313A - A method of extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus - Google Patents
A method of extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus Download PDFInfo
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- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
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Abstract
The method that the present invention relates to a kind of to extract the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, belongs to technical field of phytochemistry.It after this method crushes Pachyrhizua angulatus, using water as Extraction solvent, is extracted at 40 DEG C -60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10 ~ 16g/mL, and after extraction time is 2-2.5h, decompression is filtered, and collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；It is 40-50mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.The method of the present invention is simple and reliable, and recovery rate is high, provides reference frame to improve the utilization rate of Puerarin in Pachyrhizua angulatus in food production；Obtained Puerarin has compared with high anti-oxidation activity, application easy to spread.
Description
Technical field
The invention belongs to technical field of phytochemistry, and in particular to one kind extracts the active pueraria lobata of high anti-oxidation from Pachyrhizua angulatus
The method of element.
Background technique
Pueraria lobata is recorded in Shennong's Herbal earliest, is brought down a fever with expelling pathogenic factors from muscles and skin, is promoted the production of body fluid to quench thirst, relieving alcoholism and other effects.It is common
It has a headache in fever caused by exogenous pathogens, it is thirsty, it quenches one's thirst, dizziness and headache, wine poison hurts medium symptom.Pueraria lobata is mainly derived from legume pueraria lobata
(Pueraria lobata(Willd.) Ohwi) and Pachyrhizua angulatus (sweet kudzuPueraria thomsoniiBenth. drying)
Root, because the two active constituent content is distinguished obvious (mainly Puerarin), 2005 editions pharmacopeia are by Pachyrhizua angulatus and pueraria lobata (elegant jessaminePueraria lobata(Willd.) Ohwi) it distinguishes.Puerarin is peculiar active constituent in elegant jessamine and Pachyrhizua angulatus, has drop blood
Pressure, coronary artery dilator adjust alcohol metabolism, improve the multiple biological activities such as microcirculation, reducing blood sugar and blood fat, anti-oxidant；Face
Bed application based on elegant jessamine, Pachyrhizua angulatus eat it is more, and Pachyrhizua angulatus be incorporated in defend planning commission promulgation dual-purpose of drug and food list in, but
Elegant jessamine can only be medicinal, and there are many health care products based on Pachyrhizua angulatus exploitation currently on the market.
Modern study is the results show that temperature, Cu2+, reducing agent Na2SO3It is larger to Puerarin stability influence, and Puerarin
It is water-soluble and fat-soluble lower, greatly limit exploitation and utilization of the Pachyrhizua angulatus as integration of drinking and medicinal herbs product.Needle at present
Relatively fewer to the research of Puerarin extraction and application in Pachyrhizua angulatus, it is current coating skill that the active Puerarin of high anti-oxidation, which how is made,
The problem of art field urgent need to resolve.
Summary of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide one kind to extract high anti-oxidation activity from Pachyrhizua angulatus
Puerarin method, this method is simple and reliable, and recovery rate is high, and obtained Puerarin has compared with high anti-oxidation activity, is easy to
It promotes and applies.
To achieve the above object, The technical solution adopted by the invention is as follows:
A method of it extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 40 ~ 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10 ~ 16g/
ML, after extraction time is 2-2.5h, decompression is filtered, and collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 40-50mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
There is no limit for present invention partial size smashed for Pachyrhizua angulatus.
It is further preferred that the extraction time is multiple.
It is further preferred that the extraction time is 2 times.
It is further preferred that it is 45mg/ that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extracting
mL。
It is further preferred that the method that the active Puerarin of high anti-oxidation is extracted from Pachyrhizua angulatus, including it is as follows
Step:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:16g/mL, is extracted
Twice, each extraction time for after 2.5h, decompression is filtered, collects filtrate, is concentrated and dried to get the active pueraria lobata of high anti-oxidation
Element；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
It is further preferred that the method that the active Puerarin of high anti-oxidation is extracted from Pachyrhizua angulatus, including such as
Lower step:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10g/mL, is extracted
Twice, each extraction time for after 2h, decompression is filtered, collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
The present invention protects the active Puerarin of high anti-oxidation made from said extracted method simultaneously.
Compared with prior art, the present invention has the advantages that:
The present invention provides a kind of method that the active Puerarin of high anti-oxidation is extracted from Pachyrhizua angulatus, and this method is simple and reliable, extracts
Rate is high, provides reference frame to improve the utilization rate of Puerarin in Pachyrhizua angulatus in food production；
The present invention is had using the Puerarin that low temperature method obtains compared with high anti-oxidation activity, to the clearance rate of OH be 41.68 ±
0.85%, the clearance rate (%) to O2- is 21.36 ± 0.44, application easy to spread.
Detailed description of the invention
Fig. 1 is Puerarin standard items and kudzu root extract liquid chromatogram；Wherein A is Puerarin standard items；B mentions for pueraria lobata
Take object；
Fig. 2 is the change curve of Puerarin recovery rate when different temperatures water mentions；
Fig. 3 is influence column diagram of the different cosolvents to Puerarin recovery rate；
Fig. 4 is influence curve figure of the PVP concentration to Puerarin recovery rate；
Fig. 5 is influence curve figure of the extraction time to Puerarin recovery rate；
Fig. 6 is influence curve figure of the Extracting temperature to Puerarin recovery rate；
Fig. 7 is influence curve figure of the solid-liquid ratio to Puerarin recovery rate；
Fig. 8 is influence curve figure of the extraction time to Puerarin recovery rate.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Production firm person is not specified in material therefor or equipment, is that can be obtained by purchase
Conventional products.
Heretofore described traditional water extraction is high temperature extraction, is completed in boiling water bath.
Embodiment 1
A kind of preparation method of the active Puerarin of high anti-oxidation, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 40 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10g/mL, is extracted
After time is 2h, decompression is filtered, and collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 40mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
Embodiment 2
A kind of preparation method of the active Puerarin of high anti-oxidation, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 50 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:16g/mL, is extracted
Twice, each extraction time for after 2.5h, decompression is filtered, collects filtrate, is concentrated and dried to get the active pueraria lobata of high anti-oxidation
Element；
It is 50mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
Embodiment 3
A kind of preparation method of the active Puerarin of high anti-oxidation, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:14g/mL, is extracted
Three times, each extraction time for after 2.3h, decompression is filtered, collects filtrate, is concentrated and dried to get the active pueraria lobata of high anti-oxidation
Element；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
Embodiment 4
A kind of preparation method of the active Puerarin of high anti-oxidation, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:16g/mL, is extracted
Twice, each extraction time for after 2.5h, decompression is filtered, collects filtrate, is concentrated and dried to get the active pueraria lobata of high anti-oxidation
Element；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
Embodiment 5
A kind of preparation method of the active Puerarin of high anti-oxidation, includes the following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10g/mL, is extracted
Twice, each extraction time for after 2h, decompression is filtered, collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
Analysis and detection
1 materials and methods
1.1 materials and reagent
Pachyrhizua angulatus is provided by Yunnan Bo Shang Biotechnology Co., Ltd, is accredited as through Yunnan Province Agriculture Academy of Science Medicine Plant Institute
Leguminous plant Pachyrhizua angulatus (Pueraria thomsoniiBenth. dry root).
Puerarin (analytical standard product, HPLC >=98%) Nanjing Sen Beijia Biotechnology Co., Ltd；
Lysine, niacinamide, the native chemical industry in the Shanghai polyvinylpyrrolidone (PVP) east；
Methanol (chromatographically pure) Sigma Co., USA；
Phen, ferrous sulfate, pyrogallol, seamount Pu Chemical Co., Ltd. on trishydroxymethylaminomethane (Tris).
1.2 instrument and equipment
Dai An company, the U3000 high performance liquid chromatograph U.S.；
Millipo company, Drict-Q5 ultrapure water machine France；
BUCHI company, R-215 Rotary Evaporators Switzerland；
Mettler Toledo company, AL104-IC electronic balance Switzerland；
It pleases time-sharing environment and detects Co., Ltd in the new century ultraviolet-uisible spectrophotometer Yunnan T6；
The long automation equipment Co., Ltd in HS-10260D ultrasonic cleaner Shanghai five.
1.3 method
1.3.1 the measurement of puerarin content
1.3.1.1 chromatographic condition
Welch materials C18 chromatographic column (5 μm, 4.6 × 250nm), mobile phase methanol: water=25: 75(volume ratio)；Inspection
Survey wavelength 250nm；Flow velocity 1mL/min.20 μ L of sample volume.
1.3.1.2 the preparation of reference substance solution
Puerarin standard items 10mg accurately is weighed in 10mL volumetric flask, is settled to scale after being dissolved with methanol, it is dense to obtain Puerarin
Degree is the reference substance solution of 1mg/mL, crosses 0.45m miillpore filter, spare.
1.3.1.3 sample preparation
It takes 10g Pachyrhizua angulatus sample in triangular flask, 30% ethyl alcohol of 200mL is added, ultrasonic extraction 40min is filtered, and filtrate is dense with volume
Degree is sample introduction after 30% ethyl alcohol dilutes 5 times, calculates the content of Puerarin in extracting solution after measurement peak area by standard curve.
1.3.1.4 the drafting of standard curve
Precision draws 1,2.5,5,10,15 μ L of Puerarin standard solution, successively sample introduction, by above-mentioned chromatographic condition respectively to standard items
Solution carries out content detection, and with integrating peak areas value (Y) for ordinate, puerarin content (X) is that abscissa draws standard curve.
1.3.1.5 the recovery rate of Puerarin in Pachyrhizua angulatus extract is calculated
Pachyrhizua angulatus extract sample acquires the puerarin content in sample after HPLC is detected, through standard curve regression equation, then
The recovery rate of Puerarin is calculated according to following equation.
In formula:
T-Puerarin recovery rate (100%)；
WyPuerarin content (mg) in-extracting solution；
W0The weight (g) of-Pachyrhizua angulatus raw material
1.3.2 the Antioxidative Activity Determination of extract
1.3.2.1 the scavenging effect to OH free radical:
Scavenging effect using Fenton reaction detection extract to OH free radical, experimental method are as follows:
1.5 mL of Phen solution that concentration is 5 mmol/L is measured, the phosphate buffer (PBS) that pH value is 7.4 is then added
4.0 mL, mix well, and 1.0 mL of ferrous sulfate solution that concentration is 7.5 mmol/L is added, mixes immediately, 2.5 mL are added
Deionized water is eventually adding the H that 1.0mL mass concentration is 1%2O2, reaction system keeps the temperature 1h under the conditions of 37 DEG C, and use is ultraviolet
Spectrophotometer measures absorbance A at 536 nm wavelength1。
According to the above experimental procedure, water is replaced with into kudzu root extract, measures absorbance A2；
According to the above experimental procedure, the H for being 1% by mass concentration2O2Water is replaced with, absorbance A is measured3。
The clearance rate of apparent OH is calculated as follows:
1.3.2.2 to O2 -The scavenging effect of free radical
By assay NBT photoreduction Detection and Extraction object to O2 - Scavenging effect, experimental method is as follows:
4.5 mL of Tris-HCl buffer solution (pH value 8.2) that concentration is 50 mmol/L is measured, 4.2 mL deionizations are added
Water is added concentration and replaces pyrogallol as blank pair by the HCl of pyrogallol 0.3 mL(using 10 mmol/L of 3mmol/L
According to), after solution is mixed, cuvette is poured into immediately, carries out time sweep using ultraviolet specrophotometer at 325 nm wavelength,
5 min are surveyed altogether, are obtained absorbance and are changed with time rate F0。
According to the above experimental procedure, 4.2 mL deionized waters are replaced with and are added after 1.0 mL Pachyrhizua angulatus extract solutions are first added
Enter 3.2 mL deionized waters, show that absorbance changes with time rate Fx。
By F0With FxFollowing formula is substituted into calculate extract to O2 - Clearance rate.
1.3.3 the single factor experiment of low temperature extraction Puerarin
1.3.3.1 the variation of Puerarin recovery rate when different temperatures water mentions
Experiment of single factor is carried out to the Puerarin recovery rate variation of water extraction traditional under different temperatures first.Take 10g smashed
Pachyrhizua angulatus is put into triangular flask, solid-liquid ratio 1:8, after extracting 2h under 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C of water-baths respectively
Decompression filters, and collects filtrate, is concentrated and dried, and measures puerarin content by HPLC and calculates Puerarin recovery rate.
1.3.3.2 influence of the different cosolvents to Puerarin recovery rate
It takes the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, is separately added into three kinds of PVP, lysine, niacinamide cosolvents, cosolvent
Concentration is 0.04g/mL, and one group of blank group that cosolvent is not added, solid-liquid ratio 1:8, after extracting 2h under 40 DEG C of water-baths is arranged
Decompression filters, and collects filtrate, is concentrated and dried, and measures puerarin content by HPLC and calculates Puerarin recovery rate.
1.3.3.3 influence of the co-solvent concentration to Puerarin recovery rate
It takes the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, the cosolvent (20,30,40,50,60mg/mL) of various concentration is added, expect
Liquor ratio is 1:8, depressurizes suction filtration after 2h is extracted under 40 DEG C of water-baths, collects filtrate, is concentrated and dried, and measures Puerarin by HPLC and contains
It measures and calculates Puerarin recovery rate.
1.3.3.4 influence of the extraction time to Puerarin recovery rate
It takes the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, suitable cosolvent is added, solid-liquid ratio 1:8 is extracted under 40 DEG C of water-baths
It is filtered under diminished pressure after 2h, collects filtrate, setting extraction time is 1 time, 2 times, 3 three levels, is concentrated and dried, is measured by HPLC
Puerarin content simultaneously calculates Puerarin recovery rate.
1.3.3.5 influence of the Extracting temperature to Puerarin recovery rate
It takes the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, suitable cosolvent, solid-liquid ratio 1:8, in different temperatures (30 is added
DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C) under extract 2h after depressurize filter, collect filtrate, be concentrated and dried, pueraria lobata is measured by HPLC
Cellulose content simultaneously calculates Puerarin recovery rate.
1.3.3.6 influence of the solid-liquid ratio to Puerarin recovery rate
Take the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, suitable cosolvent be added, using different solid-liquid ratio (1:5,1:10,1:
15,1:20), water-bath depressurizes suction filtration after extracting 2h at a proper temperature, collects filtrate, is concentrated and dried, measures Pueraria lobota by HPLC
Root cellulose content simultaneously calculates Puerarin recovery rate.
1.3.3.7 influence of the extraction time to Puerarin recovery rate
It takes the smashed Pachyrhizua angulatus of 10g to be put into triangular flask, suitable cosolvent is added, using solid-liquid ratio appropriate, in proper temperature
Different time (1h, 2h, 3h, 4h) is extracted in lower water-bath, and decompression filters afterwards, collects filtrate, is concentrated and dried, measures pueraria lobata by HPLC
Cellulose content simultaneously calculates Puerarin recovery rate.
When HPLC is measured, sample introduction is measured after being configured to solution using 30% ethyl alcohol.
1.3.4 orthogonal experiment
On the basis of single factor experiment, the concentration of high spot reviews cosolvent, Extracting temperature, extraction time and solid-liquid ratio four because
Influence of the element to Puerarin recovery rate designs L9(34) orthogonal test, by carrying out intuitive analysis and variance point to experimental data
Analysis, seeks optimum extraction scheme.
1.4 data processing
Every group of experiment is repeated 3 times, data with `x ±sIt indicates, statistical is carried out to experimental data with 17.0 software of SPSS
Analysis,P< 0.05 is significant difference,P< 0.01 is extremely significant for difference.
2 results and analysis
The measurement of 2.1 puerarin contents
2.1.1 linear relationship is investigated
The puerarin content in Puerarin standard specimen and Pachyrhizua angulatus extract sample, the result is shown in Figure 1 are measured respectively by the method for 1.3.1.
Using puerarin content as abscissa, standard curve is drawn as ordinate using integrating peak areas value and calculates recurrence side
Journey.To data carry out linear fit analysis can obtain equation of linear regression Y=79.299X-0.3775 (R 2=0.9996, n=5).Pueraria lobota
Root element detection level linear relationship in 1-15 μ g range is good.
2.1.2 precision test
Precision draws 20 μ L of reference substance solution, continuous sample introduction 6 times, records peak area, calculates RSD(n=6 of puerarin peak area)
It is 0.15%, shows that this method meets the requirements.
2.1.3 repetitive test
It takes with a collection of 6 parts of raw material of Pachyrhizua angulatus, sample test solution is prepared according to the method for " 1.3.1.3 " item, according to " 1.3.1.1 " item
Chromatographic condition is measured respectively, records peak area, and the RSD (n=6) for calculating puerarin peak area is 2.34%, shows this method
Accuracy is good.
2.1.4 stability test
Precision draws 20 μ L of test solution, is measured respectively in the 0th, 2,4,6,8,10,12h, records peak area, calculates Pueraria lobota
The RSD (n=6) of root element peak area is 0.46%, shows that test solution is good in 12h internal stability.
The single factor experiment of Puerarin in Pachyrhizua angulatus is extracted under 2.2 low temperature
2.2.1 different temperatures proposes the influence of Puerarin in Pachyrhizua angulatus to traditional water
As shown in Figure 2, when extracting Pachyrhizua angulatus using traditional water extraction, Puerarin recovery rate is in rising trend with temperature.It is warm when extracting
When degree is 80 DEG C, Puerarin recovery rate highest has reached 1.38%.When Extracting temperature reaches 90 DEG C, Puerarin recovery rate has been
It reduces (1.21%), considers that environment temperature Puerarin at 80 DEG C or more starts to decompose, so as to cause recovery rate reduction.
2.2.2 influence of the different cosolvents to Puerarin recovery rate
It can be seen that by the experimental data of comparison blank group data and other three groups of addition cosolvents (see Fig. 3), in 40 DEG C of low temperature
Under the conditions of when the direct water-bath of cosolvent be not added extracting, the recovery rate of Puerarin is very low.And add PVP, lysine, niacinamide this
Three kinds of cosolvents can improve the recovery rate of Puerarin by improving the solubility of Puerarin in water.Add different cosolvents
It is found that PVP group data have a distinct increment with respect to blank group data compared with blank group data, the influence to Puerarin recovery rate is most
Greatly.Therefore, it selects the optimal PVP of solubilization-aid effect as cosolvent in subsequent experiment, inquires into warm Extraction technique.
2.2.3 influence of the PVP co-solvent concentration to Puerarin recovery rate
With PVP be best cosolvent, control its dependent variable it is constant on the basis of, inquire into PVP concentration variation to Puerarin recovery rate
Influence.Experiment of single factor result is shown in Fig. 4.As shown in Figure 4, in the interval range of 20-50mg/mL, Puerarin mentions PVP concentration
Take rate that ascendant trend is presented as PVP concentration increases, when PVP concentration reaches 50mg/mL, Puerarin recovery rate reaches maximum
Value, continues growing PVP concentration, Puerarin recovery rate is declined instead, illustrates to work as PVP there may be a saturation PVP concentration
Concentration be more than this value when, the dissolution assistant effect of Puerarin can not be continued growing.When being higher than 40mg/mL due to PVP concentration, pueraria lobata
Plain recovery rate increases unobvious, and for cost consideration, when extraction, PVP concentration, which is chosen to be 40mg/mL, is advisable.
2.2.4 influence of the extraction time to Puerarin recovery rate
Using the PVP of 40mg/mL concentration as cosolvent, influence of the different extraction times to Puerarin recovery rate is investigated, as a result sees figure
5.The data from Fig. 5 are it is found that extraction time differs larger with the data of 2 recovery rates for 1 recovery rate, after illustrating extraction 1 time
Still there is quite a few Puerarin to remain in raw material；Conversely, 2 recovery rates are not much different with 3 recovery rate data, explanation
Puerarin residual is few after 2 extractions, therefore extracting twice substantially can be by effective component extraction completely.In addition, experimentation
In observe, the extracting solution obtained in third time extraction operation is more shallow compared to the preceding extracting solution color obtained twice, explanation
All extract concentrations including pigment are substantially reduced, this is consistent with the conclusion that experimental data obtains.Therefore it extracts
Number is chosen to be twice.
2.2.5 influence of the Extracting temperature to Puerarin recovery rate
Using the PVP of 40mg/mL concentration as cosolvent, extracts twice, the experiment of single factor of Extracting temperature is set, as a result sees Fig. 6.Figure
Data are shown in 6, and Puerarin recovery rate can rise with the raising of Extracting temperature.Because temperature raising leads to Puerarin in water
Solubility increase, to improve recovery rate, in addition, temperature height will lead to diffusion coefficient increase, can also accelerate extraction efficiency.
As can be seen from Figure 6, when Extracting temperature is 40 DEG C, the recovery rate of Puerarin is 0.9%, and with this condition, traditional water extraction pueraria lobata
Only 0.1%(is shown in Fig. 2 to the recovery rate of element).From experiment of single factor the result shows that, temperature be higher than 40 DEG C after, the increasing of Puerarin recovery rate
Acceleration tends towards stability, it is contemplated that high temperature also results in Puerarin and other Flavonoid substances decompose, and can destroy extract
Inoxidizability.In addition extraction cost also will increase, and Extracting temperature, which selects 40 DEG C, to be advisable.Therefore, it is extracted using PVP hydrotropy method
Puerarin belongs to low temperature extraction method in Pachyrhizua angulatus, avoids the shortcomings that recovery rate low and high temperature is decomposed under traditional handicraft low temperature.
2.2.6 influence of the solid-liquid ratio to Puerarin recovery rate
It using the PVP of 40mg/mL concentration as cosolvent, is extracted under the conditions of 40 DEG C twice, investigates different feed liquid comparison Puerarin and mention
The influence of rate is taken, experimental result is shown in Fig. 7.As solid-liquid ratio increases, i.e., quantity of solvent increases, and Puerarin recovery rate can rise with it；
The recovery rate of pueraria lobata dramatically increases when pueraria lobata recovery rate when solid-liquid ratio is 1:10 than solid-liquid ratio is 1:5, and when solid-liquid ratio is more than
When 1:10, Puerarin recovery rate increase, which is not obvious, then downward trend.In view of solvent increase can bring increased costs, dense
Contracting, the problems such as recycling design is more difficult, it is proper that solid-liquid ratio is set to 1:10.
2.2.7 influence of the extraction time to Puerarin recovery rate
Using the PVP of 40mg/mL concentration as cosolvent, solid-liquid ratio 1:10 extracts twice under the conditions of 40 DEG C, investigates different extractions
Influence of the time to Puerarin recovery rate, is as a result shown in Fig. 8.As seen from Figure 8, under the same conditions, extraction time is longer, Puerarin
Recovery rate it is higher.This is because the time is longer, the diffusion and dissolution of Puerarin are more abundant.It can by curvilinear trend in Fig. 8
Know, upon extracting between more than 2 hours after, recovery rate improve slow.Therefore extraction time selection 2 hours more appropriate.
The low temperature extraction process of Puerarin in 2.3 optimization of orthogonal test Pachyrhizua angulatus
2.3.1 orthogonal experiments
According to single factor experiment as a result, selecting PVP as cosolvent, fixed extraction number is 2 times, is investigated and is expected by orthogonal test
The influence of liquor ratio, Extracting temperature, extraction time, PVP concentration to the Puerarin recovery rate of Pachyrhizua angulatus extract；In single factor experiment
On the basis of, preferred processing condition.By L16(45) orthogonal test table tested, quadrature factor and level are shown in Table 1, orthogonal test knot
Fruit is shown in Table 2, and the results of analysis of variance is shown in Table 3.From intuitively analyzing available conclusion: factor A(solid-liquid ratio) K value size order
Are as follows: K4>K3>K1>K2；Factor B(Extracting temperature) K value size order are as follows: K4>K3>K2>K1；Factor C(extraction time) K value it is big
Small sequence are as follows: K3>K4>K2>K1；Factor D(PVP concentration) K value size order are as follows: K3>K2>K4>K1.Therefore each factor is more excellent
Level should be A4B4C3D3, i.e. solid-liquid ratio is 1:16, extracts 2.5 hours at 60 DEG C, and PVP concentration is 45mg/mL.By very poor
Analysis it is found that four factors it is very poorRSize order is D > B > C > A.It can be seen that each factor to pueraria lobata according to orthogonal experiment data
The influence of plain recovery rate is respectively from big to small: PVP concentration, Extracting temperature, extraction time, solid-liquid ratio.By variance analysis it is found that
Factor B and factor D is more significant factor, and factor A and factor C be not significant, this is consistent with the conclusion that range analysis obtains.
1 quadrature factor of table and water-glass
2 orthogonal experiments of table
| Tested number | A | B | C | D | E(blank) | Puerarin recovery rate (%) |
| 1 | 1 | 1 | 1 | 1 | 1 | 0.312 |
| 2 | 1 | 2 | 2 | 2 | 2 | 0.910 |
| 3 | 1 | 3 | 3 | 3 | 3 | 1.387 |
| 4 | 1 | 4 | 4 | 4 | 4 | 1.125 |
| 5 | 2 | 1 | 2 | 3 | 4 | 0.723 |
| 6 | 2 | 2 | 1 | 4 | 3 | 0.922 |
| 7 | 2 | 3 | 4 | 1 | 2 | 0.632 |
| 8 | 2 | 4 | 3 | 2 | 1 | 1.274 |
| 9 | 3 | 1 | 3 | 4 | 2 | 0.873 |
| 10 | 3 | 2 | 4 | 3 | 1 | 1.230 |
| 11 | 3 | 3 | 1 | 2 | 4 | 1.097 |
| 12 | 3 | 4 | 2 | 1 | 3 | 0.835 |
| 13 | 4 | 1 | 4 | 2 | 3 | 0.943 |
| 14 | 4 | 2 | 3 | 1 | 4 | 0.803 |
| 15 | 4 | 3 | 2 | 4 | 1 | 1.288 |
| 16 | 4 | 4 | 1 | 3 | 2 | 1.240 |
| k1 | 0.933 | 0.713 | 0.893 | 0.645 | 1.026 | |
| k2 | 0.888 | 0.966 | 0.939 | 1.056 | 0.914 | |
| k3 | 1.009 | 1.101 | 1.084 | 1.145 | 1.022 | |
| k4 | 1.069 | 1.119 | 0.983 | 1.052 | 0.937 | |
| R | 0.181 | 0.367 | 0.191 | 0.500 | 0.112 |
3 orthogonal test variance analysis of table
| Factor | Sum of square of deviations | Freedom degree | F value | Conspicuousness |
| A | 0.077 | 3 | 1.925 | It is not significant |
| B | 0.421 | 3 | 10.525 | Significantly |
| C | 0.080 | 3 | 2.000 | It is not significant |
| D | 0.600 | 3 | 15.000 | Significantly |
| Error E | 0.04 | 3 |
Note: α=0.05
2.3.2 confirmatory experiment
According to above-mentioned analysis, optimal extracting factor is that solid-liquid ratio is 1:16, extracts 2.5 hours at 60 DEG C, PVP concentration
For 45mg/mL；Again because variance analysis shows that solid-liquid ratio and extraction time are no conspicuousness influence factor, so considering to pass through
On the basis of Ji and time, and experiment of single factor is combined as a result, solid-liquid ratio 1:10, extraction time 2 hours economic items may be selected
Part；Verification test is carried out with this condition, and the results are shown in Table 4.Verification result shows the Puerarin recovery rate under optimal conditions
Up to 1.6%, the Puerarin recovery rate under economic condition also has 1.49%, and optimization low temperature can be selected to extract Pachyrhizua angulatus according to actual needs
Technique.
4 orthogonal test confirmatory experiment of table
| Experimental condition | 1 Puerarin recovery rate (%) | 2 Puerarin recovery rates (%) | 3 Puerarin recovery rates (%) | Average value |
| Optimal conditions | 1.594 | 1.610 | 1.608 | 1.604±0.007 |
| Economic condition | 1.477 | 1.493 | 1.501 | 1.490±0.013 |
2.4 antioxidant activity
By orthogonal test we determined that in Pachyrhizua angulatus Puerarin low temperature extraction process, while having investigated under this process condition
Pachyrhizua angulatus extract antioxidant activity, the results are shown in Table 5.The result shows that the extract of low temperature hydrotropy group is to OH and O2 -It is clear
Except rate is apparently higher than other two groups of control groups.The inoxidizability property basic source of Pachyrhizua angulatus extract is in wherein using Puerarin as generation
The isoflavone like substance of table.Low temperature control group does not add cosolvent when extracting, therefore the recovery rate of its Puerarin is lower, extract
The content of middle Puerarin is lower, therefore its extract is to OH and O2 -Clearance rate it is lower.And high temperature control group was extracting
Cheng Zhong, a part of Puerarin decompose loss because of high temperature, and in addition high temperature can also destroy the inoxidizability of kudzu root extract, therefore most
Its extract is to OH and O eventually2 -Clearance rate to be also lower than low temperature hydrotropy group.Therefore, using the low temperature after this research optimization
The kudzu root extract that hydrotropy extraction process is extracted has good inoxidizability compared with conventional high-temperature extracting method.
5 Pachyrhizua angulatus extract of table is to OH and O2 -Scavenging effect
| Experimental group | To the clearance rate (%) of OH | To O2 -Clearance rate (%) |
| 1(40 DEG C of hydrotropy group) | 40.98±0.83 | 20.87±0.92 |
| 2(60 DEG C of hydrotropy group) | 41.68±0.85 | 21.36±0.44 |
| 3(40 DEG C of control group) | 5.53±1.24 | 3.98±0.93 |
| 4(60 DEG C of control group) | 8.92±1.15 | 6.75±0.78 |
| 5(90 DEG C of control group) | 25.57±0.65 | 16.28±1.00 |
1(40 DEG C of hydrotropy group): it solid-liquid ratio 1:16, is extracted 2.5 hours at 40 DEG C, the PVP that concentration is 45mg/mL is added and helps
Solvent.
2(60 DEG C of hydrotropy group): it solid-liquid ratio 1:16, is extracted 2.5 hours at 60 DEG C, the PVP that concentration is 45mg/mL is added
Make cosolvent.
3(40 DEG C of control group): it solid-liquid ratio 1:16, is extracted 2.5 hours at 40 DEG C, cosolvent is not added.
4(60 DEG C of control group): it solid-liquid ratio 1:16, is extracted 2.5 hours at 60 DEG C, cosolvent is not added.
5(90 DEG C of control group): it solid-liquid ratio 1:16, is extracted 2.5 hours at 90 DEG C, cosolvent is not added.
3 conclusions
Compared with traditional water extraction, Puerarin in Pachyrhizua angulatus is extracted using PVP hydrotropy method and belongs to low temperature extraction method, avoids tradition
The shortcomings that recovery rate low and high temperature is decomposed under technique low temperature.And PVP has excellent biocompatibility, is highly soluble in water, safe nothing
Poison is widely used in medicine with field of food；But PVP has not been reported yet the hydrotropy mechanism of Puerarin, may be with PVP points
Sub physics and chemical association effect between drug molecule is related.On the basis of single factor experiment, determine that selecting PVP to be used as helps
Solvent, extraction time are 2 times, investigate solid-liquid ratio, Extracting temperature, extraction time, PVP concentration to Pueraria lobota in Pachyrhizua angulatus by orthogonal test
The low temperature extraction of root element optimizes.Wherein influence of each factor to Puerarin recovery rate is respectively from big to small: PVP concentration,
Extracting temperature, extraction time, solid-liquid ratio.Optimised process after optimization of orthogonal test is that solid-liquid ratio is 1:16, at 60 DEG C
It extracts 2.5 hours, PVP concentration is 45mg/mL；Economic condition technique is that solid-liquid ratio is 1:10, extracts 2 hours at 60 DEG C, PVP
Concentration is 45mg/mL, and under this optimal conditions, Puerarin recovery rate is respectively 1.6% and 1.49%.Meanwhile it is low using PVP hydrotropy
The Pachyrhizua angulatus extract that temperature is extracted, which has, improves OH and O2 -Clearance rate effect.This process is to improve food production
The utilization rate of Puerarin provides reference frame in Pachyrhizua angulatus in journey.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (7)
1. a kind of method for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which comprises the steps of:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 40 ~ 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10 ~ 16g/
ML, after extraction time is 2-2.5h, decompression is filtered, and collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 40-50mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
2. the method according to claim 1 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which is characterized in that institute
The extraction time stated is multiple.
3. the method according to claim 2 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which is characterized in that institute
The extraction time stated is 2 times.
4. the method according to claim 1 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which is characterized in that mention
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when taking.
5. the method according to claim 1 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which is characterized in that packet
Include following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:16g/mL, is extracted
Twice, each extraction time for after 2.5h, decompression is filtered, collects filtrate, is concentrated and dried to get the active pueraria lobata of high anti-oxidation
Element；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
6. the method according to claim 1 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus, which is characterized in that packet
Include following steps:
It after Pachyrhizua angulatus is crushed, using water as Extraction solvent, is extracted at 60 DEG C, the solid-liquid ratio of Pachyrhizua angulatus and water is 1:10g/mL, is extracted
Twice, each extraction time for after 2h, decompression is filtered, collects filtrate, is concentrated and dried to get the active Puerarin of high anti-oxidation；
It is 45mg/mL that polyvinylpyrrolidone to polyvinylpyrrolidoneconcentration concentration is added when extraction.
7. height made from the method as claimed in any one of claims 1 to 6 for extracting the active Puerarin of high anti-oxidation from Pachyrhizua angulatus
The Puerarin of antioxidant activity.
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