CN104644815B - A kind of method of utilization double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids - Google Patents
A kind of method of utilization double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids Download PDFInfo
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Abstract
The invention discloses a kind of method of utilization double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids；Methods described first crushes Chinese wampee leaf, add certain density isopropanol and ammonium sulfate solution formation double-aqueous phase system, with general flavone in ultrasonic extraction Chinese wampee leaf, methods described designs orthogonal test on the basis of single factor experiment result, preferably goes out the extracting factor of general flavone；Optimum extraction condition is defined as ammonium sulfate concentrations 310mg/mL, 40% aqueous isopropanol, ultrasonic time 20min, solid-liquid ratio 1: 40；The method of the present invention is easy to operate, shortens extraction time, effectively raises extraction efficiency.
Description
Technical field
It is more particularly to a kind of to utilize isopropanol-ammonium sulfate aqueous two-phase body the invention belongs to extract the technical field of general flavone
System and the method for ultrasonic integrated extraction Calusena lansium leaf flavonoids.
Background technology
Calusena lansium [Clausena lansium (Lour.) Skeels] belongs to Rutaceae Aurantiae plant, is perennial evergreen
Big shrub or dungarunga, originate in south China, Taiwan, Fujian, Guangdong, Hainan, Guangxi, South of Guizhou, Yunnan and Sichuan Jinsha
River Valley has cultivation.Branches and leaves can do medicinal material, with inducing diaphoresis radiating, qi-regulating and phlegm-dissipating, sterilization, hemostasis the effects such as.Flavonoid
Thing is widely present in plant tissue, and abundant flavone compound is contained in Chinese wampee leaf.Flavone compound can prevent many
The burst of disease, such as angiocardiopathy.The method that tradition extracts flavones in plant has supercritical CO2Extraction, extraction
Method etc..Aqueous two-phase extraction (Aqueous two-phase extraction, ATPE) technology is a kind of new separation technology, its side
Method is that one or more of materials are dissolved in water with appropriate concentration, the immiscible water solution system formed under certain condition
System, reaches according to the difference that pre-separation material is distributed in double-aqueous phase system (Aqueous two-phase system, ATPS)
To extraction purpose.ATPE has easy to operate, mild condition, product purity height, bioaffinity is good, solvent is easily reclaimed, be easy to
The advantages of with other Integration ofTechnologies.
The method that CN103211926A proposes general flavone in a kind of double-aqueous phase system extraction grape pip, this method is first with carrying
Take agent to extract plant component, extract solution, then addition ethanol, ammonium sulfate or propyl alcohol, ammonium sulfate shape into extract solution are obtained after filtering
Into double-aqueous phase system, extraction, point liquid obtain main component, and step is more, and the time is long.
In view of the shortcomings of the prior art, the present invention proposes a kind of method of new extraction Calusena lansium leaf flavonoids, directly uses
Isopropanol and a certain amount of ammonium sulfate are made into double-aqueous phase system solution, and plant component is extracted as extractant, are extracted when extracting,
Step is few, the time is short, efficiency high.
The content of the invention
Isopropanol-ammonium sulfate double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids are utilized the invention discloses one kind
Method, the described method comprises the following steps：
S100：Fresh Chinese wampee leaf is crushed after drying in the shade, 40 mesh sieves are crossed, sealed type storage is standby；
S200：Double-aqueous phase system is added into the Chinese wampee leaf of crushing；
S300：With general flavone in ultrasonic extraction Chinese wampee leaf, orthogonal test is designed on the basis of single factor experiment result, is selected
The extracting factor of general flavone.
Double-aqueous phase system is the double-aqueous phase system that inorganic salts are constituted with isopropanol in the step S200.
Described inorganic salts are preferably any of ammonium sulfate, sodium sulphate, sodium chloride, sodium carbonate, ammonium carbonate, are preferably
Ammonium sulfate.
In the double-aqueous phase system, the concentration of the inorganic salts is preferably 200-400mg/mL, and the isopropanol is preferred
For 20-40% isopropanol water solutions.
Ultrasonic wave rated power is 250W in the step S300, and the ultrasonic extraction time is in 20min.
The volume ratio of the Calusena lansium leaf quality and double-aqueous phase system is 1: 30.
The inorganic salt concentration 310mg/mL, the isopropanol is 40% isopropanol water solution, Chinese wampee leaf and aqueous two-phase body
The solid-liquid ratio 1: 40 (g/mL) of system, ultrasonic time 20min.
The method of the present invention is directly made into double-aqueous phase system solution with isopropanol and a certain amount of ammonium sulfate, is used as extractant
Plant component is extracted, is extracted when extracting, step is few, the time is short, efficiency high.
Isopropyl alcohol part is taken after extracting Calusena lansium leaf composition, filtering, point liquid with the method for the present invention, low-temperature evaporation is dried always
Extract, weighs, and analysis wherein flavones content is 71.34%.
Brief description of the drawings
Fig. 1 is influence of the double-aqueous phase system that constitutes of different salt and isopropanol to Chinese wampee leaf extracting flavonoids rate；In figure：A is
(NH4)2SO4, b is Na2SO4, c is (NH4)2CO3, d is NaCl, and e is Na2CO3；
Fig. 2 is influence of the ammonium sulfate concentrations to extracting flavonoids rate；
Fig. 3 is influence of the isopropyl alcohol concentration to extracting flavonoids rate；
Fig. 4 is influence of the ultrasonic time to extracting flavonoids rate；
Fig. 5 is influence of the solid-liquid ratio to extracting flavonoids rate.
Embodiment
Embodiment 1
A kind of method of utilization isopropanol-ammonium sulfate double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids, it is described
Method comprises the following steps：
S100：Fresh Chinese wampee leaf is crushed after drying in the shade, 40 mesh sieves are crossed, sealed type storage is standby；Chinese wampee leaf, picks up from Liuzhou Sha Tang
Town, Clausena Rutaceae Aurantiae plant fresh leaf is accredited as through doctor Qin Yiming.
S200：Double-aqueous phase system is added into the Chinese wampee leaf of crushing；Accurately weighing Chinese wampee leaf powder 1.00g is placed in 100mL
In round-bottomed flask, the aqueous isopropanol of various concentrations and the ammonium sulfate formation isopropyl of various concentrations are added by certain solid-liquid ratio respectively
Alcohol-ammonium sulfate aqueous two-phase solution.
S300：With general flavone in ultrasonic extraction Chinese wampee leaf, orthogonal test is designed on the basis of single factor experiment result, is selected
The extracting factor of general flavone.
By the aqueous two-phase solution containing Chinese wampee leaf ultrasonic certain time, Chinese wampee leaf is filtered, filtrate is placed in a point liquid leakage
2h is stood in bucket, upper solution is taken after point liquid, is settled to 25mL with 30% isopropanol, shakes up.
Embodiment 2
The present invention presses formula and calculates general flavone recovery rate
Quality (g) × 100% of general flavone recovery rate (%)=general flavone quality (g)/Chinese wampee leaf powder
Embodiment 3
The selection of salt
30mL 30% aqueous isopropanol is taken, ammonium sulfate, sodium sulphate, sodium chloride, carbonic acid is added by 300mg/mL respectively
Sodium, ammonium carbonate solid, concussion dissolving, form double-aqueous phase system, accurately weigh Chinese wampee leaf powder 1.00g, ultrasonic extraction 15min,
Filtering, filter residue is washed several times with a small amount of 30% aqueous isopropanol, and collection filtrate, which is placed in separatory funnel, stands 2h, takes upper strata molten
Liquid, is settled to 25mL with 30% isopropanol, shakes up, and flavones content is determined as described in Example 2, calculates the recovery rate of flavones,
Experimental result is shown in Fig. 1.
As a result show, the double-aqueous phase system constituted with ammonium sulfate and isopropanol is as the recovery rate of extract solution flavones than other
Four kinds of salt height, its extracting flavonoids rate is 2.68%, therefore experiment constitutes double-aqueous phase system to flavones from ammonium sulfate and isopropanol
Extracted.
Embodiment 4
Experiment of single factor
Influence of the ammonium sulfate consumption to recovery rate
Accurately weigh 5 parts of Chinese wampee leaf powder 1.00g, take 30mL 30% aqueous isopropanol, respectively by 240,260,
280th, 300,320mg/mL adds ammonium sulfate solids, concussion dissolving, forms stable double-aqueous phase system, ultrasonic 15min, filtering,
Washed with a small amount of 30% aqueous isopropanol several times, collection filtrate, which is placed in separatory funnel, stands 2h, takes upper solution, different with 30%
Propyl alcohol is settled to 25mL, shakes up, and flavones content is determined as described in Example 2, calculates extracting flavonoids rate.
Influence of the isopropyl alcohol concentration to recovery rate
6 parts of 1.00g Chinese wampee leaves powder is weighed, is respectively that 300mg/mL adds ammonium sulfate by concentration, is with solid-liquid ratio (m: V)
Be separately added into 20%, 25%, 30%, 35%, 40%, 45% aqueous isopropanol at 1: 30, and ultrasonic time 15min is filtered, used
Several times, collection filtrate, which is placed in separatory funnel, stands 2h, takes upper solution, uses 30% isopropyl for a small amount of 30% aqueous isopropanol washing
Alcohol is settled to 25mL, shakes up, and flavones content is determined as described in Example 2, calculates extracting flavonoids rate.
Influence of the ultrasonic time to recovery rate
6 parts of 1.00g Chinese wampee leaves powder is weighed, is respectively 1: 30 aqueous isopropanol for adding 40% by solid-liquid ratio (m: V), point
Not ultrasound 5,10,15,20,25,30min, filtering, is washed several times with a small amount of 30% aqueous isopropanol, is collected filtrate and is placed in a point liquid
2h is stood in funnel, divides and takes upper solution, be settled to 25mL with 30% isopropanol, shake up, flavones is determined as described in Example 2
Content, calculates extracting flavonoids rate.
Influence of the solid-liquid ratio to recovery rate
7 parts of 1.00g Chinese wampee leaves powder is weighed, is added respectively by 1: 15,1: 20,1: 25,1: 30,1: 35,1: 40,1: 45
The double-aqueous phase system solution of 40% aqueous isopropanol and 300mg/mL ammonium sulfate composition, ultrasonic 20min, filtering, with a small amount of 30%
Aqueous isopropanol is washed several times, and collection filtrate, which is placed in separatory funnel, stands 2h, and point liquid takes upper solution, uses 30% isopropanol
25mL is settled to, is shaken up, flavones content is determined as described in Example 2, extracting flavonoids rate is calculated.
Experiment of single factor result
Influence of the ammonium sulfate concentrations to extracting flavonoids rate
Experimental result is as shown in Figure 2.
Fig. 2 shows that general flavone recovery rate first increases with ammonium sulfate concentrations to be declined afterwards, and ammonium sulfate concentrations are in 240~300mg/
Between mL, general flavone recovery rate increases with the increase of ammonium sulfate concentrations, and ammonium sulfate concentrations are reached after 320mg/mL, general flavone
Recovery rate starts reduction, it can be seen that general flavone recovery rate is maximum when ammonium sulfate concentrations are 300mg/mL or so.
Influence of the isopropyl alcohol concentration to extracting flavonoids rate
Experimental result is as shown in Figure 3.
As seen from Figure 3, with the increase of isopropyl alcohol concentration, general flavone, which is extracted, to take the lead in subtracting after increasing；Its concentration 20%~
When 40%, general flavone recovery rate increases and increased with isopropyl alcohol concentration；General flavone recovery rate declines when isopropanol is 45%.Cause
This, the isopropanol effect of experimental selection 40% is preferable.
Influence of the ultrasonic time to extracting flavonoids rate
Experimental result is as shown in Figure 4.
From fig. 4, it can be seen that with the increase of ultrasonic time, general flavone recovery rate progressively increases, increase very fast in 5~20min,
Increasing degree is smaller after 20min, because extracting flavonoids are close complete, therefore ultrasonic time is to be in 20min or so
Preferably.
Influence of the solid-liquid ratio to extracting flavonoids rate
Experimental result is as shown in Figure 5.
As seen from Figure 5, solid-liquid ratio increases soon at 1: 15~1: 30, is begun to decline after 1: 35, it can be seen that general flavone
Recovery rate is maximum at solid-liquid ratio 1: 30, therefore solid-liquid ratio is that 1: 30 extraction effect is preferable.
Embodiment 5
Orthogonal experiment
According to experiment of single factor result, L is used9(34) table design arranges orthogonal experiment, empirical factor level is shown in Table 1, orthogonal reality
Test and arrange and the results are shown in Table 2.
The orthogonal experiment factor level table of table 1
The experiment of single factor in embodiment 4, during the integrated extraction Calusena lansium leaf flavonoids of ultrasound-aqueous two-phase, its recovery rate
Carried out by ammonium sulfate concentrations, isopropyl alcohol concentration, ultrasonic time, four factors of solid-liquid ratio, be that further extracting flavonoids condition is entered
One-step optimization, carries out the orthogonal experiment of the level of 4 factor 3, and orthogonal experiment factor level is shown in Table 1, experimental arrangement and the results are shown in Table 2.
The orthogonal experiment arrangement of table 2 and experimental result
Each factor can be obtained according to the extreme difference R of Orthogonal experiment results B is followed successively by from big to small to the primary and secondary of general flavone recovery rate
＞ C ＞ D ＞ A, i.e. isopropyl alcohol concentration ＞ solid-liquid ratios ＞ ultrasonic times ＞ ammonium sulfate consumptions；Optimum extraction is determined according to k value sizes
Condition is A3B3C3D3, i.e. 40% isopropanol, solid-liquid ratio 1: 40, ultrasonic time 20min, ammonium sulfate concentrations 310mg/mL.
Embodiment 6
The assay method of flavones content,：
Accurate to weigh 10.00mg control substance of Rutin, plus methanol 10mL, low-grade fever dissolving is cooled to room temperature, is transferred to 50mL appearances
Measuring bottle, uses methanol solution constant volume, shakes up, and obtains concentration for 0.20mg/mL control substance of Rutin solution, pipettes 0.0,2.0,4.0,6.0,
8.0,10.0mL control substance of Rutin solution add water to 10ml or so, plus 5%NaNO21.00mL, shaken up in 25mL volumetric flasks,
5min, plus 10%Al (NO3) 31.0mL are placed, is shaken up, 5min, plus 4%NaOH 10mL is placed, adds water to graduation mark, shake up,
15min is placed, using blank as reference, absorbance is surveyed at maximum absorption wavelength 510nm.Using the concentration of rutin standard liquid as
Ordinate, using absorbance A as abscissa, draws standard curve, is returned with least square method, obtain absorbance and rutin mark
The regression equation of the concentration c (mg/mL) of quasi- solution：C=0.1825A+0.001, correlation coefficient r=0.9973, this shows the party
Method has good linear relationship in 0.00-0.08mg/mL concentration ranges, the measure available for general flavone content in Chinese wampee leaf.
Measure need testing solution a certain amount of, solution is separately added into as stated above and absorbance is determined, by absorbance
General flavone quality in regression equation calculation general flavone concentration and sample is substituted into, general flavone recovery rate is calculated by the method for example 2.
Embodiment 7
A kind of method of utilization isopropanol-ammonium sulfate double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids, it is several
General flavone extracts the comparison of yield under different condition：
S100：Accurately Chinese wampee leaf powder is weighed to be placed in 100mL round-bottomed flasks；
S200：Ammonium sulfate with finite concentration (20%~45%) isopropanol water solution is made into double-aqueous phase system solution (dense
Spend for 240~320mg/mL)；
S300：Double-aqueous phase system solution accurately is measured by certain solid-liquid ratio (between 1: 15 to 1: 40), above-mentioned round bottom is poured into and burns
Mixed in bottle with Chinese wampee leaf powder；
S400：After ultrasonic extraction certain time (5~30min), filtering, point liquid, measuring general flavone extraction yield is
1.62%.
Change ammonium sulfate concentrations, isopropyl alcohol concentration, solid-liquid ratio and ultrasonic time, measure under different condition several results such as
Under：
General flavone extracts the comparison of yield under the different condition of table 3
Note：Experiment 5 is the extraction effect of this method optimal conditions.
Claims (4)
1. a kind of method of utilization double-aqueous phase system and ultrasonic integrated extraction Calusena lansium leaf flavonoids, it is characterised in that methods described
Comprise the following steps：
S100：Crushed after fresh Chinese wampee leaf is dried in the shade, sealed type storage is standby；
S200：Double-aqueous phase system is added into the Chinese wampee leaf of crushing；
S300：By the ultrasonic certain time of the double-aqueous phase system containing Chinese wampee leaf, Chinese wampee leaf is filtered, filtrate is placed in separatory funnel
2h is stood, upper solution is taken after point liquid, obtains Calusena lansium leaf flavonoids；
Wherein：
Double-aqueous phase system is the double-aqueous phase system that inorganic salts are constituted with isopropanol in the step S200；
In the double-aqueous phase system, the concentration of the inorganic salts is 200-400mg/mL, and the isopropanol is 20-40% isopropyls
Alcohol solution；
Ultrasonic time is 20min in the step S300；
The inorganic salts are ammonium sulfate.
2. according to the method described in claim 1, it is characterised in that the recovery rate calculation formula of general flavone in the step S300
It is as follows：
Quality (g) × 100% of general flavone recovery rate (%)=general flavone quality (g)/Chinese wampee leaf powder.
3. according to the method described in claim 1, it is characterised in that the volume ratio of the Calusena lansium leaf quality and double-aqueous phase system
For 1: 30 (g/ml).
4. according to the method described in claim 1, it is characterised in that the inorganic salt concentration 310mg/mL, the isopropanol is
The solid-liquid ratio 1: 40 (g/mL) of 40% isopropanol water solution, Chinese wampee leaf and double-aqueous phase system.
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| CN112876443B (en) * | 2021-02-05 | 2023-02-03 | 许昌学院 | Method for extracting flavonoid active substances in honey |
| CN114796331A (en) * | 2022-04-07 | 2022-07-29 | 福建师范大学 | Method for preparing total flavonoids of fructus aurantii |
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| CN103211926A (en) * | 2013-05-02 | 2013-07-24 | 甘肃凯源生物技术开发中心 | Method for extracting total flavonoids in grape seeds in double-aqueous phase system |
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| Title |
|---|
| 双水相萃取法分离纯化黄酮类化合物的研究进展;陈丛瑾等;《应用化工》;20101031;第39卷(第10期);第1587-1589、1596页 * |
| 超声波法提取黄皮叶中黄酮工艺的优化;周燕芳;《湖北农业科学》;20120430;第51卷(第7期);第1433-1435页 * |
| 超声集成丙醇-硫酸铵双水相体系从苦荞麦苗中提取总黄酮及其抗氧化活性研究;高云涛等;《食品科学》;20091231;第30卷(第2期);第110-113页 * |
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