CN107050161A - A kind of extraction of one side pin total alkaloid and isolation and purification method - Google Patents
A kind of extraction of one side pin total alkaloid and isolation and purification method Download PDFInfo
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Abstract
The invention discloses a kind of extraction of one side pin total alkaloid and isolation and purification method, the extraction of one side pin total alkaloid is optimized using response method and isolated and purified, this method fast, economical, the perfect extraction and preparation technique of one side pin total alkaloid, the extraction process reported is more efficiently quick, and product purity is higher.
Description
Technical field
One side pin total alkaloid is extracted the present invention relates to a kind of response phase method, and product purification point is carried out using macroreticular resin
From extraction preparation method, belong to effective ingredients in plant extractive technique field.
Background technology
One side pin (Zanthoxylum Dissitum Hemsl) also known as a species of small clam living in fresh water shell Chinese prickly ash.Root, stem and leaf can be used as medicine, clinical
On have the effect of blood stasis removing analgesic, removing toxicity for detumescence, it is excessive to cure mainly toothache, pain in the back, woman in menstrual period, the disease such as postpartum irregular menstruation；Research
It is primary medicinal component to show in one side pin active alkaloid, wherein alkaloid contained by the one side pin being currently known have dictaminine,
A variety of alkaloids such as nitidine, fagarine, skimmianine, sanguinarine, Chinese prickly ash canopy alkali are all effective to inflammation and suppression tumour.
In recent years, because Chinese patent drug production scale expands increasingly, and the natural propagation rate of one side pin is low, and growth is also more slow, causes
Make that the natural resources are increasingly poor, supply falls short of demand, and gradually threatens the normal production in related pharmaceutical factory.Therefore, seek suitable
Extracts active ingredients preparation technology is most important to making full use of for its resource.
Response phase method extraction optimization, be a kind of effective ways of optimization of process conditions, set up continuous variable surface model,
Regression equation investigates different tests factor and its interaction come the relation being fitted between multiple response variables and a series of experiments variable
Act on the influence to target response value.This method can optimum combination condition that is economic, effectively and quickly determining many experimental factors.
This research extracts solution as solvent to mix, and examines different volumes fraction mixed solution, liquid ratio, Microwave Extraction time, temperature
The affecting laws to one side pin total alkaloid are spent, optimum extraction process is obtained using response phase method analysis.
Macroreticular resin is a kind of polymer absorbant, the high molecular polymer to organic matter with concentration, centrifugation, choosing
The separating-purifying effect of specific organic matter can be effectively played by selecting suitable resinous type, by examining or check during separating-purifying
The adsorption rate of pH, mixed extract volume fraction, loading flow velocity, eluent flow rate, eluent consumption to selected macroreticular resin
With the influence of desorption.While obtaining the optimize technique of a species of small clam living in fresh water shell Chinese prickly ash Microwave Extraction, its also perfect separation purifying technique.
And the comparison of the active alkaloid bacteriostatic experiment after separating-purifying is made, verify that its purifying process can be effectively obtained purity higher
One side pin total alkaloid.
The content of the invention
The present invention is directed in existing one side pin active ingredient alkaloid extractive technique：Extraction efficiency is low, active ingredient is lived
Property the defect such as low, impurity is higher there is provided the microwave loss mechanisms that a kind of response phase method optimizes one side pin total alkaloid, and separation
Purifying process.
A kind of extraction of one side pin total alkaloid and isolation and purification method, one side crown stem is first mixed and is milled, add second
Alcohol-acetone mixed solution, carries out Microwave Extraction；Gained extract solution recycles resin isolate and purify, and to obtain one side pin always biological
Alkali.
As a further improvement, in the above method one side crown, stem according to mass ratio 1:2 mixing.
As a further improvement, powder is beaten into the mixing of one side crown stem in the above method and crosses 60 mesh sieves, obtain rhizome powder.
As a further improvement, the volume ratio of ethanol and acetone is 1 in the above method:1.
As a further improvement, in the above method during Microwave Extraction, the percent by volume of ethanol-acetone mixed solution is
70~90%, remaining is water；Solid-liquid ratio is 15:1~25:1mL.g-1；Extracting temperature is 50~70 DEG C；Extraction time be 15~
25min。
As a further improvement, it is preferred during Microwave Extraction in the above method, the volume basis of ethanol-acetone mixed solution
Than for 89%, remaining is water；Solid-liquid ratio is 21:1mL.g-1；Extracting temperature is 70 DEG C；Extraction time is 20min.
As a further improvement, microwave power is 300W in the above method.
As a further improvement, Microwave Extraction number of times is 2 times in the above method, and waits extraction system recovery for the first time
Carry out extracting for second after room temperature.The temporal summation extracted twice, is 40 minutes except the intercooled time.
As a further improvement, resin selects NKA-9 macroporous absorbent resins in the above method.
As a further improvement, the actual conditions isolated and purified in the above method is：Crude drug is 1 with resin quality ratio:
1, regulation sample solution pH is 7.0, loading flow velocity 2BV/h, with the 1 of 6BV 80% during elution:The ethanol-acetone mixed liquor of 1 volume
Eluted with 2BV/h speed.
The present invention, which dries perennial one side crown stem, to be smashed after 60 mesh sieves by rhizome powder 1:2 mixing, are handled
To mixing rhizome powder, weigh a certain amount of one side crown stem powder and add in container, the mixing for being separately added into different volumes fraction is molten
Liquid, setting different temperatures, solid-liquid ratio carry out Microwave Extraction.Gained extract solution is being adsorbed and desorption reaches using macroreticular resin
The purpose of one side pin total alkaloid is isolated and purified, one side pin total alkaloid extract solution after purification is obtained；
The variable parameter that above-mentioned response phase method setting is extracted passes through following regression equation：Y=-43.05333+0.93982A+
0.89240B+13.24900C-0.36270D+3.49700AB+0.049500AC+7.197AD-6.997BC-2.496BD+
0.011500CD-9.15250A2-0.027560B2-4.1010C2-1.912D2。
In above-mentioned, Y is the extracted amount of response and one side pin total alkaloid；Variables A is set as mixed extract volume integral
Number, B are solid-liquid ratio；C is the microwave time；D is Extracting temperature；The A is 70~90%；The B is 15:1~25:1mL.g-1；
The C is 15~25min；The D is 50~70 DEG C.
Determined after one side pin Determination of Total Alkaloid, the total alkaloid extract solution dilution respective volume according to obtained by experiment
Light absorption value, compares dictaminine standard curve, draws the concentration of total alkaloid in extract solution, calculates gained total alkaloid quality,
Finally calculate the extracted amount of one side pin total alkaloid.Calculation formula is as follows；
The standard curve of above-mentioned dictaminine and obtaining for regression equation：Precision weighs dictaminine standard items 1.2mg and is dissolved in
In 25mL absolute ethyl alcohols, be configured to 48 μ g/mL titer, respectively it is accurate draw dictaminine titer 0.5,1.0,1.5,2.0,
2.5mL being placed in 10mL volumetric flasks, absolute ethyl alcohol is settled to scale.Using absolute ethyl alcohol as blank control, determine each at 310nm
Solution absorbance, with concentration C (ug/ml) for abscissa, absorbance A is ordinate, draws standard curve and draws linear regression side
Journey A=32.5C-0.0085, r=0.9991.
The beneficial effects of the invention are as follows：Compared with prior art, the present invention uses multistage microwave amplifier one side pin total alkaloid
Obtained Optimized Simulated equation：Y=-43.05333+0.93982A+0.89240B+13.24900C-0.36270D+
3.49700AB+0.049500AC+7.197AD-6.997BC-2.496BD+0.011500CD-9.15250A2-0.027560B2-
4.1010C2-1.912D2.The Optimized Extraction Process obtained by response surface analysis is the one side pin total alkaloid optimum extraction of optimization
Process conditions are:Ethanol-acetone (1:1) mixing liquid fraction 89%, solid-liquid ratio 21mL/g, microwave time 20min, extraction temperature
It is 12.35mg/g that one side pin total alkaloid amount is extracted under 70 DEG C of degree, this process conditions, is differed only by with model prediction theoretical value
0.032mg/g.Later stage NKA-9 macroreticular resin is used for isolating and purifying for one side pin total alkaloid, and crude drug is 1 with resin quality ratio:1
(g/g), regulation sample solution pH is 7.0, loading flow velocity 2BV/h, during elution, with the ethanol-acetones (1 of 6BV 80%：1) mixed solution
Eluted with 2BV/h speed, gained total alkaloid average purity is 70.9%, and bacteriostatic experiment shows carried alkaloid to spy
Determining bacterium has good biocidal property.Its extraction separation and purification fast, economical, the extraction of perfect one side pin total alkaloid prepares work
Skill, the extraction process reported is more efficiently quick.
Brief description of the drawings
Fig. 1 is influence of the Extraction solvent concentration to extraction effect in embodiment 3；
Fig. 2 is influence of the solid-liquid ratio to total alkaloid extraction effect in embodiment 4；
Fig. 3 is influence of the microwave time to total alkaloid extraction effect in embodiment 5；
Fig. 4 is influence of the temperature to total alkaloid recovery rate effect in embodiment 6；
Fig. 5 is to interact in embodiment 7 to influence total alkaloid extraction effect two-by-two between each extraction factor；
Fig. 6 is influences of the sample liquid pH to Adsorbent rate during total alkaloid is isolated and purified in embodiment 9；
Fig. 7 is crude drug and shadow of the resin ratio to Adsorbent rate during total alkaloid is isolated and purified in embodiment 10
Ring；
Fig. 8 is shadow of the mixing liquid fraction to resin desorption rate during total alkaloid is isolated and purified in embodiment 11
Ring；
Fig. 9 is influence of the loading flow velocity to Adsorbent rate during total alkaloid is isolated and purified in embodiment 12；
Figure 10 is influence of the eluent flow rate to resin desorption rate during total alkaloid is isolated and purified in embodiment 13；
Figure 11 is influence of the eluent consumption to resin desorption rate during total alkaloid is isolated and purified in embodiment 14.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment, the present invention is furture elucidated, and its embodiment is only used for this
Invention rather than the limitation present invention.
Example 1 below selects for the solvent of microwave loss mechanisms, and embodiment 2 is that rhizome mixed proportion is selected to list with particle diameter
The influence that face pin total alkaloid is extracted, operates the most suitable rhizome mixed proportion of selection with particle diameter to ensure to carry according to actual industrial
Cost is reduced while taking effect.Embodiment 3 to 6 selects different extract concentrations, solid-liquid ratio, microwave time and temperature respectively
Degree carries out experiment of single factor, examines or check influence of the single factor to the extracted amount of one side pin total alkaloid.Embodiment 7 is in single factor test
On the basis of, carry out experimental design using Design-Expert.V8.0.6 softwares and response phase method is analyzed, with total alkaloid
Extracted amount (Y) is response, and mixed liquid concentration (A), solid-liquid ratio (B), extraction time (C), Extracting temperature (D) are variable parameter,
Extraction process to one side pin total alkaloid is optimized.Embodiment 8 to 14 screens most suitable macroreticular resin from 5 kinds of resin materials
The separating-purifying of total alkaloid is carried out, and passes through the pH of first extract, crude drug and resin ratio, mixing liquid fraction, sample solution
Flow velocity, eluent flow rate and consumption progressively establish the i.e. optimal purifies and separates bar of optimal adsorption and resolution factor of this macroreticular resin
Part, embodiment 15 carries out In Vitro Bacteriostasis test to the one side pin total alkaloid after separating-purifying, verifies its fungistatic effect.
Embodiment 1
Influence of the different solvents to Microwave Extraction effect
Weigh 10 parts it is vacuum dried after the one side crown stem mixing each 10g of crude drug powder, be separately added into 20mL 1% (v/v)
Ammoniacal liquor infiltration, then with distilled water, absolute ethyl alcohol, n-butanol, chloroform, acetone, ethyl acetate, EtOH-EtOAc (v: v
=1：1), ethanol-n-butanol (v: v=1: 1), ethanol-chloroform (v: v=1: 1), ethanol-acetone (v: v=1: 1) are molten to extract
Agent, the solvent of crude drug powder and 10 times of amounts is loaded in flask and mixed, and 70 DEG C of microwaves carry 20min, extract solution concentration system after immersion 15h
Medicinal extract is obtained, water-soluble alkaloid extracted amount is calculated, as a result as shown in table 1.So selection ethanol-acetone (1:1) mixed solution is
Extractant.
The different solvents Microwave Extraction one side pin effect of table 1
Embodiment 2
The influence of different-grain diameter and the rhizome powder of mixed proportion to Microwave Extraction effect
5 parts of dry one side crowns and stem powder are weighed respectively, 40,60,80,100 mesh sieves are crossed respectively, according to 1 after sieving:
1、1:2、1:3 ratio is mixed into 5g aggregate samples.80% mixing Extraction solvent 50mL, solid-liquid ratio 20g/mL are separately added into, is soaked
15h is steeped after 70 DEG C of Microwave Extraction 20min, and filtering, filter residue is repeated to extract once with same procedure, is merged filtrate twice, is calculated
Total alkaloid extracted amount, as a result as shown in table 2, different-grain diameter, different rhizome mixed proportions equally can be to one side pin total alkaloids
Extraction effect produce influence.
The different rhizome mixed proportions of table 2 are with sieving number to extracting one side pin effect
| Rhizome mixed proportion 1:1 | Rhizome mixed proportion 1:2 | Rhizome mixed proportion 1:3 | |
| 40 mesh sieves | 9.02mg/g | 8.11mg/g | 8.21mg/g |
| 60 mesh sieves | 11.25mg/g | 11.21mg/g | 8.24mg/g |
| 80 mesh sieves | 11.31mg/g | 11.23mg/g | 8.52mg/g |
| 100 mesh sieves | 11.56mg/g | 11.32mg/g | 8.54mg/g |
One side crown stem is beaten during powder, and the hardness of root is higher, is often needed to carry out repeatedly to beat powder and just be can obtain root powder, and root
In alkaloid be also relatively higher than stem.Extracted to meet actual industrial, thus adjustment rhizome powder mixed proportion, carry as far as possible
The consumption that the utilization rate of high stem can save material reduces waste, while in order to obtain higher total alkaloid recovery rate, suitable
Particle size can also improve Microwave Extraction efficiency, and in summary factor drops, and the loss of low material and extraction cost are ensured simultaneously
It is about 250 microns of rhizome powder by 1 that certain extraction efficiency, which selected the rhizome powder and particle diameter of 60 mesh sieves,:2 ratio is mixed
Close and extract, though this mixed proportion is not optimal alkaloid withdrawal ratio, relatively other mixed proportions are with Particle size requirements in cost
It is relative with material consumption reverse side minimum and close to optimum value.
Embodiment 3
Influence of the Extraction solvent concentration to extraction effect
Weigh 5 parts of dry one side crown stem powder, every part of 5g.It is separately added into 50%, 60%, 70%, 80%, 90%
Extraction solvent 50mL, solid-liquid ratio 20g/mL are mixed, immersion 15h is after 70 DEG C of Microwave Extraction 20min, filtering, filter residue phase Tongfang
Method repeats to extract once, merges filtrate twice, calculates total alkaloid extracted amount.Mixed extract volume fraction be 50%~
In the range of 90%, with the increase of mixed extract volume fraction, total alkaloid recovery rate gradually increases.When mixed extract volume
When fraction is more than 80%, total alkaloid recovery rate reduction (Fig. 1).Therefore when mixing liquid fraction is 80%, the pole of extract solution
The polarity of property and one side pin total alkaloid is closest, the dissolution rate highest of alkaloid.
Embodiment 4
Influence of the solid-liquid ratio to total alkaloid extraction effect
Weigh 5 parts of dry one side crown stem powder, every part of 5g, be separately added into 80% mixed extract 25,50,75,
100th, 125mL, immersion 15h are repeated to extract once after 70 DEG C of ultrasonic extraction 20min, filtering, filter residue with same procedure, merge two
Secondary filtrate, calculates total alkaloid extracted amount.With the increase of solid-liquid ratio, two alternate concentration difference increases are conducive to total alkaloid
Diffusion dissolution, the recovery rate of total alkaloid is continuously increased；When solid-liquid ratio reaches 1:When 20, the dissolution of total alkaloid has tended to
Entirely, extracted amount with solid-liquid ratio increase Long-term change trend less (Fig. 2), and solid-liquid ratio cross conference cause production cost increase and after
Continuous filtering and concentrating time long impurity increases, and total alkaloid loss is more etc., therefore it is 1 to choose suitable solid-liquid ratio:20.
Embodiment 5
Influence of the microwave time to total alkaloid extraction effect
Weigh 5 parts of dry one side crown stem powder, every part of 5g, be separately added into 80% mixed extract 50mL, immersion
Respectively at 70 DEG C of Microwave Extractions 5,10,15,20,25min after 15h, filtering, filter residue is repeated to extract once with same procedure, is merged
Filtrate, calculates total alkaloid extracted amount twice.As extraction time increases, the recovery rate of total alkaloid is in increase trend, when carrying
Take the time more than 20min, extracted amount increase is not obvious, now, the basic dissolution of total alkaloid, extraction time is long may to cause life
Alkaloids are unstable under higher microwave energy, and composition is gradually destroyed by microwave heat temperature raising, and the extraction total amount of alkaloid is on the contrary
It can descend (Fig. 3), and the microwave energy of long period can make cell is greatly broken to be allowed to more contents releases
Out cause impurity to improve, be difficult and later-period purification, therefore ultrasonic time 20min is optimal.
Embodiment 6
Influence of the temperature to total alkaloid recovery rate
Weigh 5 parts of dry one side crown stem powder, every part of 5g, be separately added into 80% mixed solvent 50mL, soak 15h
Afterwards respectively at 50,60,70,80,90 DEG C of Microwave Extraction 15min, filtering, filter residue repeats to extract once with same procedure, merges two
Secondary filtrate, calculates total alkaloid extracted amount.As temperature is raised, total alkaloid extracted amount is raised and increased with temperature；Temperature is big
In 50 DEG C, total alkaloid extracted amount increase is not notable, and peak value (Fig. 4) is reached at 60 DEG C.Total alkaloid is carried under the conditions of illustrating this
Take substantially completely.The too high meeting accelerating alcohol of temperature-acetone volatilization makes its biological alkali-solubility reduction, influences extraction effect, simultaneously
Alkaloid can be caused to cross thermal decomposition, extracted amount is reduced.Therefore optimum extraction temperature 60 C is selected.
Embodiment 7
Response phase method optimizes the extraction process of one side pin total alkaloid
On the basis of experiment of single factor, using Design-Expert.V8.0.6 software data processings.Choose mixed liquor
Volume fraction, solid-liquid ratio, Microwave Extraction time, 4 factors of Extracting temperature make empirical factor, using total alkaloid extracted amount as response
Value, contrived experiment.Response surface experimental design and it the results are shown in Table 3.
The response surface experiments result of table 3
Note：Data are 3 repetition experiment average values in table.
Note：The date in table is the mean of three repeated experiments.
It is certainly with A (mixed extract volume fraction %), B (solid-liquid ratio mL/g), C (microwave time min), D (temperature DEG C)
Variable, one side pin total alkaloid extracted amount is response, and 29 groups of response surface analysis experiments are carried out altogether, wherein 5 center experiments, are used
With evaluated error, 24 are that factorial experiment is analyzed test result by response surface software, obtain always biological with one side pin
Alkali carries taken amount is the regression equation of response：Y=-43.05333+0.93982A+0.89240B+13.24900C-0.36270D+
3.49700AB+0.049500AC+7.197AD-6.997BC-2.496BD+0.011500CD-9.15250A2-0.027560B2-
4.1010C2-1.912D2.To the data analysis of table 2, regression model P values are less than 0.05, its coefficient of determination R2=for 0.0319
0.8369, pure error is not notable, illustrates that the fit equation has a significant impact and is consistent with actual conditions.
The response surface experiments variance analysis of table 4
Note：* represent notable, * * represent extremely notable.
Note：*and**indicated significance at 0.05and 0.01levels,respectively
Lose plan item P values and show that Orthogonal experiment results and the good error of the Model fitting are smaller more than 0.1.The fitting side
Journey can fully reflect the relation between response and each factor, and it influences not simple linear relationship.Exist between each factor
Certain reciprocation, wherein A, B2, C2 are that pole significantly affects P values respectively less than 0.01, and C, AC, AD, BC, A2 are to significantly affect.
And then can show that its influence order is from the point of view of single factor test level:A (mixed extract volume fraction %)>C (the microwave times
min)>B (solid-liquid ratio mL/g)>D (temperature DEG C).In the presence of reciprocation, the influence to one side pin total alkaloid extracted amount
Sequentially it is:AC>BC>AD>AB>CD>BD.
Network analysis is carried out using Design-Expert.V8.0.6 softwares, optimum extraction one side pin total alkaloid work is drawn
Skill condition is:Mixed extract volume fraction 88.87%, solid-liquid ratio 21.23mL/g, microwave time 20min, 70 DEG C of Extracting temperature
When one side pin total alkaloid extracted amount can reach 12.382mg/g, limitation during with reference to practical operation finally draws and most preferably carried
Taking technique condition is:Mixing liquid fraction 89%, solid-liquid ratio 21mL/g, microwave time 20min, 70 DEG C of Extracting temperature, by this
Optimized Extraction Process carries out 3 repetitions and tested, and it is 12.35mg/g to average as total alkaloid extracted amount, is differed with theoretical value
0.032mg/g, it was demonstrated that this response surface optimization extracting factor is reliable.
Embodiment 8
The selection of 5 kinds of macroreticular resins
The 5 kinds of each 1g of model macroreticular resin handled well are taken, are respectively placed in 50mL conical flasks, be prepared by each above method that adds
Total alkaloid stoste 30mL, 120r/min shaking tables 24h is to adsorption equilibrium at room temperature.Filtering, determines total alkaloid in filtrate dense
Degree, calculates the adsorption rate of macroreticular resin.It is placed in 50mL conical flasks, adds respectively after macroreticular resin after filtering is blotted with filter paper
Enter 70% ethanol-acetone (1:1) mixed liquor 30mL, 120r/min shaking tables 24h is to desorbing balance at room temperature.Determine total in stripping liquid
Alkaloid concentrations, calculate desorption efficiency.Adsorption rate and desorption efficiency using total alkaloid filters out as index and is most suitable for total alkaloid point
From the resinous type of purifying.
Above-mentioned adsorption rate and desorption efficiency computational methods：Adsorption rate P=((C0-C1) × 100%)/C0 desorption efficiencies D=(C ×
V0)/adsorbance of (Q × M) × 100%=((C0-C1) × V)/M
In above-mentioned formula：Q is adsorbance (mg/g), and C0 is total alkaloid extract solution initial concentration (mg/mL), after C1 is absorption
Total alkaloid solution concentration, V is total alkaloid liquor capacity, and M is resin quality, and C is stripping liquid concentration (mg/mL), and V0 is solution
Imbibition volume.As a result it is as shown in table 5
55 kinds of macroreticular resins of table are to total alkaloid Static Adsorption and desorption result
From the result of table 4,3 indexs of absorption ration, adsorption rate, desorption efficiency of NKA-9 type resins are obviously higher than it
His 4 kinds of resins, illustrate that NKA-9 types resin is applied to isolating and purifying for one side pin total alkaloid, therefore this process choice NKA-9 types
Resin is used for isolating and purifying for one side pin total alkaloid.
Embodiment 9
Influences of the sample liquid pH to Adsorbent rate
Precision weighs 5 parts of the NKA-9 resins that 1.0g has been pre-processed, and is respectively placed in 50mL conical flasks, respectively adds pH value and (uses
Hydrochloric acid and ammoniacal liquor regulation) be 4.0,5.0,6.0,7.0,8.0 total alkaloid extract solution 30mL, 120r/min shaking tables at room temperature
24h is to adsorption equilibrium.Filtering, determines total alkaloid concentration in filtrate, calculates the adsorption rate of macroreticular resin.Such as Fig. 6 sample liquid pH value
Absorption of the macroreticular resin to total alkaloid is significantly affected, the existence of total alkaloid is not under the conditions of different pH, in sample liquid
Together, the binding ability with resin has differences, therefore the absorption of influence resin.In the range of pH4.0-7.0, with pH increasing
Plus, macroporous resin adsorption rate gradually increases, and when pH is 8.0, adsorption rate decreases, and illustrates pH when being 7.0 most beneficial for macropore
Absorption of the resin to total alkaloid, accordingly, it is determined that sample solution pH is 7.0.
Embodiment 10
Crude drug and influence of the resin ratio to Adsorbent rate
Precision weighs 5 parts of the NKA-9 resins that 1.0g has been pre-processed, and is respectively placed in conical flask, is separately added into total alkaloid
Extract solution 10,20,30,40,50mL (equivalent to the ratio between crude drug and resin quality be 0.5,1,1.5,2,2.5g/g), adjust pH value
For 7.0,120r/min shaking tables 24h is to adsorption equilibrium at room temperature.Filtering, determines total alkaloid concentration in filtrate, calculates macropore tree
The adsorption rate of fat.During such as Fig. 7 crude drugs and resin ratio for 0.5-1, resin is higher to the adsorption rate of total alkaloid, will almost extract
Alkaloid in liquid is adsorbed completely；With the increase of ratio, the adsorption rate of alkaloid is substantially reduced, and crude drug is with resin ratio
When 2.5, alkaloid adsorption rate only has 54.3%, therefore, and in order to reach optimal adsorption effect, crude drug is defined as 1 with resin ratio:
1。
Embodiment 11
Influence of the ethanol-acetone mixing liquid fraction to resin desorption rate
The NKA-9 resins of above-mentioned adsorption saturation are taken, with being placed in after the 20mL distilled water swashes of wave in 50mL conical flasks, are separately added into
30mL volume fractions are 50%, 60%, 70%, 80%, 90% ethanol-acetone (1:1) mixed liquor, at room temperature 120r/min shake
Bed 24h is to desorbing balance.Total alkaloid concentration in stripping liquid is determined, desorption efficiency is calculated.Total life is isolated and purified using macroreticular resin
During alkaloids desorption, desorption efficiency would generally increase with the increase of concentration of alcohol, and the alkaloid in Chinese prickly ash is primarily present in
70%-90% ethanol-acetone mixed liquor eluents part.As seen from Figure 8, resin when ethanol-acetone mixed liquid concentration is 80%
Desorption efficiency highest, during excessive concentration, the content of impurity can increase in desorbed solution, therefore, select 80% ethanol-acetone mixed liquor
It is used as the experiment eluent.
Embodiment 12
Influence of the loading flow velocity to Adsorbent rate
Take 5 parts of the pretreated NKA-9 resins of 1.0g, wet method dress post.Sample solution is flowed through with 1,2,3,4,5BV/h respectively
Resin column carries out Dynamic Adsorption.Efflux is collected, total alkaloid concentration is determined, adsorption rate is calculated.As seen from Figure 9, with loading
The increase of flow velocity, the adsorption rate of total alkaloid illustrates that flow velocity is unfavorable for greatly the absorption of alkaloid in lower by trend, it may be possible to because
For flow velocity it is big when alkaloid can not fully with resin contact, cause adsorption rate to reduce.In comprehensive adsorption rate and time cost, selection
Sample liquid flow velocity is 2BV/h.
Embodiment 13
Influence of the eluent flow rate to resin desorption rate
5 parts of 80% ethanol-acetone mixed liquors of 5BV are taken respectively with 1,2,3,4,5BV/h flow velocitys by having reached that absorption is full
The resin column (first washing away impurity with 2BV distilled water) of sum carries out dynamic resolution absorption, investigates influence of the eluent flow rate to desorption efficiency.
As seen from Figure 10, eluent flow rate has certain influence on resin desorption rate, desorption efficiency highest when eluent flow rate is 2BV/h, with
Flow velocity increase, resin eluting rate is gradually reduced, therefore, eluent flow rate selection 2BV/h.
Embodiment 14
The influence attached to resin desorption of eluent consumption
The resin column of adsorption saturation is taken, impurity, 80% ethanol-acetone (1 are washed away with 2BV distilled water:1) mixed liquor is with 2BV/
H is eluted, and collecting eluent every 1BV determines alkaloid concentrations, determines eluting agent.As seen from Figure 11, with elution
The increase of liquid consumption, resin accumulation desorption efficiency gradually increases, and when eluent consumption reaches 6BV, accumulation desorption efficiency reaches
86.6%, accumulation desorption efficiency almost no longer increases, and alkaloid is also nearly no detectable in the eluent of collection, illustrates desorption
It is basically completed, therefore, eluent consumption is defined as 6BV.
Embodiment 15
One side pin total alkaloid In Vitro Bacteriostasis is studied after extraction purification
After the one side pin total alkaloid that the optimum extraction process as obtained by response surface is extracted is isolated and purified, sterile distilled water is used
Concentrate is diluted to 0.10,0.15,0.2,0.25,0.30,0.35,0.40mg/mL7 group concentration gradient, separately makees positive and cloudy
Property control be measured, as a result such as table 6.
The one side pin total alkaloid of table 6 extracts concentrate minimal inhibitory concentration and minimum bactericidal concentration
Note：Data are the average value of 3 repetition experiments in table."+" indicates bacteria growing；" ++ " represents that bacterial growth is more luxuriant
Contain；" +++ " represents that bacterial growth is luxuriant；"-" represents asepsis growth.
One side pin total alkaloid after isolating and purifying is to Escherichia coli, staphylococcus aureus, 3 kinds of Candida albicans for trying
The minimal inhibitory concentration of bacterium is respectively 0.35,0.20,0.25mg/mL, and minimum bactericidal is 0.30,0.25,0.30mg/mL respectively.3
Planting Candida albicans in bacterium has stronger drug-resistant organisms film feature, and conventional antibiotic and bactericide is difficult to penetrate fungal organism
Film touches candida albicans surface and played a role, and dictaminine is inhibited to Candida albicans biomembrane makes Candida albicans
Growth retardation, it is therefore, dittany alkali content in the one side pin total alkaloid extracted under side light response surface optimization process conditions, pure
Degree is higher, and this extraction and purification process is reliable and stable.
Embodiment of above is merely to illustrate the present invention, not limitation of the present invention, the common skill of correlative technology field
Art personnel, without departing from the spirit and scope of the present invention, can also make a variety of changes and modification, therefore all etc.
Same technical scheme falls within scope of the invention, and scope of patent protection of the invention should be defined by the claims.
Claims (10)
1. extraction and the isolation and purification method of a kind of one side pin total alkaloid, it is characterised in that：
First one side crown stem is mixed and is milled, ethanol-acetone mixed solution is added, Microwave Extraction is carried out；Gained extract solution is sharp again
With resin isolate and purify and obtain one side pin total alkaloid.
2. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：One side pin
Root, stem are according to mass ratio 1:2 mixing.
3. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：By one side
The mixing of crown stem beats powder and crosses 60 mesh sieves, obtains rhizome powder.
4. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：Ethanol with
The volume ratio of acetone is 1:1.
5. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：Microwave is carried
When taking, the percent by volume of ethanol-acetone mixed solution is, 70~90%, remaining is water；Solid-liquid ratio is 15:1~25:
1mL.g-1；Extracting temperature is 50~70 DEG C；Extraction time is 15~25min.
6. extraction and the isolation and purification method of one side pin total alkaloid according to claim 5, it is characterised in that：Microwave is carried
When taking, the percent by volume of ethanol-acetone mixed solution is 89%, and remaining is water；Solid-liquid ratio is 21:1mL.g-1；Extracting temperature
For 70 DEG C；Extraction time is 20min.
7. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：Microwave work(
Rate 300W.
8. extraction and the isolation and purification method of the one side pin total alkaloid according to claim any one of 1-7, its feature exist
In：The Microwave Extraction number of times is 2 times, and waits first time to carry out second of extraction after extracting system recovery room temperature；Carry twice
The temporal summation taken, is 40 minutes except the intercooled time.
9. extraction and the isolation and purification method of one side pin total alkaloid according to claim 1, it is characterised in that：Resin is selected
Use NKA-9 macroporous absorbent resins.
10. extraction and the isolation and purification method of the one side pin total alkaloid according to claim 1 or 9, it is characterised in that：Point
Actual conditions from purifying is：Crude drug is 1 with resin quality ratio:1, regulation sample solution pH are 7.0, loading flow velocity 2BV/h, elution
When with the 1 of 6BV 80%:The ethanol-acetone mixed liquor of 1 volume is eluted with 2BV/h speed.
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| CN111380973A (en) * | 2018-12-29 | 2020-07-07 | 株洲千金药业股份有限公司 | Quality identification control method for Chinese medicinal material zanthoxylum dissitum decoction pieces |
| CN112748213A (en) * | 2019-10-31 | 2021-05-04 | 株洲千金药业股份有限公司 | Single-side needle thin-layer chromatography identification method |
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| CN101513466A (en) * | 2009-03-18 | 2009-08-26 | 裴刚 | Method for preparing quinoline alkaloid from zanthoxylum dissitum Hemsl |
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| CN101513466A (en) * | 2009-03-18 | 2009-08-26 | 裴刚 | Method for preparing quinoline alkaloid from zanthoxylum dissitum Hemsl |
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| CN111380973A (en) * | 2018-12-29 | 2020-07-07 | 株洲千金药业股份有限公司 | Quality identification control method for Chinese medicinal material zanthoxylum dissitum decoction pieces |
| CN112748213A (en) * | 2019-10-31 | 2021-05-04 | 株洲千金药业股份有限公司 | Single-side needle thin-layer chromatography identification method |
| CN112748213B (en) * | 2019-10-31 | 2023-11-24 | 株洲千金药业股份有限公司 | Thin-layer chromatography identification method for single needle |
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