CN111991478A - Method for separating and purifying callicarpa nudiflora phenylethanoid glycoside - Google Patents
Method for separating and purifying callicarpa nudiflora phenylethanoid glycoside Download PDFInfo
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- CN111991478A CN111991478A CN202010998009.1A CN202010998009A CN111991478A CN 111991478 A CN111991478 A CN 111991478A CN 202010998009 A CN202010998009 A CN 202010998009A CN 111991478 A CN111991478 A CN 111991478A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/85—Verbenaceae (Verbena family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses a method for separating and purifying callicarpa nudiflora phenylethanoid glycoside, belonging to the technical field of biology. The invention discloses a method for separating and purifying callicarpa nudiflora phenylethanoid glycoside, which comprises the steps of equivalently replacing the total phenylethanoid glycoside content in callicarpa nudiflora with verbascoside content as a content investigation index, taking the adsorption rate-desorption rate as a comprehensive investigation index, screening out the optimal type of macroporous resin for purifying callicarpa nudiflora phenylethanoid glycoside, investigating the adsorption kinetics and thermodynamic characteristics of the macroporous resin for purifying callicarpa nudiflora phenylethanoid glycoside, and providing reference for the research of the extraction and purification process of phenylethanoid glycoside.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a separation and purification method of callicarpa nudiflora phenylethanoid glycoside.
Background
Callicarpa nudiflora hook ex Arn is a dry aerial part of a plant of Callicarpa of Verbenaceae, and is mainly produced in China such as Hainan, Guangdong and Jiangxi; has antibacterial, hemostatic, antiinflammatory, toxic materials clearing away, blood stasis dispelling, repercussive, pathogenic wind expelling, and dampness removing effects; can be used for treating suppurative inflammation, acute infectious hepatitis, hemorrhage of respiratory tract and digestive tract, and hemorrhage due to wound; it is externally used for treating burn, scald and traumatic hemorrhage. The callicarpa nudiflora contains a plurality of chemical components: flavones, phenethyl alcohol glycosides, terpenes, phenolic acids and volatile oils, and has antiinflammatory, hemostatic, lipid peroxidation resisting, and antibacterial effects.
The callicarpa nudiflora contains high content of phenylethanoid glycosides, mainly including Forsythoside B (FB), acteoside (acteoside, AS), Isoacteoside (IAS), etc., and the phenylethanoid glycosides have pharmacological activities of resisting bacteria, resisting inflammation, resisting tumor, resisting oxidation, regulating immunity, enhancing memory, etc. Pharmacological research on callicarpa nudiflora tablets by using a test tube in-vitro antibacterial test method shows that the callicarpa nudiflora tablets have different degrees of inhibitory action on pneumococcus, salmonella typhi, staphylococcus aureus and the like. The callicarpa nudiflora tablet is a better antibacterial drug.
In addition, researches show that the plant extract containing acteoside has obvious inhibition effect on platelet aggregation and 5-hydroxytryptamine release; in the excision wound model, acteoside and Teupoloside can accelerate wound healing, and the effects are not only related to the activity of removing free radicals, but also related to the effective inhibition of the release of some inflammatory factors (such as TNF-alpha and IFN-gamma), thereby showing good anti-inflammatory effect. Phenylethanoid glycosides compounds such as acteoside, isoacteoside, echinacoside, etc. separated from Chinese herbal medicine pedicularis parturiens have obvious inhibitory activity on liver cancer SMMC-7721, lung adenocarcinoma L342 and gastric adenocarcinoma MGC-803 cells. In addition, the acteoside can intervene the differentiation and apoptosis of cells by regulating telomerase and cell circulation, and inhibit the generation of telomerase to generate antitumor activity, which shows that the acteoside has obvious antitumor effect. In addition, the phenylethanoid glycosides compounds have strong antioxidant activity and strong free radical scavenging effect. Phenylethanoid glycosides such as acteoside and isoacteoside separated from rehmanniae radix have good immunosuppressive effect. Piao JH and the like find that in a mouse dysmnesia model caused by scopolamine, acteoside can prolong the platform retention period of a jump bench experiment and reduce the error times; in the water maze experiment, the percent of correct reaction can be improved, and the increase of cerebral cortex acetylcholinesterase and the reduction of the maximum binding force of cortex and striatum M receptors caused by scopolamine can be antagonized, which shows that the compound has the function of enhancing memory.
Currently, the study of the adsorption behavior and separation theory of the active ingredients on the macroporous resin by scholars mainly focuses on the two aspects of thermodynamics and kinetics. The study on the dynamics of the macroporous adsorption resin mainly relates to the contents of adsorption mechanism, activation energy, speed determining step, diffusion behavior mode of target molecules in particles and the like in the adsorption process of the target molecules on the surface of the macroporous adsorption resin. The adsorption process is usually fitted using quasi-first order kinetic equations, quasi-second order kinetic equations and intra-particle diffusion equations. The research on the adsorption thermodynamics of the macroporous adsorption resin mainly judges the adsorption mode of a target molecule on the surface of the macroporous adsorption resin by observing the adsorption isotherm of the target molecule on the surface of the resin and combining Langmuir, Freundlich and other models.
Quality of dun, etc. with adsorption rate, elution rate as the comprehensive evaluation index, through static adsorption-elution test, select the macroporous resin type suitable for purifying fructus Schisandrae Sphenantherae total triterpene from 11 macroporous adsorption resins, select AB-8 type macroporous resin finally, the dynamics research shows: the adsorption behavior of AB-8 type macroporous resin on the total triterpenoids of the schisandra sphenanthera conforms to a quasi-second order kinetic equation, and the adsorption rate is jointly controlled by liquid membrane diffusion and intra-particle diffusion; the equilibrium adsorption data conforms to Freundlich isothermal equation, belongs to multi-molecular layer adsorption, and high temperature is favorable for adsorption. The Sunli et al uses vanillin-glacial acetic acid-perchloric acid as a color developing agent, and adopts a visible light spectrophotometry to measure adsorption isotherms at different temperatures and adsorption kinetic curves at different initial concentrations, and the results show that the adsorption balance data conform to a Freundlich equation, a Lagergren first-order rate equation and a Dumwald-Wagner intra-particle diffusion equation, and the correlation is good. The adsorption process is proved to be a heat absorption process which is carried out spontaneously, the high temperature is favorable for adsorption, and the adsorption rate is mainly controlled by the diffusion in the particles.
Because the phenylethanoid glycosides compounds have various pharmacological activities and have wide application prospects, the extraction and purification technology of the phenylethanoid glycosides compounds is more and more a hot point of research. At present, the domestic research on callicarpa nudiflora phenylethanoid glycoside mostly only stays in component analysis and content determination, and the research on the purification process is less. Therefore, it is an urgent need to solve the problems of the art to provide a method for separating and purifying callicarpa nudiflora phenylethanoid glycoside.
Disclosure of Invention
In view of the above, the invention provides a method for separating and purifying callicarpa nudiflora phenylethanoid glycoside.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for separating and purifying callicarpa nudiflora phenylethanoid glycoside comprises the following specific steps:
(1) preparing beautyberry leaf test solution
Weighing 300g of dry leaves of callicarpa nudiflora, cutting into pieces, placing the cut leaves into a round-bottom flask, respectively heating and refluxing with 20 and 15 times of water for two times, performing extraction for 2 hours for the first time and 1 hour for the second time, filtering, combining filtrates, performing vacuum concentration to about 0.25g/mL, adding 1% of chitosan according to 12% for clarification, centrifuging at 3000r/min for 10min, taking supernatant, and refrigerating at 4 ℃ for later use;
(2) pretreatment of macroporous resin
Soaking SP-825 type or SP-207 type macroporous resin with 95% ethanol overnight, wet packing, washing with 95% ethanol until effluent liquid is white and turbid, and washing with water until no ethanol smell;
(3) precisely adding the beautyberry test solution at a ratio of 1:12.5, oscillating at 32 deg.C for 6 hr for adsorption, measuring absorbance at 332nm with ultraviolet spectrophotometer, and calculating total phenylethanoid glycoside adsorption amount.
Further, the SP-825 type or SP-207 type macroporous resin in the step (2) is obtained by screening by taking the adsorption rate and the desorption rate as comprehensive investigation indexes, and static adsorption kinetics and static adsorption thermodynamics research are carried out on the macroporous resin.
According to the technical scheme, compared with the prior art, the method for separating and purifying callicarpa nudiflora phenylethanoid glycoside disclosed by the invention has the advantages that the content of verbascoside is used as a content investigation index to equivalently replace the content of total phenylethanoid glycoside in callicarpa nudiflora, the adsorption rate-desorption rate is used as a comprehensive investigation index, the optimal type of macroporous resin for purifying callicarpa nudiflora phenylethanoid glycoside is screened out, the adsorption kinetics and thermodynamic characteristics of the macroporous resin for purifying callicarpa nudiflora phenylethanoid glycoside are investigated, and reference is provided for the research of the extraction and purification process of phenylethanoid glycoside.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a scanning ultraviolet absorption spectrum of a reference verbascoside of the present invention;
FIG. 2 is a scanning ultraviolet absorption spectrum of a test solution of Callicarpa nudiflora according to the present invention;
FIG. 3 is a graph showing the effect of adsorption of phenylethanoid glycosides at different oscillation rates in accordance with the present invention;
FIG. 4 is a graph showing the static adsorption kinetics curves of SP-825 and SP-207 according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Callicarpa nudiflora (leaf, flower, branch, stem) is collected from Baisha county of Hainan province, and is identified as dry aerial part of Callicarpa nudiflora (Callicarpa nudiflora Hook EtArn.) of Callicarpa of Verbenaceae by Proc. of medical college of Hainan medical institute.
Macroporous adsorption resin: AB-8, X-5 (Hangzhou Prorepair Biotechnology science and technology Co., Ltd.), SP-207, SP-825, HP2MGL (Shanghai Mokushi scientific Equipment Co., Ltd.), D101 (national drug group chemical reagent Co., Ltd.), HP-20 (Sichuan Baister chemical reagent Co., Ltd.), DA-201 (Tianjin Hitsu chemical Co., Ltd.); comparison products: verbascoside (batch 120804) (Sichuan Vickqi Biotechnology Co., Ltd.); ethanol is analytically pure, and water is ultrapure water.
EXAMPLE 1 determination of the Total Phenylethanoid glycoside content
Determining total phenylethanoid glycoside content in Callicarpa nudiflora by ultraviolet spectrophotometry with verbascoside as reference.
(1) Preparation of verbascoside reference solution
Precisely weighing acteoside 2.68mg, placing in 50mL measuring flask, adding 50% methanol, ultrasonic dissolving for 0.5 hr, diluting to desired volume, and refrigerating at 4 deg.C for use.
(2) Preparation of test solutions
Weighing 300g of dry leaves of callicarpa nudiflora, cutting into pieces, placing the cut leaves into a round-bottom flask, respectively heating and refluxing with 20 and 15 times of water for two times, carrying out 2 hours for the first time and 1 hour for the second time, filtering, combining filtrates, concentrating under reduced pressure to about 0.25g/mL (based on crude drug), adding 1% of chitosan for clarification according to 12%, centrifuging at 3000r/min for 10min, taking supernatant, and refrigerating at 4 ℃ for standby.
(3) Determination of absorption wavelength
Taking a verbascoside reference substance solution, diluting with 50% methanol to a constant volume, scanning with ultraviolet spectrophotometer at full wavelength, scanning with 50% methanol as blank at wavelength of 200-400nm, and showing that the verbascoside has an obvious absorption peak near 332nm, as shown in figure 1. The sample solution is taken and scanned under the same conditions by using an ultraviolet spectrophotometer at full wavelength, and the result shows that the sample solution has an obvious absorption peak at 330nm, which is shown in figure 2. 332nm can be selected as the detection wavelength for determining the content of the total phenylethanoid glycoside in the callicarpa nudiflora.
(4) Drawing of standard curve
Precisely sucking the acteoside control solution 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0mL to 10mL, respectively adding 50% methanol to dilute to scale, shaking, and respectively measuring absorbance at 332 nm. Drawing a standard curve by taking the content (mu g/mL) of the verbascoside as an abscissa and the absorbance value (A) as an ordinate to obtain a linear regression equation A of the verbascoside of 0.0283C-0.0506 and r of 0.9998; shows that the verbascoside has good linear relation in the range of 5.36-37.52 mu g/mL.
(5) Precision test
Taking the reference solution of acteoside, diluting with 50% methanol to constant volume, measuring absorbance at 332nm, and continuously measuring for 6 times with RSD of 0.29%, which indicates good precision.
(6) Stability test
Taking a test solution, adding water for dilution, diluting to a constant volume, measuring the absorbance values at 0min, 10min, 20 min, 30 min, 40 min, 50 min, 60min and 70min respectively, wherein the RSD is 0.21%, which indicates that the solution has good stability.
EXAMPLE 2 purification of Total Phenylethanolic glycosides by macroporous resin
The formula:
adsorption rate: a (%) ═ Co-Ce)/Co (1)
Desorption rate: d (%) ═ Cd/(Co-Ce) (2)
Specific desorption rate: r (%) ═ a (%) × D (%) (3)
Adsorption amount at time t: q. q.st＝(Co-Ct)×V/W (4)
A quasi-first order kinetic model: ln (q)e-qt)＝-k1t+ln qe (5)
A quasi-second order kinetic model: 1/qt＝1/k2 qe 2×1/t+1/qe (6)
Intraparticle diffusion kinetics model: q. q.st＝kd×t1/2+C (7)
Langmuir equation: ce/qe＝1/qm∙Ce+1/qmkL (8)
Freundlich equation: ln qe＝1/n∙ln Ce+ln kF (9)
In the above formula, CoThe concentration (mg/mL) of the total phenylethanoid glycosides in the test solution before the oscillatory adsorption; ceThe concentration of the residual total phenylethanoid glycosides in the solution after the oscillation adsorption; cdIs the concentration of total phenylethanoid glycosides in the solution obtained after desorption; ctThe concentration of total phenylethanoid glycosides in the solution at time t; q. q.stIs the adsorption capacity (mg/g) of the macroporous resin in t time; q. q.seThe equilibrium adsorption capacity of the resin after adsorption equilibrium; q. q.smThe maximum adsorption capacity of single-layer adsorption; v is the volume (mL) of the test solution added in the experiment; a (%) is an adsorption rate (%); d (%) is the desorption rate; r (%) is the specific desorption rate, i.e. the product of the adsorption rate and the desorption rate; w is the weight of dry resin (g); k is a radical of1、k2、kdRespectively is a rate constant in the processes of a quasi-first-stage kinetic model, a quasi-second-stage kinetic model and a particle internal diffusion model; k is a radical ofLIs the binding constant (mL/mg) between the resin and the phenylethanoid glycosides; k is a radical ofFIn order to balance the adsorption coefficient, the magnitude of the adsorption amount was expressed, and 1/n was a characteristic constant, indicating the magnitude of the relative intensity and the nonuniformity of the adsorbent surface.
(1) Pretreatment of macroporous resin
Soaking the macroporous resin with 95% ethanol overnight, packing with wet method, washing with 95% ethanol until the effluent liquid is white and turbid, and washing with water until no alcohol smell.
(2) Ratio of material to liquid
Selecting SP-825 type macroporous resin, precisely adding Callicarpa nudiflora test solution at a material-to-liquid ratio of 1:12.5, oscillating at 32 deg.C for 6h at 120r/min, and adsorbing. After adsorption, the supernatant was diluted with water, and the absorbance at 332nm was measured and the adsorption amount and adsorption rate were calculated using water as a blank, the results are shown in Table 1.
Selecting SP-825 type macroporous resin, precisely adding Callicarpa nudiflora test solution at a ratio of material to liquid of 1:5 and 1:25 respectively as a comparative example, performing constant temperature oscillation adsorption for 6h at 32 deg.C and 120 r/min. And (4) after adsorption, taking the supernatant, adding water for dilution, taking water as a blank, measuring the absorbance value at 332nm, and calculating the adsorption quantity and the adsorption rate. The feed-liquid ratio and the corresponding adsorption rate and adsorption amount are shown in Table 1.
TABLE 1 feed-to-liquid ratio
The results in table 1 show that the adsorption rate and the adsorption amount under different feed-liquid ratios are significantly different, and the adsorption amount is the largest but the adsorption rate is the smallest when the feed-liquid ratio is 1: 5; the adsorption rate is the largest but the adsorption quantity is the smallest when the feed-liquid ratio is 1:25, and the adsorption rate and the adsorption quantity are higher when the feed-liquid ratio is 1:12.5, which shows that the scheme of selecting 1:12.5 as the feed-liquid ratio to carry out static adsorption and desorption experiments is correct.
(3) Rate of oscillation
Selecting SP-825 type macroporous resin, performing constant temperature oscillation adsorption at a speed of 120r/min at 32 deg.C, sucking the solution in a conical flask every 10min, adding water to dilute by a certain times, and measuring the residual concentration of total phenylethanoid glycosides with an ultraviolet spectrophotometer at 332nm, the result is shown in FIG. 3.
Selecting SP-825 type macroporous resin, performing constant temperature oscillation adsorption at the speed of 80r/min and 160r/min at 32 deg.C respectively as comparative example, sucking the solution in a conical flask every 10min, adding water to dilute by a certain times, and measuring the residual concentration of total phenylethanoid glycosides at 332nm with an ultraviolet spectrophotometer, wherein the result is shown in FIG. 3.
The result of figure 3 shows that the residual concentration of the phenylethanoid glycosides in the solution is high at the rate of 80r/min, the adsorption rate is low, the residual concentration of the phenylethanoid glycosides in the solution is low and almost close at the rates of 120r/min and 160r/min, and from the perspective of adsorption rate and energy conservation, the scheme that 120r/min is selected as the oscillation rate of the macroporous resin for adsorbing the callicarpa nudiflora phenylethanoid glycosides is correct.
(4) Static adsorption and desorption
The application of the macroporous adsorption resin in separation and purification depends on the reversibility of adsorption. The adsorption force intensity of the resin on the substances to be separated is different according to the polarity of the substances to be separated, and the desorption difficulty degree is also different. Therefore, the determination of the adsorption rate and the desorption rate is an important link for proving the reasonable selection of the resin model.
Pumping the pretreated resins, precisely weighing 2g of the resins, placing the resins in a 50mL dry conical flask, precisely adding 25mL of a test solution, placing the test solution in a constant-temperature oscillator, carrying out constant-temperature oscillation adsorption for 6h at 32 ℃ and 120r/min, respectively and precisely absorbing supernatant after adsorption, adding water to dilute the solution to a constant volume, measuring the absorbance value of the solution at 332nm by taking water as a blank, washing the solution with water, pumping the resins, precisely adding 25mL of 70% ethanol, carrying out oscillation desorption for 6h under the same condition, measuring the absorbance value, simultaneously taking the test solution, measuring the content of the total phenylethanoid glycoside by the same method, and calculating the adsorption rate and the desorption rate of each type of resin, wherein the results are shown in Table 2.
TABLE 2 adsorption-desorption of Callicarpa nudiflora total phenylethanoid glycosides by different types of macroporous resins
The results in Table 2 show that the three resins X-5, SP-207 and SP-825 have high specific desorption rates, i.e. high comprehensive adsorption rate and desorption rate, so that the resin can be used as a resin type suitable for purifying callicarpa nudiflora phenylethanoid glycosides, and in the following experiments, two macroporous resins SP-207 and SP-825 are selected for kinetic and thermodynamic research.
(5) Static adsorption kinetics
Precisely weighing 2g of pretreated and drained SP-825 and SP-207 type macroporous resin, placing the pretreated and drained SP-825 and SP-207 type macroporous resin into a 50mL conical flask, precisely adding 20mL of a sample solution, oscillating at a constant temperature of 32 ℃ and 120r/min for 18h, sampling at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 1080min respectively, adding water to dilute by a certain multiple, measuring an absorbance value at 332nm with water as a blank, and calculating the adsorption capacity (q) at t timet) And saturated adsorption capacity (q)e). With the adsorption time as abscissa and the adsorption amount as ordinate, a static adsorption curve was plotted, and the change of the adsorption amount with the adsorption time is shown in fig. 4.
The results in FIG. 4 show that the adsorption of Callicarpa nudiflora total phenylethanoid glycosides by two resins, SP-825 and SP-207, have roughly consistent adsorption tendency, which can be divided into 3 processes in general: a fast adsorption process (0-60min), a slow adsorption process (60-360min) and an adsorption equilibrium process (360-.
The macroporous adsorption resin dynamics research usually adopts a quasi-first-order equation, a quasi-second-order equation and an intra-particle diffusion model to fit the adsorption process, and the fitting result is shown in table 3.
TABLE 3 quasi-first order kinetics equation, quasi-second order kinetics, intraparticle diffusion model and kinetic parameters
It is generally believed that the rate of adsorption is primarily affected by liquid film diffusion or intraparticle diffusion or both. The results in Table 3 show that the quasi-second order kinetic equation can well simulate the adsorption kinetic process (r) of the SP-825 and SP-207 type macroporous resin on callicarpa nudiflora phenylethanoid glycoside20.9418, 0.9474), respectively), obtainedeThe theoretical value is closer to the experimental value; when SP-825 and SP-207 adsorb callicarpa nudiflora phenylethanoid glycoside for 0-60mintFor t1/2In a good linear relationship (r)2> 0.99), but the linear relationship is not good (r) at 60-1080min20.7748 and 0.6866 respectively), and the fitted diffusion equations do not pass through the origin, so that the SP can be preliminarily determinedThe adsorption rate of the-825 and SP-207 type macroporous resin on the callicarpa nudiflora phenylethanoid glycoside component is influenced by liquid membrane diffusion and intra-particle diffusion.
(6) Static adsorption thermodynamic investigation
Precisely weighing 1g of pretreated and drained SP-825 and SP-207 type macroporous resin, placing in a 50mL conical flask, concentrating the test solution, respectively diluting to 23.859, 13.581, 5.505 and 2.499mg/mL total 4 concentrations, precisely measuring 20mL of each concentration test solution, placing in a constant temperature oscillator, respectively oscillating and adsorbing at 23, 30 and 37 ℃ at 120r/min for 12h, taking the supernatant after oscillating and adsorbing, adding water to dilute by a certain multiple, taking water as a blank, measuring the absorbance value at 332nm, and calculating the equilibrium adsorption quantity qe。
The fitting of the isothermal adsorption model often uses the Langmuir equation (C)e/qe＝1/qm∙Ce+1/qm kL) And Freundlich equation (ln q)e＝1/n∙lnCe+ln kF). Q in Langmuir equationmAnd kLIs composed of Ce/qeTo CeThe line graphs of (a) were calculated and the results are shown in Table 4.
TABLE 4 Langmuir isothermal equation parameters for SP-825, SP-207 type macroporous resin
According to linear regression equation coefficient (r)2) It can be known that the Langmuir equation can well simulate the adsorption process of SP-825 and SP-207 type macroporous resin purified callicarpa nudiflora total phenylethanoid glycoside, and a dimensionless constant is introduced to the separation coefficient RL(used for reflecting the type of Langmuir adsorption isotherm) to illustrate the adsorption performance of the macroporous resin on the phenylethanoid glycoside[16]。RLThe expression is as follows:
in the formula C0Is the highest of total phenylethanoid glycosidesInitial concentration (mg/mL), KLIs the parameter k in the Langmuir equationL，RLThree different zone sizes are indicative of different adsorption behavior for an indicative quantity in an isothermal adsorption model: rL> 1 indicates that certain types of macroporous resins are not suitable for adsorbing the target compound; rLLinear adsorption is shown by 1, 0 < RL< 1 > indicates the type of macroporous resin suitable as a resin for purifying the target compound, R L0 indicates that the adsorption process is irreversible adsorption. As can be seen from the results in Table 4, RLThe values of (A) are all between 0 and 1, indicating that the SP-825 and SP-207 type macroporous resins belong to the type of resins suitable for purifying callicarpa nudiflora phenylethanoid glycoside.
Parameter k in Freundlich equationFAnd n is from ln qeTo ln CeCalculated from the linear regression equation obtained, the results are shown in table 5.
TABLE 5SP-825, SP-207 type macroporous resin Freundlich isothermal equation parameters
Based on the coefficient of the linear regression equation (r)2) The magnitude of the values in (A) shows that the Freundlich model also shows good consistency with the adsorption isotherm. The value n can indicate the size of the adsorption strength or the heterogeneity of the resin surface, and the calculated n values are all larger than 1, which indicates that the adsorption process of the SP-825 and SP-207 type macroporous resin purified callicarpa nudiflora phenylethanoid glycoside is preferential adsorption.
Through a static adsorption-desorption test, SP-827 and SP-207 type macroporous adsorption resins are selected for kinetic and thermodynamic research. The results show that the two resins have roughly the same adsorption process: the rapid adsorption stage is carried out for 0-60 min; the slow adsorption stage is carried out for 60-360 min; 360-1080min is the adsorption equilibrium stage. The quasi-second order kinetic equation can well simulate the adsorption kinetic process of SP-825 and SP-207 type macroporous resin on callicarpa nudiflora phenylethanoid glycoside, and the adsorption rate is influenced by liquid membrane diffusion and intra-particle diffusion; the Langmuir model and the Freundlich model can better fit adsorption isotherm data, the SP-825 and SP-207 type macroporous resins have good adsorption performance on callicarpa nudiflora phenylethanoid glycosides, and the adsorption process belongs to preferential adsorption. The dynamic model and the thermodynamic model can be well fitted with the adsorption process of the SP-825 and SP-207 type macroporous resin purified callicarpa nudiflora total phenylethanoid glycoside components, are two excellent resins for purifying callicarpa nudiflora phenylethanoid glycoside components, and provide certain reference for further separation and purification process research of callicarpa nudiflora total phenylethanoid glycoside.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. A separation and purification method of callicarpa nudiflora phenylethanoid glycoside is characterized by comprising the following specific steps:
(1) preparing beautyberry leaf test solution
Weighing 300g of dry leaves of callicarpa nudiflora, cutting into pieces, placing the cut leaves into a round-bottom flask, respectively heating and refluxing with 20 and 15 times of water for two times, performing extraction for 2 hours for the first time and 1 hour for the second time, filtering, combining filtrates, performing vacuum concentration to about 0.25g/mL, adding 1% of chitosan according to 12% for clarification, centrifuging at 3000r/min for 10min, taking supernatant, and refrigerating at 4 ℃ for later use;
(2) pretreatment of macroporous resin
Soaking SP-825 type or SP-207 type macroporous resin with 95% ethanol overnight, wet packing, washing with 95% ethanol until effluent liquid is white and turbid, and washing with water until no ethanol smell;
(3) precisely adding the beautyberry test solution according to the ratio of material to liquid of 1:12.5, oscillating at 32 deg.C for 6h at 120r/min, measuring absorbance values of the solution before and after adsorption at 332nm with ultraviolet spectrophotometer, and calculating total phenylethanoid glycoside adsorption amount.
2. The method for separating and purifying callicarpa nudiflora phenylethanoid glycoside according to claim 1, characterized in that the SP-825 type or SP-207 type macroporous resin in step (2) is obtained by screening using adsorption rate-desorption rate as a comprehensive investigation index, and static adsorption kinetics and static adsorption thermodynamics research are performed on the macroporous resin.
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