CN110658272A - Method for detecting encapsulation rate of composite polysaccharide liposome - Google Patents
Method for detecting encapsulation rate of composite polysaccharide liposome Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 113
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- 239000002502 liposome Substances 0.000 title claims abstract description 48
- 239000002131 composite material Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000005538 encapsulation Methods 0.000 title claims abstract description 19
- -1 compound polysaccharide Chemical class 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 27
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- 238000004811 liquid chromatography Methods 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 230000001419 dependent effect Effects 0.000 claims description 6
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- 238000002360 preparation method Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
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- 238000001914 filtration Methods 0.000 claims description 3
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- 239000007924 injection Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000006386 neutralization reaction Methods 0.000 claims description 3
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- 239000000047 product Substances 0.000 claims description 3
- 241001061264 Astragalus Species 0.000 claims description 2
- 235000006533 astragalus Nutrition 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
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- 230000035945 sensitivity Effects 0.000 abstract description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention discloses a method for detecting the encapsulation efficiency of a composite polysaccharide liposome. And substituting the peak area of the monosaccharide into a regression equation, and calculating to obtain the mass of the unencapsulated composite polysaccharide and the mass of the total composite polysaccharide of the composite polysaccharide liposome, so as to obtain the encapsulation rate of the composite polysaccharide liposome. According to the invention, by establishing a regression equation of the peak area of the chromatogram of the hydrolyzed monosaccharide of the compound polysaccharide and the concentration of the compound polysaccharide, the direct detection of the encapsulation rate of the compound polysaccharide liposome by the HPLC method is realized, the problems of low accuracy, low sensitivity and higher detection limit of the traditional spectrophotometry are solved, and the method has a great prospect in the field of traditional Chinese medicine compound polysaccharide.
Description
Technical Field
The invention relates to the field of detection of the encapsulation rate of composite polysaccharide liposome, in particular to a detection method of the encapsulation rate of composite polysaccharide liposome.
Background
The compound polysaccharide represented by the yupingfeng compound polysaccharide plays an important role in the aspects of regulating immunity, resisting stress, resisting virus, resisting fatigue and the like. However, the traditional Chinese medicine compound polysaccharide preparation has the defects of weak penetrating power to cells, easy degradation, low oral bioavailability, poor stability and the like. The liposome is composed of phospholipid and cholesterol, has a ultramicro spherical carrier with a similar biomembrane bilayer structure, is harmless and nontoxic, and has the advantages of targeted drug release, slow release, biological tissue compatibility and the like. Therefore, the traditional Chinese medicine preparation using liposome as the compound polysaccharide represented by Yupingfeng compound polysaccharide is an effective means for solving the problems of the traditional Chinese medicine compound polysaccharide preparation, and the entrapment rate of the liposome is one of the key indexes for controlling the quality of the liposome.
At present, a method for detecting liposome entrapment rate mainly adopts a spectrophotometer method, although the spectrophotometric method is simple to operate, the accuracy is not as high as that of a high performance liquid chromatography (HPLC method), the HPLC method has the advantages of high sensitivity and low detection value (up to 10 mug/mL), but each substance is detected by the HPLC method, a corresponding standard substance is needed, the HPLC method is not suitable for analyzing unknown components or complex components, the traditional Chinese medicine compound polysaccharide has a complex structure, and no corresponding standard substance exists, so that the content analysis cannot be performed by the HPLC method. Therefore, it is very significant to provide a method for directly detecting the encapsulation efficiency of the liposome of the complex polysaccharide represented by the yupingfeng complex polysaccharide by combining with the high performance liquid chromatography.
Disclosure of Invention
In order to solve the technical problem that the encapsulation efficiency of the complex polysaccharide liposome can not be directly detected by using an HPLC method, the invention provides a detection method of the encapsulation efficiency of the complex polysaccharide liposome, which comprises the following steps:
step 1: centrifuging the composite polysaccharide liposome to be detected, and taking supernatant B;
step 2: taking the composite polysaccharide liposome to be detected with the same volume as the step 1, adding anhydrous methanol for ultrasonic demulsification, adding water, centrifuging, and taking supernatant C;
and step 3: respectively hydrolyzing the supernatant B and the supernatant C and then respectively performing pre-column derivatization;
and 4, step 4: respectively carrying out liquid chromatography detection on the supernatant B and the supernatant C after pre-column derivatization to respectively obtain a chromatogram D and a chromatogram E;
and 5: respectively substituting peak areas of F monosaccharide in the chromatogram D and the chromatogram E into a regression equation to obtain the concentration of unencapsulated composite polysaccharide in the composite polysaccharide liposome to be detected and the total composite polysaccharide concentration of the composite polysaccharide liposome to be detected, and calculating the mass m of unencapsulated composite polysaccharide in the composite polysaccharide liposome to be detected according to the volumes in the step 1 and the step 2Is prepared fromAnd the mass m of the total complex polysaccharide of the complex polysaccharide liposome to be testedGeneral assemblyThe encapsulation efficiency of the compound polysaccharide liposome is (m)General assembly-mIs prepared from)/mGeneral assembly*100％；
The regression equation is obtained by the following process: and (3) respectively taking the compound polysaccharide A with gradient concentration, wherein the compound polysaccharide A is the same as the compound polysaccharide in the compound polysaccharide liposome to be detected, respectively carrying out hydrolysis and pre-column derivatization which are the same as those in the step (3), then respectively carrying out liquid chromatography detection to obtain a chromatogram of the compound polysaccharide A with each gradient concentration, selecting the peak area of F monosaccharide after hydrolysis as a dependent variable, taking the concentration of the compound polysaccharide A as an independent variable, and establishing a regression equation.
Through years of research, the inventor discovers that chromatographic analysis is carried out after the complex polysaccharide with a complex structure is hydrolyzed into monosaccharides (the monosaccharides forming the complex polysaccharide have various types), and the result of a chromatograph discovers that the peak area of each monosaccharide forming the complex polysaccharide is in positive correlation with the concentration of the complex polysaccharide, so that a regression equation can be obtained by selecting one monosaccharide and taking the concentration as an independent variable and the peak area as a dependent variable, and then the content of the complex polysaccharide is reversely deduced through the peak area of the monosaccharide with unknown concentration, which is the key point for determining the encapsulation rate of the liposome of the complex polysaccharide by using the method.
Wherein, when the monosaccharide F is the monosaccharide with the maximum concentration after the hydrolysis of the compound polysaccharide A, the detection effect is better; the complex polysaccharide in the complex polysaccharide liposome to be detected can be at least one of jade screen complex polysaccharide, astragalus complex polysaccharide, divaricate saposhnikovia root complex polysaccharide and atractylodes macrocephala complex polysaccharide. When the compound polysaccharide is the Yupingfeng compound polysaccharide, researches show that the peak area of each monosaccharide is in positive correlation with the concentration of the Yupingfeng compound polysaccharide at 125-4000 mu g/mL, so that in the preparation process of a regression equation, the gradient concentration is 125, 250, 500, 1000, 2000 and 4000 mu g/mL, and the centrifugation condition of the step 1 is as follows: centrifuging at 12000r/min for 10 min; the centrifugation conditions of step 2 were: centrifuging at 3000r/min for 10 min;
in step 3, the hydrolysis process comprises the following specific steps: respectively carrying out reduced pressure evaporation on the supernatant B and the supernatant C, adding 15mL of 2mol/L trifluoroacetic acid respectively, placing the mixture in a closed container after complete dissolution, treating the mixture for 120min at the temperature of 121 ℃, then removing the trifluoroacetic acid through reduced pressure evaporation, adding 10mL of deionized water respectively, and fully dissolving the deionized water;
in step 3, the specific process of pre-column derivatization is as follows: respectively putting 100 mu L of each of the hydrolyzed supernatant B and the hydrolyzed supernatant C into two centrifuge tubes, respectively adding 50 mu L of 0.5mol/L PMP (1-phenyl-3-methyl-5-pyrazolone) solution and 50 mu L of 0.3mol/L NaOH solution, fully and uniformly mixing, carrying out water bath at 80 ℃ for 30min, cooling, respectively adding 50 mu L of 0.3mol/L HCl solution for neutralization, respectively adding 1mL of chloroform for extraction, centrifuging at 3000r/min for 10min, removing the supernatant, repeatedly extracting for 3 times, filtering through a 0.22 mu m filter membrane, and storing the filtrate at-20 ℃ for later use;
in the step 4, the chromatographic conditions of the liquid chromatogram are as follows: 4.6 x 250mm, 5 μm-Wondasil C18 column; phosphate buffer solution-acetonitrile is used as a mobile phase, the pH of the phosphate buffer solution is 6.8, and the volume ratio of the phosphate buffer solution to the acetonitrile is 83: 17; the column temperature was 25 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 20 mu L; the detection wavelength was 254 nm.
The invention has the beneficial effects that: according to the invention, by establishing a regression equation of the peak area of the chromatogram of the hydrolyzed monosaccharide of the compound polysaccharide and the concentration of the compound polysaccharide, the direct detection of the encapsulation rate of the compound polysaccharide liposome by the HPLC method is realized, the problems of low accuracy, low sensitivity and higher detection limit of the traditional spectrophotometry are solved, and the method has a great prospect in the field of traditional Chinese medicine compound polysaccharide.
Drawings
FIG. 1 is a chromatogram of unencapsulated Yupingfeng complex polysaccharide;
FIG. 2 is a chromatogram of Yupingfeng complex polysaccharide liposome total polysaccharide;
FIG. 3 is a chromatogram of Jade-screen complex polysaccharide;
FIG. 4 is a chromatogram of a D-glucose standard;
FIG. 5 is a plot of a regression equation plotted.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described in the following embodiments to fully understand the objects, aspects and effects of the present invention. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
Example 1:
the detection method of the encapsulation rate of the jade screen composite polysaccharide liposome comprises the following steps:
step 1 (separation of unencapsulated yupingfeng complex polysaccharide and liposome): centrifuging 1ml of Jade-screen complex polysaccharide liposome at 12000r/min for 10min, and collecting supernatant B, which is unencapsulated Jade-screen complex polysaccharide;
step 2 (liposome demulsification): taking 1mL of Yupingfeng compound polysaccharide liposome same as the Yupingfeng compound polysaccharide liposome obtained in the step 1, adding 4mL of anhydrous methanol for ultrasonic demulsification, adding deionized water to a constant volume of 10mL, oscillating and uniformly mixing, centrifuging at 3000r/min for 10min, and taking supernatant C, namely the total compound polysaccharide of the Yupingfeng compound polysaccharide liposome;
and step 3: respectively carrying out reduced pressure evaporation on the supernatant B and the supernatant C, adding 15mL of 2mol/L trifluoroacetic acid respectively, placing the mixture in a closed container after complete dissolution, heating the mixture at the temperature of 121 ℃ for 120min, then carrying out reduced pressure evaporation on the trifluoroacetic acid for removal, adding 10mL of deionized water respectively, and fully dissolving to obtain a sample 1 and a sample 2; then respectively carrying out pre-column derivatization, wherein the specific process of the pre-column derivatization is as follows: respectively placing 100 mu L of each sample 1 and 2 in two centrifuge tubes, respectively adding 50 mu L of 0.5mol/L PMP solution and 50 mu L of 0.3mol/L NaOH solution, fully and uniformly mixing, carrying out water bath at 80 ℃ for 30min, cooling, respectively adding 50 mu L of 0.3mol/L HCl solution for neutralization, respectively adding 1mL of chloroform for extraction, centrifuging at 3000r/min for 10min, removing supernatant, repeatedly extracting for 3 times, filtering by a 0.22 mu m filter membrane, and storing the filtrate at-20 ℃ for later use;
and 4, step 4: respectively carrying out liquid chromatography detection on the pre-column derivatized sample 1 and the pre-column derivatized sample 2 to respectively obtain a chromatogram D (shown in figure 1) and a chromatogram E (shown in figure 2); the chromatographic conditions are as follows: 4.6 x 250mm, 5 μm-monodasil C18 chromatography column; phosphate buffer solution-acetonitrile is used as a mobile phase, the pH of the phosphate buffer solution is 6.8, and the volume ratio of the phosphate buffer solution to the acetonitrile is 83: 17; the column temperature was 25 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 20 mu L; the detection wavelength is 254 nm;
and 5: substituting peak areas of D-glucose in chromatogram D and chromatogram E (as shown in FIG. 1 and FIG. 2, the peak of D-glucose is highest, and the concentration of D-glucose is highest) into regression equation, and calculating to obtain mass m of unencapsulated Jade-Screen complex polysaccharideIs prepared fromMass m of Yupingfeng complex polysaccharide combined with Yupingfeng complex polysaccharide liposomeGeneral assemblyThe encapsulation rate of the Yupingfeng compound polysaccharide liposome is (m)General assembly-mIs prepared from)/mGeneral assembly*100％；
The regression equation is obtained by the following process: respectively taking Jade-screen wind composite polysaccharide with gradient concentration (125, 250, 500, 1000, 2000 and 4000 mu g/mL) which is the same as that in the Jade-screen wind composite polysaccharide lipidosome in the step 1, respectively carrying out hydrolysis and pre-column derivatization which are the same as those in the step 3, then respectively carrying out liquid chromatography detection to obtain chromatograms of the Jade-screen wind composite polysaccharide with each gradient concentration, as shown in figure 3, selecting D-glucose (carrying out liquid chromatography detection after carrying out pre-column derivatization which is the same as that in the step 3 on a standard product of the D-glucose to obtain the chromatogram of the D-glucose standard product, as shown in figure 4, as the same as the peak emergence time in figures 1 to 3, the peak area of the position marked 1 in figures 1 to 3 can be determined as the D-glucose) is used as a dependent variable, and the concentration of the Jade-screen wind composite polysaccharide is the dependent variableIndependent variable, drawing regression equation to obtain y-1196.9 x +192723, R20.9957 (where the data values for the dependent and independent variables are shown in table 1 and the regression equation is shown in fig. 5).
Taking the peak areas of D-glucose marked by 1 in the graphs of 2 and 3, substituting the peak areas into a regression equation, wherein the peak area of the chromatographic peak of D-glucose generated by hydrolysis of unencapsulated Jade Screen complex polysaccharide is 955234, and the peak area of the chromatographic peak of D-glucose generated by hydrolysis of total Jade Screen complex polysaccharide is 3797224; calculating by combining the volume of 1mL to obtain the unencapsulated Yupingfeng compound polysaccharide mIs prepared from637.07 μ g of total Jade screen complex polysaccharide mGeneral assembly3011.53 μ g, encapsulation efficiency (m)General assembly-mIs prepared from)/m General assembly100%, the encapsulation yield was 78.84%.
TABLE 1 Yupingfeng complex polysaccharide concentration and corresponding D-glucose chromatographic peak area
Claims (8)
1. The method for detecting the encapsulation efficiency of the composite polysaccharide liposome is characterized by comprising the following steps of:
step 1: centrifuging the composite polysaccharide liposome to be detected, and taking supernatant B;
step 2: taking the composite polysaccharide liposome to be detected with the same volume as the step 1, adding anhydrous methanol for ultrasonic demulsification, adding water, centrifuging, and taking supernatant C;
and step 3: respectively hydrolyzing the supernatant B and the supernatant C and then respectively performing pre-column derivatization;
and 4, step 4: respectively carrying out liquid chromatography detection on the supernatant B and the supernatant C after pre-column derivatization to respectively obtain a chromatogram D and a chromatogram E;
and 5: respectively substituting peak areas of F monosaccharide in chromatogram D and chromatogram E into regression equation to obtain concentration of unencapsulated complex polysaccharide in the composite polysaccharide liposome to be detected and total complex polysaccharide concentration of the composite polysaccharide liposome to be detected, and obtaining the final product according to the product obtained in step 1 and step 2Calculating to obtain the mass m of unencapsulated complex polysaccharide in the complex polysaccharide liposome to be detectedIs prepared fromAnd the mass m of the total complex polysaccharide of the complex polysaccharide liposome to be testedGeneral assemblyThe encapsulation efficiency of the compound polysaccharide liposome is (m)General assembly-mIs prepared from)/mGeneral assembly*100％；
The regression equation is obtained by the following process: and (3) respectively taking the compound polysaccharide A with gradient concentration, wherein the compound polysaccharide A is the same as the compound polysaccharide in the compound polysaccharide liposome to be detected, respectively carrying out hydrolysis and pre-column derivatization which are the same as those in the step (3), then respectively carrying out liquid chromatography detection to obtain a chromatogram of the compound polysaccharide A with each gradient concentration, selecting the peak area of F monosaccharide after hydrolysis as a dependent variable, taking the concentration of the compound polysaccharide A as an independent variable, and establishing a regression equation.
2. The detection method according to claim 1, wherein the complex polysaccharide in the complex polysaccharide liposome to be detected is one of yupingfeng complex polysaccharide, astragalus complex polysaccharide, ledebouriella complex polysaccharide and atractylodes complex polysaccharide.
3. The detection method according to claim 1, wherein in the preparation process of the regression equation, the monosaccharide F is the monosaccharide with the highest concentration after the hydrolysis of the complex polysaccharide A.
4. The detection method according to claim 2, wherein the complex polysaccharide in the complex polysaccharide liposome to be detected is yupingfeng complex polysaccharide, and the centrifugation conditions in step 1 are as follows: centrifuging at 12000r/min for 10 min; the centrifugation conditions of step 2 were: centrifuging at 3000r/min for 10 min.
5. The detection method according to claim 4, wherein in the step 3, the hydrolysis is performed by the following specific processes: and (3) respectively carrying out reduced pressure evaporation on the supernatant B and the supernatant C, adding 15mL of 2mol/L trifluoroacetic acid, placing the mixture into a closed container after complete dissolution, heating the mixture at the temperature of 121 ℃ for 120min, then carrying out reduced pressure evaporation on the trifluoroacetic acid for removal, adding 10mL of deionized water, and fully dissolving.
6. The detection method according to claim 5, wherein in step 3, the specific process of pre-column derivatization is as follows: respectively putting 100 mu L of each hydrolyzed supernatant B and C into two centrifuge tubes, respectively adding 50 mu L of 0.5mol/L PMP solution and 50 mu L of 0.3mol/L NaOH solution, fully mixing, carrying out water bath at 80 ℃ for 30min, cooling, respectively adding 50 mu L of 0.3mol/L HCl solution for neutralization, respectively adding 1mL of chloroform for extraction, centrifuging at 3000r/min for 10min, removing the supernatant, repeatedly extracting for 3 times, filtering by a 0.22 mu m filter membrane, and storing the filtrate at-20 ℃ for later use.
7. The detection method according to claim 6, wherein in the step 4, the chromatographic conditions of the liquid chromatography are as follows: 4.6 x 250mm, 5 μm-Wondasil C18 column; phosphate buffer solution-acetonitrile is used as a mobile phase, the pH of the phosphate buffer solution is 6.8, and the volume ratio of the phosphate buffer solution to the acetonitrile is 83: 17; the column temperature was 25 ℃; the flow rate is 1.0 mL/min; the sample injection amount is 20 mu L; the detection wavelength was 254 nm.
8. The detection method according to claim 7, wherein the gradient concentration in the regression equation is 125, 250, 500, 1000, 2000, 4000 μ g/mL.
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| CN113358836A (en) * | 2021-04-30 | 2021-09-07 | 北京诺康达医药科技股份有限公司 | Method for measuring encapsulation efficiency of drug-loaded fat emulsion |
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