CN110632190A - Method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary - Google Patents
Method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary Download PDFInfo
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Abstract
The invention provides a method for simultaneously determining rosmarinic acid, carnosic acid and carnosol in rosemary, relates to the technical field of plant component detection, can shorten the detection time, and uses a non-toxic and harmless extraction reagent; the method comprises the steps of S1, taking a required amount of rosemary extract, carrying out ultrasonic extraction by using ethanol, cooling, fixing the volume, and shaking up to obtain a solution to be analyzed; s2, filtering the solution to be analyzed by adopting a 0.45-micron organic microporous filter membrane, and analyzing by using a liquid chromatography; compared with the content measured by the method in GB1886.172-2016, the content measured by the method provided by the application is as follows: the content error of the rosmarinic acid is within 3.1 percent, the content error of the carnosic acid is within 0.5 percent, and the content error of the carnosol is within 4 percent. The technical scheme provided by the invention is suitable for the process of measuring the content of the components in the rosemary extract.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of plant component detection, in particular to a method for simultaneously determining rosmarinic acid, carnosic acid and carnosol in rosemary.
[ background of the invention ]
GB1886.172-2016 food safety national standard food additive rosemary extract appendix A.3 records the determination of rosmarinic acid in rosemary extract: accurately weighing about 200mg of sample, dissolving with mobile phase, metering to 100mL, and filtering with 0.45 μm microporous membrane to obtain sample solution. A chromatographic column: a chromatographic column with octadecylsilane as filler; mobile phase: methanol: phosphoric acid solution (0.5%) 45: 55; flow rate: 0.6 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; the detection wavelength is 283 nm; retention time: and (4) 18 min.
GB1886.172-2016 food safety national Standard food additive Rosemary extract appendix A.2 records the determination of carnosic acid and carnosol in Rosemary extracts: weighing 140-180 mg of sample, dissolving in 20mL of acetone, fixing the volume to a 25mL volumetric flask, fully and uniformly mixing, and filtering through a 0.22 mu m microporous membrane. A chromatographic column: c18 reverse phase chromatography column; the mobile phase A is water + phosphoric acid solution (0.1%), the mobile phase B is acetonitrile + phosphoric acid solution (0.1%), and the gradient elution condition is shown in GB1886.172-2016 Table A.1; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; the detection wavelength is 280 nm; retention time: and (5) 35 min. Meanwhile, the peak area of the carnosol with the same concentration is 1.36 times of that of the carnosic acid under 280nm, so that the concentration of the carnosol can be calculated according to a standard curve of the carnosic acid.
The method for detecting the rosmarinic acid, the carnosic acid and the carnosol in the rosemary extract needs to be respectively pretreated and then respectively subjected to liquid chromatography detection, the time consumption is long, and the used extraction reagents, namely the methanol and the acetone, are volatile and toxic organic reagents, so that the method causes harm to a human body and pollutes the environment.
Therefore, there is a need to develop a method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary to overcome the disadvantages of the prior art, so as to solve or alleviate one or more of the above problems.
[ summary of the invention ]
In view of the above, the invention provides a method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary extract, which can shorten the detection time and use non-toxic and harmless extraction reagents.
In one aspect, the present invention provides a method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary, wherein the rosemary comprises rosmarinic acid, carnosic acid and carnosol, and the method comprises the following steps:
s1, taking a required amount of rosemary extract, carrying out ultrasonic extraction by using ethanol, cooling, fixing the volume, and shaking up to obtain a solution to be analyzed;
s2, filtering the solution to be analyzed, and analyzing and calculating by using a liquid chromatography;
the error between the content of each component measured by the simultaneous measurement method and the content of each component measured by the measurement method in GB1886.172-2016 is as follows: the content error of the rosmarinic acid is within 3.1 percent, the content error of the carnosic acid is within 0.5 percent, and the content error of the carnosol is within 4 percent.
The above aspect and any possible implementation further provides an implementation, and the conditions for performing the analysis by liquid chromatography include:
a) the instrument comprises the following steps: a binary liquid chromatograph;
b) specification of chromatographic column: c18 chromatographic column with length of 250mm, inner diameter of 4.6mm and aperture of 5 μm;
c) column temperature: 35-40 ℃;
d) flow rate of the column: 0.8-1.0 mL/min;
e) mobile phase: methanol + phosphoric acid solution with the concentration of 0.5%, wherein the volume ratio of the methanol to the phosphoric acid solution is 65: 35-72: 28;
f) detection wavelength: 270-290 nm.
In the above aspect and any possible implementation manner, there is further provided an implementation manner, in S2, the solution to be analyzed is filtered by using a 0.40-0.50 μm organic microporous filter membrane.
The above aspects and any possible implementations further provide an implementation that the solution to be analyzed is filtered by using a 0.45 μm organic microporous filter membrane.
The above aspect and any possible implementation manner further provide an implementation manner that the amount of ethanol in the ultrasonic extraction with ethanol in S1 is 30-40 mL.
The above aspect and any possible implementation manner further provide an implementation manner, and the time of ultrasonic extraction in S1 is 10-20 min.
The above aspects and any possible implementation manner further provide an implementation manner, wherein the amount of the rosemary extract in the S1 is 0.15-0.20 g.
The above-described aspects and any possible implementation further provide an implementation, and the binary liquid chromatograph is specifically an Alltech Uvis-20i liquid chromatograph.
The above aspects and any possible implementations further provide an implementation where the liquid chromatography analysis of rosemary extract results in: the peak emergence time of the rosmarinic acid is 2.860-2.872 min, the peak emergence time of the carnosol is 17.180min, and the peak emergence time of the carnosic acid is 24.113-24.127 min.
The above-described aspects and any possible implementation further provide an implementation in which the standard curve equation for calculating the rosmarinic acid content is: y 522.0x +188, r2Is 0.9998; the standard curve equation used to calculate carnosic acid and carnosol content is: y 16107x 9284, r20.9995; substituting the peak areas of the rosmarinic acid and the carnosic acid into respective standard curve equations to obtain the contents of the rosmarinic acid and the carnosic acid; substituting the peak area of the carnosol into the standard curve equation, and dividing the obtained value by the conversion coefficient between the carnosic acid and the carnosol to obtain the content of the carnosol.
The above aspect and any possible implementation manner further provide an implementation manner that the error between the content of each component measured by the simultaneous measurement method and the content of each component measured by the measurement method in GB1886.172-2016 is: the content error of the rosmarinic acid is within 3.1 percent, the content error of the carnosic acid is within 0.5 percent, and the content error of the carnosol is within 4 percent.
Compared with the prior art, the invention can obtain the following technical effects: the invention solves the problems that the rosmarinic acid, the carnosic acid and the carnosol can not be extracted at the same time and can not be subjected to on-machine analysis at the same time; the detection method can shorten the detection time, changes the extraction reagent from toxic methanol and acetone into nontoxic ethanol, and is economical and environment-friendly; the detection method provided by the invention is simple, rapid and accurate to operate, has the detection precision consistent with that of GB1886.172-2016, and is good in repeatability.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a liquid chromatogram of a mixed standard solution of rosmarinic acid and carnosic acid according to an embodiment of the invention;
FIG. 2 is a liquid chromatogram of a sample of rosemary extract according to an embodiment of the present invention;
fig. 3 is a graph of a standard rosmarinic acid solution according to an embodiment of the present invention;
FIG. 4 is a standard curve diagram of a carnosic acid standard solution according to an embodiment of the present invention.
[ detailed description ] embodiments
For better understanding of the technical solutions of the present invention, the following detailed descriptions of the embodiments of the present invention are provided with reference to the accompanying drawings.
It should be understood that the described embodiments are only some embodiments of the invention, and not all 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.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the present invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary extract comprises the following specific steps:
step 1: transferring 0.15-0.20 g (accurate to 0.0001g) of rosemary extract, putting into a 50mL volumetric flask, immediately adding 30-40 mL of chromatographic pure ethanol, carrying out ultrasonic treatment for 10-20 min, cooling, fixing the volume, and shaking up to obtain a solution to be analyzed;
step 2: filtering with 0.4-0.5 μm organic microporous filter membrane, and analyzing with liquid chromatography; preferably 0.45 μm organic microporous filter membrane;
wherein, the liquid chromatographic analysis conditions are as follows:
a) the instrument comprises the following steps: a binary high performance liquid chromatograph; the application selects an Alltech Uvis-20i liquid chromatograph;
b) specification of chromatographic column: a C18 chromatographic column with the length of 250mm, the inner diameter of 4.6mm and the aperture of 5 μm; the chromatographic column selected by the application is Thermo BDS HYPERSIL C18;
c) column temperature: 35-40 ℃;
d) flow rate of the column: 0.8-1.0 mL/min;
e) mobile phase: methanol + 0.5% phosphoric acid solution, wherein the volume ratio of the methanol to the phosphoric acid solution is 65: 35-72: 28;
f) detection wavelength: 270-290 nm.
Example (b):
1. liquid chromatography analysis:
shaking herba Rosmarini officinalis extract, sampling with 1.0mL of Dachang plastic pipette, accurately weighing 0.16g (to 0.0001g), placing into 50mL volumetric flask, immediately adding 40mL of chromatographic pure ethanol, ultrasonic treating for 10min, cooling, diluting to desired volume, and shaking.
The resulting mixture was filtered through a 0.45-. mu.m organic microfiltration membrane and analyzed by liquid chromatography.
The analysis method is liquid chromatography (ultraviolet detector), wherein the analysis conditions of the liquid chromatography are as follows:
a) the instrument comprises the following steps: a binary liquid chromatograph;
b) specification of chromatographic column: c18 chromatographic column, 250X 4.6mm, 5 μm;
c) column temperature: 35 ℃;
d) flow rate: 1.0 mL/min;
e) mobile phase: methanol + phosphoric acid solution (0.5%), volume ratio 72: 28;
f) detection wavelength: 280 nm.
Wherein, the liquid chromatogram of the mixed standard solution of rosmarinic acid and carnosic acid is shown in figure 1, wherein: the peak emergence time is 2.872min for rosmarinic acid, and 24.127min for carnosic acid. The liquid chromatogram of rosemary extract sample is shown in figure 2, wherein: the peak time of 2.860min is rosmarinic acid, the peak time of 17.180min is carnosol, and the peak time of 24.113min is carnosic acid.
2. Drawing a standard curve:
a) accurately weighing 40mg of a rosmarinic acid standard substance, dissolving with ethanol, and diluting to 10mL to prepare 4mg/mL of rosmarinic acid single-standard stock solution A;
b) transferring 5mL of the rosmarinic acid single-standard stock solution A by using a big belly suction pipe, diluting with ethanol, and diluting to 50mL to prepare 0.4mg/mL of rosmarinic acid intermediate use solution B;
c) transferring 5mL of the rosmarinic acid intermediate use solution B by using a big belly suction pipe, diluting with ethanol, and diluting to a constant volume of 50mL to prepare 0.04mg/mL of rosmarinic acid intermediate use solution C;
d) accurately weighing 40mg of carnosic acid, dissolving with ethanol, and diluting to a constant volume of 10mL to prepare 4mg/mL of carnosic acid single-standard stock solution D;
e) respectively transferring 0.25mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL of the rosmarinic acid intermediate use solution C and the carnosic acid single-standard stock solution D by using a graduated pipette, and diluting the volume to 10mL by using ethanol to prepare a mixed standard use solution F; the concentrations of rosmarinic acid in the mixed standard use solution F are respectively 1ng/mL, 2ng/mL, 4ng/mL, 6ng/mL, 8ng/mL and 10ng/mL, and the concentrations of carnosic acid are respectively 0.1 mu g/mL, 0.2 mu g/mL, 0.4 mu g/mL, 0.6 mu g/mL, 0.8 mu g/mL and 1.0 mu g/mL;
f) transferring 10 μ L of each mixed standard solution, performing liquid chromatography, performing parallel sample introduction for 3 times, drawing standard curves of rosmarinic acid and carnosic acid by using absolute sample introduction amount as abscissa and average value of peak area of three parallel sample introduction as ordinate, and obtaining results shown in Table 1, FIG. 3 and FIG. 4.
TABLE 1
The retention time of the rosmarinic acid is 2.872min, the concentration of the rosmarinic acid is taken as an abscissa, the response value of the rosmarinic acid is taken as an ordinate to draw a standard curve, and the standard curve equation is as follows: y 522.0x +188, r2Is 0.9998; the carnosic acid retention time was 24.127min, and the standard curve equation was: y 16107x 9284, r20.9995; the retention time of carnosol is 17.180 min. The x of the standard curve represents the absolute sample amount (unit: ng) of the standard sample, and y represents the peak area (no unit) of the standard sample.
Substituting the peak areas of the rosmarinic acid and the carnosic acid of the sample into the y value of each standard curve to obtain the content of the rosmarinic acid and the carnosic acid, namely the value of x. Substituting the peak area of carnosol of the sample into the y value of the standard curve of carnosic acid, and dividing the obtained value by 1.36 to obtain the content of carnosol (1.36 is the conversion coefficient between carnosic acid and carnosol).
3. And (3) precision verification:
a sample (lot: 2017071501) of the same lot number 0.16g (to the nearest 0.0001g) was subjected to 6 parallel tests in the pretreatment method and pretreatment conditions described above, and the results are shown in Table 2.
TABLE 2
The method is used for detecting the content of rosmarinic acid, carnosic acid and carnosol in the rosemary extract, and meanwhile, the detection is carried out according to the method of GB1886.172-2016, and the result is shown in Table 3. Rosmarinic acid code M, carnosic acid code S, carnosol code F.
TABLE 3
The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary provided in the embodiments of the present application is described in detail above. The above description of the embodiments is only for the purpose of helping to understand the method of the present application and its core ideas; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.
As used in the specification and claims, certain terms are used to refer to particular components. As one skilled in the art will appreciate, manufacturers may refer to a component by different names. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include/include, but not limited to. "substantially" means within an acceptable error range, within which a person skilled in the art can solve the technical problem and substantially achieve the technical result. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.
It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, the use of the phrase "comprising a. -. said" to define an element does not exclude the presence of other like elements in a commodity or system that comprises the element.
It should be understood that the term "and/or" as used herein is merely one type of association that describes an associated object, meaning that three relationships may exist, e.g., a and/or B may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the word "/", herein, generally indicates that the objects associated therewith are in an "or" relationship.
The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the application as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.
Claims (10)
1. A method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary, wherein the rosemary comprises rosmarinic acid, carnosic acid and carnosol, and is characterized by comprising the following steps:
s1, taking a required amount of rosemary extract, carrying out ultrasonic extraction by using ethanol, cooling, fixing the volume, and shaking up to obtain a solution to be analyzed;
s2, filtering the solution to be analyzed, and analyzing and calculating by using a liquid chromatography;
the error between the content of each component measured by the simultaneous measurement method and the content of each component measured by the measurement method in GB1886.172-2016 is as follows: the content error of the rosmarinic acid is within 3.1 percent, the content error of the carnosic acid is within 0.5 percent, and the content error of the carnosol is within 4 percent.
2. A method of simultaneous determination of rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 1, wherein the conditions for analysis by liquid chromatography comprise:
a) the instrument comprises the following steps: a binary liquid chromatograph;
b) specification of chromatographic column: c18 chromatographic column with length of 250mm, inner diameter of 4.6mm and aperture of 5 μm;
c) column temperature: 35-40 ℃;
d) flow rate of the column: 0.8-1.0 mL/min;
e) mobile phase: methanol + phosphoric acid solution with the concentration of 0.5%, wherein the volume ratio of the methanol to the phosphoric acid solution is 65: 35-72: 28;
f) detection wavelength: 270-290 nm.
3. The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 1, wherein the solution to be analyzed is filtered by using a 0.40-0.50 μm organic microporous membrane in S2.
4. A method of simultaneous determination of rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 3, wherein the solution to be analysed is filtered using a 0.45 μm organic microfiltration membrane.
5. The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 1, wherein the time for ultrasonic extraction in S1 is 10-20 min.
6. The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 1, wherein the amount of rosemary extract in S1 is 0.15-0.20 g.
7. The method for simultaneously measuring rosmarinic acid, carnosic acid and carnosol in rosemary according to claim 6, wherein the amount of ethanol in the ultrasonic extraction with ethanol in S1 is 30-40 mL.
8. A method of simultaneous determination of rosmarinic acid, carnosic acid and carnosol from rosemary according to claim 2, wherein the binary liquid chromatograph is in particular an Alltech Uvis-20i liquid chromatograph.
9. A method of simultaneous determination of rosmarinic acid, carnosic acid and carnosol in rosemary according to any of claims 1 to 8, wherein the results of analysis of rosemary extract by liquid chromatography are: the peak emergence time of the rosmarinic acid is 2.860-2.872 min, the peak emergence time of the carnosol is 17.180min, and the peak emergence time of the carnosic acid is 24.113-24.127 min.
10. The method of claim 9, wherein the standard curve equation for calculating the rosmarinic acid content is: y 522.0x +188, r2Is 0.9998; the standard curve equation used to calculate carnosic acid and carnosol content is: y 16107x 9284, r20.9995; substituting the peak areas of the rosmarinic acid and the carnosic acid into respective standard curve equations to obtain the contents of the rosmarinic acid and the carnosic acid; substituting the peak area of the carnosol into the standard curve equation, and dividing the obtained value by the conversion coefficient between the carnosic acid and the carnosol to obtain the content of the carnosol.
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