CN117110456A - Method for measuring volatile oil content in patchouli leaf - Google Patents

Method for measuring volatile oil content in patchouli leaf Download PDF

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CN117110456A
CN117110456A CN202310931286.4A CN202310931286A CN117110456A CN 117110456 A CN117110456 A CN 117110456A CN 202310931286 A CN202310931286 A CN 202310931286A CN 117110456 A CN117110456 A CN 117110456A
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concentration
patchouli
volatile oil
beta
caryophyllene
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欧阳慧子
何俊
曹秀楠
白宇
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FIRST AFFILIATED HOSPITAL OF TIANJIN UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
Tianjin University of Traditional Chinese Medicine
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FIRST AFFILIATED HOSPITAL OF TIANJIN UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
Tianjin University of Traditional Chinese Medicine
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Abstract

The invention belongs to the technical field of medicine detection, and provides a method for measuring the content of volatile oil in patchouli leaf. According to the invention, the GC-MS analysis method is adopted to measure the contents of 5 volatile oils of beta-elemene, beta-caryophyllene, patchouli alcohol and farnesol in patchouli leaves in different producing areas, and the quality of patchouli medicinal materials can be more comprehensively evaluated through the 5 volatile oils, so that a more reliable basis is provided for the quality evaluation of patchouli leaves.

Description

Method for measuring volatile oil content in patchouli leaf
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for measuring the content of volatile oil in patchouli leaf.
Background
Herba Agastaches is perennial aromatic herb or half shrub, has wide production area distribution, is planted in India, malaysia and Philippines, and is widely cultivated in Guangdong, hainan, guangxi, fujian and other places in China. Herba Agastaches contains abundant volatile oil, and has remarkable effects of improving gastrointestinal function, relieving cough, relieving nerve, expelling parasites, killing bacteria, and improving skin problems. The patchouli alcohol specified in the 'Chinese pharmacopoeia' of 2020 edition is a quality evaluation index of patchouli, and most of the current literature research is focused on the component, but the quality of the patchouli is uneven due to numerous chemical components in the patchouli by the existing evaluation method. Therefore, how to provide a more comprehensive method for evaluating the quality of patchouli medicinal materials is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides a method for determining the volatile oil content of patchouli leaf. The method aims to solve the technical problem that the quality of patchouli is uneven due to the existing evaluation method.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a method for determining the content of volatile oil in patchouli leaf, wherein the volatile oil comprises patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllin and farnesol;
the method comprises the following steps:
s1, respectively mixing patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllene and farnesol with ethyl acetate to obtain stock solution;
s2, mixing all stock solutions to obtain reference substance solutions, respectively carrying out GC-MS analysis on the reference substance solutions with different concentrations, and drawing a standard curve by taking the peak area of the volatile oil as an ordinate and the concentration of the volatile oil as an abscissa; the concentration of the volatile oil is the lowest quantitative limit when the signal to noise ratio S/N=10;
s3, mixing patchouli leaf powder with ethyl acetate, sequentially performing ultrasonic extraction, volume fixing, centrifugation and filtration to obtain a test solution, performing GC-MS analysis on the test solution, and reading the concentration of the volatile oil on a standard curve.
Further, in the step S1, the concentration of the stock solution is independently 0.5 to 2mg/mL.
Further, in the step S2, in the reference substance solution, the concentration of patchouli alcohol is 120-160 mug/mL, the concentration of beta-elemene is 20-30 mug/mL, the concentration of beta-caryophyllene is 35-45 mug/mL, the concentration of caryophyllene is 90-100 mug/mL, and the concentration of farnesol is 75-85 mug/mL.
Further, in the step S2, the total concentration of the control solutions with different concentrations is 9, including the initial concentration, and the control solutions are diluted 2 times, 2.5 times, 2 times and 2 times in sequence.
Further, in the step S3, the particle size of the patchouli leaf powder is more than or equal to 10 meshes; the concentration of the sample solution is 0.01-0.1 g/mL.
Further, in the step S3, the power of ultrasonic extraction is 200-400W, the frequency is 30-50 KHz, and the time is 30-50 min.
Further, in the step S3, the rotational speed of centrifugation is 10000-20000 r/min, and the time of centrifugation is 10-20 min.
Further, in the step S2 and the step S3, the chromatographic conditions of GC-MS are independently:
the chromatographic column is HP5-MS (0.25 μm. Times.0.25 mm. Times.30 m) capillary chromatographic column; the carrier gas is helium, and the purity is more than or equal to 99.99%; the split ratio of the split mode is 20:1, a step of; the flow rate is 1.0-2.0 mL/min; the sample injection amount is 0.5-2 mu L; the temperature of the sample inlet is 240-280 ℃; chromatographic column temperature procedure: the initial temperature is 140 ℃; heating to 150 ℃ at 2 ℃/min; then heating to 180 ℃ at 12 ℃/min, and keeping for 2min.
Further, in the step S2 and the step S3, the mass spectrum conditions of the GC-MS are independently:
the ion source is an EI source; the scanning mode is a Scan monitoring mode; the scanning range is 35-500 m/z; the detector voltage is 1.3kV; the temperature of the ion source is 200-250 ℃; the interface temperature is 220-260 ℃; the solvent delay was 2.5min.
Compared with the prior art, the invention has the following beneficial effects:
the technical scheme of the invention has the advantages of simplicity, high sensitivity, high analysis speed, strong specificity and the like, so that the method can be more comprehensively and accurately used for quality control of the patchouli medicinal material, and the technical problem that the quality of the patchouli is uneven due to the existing evaluation method is solved.
Drawings
FIG. 1 is a GC-MS TIC diagram of example 2.
Detailed Description
The invention provides a method for determining the content of volatile oil in patchouli leaf, wherein the volatile oil comprises patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllin and farnesol;
the method comprises the following steps:
s1, respectively mixing patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllene and farnesol with ethyl acetate to obtain stock solution;
s2, mixing all stock solutions to obtain reference substance solutions, respectively carrying out GC-MS analysis on the reference substance solutions with different concentrations, and drawing a standard curve by taking the peak area of the volatile oil as an ordinate and the concentration of the volatile oil as an abscissa; the concentration of the volatile oil is the lowest quantitative limit when the signal to noise ratio S/N=10;
s3, mixing patchouli leaf powder with ethyl acetate, sequentially performing ultrasonic extraction, volume fixing, centrifugation and filtration to obtain a test solution, performing GC-MS analysis on the test solution, and reading the concentration of the volatile oil on a standard curve.
In the present invention, in the step S1, the concentration of the stock solution is independently 0.5 to 2mg/mL, preferably 0.8 to 1.5mg/mL, and more preferably 1.0mg/mL.
In the present invention, in the step S2, the concentration of patchouli alcohol in the control solution is 120 to 160. Mu.g/mL, preferably 140 to 155. Mu.g/mL, and more preferably 150. Mu.g/mL; the concentration of the beta-elemene is 20-30 mug/mL, preferably 22-26 mug/mL, and more preferably 25 mug/mL; the concentration of the beta-caryophyllene is 35-45 mug/mL, preferably 37-42 mug/mL, and more preferably 40 mug/mL; the concentration of the carnosine is 90-100 mug/mL, preferably 94-99 mug/mL, and more preferably 98 mug/mL; the concentration of farnesol is 75-85. Mu.g/mL, preferably 78-82. Mu.g/mL, more preferably 80. Mu.g/mL.
In the present invention, in the step S2, the total concentration of the control solutions with different concentrations is 9, including the initial concentration, and the control solutions are diluted 2 times, 2.5 times, 2 times and 2 times in sequence.
In the invention, in the step S3, the particle size of the patchouli leaf powder is more than or equal to 10 meshes, preferably more than or equal to 15 meshes, and more preferably more than or equal to 20 meshes; the concentration of the sample solution is 0.01 to 0.1g/mL, preferably 0.04 to 0.06g/mL, and more preferably 0.05g/mL.
In the present invention, in the step S3, the power of the ultrasonic extraction is 200 to 400W, preferably 250 to 350W, and more preferably 300W; the frequency is 30-50 KHz, preferably 35-45 KHz, and more preferably 40KHz; the time is 30 to 50 minutes, preferably 35 to 45 minutes, and more preferably 40 minutes.
In the present invention, in the step S3, the rotational speed of the centrifugation is 10000 to 20000r/min, preferably 12000 to 16000r/min, and more preferably 14000r/min; the centrifugation time is 10 to 20 minutes, preferably 12 to 18 minutes, and more preferably 12 to 14 minutes.
In the present invention, in the step S2 and the step S3, the chromatographic conditions of GC-MS are independently:
the chromatographic column is HP5-MS (0.25 μm. Times.0.25 mm. Times.30 m) capillary chromatographic column; the carrier gas is helium, and the purity is more than or equal to 99.99%; the split ratio of the split mode is 20:1, a step of; the flow rate is 1.0 to 2.0mL/min, preferably 1.2 to 1.8mL/min, and more preferably 1.4 to 1.6mL/min; the sample amount is 0.5 to 2. Mu.L, preferably 0.8 to 1.5. Mu.L, and more preferably 1.0. Mu.L; the temperature of the sample inlet is 240-280 ℃, preferably 250-270 ℃, and further preferably 260 ℃; chromatographic column temperature procedure: the initial temperature is 140 ℃; heating to 150 ℃ at 2 ℃/min; then heating to 180 ℃ at 12 ℃/min, and keeping for 2min.
In the present invention, in the step S2 and the step S3, the mass spectrometry conditions of the GC-MS are independently:
the ion source is an EI source; the scanning mode is a Scan monitoring mode; the scanning range is 35-500 m/z; the detector voltage is 1.3kV; the ion source temperature is 200-250 ℃, preferably 220-240 ℃, and more preferably 230 ℃; the interface temperature is 220-260 ℃, preferably 230-250 ℃, and further preferably 240 ℃; the solvent delay was 2.5min.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The place of origin and lot numbers of patchouli leaf used in the examples of the present invention are shown in Table 1.
Table 1 Source information Table of patchouli leaf used in examples
Example 1
Respectively weighing beta-elemene, beta-caryophyllene, caryophyllene and autumn Li Chunhe farnesol, adding ethyl acetate to constant volume to 10mL, respectively preparing into 1mg/mL stock solution, and storing in a refrigerator at 4 ℃ for later use.
The sources and lot numbers of the above raw materials are as follows: patchouli alcohol (lot number: DSTDB 004102), beta-elemene (lot number: DSTDL 026701), beta-caryophyllene (lot number: DSTDS 009501), caryophyllene (lot number: DSTDS 021001) were purchased from Dou Desai Tebiotech Co., ltd; farnesol (lot number: M27GS 143435) was purchased from Shanghai Seiyaku Biotechnology Co., ltd; ethyl acetate (analytically pure) is supplied by Kangkode technologies, inc. of Tianjin.
Mixing all stock solutions, regulating and preparing into a reference substance solution with the concentration of beta-elemene of 25 mug/mL, the concentration of beta-caryophyllene of 40 mug/mL, the concentration of caryophyllene of 98 mug/mL, the concentration of patchouli alcohol of 150 mug/mL and the concentration of farnesol of 80 mug/mL by ethyl acetate, sequentially diluting the reference substance solution by 2 times, 2.5 times, 2 times and 2 times by ethyl acetate to obtain 9 reference substance solutions with different concentrations, taking 1 mug of each reference substance solution for GC-MS analysis, taking the peak area of volatile oil as an ordinate (Y), taking the concentration of the volatile oil as an abscissa (X), and drawing a standard curve; the concentration of volatile oil at signal to noise ratio S/n=10 was the lowest limit of quantification (LLOQ) at stepwise dilution of the control solution, and the results are shown in table 2.
TABLE 2 Standard Curve regression equation for different volatile oils, lowest quantitative limit
Pulverizing herba Agastaches leaf of batch 1 with pulverizer, sieving with 10 mesh sieve, weighing 0.5g herba Agastaches coarse powder, placing in 10mL volumetric flask, adding ethyl acetate to constant volume to scale, extracting with ultrasound (power 300W, frequency 40 KHz) for 40min, taking out, standing to room temperature, supplementing to scale, taking 1mL of the solution, centrifuging at 14000r/min for 10min, collecting supernatant, filtering with 0.22 μm organic microporous membrane, collecting filtrate as sample solution, performing GC-MS analysis on the sample solution, and reading the concentration of volatile oil on standard curve.
The chromatographic conditions of the GC-MS used in this example were:
the chromatographic column is HP5-MS (0.25 μm. Times.0.25 mm. Times.30 m) capillary chromatographic column; the carrier gas is helium, and the purity is more than or equal to 99.99%; the split ratio of the split mode is 20:1, a step of; the flow rate is 1.8mL/min; the sample injection amount is 1 mu L; the temperature of the sample inlet is 260 ℃; chromatographic column temperature procedure: the initial temperature is 140 ℃; heating to 150 ℃ at 2 ℃/min; then heating to 180 ℃ at 12 ℃/min, and keeping for 2min.
The mass spectrometry conditions of the GC-MS were:
the ion source is an EI source; the scanning mode is a Scan monitoring mode; the scanning range is 35-500 m/z; the detector voltage is 1.3kV; the ion source temperature is 230 ℃; the interface temperature is 240 ℃; the solvent delay was 2.5min.
Example 2
Batch 1 was replaced with Pogostemon cablin leaves of batch 2, and the rest of the procedure was the same as in example 1.
FIG. 1 is a GC-MS TIC diagram of the present example, as can be obtained from FIG. 1, the 5 chemical components of the mixed control solution (FIG. 1A) and the patchouli leaf of batch 2 (FIG. 1B) do not interfere with each other, the symmetry of the chromatographic peaks of each chemical component is good, and the degree of separation is high.
Example 3
Batch 1 was replaced with Pogostemon cablin leaves of batch 3, and the rest of the procedure was the same as in example 1.
Example 4
Batch 1 was replaced with patchouli leaves of batch 4, the remainder of the procedure being as in example 1.
Example 5
Batch 1 was replaced with Pogostemon cablin leaves of batch 5, and the rest of the procedure was the same as in example 1.
Example 6
Batch 1 was replaced with Pogostemon cablin leaves of batch 6, and the rest of the procedure was the same as in example 1.
Example 7
Batch 1 was replaced with Pogostemon cablin leaves of batch 7, and the rest of the procedure was the same as in example 1.
Example 8
Batch 1 was replaced with patchouli leaves of batch 8, the rest of the procedure being the same as in example 1.
Example 9
Batch 1 was replaced with patchouli leaves of batch 9, the rest of the procedure being the same as in example 1.
Example 10
Batch 1 was replaced with Pogostemon cablin leaves of batch 10, and the rest of the procedure was the same as in example 1.
Example 11
Batch 1 was replaced with patchouli leaves of batch 11, the remainder of the procedure being as in example 1.
Example 12
Batch 1 was replaced with patchouli leaves of batch 12, the remainder of the procedure being as in example 1.
The measurement results of examples 1 to 12 are shown in Table 3.
TABLE 3 content of 5 chemical Components in different batches of Agastache rugosa leaves (μg/g, n=3)
And (3) precision testing:
precision (within day): the sample solution was continuously sampled 6 times according to the method of example 2, the peak area of each chemical component was recorded, RSD values of 5 chemical component contents were calculated, and the results are shown in table 4.
Table 4 daily precision of chemical composition (n=6)
As shown in Table 4, the detection method according to the present invention has good within-day precision.
Precision (daytime): the sample solution was repeatedly sampled 2 times according to the method of example 2, continuously sampled for 3 days, the peak area of each chemical component was recorded, and RSD values of 5 chemical component contents were calculated, respectively, and the results are shown in table 5.
Table 5 daytime precision of chemical composition (n=6)
As shown in Table 5, the detection method according to the present invention has good daytime precision.
Repeatability test:
the measurement method of example 2 was repeated 6 times, and RSD values of 5 kinds of chemical component contents were calculated, respectively, and the results are shown in table 6.
Table 6 repeatability of chemical composition (μg/g, n=6)
Chemical composition Beta-elemene Beta-caryophyllene Caryophyllin Patchouli alcohol Acanthol alcohol
1 53.73 88.38 182.25 41.54 13.3
2 57.72 94.17 204.78 42.82 13.31
3 59.6 97.42 239.13 49.26 15.88
4 66.11 108.51 229.96 51.46 15.54
5 67.11 109.43 246.16 52.06 17.39
6 60.3 98.1 223.32 45.99 15.28
Average value of 60.76 99.34 220.94 47.19 15.12
RSD(％) 8.4 8.3 10.7 9.4 10.5
As can be seen from Table 6, the detection method provided by the invention has good repeatability.
Stability test:
the samples were taken at 3 hour intervals, 6 times in total, the peak areas of the chemical components were recorded, the stability under room temperature conditions was examined, and the RSD values thereof were calculated according to the test method of example 2, and the results are shown in table 7.
Table 7 stability of chemical composition (n=6)
Chemical composition Beta-elemene Beta-caryophyllene Caryophyllin Patchouli alcohol Acanthol alcohol
1 21439 83897 42803 512975 91583
2 21397 82604 44458 521510 92597
3 22644 89021 46319 550053 96908
4 23290 88758 47368 560564 97172
5 24699 95373 49667 586150 104116
6 25958 97827 55295 622417 112301
Average value of 23237.8 89580.0 47651.9 558944.9 99112.9
RSD(％) 7.8 6.8 9.3 7.3 7.9
As can be seen from Table 7, the 5 chemical components in patchouli leaf were stable at room temperature for 15 hours. Sample addition recovery rate experiment:
and respectively weighing beta-elemene, beta-caryophyllene, caryophyllene and patchouli Li Chunhe farnesol, adding ethyl acetate to a constant volume of 10mL to prepare a stock solution, wherein the concentrations of the beta-elemene, the beta-caryophyllene, the caryophyllene and the farnesol are 1mg/mL, the concentration of the patchouli alcohol is 10mg/mL, and storing in a refrigerator at 4 ℃ for later use.
The sources and lot numbers of the above raw materials are as follows: patchouli alcohol (lot number: DSTDB 004102), beta-elemene (lot number: DSTDL 026701), beta-caryophyllene (lot number: DSTDS 009501), caryophyllene (lot number: DSTDS 021001) were purchased from Dou Desai Tebiotech Co., ltd; farnesol (lot number: M27GS 143435) was purchased from Shanghai Seiyaku Biotechnology Co., ltd; ethyl acetate (analytically pure) is supplied by Kangkode technologies, inc. of Tianjin.
Pulverizing herba Agastaches leaf of batch 2 with pulverizer, sieving with 10 mesh sieve, weighing 0.25g herba Agastaches coarse powder, placing in 10mL volumetric flask, adding ethyl acetate to volume to scale, extracting with ultrasound (power 300W, frequency 40 KHz) for 40min, taking out, standing to room temperature, supplementing to scale, taking 1mL of the solution, centrifuging at 14000r/min for 10min, collecting supernatant, filtering with 0.22 μm organic microporous membrane, collecting filtrate as sample solution, and storing in refrigerator at 4deg.C for use.
Taking 14 mu L of beta-elemene stock solution, 23 mu L of beta-caryophyllene stock solution, 43 mu L of caryophyllene stock solution, 4 mu L of farnesol stock solution and 118 mu L of patchouli alcohol stock solution, adding into a sample solution, fixing the volume to 10mL by using methanol, performing GC-MS analysis, reading the concentration of volatile oil on a standard curve, and performing 5 groups of parallel experiments, wherein the experimental results are shown in Table 8.
Table 8 recovery of chemical components on sample (μg, n=6)
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. A method for determining the content of volatile oil in patchouli leaf, which is characterized in that the volatile oil comprises patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllin and farnesol;
the method comprises the following steps:
s1, respectively mixing patchouli alcohol, beta-elemene, beta-caryophyllene, caryophyllene and farnesol with ethyl acetate to obtain stock solution;
s2, mixing all stock solutions to obtain reference substance solutions, respectively carrying out GC-MS analysis on the reference substance solutions with different concentrations, and drawing a standard curve by taking the peak area of the volatile oil as an ordinate and the concentration of the volatile oil as an abscissa; the concentration of the volatile oil is the lowest quantitative limit when the signal to noise ratio S/N=10;
s3, mixing patchouli leaf powder with ethyl acetate, sequentially performing ultrasonic extraction, volume fixing, centrifugation and filtration to obtain a test solution, performing GC-MS analysis on the test solution, and reading the concentration of the volatile oil on a standard curve.
2. The method according to claim 1, wherein in the step S1, the concentration of the stock solution is independently 0.5 to 2mg/mL.
3. The method according to claim 2, wherein in the step S2, in the control solution, the concentration of patchouli alcohol is 120 to 160 μg/mL, the concentration of β -elemene is 20 to 30 μg/mL, the concentration of β -caryophyllene is 35 to 45 μg/mL, the concentration of caryophyllene is 90 to 100 μg/mL, and the concentration of farnesol is 75 to 85 μg/mL.
4. A method according to claim 2 or 3, wherein in step S2, 9 total concentrations of the control solutions of different concentrations are included, including the initial concentration and dilution of the control solution by 2-fold, 2.5-fold, 2-fold and 2-fold in sequence.
5. The method according to claim 4, wherein in the step S3, the particle size of patchouli leaf powder is not less than 10 mesh; the concentration of the sample solution is 0.01-0.1 g/mL.
6. The method according to claim 2 or 5, wherein in the step S3, the power of the ultrasonic extraction is 200-400W, the frequency is 30-50 KHz, and the time is 30-50 min.
7. The method according to claim 6, wherein in the step S3, the rotational speed of centrifugation is 10000-20000 r/min, and the time of centrifugation is 10-20 min.
8. The method according to claim 3, 5 or 7, wherein in step S2 and step S3, the chromatographic conditions of GC-MS are independently:
the chromatographic column is HP5-MS (0.25 μm. Times.0.25 mm. Times.30 m) capillary chromatographic column; the carrier gas is helium, and the purity is more than or equal to 99.99%; the split ratio of the split mode is 20:1, a step of; the flow rate is 1.0-2.0 mL/min; the sample injection amount is 0.5-2 mu L; the temperature of the sample inlet is 240-280 ℃; chromatographic column temperature procedure: the initial temperature is 140 ℃; heating to 150 ℃ at 2 ℃/min; then heating to 180 ℃ at 12 ℃/min, and keeping for 2min.
9. The method according to claim 8, wherein in the step S2 and the step S3, the mass spectrometry conditions of the GC-MS are independently:
the ion source is an EI source; the scanning mode is a Scan monitoring mode; the scanning range is 35-500 m/z;
the detector voltage is 1.3kV; the temperature of the ion source is 200-250 ℃; the interface temperature is 220-260 ℃;
the solvent delay was 2.5min.
CN202310931286.4A 2023-07-27 2023-07-27 Method for measuring volatile oil content in patchouli leaf Pending CN117110456A (en)

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