Quantitative Analysis Method for Active Components of Cinnamomum camphora Leaf Hydrosol

Abstract

The present invention belongs to the technical field of analysis and detection, and specifically is a quantitative analysis method for active components of camphor leaf hydrosol. The quantitative analysis method for active components of camphor leaf hydrosol comprises the following steps: accurately weigh the camphor leaf hydrosol, then add a certain amount of internal standard containing hydroxyl group to the camphor leaf hydrosol and fully dissolve it; use gas chromatography-mass spectrometry to test the mixed solution to determine the quantitative relationship between the mass concentration of the main active components in the camphor leaf hydrosol and the internal standard; the internal standard containing hydroxyl group is phenol. In the method provided by the present invention, the internal standard does not react with the effective components in the hydrosol, and the difference between its retention time and the retention time of the main component is only 0.9 minutes, which effectively avoids the influence of baseline fluctuation on concentration determination. The current problem of quantitative determination of effective components of camphor leaf hydrosol is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a quantitative analysis method for active components of camphor leaf hydrosol. Background Art

[0002] Cinnamomum camphora is an evergreen tree native to China, distributed in Hunan, Guangdong, Sichuan, Yunnan and other provinces. Camphor oil essential oil can be extracted from the roots, branches and leaves of Cinnamomum camphora, among which the leaves have the highest oil yield. The production of Cinnamomum camphora essential oil in Yibin, Sichuan accounts for 75% of the country's total and 90% of Sichuan Province, making it the largest producer of Cinnamomum camphora essential oil in the country. The main components of Cinnamomum camphora essential oil are 1,8-cineole, α-terpineol, sabinene, 4-terpineol, pinene, γ-terpinene, caryophyllene, α-terpinene, etc. in descending order of content. Yin Hao et al. (Preservation and Processing, 2020, 20, 183-191) showed that camphor essential oil has a good inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus; Li et al. (Int. J. Clin. Exp. Med. 2014, 7, 1721-1727) studied the minimum inhibitory concentration and minimum bactericidal concentration of the active ingredients of camphor essential oil 1,8-cineole, α-terpineol, terpinene-4-ol, and γ-terpinene against Escherichia coli ATCC25922, Salmonella enteritidis 50041, and Staphylococcus aureus ATCC25923, respectively. Among them, α-terpineol showed the best antibacterial and bactericidal activity against the three bacteria.

[0003] At present, researchers at home and abroad have reported methods for extracting camphor essential oil by steam distillation, enzyme-assisted extraction, and supercritical fluid extraction. However, steam distillation for extracting camphor essential oil has the advantages of simple production equipment and good oil yield, and is currently the main method for extracting camphor essential oil in the industry. During steam distillation, the essential oil will inevitably carry a large amount of water vapor to be evaporated and condensed, with a water-oil ratio of about 8:1. The aqueous phase is separated to become "camphor leaf hydrosol". In 2021, Wei Qin et al. (Anhui Agricultural Sciences, 2021, 49, 190-192) took the lead in qualitatively determining the composition of camphor leaf hydrosol in different seasons and studied its antibacterial activity. It was found that camphor leaf hydrosol mainly contains 1,8-cineole, α-terpineol, terpinene-4-ol and other substances. The hydrosol has good antibacterial and bactericidal activity against Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Salmonella enteritidis. Considering that many plant hydrosols have been used in the cosmetics or food industries due to their antibacterial or antioxidant properties, the effective use of camphor leaf hydrosol has become an important means to increase the economic added value of camphor. However, the content of various essential oil components in camphor leaves from different origins and seasons is different. Accurately determining the content of active ingredients in the hydrosol has become an important technical problem that needs to be overcome in the scientific use of camphor leaf hydrosol. Summary of the invention

[0004] The purpose of the present invention is to quickly and accurately determine the content of active ingredients in the oily camphor leaf hydrosol, and the present invention provides a quantitative analysis method for the active components of the oily camphor leaf hydrosol. The method is simple and easy to implement, and can quickly and accurately determine the content of active ingredients in the oily camphor leaf hydrosol.

[0005] In order to achieve the above invention object, the specific technical solution of the present invention is:

[0006] The quantitative analysis method of the active components of camphor leaf hydrosol comprises the following steps:

[0007] (1) Add a certain amount of internal standard containing hydroxyl groups to a known weight of hydrosol and allow to fully dissolve.

[0008] (2) The mixed solution was tested by gas chromatography-mass spectrometry to determine the quantitative relationship between the mass concentrations of the main active components in the Cinnamomum camphora leaf hydrosol and the internal standard.

[0009] Furthermore, the effective ingredients of camphor essential oil mainly include groups containing alkyl, ether, alkenyl, alcohol hydroxyl, cycloalkyl and the like; and camphor leaf hydrosol mainly contains 1,8-cineole, α-terpineol, terpinene-4-ol, α, α-dimethyl-4-methylenecyclohexane, 2-hydroxy-1,8-cineole and the like. That is, the present application is equivalent to providing a method for simultaneously quantitatively determining active components such as 1,8-cineole, α-terpineol, terpinene-4-ol, α, α-dimethyl-4-methylenecyclohexane, 2-hydroxy-1,8-cineole and the like in camphor leaf hydrosol.

[0010] As a better implementation in the present application, in the quantitative analysis method of the active components of the camphor leaf hydrosol, the internal standard containing hydroxyl group is phenol.

[0011] As a better implementation method in the present application, in the quantitative analysis method of the active components of the camphor leaf hydrosol, the dosage relationship between the camphor leaf hydrosol and the internal standard is 20g / 1mmol.

[0012] As a better implementation in the present application, in the quantitative analysis method of the active components of the camphor leaf hydrosol, dissolution is performed by mixing under ultrasonic vibration.

[0013] As a better implementation in the present application, in the quantitative analysis method of the active components of the oil camphor leaf hydrosol, the setting parameters of the gas chromatography-mass spectrometry chromatograph are: using GCMS-QP2010plus for injection testing. Test conditions: column box temperature: 50.0 ° C; injection temperature: 280.00 ° C; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0 mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The main difficulty faced by the present invention in quantitatively determining the content of the main components in the sample by adding an internal standard method using gas chromatography-mass spectrometry is how to ensure that the internal standard can be completely dissolved in the hydrosol solution, but does not react with the effective components in the hydrosol, and its chromatographic retention time is close to the retention time of the sample component but does not interfere with the normal component integration. Through experiments, the internal standard in the method provided by the present invention does not react with the effective components in the hydrosol, and the difference between its retention time and the retention time of the main component is only 0.9 minutes, which effectively avoids the influence of baseline fluctuations on concentration determination. The current problem of quantitative determination of the effective components of oil camphor leaf hydrosol is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a GC-MS component peak result diagram using phenol as an internal standard in the quantitative analysis method of the active components of the camphor leaf hydrosol of the present invention;

[0017] Figure 2 It is a locally enlarged peak table diagram of the GC-MS component peak results using phenol as an internal standard in the quantitative analysis method of the active components of the camphor leaf hydrosol of the present invention;

[0018] Figure 3 The GC-MS mass spectra and standard spectrum library diagram of the target components with retention times of 6.335 and 7.235 using phenol as the internal standard in the quantitative analysis method of the active components of the camphor leaf hydrosol of the present invention;

[0019] Figure 4 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 9.375 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0020] Figure 5 The GC-MS retention time of the target component with phenol as the internal standard is 9.540 and the standard spectrum library diagram in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0021] Figure 6 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 9.745 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0022] Figure 7 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 10.075 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0023] Figure 8 This is a GC-MS component peak result diagram of a mixture of phenol and α-terpineol in an accuracy confirmation experiment of a quantitative analysis method for active components of a Cinnamomum camphora leaf hydrosol of the present invention;

[0024] Fig. 9 The GC-MS retention time of the phenol and α-terpineol mixture in the accuracy confirmation experiment of the quantitative analysis method of the active components of the camphor leaf hydrosol of the present invention is 6.295 target component mass spectrum and standard spectrum library diagram;

[0025] Fig.10 The GC-MS retention time of the phenol and α-terpineol mixture in the accuracy confirmation experiment of the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention is 9.745, and the target component mass spectrum and standard spectrum library diagram;

[0026] Fig.11 The GC-MS component peak results and peak table diagram using triethylene glycol as an internal standard in the quantitative analysis method of the active components of the camphor leaf hydrosol of the present invention;

[0027] Fig.12 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 9.065 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0028] Fig.13 The GC-MS retention time of the target component with phenol as the internal standard is 9.380 and the standard spectrum library diagram in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0029] Fig.14 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 9.545 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0030] Fig.15 The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 9.755 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0031] Fig.16The GC-MS mass spectrum and standard spectrum library diagram of the target component with a retention time of 10.110 using phenol as an internal standard in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0032] Fig.17 The GC-MS retention time of the target component with phenol as the internal standard is 10.580 and the standard spectrum library diagram in the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0033] Fig.18 This is a GC-MS component peak result diagram of a mixture of triethylene glycol and α-terpineol in an accuracy confirmation experiment of a quantitative analysis method for active components of a Cinnamomum camphora leaf hydrosol of the present invention;

[0034] Fig.19 The GC-MS retention time of the target component of the mixture of triethylene glycol and α-terpineol is 9.740 and the standard spectrum library diagram in the accuracy confirmation experiment of the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention;

[0035] Fig. 20 The present invention provides a mass spectrum diagram of the target component with a GC-MS retention time of 10.440 in the accuracy confirmation experiment of the quantitative analysis method of the active component of the camphor leaf hydrosol of the present invention and a standard spectrum library diagram. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes and modifications to the present invention, but these equivalent forms also fall within the scope defined by the claims attached to the application.

[0037] Embodiment 1:

[0038] The quantitative analysis method of the active components of camphor leaf hydrosol comprises the following steps:

[0039] Weigh 20.0000g of Cinnamomum camphora leaf hydrosol accurately with a 1 / 10,000 balance, add 0.1200g of isopropanol and mix well under ultrasonic vibration. Use GCMS-QP2010plus for injection test. Test conditions: column box temperature: 50.0℃; injection temperature: 280.00℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium. The test shows that there are 5 main organic components in Cinnamomum camphora leaf hydrosol. The retention time of 1,8-cineole was 7.165 min; the retention time of α, α-dimethyl-4-methylenecyclohexane methanol was 9.330 min; the retention time of terpinen-4-ol was 9.500 min; the retention time of α-terpineol was 9.700 min, and the retention time of 2-hydroxy-1,8-cineole was 10.031 min. However, the internal standard isopropanol was not detected, so the content of the active ingredient in the oil camphor leaf hydrosol could not be determined using this internal standard.

[0040] Embodiment 2:

[0041] The quantitative analysis method of the active components of camphor leaf hydrosol comprises the following steps:

[0042] Weigh 20.0000g of Cinnamomum camphora leaf hydrosol accurately with a 1 / 10,000 balance, add 0.1080g of benzyl alcohol and mix well under ultrasonic vibration. Use GCMS-QP2010plus for injection test. Test conditions: column box temperature: 50.0℃; injection temperature: 280.00℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium. The test shows that there are 5 main organic components in Cinnamomum camphora leaf hydrosol. The retention time of 1,8-cineole is 7.235 min; the retention time of α, α-dimethyl-4-methylenecyclohexane methanol is 9.395 min; the retention time of terpinene-4-ol is 9.560 min; the retention time of α-terpineol is 9.765 min, and the retention time of 2-hydroxy-1,8-cineole is 10.150min. The retention time of the internal standard benzyl alcohol is between 7.300-7.500 min, which is too close to the retention time of the active component 1,8-cineole (7.235 min), which may have a certain impact on the accuracy of integration. In addition, benzaldehyde appears at the retention time of 6.105min in the spectrum, which is the oxide of benzyl alcohol. Therefore, although this method can calculate the content of each component, since the retention time of the internal standard is too close to the active component and is easily oxidized, the internal standard is not suitable for quantitative determination of the active ingredients of the hydrosol.

[0043] Embodiment 3:

[0044] The quantitative analysis method of the active components of camphor leaf hydrosol comprises the following steps:

[0045] Weigh 20.0000g of Cinnamomum camphora leaf hydrosol accurately with a 1 / 10,000 balance, add 0.0942g of phenol and mix well under ultrasonic vibration. Use GCMS-QP2010plus for injection testing. Test conditions: column box temperature: 50.0℃; injection temperature: 280.00℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0 mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium. According to the chromatographic Figure 1-Figure 7As shown, the retention time of the internal standard phenol is 6.335 min (only 0.9 minutes different from the retention time of the first organic component of the oil camphor leaf hydrosol), and the peak area is 77.41%. There are 5 main organic components of the oil camphor leaf hydrosol. Among them, the retention time of 1,8-cineole is 7.235 min, and the peak area is 11.08%; the retention time of α, α-dimethyl-4-methylenecyclohexane methanol is 9.375 min, and the peak area is 0.57%; the retention time of terpinene-4-ol is 9.540 min, and the peak area is 1.66%; the retention time of α-terpineol is 9.745 min, and the peak area is 6.19%; the retention time of 2-hydroxy-1,8-cineole is 10.077 min, and the peak area is 0.87%.

[0046] Based on the fact that the added internal standard phenol is 0.0942 g, it is deduced that the content of 1,8-cineole in 20.0000 g of camphor oil hydrosol is 13.484 mg, and the content is 6.74‱; the content of α, α-dimethyl-4-methylenecyclohexane is 0.692 mg, and the content is 0.35‱; the content of terpinen-4-ol is 2.016 mg, and the content is 1.01‱; the content of α-terpineol is 7.517 mg, and the content is 3.76‱; and the content of 2-hydroxy-1,8-cineole is 1.057 mg, and the content is 0.53‱.

[0047] Confirmation of quantitative measurement accuracy:

[0048] (1) To verify the accuracy of the internal standard detection, 20 g of distilled water was weighed, and 94.8 mg of phenol and 14.3 mg of α-terpineol accurately weighed with a 1 / 10,000 balance were added thereto, and the mixture was mixed evenly under ultrasonic vibration.

[0049] (2) GCMS-QP2010plus was used for injection test. Test conditions: column box temperature: 50.0 ℃; injection temperature: 280.00 ℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0 mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium.

[0050] According to the chromatogram Figure 8-Figure 10As shown, there are two main organic components in the confirmatory mixed solution. Among them, the retention time of internal standard phenol is 6.295 min (very close to the retention time of phenol in Example 3, 6.335 min), and the peak area is 86.85%. The retention time of α-pinene alcohol is 9.744 min (almost consistent with the retention time of phenol in Example 3, 9.745 min), and the peak area is 13.15%. The test shows that the mass ratio of phenol to α-pinene alcohol is 86.85% / 13.15% = 6.605, which is very close to the ratio of internal standard phenol to α-pinene alcohol addition, 6.629, and the accuracy rate reaches 99.64%. Considering that the retention time of the effective components of the hydrosol is basically between 7.2 min – 10.1 min, phenol can be used as an internal standard to quantitatively determine the effective components of the hydrosol.

[0051] Embodiment 4:

[0052] The quantitative analysis method of the active components of camphor leaf hydrosol comprises the following steps:

[0053] (1) Accurately weigh 20.0000 g of Cinnamomum camphora leaf hydrosol using a 1 / 10,000 balance, add 0.1501 g of triethylene glycol and mix well under ultrasonic vibration.

[0054] (2) GCMS-QP2010plus was used for injection test. Test conditions: column box temperature: 50.0 ℃; injection temperature: 280.00 ℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0 mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium.

[0055] According to the chromatogram Fig.11 – As shown in Figure 17, the retention time of internal standard triethylene glycol is 10.580 min, and the peak area is 86.53%. The retention time of 1,8-cineole is 7.221 min, and the peak area is 7.13%; the retention time of α, α-dimethyl-4-methylenecyclohexane methanol is 9.379 min, and the peak area is 0.38%; the retention time of terpinen-4-ol is 9.548 min, and the peak area is 1.27%; the retention time of α-terpineol is 9.755 min, and the peak area is 3.86%; the retention time of 2-hydroxy-1,8-cineole is 10.109 min, and the peak area is 0.37%.

[0056] Based on the fact that the added internal standard triethylene glycol is 0.1501 g, it is deduced that the content of 1,8-cineole in 20.0000 g of camphor oil hydrosol is 12.442 mg, and the content is 6.22‱; the content of α, α-dimethyl-4-methylenecyclohexane is 0.663 mg, and the content is 0.33‱; the content of terpinen-4-ol is 2.216 mg, and the content is 1.11‱; the content of α-terpineol is 6.736 mg, and the content is 3.37‱; and the content of 2-hydroxy-1,8-cineole is 0.646 mg, and the content is 0.32‱.

[0057] Quantitative determination accuracy confirmation

[0058] (1) To verify the accuracy of the internal standard detection, 20 g of distilled water was weighed, and 150.0 mg of triethylene glycol and 15.1 mg of α-terpineol were accurately weighed using a 1 / 10,000 balance. The mixture was mixed evenly under ultrasonic vibration.

[0059] (2) GCMS-QP2010plus was used for injection test. Test conditions: column box temperature: 50.0 ℃; injection temperature: 280.00 ℃; injection mode: split; flow control mode: linear velocity; pressure: 53.6 kPa; total flow: 14.0 mL / min; column flow: 1.00 mL / min; linear velocity: 36.3 cm / sec; purge flow: 3.0 mL / min; split ratio: 10.0; gas: helium.

[0060] According to the chromatogram Fig.18 – Fig. 20 As shown, there are two main organic components in the confirmatory mixed solution. Among them, the retention time of triethylene glycol is 10.441 min, and the peak area is 87.53%. The retention time of α-pineneol is 9.740 min, and the peak area is 12.47%. The test shows that the mass ratio of triethylene glycol to α-pineneol is 87.53% / 12.47% = 7.02, which is quite different from the ratio of the internal standard triethylene glycol to α-pineneol added of 9.93, and the accuracy is only 70.69%. Therefore, the accuracy of using triethylene glycol as the internal standard for quantitative determination of the active ingredients of the hydrosol is not good.

[0061] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

[0062] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of this section that did not constitute prior art at the time of application are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A quantitative analysis method for the active components of camphor leaf hydrosol, characterized in that The following steps are involved: Accurately weigh the camphor leaf hydrosol, then add a certain amount of internal standard containing hydroxyl group into the camphor leaf hydrosol and fully dissolve it; use gas chromatography-mass spectrometry to test the mixed solution to determine the quantitative relationship between the mass concentration of the main active components in the camphor leaf hydrosol and the internal standard; the internal standard containing hydroxyl group is phenol; the main active components in the camphor leaf hydrosol are 1,8-cineole, α-terpineol, terpinene-4-ol, α, α-dimethyl-4-methylenecyclohexane, and 2-hydroxy-1,8-cineole; the setting parameters of the gas chromatography-mass spectrometry are: using GCMS-QP2010plus for injection testing; the column box temperature of the test is 50.0℃; the injection temperature is 280.00℃; the injection mode of the test is split; the flow control mode of the test is linear velocity; the test pressure is 53.6kPa; the total flow rate is 14.0 mL / min; the column flow rate is 1.00 mL / min; the linear velocity is 36.3 cm / sec; the purge flow rate of the test was 3.0 mL / min; the split ratio was 10.0; and the gas was helium.

2. The quantitative analysis method of the active components of the camphor leaf hydrosol as claimed in claim 1, characterized in that: The dosage relationship between Cinnamomum camphora leaf hydrosol and internal standard is 20g / 1mmol.

3. The quantitative analysis method of the active components of the camphor leaf hydrosol as claimed in claim 1, characterized in that: Dissolution was performed by mixing under ultrasonic vibration.

Images