Method for determining contents of various components in fructus cinnamoii and detection method of UHPLC (Ultra High Performance Liquid Chromatography) specific chromatogram

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for determining the content of multiple components in fructus cinnamoii and a method for detecting a UHPLC (Ultra High Performance Liquid Chromatography) specific chromatogram, the UHPLC method is adopted for determination, and the specific chromatographic conditions are as follows: a ZORBAX Eclipse Plus C18 chromatographic column (the column length is 100mm, the inner diameter is 2.1 mm and the particle size is 1.8 mu m) is adopted; acetonitrile is used as a mobile phase A, and a 0.2% formic acid solution is used as a mobile phase B; gradient elution: acetonitrile: 5%-7% in 0-1 min; 1-5 min: 7%-8%; 5-7 min: 8%-9%; 7-11 min: 9%-11%; 11%-17% in 11-19 min; 19-20 min: 17%; 20-22 min: 17%-19% of the raw materials; the flow velocity is 0.3 mL / min; the sample size is 2 mu L; the column temperature is 35 DEG C; the detection wavelength is 276 nm.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and specifically to a method for determining the contents of multiple components in Litsea cubeba and a method for detecting a UHPLC characteristic spectrum. Background Art

[0002] Litsea cubeba is the dried mature fruit of Cinnamomum migao H.W.Li, a plant of the Lauraceae family. It is distributed in Luodian, Wangmo, Sandu and other places in Guizhou, and is also cultivated in Guangxi and Yunnan. It has the effects of warming the middle and regulating qi, relieving pain and drying dampness, and strengthening the spleen and digestion. It is included in the 2019 edition of the "Quality Standards for Traditional Chinese Medicine and Ethnic Medicinal Materials in Guizhou Province" and was first recorded in the "Supplement to Compendium of Materia Medica", which called it "Camphor Pear", that is, camphor tree seeds. Migao Xinle Drop Pills, Xinnaoning Capsules and other marketed preparations all use Litsea cubeba as raw materials, with significant clinical efficacy and good social and economic benefits.

[0003] As one of the top ten Miao medicines in Guizhou, Litsea cubeba is widely used in traditional medicinal materials and new drug research and development of the Miao and Buyi ethnic groups. However, current research on Litsea cubeba is mostly focused on the chemical composition identification of volatile oils, extraction process optimization and pharmacological effects, and the quality standard only controls the volatile component eucalyptol. The single indicator fails to reflect the overall efficacy and its quality cannot be fully controlled. The reason is that the quality research of medicinal materials from different sources is not sufficient and the basic research is relatively weak.

[0004] Therefore, UHPLC fingerprint and multi-component content determination combined with chemical model analysis of Litsea cubeba from different origins were used to provide a reference for the quality control of Litsea cubeba. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for determining the content of multiple components in Litsea cubeba and a method for detecting the UHPLC characteristic spectrum, which are as follows:

[0006] The invention discloses a method for determining the contents of multiple components in Litsea cubeba, which adopts a UHPLC method for determination, and the specific chromatographic conditions are as follows: a ZORBAX Eclipse Plus C18 chromatographic column (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm) is adopted; acetonitrile is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B.

[0007] Furthermore, the specific elution procedure of the UHPLC method is as follows: gradient elution: acetonitrile: 0-1 min: 5%-7%; 1-5 min: 7%-8%; 5-7 min: 8%-9%; 7-11 min: 9%-11%; 11-19 min: 11%-17%; 19-20 min: 17%; 20-22 min: 17%-19%.

[0008] Furthermore, the flow rate of the UHPLC method was 0.3 mL / min; the injection volume was 2 μL; the column temperature was 35° C.; and the detection wavelength was 276 nm.

[0009] Furthermore, the contents of the multiple components are specifically determined to be salidroside, chlorogenic acid, magnolamine, and naringin.

[0010] Furthermore, the test solution is prepared by the following steps: take 1 g of Litsea cubeba medicinal material powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, record the weight, heat and reflux for 30 minutes, cool to room temperature, weigh it again, make up the weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0011] Furthermore, the mixed reference solution is a precisely weighed reference solution of salidroside, chlorogenic acid, magnolidine and naringin with a mass concentration of 1 mg / mL using methanol as a solvent.

[0012] A method for detecting a UHPLC characteristic spectrum of Litsea cubeba, comprising sample injection and analysis according to the above chromatographic conditions.

[0013] Furthermore, after the sample injection and analysis according to the chromatographic conditions in claim 1, the data is imported into the "Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", with the chromatogram of sample S1 as the reference spectrum, and the time window width is 0.1 min.

[0014] Furthermore, the UHPLC characteristic spectrum of the obtained Litsea cubeba had 13 common peaks calibrated, and by comparison with the reference substance, a total of 6 components were identified, namely Peak 1 (protocatechuic acid), Peak 3 (salidroside), Peak 6 (chlorogenic acid), Peak 9 (cryptochlorogenic acid), Peak 11 (magnolin), and Peak 13 (naringin).

[0015] The method for determining the contents of multiple components in Litsea cubeba and the detection method of UHPLC characteristic spectrum are applied to control the quality of Litsea cubeba. The fingerprint spectrum and content determination method of Litsea cubeba established based on UHPLC in this study are simple and easy to operate, which can provide a reference for quality control and identification of Litsea cubeba.

[0016] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0017] The present invention establishes a fingerprint of Litsea cubeba based on UHPLC technology, and simultaneously determines the contents of salidroside, chlorogenic acid, magnolone and naringin, providing a basis for quality control of Litsea cubeba. Specifically, a ZORBAX Eclipse Plus C18 chromatographic column was used; 15 batches of Litsea cubeba medicinal material fingerprints were established, with similarities between batches of 0.933 to 1.000, 13 common peaks were calibrated, and 6 chemical components were identified, namely protocatechuic acid, salidroside, chlorogenic acid, cryptochlorogenic acid, magnolone and naringin. The content determination results showed that the content ranges of salidroside, chlorogenic acid, magnolone and naringin were 0.0822 to 1.9372 mg / g, 0.2944 to 0.7835 mg / g, 0.4243 to 1.0388 mg / g, and 0.2326 to 0.5417 mg / g, respectively. The fingerprint spectrum and content determination method of Litsea cubeba established in this application based on UHPLC are simple and easy to operate, and can provide a reference for the quality control and identification of Litsea cubeba. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the superposition of fingerprint spectra of 15 batches of Litsea cubeba and the reference fingerprint spectra (R).

[0019] Figure 2 This is the UHPLC chart of the mixed reference solution and Litsea cubeba test solution.

[0020] Figure 3 The MS1 and MS2 spectra of peak 3 (RT = 4.21)

[0021] Figure 4 The MS1 and MS2 spectra of peak 6 (RT = 6.31)

[0022] Figure 5 The MS1 and MS2 spectra corresponding to peak 9 (RT = 7.44)

[0023] Figure 6 The MS1 and MS2 spectra of peak 11 (RT = 11.27)

[0024] Figure 7 The MS1 and MS2 spectra of peak 13 (RT = 22.23)

[0025] Figure 8 This is the UV absorption graph of protocatechuic acid in the test sample and the reference sample.

[0026] Fig. 9This is the UHPLC chart for the specificity investigation of Litsea cubeba: Figure A is blank solvent; Figure B is mixed reference solution; Figure C is test solution. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0028] Example

[0029] 1. Materials and Methods

[0030] 1.1 Instrument

[0031] 1290 InfinityⅡ ultra-high performance liquid chromatograph (Agilent, USA); EL-240 electronic balance (Mettler-Toledo Instrument Co., Ltd.); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); WP-UP-IV-20 ultrapure water machine (Sichuan Water Technology Development Co., Ltd.); DRHH-D4 constant temperature water bath (Shanghai Shuangjie Experimental Equipment Co., Ltd.).

[0032] 1.2 Drug testing

[0033] A total of 15 batches of Litsea cubeba were collected, and the sources are shown in Table 1. They were identified as the dried mature fruits of Cinnamomum migao H. W. Li, a plant of Lauraceae, by Associate Professor Liu Chunhua of the School of Pharmacy of Guizhou Medical University. Salidroside (batch number: AF21060954, purity 99.7%) and chlorogenic acid (batch number: 110753-202119, purity 96.3%) were purchased from the China Food and Drug Inspection Institute; magnolia alkaloids (batch number: AFBJ1903, purity 99.62%) and naringin (batch number: AFCI2155, purity 93.5%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0034] Table 1 Information on the source of Litsea cubeba medicinal materials

[0035]

[0036] 2. Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] A ZORBAX Eclipse Plus C18 chromatographic column (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used; acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, gradient elution (acetonitrile: 0-1 min: 5%-7%; 1-5 min: 7%-8%; 5-7 min: 8%-9%; 7-11 min: 9%-11%; 11-19 min: 11%-17%; 19-20 min: 17%; 20-22 min: 17%-19%) was used; the flow rate was 0.3 mL / min; the injection volume was 2 μL; the column temperature was 35°C; and the detection wavelength was 276 nm.

[0039] 2.2 Solution preparation

[0040] Accurately weigh appropriate amounts of salidroside, chlorogenic acid, magnolamine, and naringin reference substances, and add methanol to obtain a reference substance stock solution with a reference substance mass concentration of approximately 1 mg / mL.

[0041] Take 1 g of Litsea cubeba powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, record the weight, heat and reflux for 30 minutes, cool to room temperature, weigh it again, make up the weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0042] 2.3 Establishment of fingerprint of Litsea cubeba

[0043] 2.3.1 Methodological investigation

[0044] Take the powder of Litsea cubeba (batch number: DG-YC-3), prepare the sample according to the method of "2.2", analyze the sample according to the chromatographic conditions of "2.1", take the chlorogenic acid of peak 6 as the reference peak, the relative retention time RSD of precision is ≤0.09%, the relative peak area RSD is ≤1.99%; the relative retention time RSD of repeatability is ≤0.10%, the relative peak area RSD is ≤2.28%; the relative retention time RSD of stability is ≤0.44%, the relative peak area RSD is ≤2.86%. In summary, the results show that this method is stable and reliable, with high instrument precision, good repeatability and good stability, and can be used as the conditions for subsequent experiments.

[0045] 2.3.2 Common peak calibration

[0046] The chromatogram of sample S1 was used as the reference spectrum, the time window width was set to 0.1 min, and the spectra of 15 batches of samples were subjected to multi-point correction and Mark peak matching. The median method was used to generate the reference spectrum (R) and the fingerprint spectrum overlay of 15 batches of Litsea cubeba slices. A total of 13 common peaks were calibrated, see Figure 1 .

[0047] 2.3.3 Reference peak selection

[0048] Among the 13 common peaks calibrated, the retention time of chromatographic peak No. 6 (chlorogenic acid) was moderate, the separation was good, and the peak area was large. Therefore, peak No. 6 was selected as the reference peak (S), and the relative peak area and relative retention time of the 13 common peaks between batches were calculated. The results showed that the RSD of the relative retention time of each common peak between batches was between 0.04% and 0.22%, and the RSD of the relative peak area was between 18.07% and 61.45%, indicating that the retention time of each common peak between batches was relatively stable, but there were differences in the content of chemical components.

[0049] 2.3.4 Identification of common peaks

[0050] Each chromatographic peak was analyzed by LC-MS / MS. By comparing the primary and secondary mass spectra with the database, a total of 5 components were identified, namely peak 3 (salidroside), peak 6 (chlorogenic acid), peak 9 (cryptochlorogenic acid), peak 11 (magnolin), and peak 13 (naringin). The results were then verified with reference substances. Figure 2 The mass spectrum fragment information of each component is shown in Table 2. Figure 3 to Figure 7 At the same time, by comparing the retention time and UV absorption of the reference substance and the sample, peak 1 was identified as protocatechuic acid. Figure 8 .

[0051] Table 2 Characteristic peak fragment information

[0052]

[0053] 2.3.5 Similarity evaluation

[0054] The similarity of fingerprint data of 15 batches of Litsea cubeba slices was calculated using the “Chinese Medicine Fingerprint Similarity Evaluation System” (2012 Edition). The results are shown in Table 3. The similarity between the 15 batches of Litsea cubeba slices samples ranged from 0.933 to 1.000, indicating that the similarity between the batches was high and the quality was relatively stable.

[0055] Table 3 Similarity evaluation results of fingerprint spectra of 15 batches of Litsea cubeba

[0056]

[0057]

[0058] Note: (S1~S15: DGMJZ-YC-1~15, R: reference fingerprint)

[0059] 2.4 Determination of the content of multiple components in Litsea cubeba

[0060] 2.4.1 Exclusivity

[0061] Take blank solvent, mixed reference solution, and Litsea cubeba test solution and analyze them according to the chromatographic conditions under "2.1". The results are shown in Fig. 9 As shown, the blank solvent did not produce a peak at the corresponding position of the reference substance, there was no obvious interference, and the four components were well separated, indicating that the method had good specificity.

[0062] 2.4.2 Investigation of linear relationship

[0063] Take the mixed reference solution under "2.2" respectively, and inject and measure according to the chromatographic conditions under "2.1". Linear regression is performed on the mass concentration (x) of the peak area (y) of the component to be measured. The results show that the four components including salidroside have good linearity (r≥0.9997) within their respective concentration ranges. The results are shown in Table 4.

[0064] Table 4 Linear equations of the four components

[0065]

[0066] 2.5.3 Precision test

[0067] Take the mixed reference solution under "2.2" respectively, and inject and measure according to the chromatographic conditions under "2.1". Inject 6 times in succession. The RSD of the peak area of ​​4 components including salidroside is 0.64%~0.76%, indicating that the method has good precision.

[0068] 2.5.4 Repeatability test

[0069] For the six samples prepared in parallel under item "2.2", the RSD of the peak areas of four components including salidroside was 1.52% to 2.75%, indicating that the method had good repeatability.

[0070] 2.5.5 Stability test

[0071] The test solution was prepared according to item "2.2" and injected at 0h, 2h, 4h, 8h, 12h and 24h. The RSD of the peak areas of salidroside and other four components were 0.45% to 1.79%, indicating that the method had good stability within 24h.

[0072] 2.5.6 Sample recovery test

[0073] Take about 0.5 g of Litsea cubeba powder and add mixed reference solution to each sample in a ratio of 1:1, for a total of 6 samples. Analyze according to the chromatographic conditions under "2.1". The results are shown in Table 5. The average recovery rates of 4 components including salidroside are 97.42% to 103.98%, and the RSD is 1.64% to 2.58%, indicating that this method has a good recovery rate.

[0074] Table 5 Recovery results of 4 components (n=6)

[0075]

[0076] 2.5.7 Content determination

[0077] Take 15 batches of Litsea cubeba and prepare the test solution according to item "2.2", and determine it according to the chromatographic conditions under item "2.1". The external standard method is used for calculation, and the results are shown in Table 6. Calculated on the basis of dry product, the contents of salidroside, chlorogenic acid, magnolamine, and naringin in 15 batches of Litsea cubeba are in the range of 0.0822-1.9372 mg / g, 0.2944-0.7835 mg / g, 0.4243-1.0388 mg / g, and 0.2326-0.5417 mg / g, respectively.

[0078] Table 6 Results of determination of the contents of four components (n=3, mg / g)

[0079]

[0080] 3. Discussion

[0081] In the preliminary experimental method investigation, different extraction solvents (30% methanol, 50% methanol, 70% methanol, methanol), different extraction methods (ultrasound, reflux), and different extraction times (0.25h, 0.5h, 0.75h, 1h) of the test solution were investigated. The extraction efficiency under different conditions was compared by the content of 4 components such as salidroside and "total peak area / sample weight". After single-factor investigation and orthogonal analysis, the optimal extraction method was obtained: 70% methanol heated reflux extraction for 0.5h. Under this method, the extraction of each component is relatively complete and the spectral information is maximized.

[0082] The similarity results of 15 batches of samples were obtained by fingerprint similarity evaluation software, indicating that the similarity between batches of samples was high, but there were large differences in the peak areas of each component between different batches, which may be related to the natural environment such as the soil environment of the origin and storage conditions. The content of 4 components such as salidroside identified by the reference substance was determined to characterize the quality differences between medicinal materials from different origins by the content of the components.

[0083] The index components selected for content determination comprehensively characterize the chemical information of Litsea cubeba. Among them, the content of salidroside is relatively high, which can be considered as the focus of investigation. Salidroside has significant effects in the prevention and treatment of cardiovascular diseases, and has anti-myocardial damage and anti-arrhythmic effects, which is consistent with the clinical application of Litsea cubeba; chlorogenic acid can increase and improve intestinal flora, and has antibacterial, antioxidant, and immunomodulatory pharmacological activities; magnolia alkaloids can be used to treat ulcerative colitis and RA model rats; naringin can activate mouse airway Tas2rs and signal transduction, relieve lung inflammation and relieve asthma; cryptochlorogenic acid and protocatechuic aldehyde are less in the sample and difficult to detect, so they were not quantitatively analyzed.

[0084] In summary, this experiment established a rapid and stable UHPLC fingerprint and multi-index content determination method for Litsea cubeba, which can provide a strong basis for the quality control of Litsea cubeba and a reference for its quality control evaluation.

[0085] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for determining the contents of multiple components in Litsea cubeba, characterized in that: The UHPLC method was used for determination, and the specific chromatographic conditions were as follows: a ZORBAX Eclipse Plus C18 chromatographic column (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.8 μm) was used; acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B.

2. The method for determining the contents of multiple components in Litsea cubeba according to claim 1, characterized in that: The specific elution procedure of the UHPLC method is as follows: gradient elution: acetonitrile: 0-1 min: 5%-7%; 1-5 min: 7%-8%; 5-7 min: 8%-9%; 7-11 min: 9%-11%; 11-19 min: 11%-17%; 19-20 min: 17%; 20-22 min: 17%-19%.

3. The method for determining the contents of multiple components in Litsea cubeba according to claim 1, characterized in that: The contents of the various components are specifically determined for salidroside, chlorogenic acid, magnolamine, and naringin.

4. The method for determining the contents of multiple components in Litsea cubeba according to claim 1, characterized in that: The test solution is prepared by the following steps: take 1 g of Litsea cubeba powder, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh it, record the weight, heat and reflux for 30 minutes, cool to room temperature, weigh it again, make up the weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

5. The method for determining the contents of multiple components in the seedling medicine Litsea cubeba according to claim 1, characterized in that: The mixed reference solution is a precisely weighed 1 mg / mL mass concentration of salidroside, chlorogenic acid, magnolidine, and naringin reference solution using methanol as the solvent.

6. A method for detecting the UHPLC characteristic spectrum of Litsea cubeba, characterized in that: Inject and analyze according to the chromatographic conditions in claim 1.

7. The method for detecting the UHPLC characteristic spectrum according to claim 6, characterized in that: After the sample injection and analysis according to the chromatographic conditions in claim 1, the data is imported into the "Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", with the chromatogram of sample S1 as the reference spectrum, and the time window width is 0.1min.

8. The method for detecting the UHPLC characteristic spectrum according to claim 6, characterized in that: The UHPLC characteristic spectrum of the obtained Litsea cubeba has 13 common peaks calibrated in total, and by comparison with the reference substance, a total of 6 components were identified, namely Peak 1 (protocatechuic acid), Peak 3 (salidroside), Peak 6 (chlorogenic acid), Peak 9 (cryptochlorogenic acid), Peak 11 (magnolin), and Peak 13 (naringin).

9. Use of the method for determining the contents of multiple components in Litsea cubeba as claimed in claim 1 in controlling the quality of Litsea cubeba.

10. Use of the UHPLC characteristic spectrum detection method according to claim 6 in controlling the quality of Litsea cubeba.

Images