A method for determining the content of multiple components in a large fruit litsea and a detection method of UHPLC characteristic spectrum

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically a method for determining the content of multiple components in Litsea cubeba and a method for detecting them using UHPLC characteristic chromatograms. The determination is performed using UHPLC, with the following specific chromatographic conditions: a ZORBAX Eclipse Plus C18 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size); acetonitrile as mobile phase A and 0.2% formic acid solution 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%; flow rate: 0.3 mL / min; injection volume: 2 μL; column temperature: 35℃; detection wavelength: 276 nm.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically a method for determining the content of multiple components in Litsea cubeba and a method for detecting them using UHPLC characteristic chromatograms. Background Technology

[0002] Litsea cubeba fruit is the dried, mature fruit of *Cinnamomum migao* H.W.Li, a plant belonging to the Lauraceae family. It is distributed in Luodian, Wangmo, and Sandu counties of Guizhou Province, and is also cultivated in Guangxi and Yunnan. It has the effects of warming the middle jiao (spleen and stomach), regulating qi, relieving pain, drying dampness, strengthening the spleen, and promoting digestion. It is included in the 2019 edition of the *Guizhou Provincial Standards for the Quality of Traditional Chinese and Ethnic Medicines*, and was first recorded in *Compendium of Materia Medica Supplement*, where it is referred to as "Zhangli," meaning camphor tree fruit. Marketed preparations such as Migao Xinle Dripping Pills and Xinnaoning Capsules all use Litsea cubeba fruit as a raw material, demonstrating significant clinical efficacy and generating good social and economic benefits.

[0003] Litsea cubeba, one of the top ten Miao medicinal herbs in Guizhou, is widely used in traditional medicine and new drug research and development by the Miao and Buyi ethnic groups. However, current research on Litsea cubeba mainly focuses on the identification of the chemical composition of volatile oils, optimization of extraction processes, and pharmacological effects. Moreover, the quality standards only control the volatile component eucalyptol, and the single indicator fails to reflect the overall efficacy and cannot fully control its quality. The reason for this is that the quality research on medicinal materials from different sources is insufficient and the basic research is relatively weak.

[0004] Therefore, by combining UHPLC fingerprinting and multi-component content determination with chemical model analysis, we can provide a reference for the quality control of Litsea cubeba medicinal materials from different producing areas. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a method for determining the content of multiple components in Litsea cubeba and a method for detecting them using UHPLC characteristic chromatograms, as detailed below:

[0006] A method for determining the content of multiple components in Litsea cubeba is disclosed. The determination is performed using UHPLC, and the specific chromatographic conditions are as follows: a ZORBAX Eclipse Plus C18 column (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm) is used; 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 for 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 content of the aforementioned multiple components was determined, specifically rhodioloside, chlorogenic acid, magnoflorine, and naringin.

[0010] Furthermore, the test solution is prepared by the following steps: 1g of Litsea cubeba powder is accurately weighed and placed in a stoppered conical flask. 25mL of 70% methanol is accurately added, the weight is measured and recorded, the mixture is heated under reflux for 30min, cooled to room temperature, weighed again, and the weight is made up with 70% methanol. The mixture is shaken well, filtered, and the filtrate is collected to obtain the test solution.

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

[0012] A UHPLC characteristic chromatogram detection method for Litsea cubeba, using the above-described chromatographic conditions for injection and analysis.

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

[0014] Furthermore, the UHPLC characteristic chromatogram of Litsea cubeba obtained therein identified a total of 13 common peaks, and by comparison with the reference standard, a total of 6 components were identified, namely peak 1 (protocatechuic acid), peak 3 (rhodioloside), peak 6 (chlorogenic acid), peak 9 (cryptochlorogenic acid), peak 11 (magnolidinyl alkaloid), and peak 13 (naringin).

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

[0016] Compared with the prior art, the technical effects of this invention are reflected in:

[0017] This invention establishes a fingerprint chromatogram of Litsea cubeba based on UHPLC technology, and simultaneously determines the contents of rhodioloside, chlorogenic acid, magnoflorine, and naringin, providing a basis for the quality control of Litsea cubeba. Specifically, a ZORBAX Eclipse Plus C18 column was used; fingerprint chromatograms of 15 batches of Litsea cubeba were established, with a batch similarity of 0.933–1.000. A total of 13 common peaks were identified, and 6 chemical components were identified: protocatechuic acid, rhodioloside, chlorogenic acid, cryptochlorogenic acid, magnoflorine, and naringin. The content determination results showed that the content ranges of rhodioloside, chlorogenic acid, magnoflorine, and naringin were 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. The fingerprint chromatogram and content determination method for Litsea cubeba established by 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. Attached Figure Description

[0018] Figure 1 These are overlay images of fingerprint spectra from 15 batches of Litsea cubeba and a control fingerprint spectrum (R).

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

[0020] Figure 3 These are the MS1 and MS2 spectra corresponding to peak 3 (RT = 4.21).

[0021] Figure 4 These are the MS1 and MS2 spectra corresponding to peak 6 (RT = 6.31).

[0022] Figure 5 These are the MS1 and MS2 spectra corresponding to peak 9 (RT = 7.44).

[0023] Figure 6 These are the MS1 and MS2 spectra corresponding to peak 11 (RT = 11.27).

[0024] Figure 7 These are the MS1 and MS2 spectra corresponding to peak 13 (RT = 22.23).

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

[0026] Figure 9The following are UHPLC chromatograms for the specificity study of Litsea cubeba: Figure A: Blank solvent; Figure B: Mixed reference solution; Figure C: Test solution. Detailed Implementation

[0027] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0028] Example

[0029] 1. Materials and Methods

[0030] 1.1 Instruments

[0031] 1290InfinityⅡ Ultra-High Performance Liquid Chromatograph (Agilent Technologies, USA); EL-240 Electronic Balance (Mettler-Toledo Instruments, Ltd.); KQ-500DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); WP-UP-IV-20 Ultrapure Water System (Sichuan Water Technology Development Co., Ltd.); DRHH-D4 Thermostatic Water Bath (Shanghai Shuangjie Experimental Equipment Co., Ltd.).

[0032] 1.2 Test Drugs

[0033] A total of 15 batches of Litsea cubeba medicinal materials were collected, and their sources are shown in Table 1. Associate Professor Liu Chunhua of the School of Pharmacy, Guizhou Medical University, identified them as the dried, mature fruits of *Cinnamomum migao* H.W.Li, a plant in the Lauraceae family. Rhodioloside (batch number: AF21060954, purity 99.7%) and chlorogenic acid (batch number: 110753-202119, purity 96.3%) reference standards were purchased from the China National Institutes for Food and Drug Control; magnoflorin (batch number: AFBJ1903, purity 99.62%) and naringin (batch number: AFCI2155, purity 93.5%) reference standards were purchased from Chengdu Efa Biotechnology Co., Ltd.

[0034] Table 1. Source information of Litsea cubeba medicinal material

[0035]

[0036] 2. Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] A ZORBAX Eclipse Plus C18 column (100 mm length, 2.1 mm inner diameter, 1.8 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, with 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%). 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 rhodioloside, chlorogenic acid, magnoflorin, and naringin reference standards, and add methanol to obtain a reference standard stock solution with a mass concentration of approximately 1 mg / mL.

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

[0042] 2.3 Establishment of fingerprint spectrum for Litsea cubeba

[0043] 2.3.1 Methodological Examination

[0044] Powdered Litsea cubeba (batch number: DG-YC-3) was prepared according to method "2.2" and analyzed under the chromatographic conditions "2.1". Using peak 6 (chlorogenic acid) as the reference peak, the relative retention time RSD was ≤0.09% and the relative peak area RSD was ≤1.99% for precision; the relative retention time RSD was ≤0.10% and the relative peak area RSD was ≤2.28% for repeatability; and the relative retention time RSD was ≤0.44% and the relative peak area RSD was ≤2.86% for stability. 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 Calibration of common peaks

[0046] Using the chromatogram of sample S1 as the reference chromatogram, with a time window width of 0.1 min, after multi-point correction of the chromatograms of 15 batches of samples, Mark peak matching was performed. A superimposed chromatogram of the control chromatogram (R) and the fingerprint chromatograms of 15 batches of Litsea cubeba slices was generated using the median method. A total of 13 common peaks were identified. (See...) Figure 1 .

[0047] 2.3.3 Selection of Reference Peak

[0048] Of the 13 common peaks identified, peak 6 (chlorogenic acid) exhibited a moderate retention time, good resolution, and a large peak area. Therefore, peak 6 was selected as the reference peak (S), and the relative peak areas and relative retention times of the 13 common peaks were calculated among different batches. The results showed that the RSD of the relative retention times of the common peaks among different batches ranged from 0.04% to 0.22%, and the RSD of the relative peak areas ranged from 18.07% to 61.45%, indicating that the retention times of the common peaks were relatively stable among different batches, but the content of chemical components varied.

[0049] 2.3.4 Identification of Common Peaks

[0050] The chromatographic peaks were analyzed by LC-MS / MS. Five components were identified by comparing the primary and secondary mass spectra with the database: peak 3 (rhodioloside), peak 6 (chlorogenic acid), peak 9 (cryptochlorogenic acid), peak 11 (magnolianin), and peak 13 (naringin). The results were then verified using reference standards. Figure 2 The mass spectrometry fragment information of each component is shown in Table 2. Figures 3-7 Simultaneously, by comparing the retention time and UV absorption of the reference standard 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 fingerprint data of 15 batches of Litsea cubeba decoction pieces were similarized using the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System" (2012 version). The results are shown in Table 3. The similarity among the 15 batches of Litsea cubeba decoction pieces ranged from 0.933 to 1.000, indicating that the similarity among the batches was high and the quality was relatively stable.

[0055] Table 315 shows the similarity evaluation results of fingerprint spectra of Litsea cubeba.

[0056]

[0057]

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

[0059] 2.4 Determination of Multiple Components in Litsea cubeba

[0060] 2.4.1 Specificity

[0061] Take blank solvent, mixed reference solution, and Litsea cubeba test solution, and analyze them according to the chromatographic conditions under section "2.1". The results are shown in [Figure 2]. Figure 9 As shown, the blank solvent did not elute at the corresponding position of the reference standard, indicating no significant interference. The four components were well separated, demonstrating the good specificity of this method.

[0062] 2.4.2 Examination of Linear Relationships

[0063] Take the mixed reference solution from section "2.2" and inject it according to the chromatographic conditions in section "2.1". Perform linear regression on the peak area (y) of the analyte against the mass concentration (x). The results show that the four components, including rhodioloside, have good linearity within their respective concentration ranges (r≥0.9997), as shown in Table 4.

[0064] Table 4. Linear equations for the four components

[0065]

[0066] 2.5.3 Precision Test

[0067] Take the mixed reference solution from section "2.2" and inject it according to the chromatographic conditions in section "2.1". After six consecutive injections, the RSD of the peak areas of the four components, including rhodioloside, was 0.64% to 0.76%, indicating that the method has good precision.

[0068] 2.5.4 Repeatability Test

[0069] The RSD of the peak areas of the six samples prepared in parallel according to section "2.2" ranged from 1.52% to 2.75%, indicating that the method has good repeatability.

[0070] 2.5.5 Stability Test

[0071] The test solution was prepared according to section “2.2” and injected at 0h, 2h, 4h, 8h, 12h and 24h. The RSD of the peak areas of the four components, including rhodioloside, was 0.45% to 1.79%, indicating that the method has good stability within 24h.

[0072] 2.5.6 Recovery Test

[0073] Take about 0.5g of Litsea cubeba powder, add each sample to the mixed reference solution at a 1:1 ratio, for a total of 6 samples, and analyze them according to the chromatographic conditions in section "2.1". The results are shown in Table 5. The average recovery rates of the four components, including rhodioloside, were 97.42% to 103.98%, and the RSDs were 1.64% to 2.58%, indicating that the method has good recovery rate.

[0074] Table 5. Recovery rates of the four components (n=6)

[0075]

[0076] 2.5.7 Content Determination

[0077] Fifteen batches of Litsea cubeba fruit were used to prepare test solutions according to section "2.2", and the chromatographic conditions in section "2.1" were used for determination. The results, calculated using the external standard method, are shown in Table 6. Based on dried samples, the contents of rhodioloside, chlorogenic acid, magnoflorin, and naringin in the 15 batches of Litsea cubeba fruit ranged from 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.

[0078] Table 6. Results of content determination 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, and methanol), different extraction methods (ultrasound and reflux), and different extraction times (0.25 h, 0.5 h, 0.75 h, and 1 h) of the test sample solution were investigated. The extraction efficiency under different conditions was compared by the content of four components, including rhodioloside, and the ratio of "total peak area / sample weight". After single-factor investigation and orthogonal analysis, the optimal extraction method was obtained: 70% methanol reflux extraction for 0.5 h. This method resulted in more complete extraction of each component and maximized spectral information.

[0082] Similarity results for 15 batches of samples were obtained using fingerprint spectral similarity evaluation software, indicating high similarity among batches. However, significant differences existed in the peak areas of various components between different batches, which may be related to natural environmental factors such as soil conditions and storage conditions. The content of four components, including rhodioloside, identified by reference standards, was determined to characterize the quality differences between medicinal materials from different origins through component content analysis.

[0083] The selected indicators for content determination comprehensively characterize the chemical information of Litsea cubeba. Rhodioloside was found to be present in high amounts and should be considered a key focus of investigation. Rhodioloside also has significant effects in the prevention and treatment of cardiovascular diseases, exhibiting anti-myocardial damage and anti-arrhythmic effects, which aligns with the clinical applications of Litsea cubeba. Chlorogenic acid can improve gut microbiota and possesses pharmacological activities such as antibacterial, antioxidant, and immunomodulatory effects. Magnoliacin can be used to treat ulcerative colitis and RA model rats. Naringin can activate Tas2rs and signal transduction in mouse airways, alleviating lung inflammation and relieving asthma. Cryptochlorogenic acid and protocatechuic aldehyde were present in low amounts in the sample and difficult to detect, therefore, they were not quantitatively analyzed.

[0084] In summary, this experiment established a rapid and stable UHPLC fingerprinting method 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 noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for determining the content of multiple components in Litsea cubeba, characterized in that, The determination was performed using UHPLC, with the following specific chromatographic conditions: a ZORBAX Eclipse Plus C18 column was used, with specifications of 100 mm column length, 2.1 mm inner diameter, and 1.8 µm particle size; acetonitrile was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B. The specific elution procedure for 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%; The content determination of the various components specifically includes rhodioloside, chlorogenic acid, magnoflorin, and naringin. The test solution was prepared by the following steps: 1 g of Litsea cubeba powder was accurately weighed and placed in a stoppered conical flask. 25 mL of 70% methanol was accurately added, the weight was recorded, and the mixture was heated under reflux for 30 min. After cooling to room temperature, the mixture was weighed again, and the weight was made up with 70% methanol. The mixture was shaken well, filtered, and the filtrate was collected to obtain the test solution.

2. The method for determining the content of multiple components in Litsea cubeba according to claim 1, characterized in that, The mixed reference solution is a precisely weighed reference solution of rhodioloside, chlorogenic acid, magnoflorin, and naringin with a mass concentration of 1 mg / mL, using methanol as the solvent.

3. A method for detecting the characteristic chromatograms of Litsea cubeba, characterized in that, The content determination method according to claim 1 is used for sample injection analysis.

4. The detection method of UHPLC characteristic chromatograms according to claim 3, characterized in that, After sample injection and analysis, the data were imported into the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", with the chromatogram of sample S1 as the reference chromatogram and a time window width of 0.1 min.

5. The detection method of UHPLC characteristic chromatograms according to claim 3, characterized in that, The UHPLC characteristic chromatogram of Litsea cubeba was obtained, and a total of 13 common peaks were identified. By comparing with the reference standard, a total of 6 components were identified: peak 1 is protocatechuic acid, peak 3 is rhodioloside, peak 6 is chlorogenic acid, peak 9 is cryptochlorogenic acid, peak 11 is magnoflorine, and peak 13 is naringin.

6. The application of the method for determining the content of multiple components in Litsea cubeba as described in claim 1 in controlling the quality of Litsea cubeba.

7. The application of the UHPLC characteristic chromatogram detection method according to claim 3 in controlling the quality of Litsea cubeba.

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