A method for simultaneous determination of multiple chemical components in Cymbidium heterophyllum

Abstract

The present invention discloses a method for simultaneously determining multiple chemical components in Cymbidium heterophyllum, and relates to the technical field of separation and analysis of natural products. Ultra-high performance liquid chromatography is used to simultaneously determine the chemical components of Cymbidium heterophyllum, and the chemical components include at least neochlorogenic acid, cryptochlorogenic acid, and diosgenin-7-O-glucuronide; the detection conditions of ultra-high performance liquid chromatography include: chromatographic column stationary phase: diisobutyl group is bonded to the silica gel surface through siloxane; mobile phase A is selected from methanol or acid-containing methanol, and mobile phase B is selected from water or acid-containing aqueous solution; mobile phase A and mobile phase B are used as a mixed mobile phase for gradient elution. The present invention establishes a UPLC content determination method for multiple chemical components in Cymbidium heterophyllum, which is simple to operate, has reliable results, and good repeatability, and can provide a certain reference for the determination of the content of chemical components in Cymbidium heterophyllum and the improvement of existing quality standards.

Description

Technical Field

[0001] The invention relates to the technical field of natural product separation and analysis, in particular to a method for simultaneously determining multiple chemical components in Cymbidium heterophyllum. Background Art

[0002] Dracocephalum heterophyllum, also known as "white flower branch flower," is the dried aerial part of the perennial herbaceous plant Dracocephalumheterophyllum Benth. Its Tibetan name, "Jizi Qingbao," is included in the 1995 edition of the Ministry of Health's Drug Standards. The entire plant is used as medicine and is a common medicinal herb in ethnic minority areas. It grows primarily on hillside grasslands and riverbanks at altitudes of 2,000-5,000 meters. It is distributed in Qinghai, Tibet, Gansu, northwestern Sichuan, and Xinjiang. The "Compendium of Traditional Chinese Medicine" records that Dracocephalum heterophyllum has liver-calming and heat-clearing properties, and is used to treat icteric hepatitis, gingival swelling and pain caused by liver fire, and oral ulcers. The Tibetan medicine classic "Jingzhu Bencao" also records that Dracocephalum heterophyllum can treat oral and dental diseases. Modern research has revealed that Dracocephalum heterophyllum contains a variety of chemical components, including flavonoids, volatile oils, alkaloids, triterpenes, and steroids, exhibiting antioxidant, anti-inflammatory, antitussive, anti-asthmatic, liver-protective, and cardiovascular protective properties.

[0003] As a formula herb, Cynanchum heterophyllum is widely used in classic Tibetan medicine formulas such as Seventy Flavors Songshi Pills, Three Flavors Ganlu Powder, and Shenhe Pingchuan Capsules. However, its chemical composition and quality control indicators are lacking. Currently, the only standard for Cynanchum heterophyllum is a ministerial-issued standard, which fails to effectively control the quality of the medicinal material. Studies have shown that Cynanchum heterophyllum contains chemical components such as luteolin, rosmarinic acid, oleanolic acid, methyl ursula, chlorogenic acid, and luteolin. Currently, only literature reports have simultaneously determined the content of two or four of these components. Summary of the Invention

[0004] The object of the present invention is to provide a method for the simultaneous determination of multiple chemical components in Cymbidium heterophyllum. A method for the simultaneous determination of nine chemical components, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, verbascoside, luteolin, rosmarinic acid, diosmetin-7-O-glucuronide, quercetin, and luteolin, was established using ultra-performance liquid chromatography (UPLC). The method was applied to the determination of the chemical components in Cymbidium heterophyllum from different origins, in order to provide a certain reference for the quality standard research and comprehensive utilization of the chemical components in Cymbidium heterophyllum.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for simultaneously determining multiple chemical components in Cymbidium heterophyllum, using ultra-high performance liquid chromatography to determine the chemical components of Cymbidium heterophyllum, wherein the chemical components include at least neochlorogenic acid, cryptochlorogenic acid, and diosmetin-7-O-glucuronide;

[0007] ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) detection conditions include:

[0008] Column stationary phase: diisobutyl group is bonded to the silica surface via siloxane;

[0009] Mobile phase A is selected from methanol or methanol containing acid, and mobile phase B is selected from water or aqueous solution containing acid;

[0010] Gradient elution was used, and the gradient elution program for the first 60 min was as follows:

[0011] 0-15min, 25%-25% A; 15-25min, 25%-35% A; 25-26min, 35%-38% A; 26-40min, 38%-45% A; 40-42min, 45%-45% A; 42-50min, 45%-60% A; 50-60min, 60%-90% A.

[0012] Furthermore, the chromatographic column is Agilent ZORBOX SB-C18.

[0013] Furthermore, the mobile phase A is methanol containing 0.1-0.3% formic acid, and the mobile phase B is a 0.1-0.3% formic acid aqueous solution.

[0014] Furthermore, the gradient elution program further includes: 60-62 min, 90%-100% A; 62-65 min, 100%-100% A.

[0015] Furthermore, the ultra-high performance liquid chromatography conditions further include one or more of the following i to iv:

[0016] i Column specifications: 4.6×100mm, 1-5μm;

[0017] ii. Column temperature: 30-40°C;

[0018] iii. Flow rate: 0.1-0.5 mL min -1 ;

[0019] iv detection wavelength: 210-360nm;

[0020] Furthermore, the ultra-high performance liquid chromatography conditions further include one or more of the following i to iv:

[0021] i Column specifications: 4.6×100 mm, 1.8 μm;

[0022] ii Column temperature: 35°C;

[0023] iii. Flow rate: 0.3 mL min -1 ;

[0024] IV detection wavelength: 340nm

[0025] Furthermore, the chemical components also include chlorogenic acid, verbascoside, luteolin, rosmarinic acid, quercetin or luteolin.

[0026] Neochlorogenic acid has multiple biological activities such as antioxidant, anti-inflammatory, antibacterial and antiviral. In the medical field, neochlorogenic acid is widely used in the research and development of anti-tumor, anti-cancer, anti-diabetic and other drugs; verbascoside has multiple physiological functions such as antioxidant, anti-inflammatory response, neuroprotection, immunomodulation, anti-tumor and wound healing, and has made certain progress in neuroprotection in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease; cryptochlorogenic acid has anti-platelet aggregation, antibacterial and anti-inflammatory effects; quercetin has antioxidant, anti-inflammatory, anti-cancer, cardiovascular protection and anti-allergic effects; dioscorea ginsenoside-7-O-glucuronide, as the main component of the effective part of dianthus, may have the pharmacological effects of the effective part of dianthus against myocardial ischemia and atherosclerosis.

[0027] The present invention analyzes and identifies neochlorogenic acid, cryptochlorogenic acid and diosmetin-7-O-glucuronide from Cymbidium heterophyllum for the first time. The determination of the above components can more intuitively reflect the medicinal material quality of Cymbidium heterophyllum.

[0028] Furthermore, the preparation method of the test solution is as follows: the sample to be tested is extracted with 70-80% methanol, filtered, and the filtrate is filtered through a membrane to prepare the test solution.

[0029] Furthermore, the solvent is 75% methanol solution.

[0030] The extraction method can be conventional methods such as reflux, immersion, and ultrasound.

[0031] Using a membrane filter is a common procedure before liquid phase injection to prevent clogging of the chromatographic column. The pore size of the conventional membrane filter is 0.45 μm, but smaller pore sizes can also achieve the same goal.

[0032] The present invention adopts UPLC content determination method, uses ZORBOX SB-C18 chromatographic column, uses 0.1% formic acid-methanol (A)-0.1% formic acid aqueous solution (B) as mobile phase, the specification of chromatographic column is 4.6×100mm, 1.8μm, and the flow rate is 0.3mL·min -1, detection wavelength of 340 nm, column temperature of 35°C, injection volume of 2 μL, under the conditions of gradient elution: gradient elution (0-15 min, 25%-25% A; 15-25 min, 25%-35% A; 25-26 min, 35%-38% A; 26-40 min, 38%-45% A; 40-42 min, 45%-45% A; 42-50 min, 45%-60% A; 50-60 min, 60%-90% A; 60-62 min, 90%-100% A; 62-65 min, 100%-100% A), the separation of the target chromatographic peaks was good. The method is simple to operate, the results are reliable, and the repeatability is good.

[0033] The solution concentrations or percentages described in the present invention are all volume ratios.

[0034] The beneficial effects of the present invention are:

[0035] The present invention establishes a UPLC content determination method for 9 chemical components in Cymbidium heterophyllum. The method is simple to operate, has reliable results, and has good reproducibility. It can provide a certain reference for the determination of the content of chemical components in Cymbidium heterophyllum and the improvement of existing quality standards. It is of great significance for the quality control of Cymbidium heterophyllum and the formulation of quality standards for related medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 UPLC chromatogram of mixed reference solution;

[0037] Figure 2 UPLC chromatogram obtained under elution program No. 1;

[0038] Figure 3 UPLC chromatogram obtained under elution program No. 2;

[0039] Figure 4 UPLC chromatogram obtained under elution program No. 3;

[0040] Figure 5 UPLC chromatogram of the test solution (obtained under elution program No. 4);

[0041] Reference numerals: 1-neochlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-verbascoside, 5-luteolin, 6-rosmarinic acid, 7-diosmin-7-O-glucuronide, 8-quercetin, 9-luteolin. DETAILED DESCRIPTION

[0042] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described here are only part of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0043] Example 1

[0044] 1. Instruments and test drugs

[0045] An Aglient-G6500 ultra-high performance liquid chromatograph (Agilent, USA) was used. A JP100-S ultrasonic cleaner was used (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.). Reference standards for neochlorogenic acid, cryptochlorogenic acid, and diosmin-7-O-glucuronide were purchased from Beijing Beina Chuanglian Biotechnology Research Institute; reference standards for verbascoside, luteolin, rosmarinic acid, luteolin, and quercetin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and a reference standard for chlorogenic acid was purchased from Beijing North Weiye Metrology Technology Research Institute. The purity of all purchased standards was greater than 98%. Water was purified, methanol was chromatographic grade, and all other reagents were analytical grade.

[0046] The samples of Orchidaceae were collected from various regions of Qinghai Province from July to August 2023. The sample information is shown in Table 1. The whole plant was naturally dried and then crushed.

[0047] Table 1 Sample information of Cymbidium heterophyllum

[0048]

[0049] 2. Methods and Results

[0050] 2.1 Chromatographic conditions

[0051] A ZORBOX SB-C18 column (4.6 × 100 mm, 1.8 μm) was used with a mobile phase consisting of 0.1% formic acid in methanol (A)-0.1% formic acid in water (B) in a gradient elution mode (0-15 min, 25%-25% A; 15-25 min, 25%-35% A; 25-26 min, 35%-38% A; 26-40 min, 38%-45% A; 40-42 min, 45%-45% A; 42-50 min, 45%-60% A; 50-60 min, 60%-90% A; 60-62 min, 90%-100% A; 62-65 min, 100%-100% A) at a flow rate of 0.3 mL min. -1 , detection wavelength 340nm, column temperature 35℃, injection volume 2μL. Take the mixed reference substance and test solution and inject them under the above chromatographic conditions. The UPLC chromatogram of the mixed reference substance is as follows Figure 1As shown, the UPLC chromatogram of the test solution is as follows Figure 5 As shown by Figure 5 It can be seen that the separation of the target chromatographic peaks is good.

[0052] 2.2 Solution preparation

[0053] Nine standard reference substances, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, verbascoside, luteolin, rosmarinic acid, diosmin-7-O-glucuronide, quercetin, and luteolin, were accurately weighed and prepared with methanol to concentrations of 0.030, 0.180, 0.030, 0.090, 0.110, 0.200, 0.070, 0.070, and 0.030 mg mL, respectively. -1 Mixed reference stock solution.

[0054] Accurately weigh 1.000 g of the whole plant sample powder of Orchidaceae and place it in a conical flask, accurately add 10 mL of 75% methanol, weigh the weight, ultrasonicate for 30 minutes, cool to room temperature, and make up the missing weight with 75% methanol, shake well, filter, and pass the filtrate through a 0.45 μm filter membrane to obtain the test solution.

[0055] 2.3 Linear relationship investigation

[0056] 0.2, 0.5, 2, 4, and 6 μL of the mixed reference solution were drawn respectively and injected according to the chromatographic conditions in "2.1". The concentration represented by the reference substance in the injected sample (μg·mL) was used. -1 ) as the abscissa and the peak area as the ordinate for linear regression, and the regression equations and linear parameters of each component were obtained as shown in Table 2.

[0057] Table 2 Linear regression equation, correlation coefficient, and linear range of 9 chemical components

[0058]

[0059] 2.4 Precision, repeatability and stability tests

[0060] The mixed reference solution was injected continuously 6 times within 1 day according to the chromatographic conditions in "2.1". The final calculated RSD values ​​of the peak areas of each reference substance were 1.89%, 0.90%, 1.40%, 0.96%, 1.69%, 1.04%, 1.07%, 0.86% and 2.44% (n=6), respectively, indicating that the instrument had good precision.

[0061] Take S3 heterophylla sample and prepare 6 test solutions in parallel according to the method in "2.2". The samples were injected and determined according to the chromatographic conditions in "1.2.1". The final calculated peak area RSDs of the 9 chemical components were 2.61%, 1.02%, 0.62%, 1.52%, 1.90%, 0.38%, 0.84%, 2.71%, and 1.12% (n=6), respectively, indicating that the method has good repeatability.

[0062] The S3 heterophylla test solution was injected according to the chromatographic conditions of "1.2.1" at 0, 2, 4, 6, 8 and 12 h, and the final calculated peak area RSDs of the nine chemical components were 0.62%, 0.21%, 3.61%, 0.16%, 0.93%, 0.32%, 0.31%, 1.53% and 0.46% (n=6), respectively, indicating that the test solution had good stability within 12 h.

[0063] 2.5 Sample recovery test

[0064] Accurately weigh 1.000 g of S3 heterophylla whole plant sample powder, accurately add chlorogenic acid (0.020 mg mL) prepared with 75% methanol (v / v) -1 ), chlorogenic acid (0.130 mg mL -1 ), cryptochlorogenic acid (0.050 mg mL -1 ), verbascoside (0.100 mg mL -1 ), luteolin (0.080 mg mL -1 ), rosmarinic acid (0.180 mg mL -1 ), diosmin-7-O-glucuronide (0.070 mg mL -1 ), quercetin (0.030 mg mL -1 ), luteolin (0.050 mg mL -1 ) Mix 10 mL of the reference solution and prepare the test solution according to the method in "2.2". Inject the samples according to the chromatographic conditions in "2.1" and calculate the sample recovery. The average sample recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, verbascoside, luteolin, rosmarinic acid, diosmin-7-O-glucuronide, quercetin, and luteolin were 100.9%, 93.18%, 103.3%, 112.61%, 90.25%, 96.67%, 97.02%, 101.11%, and 97.07%, respectively. The corresponding RSD values ​​were all less than 1.25% (n=6).

[0065] 2.6 Determination of sample content

[0066] Ten batches of whole-plant samples of Cymbidium heterophyllum were weighed from nine production areas. Test solutions were prepared according to 2.2 and assayed according to the chromatographic conditions in 2.1. The contents of nine chemical components in Cymbidium heterophyllum were calculated. The assay results are shown in Table 3.

[0067] Table 3 Determination of chemical components of Cymbidium heterophyllum from different origins (mg·g -1 , n=3)

[0068]

[0069] The results in Table 3 show that the rosmarinic acid content was the highest in the 10 samples of Cymbidium heterophyllum from different origins, followed by chlorogenic acid. Comparison of the chemical components of Cymbidium heterophyllum from different origins revealed that the differences in species were small, but the differences in content were more obvious. Among them, the difference in rosmarinic acid content was the largest, with the rosmarinic acid content in the Ledu S4 sample reaching 13.01 mg·g -1 , while the rosmarinic acid content in Gonghe S1 sample was only 3.455 mg·g -1 The content of chlorogenic acid in Tongde S2 sample was the highest, reaching 3.043 mg·g -1 , while the chlorogenic acid content in Minhe S5 sample was only 0.009 mg·g -1 The content of neochlorogenic acid in Tongde S2 sample was the highest, at 0.464 mg·g -1 , while in Nangqian S10 sample it was only 0.015 mg·g -1 The content of cryptochlorogenic acid in Tongde S2 sample was the highest, at 0.864 mg·g -1 , while the content in Nangqian S10 sample was only 0.119 mg·g -1 The concentration of verbascoside in Minhe S5 sample reached 1.029 mg·g -1 , while in Nangqian S10 sample it was only 0.016 mg·g -1 The content of luteolin was the highest in Minhe S5 sample, reaching 1.052 mg·g -1 , while the content in Gonghe S1 sample was the lowest, only 0.170 mg·g -1 The content of diosmin-7-O-glucuronide in Minhe S5 sample reached 1.070 mg·g -1 , while in the Menyuan S8 sample it was only 0.051 mg·g -1 The quercetin content in Minhe S5 sample was as high as 1.822 mg·g -1 , while in Gonghe S1 sample it was only 0.019 mg·g -1 The highest content of luteolin was found in the Menyuan S9 sample, which was 0.141 mg·g -1 , while the content in Gonghe S1 sample was the lowest, only 0.041 mg·g -1The differences in the contents of chemical components in C. heterophylla indicate that different habitats determine the contents of the components.

[0070] 2 Conclusion

[0071] In terms of wavelength selection, the analysis method for the nine chemical components established in the present invention uses detection wavelengths such as 254, 280, 340, and 360 nm for analysis. The maximum absorption wavelengths of the nine selected chemical components are different. After comprehensive consideration, 340 nm, which has a large absorption, was selected as the detection wavelength.

[0072] Tan Jinhua et al. proposed in the draft of the quality standard for Cymbidium heterophyllum that the rosmarinic acid content in the dried Cymbidium heterophyllum should not be less than 0.5% (i.e. 5 mg·g -1 ), the experiment used 10 batches of samples, except for the rosmarinic acid content of the S1 batch sample of Gonghe, which was 3.455 mg·g -1 , the rest met the standards; Zhao Lin et al. suggested that the content of luteolin in the dried product of Heterophyllum should not be less than 0.03% (i.e. 0.3 mg·g -1 ), the contents of luteolin in the 10 samples were 0.170 mg·g -1 , 0.233mg·g -1 , 0.282mg·g -1 The luteolin content in the remaining seven batches of C. heterophylla met the standard. Environmental factors are important factors affecting plant growth and the accumulation of secondary metabolites. It is speculated that the differences in the chemical component contents among the 10 batches of samples are mainly caused by the large differences in the altitude and habitat of their growth sites.

[0073] A UPLC method for the determination of nine chemical components in Cymbidium heterophyllum was invented and established. The method is simple to operate, has reliable results and good reproducibility, and can provide a certain reference for the determination of the content of chemical components in Cymbidium heterophyllum and the improvement of existing quality standards.

[0074] Example 2

[0075] An exploratory experiment was conducted to determine the 9 chemical components in Cymbidium heterophyllum by ultra-high performance liquid chromatography (UPLC) conditions: a ZORBOX SB-C18 column was used with a mobile phase of 0.1% formic acid-methanol (A)-0.1% formic acid aqueous solution (B). The column size was 4.6 × 100 mm, 1.8 μm, and the flow rate was 0.3 mL min. -1 , under the conditions of detection wavelength of 340nm, column temperature of 35℃, and injection volume of 2μL, the elution program parameters of ultra high performance liquid chromatography were adjusted, and the test solution of Cymbidium orchid (combined with S1) was measured respectively. No. 1-3 were comparative elution programs, and No. 4 was the elution program of the present invention. The specific parameter adjustments are shown in Table 4.

[0076] Table 4 Exploration of ULHPLC elution program parameters

[0077] Serial number Retention time (minutes) Mobile phase (volume %) Corresponding spectrum 1 0-10-15-25-35-40-50-55 10-10-25-40-60-80-100-100％A Figure 2 2 0-10-20-30-40-50-55 25-25-35-50-70-100-100％A Figure 3 3 0-10-20-21-35-37-45-55-57-60 25-25-35-38-45-45-60-90-100-100％A Figure 4 4 0-15-25-26-40-42-50-60-62-65 25-25-35-38-45-45-60-90-100-100％A Figure 5

[0078] From Table 4, we can see that Figure 2-Figure 4 In the samples, peak 7 was co-eluted with the previous peak to form a peak, which did not achieve the purpose of separation. Figure 5 Peak 7 in the image is baseline-resolved from the preceding peaks.

[0079] Figure 2 and Figure 4 Peak 4 in the figure also co-eluted with the previous chromatographic peak. Figure 5 Peak 4 in the figure is significantly separated from the previous peaks and can be used for quantitative analysis.

Claims

1. A method for simultaneously determining multiple chemical components in Cymbidium heterophyllum, characterized in that: The chemical components of the blue lily were determined by ultra-high performance liquid chromatography, and the chemical components at least included neochlorogenic acid, cryptochlorogenic acid, and diosmetin-7-O-glucuronide; ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) detection conditions include: Mobile phase A is selected from methanol or methanol containing acid, and mobile phase B is selected from water or aqueous solution containing acid; Gradient elution was used, and the gradient elution program for the first 60 min was as follows: 0-15 min, 25%-25% A; 15-25 min, 25%-35% A; 25-26 min, 35%-38% A; 26-40 min, 38%-45% A; 40-42 min, 45%-45% A; 42-50 min, 45%-60% A; 50-60 min, 60%-90% A; The chromatographic column was Agilent ZORBAX SB-C18; The mobile phase A is methanol containing 0.1-0.3% formic acid, and the mobile phase B is a 0.1-0.3% formic acid aqueous solution; Preparation method of test solution: extract the sample to be tested with 70-80% methanol, filter, and filter the filtrate through a filter membrane to prepare the test solution; The specifications of the chromatographic column of the ultra-high performance liquid chromatography are: 4.6×100 mm, 1.8 μm, and the detection wavelength is: 340 nm.

2. The method according to claim 1, characterized in that The gradient elution program further includes: 60-62 min, 90%-100% A; 62-65 min, 100%-100% A.

3. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions further include one or more of the following ii to iii: ii. Column temperature: 30-40°C; iii. Flow rate: 0.1-0.3 mL min -1 .

4. The method according to claim 3, characterized in that The ultra-high performance liquid chromatography conditions further include one or more of the following ii to iii: ii Column temperature: 35°C; iii. Flow rate: 0.3 mL min -1 .

5. The method according to claim 1, wherein The chemical components also include chlorogenic acid, verbascoside, rosmarinic acid, quercetin or luteolin.

6. The method according to claim 1, characterized in that In the preparation of the test solution, the concentration of methanol is 75%.

Images