Quality control method of Cyperus rotundus based on UPLC fingerprint and content determination

Abstract

The quality control method of Cyperus rotundus based on UPLC fingerprint spectrum comprises the following steps: drying Cyperus rotundus and then crushing it with a grinder, passing it through a 60-mesh sieve of the Chinese Pharmacopoeia, accurately weighing 5.0 g of Cyperus rotundus from different origins, refluxing and extracting it three times with a 75% ethanol solution, filtering, combining, concentrating, and then extracting it with ethyl acetate, concentrating it with a rotary evaporator to obtain an ethyl acetate extract, and dissolving it with methanol to make up the volume; drawing the test solution, injecting it into an ultra-high performance liquid chromatograph, and performing chromatography on a column: Ascentis-C18 (250 mm× The fingerprint spectrum of Cyperus rotundus was recorded under the following chromatographic conditions: column temperature: 4.6mm, 5µm); mobile phase: acetonitrile (A), 0.1% formic acid aqueous solution (B); detection wavelength: 330nm; column temperature: 25℃; flow rate: 0.5mL / min; injection volume: 2μL; the obtained fingerprint spectrum data was imported into the spectrum similarity evaluation system, and the median method was used. The time window was set to 0.1min. After multi-point correction, Mark peak matching was performed, and the system generated the chromatographic overlay diagram and control fingerprint spectrum of Cyperus rotundus.

Description

Technical Field

[0001] The invention relates to a quality control method of Cypress Grove based on UPLC fingerprint and content determination. Background Art

[0002] Cypress on the stone ( Selaginella doederleinii Hieron.) belongs to the whole herb of Selaginella serrata, a plant of the genus Selaginella in the family Selaginella of the phylum Pteridophyte, and is a perennial herb, also known as large-leafed vegetable, ground arborvitae, etc. [1] . It is widely distributed in Guizhou, Sichuan, Guangdong, Guangxi and other places in my country. The drug tastes sweet and astringent, is mild in nature, and has the effects of anti-cancer hemostasis, clearing away heat and detoxification, and removing wind and dampness. It is mainly used clinically to treat various diseases such as sore throat, rheumatic pain, nasopharyngeal carcinoma, choriocarcinoma, lung cancer, cervical cancer, etc. It also has significant effects on various infectious inflammations, especially upper respiratory tract, acute tonsillitis, pneumonia, etc. [2，3] .

[0003] Modern chemical composition research shows that Selaginella mainly contains biflavonoids, alkaloids, sterols, saponins, amino acids and other components. The most significant discovery in the chemical composition research of Selaginella medicinal plants is the discovery of biflavonoid chemical components. Currently, 8 flavonoids have been isolated from Selaginella selaginella. [4-7] Among them, five kinds of biflavonoids, such as tachyphylla biflavonoids, tachyphylla biflavonoids, roposta biflavonoids, ginkgo biflavonoids, cypress biflavonoids, and rubber tree biflavonoids, have good biological activity. [8-12] .

[0004] Jiang Yongmei et al

[13] The contents of three kinds of biflavonoids were determined by high performance liquid chromatography (HPLC). Huang Jianyong et al.

[14] HPLC was used to simultaneously determine the contents of apigenin, apigenin, 2",3"-dihydro-3',3‴-bis-apigenin and 3',3‴-bis-apigenin. The five bis-flavonoids have good anti-tumor effects and are the active ingredients of Cypress. After searching, there is no literature that uses UPLC to simultaneously determine the contents of the five bis-flavonoids in Cypress. Summary of the invention

[0005] The present invention aims to solve the technical problem of establishing a content analysis method for simultaneously determining the five main active ingredients of Metasequoia glyptostroboides, Robusta sphaerocephala flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia flavonoids and Hevea brasiliensis flavonoids by using the UPLC method, and successfully applying the method to the content analysis of active ingredients in Cephalotaxus sphaerocephala medicinal materials.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The quality control method of Cyperus rotundus based on UPLC fingerprint and content determination includes the following steps:

[0008] Step 1, preparation of test solution;

[0009] After drying, the cypress was crushed in a grinder, passed through a 60-mesh sieve in the Chinese Pharmacopoeia, 5.0 g of cypress from different origins were accurately weighed, extracted three times with 75% ethanol solution, filtered, combined, concentrated, and then extracted with ethyl acetate. The ethyl acetate extract was concentrated by a rotary evaporator, and dissolved with methanol to make up the volume.

[0010] Step 2, determination method: draw the test solution, inject it into the ultra-high performance liquid chromatograph, and record the fingerprint spectrum of Shi Shangbai according to the chromatographic conditions of chromatographic column: Ascentis-C18 (250mm×4.6mm, 5µm); mobile phase: acetonitrile (A), 0.1% formic acid aqueous solution (B); detection wavelength: 330nm; column temperature: 25℃; flow rate: 0.5mL / min; injection volume: 2μL;

[0011] The obtained fingerprint data were then imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", using the median method, with the time window set to 0.1 min. After multi-point correction, Mark peak matching was performed, and the system generated a chromatographic overlay and a control fingerprint of the stone cypress medicinal material.

[0012] The present invention establishes the UPLC method for the first time to simultaneously determine the contents of 5 biflavonoid components (Aquifolium biflavonoids, Robusta biflavonoids, Ginkgo biflavonoids, Chamaecyparis biflavonoids, and Rubber tree biflavonoids) of 13 batches of Chinese herbal medicines, and investigates the precision, repeatability, stability, and sample recovery rate. The results of the methodological investigation show that the relative standard deviations of all evaluation indicators are <2.0%, indicating that under this chromatographic condition, the instrument precision is good, the sample stability is good within 24 hours, and it is suitable for the content determination of 5 biflavonoids in Aquifolium. The content determination results show that the content distribution of the 5 biflavonoids in Aquifolium presents a certain regularity, that is, Aquifolium biflavonoids> Robusta biflavonoids> Chamaecyparis biflavonoids> Rubber tree biflavonoids> Ginkgo biflavonoids. Combined with principal component analysis, the quality of Aquifolium biflavonoids can be comprehensively judged from multiple angles. There are certain differences in the contents of the 5 biflavonoids between different origins of Aquifolium, which may be related to different regions, different harvesting times, and different processing methods. The method is simple to operate and accurate in determination, and can be used for the quality analysis of the traditional Chinese medicine Ligusticum chuanxiong. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the fingerprint maps of Cypressus cycadus from different origins.

[0014] Figure 2 This is a picture of the Ishigaki cypress sample and mixed standard.

[0015] Figure 3 This is the fingerprint score diagram of Ishigamibai. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Embodiment 1:

[0018] 1. Materials and Methods

[0019] 1.1 Materials and Instruments

[0020] Waters Acquity UPLCH-Class ultra-high performance liquid chromatograph (Waters, USA); FA1004N electronic balance (Jinghai Instrument Company); KQ118 multi-frequency ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); DLSB-2L / -20 low-temperature coolant circulation pump (Shanghai Yukang Science and Education Instrument Equipment Co., Ltd.); FW177 pulverizer (Test Instrument Company).

[0021] Metasequoia glyptostroboides flavonoids (purity ≥ 99%), Robusta flavonoids (purity ≥ 96%), Ginkgo biflavonoids (purity ≥ 99%), Chamaecyparis obtusifolia flavonoids (purity ≥ 99%), and Hevea brasiliensis flavonoids (purity ≥ 99%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; methanol (HPLC) and acetonitrile (HPLC) were purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.

[0022] The medicinal materials of Cyperus sphenanthera were purchased from medicinal material markets of different origins and identified as Cyperus sphenanthera by Associate Professor Zhang from the Department of Pharmacy of Zunyi Medical University. The sample information is shown in Table 1.

[0023] Table 1 Information on samples of Cypress

[0024]

[0025] 1.2.1 Preparation of reference solution

[0026] Accurately weigh appropriate amounts of tachycarpus flavonoids, Robusta flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia, and Hevea brasiliensis flavonoids into a volumetric flask, add methanol to the scale line, and dissolve them by ultrasonication to obtain the stock solutions of each reference substance. The mass concentrations of the tachycarpus flavonoids, Robusta flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia, and Hevea brasiliensis flavonoids reference substance solutions were 0.340 mg / mL, 0.324 mg / mL, 0.344 mg / mL, 0.308 mg / mL, and 0.362 mg / mL, respectively.

[0027] 1.2.2 Preparation of test solution

[0028] After drying, the stone cypress is crushed in a grinder and passed through a 60-mesh sieve in the Chinese Pharmacopoeia. 5.0 g of stone cypress from different origins are accurately weighed, extracted three times with 75% ethanol solution under reflux, filtered, combined, concentrated, and then extracted with ethyl acetate. The ethyl acetate extract is concentrated on a rotary evaporator and dissolved in methanol to obtain the final product.

[0029] 1.2.3 Chromatographic conditions

[0030] Chromatographic column: Ascentis-C18 (250mm×4.6mm, 5µm); mobile phase: acetonitrile (A), 0.1% formic acid aqueous solution (B); detection wavelength: 330nm; column temperature: 25℃; flow rate: 0.5mL / min; injection volume: 2μL.

[0031] Table 2 UPLC chromatographic conditions

[0032]

[0033] 1.2.4 Establishment of fingerprint

[0034] The separation degree of aconitum biflavonoids in each sample spectrum is good, the peak area is high and the stability is good, so it is used as the chromatographic reference peak to calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[0035] Thirteen batches of Cyperus rotundus medicinal materials were prepared into test solutions according to the method under "1.2.2", and then measured according to the chromatographic conditions under "1.2.3", and the chromatograms of each sample were recorded. The obtained chromatographic data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", with S1 as the reference spectrum, the median method was used, the time window was set to 0.1min, and after multi-point correction, Mark peak matching was performed, and the system generated the chromatographic overlay diagram and control fingerprint spectrum of Cyperus rotundus medicinal materials.

[0036] 1.2.5 Linear investigation

[0037] Accurately pipette 1, 2, 3, 4, and 5 mL of reference solutions of Metasequoia glyptostroboides, Robusta sphaerocephala, Ginkgo biflavonoids, Chamaecyparis obtusifolia, and Hevea brasiliensis flavonoids into 5 mL volumetric flasks, add methanol to make up to volume, shake well, and obtain a series of reference solutions. According to the chromatographic conditions under "1.2.3", inject 2 μL of each solution and determine the peak area.

[0038] 1.2.6 Stability study

[0039] Take the test solution of S1 sample, and inject it at 0, 2, 4, 8, 12, and 24 hours according to the chromatographic conditions under "1.2.3", and calculate the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of each common peak of the sample is less than 0.61%, and the RSD of the relative peak area is less than 2.0%, indicating that the test solution is stable within 24 hours.

[0040] 1.2.7 Precision investigation

[0041] Take the test solution of S1 sample, and inject it 6 times continuously according to the chromatographic conditions under "1.2.3", and calculate the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of each common peak of the sample is less than 0.40%, and the RSD of the relative peak area is less than 2.0%, indicating that the instrument has good precision.

[0042] 1.2.8 Repeatability study

[0043] Take 6 portions of the powder of the S1 sample, prepare the test solution according to the method under "1.2.2", and then measure according to the chromatographic conditions under "1.2.3", and calculate the RSD of the relative peak area and relative retention time of each common peak. The results show that the RSD of the relative retention time of each common peak of the sample is less than 0.44%, and the RSD of the relative peak area is less than 2.1%, indicating that the test repeatability is good.

[0044] 1.2.9 Sample recovery test

[0045] Take the powder of S1 sample and weigh it accurately. Prepare the test solution according to the method in "1.2.2". Add a certain amount of standard solutions of Metasequoia glyptostroboides, Robusta flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia and Hevea brasiliensis flavonoids. Then measure according to the chromatographic conditions in "1.2.3" and calculate the recovery rate.

[0046] 1.2.10 Contents of five biflavonoids in Cypress from different origins

[0047] Take 5.0g of Cypress from 13 origins, determine the peak areas of 5 kinds of biflavonoids according to the method of "1.2.3", and calculate their extraction yields.

[0048] In the formula: C is the concentration of different biflavonoids in Lithospermum officinale, mg / mL; V is the volume of Lithospermum officinale extract, mL; m is the total mass of Lithospermum officinale medicinal materials, g.

[0049] 2. Results

[0050] 2.1 Establishment of fingerprint

[0051] Ten common peaks were calibrated, and five chromatographic peaks were identified by comparing the PDA spectra and retention times with those of the reference substances, namely, flavonoids of Aquilegia sutchuenensis (peak 1), flavonoids of Robusta sutchuenensis (peak 2), flavonoids of Ginkgo biflavonoids (peak 3), flavonoids of Chamaecyparis obtusifolia (peak 9), and flavonoids of Hevea brasiliensis (peak 10). Figure 1 , Figure 2 .

[0052] 2.2 Similarity Analysis

[0053] The similarity evaluation of 13 batches of Cyperus rotundus medicinal materials was performed with the reference spectrum, and the similarity evaluation results are shown in Table 3. The similarity results of each batch of Cyperus rotundus medicinal materials were 0.989~0.999, and the similarities were all higher than 0.98, indicating that the established fingerprint method can be used for the overall quality control of Cyperus rotundus medicinal materials.

[0054] Table 3 Similarity evaluation results

[0055]

[0056] 2.3 Linear investigation

[0057] With the peak area of ​​the reference substance as the ordinate and the concentration as the abscissa, the linear regression equations, correlation coefficients and linear ranges of Metasequoia glyptostroboides, Robusta sphaerocephala flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia flavonoids and Hevea brasiliensis flavonoids were obtained. As shown in Table 4, the correlation coefficients were all >0.999, indicating that the five flavonoids had a good linear relationship in the concentration ranges of 3.342-250.32, 1.534-15.34, 1.694-43.36, 1.644-30.48 and 3.016-124.92 μg / mL, respectively.

[0058] Table 4 Linear relationships of 5 biflavonoid standards

[0059]

[0060] 2.4 Precision, stability and repeatability test results

[0061] According to the test results, the RSDs of Aquilegia sutchuenensis flavonoids, Robusta flavonoids, Ginkgo biflavonoids, Chamaecyparis obtusifolia flavonoids, and Hevea brasiliensis flavonoids were calculated (n=6). The results are shown in Table 5. As can be seen from Table 5, their RSDs are all <2.0%, indicating that the precision of the instrument is good, the test solution is stable within 8 hours, and the method has good stability.

[0062] Table 5 Test results of precision, stability and repeatability of 5 kinds of biflavonoids

[0063]

[0064] 2.5 Sample recovery test

[0065] Results The average recoveries of aconitum flavonoids, roposta flavonoids, ginkgo flavonoids, cypress flavonoids, and rubber tree flavonoids were 98.26%, 99.13%, 98.56%, 97.88%, and 98.66%, respectively.

[0066] Contents of five biflavonoids in Cypress from 2.613 origins

[0067] The contents of flavonoids of schizonepeta (X1), flavonoids of Robusta (X2), flavonoids of ginkgo (X3), flavonoids of Chamaecyparis obtusifolia (X4), and flavonoids of rubber tree (X5) in each sample were calculated, and descriptive statistical analysis was performed using SPSS18.0 to obtain the minimum, maximum, mean, and standard deviation of the contents of each component in the samples of Cypress. The results are shown in Table 6. The results show that the content of flavonoids of schizonepeta is 0.096%~0.309%, the content of flavonoids of Robusta is 0.031%~0.135%, the content of flavonoids of ginkgo is 0.005%~0.029%, the content of flavonoids of Chamaecyparis obtusifolia is 0.008%~0.069%, and the content of flavonoids of rubber tree is 0.003%~0.043%. The results show that the contents of the five flavonoid compounds in Cypress from different origins are somewhat different, which may be related to different regions, different harvesting times, and different processing methods.

[0068] Table 6 Determination of the content of Cypress in different origins

[0069]

[0070] 2.7 Principal Component Analysis

[0071] The original data were standardized by factor analysis in SPSS18.0 software, and the characteristic roots and contribution rates of the principal components were the basis for selecting the principal components. After calculation, the characteristic values ​​of the first three components A1, A2 and A3 were greater than 1, i.e. 4.939, 1.770, 1.340, and the cumulative contribution rate to the total variance reached 80.481%, so A1, A2 and A3 were selected as the first, second and third principal components, and the results are shown in Table 7.

[0072] Table 7 Principal component eigenvalues ​​and contribution rates

[0073]

[0074] The rotated common factor loading matrix is ​​shown in Table 8. It can be concluded that chromatographic peaks 1, 2, 3, 4, 5, 7, 8, 9, and 10 have obvious positive loads in principal component 1, indicating that the information of the first principal component mainly comes from these 9 peaks, while the other chromatographic peaks have relatively little influence on the first principal component. The information of the second principal component mainly comes from chromatographic peaks 4, 5, 6, 7, 8, and 10, while the other chromatographic peaks have relatively little influence on the second principal component. The information of the second principal component mainly comes from chromatographic peaks 2, 3, 6, 7, 9, and 10, while the other chromatographic peaks have relatively little influence on the second principal component.

[0075] Table 8 Common factor loading matrix after rotation

[0076]

[0077] The principal component analysis of the common factor loading matrix was performed, and the two-dimensional projection was obtained with the first and second principal components as variables. Figure 3 It can be seen that the internal relationships of the test samples from different origins can be well expressed, and then the classification between different samples can be achieved. From the figure, it can be seen that 10 batches of stone cypress medicinal materials from different origins are divided into 6 categories. The first category is the stone cypress produced in Fujian, the second category is the stone cypress produced in Hainan, the third category is the stone cypress produced in Hubei, the fourth category is the stone cypress produced in Guizhou, the fifth category is the stone cypress produced in Yunnan, and the sixth category is the stone cypress produced in Guangxi.

[0078] 3. Conclusion

[0079] In this study, the UPLC method was established for the first time to simultaneously determine the contents of five biflavonoid components (Aquifolium sutchuenensis biflavonoids, Robusta biflavonoids, Ginkgo biflavonoids, Chamaecyparis biflavonoids, and Hevea biflavonoids) in 13 batches of Chinese herbal medicine, and the precision, repeatability, stability, and sample recovery were investigated. The results of the methodological investigation showed that the relative standard deviations of all evaluation indicators were <2.0%, indicating that under this chromatographic condition, the instrument precision was good and the sample stability was good within 24 hours, which was suitable for the determination of the five biflavonoids in Aquifolium sutchuenensis. The content determination results showed that the content distribution of the five biflavonoids in Aquifolium sutchuenensis showed a certain regularity, that is, Aquifolium sutchuenensis biflavonoids> Robusta biflavonoids> Chamaecyparis biflavonoids> Hevea biflavonoids> Ginkgo biflavonoids. Combined with principal component analysis, the quality of Aquifolium sutchuenensis biflavonoids can be comprehensively judged from multiple angles. There are certain differences in the contents of the five biflavonoids in Aquifolium sutchuenensis from different origins, which may be related to different regions, different harvesting times, and different processing methods. The method is simple to operate and accurate in determination, and can be used for the quality analysis of the traditional Chinese medicine Ligusticum chuanxiong.

[0080] References

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Claims

1. A quality control method for Cypress sphaerocarpa based on UPLC fingerprint and content determination, characterized in that: It includes the following determination methods: Step 1: Crush the Cypress, pass it through a 60-mesh sieve, accurately weigh 5.0 g of Cypress from different origins, extract it three times with 75% ethanol reflux, filter, combine, concentrate, extract it with ethyl acetate, concentrate it to obtain an extract, dissolve it with methanol to make up the volume, and obtain the test solution; Step 2: Accurately weigh appropriate amounts of flavonoids of Metasequoia glyptostroboides, flavonoids of Robusta, flavonoids of Ginkgo biloba, flavonoids of Chamaecyparis obtusifolia, and flavonoids of Hevea brasiliensis, put them into a volumetric flask, add methanol to the scale, dissolve them by ultrasonication, and obtain the stock solutions of the reference substances; Step 3: Determine by ultra-high performance liquid chromatography, using the chromatographic column: Ascentis-C18, 250 mm × 4.6 mm, 5 µm; The liquid phase data were obtained by chromatographic determination under the following conditions: mobile phase: acetonitrile for phase A, 0.1% formic acid aqueous solution for phase B; detection wavelength: 330 nm; column temperature: 25 °C; flow rate: 0.5 mL / min; injection volume: 2 μL; The gradient elution program of the mobile phase was: Step 4: The obtained data is then imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for analysis. The similarity between the test sample fingerprint and the control fingerprint is calculated, and the similarity shall not be less than 0.90; Step 5: Calculate the contents of flavonoids of Metasequoia ginkgo, flavonoids of Robusta, flavonoids of Ginkgo biflavonoids, flavonoids of Chamaecyparis obtusifolia, and flavonoids of Hevea brasiliensis in each sample; Step six: by calculation, the total content of flavonoids of cypress, flavonoids of Robusta, flavonoids of ginkgo, flavonoids of cypress and flavonoids of rubber tree is not less than 0.1%.

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