TY - JOUR
AU1 - Feng, Yufei
AU2 - Teng, Lin
AU3 - Wang, Yanli
AU4 - Gao, Yanyu
AU5 - Ma, Yuxuan
AU6 - Zhou, Haichun
AU7 - Cai, Guofeng
AU8 - Li, Ji
AB - Abstract This research explored the HPLC fingerprints of Hypericum attenuatum Choisy, which has anti-arrhythmic activity. HPLC was adopted to perform a determination of chemical fingerprints of H. attenuatum specimens acquired through seven distinct sources. The anti-arrhythmic activity of each H. attenuatum sample was obtained through pharmacodynamics experiments in animals. A regression analysis and correlation analysis were utilized to calculate the relationship of the peak and pharmacological effectiveness with the identified peak. Peaks numbered 5, 7, 13 and 14 in the fingerprint were regarded as the likely anti-arrhythmic agents. The fingerprint was compared with reference standards for identification of the correlative peaks. Liquid chromatography–time-of-flight–mass spectrometry was applied to identify its structure. As a consequence, a universal model was established for the utilization of HPLC to investigate anti-arrhythmic activity and the spectrum-effect relationship among H. attenuatum. This model is available for the discovery of the major bioactive constituents of Hypericum. Introduction Hypericum attenuatum Choisy is a commonly used Chinese herbal medicine that has a history spanning more than 2,400 years. It is commonly used in southern China to treat burning injuries and hyperhidrosis. In the northeast border region (Lvshun, Dalian), the whole herb is used as an internal medicine to relieve heat or treat menstrual cramps (Jianchang County). In the northeast region, people often use the whole herb instead of tea to treat heart disease (1). Recently, an increasing interest has been seen in H. attenuatum because of its wide pharmacological actions ranging from anti-myocardial ischemia effects, anti-tumor activity, anti-depressant effects and anti-diabetic actions (2–6). H. attenuatum comprises numerous important medical phytoconstituents including flavonoids, volatile oils and phloroglucinol derivatives. Therefore, H. attenuatum is considered to be of great medicinal value for further rational development and utilization of plant resources. Worldwide, 17 million people die from heart disease every year. In China, one person dies of cardiovascular disease every 20 s (7). For a long time, domestic and foreign scholars have been committed to seeking effective measures to treat arrhythmia, and drug treatment has been the main method to treat arrhythmia. At present, the Western pharmaceuticals that are commonly used to treat arrhythmia include Na+ channel blockers, β-glucoepinephrine receptor blockers, Ca2+ channel blockers and action potential prolonging drugs. Although these pharmaceuticals are widely used in clinical practice, they all have different degrees of side effects. In contrast, the treatment of arrhythmia symptoms by Chinese herbal medicine shows many advantages, thus it has attracted extensive attention due to its unique curative effect, limited toxic side effects and huge development potential. Therefore, further research into this Chinese herbal medicine is an effective way to develop arrhythmia drugs. H. attenuatum can increase the activities of Na+–K+–ATP enzymes as well as Ca2+–Mg 2+–ATP enzymes in the myocardial cell membrane of tachyarrhythmia rats, and has significant anti-arrhythmic effects (8). Previous studies have shown that the total flavonoids in H. attenuatum could postpone calcium chloride induced heart disease in mice, the occurrence time of arrhythmia and the duration of arrhythmia. In addition, the incidence of ventricular fibrillation induced by chloroform in mice was shortened (9). Total flavonoids in H. attenuatum have a positive protective effect on arrhythmia in mice induced by calcium chloride and chloroform (9); total flavonoids are important active sites for anti-arrhythmia. Nevertheless, the systematic quality evaluation of H. attenuatum as well as the main active ingredients in the anti-arrhythmic actions are still unknown. In biological activity, pharmacology or pharmacological activity describes the advantageous or unfavorable effects of Traditional Chinese Medicine (TCM) on living organisms. A number of TCMs contain multiple chemical compounds, exerting various functions such as the features of multiple channels, multiple levels and multiple targets. The concept of the spectrum-effect (fingerprint-effect) relationship was proposed by Li et al. (10) in 2002. To explore the correlation of fingerprints with pharmacological effectiveness, the spectrum-effect relationship is usually employed for clarification of the pharmacodynamic foundation for the effectiveness of a TCM (11–15). In addition, the approach can be used for the control of TCM quality. As a result, the spectrum-effect relationship is an important component of TCM research. Through the establishment of a fingerprint, the pharmacodynamic assessment and information processing serve as significant factors for studies on the spectrum-effect relationship. Because of the severe non-linearity between ingredients in the fingerprint chromatogram and the efficacy of TCMs, the application of various methods, including clustering analysis, correlation analysis, gray relational analysis and multiple linear regression, are used to explain the relationships (16–18). The chemical fingerprints of various TCMs are attainable using HPLC (19, 20), HPTLC (21), GC–MS (22), IR (23), HPCE (24) and other new technology (25–27). Liquid chromatography–mass spectrometry (LC–MS) detection makes it possible to obtain structural information, distinguish between compounds with the same molecular weights, and perform an identification of tentative compounds, in view of the unavailability of standard reference compounds. LC–MS is an important tool to map the chemical profiles of herbal medicines (28). Consequently, an investigation of the spectrum-effect relationships between H. attenuatum was performed to screen the effective compounds possessing anti-arrhythmic activities. With the application of liquid chromatography–time-of-flight–mass spectrometry (LC–TOF–MS), determination of the chemical structures was performed for screening effective ingredients. The aim of this research was to reveal the effective compounds of H. attenuatum for anti-arrhythmic therapies and the quality control of H. attenuatum, which also offers an essential model to screen effective ingredients in herbal medicine. Experimental Animals and materials Seven samples of H. attenuatum were bought or collected from different locations including Hailin City, Heilongjiang Province (July 2015); Jingpo Lake scenic area in Mudanjiang City, Heilongjiang Province (July 2015); Zuojia Town, Jilin City (July 2014), Jilin Province; Mishan City, Heilongjiang Province; Harbin City, Heilongjiang Province (July 2015); Daxinganling Mountain, Heilongjiang Province (July 2016) and Huma County, Neimonggu Autonomous Region (July 2016). Identification of all the herbs was performed by Professor Wang Zhenyue (Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China). Male and female Kunming mice [(20 ± 2) g] and male and female Wistar rats [(180 ± 20) g] were obtained from the Safety Evaluation Center of Heilongjiang University of Chinese Medicine, all of which were free of pathogens and reared for at least 5 days in a specific pathogen-free environment. Mice and rats were fed open formula grain-based diets with running water containing no fluoride. The protocol of this experiment was reviewed by the National Institute of Health and Nutrition Guide for Care and Use of Laboratory Animals and received approval from the Animal Ethics Committee of Heilongjiang University of Chinese Medicine. The HPLC grade approach was procured from Dikma Technologies, Inc. (Beijing, China). Reference samples, including hyperoside, hypericin, rutin, quercetin and kaempferol, were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The following materials were used in this study: Verapamil Hydrochloride Tablets (Guangzhou Meitxing Pharmaceutical Co., Ltd, batch number: 20150609, specifications 40 mg/tablet), Calcium Chloride Injection (Deyang Huakang Pharmaceutical Co., Ltd, batch number: 20150703), Methylene Chloride (Guangzhou Suchen Chemical Co., Ltd), Biological Signal Acquisition and Processing System (Nanjing Meiyi Technology Co., Ltd, Med, Lad-U/4CS), Xbrige C18 column (250 x 4.6 mm, 5 μm, Waters Corporation, Milford, MA, USA), Waters 2695 HPLC system, Waters 2996 Photodiode Array (PDA) detector (Waters Corporation, Milford, MA, USA), Xevo QTofMS Mass Spectrometer (Waters Corporation, Milford, MA, USA), GL-2 isolated heart perfusion system (Chengdu Tai Meng Technology Co., Ltd), IX53 inverted microscope (Japan OLYMPUS Optical Co., Ltd), EPC10 USB patch clamp amplifier (HEKA, Germany), model p-97 Electrode puller (SUTTER instruments, USA), XevoQ-Tof-MS mass spectrometry system (Waters, USA) used an electrospray ion source (ESI). Type II collagenase, HEPES, Na2ATP, EGTA, L-glutamic acid, Taurine and BSA were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Establishment of HPLC fingerprint of total flavonoids in H. attenuatum extracts Preparation of total flavonoid extracts from H. attenuatum In order to systematically investigate the extraction parameters of the total flavonoids of H. attenuatum, solvent extraction time, number of extractions, extraction concentration, material–liquid ratio and the index of investigation were conducted and the experimental design followed an L9 (34) orthogonal test. After determining the optimal extraction process, the main factors affecting the purification were found to be resin type, sample concentration, sample volume, elution rate, elution concentration and elution volume. Analysis of orthogonal experimental results showed that the best technology for extracting the total flavonoids from H. attenuatum is 3 g of sample from different batches of H. attenuatum that ground and sieved through a 40 mesh screen, then reflux extracted twice with 30 mL 80% ethanol for 2 h each time. Samples were then centrifuged to obtain a supernatant; the supernatant was concentrated to 10 mL by rotary evaporation. The results showed that the extraction rate of total flavonoids was 10.11%. Four types of resins were selected for the purification experiment of H. attenuatum. D101 resin was found to be the best using an analysis of adsorption rate and resolution. The best loading concentration was 22.8 mg/mL, which is equivalent to 1 mL containing 0.2 g crude drug. The sample was loaded on a D101 macroporous adsorption resin column (column diameter × column length: 1.0 cm × 15.0 cm) and eluted with pure water at a flow rate of 8 BV/h for 350 mL, followed by 40% ethanol at a flow rate of 8 BV/h for 300 mL. Using these steps, the purity of the total flavonoid content of H. attenuatum reached 51.7%. HPLC conditions An HPLC system was applied to acquire the HPLC fingerprint of total flavonoid extracts from H. attenuatum obtained from seven distinct sources. The HPLC had a PDA detector and was connected to LC solution software. The chromatographic separation was conducted by using an Xbrige C18 column, which operated at the temperature of 30°C by using gradient elution. The mobile phases A and B were 0.1% aqueous formic acid solution and acetonitrile, respectively, with the linear gradients ranging from 0 to 3 min, 15% B; 3 to 38 min, 15% B → 90% B; 38 to 43 min, 90% B; 43 to 46 min and 90% B → 15% B to 46 to 50 min, 15% B. Analysis of fingerprints The HPLC approach was assessed for its repeatability, precision and accuracy for acquisition of a reproducible and stable chemical fingerprint. Similarity of HPLC analysis With the adoption of the software of the HPLC similarity evaluation system of the Chinese Pharmacopoeia Committee, calculation of the similarity value was performed among reference fingerprints and chromatograms from chicory extracts (Version 2004A). Anti-arrhythmic experiment of H. attenuatum Grouping and administration of animals After 5 days of adaptive feeding, the mice were tested with lead electrocardiogram (ECG), and normal ECGs were obtained from mice. The qualified mice were randomly divided into nine groups. The treatments were separated into three groups, namely, a treatment group consisting of mice treated with one of the seven batches of total flavonoid extracts (total concentration of flavonoids 2.82 mg/kg) from H. attenuatum, a model group, and a positive control group (verapamil hydrochloride, 65 mg/kg). The model group was given an equal volume of normal saline. Calcium chloride induced arrhythmia in mice Animals were continuous administered the above nine groups of drugs once per day for 7 days. One hour after the last administration, the mice were anesthetized by intraperitoneal injection of 20% urethane and fixed on their backs. Needle-like electrodes were inserted into the right forelimbs, left hind limbs and right hind limbs. The standard lead II normal ECG was recorded, and 3.5% calcium chloride (120 mg/kg) was injected into the tail vein 10 min later. The ECG changes were recorded within 10 s. The occurrence time and the recovering time of arrhythmia after the first injection of calcium chloride were counted. Effect of chloroform on ventricular fibrillation in mice Animals were continuous administered the above nine groups of drugs once per day for 7 days. One hour after the last administration, each mouse was placed in an inverted beaker containing cotton balls with 3–4 mL chloroform, until the mice stopped breathing. The thoracic cavity of mice was opened immediately, the beating of the heart was observed by naked eye and the incidence of ventricular fibrillation was calculated. Statistical methods SPSS 19.0 was utilized for data analysis. The mean ± standard deviation |$(\overline{X}\pm S)$| was used to express the experimental data, with differences considered statistically different at P < 0.05. Analysis of spectrum-effect relationship Through analysis under chromatographical conditions, the peak areas in the fingerprints were marked respectively as X1, X2, X3…Xn, which were in association with the pharmacological results of the occurrence time of arrhythmia (W), arrhythmia duration (S) and ventricular fibrillation rate (R), constituting the data matrix for the regression analysis and correlation analysis. The calculation of these ingredients was correlated with pharmacological outcomes by using SPSS statistical software (SPSS for Windows 19.0, SPSS Inc., USA) for the acquisition of the union and intersection outcomes in both analyses. Regression analysis Regression, as a common statistical analysis approach, can verify whether there is a linear or nonlinear relationship of independent variables and dependent variables. There are many methods to calculate multivariate regressions, such as REMOVE, ENTER, FORWARD, BACKWARD and STEPWISE (29, 30). The stepwise regression method was applied in this study to carry out regression analysis using SPSS19.0 statistical software. Correlation analysis Correlation analyses are widely used in statistics to study the degree of relatedness between variables. Correlation analyses include Distance analysis, Bivariate analysis and Partial analysis (31). This experiment is intended for the analysis of the relationship of compatibility components with pharmacodynamic indexes, and the connotation of this relationship is actually the quantitative relationship between variables, which accords with the application of bivariate correlation analysis methods, so it can be used to make statistical inferences. The Pearson correlation coefficients obtained by the bivariate relevance analysis approach in SPSS 19.0 were used to reflect the correlation between the characteristic peaks and the pharmacodynamic results. Assignments of correlated peaks The compound standard solution containing 12.9 μg/mL hypercin and 10.1 μg/mL hyperoside was prepared by adding a precise quantity of every standard stock to a volumetric flask and dissolved with 10 mL ethanol. Specimens together with reference substances were prepared and injected into the HPLC system for assignment of correlative peaks, under the same chromatographic conditions as shown in “HPLC conditions.” For identification of the structure of correlative peaks, an LC–TOF–MS analysis was conducted by using Xevo Q-Tof Mass Spectrometer (Waters Corporation, Milford, MA, USA). MSE positive ion scanning mode detection was deployed, with the data format of MSE centroid. The expression collision energy was 10 V by using low energy scanning. The expression collision energy, flow rate of atomizing gas (N2), air curtain flow rate, capillary voltage, sample Cone voltage and extraction cone voltage were 20–40 V under high energy scanning, 650 L·H−1, 50 L·H−1, 3.0 kV, 40 V and 4 V, respectively. The temperatures of de-solvent gas and ion source were 150 and 120°C, respectively. Spectra were recorded above the m/z scope, ranging from 100 to 1,500 Da, to acquire full scan data, with a scanning time of 0.2 S. Verification of anti-arrhythmic effects of related peaks of H. attenuatum Acute isolation of rat myocardial cells Adult rats were injected intraperitoneally with a dose of 5 mL/kg 20% urethane, then 10–15 min after the intraperitoneal injection, rats were given a dose of 1,000 U/kg of heparin sodium. After successful anesthesia, the rats were fixed into position. The chest was disinfected with iodine and the heart and lung tissue was removed quickly. The blood on the surface of the heart tissue was washed with normal saline, put it into normal Tye’s solution at 0 °C, and the heart was squeezed gently three to five times to remove the residual blood. The aorta of rats was dissected, and ~1 cm in length was left. The heart of rats was fixed on the perfusion apparatus with surgical line to make the perfusion fluid retrograde from the aorta to the heart, and the lungs and other redundant tissues were removed after fixing. The heart was removed from the chest and hung on the improved perfusion device within 3 min. About 3–5 mL of normal Tai’s fluid was perfused to make the heart beat again, and then the calcium free perfusion of the liquid was used to stop the heart. The enzyme solution was quickly circulated after the heart stopped. The heart was taken off and the left ventricular myocardium was dissected. The epicardium was carefully dissected with ophthalmic scissors and forceps. The myocardium was cut into small pieces and put into 4°C KB solution. To obtain the cell suspension, the tissue was blown to flocculent. The cell suspension was placed at 4°C for 1 h. The whole perfusion process was conducted at 37°C with the continual passing of oxygen. About 10 mL of 1.8 mM CaCl2 was added to the cell suspension. Approximately 30% of the cells were long rods with intact edges, clear surface texture and no bubbles. This meets the requirements of Patch clamp experiments. Whole cell record After electrode infusion, sealing and membrane breaking treatment of myocardial cells, the whole cell was recorded under the stimulation procedure. The clamping current was set at 0 nA, and the stimulation current jumped from 0 to 10 nA in steps of 0.5 nA. The pulse lasted for 3 ms, and the stimulation frequency was 0.1 Hz. Recording and analyzing action potentials The electrophysiological characteristics of the myocardium are determined by the movement of ions such as Na+, Ca2+ and K+ across the myocardial cell membrane. Therefore, to a certain extent, the relevant parameters of the action potential can reflect the changes of the ion current across the membrane. Observing the changes of AP parameters is also an important method that can be used to study the effects of drugs. The action potential amplitude (APA) in the action potential mainly reflects the 0-phase Na + influx, and the decrease in APA may be caused by the 0-phase Na + influx. APD50 represents the prophase of repolarization and mainly reflects the length of the effective refractory period. The duration of the action potential is mainly determined by the length of the plateau period. APD50 reflects the length of the plateau period, which is affected by the influx of Ca2+ and efflux of K+. The inhibition of Ca2+ influx and factors that promote the efflux of K+ can shorten the plateau period. The part of APD90 excluding APD50 represents the late repolarization process, indicating the length of the relative refractory period (32). In this study, APA, APD90 and APD50 were determined to explore the recovery effect of the samples on the model cell action potential and to explore the effect of these components on arrhythmia from the perspective of electrophysiological mechanism. Drugs were administered in groups. Data of the blank control group were recorded after 5 min of cell suspension was perfused with extracellular fluid. After the results of the model group were recorded in the blank control group, 10 μL of aconitine was added to the extracellular fluid, and the final concentration of aconitine in the cell suspension was 1 mol/L. Rutin, hyperoside, quercetin and kaempferol were added into the extracellular solution (2.03 mg/mL rutin, 1.51 mg/mL hyperoside, 3.52 mg/mL quercetin, 2.01 mg/mL kaempferol, 10 μL). After the results of the model group were recorded, the final rutin, hyperoside, quercetin and kaempferol concentration in the cell suspension was 20.3, 15.1, 35.2, 20.1 μg/mL, respectively, and the compounds with anti-arrhythmic effects were recorded after 5 min of administration. Results of the amiodarone group were recorded at the end of the model group; 2.424 mmol/L amiodarone was added to 10 L extracellular solution, so that the final concentration of amiodarone in the cell suspension was 24.24 mol/L, and data was recorded 5 min after administration. After the formation of the whole cell configuration, recording of the action potential was performed under the mode of the current clamp. Before this experiment, 900 pA, 10 ms and 1 Hz were applied for 5 min, and cells with resting membrane potential (RMP) of approximately −70 mV were selected for the experiment. The stimulation procedure followed that described in section of whole cell record. The change of internal potential within 1 s was measured, and the curve at 1.5 times threshold was selected as the action potential. RMP, APA, APD50 of repolarization and APD90. Statistical analysis Data were processed by independent t-test, with differences considered statistically significance at P < 0.05. Results Chromatographic fingerprint analysis According to the methodology validation outcomes, the relative standard deviation (RSD) values including precision, stability and repeatability for relative retention time and relative peak area were <1.46% and <4.98%; <1.15% and <4.80%; <1.19% and <4.83%, respectively. The outcomes prove the stability and reliability of the HPLC fingerprint analysis method. Determination of fingerprints Through the comparison of fingerprints of the total flavonoid extracts from seven samples of H. attenuatum (Figure 1), the relative retention time was used to describe the characteristics of 15 chromatographic peaks (Figure 2). Figure 1 Open in new tabDownload slide Fingerprint of total flavonoid extracts from seven samples of H. attenuatum Choisy. Figure 1 Open in new tabDownload slide Fingerprint of total flavonoid extracts from seven samples of H. attenuatum Choisy. Figure 2 Open in new tabDownload slide Chromatograms of total flavonoids from H. attenuatum. Figure 2 Open in new tabDownload slide Chromatograms of total flavonoids from H. attenuatum. Similarity of fingerprints A comparison of the similarities was performed among the reference standard fingermarks and chromatographic fingerprints through total flavonoid extracts from seven samples of H. attenuatum, with similarity values of 0.653, 0.826, 0.655, 0.663, 0.759, 0.761 and 0.737, respectively. The difference of correlation coefficients further demonstrated that variations existed in the fingerprints and internal quality of the specimens. Results of the anti-arrhythmic experiments To study the spectrum-effect relationship, Chinese herbal formulas are chiefly divided into various batches and specimens are prepared by using either multiple extraction approaches or multiple combinations, which refers to the orthogonal compatibility combination of herbs, extracts or compositions (33, 34). Preparation and determination of total flavonoid extracts from seven samples of H. attenuatum were made in this research by using an established approach. In this study, after injection of calcium chloride, the heart rate of the mice increased and the ECG changed, indicating that the model was successful. The normal ECG before injection of calcium chloride and ECG for arrhythmia after injection of calcium chloride are shown in Figures 3 and 4. Occurrence time of arrhythmia (W), the recovery time of arrhythmia (S) and incidence of ventricular fibrillation (R) of all the groups are indicated in Figure 5 (n = 10). Figure 3 Open in new tabDownload slide Normal ECG. Figure 3 Open in new tabDownload slide Normal ECG. Figure 4 Open in new tabDownload slide Arrhythmia ECG after injection of calcium chloride. Figure 4 Open in new tabDownload slide Arrhythmia ECG after injection of calcium chloride. Figure 5 Open in new tabDownload slide Effects of total flavonoid extracts from H. attenuatum on calcium chloride induced arrhythmia and ventricular fibrillation induced by chloroform on mice (Group: 1, model group, normal saline administration; 2, positive control group, verapamil hydrochloride administration dosage is 65 mg/kg; 3–9, total flavonoid extracts from H. attenuatum, administration dosage is according to the amount of total flavonoids with 2.82 mg/kg). Figure 5 Open in new tabDownload slide Effects of total flavonoid extracts from H. attenuatum on calcium chloride induced arrhythmia and ventricular fibrillation induced by chloroform on mice (Group: 1, model group, normal saline administration; 2, positive control group, verapamil hydrochloride administration dosage is 65 mg/kg; 3–9, total flavonoid extracts from H. attenuatum, administration dosage is according to the amount of total flavonoids with 2.82 mg/kg). Analysis of spectrum-effect relationships Selection of 15 characteristic peaks marked from X1 to X15 in this fingerprint was performed for investigation of the spectrum-effect relationship of the ingredients with a pharmacological effect. Eventually, the peak areas of seven samples were associated with all the occurrence time of arrhythmia (W), the recovering time of arrhythmia (S) and incidence of ventricular fibrillation (R), which formed a 7*18 data matrix for subsequent statistical analysis as indicated in Table I. Additionally, the data processing approaches used in this research of “spectrum-effect relationship” comprise correlation analyses as well as regression analyses. Distinct methods have various focuses. Hence, one or a combination of multiple data processing approaches were generally adopted. Table I A 7*18 Data Matrix of 15 Common Peak Areas of Seven Batch of H. attenuatum Choisy Total Flavonoids Extract (S1, S2, S3, S4, S5, S6, S7) Associated with Each of Occurrence Time of Arrhythmia (W), the Recovering Time of Arrhythmia (S) and Incidence of Ventricular Fibrilation (R) Peak number . Seven batch of H. attenuatum Choisy total flavonoids extract . S1 . S2 . S3 . S4 . S5 . S6 . S7 . 1 397.200 295.800 513.350 529.090 713.670 505.640 793.740 2 144.470 141.290 315.310 170.22 491.310 464.320 393.800 3 199.01 189.76 262.050 257.370 312.080 137.680 122.760 4 168.080 172.640 260.310 330.980 645.54 589.56 718.810 5 346.950 341.260 430.350 294.290 318.110 259.72 452.890 6 115.360 113.480 137.800 270.880 402.73 500.85 563.800 7 2121.070 2280.89 1186.560 1337.220 2116.410 2247.680 3158.280 8 427.430 418.260 339.780 367.070 503.880 437.820 520.950 9 183.660 180.400 124.690 81.370 149.470 112.820 414.730 10 197.350 192.620 161.650 111.860 327.390 280.260 309.100 11 254.920 250.480 265.970 249.970 389.470 524.150 568.410 12 175.56 177.85 254.720 159.920 133.030 223.560 180.430 13 2035.360 1989.370 1162.900 1311.210 3663.940 1751.800 3769.470 14 6866.760 6716.160 4016.920 3091.110 3743.340 4908.56 2899.100 15 1217.610 1221.370 1935.060 1562.640 1771.800 1856.340 1938.950 W 1.98 2.16 2.52 2.08 2.31 1.95 2.04 S 15.29 15.75 15.85 15.16 15.21 16.02 15.99 R 33.3 33.3 33.3 41.7 33.33 41.7 41.7 Peak number . Seven batch of H. attenuatum Choisy total flavonoids extract . S1 . S2 . S3 . S4 . S5 . S6 . S7 . 1 397.200 295.800 513.350 529.090 713.670 505.640 793.740 2 144.470 141.290 315.310 170.22 491.310 464.320 393.800 3 199.01 189.76 262.050 257.370 312.080 137.680 122.760 4 168.080 172.640 260.310 330.980 645.54 589.56 718.810 5 346.950 341.260 430.350 294.290 318.110 259.72 452.890 6 115.360 113.480 137.800 270.880 402.73 500.85 563.800 7 2121.070 2280.89 1186.560 1337.220 2116.410 2247.680 3158.280 8 427.430 418.260 339.780 367.070 503.880 437.820 520.950 9 183.660 180.400 124.690 81.370 149.470 112.820 414.730 10 197.350 192.620 161.650 111.860 327.390 280.260 309.100 11 254.920 250.480 265.970 249.970 389.470 524.150 568.410 12 175.56 177.85 254.720 159.920 133.030 223.560 180.430 13 2035.360 1989.370 1162.900 1311.210 3663.940 1751.800 3769.470 14 6866.760 6716.160 4016.920 3091.110 3743.340 4908.56 2899.100 15 1217.610 1221.370 1935.060 1562.640 1771.800 1856.340 1938.950 W 1.98 2.16 2.52 2.08 2.31 1.95 2.04 S 15.29 15.75 15.85 15.16 15.21 16.02 15.99 R 33.3 33.3 33.3 41.7 33.33 41.7 41.7 Open in new tab Table I A 7*18 Data Matrix of 15 Common Peak Areas of Seven Batch of H. attenuatum Choisy Total Flavonoids Extract (S1, S2, S3, S4, S5, S6, S7) Associated with Each of Occurrence Time of Arrhythmia (W), the Recovering Time of Arrhythmia (S) and Incidence of Ventricular Fibrilation (R) Peak number . Seven batch of H. attenuatum Choisy total flavonoids extract . S1 . S2 . S3 . S4 . S5 . S6 . S7 . 1 397.200 295.800 513.350 529.090 713.670 505.640 793.740 2 144.470 141.290 315.310 170.22 491.310 464.320 393.800 3 199.01 189.76 262.050 257.370 312.080 137.680 122.760 4 168.080 172.640 260.310 330.980 645.54 589.56 718.810 5 346.950 341.260 430.350 294.290 318.110 259.72 452.890 6 115.360 113.480 137.800 270.880 402.73 500.85 563.800 7 2121.070 2280.89 1186.560 1337.220 2116.410 2247.680 3158.280 8 427.430 418.260 339.780 367.070 503.880 437.820 520.950 9 183.660 180.400 124.690 81.370 149.470 112.820 414.730 10 197.350 192.620 161.650 111.860 327.390 280.260 309.100 11 254.920 250.480 265.970 249.970 389.470 524.150 568.410 12 175.56 177.85 254.720 159.920 133.030 223.560 180.430 13 2035.360 1989.370 1162.900 1311.210 3663.940 1751.800 3769.470 14 6866.760 6716.160 4016.920 3091.110 3743.340 4908.56 2899.100 15 1217.610 1221.370 1935.060 1562.640 1771.800 1856.340 1938.950 W 1.98 2.16 2.52 2.08 2.31 1.95 2.04 S 15.29 15.75 15.85 15.16 15.21 16.02 15.99 R 33.3 33.3 33.3 41.7 33.33 41.7 41.7 Peak number . Seven batch of H. attenuatum Choisy total flavonoids extract . S1 . S2 . S3 . S4 . S5 . S6 . S7 . 1 397.200 295.800 513.350 529.090 713.670 505.640 793.740 2 144.470 141.290 315.310 170.22 491.310 464.320 393.800 3 199.01 189.76 262.050 257.370 312.080 137.680 122.760 4 168.080 172.640 260.310 330.980 645.54 589.56 718.810 5 346.950 341.260 430.350 294.290 318.110 259.72 452.890 6 115.360 113.480 137.800 270.880 402.73 500.85 563.800 7 2121.070 2280.89 1186.560 1337.220 2116.410 2247.680 3158.280 8 427.430 418.260 339.780 367.070 503.880 437.820 520.950 9 183.660 180.400 124.690 81.370 149.470 112.820 414.730 10 197.350 192.620 161.650 111.860 327.390 280.260 309.100 11 254.920 250.480 265.970 249.970 389.470 524.150 568.410 12 175.56 177.85 254.720 159.920 133.030 223.560 180.430 13 2035.360 1989.370 1162.900 1311.210 3663.940 1751.800 3769.470 14 6866.760 6716.160 4016.920 3091.110 3743.340 4908.56 2899.100 15 1217.610 1221.370 1935.060 1562.640 1771.800 1856.340 1938.950 W 1.98 2.16 2.52 2.08 2.31 1.95 2.04 S 15.29 15.75 15.85 15.16 15.21 16.02 15.99 R 33.3 33.3 33.3 41.7 33.33 41.7 41.7 Open in new tab Regression analysis The independent variable of peak area as well as the dependent variables of W, S and R were analyzed to establish the ENTER regression equation of W, S and R, respectively. Six peaks (X5, X7, X8, X11, X13 and X14) were described in the equations, including. W = –0.001X5 + 0.005X7–0.000326X8–07.540E-5X11 + 0.002X13 + 0.000258X14 + 1.652; S = –0.005X5 + 0.031X7–0.001X8 + 0.000405X11 + 0.041X13 + 0.001X14 + 11.064; R = –0.018X5 + 0.108X7 + 0.000449X8–0.000347X11 + 0.045X13 + 0.001X14 + 33.2. It was reflected from the residual statistics of Durbin–Watson that the independence between residuals was within the range of 2 ± 1.5. The determination coefficient was 0.771 in W, 0.830 in S and 0.728 in R. These outcomes from the variance analysis indicated statistically significant differences (P < 0.05). Correlation analysis Each of the peaks was respectively analyzed with W, S and R. In the canonical correlation analysis, SPSS19.0 statistical software was used to calculate the Pearson’s correlation coefficient, which showed the correlation between characteristic peaks and anti-arrhythmic effects. All the variables were found to be normally distributed and could be analyzed by using a bivariate analysis. The Pearson’s correlation coefficients of the original matrix data are shown in Figure 6. It was shown in the results that all of the peaks X7, X13, X14 to W, the peaks X5, X7, X13, X14 to S and X5, X7, X13, X14 had statistically significant differences (P < 0.05). Figure 6 Open in new tabDownload slide Pearson correlation between 15 peak areas in the fingerprint of total flavonoid extracts from H. attenuatum and occurrence time of arrhythmia (W), the recovering time of arrhythmia (S) and incidence of ventricular fibrillation (R). *P < 0.05, **P < 0.01. Figure 6 Open in new tabDownload slide Pearson correlation between 15 peak areas in the fingerprint of total flavonoid extracts from H. attenuatum and occurrence time of arrhythmia (W), the recovering time of arrhythmia (S) and incidence of ventricular fibrillation (R). *P < 0.05, **P < 0.01. Integration of the analytical results The results of the forced induction regression analysis showed that the components closely related to the efficacy were X5, X7, X8, X11, X13 and X14. The results of the bivariate correlation analysis showed that the components closely related to efficacy were X5, X7, X13 and X14. The intersections of the regression and the correlation analysis outcomes were the peaks of X5, X7, X13 and X14. Determination of the main chromatographic peaks in total flavonoid extract from H. attenuatum Assignments of the correlated peaks Fifteen common peaks were discriminated in the fingerprint resemblance for all the groups. The outcomes of the total flavonoid extracts from H. attenuatum chromatograms were acquired through comparison of the chromatograms of reference substances (Figure 7), which indicated that two peaks were identified through comparison with a reference substance: peak 7, hyperoside; peak 15, hypericin. Figure 7 Open in new tabDownload slide Typical chromatogram of total flavonoids and the chromatogram of mixed reference substances in H. attenuatum peak 7, hyperin and peak 15, hypericin. Figure 7 Open in new tabDownload slide Typical chromatogram of total flavonoids and the chromatogram of mixed reference substances in H. attenuatum peak 7, hyperin and peak 15, hypericin. Structural identification of the correlated peaks analyzed by LC–TOF–MS LC–TOF–MSE was used to scan the total flavonoid extract peaks from H. attenuatum under the MSE mode. The total ion flow diagram is shown in Figure 8. Fifteen peaks were clearly visible and the four target components were analyzed by using MSE fragmentation. By comparison with the reference substance, peaks 7 and 15 are hyperoside and hypericin, respectively, which is consistent with the HPLC results. The structures and fragmentation patterns of the four target components (peaks 5, 7, 13 and 14) are shown in Figures 9 and 10. The structural identification of the correlated peaks analyzed by LC–TOF–MS showed that X5 was rutin, X7 was hyperoside, X13 was quercetin and X14 was kaempferol. Figure 8 Open in new tabDownload slide Total ion flow graph of total flavonoid extracts in H. attenuatum. Figure 8 Open in new tabDownload slide Total ion flow graph of total flavonoid extracts in H. attenuatum. Figure 9 Open in new tabDownload slide Mass spectrogram fragmentation and chemical structure of four potential antiarrhythmic components. Figure 9 Open in new tabDownload slide Mass spectrogram fragmentation and chemical structure of four potential antiarrhythmic components. Figure 10 Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Pattern fragments of four potential anti-arrhythmic components. Figure 10 Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Pattern fragments of four potential anti-arrhythmic components. Effects of potential anti-arrhythmic compounds from H. attenuatum on action potential of rat cardiac myocytes The results of the anti-arrhythmic effect of rutin, hyperoside, quercetin and kaempferol from H. attenuatum on the action potential of rat cardiac myocytes are shown in Figure 11. In contrast with the blank control group, the APA of the model group increased significantly from 106.39 ± 4.59 to 116.78 ± 5.66 mV (Figure 11). The APD90 and APD50 were significantly prolonged; APD90 increased from 38.51 ± 1.70 to 61.11 ± 1.99 ms, and APD50 was increased from 19.45 ± 1.24 to 26.53 ± 2.39 ms after 5 min of aconitine treatment. After 5 min of rutin, hyperoside, quercetin and kaempferol intervention, APA recovered to 112.32 ± 3.53, 110.26 ± 3.06, 109.32 ± 3.25 and 109.98 ± 3.76 mV, respectively, while APD90 recovered to 45.76 ± 1.53, 44.80 ± 1.50, 44.89 ± 1.91 and 43.96 ± 1.93 ms; APD50 recovered to 22.30 ± 2.03, 23.05 ± 2.04, 22.54 ± 2.36 and 22.98 ± 2.07 ms, respectively. In contrast with the model group (P < 0.01, P < 0.05), the action potential changes that were evaluated by RMP did not change significantly. Figure 11 Open in new tabDownload slide Effect of potential anti-arrhythmic components on action potentials of rat cardiac myocytes compared with the model group (±S, n = 6), *P < 0.05, **P < 0.01. 1, Blank control group; 2, model group; 3, rutin; 4, hyperin; 5, quercetin; 6, kaempferol, 7, amiodarone group. Table 1. A 7*18 data matrix of 15 common peak areas of seven samples of total flavonoid extract from H. attenuatum associated with occurrence time of arrhythmia (W), the recovering time of arrhythmia (S) and incidence of ventricular fibrillation (R). Figure 11 Open in new tabDownload slide Effect of potential anti-arrhythmic components on action potentials of rat cardiac myocytes compared with the model group (±S, n = 6), *P < 0.05, **P < 0.01. 1, Blank control group; 2, model group; 3, rutin; 4, hyperin; 5, quercetin; 6, kaempferol, 7, amiodarone group. Table 1. A 7*18 data matrix of 15 common peak areas of seven samples of total flavonoid extract from H. attenuatum associated with occurrence time of arrhythmia (W), the recovering time of arrhythmia (S) and incidence of ventricular fibrillation (R). The results showed that APD was decreased by four potential anti-arrhythmic components; however, the effective refractory period has been extended, and the probability of ectopic exaction in the effective refractory period was increased, which is beneficial to eliminate reentrant. The four potential anti-arrhythmic components have a good recovery effect on the action potential of the cardiac arrhythmia model in rats. Discussion The primary research method used to screen anti-arrhythmic compounds in H. attenuatum is the phytochemical separation method. After extraction, separation and structural identification, the pharmacological activity of the purified chemical components is tested on the pharmacological model. However, this method is inconsistent with the multi-component and multi-target action pathways of TCM because it does not better reflect the material basis of the interactions of the multiple compounds in TCM that exert effects. Moreover, this method does not reflect the “holistic view” of TCM. The spectrodynamics of TCM uses data processing technology to screen out the characteristic peaks that are closely related to the efficacy of Chinese herbal medicines by using the chemical fingerprint information and the efficacy information of Chinese herbal medicines to provide new ideas for elucidating the material basis of the efficacy of Chinese medicines. Compared with other research methods, spectrodynamics has outstanding advantages as it increases the amount of research on the correlation between fingerprints and pharmacological effects. Although there are many methods to analyze the spectrum-effect relationships, the accuracy of any one method cannot reach the complete determination of the pharmacodynamic substance base. Therefore, most of the investigation of the pharmacodynamic substance base is based on a variety of analysis methods and comprehensive evaluations. Forced regression analysis is used to introduce all independent variables into the equation, and stepwise regression analysis is used to add or eliminate a single variable until the equation no longer contains variables to add or remove, so the two can be combined; bivariate correlation analysis can obtain the correlation index between each independent variable and dependent variable and the pharmacological number according to the complementary evaluation of compatibility. The correlation analysis results can be combined; the comprehensive regression analysis and correlation analysis results show that the two are interrelated and intersected. Finally, the results of spectral effect correlation analysis synthesized the characteristics of each analysis method, and its error and accuracy were greatly improved. Therefore, it can be considered that the results are the pharmacodynamic basis of H. attenuatum. Fifteen common characteristic peaks with good baseline separation, peak shape and response in the fingerprint analysis of H. attenuatum were included in our investigation. Therefore, the screening results were found in 15 common peaks in the spectrum-effect construction of H. attenuatum. In order to avoid false negative results, our research group incorporated non-common peaks into fingerprint information in the later stage to further study the anti-arrhythmic material basis of H. attenuatum. Heilongjiang Province is located in northeast China. The terrain has a relatively high elevation ranging from 300 to 1,000 m and consisting mainly of mountain forests. The forest area in the province accounts for half of the entire land area and the regional climate varies greatly. The formation of the quality of medicinal plants is related to both genetic factors and is affected by the external environment. In addition to climate and soil, the external environment also includes natural factors such as organisms, topography and community environment (35). Research has shown that the active ingredients of many medicinal plants are related to secondary metabolites, and the synthesis and accumulation of secondary metabolites are often subject to changes due to the environment. For example, Scutellaria baicalensis flavonoids are secondary metabolites, and their content is greatly affected by the environment (35). In humid areas, the increase in altitude is conducive to the conversion and accumulation of flavonoids. In addition, the increase in altitude and the decrease in photosynthesis will cause a variation in flavonoids, with a reduction of flavonoids above a certain altitude. The increase in altitude and temperature affect the accumulation of flavonoids; when the threshold is exceeded, normal metabolism will be affected, eventually leading to a decrease in flavonoid accumulation (36). Liu Tong et al. determined the content of total flavonoids and rutin in different parts of wild and cultivated H. attenuatum and they found that under different growth conditions, the content of flavonoids and rutin varied. Their findings also indicated that there is a certain difference between the accumulation of hypericum and flavonoids (37). In this experiment, a total of seven batches of H. attenuatum were studied, of which six batches were collected from Heilongjiang. The H. attenuatum collected in this study were wild and cultivated, and the altitude of origin ranged from 142 to 1,000 m. The similarity values of the fingerprints of total flavonoid extracts from seven samples of H. attenuatum were all lower than 0.8, indicating that the composition of total flavonoids varies greatly. The results of this study are consistent with the results of previously published literature (37). As the resources of Hypericum decrease sharply, this suggests that we should conduct a comparative study on the chemical composition of the H. attenuatum in different regions in Heilongjiang Province, and further explore the impact of environmental factors on its flavonoid content. Using the findings from this and future studies, a scientifically based cultivation, management and utilization plan can be established to lay the foundation for further development and utilization of H. attenuatum. This study indicated that four potential anti-arrhythmic components (rutin, hyperoside, quercetin and kaempferol) in H. attenuatum had a good recovery effect on the action potential of the cardiac arrhythmia model in rats. Hyperoside has been deemed the primary active indigent in H. attenuatum (38, 39). Jia’s group reported that it could improve rapid arrhythmia in the myocardial cell membrane of rats, Na+–K+–ATP enzyme, and Ca2+–Mg2+–ATP enzyme activity, suggesting that hyperoside could inhibit Ca2+ overload of the intracellular region, maintain normal ionic gradients inside and outside of cells, and maintain the current balance of Na +, K + and Ca2 + to avoid rapid arrhythmia (40). The effects of hyperoside on the occurrence time, duration, and incidence of ventricular fibrillation in rats with arrhythmia have been reported in the literature (41). Quercetin has the effect of anti-arrhythmia, and its anti-arrhythmia effect may be related to various factors, such as reducing the autonomy of cardiac cells, prolonging the refractory period, protecting Na+–K+–ATP enzyme on cardiac cell membrane, and antagonizing the internal flow of external calcium (42).Therefore, the research results of the spectrum-effect relationship are consistent with the literature. This study is the first to show that rutin and kaempferol have anti-arrhythmia effects in terms of myocardial electrophysiology. The spectrum-effect relationship of TCM requires taking the pharmacodynamic index of the sample as a system and the peak area of the characteristic peak in the fingerprint spectrum as another system. The correlation degree of each factor is then compared in the two systems to determine which peaks are effective contributors. The method of predicting the correlation between each component and the drug efficacy focuses on the correlation between the chromatographic peak and drug efficacy index to predict the comprehensive contribution of the chromatographic peak to the drug efficacy index. In this way, a material basis can be established to explain the efficacy of TCM from the perspective of a “holistic view” to make it more in line with the theories of TCM. At the same time, it also provides references for the formulation of quality control and evaluation of TCM and accelerates the development of modern Chinese medicine (43–44). The overall effect of TCM and the characteristics of multi-component and multi-target actions determine whether any one component used as an indicator can accurately reflect and evaluate the quality of Chinese medicine. Establishment of the quality control and evaluation system for TCM should be based on its characteristics and be able to control the overall efficacy of TCM and its preparations with multiple components and multiple indicators. Based on the establishment of the spectrum-effect relationship using the intrinsic functional and proportional relationships for the effective components of TCM, a readily available reference substance component is used as an internal reference to establish the relative correction factor for the same or different components within a certain linear range. A relative correction factor is used to obtain the content of other components, realizing the quality control mode of simultaneous determination of multiple components (45–46). Conclusions The study on the relationships of HPLC fingerprints with the pharmacodynamics of herbal medicines makes it possible to perform a holistic assessment of the internal quality. Additionally, it offers an appropriate means to discover the potentially effective ingredients from plants with multiple active compounds. Most importantly, the research on the spectrum-effect relationships can be conducted as an important means to perform quality control of H. attenuatum. Funding This study was supported by the National Natural Science Foundation of China (No. 81703944), Heilongjiang Natural Science Foundation Project (YQ2019H031) and the Outstanding Innovative Talents Project from Heilongjiang University of Chinese Medicine (2018). References 1. Editorial Committee of Chinese Flora of the Chinese Academy of Sciences ; Flora of China . Science Press, Beijing , ( 2004 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 2. Li , J. , Teng , L., Gao , Y.Y.; Protective effects of Hypericum attenuatum Choisy. Extract on myocardial ischemia in mice ; Chinese Journal of Information on Traditional Chinese Medicine , ( 2009 ); 16 : 38 – 39 . Google Scholar OpenURL Placeholder Text WorldCat 3. Li , J. , Wu , Q.E., Gao , Y.Y., Shi , X., Tang , M.Z., Cao , M.M.; The effect of Hypericum attenuatum Choisy on the contents of 5-HT and 5-HIAA from hippocampus monoamine neurotransmitters of depressive rats ; Information on Traditional Chinese Medicine , ( 2012 ); 29 : 18 – 20 . Google Scholar OpenURL Placeholder Text WorldCat 4. Zhou , Z.B. , Zhang , Y.M., Pan , K., Luo , J.G., Kong , L.Y.; Cytotoxic polycyclic polyprenylated acylphloroglucinols from Hypericum attenuatum ; Fitoterapia , ( 2014 ); 95 : 1 – 7 . Google Scholar OpenURL Placeholder Text WorldCat 5. Li , D.Y. , Xue , Y.B., Zhu , H.C., Li , Y., Sun , B., Liu , J.J. et al. ; Hyperattenins A-I, bioactive polyprenylated acylphloroglucinols from Hypericum attenuatum Choisy ; RSC Advances , ( 2015 ); 5 : 5277 – 5287 . Google Scholar OpenURL Placeholder Text WorldCat 6. Yang , Y. , Chang , G.Y., Zhang , C.L., Liu , C.N., Dang , X.Y., Liu , M.Q. et al. ; Activities of alcohol soluble substance of Hypericum attenuatum in vitro ; Hunan Agricultural Sciences , ( 2015 ); 2 : 60 – 61 . Google Scholar OpenURL Placeholder Text WorldCat 7. Zhang , Y.X. , Chen , Y.J., Ma , L.N.; Depression and cardiovascular disease in elderly: Current understanding ; Journal of Clinical Neuroscience Official Journal of the Neurosurgical Society of Australasia , ( 2018 ); 47 : 1 – 5 . Google Scholar OpenURL Placeholder Text WorldCat 8. Teng , L. ; Experimental study on anti arrhythmia and myocardial ischemia of Hypericum attenuatum Choisy in mice . Heilongjiang University of Chinese Medicine, Harbin , ( 2008 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 9. Li , J. , Teng , L., Gao , Y.Y.; Experimental study of Hypericum attenuatum choisy on arrhythmia ; Journal of Chinese Medicine Information , ( 2008 ); 25 : 32 – 33 . Google Scholar OpenURL Placeholder Text WorldCat 10. Li , R. , Yan , Z.Y.; The study of spectral efficiency relationship is the key link in the quality and efficacy standard of Chinese medicine ; China News of Traditional Chinese Medicine:Guangzhou , ( 2002 ); 10 : 18 – 20 . Google Scholar OpenURL Placeholder Text WorldCat 11. Zhang , C. , Zheng , X., Ni , H., Li , P., Li , H.J.; Discovery of quality control markers from traditional Chinese medicines by fingerprint-efficacy modeling: Current status and future perspectives ; Journal of Pharmaceutical & Biomedical Analysis , ( 2018 ); 159 : 296 – 304 . Google Scholar OpenURL Placeholder Text WorldCat 12. Liu , M.Q. , Wu , Y.J., Huang , S.S., Liu , H.G., Feng , J.; Spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of Curcuma aromatica ; Biomedical Chromatography , ( 2018 ); 32 : e4220. Google Scholar OpenURL Placeholder Text WorldCat 13. Jiang , Z.M. , Zhao , C., Gong , X.J., Sun , X., Li , H.D., Zhao , Y. et al. ; Quantification and efficient discovery of quality control markers for Emilia prenanthoidea DC. By fingerprint-efficacy relationship modelling ; Journal of Pharmaceutical & Biomedical Analysis , ( 2018 ); 156 : 36 – 44 . Google Scholar OpenURL Placeholder Text WorldCat 14. Cheng , J. , He , S.Y., Wan , Q., Jing , P.; Multiple fingerprinting analyses in quality control of Cassiae semen polysaccharides ; Journal of Chromatography B , ( 2018 ); 1077–1078 : 22 – 27 . Google Scholar OpenURL Placeholder Text WorldCat 15. Zhang , X.D. , Yu , Y.G., Cen , Y.S., Yang , D.F., Qi , Z.C., Hou , Z.N. et al. ; Bivariate correlation analysis of the chemometric profiles of Chinese wild salvia miltiorrhiza based on UPLC-Qqq-MS and antioxidant activities ; Molecules , ( 2018 ); 23 : 538 . Google Scholar OpenURL Placeholder Text WorldCat 16. Zhang , Y.B. , Zhang , M., Cheng , Q.B., Li-Sha , M.A., Zhang , L.W.; Relationship between HPLC fingerprint chromatogram and antioxidant activity of forsythia suspense leaves ; Natural Product Research & Development , ( 2017 ); 29 : 629 – 634 . Google Scholar OpenURL Placeholder Text WorldCat 17. Iorgulescu , E. , Voicu , V.A., Sârbu , C., Tache , F., Albu , F., Stnculescu , I. et al. ; Holistic evaluation of gamma-irradiation effects on green teas: New linear regression based approach applied to (+/−)ESI/MS and RPLC/UV data and comparison with PCA and CA chemometric methods ; Radiation Physics & Chemistry , ( 2018 ); 149 : 126 – 133 . Google Scholar OpenURL Placeholder Text WorldCat 18. Ahmed , Z.B. , Yousfi , M., Viaene , J., Dejaegher , B., Demeyer , K., Mangelings , D. et al. ; Potentially antidiabetic and antihypertensive compounds identified from Pistacia atlantica leaf extracts by LC fingerprinting ; Journal of Pharmaceutical & Biomedical Analysis , ( 2018 ); 149 : 547 – 556 . Google Scholar OpenURL Placeholder Text WorldCat 19. Li , T. , He , X.; Quantitative analysis of Salidroside and p-Tyrosol in the traditional Tibetan medicine Rhodiola crenulata by Fourier transform near-infrared spectroscopy ; Chemical & Pharmaceutical Bulletin , ( 2016 ); 64 : 289 – 296 . Google Scholar OpenURL Placeholder Text WorldCat 20. Seal , T. ; Quantitative HPLC analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, Sonchus arvensis and Oenanthe linearis of north-eastern region in India ; Journal of Applied Pharmaceutical Science , ( 2016 ); 6 : 157 – 166 . Google Scholar OpenURL Placeholder Text WorldCat 21. Agatonovic-Kustrin , S. , Loescher , C.M.; Qualitative and quantitative high performance thin layer chromatography analysis of Calendula officinalis using high resolution plate imaging and artificial neural network data modelling ; Analytica Chimica Acta , ( 2013 ); 798 : 103 – 108 . Google Scholar OpenURL Placeholder Text WorldCat 22. Feng , S. , Liu , Y., Wang , X.Y., Li , F.; Application of multivariate statistical analysis in correlation between GC-MS chromatograms of polysaccharide hydrolysis composition acetylation and cold-heat nature of traditional Chinese medicine ; Chinese Journal of Experimental Traditional Medical Formulae , ( 2013 ); 15 : 425 – 430 . Google Scholar OpenURL Placeholder Text WorldCat 23. Sim , S.F. , Lee , T.Z., Na , M.I.L., Samling , B.; Synchronized analysis of FTIR spectra and GCMS chromatograms for evaluation of the thermally degraded vegetable oils ; Journal of Analytical Methods in Chemistry , ( 2014 ); 2014 : 271970 . Google Scholar OpenURL Placeholder Text WorldCat 24. Bian , X.L. , Zhang , J., Wang , S.Q., He , L.C.; HPCE-fingerprint analysis of the active part of radix of Ligusticum chuangxiong Hort ; Chinese Journal of Pharmaceutical Analysis , ( 2005 ); 25 : 1520 – 1523 . Google Scholar OpenURL Placeholder Text WorldCat 25. Konieczyński , P. ; Electrochemical fingerprint studies of selected medicinal plants rich in flavonoids ; Acta Poloniae Pharmaceutica , ( 2015 ); 72 : 655 . Google Scholar OpenURL Placeholder Text WorldCat 26. Vargas-Caporali , J. , Juaristi , E.; Fundamental developments of chiral phase chromatography in connection with Enantioselective synthesis of β-amino acids ; Israel Journal of Chemistry , ( 2017 ); 57 : 896 – 912 . Google Scholar OpenURL Placeholder Text WorldCat 27. Han , S.L. , Zhang , T., Huang , J., Cui , R.H., He , L.C.; New method of screening allergenic components from Shuanghuanglian injection: With RBL-2H3/CMC model online HPLC/MS system ; J Pharm Biomed Anal , ( 2014 ); 88 : 602 – 608 . Google Scholar OpenURL Placeholder Text WorldCat 28. Ciminiello , P. , Dell’Aversano , C., Iacovo , E.D., Fattorusso , E., Forino , M., Grauso , L. et al. ; High resolution LC-MS n fragmentation pattern of Palytoxin as template to gain new insights into Ovatoxin-a structure. The key role of calcium in MS behavior of Palytoxins ; Journal of the American Society for Mass Spectrometry , ( 2012 ); 23 : 952 – 963 . Google Scholar OpenURL Placeholder Text WorldCat 29. Wang , X.Y. , Zhen , Y.Q., Wang , X.G., Jiang , G.Z., Niu , L.Y.; Correlation of HPLC fingerprint chromatograms of Cinnamomi Ramulus formula granules, decoction pieces and water decoction ; Chinese Journal of Information on Traditional Chinese Medicine , ( 2017 ); 24 : 87 – 90 . Google Scholar OpenURL Placeholder Text WorldCat 30. Zushi , Y. , Gros , J., Tao , Q., Reichenbach , S.E., Hashimoto , S., Arey , J.S.; Pixel-by-pixel correction of retention time shifts in chromatograms from comprehensive two-dimensional gas chromatography coupled to high resolution time-of-flight mass spectrometry ; Journal of Chromatography A , ( 2017 ); 1508 : 121 – 129 . Google Scholar OpenURL Placeholder Text WorldCat 31. Zybailov , B. , Coleman , M.K., Florens , L., Washburn , M.P.; Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling ; Analytical Chemistry , ( 2005 ); 77 : 6218 – 6224 . Google Scholar OpenURL Placeholder Text WorldCat 32. Wang , B. ; Experimental study on the effect of epimedium flavonoids on ion channels of isolated working heart and cardiomyocytes . Jilin University, Changchun , ( 2006 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 33. Wang , Y. , Li , Y., Zhang , Y., Feng , G., Yang , Z.X., Guan , Q.X. et al. ; Multi-dimensional Spectrum-effect relationship of the impact of Chinese herbal formula Lichong Shengsui yin on ovarian cancer ; Molecules , ( 2017 ); 22 : 979 . Google Scholar OpenURL Placeholder Text WorldCat 34. Zhang , X.F. , Chen , J., Yang , J.L., Shi , Y.P.; UPLC-MS/MS analysis for antioxidant components of Lycii Fructus based on spectrum-effect relationship ; Talanta , ( 2018 ); 180 : 389 – 395 . Google Scholar OpenURL Placeholder Text WorldCat 35. Xie , J. , Ma , S.J., Wang , W.Q., Hou , J.L., Xin , B., Kang , X.F.; Effects of geographic soil and provenance on contents of flavonoids from Scutellaria Baicalensis ; Information on Traditional Chinese Medicine , ( 2015 ); 32 : 8 – 11 . Google Scholar OpenURL Placeholder Text WorldCat 36. Ji , Q.Y. , Peng , X., Hua , J.W., Cheng , K.J., Zhu , B., Fang , J. et al. ; Effect of altitude on the yield and flavonoids content in Tetrastastigrma hemsletanum ; Modern Chinese Medicine , ( 2017 ); 19 : 1004 – 1006 . Google Scholar OpenURL Placeholder Text WorldCat 37. Liu , T. , Zhang , N.Y., Chang , G.Y., Xiao , F.Y., Zhang , K.Q.; Changes of total flavonoids and rutin contents in different parts of wild and cultivated Hypericum attenuatum ; Zhong Yao Cai , ( 2014 ); 37 : 960 – 963 . Google Scholar OpenURL Placeholder Text WorldCat 38. Han , J. , Xuan , J.L., Hu , H.R., Chen , Z.W.; Protective effect against myocardial ischemia reperfusion injuries induced by hyperoside preconditioning and its relationship with PI3K/Akt signaling pathway in rats ; Zhongguo Zhong yao za zhi= Zhongguo zhongyao zazhi= China journal of Chinese materia medica , ( 2015 ); 40 : 118 – 123 . Google Scholar OpenURL Placeholder Text WorldCat 39. Li , J. , Yan , D., Pan , N.F., Cao , M.M.; Research on the mechanism of active ingredients of Hypericum perforatum resist rapid arrhythmia ; Chinese Medicine Journal , ( 2013 ); 41 : 9 – 10 . Google Scholar OpenURL Placeholder Text WorldCat 40. Hou , J.Y. , Liu , Y., Liu , L., Li , X.M.; Protective effect of hyperoside on cardiac ischemia reperfusion injury through inhibition of ER stress and activation of Nrf2 signaling ; Asian Pacific Journal of Tropical Medicine , ( 2016 ); 9 : 76 – 80 . Google Scholar OpenURL Placeholder Text WorldCat 41. Yan , D. ; Study on the material basis and mechanism of anti-tachyarrhythmia with n-butanol extract of Hypericum attenuatum Choisy; Heilongjiang University of Chinese medicine , Harbin, ( 2012 ). Google Scholar OpenURL Placeholder Text WorldCat 42. Yang , X.Y. , Wang , Z.R., Li , X.J., Jing , Z.H., Ma , S., Yu , D.Q. et al. ; Protective effect of quercetin on secondary cardiomyocyte injury induced by mechanical trauma ; Chinese Heart Journal , ( 2016 ); 28 : 512 – 517 . Google Scholar OpenURL Placeholder Text WorldCat 43. Jiang , D.J. , Du , W.F., Cai , B.C.; Application of spectrum-effect relationship in quality control of traditional Chinese medicine ; China Journal of Traditional Chinese Medicine and Pharmacy , ( 2015 ); 11 : 3811 – 3814 . Google Scholar OpenURL Placeholder Text WorldCat 44. Zeng , L.J. , Lin , B., Song , H.T.; Progress in study of spectrum-effect relationship of traditional Chinese medicine and discussions ; China Journal of Chinese Materia Medica , ( 2015 ); 40 : 1425 – 1432 . Google Scholar OpenURL Placeholder Text WorldCat 45. Zhu , J.J. , Wang , Z.M., Gao , H.M., Liu , X.Q., Yan , L.H., Feng , W.H. et al. ; Advances on quality evaluation of Chinese Materia Medica by QAMS ; Chinese Journal of Experimental Traditional Medical Formulae , ( 2016 ); 22 : 220 – 228 . Google Scholar OpenURL Placeholder Text WorldCat 46. Gao , H.M. , Song , Z.H., Wang , Z.M., Qian , Z.Z., Zhang , Q.W.; A quality evaluation model suitable for the characteristics of traditional Chinese medicine——QAMS research overview ; China Journal of Chinese Materia Medica , ( 2012 ); 4 : 8 – 19 . Google Scholar OpenURL Placeholder Text WorldCat © The Author(s) 2020. Published by Oxford University Press. All rights reserved. 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TI - Using Spectrum-Effect Relationships Coupled with LC–TOF–MS to Screen Anti-arrhythmic Components of the Total Flavonoids in Hypericum attenuatum Extracts
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa101
DA - 2021-02-15
UR - https://www.deepdyve.com/lp/oxford-university-press/using-spectrum-effect-relationships-coupled-with-lc-tof-ms-to-screen-0xqd4GC1pa
SP - 246
EP - 261
VL - 59
IS - 3
DP - DeepDyve
ER -