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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 67  |  Page : 148-155  

A rapid and efficient approach based on ultra-high liquid chromatography coupled with mass spectrometry for identification in vitro and in vivo constituents from shizao decoction


1 Qiqihar Academy of Medical Sciences, The Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar, Heilongjiang, China
2 National Chinmedomics Research Center, Sino-America Chinmedomics Technology Collaboration Center, National Traditional Chinese Medicine Key Laboratory of Serum Pharmacochemistry, Metabolomics Laboratory, Heilongjiang University of Chinese Medicine, Harbin, China

Date of Submission07-Aug-2019
Date of Decision04-Sep-2019
Date of Web Publication11-Feb-2020

Correspondence Address:
Ai-Hua Zhang
National Chinmedomics Research Center, Sino-America Chinmedomics Technology Collaboration Center, National Traditional Chinese Medicine Key Laboratory of Serum Pharmacochemistry, Metabolomics Laboratory, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin
China
Qi Liu
Qiqihar Academy of Medical Sciences, The Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar, Heilongjiang
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_329_19

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   Abstract 


Introduction: Shizao decoction (SZD) is a classic Chinese prescription which used in multiple kinds of ascites and edema caused by liver cirrhosis, chronic nephritis, etc. However, considering edema and new application of cancer ascites SZD is worth further investigating. Materials and Methods: In this paper, a powerful ultra-high liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry based on the PeakView tool combined with multivariate data processing approach was employed to clarify the chemical constituents and the ingredients absorbed into blood after oral administration of SZD. Results: As a result, a total of 91 compounds (35 ions in positive mode and 56 ions in negative mode) of SZD in vitro were successfully identified. Besides, 25 constituents absorbed into blood were tentatively characterized. Among the characterized ingredients, flavonoids, prenol lipids, coumarins, and derivatives and benzopyrans were also detected, respectively. Of note, some constituents absorbed into blood played a critical role in cancer therapy. Conclusion: Established method was suitable for the rapid analysis and characterization of SZD constituents, and it provided meaningful chemical information for further pharmacology study of SZD.

Keywords: Constituents, decoction, mass spectrometry, traditional Chinese medicine, ultra-high liquid chromatography


How to cite this article:
Wang YM, Liu Q, Fu WH, Zhang AH. A rapid and efficient approach based on ultra-high liquid chromatography coupled with mass spectrometry for identification in vitro and in vivo constituents from shizao decoction. Phcog Mag 2020;16:148-55

How to cite this URL:
Wang YM, Liu Q, Fu WH, Zhang AH. A rapid and efficient approach based on ultra-high liquid chromatography coupled with mass spectrometry for identification in vitro and in vivo constituents from shizao decoction. Phcog Mag [serial online] 2020 [cited 2020 Feb 29];16:148-55. Available from: http://www.phcog.com/text.asp?2020/16/67/148/278015



SUMMARY

  • The established ultra-high liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry analysis combined with multivariate data processing approach was suitable for the rapid identification of constituents from SZT in vitro and in vivo.




Abbreviation used: SZD: Shizao decoction; TCM: Traditional Chinese medicine; UHPLC-ESI-Q-TOF-MS: ultra-high liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry; IDA: information-dependent acquisition; IST: ion source temperature; ISVF: ion spray voltage floating; DP: declustering potential; CE: collision energy; GS 1: nebulizer gas; GS 2: auxiliary gas; CUR: curtain gas; BPCs: base peak chromatograms


   Introduction Top


Traditional Chinese medicine (TCM) has made a great contribution to the prevention and treatment of diseases for thousands of years. What makes TCM unique was that it expressed its synergistic effects through multiple components and multiple targets. Therefore, the screening of essential constituents and bioactive components was the first line to further study of TCM. Recently, with the emergence of rapid analytical techniques such as liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and gas chromatography (GC)-MS/MS, and a forceful experimental method named serum pharmacochemistry, the bioactive ingredients of TCM became possible to clarify. Hence, up to now, the bioactive components in rat serum after oral administration of multifarious TCM prescriptions such as Xiaochaihu Tang,[1] FangjiHuangqi Tang,[2] Yin-Chen-Hao-Tang,[3] Simiao Wan,[4] Zhizhu Wan,[5] Kaixin San,[6],[7] and Shengmai San [8],[9],[10] and single herbs such as leaves of Radix Astragali,[11]Phellodendri amurensis cortex,[12]Panax notoginseng,[13]Acanthopanax Senticosus stem,[14]Radix Polygalae,[15] and corn silk [16],[17],[18] have been screened successfully.

Shizao decoction (SZD), a traditional Chinese herb formula from “Shanghan Lun,” one of the four medical collections from ancient China, was composed of four kinds of active Chinese herbs, including Daphne genkwa Sieb. et Zucc. (Yuanhua), Euphorbia kansui T. N. Liou ex T. P. Wang (Gansui), Euphorbia pekinensis Rupr. (Jingdaji), and Fructus Ziziphus jujuba Mill. (Dazao). It was widely used in the treatment of exudative meningitis, tuber culouspleuritis, various kinds of hydrothorax, ascites, and even hyposarca caused by liver cirrhosis, chronic nephritis, and advanced schistosomiasis in past years. However, because of the remarkable toxicity of Gansui,[19] Yuanhua [20] and Jingdaji,[21] SZD was used with extraordinary caution in the clinical study. Recently, SZD has been attracted much more attention in clinical and scientific research fields.

As is well-known that, chemical constituents are the material basis of TCM; however, up to now, the constituents of SZDin vitro andin vivo are not very clear yet. Therefore, the comprehensive research of the constituentsin vitro andin vivo is helpful for the pharmacological and mechanism studies of SZD. In this case, we wonder whether several fulfilling compounds of SZD would be detected in rat serum or not. Hence, a rapid and sensitive ultra-high LC coupled with electrospray ionization quadrupole time-of-flight MS (UHPLC-ESI-Q-TOF-MS) technology combined with an automated multivariate analysis approach based on the serum pharmacochemistry method was employed to rapidly screen and analyze the multiple absorbed bioactive components of SZD after oral administration. These results filled the gaps in the chemical study and provided helpful data for further pharmacological and action mechanism research of SZD.


   Experimental Top


Chemicals, reagents, and materials

Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Merck Company (Darmstadt, Germany). Formic acid (FA) (HPLC grade) was purchased from Fisher Scientific Company (USA). The distilled water (18.2 MΩ) was purified by a Milli-Q system (Millipore, Bedford, MA, USA). Oasis HLB SPE C18 was purchased from Waters Corporation (Milford, USA). Yuanhua, Gansui, Jingdaji, and Dazao were purchased from Harbin Sankeshu Drugstore (Heilongjiang, China) and identified by Doctor Qi Liu, Qiqihar Medical University.

Sample preparation

The dried powder of Gansui, Yuanhua, and Jingdaji was mixed according to a ratio of 1:1:1, and then, 10 times of 50% ethanol were added to the mixture powder and extracted by ultrasonic for twice, each time lasted for 1 h. Then, 1000 g of Dazao was added to 10 times of distilled water and extracted by a reflux extractor for 2 h. Finally, the above two kinds of solutions were mixed and concentrated to 1.0 g/mL by a rotary evaporation at 35°C. At the same time, 10 g of Yuanhua, Gansui, and Jingdaji were extracted through ultrasonic by 10 times of 50% ethanol for twice each time lasted for 1 h. Besides, 10 g of Dazao powder were extracted for 2 h by a reflux by 10 times of distilled water. Then, SZD sample and the individual drug samples were obtained, respectively. Finally, after diluted and filtered through a 0.22 μm membrane, the SZD sample and the individual drug samples were finally injected into the UHPLC-ESI-Q-TOF-MS system.

Animal handling

Twenty-four male Wistar rats (weighing 200 ± 20 g, 20 w) were purchased from the experimental animal center of Qiqihar Medical University. The animals were housed under controlled environmental conditions (temperature of 24°C ± 2°C humidity of 60% ± 5%; 12 h/12 h dark/light cycle) to acclimatize for 1 week. All the rats were fed with standard diet and water ad libitum. After acclimatization, all the animals were divided into four groups of six rats each as follows: dosed Group 1, dosed Group 2, dosed Group 3, and control group. The rats of dosed groups were administrated with SZD extract (1.0 mL/100 g) orally for 7 days. At the same time, the rats of the control group were administrated with CMC-Na solution alone under the same conditions. After the administration of 30 min, 60 min, and 90 min, rats of dosed Group 1, dosed Group 2, and dosed Group 3 were deeply anesthetized by 3% pentobarbital sodium and then sacrificed, respectively. Blood was collected from the abdominal aorta and placed for 1 h after centrifuged at 4000 rpm for 20 min at 4°C; the supernatant serum was then separated. Finally, the obtained serum was stored at −80°C until analysis. All animal care and experimental procedures were carried out in compliance with the Institutional Animal Care and Use Committee of Qiqihar Medical University.

Preparation of serum sample for ultra-high liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry/mass spectrometry analysis

After thawed, all the serum samples were centrifuged at 13,000 rpm for 10 min at 4°C. Then, 40 μL of phosphoric acid was added to 1 mL of serum sample and vortex-mixed for 60 s. After that, the mixed solution was extracted by an Oasis HLB solid-phase extraction C18-column (Waters, USA), which was preactivated by 2 mL of methanol and 2 mL of distilled water. Then, 1 mL of 100% distilled water was used to wipe off the impurity, and 2 mL of 100% methanol was used to eluting the aimed ingredients. Afterward, the collected eluent was dried under a stream of nitrogen gas at 45°C, and each dried sample was further redissolved in 200 μL of 80% methanol. Finally, the solution was centrifuged at 13,000 rpm for 10 min at 4°C and then filtered through a 0.22 μm membrane, 3uL of serum sample was injected into the UHPLC-ESI-Q-TOF-MS/MS system. Besides, all the serum samples were equally mixed to gain a QC sample, which was injected each five injections during the detection.

Ultra-high liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry/mass spectrometry conditions

The analysis was performed on an ultra-performance LC-30A system (Shimadzu Company, Japan) coupled with an accurate tripleTOF 4600 mass system (AB SCIEX, USA) equipped with an electrospray ion source interface. The chromatographic separation was conducted on an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 um, Waters Corporation, Milford, USA) at 40°C with a velocity of 0.4 mL/min. The mobile phase was composed of solvent A (0.1% FA in acetonitrile) and solvent B (0.1% FA in water) under gradient elution conditions: 0.01–3.00 min, 1%–10% A; 3.00–9.00 min, 10%–30% A; and 9.00–18.00 min, 30%–100% A. The injection volume was set at 3 μL.

After chromatographic separation, the column eluent of each sample was further analyzed in positive ion mode and negative ion mode. During the experiment, a comprehensive acquisition method named information-dependent acquisition (IDA) was employed to gain TOFMS and MS/MS data in one injection. The parameters of TOFMS under a positive ion mode were as follows: ion source temperature (IST) was maintained at 600°C, ion spray voltage floating (ISVF) was set at 5.5 KV, declustering potential (DP) and collision energy (CE) were maintained at 100V and 10V, respectively. Pressures of nebulizer gas (GS 1), auxiliary gas (GS 2), and the curtain gas (CUR) were maintained at 55 psi, 55 psi, and 30 psi, respectively. The TOFMS accumulation time was 150 ms, and the complete scan was performed within m/z 100–1000 Da. Besides, the parameters of IDA-MS/MS were as below: DP, CE, and CES were 100V, 40V, and 20V, respectively. The MS/MS accumulation time was run within 80 ms. The parameters under negative ion mode were as follows: IST was maintained at 600°C, ISVF was set at −4.0KV, DP, and CE were set at 100V and 10V, pressures of GS 1, GS 2, and CUR were set at 65 psi, 65 psi, and 30 psi, respectively. CE and CES were maintained at −40V and 20V. During the IDA-MS/MS analysis, a dynamic background subtracting method which could differentiate the background and matrix-related MS/MS ions from endogenous or exogenous components intelligently was employed. Moreover, the top ten strongest fragment ions of more than 100 cps during 50–1000 amu in every accumulation time were given priority to matching with the IDA criteria. In addition, calibration was operated through a Calibrant Delivery System each five samples. All the acquisitions were conducted by Analyst TF 1.7.1 software (AB Sciex Corporation, CA, USA).

Data analysis

All obtained data files were processed using Progenesis QI 2.0 (Waters Corporation, Milford, MA, USA). After the data loading forms (POS: +H, +NH4, +Na, +K, 2M+H; NEG:-H, +Cl, +FA-H) were selected, the data were further processed by peak matching, peak alignment, and peak extraction, and then, a matrix containing retention time, mass number, and peak intensity information were obtained. Then, a list including compound's name, molecular formula and m/z of constituents from SZD were imported into Progenesis SDF Studio and a new SDF database was established successfully.. Next, after matching by MS, MS/MS information and confirmed by Progenesis QI, the chemical constituents of SZD were finally identified. As the same way, constituents of Yuanhua, Gansui, Jingdaji, and Dazao samples were identified, respectively. After comparing the identified results with each other, the constituents source was located. Next, data files of three dosed groups and a control group were imported into Progenesis QI 2.0 software, after peak matching, peak alignment and peak extraction, ions which present in dosed groups but absent in control group were regarded as the potential. Finally, the detail information of constituents migrating into blood were found out and finally identified by matching with MS, MS/MS, and network databases.


   Results and Discussion Top


Based on the satisfying acquisition conditions, the approving chromatography and mass spectrum results were orderly obtained. The base peak chromatograms (BPCs) of SZD, Yuanhua, Gansui, Jingdaji, and Dazao were shown in [Figure 1]. As the results displayed, 91 peaks in SZD containing flavonoids, fattyacyls, coumarins and derivatives, organic compounds, organooxygen compounds, carboxylic acids and derivatives, imidazopyrimidines, etc., were successfully identified in sequence using the UHPLC-ESI-Q-TOF-MS/MS technique. The multiple classes of constituents revealed the complexity of TCM prescription. Among the identified constituents of SZD, 40 of them were flavonoids, accounting for 44% of the identified constituents. In the second place, 9 of them were fatty acyls, accounting for 10%. This revealed that flavonoids and fatty acyls may play an important role in SZD. Moreover, among the identified constituents, 85 constituents were from Yuanhua, accounting for 93% of the detected compounds, indicating that Yuanhua may play a critical role in the constituents of SZD. Twenty-six constituents were from Gansui, 20 constituents were from Dazao, and 18 constituents were from Jingdaji. Besides, 12 constituents including L-Proline, 7,8-dihydroxy-4-methylcoumarin, adenine, norharmane, 9-oxo-10E,12Z,15Z-octadecatrienoic acid, heptadecanoic acid, 9,12-Octadecadiynoic acid, monolinolenin (9c, 12c, 15c), 13-keto-9Z,11E-octadecadienoic acid, oleamide, erucamide, and asperulosidic acid were the common constituents from Yuanhua, Gansui, Jingdaji, and Dazao. The detail information of the identified results of SZD was shown in Table S1.
Figure 1: The base peak chromatograms of Shizao decoction (a: POS and f: NEG), Yuanhua (b: POS and g: NEG), Gansui (c: POS and h: NEG), Jingdaji (d: POS and i: NEG), and Dazao (e: POS and j: NEG) under. POS: Positive ion mode, NEG: Negative ion mode

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The control serum sample, dosed serum of 30 min, 60 min, and 90 min were orderly analyzed, and the BPCs under positive ion mode and negative ion mode were shown in [Figure 2]. At first sight, there were no significant differences among the peaks of control serum sample and dosed serum samples in [Figure 2] at first sight, because that BPC was a simplified visual map of total ion chromatogram which owns continuous point of the strongest ions in the mass spectrum at each time point, and it was mainly employed to better understanding the contour differences among different samples. Besides, the content of constituent migrating into blood was very little and owns extremely low intensity. Hence, we could not found obvious differences in BPCs in [Figure 2]. The data of control serum, dosed serum of 30 min, 60 min, and 90 min were processed by Progenesis QI 2.0 software and PeakView software. The results of dosed serum of 30 min, 60 min, and 90 min were severally compared with control serum. Take the identification process of peak 4 detectedin vitro and in vivo, for example, under positive ion mode, the retention time was 4.07 min, and the m/z was 193.0504. The ion's molecular formula was confirmed as C10H8O4 through speculated through formula finder based on the elemental composition and fractional isotope abundance. Next, the unsaturation degree was calculated as 7, suggesting that it may be a ring compound. Moreover, the main MS/MS fragments were m/z 175, m/z 147, m/z 131, m/z 119, m/z 91, m/z 77, and m/z 131, indicating that the surplus fragments may be −C10H7O3+, −C9H7O2+, −C9H7O+, −C9H11+, −C6H3O+, −C6H5+, and −C4 HO+, respectively. At last, after matching with online and native databases, the structure was finally confirmed, and it was identified as 7,8-dihydroxy-4-methylcoumarin. The detail MS/MS fragments of 7,8-dihydroxy-4-methylcoumarin and other typical constituents were displayed in [Figure 3].
Figure 2: The base peak chromatograms of control serum sample (a: POS and e: NEG), dosed serum of 30 min (b: POS and f: NEG), 60 min (c: POS and g: NEG) and 90 min (d: POS and h: NEG) under. POS: Positive ion mode, NEG: Negative ion mode

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Figure 3: The classification and proportion of constituents and the detail mass spectrometry/mass spectrometry fragments of several constituents migrating into blood of Shizao decoction

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Based on the identification results, 25 constituents including 7,8-dihydroxy-4-methylcoumarin, phomoeuphorbin C, helioscopinolide A, neochlorogenic acid, asperulosidic acid, 7,8-dihydroxycoumarin, phomoeuphorbin A, ent-Robinetinidol, 2-hydroxycinnamic acid, luteolin-4'-O-glucoside, hyperoside, scutellarin, oroxin A, quercetin 3-O-beta-D-glucose-6'-acetate, apiin, pratensein-7-O-glucoside, baicalin, genkwanol A, luteolin 7,3'-dimethyl ether 5-glucoside, cosmosiin, kaempferol 7-O-alpha-L-rhamnopyranoside, tiliroside, 3-epilitsenolide D2, dihydroxy-2-(4-hydroxyphenyl) chromen, and hispidulin were figured out to be the constituents absorbed into blood. Among the constituents migrating into blood, 51.7% of them were flavones, 10% were coumarins and derivatives, 10% were prenol lipids, and 9% were benzopyrans, besides, there were still other kinds of constituents such as lactones, organooxygen compounds, cinnamic acids and derivatives, isoflavonoids and 2-arylbenzofuran flavonoids compounds. However, the constituents unmigrating into blood of SZD mainly containing fatty acyls, carboxylic acids and derivatives, imidazopyrimidines, indoles and derivatives, etc. The results indicate that flavones, coumarins, and derivatives, prenol lipids, and benzopyrans of SZD were much more easily migrating into blood in comparison to other kinds of constituents. The proportions of constituents migrating into blood were shown in [Figure 3]. The more detail constituents information was shown in [Figure 4].
Figure 4: The detail compounds information covering the origin, names, migrating to blood or not, and classification of constituents from Shizao decoction in rat serum

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Moreover, we are surprised to find that the contents of constituents migrating into blood among dosed serum of 30 min, 60 min, and 90 min were interesting. The contents of flavonoids including oroxin A, baicalin, kaempferol 7-O-alpha-L-rhamnopyranoside, luteolin 7,3'-dimethyl ether 5-glucoside, scutellarin, cosmosiin, genkwanol A, and hispidulin, and some other class compounds such as 3-Epilitsenolide D2, asperulosidic acid, helioscopinolide A, 7,8-dihydroxycoumarin, and 7,8-dihydroxy-4-methylcoumarin were rapidly increased to the highest and then sharply decreased in the whole period. However, several flavonoids such as apiin, tiliroside, luteolin-4'-O-glucoside, pratensein-7-O-glucoside, and ent-Robinetinidol, and other compounds such as neochlorogenic acid and 2-hydroxycinnamic acid were hardly present in the front 60 min but promptly increased between 60 min and 90 min. Besides, the two benzopyrans named phomoeuphorbin A and phomoeuphorbin C were first raised then descend and finally raised again. The constituents migrating into blood indicated that the content in different times maybe related with the compound structure and polarity. Take the overall consideration, to detect most constituents, the time point of blood collection may be selected at 60 min. The detail results were displayed in [Figure 5].
Figure 5: The content trends of constituents migrating into blood after 30, 60, and 90 min. : Flavonoids compounds; : Lactones compounds; : Prenol lipids compounds; : Organooxygen compounds; : Coumarins and derivatives compounds; : Cinnamic acids and derivatives compounds; [INSIDE:7]: Benzopyrans

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Besides, it is surprised that the majority of constituents migrating into blood showed bright-eyed similar pharmacological actions, especially like anti-cancer. Here, we gave an outline of the modern pharmacological actions of 7,8-dihydroxycoumarin, hyperoside, and scutellarin. 7,8-dihydroxycoumarin, also known as daphnetin, was an active ingredient extracted from Daphne Korean Nakai, which usually used for thromboangiitis obliterans and coronary heart diseases. Except for this, modern pharmacological studies showed that 7,8-dihydroxycoumarin expressed distinct activity action in cancer. Kumar et al.[22] investigated the antiproliferative and chemotherapeutic actions of 7,8-dihydroxycoumarin against on dimethylbenz (a)anthracene-induced mammary carcinoma SD rats. The results showed that, 20 mg/kg, 40 mg/kg, and 80 mg/kg7,8-dihydroxycoumarin had different levels in alleviating the estrogen synthesis, tumor growth, proliferation markers like Ki-67 and proliferating cell nuclear antigen, cytokines such as interleukin 10 (IL-10), IL-1beta and IL-12, MCP-1chemokine, the expressions of ERalpha, PR, EGFR, IGF1R, p-MAPK1/2, p-JNK1/2, p-Akt, and 17beta-HD1 in mammary carcinoma bearing model rats in a dose-dependent manner. Furthermore, Kumar et al.[23] also verified that 7,8-dihydroxycoumarin could against mammary cancer by Nrf-2-Keap1 pathway and NF-kappaB expressions. Besides, Fukuda et al.[24] revealed that 7,8-dihydroxycoumarin reduced the numbers of intracellular stress fibers, and filopodia obviously, decreased the expression levels of RhoA and Cdc42dramatically, so that to hold back the invasion and migration of LM8 cells to against the metastatic cancer. Besides, another hot ingredient migrating into blood is hyperoside, a flavonol glycosides often acted out various actions in anticancer,[25],[26],[27] anti-inflammation,[28],[29] anti-oxidant,[30] etc.[31],[32],[33] Zhangand Zhang [34] found that after the intervention of hyperoside, the cell viability and proliferation of titanium particle-induced damage was enhanced, the apoptosis and autophagy of MC3T3E1 cells were dramatically restrained by regulating the TWEAKp38 pathway, indicating that hyperoside may be a potential protective agent for bones. Li et al.[35] illustrated that both hyperoside and let-7a-5p had a synergetic effect on suppressing the proliferation of A549 cells, suggesting that the cooperation of hyperoside and microRNA-let7a-5p may provide a novel means for the treatment of lung cancer. Scutellarin, a flavonoid compound firstly extracted from a Chinese herb named Scutellaria altissima L., is usually applied in cerebrovascular diseases to reduce the cerebral vascular resistance, improve cerebral blood circulation, increase cerebral blood flow, and reatrain platelet aggregation. Recently, studies have shown that scutellarin owns a variety of activities such as anticancer,[36] antidiabetes,[37] and anti-atherosclerosis.[38],[39] Liu et al.[40] studied the effect of scutellarin on the proliferation and invasion of hepatocellular carcinoma cells through proliferation, colony-forming, apoptosis and cell migration assays, the result showed that scutellarin could suppress invasiveness of HepG2 and MHCC97-H cellsin vitro by remodeling their cytoskeleton through inhibiting the process of EMT, so that to down-regulated the JAK2/STAT3 pathway. Furthermore, Cao et al.[41] discussed the inhibiting effect of scutellarin on A549 lung adenocarcinoma cells, the data showed that scutellarin restrained the proliferation of A549 cells depend on concentration and time, caused important G0/G1 phase arrest and apoptosis, reduced the level of pan-AKT, phosphorylated (p)-mTOR, mTOR, BCL-XL, STAT3 and p-STAT3, increased the level of 4EBP1, indicating that scutellarin impeded the proliferation and promoted apoptosis of A549 cells through AKT/mTOR/4EBP1 and STAT3 pathways. Sun et al.[42] explored that scutellarin could significantly stimulate phosphorylation of extracellular-regulated kinases 1/2 (ERK1/2), down-regulated the expression of p-AKT to induce the apoptosis and autophagy of non-small cell lung cancer through ERK1/2 and AKT signaling pathways. The study of pharmacodynamic material basis is crucial to elucidate the effect mechanism [43],[44],[45],[46],[47],[48],[49],[50] and might provide insight into the characteristics for pharmacological effects.[51],[52],[53],[54],[55],[56] In summary, the bioactive constituents migrating into blood such as 7,8-dihydroxycoumarin, hyperoside, and scutellarin expressed multiple activities, especially in anticancer activity. This may work in concert with the modern specific application in cancer of SZD. Hence, 7,8-dihydroxycoumarin, hyperoside, and scutellarin may be the critical constituents even likely to be the pharmacodynamic material basis of SZD in anticancer. However, the suppositional argument needs to be further verified in pharmacological experiment in animals and cells.


   Conclusion Top


In this paper, a reliable and sensitive method named UHPLC-ESI-Q-TOF-MS coupled with an automated multivariate analysis approach was applied in the identification and characterization of multiple constituents from SZD. Using this method, 91 compounds of SZD were identified and 25 of them were tentatively characterized. Among the constituents of the SZD sample in vitro, most of them were flavonoids. Meantime, among the constituents migrating into blood, more than half of them were flavones as well. These flavones constituents owned multiple activities, especially like anticancer. The result showed the flavones of SZD migrating into blood, especially such as 7,8-dihydroxycoumarin, hyperoside, and scutellarin may play a critical role in pharmacodynamic material basis of SZD in anticancer. Since this paper offered a powerful basis for further pharmacological studies in animals and cells. Anything else, the results also demonstrated that UHPLCESI-Q-TOFMS was a powerful analytical tool in the study of chemical constituents of herbal medicinein vitro and in vivo. It is worth mentioning that this was the first study of chemical constituents and absorbed bioactive components of SZD. The results not only filled the gaps of the fundamental constituent research of SZD but also offered useful information for further study of pharmacology and mechanism of SZD.

Financial support and sponsorship

The present study was financially supported by Foundation of Heilongjiang Education Department (Grant No. 2018-KYYWF-0080), the projects of Qiqihar Medicinal University (QY2016B-23, QY2016M-13), Key Program of Natural Science Foundation of State (Grant No. 81973745, 81302905), Young Talent Lift Engineering Project of China Association of TCM (QNRC2-B06), Natural Science Foundation of Heilongjiang Province (YQ2019H030), Foundation of Heilongjiang University of Chinese Medicine (2018jc01).

Conflicts of interest

There are no conflicts of ineterest.



 
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