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  • 1 Department of Pharmaceutical Analysis, School of Pharmacy, Guizhou Medical University, Guiyang 550025, Guizhou, PR China
  • | 2 Department of Pharmacology, School of Basic Medicine, Guizhou Medical University, Guiyang 550025, Guizhou, PR China
  • | 3 Center for Drug Research and Innovation, Guiyang Dechangxiang Pharmaceutical Co., Ltd., Guiyang 550201, Guizhou, PR China
Open access

Abstract

Dendrobium nobile and Dendrobium officinale as the main varieties of traditional Chinese medicine Dendrobium are widely used in clinic. The study aimed to systematically explore chemical constituents and their antitumor effect of D. nobile and D. officinale by ultra-performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry (UPLC-IT-TOF), network pharmacology and cancer cell experiments. D. nobile extract and D. officinale extract could significantly inhibit the proliferation of human lung cancer A549 cells, human liver cancer HepG2 cells and human breast cancer MCF-7 cells in the dose-dependent manner (P < 0.05), the antitumor effect of D. officinale extract was stronger than that of D. nobile extract at the same drug concentration. A total of 40 chemical constituents of D. nobile and D. officinale including phenanthrenes, bibenzyls and other types of compounds had been identified by UPLC-IT-TOF, LCMSsolution and MetID software according to retention times, accurate mass, MSn fragmentation, reference compounds and natural product databases. Phenanthrenes with good antitumor activity were mainly present in D. nobile, bibenzyls were the main compounds of D. officinale. Integrated networks of Herb-Compounds-Targets-Cancer revealed that gigantol, moscatilin, tristin, moscatin and densiflorol B were regarded as key antitumor compounds of D. nobile and D. officinale, D. nobile and D. officinale shared 7 targets accounting for 70% of the antitumor core targets, more than half of their antitumor KEGG pathways were similar. The results of molecular docking and western blotting experiments indicated that the antitumor mechanisms of D. nobile and D. officinale may be through inhibiting PI3K-Akt and HIF-1α signaling pathways.

Abstract

Dendrobium nobile and Dendrobium officinale as the main varieties of traditional Chinese medicine Dendrobium are widely used in clinic. The study aimed to systematically explore chemical constituents and their antitumor effect of D. nobile and D. officinale by ultra-performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry (UPLC-IT-TOF), network pharmacology and cancer cell experiments. D. nobile extract and D. officinale extract could significantly inhibit the proliferation of human lung cancer A549 cells, human liver cancer HepG2 cells and human breast cancer MCF-7 cells in the dose-dependent manner (P < 0.05), the antitumor effect of D. officinale extract was stronger than that of D. nobile extract at the same drug concentration. A total of 40 chemical constituents of D. nobile and D. officinale including phenanthrenes, bibenzyls and other types of compounds had been identified by UPLC-IT-TOF, LCMSsolution and MetID software according to retention times, accurate mass, MSn fragmentation, reference compounds and natural product databases. Phenanthrenes with good antitumor activity were mainly present in D. nobile, bibenzyls were the main compounds of D. officinale. Integrated networks of Herb-Compounds-Targets-Cancer revealed that gigantol, moscatilin, tristin, moscatin and densiflorol B were regarded as key antitumor compounds of D. nobile and D. officinale, D. nobile and D. officinale shared 7 targets accounting for 70% of the antitumor core targets, more than half of their antitumor KEGG pathways were similar. The results of molecular docking and western blotting experiments indicated that the antitumor mechanisms of D. nobile and D. officinale may be through inhibiting PI3K-Akt and HIF-1α signaling pathways.

Introduction

Dendrobium species are famous medicinal plant all over the world, original records of their pharmacodynamic effects date back to Shen Nong's Herbal Classic (Shen Nong Ben Cao Jing) in the Eastern Han Dynasty of China, one of the four classic works of traditional Chinese medicine (TCM). The TCM theory suggests that Dendrobium species contains the effects of benefiting the stomach, moisturizing the lungs to relieve cough, promoting the production of body fluids and clearing heat. Modern medical research shows that Dendrobium species have antitumor, deglycemic and enhancing-immunity effects [1–3]. Dendrobium nobile and Dendrobium officinale are two frequently-used Dendrobium species, their chemical constituents are diverse including phenanthrenes, bibenzyls, flavonoids, alkaloids, phenols, terpenoids and polysaccharides [4–7]. D. nobile and D. officinale have been used in clinical cancer patients for adjuvant treatment of malignant tumors, D. officinale as more expensive of Dendrobium species contains more polysaccharides than D. nobile. Although previous pharmaceutical scholars had tried to investigate and compare the antitumor effect of D. nobile and D. officinale, the antitumor compounds and the pharmacological mechanisms of D. nobile and D. officinale were still unclear because of their complex, similar natural compounds, multiple target and multiple signal pathway pharmacological mechanisms.

Ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was a powerful tool for characterizing unknown compounds in complex natural medicines and clarifying the multiple compound, multiple target pharmacological mechanisms [8–10], but there were some characteristic compounds such as phenanthrenes had not been effectively distinguished and identified by less MS information, fragment ions of conventional mass spectrometry such as UPLC-QQQ, UPLC-Q-TOF. Among a variety of analytical MS platforms, the hybrid ion trap time-of-flight mass spectrometer (IT-TOF) was configured with the multistage fragmentation function in ion trap full scan mode with high resolution and sensitivity in both positive and negative modes, suggesting the potential to provide more accurate structural information for structural analysis of similar organic molecules. Natural product databases including TCMSP database, SIOC chemical database and Reaxy database were also applied to provide strong assistance to IT-TOF analysis [8]. Network pharmacology is a new product of multidisciplinary integration, a multi-level network among drugs, targets, signal pathways and diseases is constructed to fully clarify pharmacodynamic substances and potential pharmacological mechanism of complex natural medicines and TCMs through the analysis and computer simulation of high-throughput omics data in various databases [11, 12]. Molecular docking is a key tool in structural molecular biology and computer-assisted drug design, can be used to perform virtual screening and propose structural hypotheses of how the ligands inhibit the targets [13]. The above means can help us to better understand the material basis and pharmacological mechanisms of complex drugs.

Hence, the present study aimed to conduct a detailed comparison of the chemical constituents and antitumor mechanisms between D. nobile and D. officinale by integrating UPLC-IT-TOF analysis, network pharmacology, molecular docking and experimental verification. The investigation would assist us to deeply realize therapeutical effect of D. officinale and D. nobile and promote their new drug research and development.

Experimental

Materials

D. nobile in Guizhou and D. officinale in Guizhou were collected from Guiyang Taisheng Chinese herbal medicine market, Guiyang city in Oct. 2020, identified by Associate Professor Shaohuan Liu from Department of Pharmacognosy, School of Pharmacy, Guizhou Medical University. D. nobile sample and D. officinale sample were both ground into powder of the homogenous 24 mesh separatively before the experiment.

Human non-small cell lung cancer A549 cell, human liver cancer HepG2 cell and human breast cancer MCF-7 cell within 5 generations of cell algebra were purchased from China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. Anti-PI3K (cat. 4249S), anti-p-PI3K (cat. 4228T), anti-p-AKT (cat. 9271S) and anti-HIF-1α (cat. 36169T) were purchased from Cell Signaling Technology, Inc. Anti-AKT (cat. Abs131788) was supplied by Absin Bioscience, Inc. Anti-GAPDH (cat. 10494-1-AP-100) was from Proteintech Group, Inc. Naringenin, densiflorol B, moscatin and nobilone (>98% purity) were previously purified from D. nobile in our laboratory and identified by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry [14, 15]. Acetonitrile (HPLC-pure, Merck, Germany), methanol (HPLC-pure, Merck, Germany), formic acid (HPLC-pure, Sinopharm, Shanghai, China), pure water (Watsons, Guangzhou, China).

Sample preparation

About 90.0 g of D. nobile or D. officinale was soaked separately in ethanol for 24 h at a solid-liquid ratio of 1 : 6 (g : mL), then extracted three times by reflux ethanol for 2 h each time in a 90°C water bath. A total of three reflux extract solution was filtered and merged, D. nobile extract concrete and D. officinale extract concrete were finally prepared one by one through concentration under reduced pressure.

50–800 mg mL−1 extract ethanol solution of D. nobile or D. officinale were dispensed for antitumor activity screening. Before cell experiment, these extracts were diluted into 50–800 μg mL−1 extract solution in basic medium containing 0.1% ethanol.

The final concentration of 1.5 mg mL−1 D. nobile sample methanol solution and 1.5 mg mL−1 D. officinale sample methanol solution were prepared successively for HRMS analysis, filtered through 0.22-μm lipophilic microporous filters before UPLC-IT-TOF analysis. The reference standard solutions were mixed together to prepared a final mixed standard solution with the concentration of 10 μg mL−1.

Antitumor activity screening of D. nobile and D. officinale

Human non-small cell lung cancer A549 cell, human liver cancer HepG2 cell and human breast cancer MCF-7 cell were selected as this disease model for antitumor screening. These three kinds of cells in the exponential growth phase were inoculated into 96-well plates contained a cell density of 8.0 × 103 per well with five duplicates, cultured in a cell incubator at 37°C and 5%CO2 conditions. After culturing for 24 h, the cells were treated with different concentrations of D. nobile extracts and D. officinale extracts (50–800 μg mL−1) for 24h respectively, 0.1%-ethanol cell culture medium was set up as blank control group. Further culturing for 24 h in the cell incubator, 15 μL of 5% MTT solution was added to each well in the dark and the 96-well plates were continuously incubated at 37°C, 5%CO2 for 4 h. Subsequently, the supernatant was discarded and 150 μL DMSO was added to each well. The optical density (OD) of each well at the detection wavelength of 490 nm was determined by a microplate reader. The inhibition rates (%) were calculated as (OD blank ‒ OD sample)/(OD blank) × 100%.

UPLC-IT-TOF analysis of D. nobile and D. officinale

An UPLC-IT-TOF apparatus (Shimadzu, Kyoto, Japan) equipped with a diode array detector (SPD-M20A) and a tandem mass spectrometer with an electrospray ionization (ESI) source coupled to ion trap (IT) and time of flight (TOF) mass analyzers was applied for rapidly identifying the major antitumor constituents of D. nobile and D. officinale. The UPLC separation was achieved on a Dikma-C18 column (100mm × 2.1 mm, 1.8 μm) at column temperature 30°C, the mobile phase was consisted of 0.1% formic acid (A) and acetonitrile (B). A binary gradient elution was performed as follows: 0–10min, 80%(A)-20%(B) to 50%(A)-50%(B); 10–20min, 50%(A)-50%(B) to 5%(A)-95%(B); 20–35min, 5%(A)-95%(B); 35–40min, 5%(A)-95%(B) to 80%(A)-20%(B); 40–45min, 80%(A)-20%(B). The flow rate was 0.2 mL min−1, injection volume was 5 μL by automatic sampling system.

IT-TOF MS instrumental parameters were set as follows: The electrospray ionization (ESI) source was set at both positive and negative ionization modes through a single sample injection, multigrade MS data (event 1 to event 6) were collected in positive ion pattern (event 1 to event 3, +MS1 → +MS2 → +MS3) and negative ion pattern (event 4 to event 6, −MS1 → −MS2 → −MS3). Ultra-high-purity Ar was used as the collision gas and high-purity N2 as the nebulizing gas. Interface voltage +4.5 kV for the positive mode and −3.5 kV for the negative mode, detector voltage 1.65 kV, RP vacuum 70.0 Pa, IT pressure 1.8 × 10−2 Pa, TOF pressure 1.7 × 10−4 Pa, drying gas 100.0 kPa, N2 flow 1.5 mL min−1, curved desorption line (CDL) temperature 200°C, heat block temperature 200°C, equipment temperature 40°C, loop time 2.96 ms. The scanned mass range was from m/z 100 to m/z 800 using an ion accumulation time of 20 ms per spectrum, scanning threshold 2000, collision energy of the secondary CID, 50% for MS2 and 100% for MS3. The MS instrument was tuned daily by a standard solution of sodium trifluoroacetate before sample analysis, the mass deviation of the instrument was within 10 ppm. Composition Formula Predictor in LCMSsolution 3.81 was used to predict the molecular formulas with less than 15 ppm deviation limit from measured mass spectrometry values.

An intelligentized strategy was proposed for rapid qualitative analysis of antitumor extract of D. nobile and D. officinale respectively by UPLC-IT-TOF and MetID solution software. An in-house database of chemical compounds of D. nobile and D. officinale which contains names, molecular formulas, CAS, InChI Key, accurate molecular weight, chemical structures and MSn fragmentation profiles was established from TCMSP database (https://tcmsp-e.com/), SIOC chemical database (www.organchem.csdb.cn), Reaxys, CNKI, Pubchem and ChemSpider. MetID software (Shimadzu) combined with this in-house database was applied to assist us through numerous evaluation principles which were peak picking, positive and negative MS1 precursors analysis, mass defect filter (MDF) technique, isotopic peak matching, molecular formula prediction.

Network pharmacology analysis

The SMILES format of each identified compound was imported into the SwissADME database to evaluate DL (druglikeness) and GIA (gastrointestinal absorption). When the prediction results of identified chemical compounds in D. nobile or D. officinale through UPLC-IT-TOF analysis both met the rules that “high” in GIA and ≥2 of 5 filters (Lipinski, Ghose, Veber, Egan and Muegge) in DL, they would be regard as candidate compounds [16, 17]. Then the SMILES and SDF formats of the above screened compounds would be imported into the SwissTargetPrediction database and PharmMapper Server with the species limited to “Homo sapiens” for target prediction. Putative genes associated with cancer were retained from GeneCards database using “cancer” as a keyword and screening genes with “relevance score” of disease ≥5. The STRING database was applied to explore the protein-protein interaction (PPI) based on the overlapping targets between candidate compounds and the cancer-related targets. In order to ensure the credibility of the results, the score of protein interaction was set as “high Confidence ≥0.700”. Cytoscape 3.7.2 software was utilize to visualize the chemical-target network and PPI network. Then, the KEGG pathway enrichment analysis was accomplished (P<0.05) through the DAVID database. The pharmacology network of “Herb-Compounds-Targets” was finally constructed to screen the key antitumor compounds in D. nobile or D. officinale.

Molecular docking analysis

Molecular docking had been carried out by Autodock vina 1.1.2 software, the 3D structure of target proteins predicted through network pharmacology analysis were download from the RCSB protein database. The solvent molecules and original ligands in target proteins were cleaned up for the naked protein, which was further hydrogenated and checked for charge, the PDBQT files of target proteins and active compounds were also prepared by the way. Each pocket was defined for the respective original ligand active site, the optimal conformation of docked models was all visualized and analyzed by the software PyMOL 2.4.0 and Proteins Plus.

Western blotting

Experimental validation of antitumor pharmacological mechanisms was performed based on potential target proteins found from the work above, A549 cells were seeded in 6-well plates at a density of 5 × 105 cells per well. The cells were treated with different concentrations of D. nobile extract and D. officinale extract for 24 h, and then total protein was extracted and measured using a BCA Protein Assay Kit. 20 μg of protein was separated on 10% sodium dodecylsulphate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. After the membranes were blocked with 5% non-fat milk and washed three times with TBST for 5 min each, the first antibodies were added at the dilution (1:1000) and incubated at 4°C overnight. Then, the membrane was incubated with secondary antibody (1:5000) for 1h at room temperature. Finally, enhanced chemilumunescence reagents (ECL) were utilized to develop color, the protein signals were imaged using a Bio-Rad ChemiDoc XRS+ imaging system. GAPDH was used as internal controls for the amounts of target proteins.

Statistical analysis

Statistical analysis was performed using SPSS 22.0, the measurement data were expressed as the mean ± standard deviation (SD), one-way ANOVA was used to compare the group differences, statistical significance was set at P < 0.05.

Results and discussion

Antitumor effect of D. nobile and D. officinale

Different concentrations of extract solution from D. nobile and D. officinale could obviously inhibit the proliferation of A549 cell, HepG2 cell and MCF-7 cell in dose-dependent manner, this in-vitro antitumor effect of D. nobile extract or D. officinale extract showed statistically differential with P<0.01 when greater than 100 μg mL−1 of the Dendrobium extract. D. officinale extract showed better antitumor effect than D. nobile extract at the same mass concentration (Fig. 1), these two Dendrobium extracts were more inhibitory on A549 lung tumor cell than the other two tumor cells.

Fig. 1.
Fig. 1.

The inhibitory effect of D. nobile extract (50–800 μg mL−1) and D. officinale extract (50–800 μg mL−1) on the proliferation of A549 cells (A), HepG 2 cells (B) and MCF-7 cells (C) in a dose-dependent manner respectively

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Antitumor chemical constituents of D. nobile and D. officinale

All exact molecular weights, all positive ion and negative ion TICs, the multi-level daughter ion fragments of antitumor extracts of D. nobile and D. officinale have been collected by data dependent acquisition (DDA) detection module of UPLC-ESI IT-TOF (Figure S1), the deduced molecular formulas from detected exact molecular weights with 4 decimal places were then easily calculated using Accurate Mass Calculator in LCMSsolutions 3.81 and MetID 1.2 software, error margin within 15 ppm and nitrogen rule were adopted in this study, these preliminarily-identified compounds were further confirmed by chromatographic retention time (t R), matching with the MSn fragmentation patterns, reference compounds and MetID 1.2 software. Some compounds with the same molecular formula can be distinguished by chemical polarity differences and literature comparison. As a result, there were 40 natural compounds in D. nobile and D. officinale had been finally identified, including phenanthrenes, bibenzyls, glycosides, fluorenones and flavonoids.

Phenanthrenes

Phenanthrenes are characteristic type of compounds in D. nobile and D. officinale, shows diverse activities involving antitumor, anti-inflammatory and antioxidant effects. Eleven phenanthrenes were characterized from D. nobile and D. officinale.

DN-Cp.26 as a peak in extracted ion chromatogram (EIC) in t R 11.858 min, collected its multistage fragment ion in negative ion mode and positive parent ion (Figure S2), the positive protonated precursor ion +MS1 peak m/z 255.0643 [M+H]+ (calcd. C15H11O4 +, mass error 3.48 ppm), the negative deprotonated precursor ion 253.0511 [M−H] (calcd. C15H9O4 , mass error 1.84 ppm). −MS2 m/z 238.0266 and −MS2 m/z 225.0582 as primary fragment ions were produced by the loss of −CH3, −CO from [M−H], the latter was further cleaved −CH2, −CH3 to obtain −MS2 m/z 211.0385 and −MS2 m/z 210.0315, −MS3 m/z 182.0351 was derived from 211.0385 by cleavage of −CHO. DN−Cp.26 was confirmed as densiflorol B. DN−Cp.27 in D. nobile was separated as a peak in t R 12.023 min, obtained its positive and negative ion mode data, the positive protonated precursor ion +MS1 peak m/z 241.0836 [M+H]+ (calcd. C15H13O3 +, mass error 9.67 ppm), +MS1 peak m/z 240.0755 [M]+ (calcd. C15H12O3 +, mass error 13.10 ppm). The positive +MS2 fragment ion m/z 225.0526 [M−CH3]+ was derived from the loss of −CH3 by positive molecular ion peak of Cp.27, it was continuously discarded −CO, +MS3 fragment ion m/z 197.0580 [M−CH3−CO]+ and +MS3 fragment ion m/z 169.0628 [M−CH3-2CO]+ were generated in turn (Fig. 2). The negative deprotonated precursor ion 239.0726 [M−H] (calcd. C15H11O3 , mass error 5.13 ppm) was fragmented into −MS2 fragment ion m/z 224.0504 [M−H−CH3] and m/z 225.0491 [M−CH2], the latter was further cleaved to produce −MS3 fragment ion m/z 196.0551 [M−CH3−CHO]. After the relevant research literature and reference substance comparison, DN−Cp.27 was inferred as moscatin (C15H12O3). With a similar fragmentation pathway as that mentioned above, the identification of other phenanthrene compounds is shown in Tables 1 and 2.

Fig. 2.
Fig. 2.

UPLC-ESI-IT-TOF extracted ion chromatogram (A), multigrade MS spectra (event 1 to event 6) (B) and proposed main fragmentation (C) of representative compound moscatin (DN-Cp.27) in D. nobile extract

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Table 1.

Identified natural compounds of D. nobile by UPLC-IT TOF

No.tR (min)IonMS1 (m/z)FormulaDiff (ppm)MS2(m/z)MS3(m/z)IdentificationCategory
DN-Cp.011.11[M-H]-341.1085aC12H22O111.27323.0816, 179.0572, 161.0506, 125.0278161.0606, 143.03397SucroseGlycoside
[M+Na]+365.1047C12H22O112.14--
DN-Cp.021.23[M+H]+268.1018C10H13N5O48.32136.0605, 135.9952b119.0017AdenosineAlkaloids
DN-Cp.031.98[M-H]-325.0902aC15H18O88.25163.0407b, 145.0319, 119.0516119.0538Cis-melilotosideGlycoside
DN-Cp.042.58[M-H]-327.1093aC15H20O82.31165.0578b, 147.0439, 121.0714147.0439, 124.0685DihydromelilotosideGlycoside
DN-Cp.052.75[M-H]-325.0971aC15H18O88.56163.0421, 119.0552-Trans-melilotosideGlycoside
DN-Cp.062.82[M-H]-197.0473aC9H10O58.85182.0260-Sryingic acidOther
DN-Cp.074.08[M-H]-579.2120aC28H36O134.46555.1236, 518.1710, 417.1527b 270.0449402.1326, 181.0539, 166.0276Acanthoside BGlycoside
DN-Cp.084.90[M-H]-165.0558aC9H10O30.11145.0466,121.0679b106.0305ApocyninOther
DN-Cp.095.61[M-H]-225.0579aC14H10O39.21197.0607b, 182.0775, 153.0122179.0361, 141.07137-Hydroxy-9,10-dihydro-1,4-phenanthrenedionePhenanthrene
DN-Cp.105.73[M-H]-227.0731aC14H12O36.72209.0620, 185.0651, 184.0721, 183.0852, 181.0657b156.06282,4,7-Trihydroxyl-9,10-dihydro-phenanthrenePhenanthrene
DN-Cp.115.74[M-H]-241.0506aC14H10O40.12226.0265b, 213.0262, 198.0269198.0310, 171.0463, 142.0396NobiloneFluorenone
DN-Cp.125.85[M-H]-282.1136aC17H17NO30.12162.0596, 145.0302, 134.0692, 119.0529b-Trans-N-coumaroyltyramineAlkaloids
[M+H]+284.12646.08--
DN-Cp.135.92[M-H]-257.0825aC15H14O42.20242.0577b, 225.0573, 207.0384225.0613, 224.0472, 214.0638, 198.0709,9,10-Dihydro-2,4,7-phenanthrenetriolPhenanthrene
DN-Cp.146.09[M+H]+314.13802.19177.0537b, 145.0279145.0273, 117.0336Trans-N-feruloyltyramineAlkaloids
[M-H]-312.1222aC18H19NO46.49297.0981, 270.1100, 178.0515, 176.0395, 160.0493, 148.0544b 135.0456106.0256
DN-Cp.156.22[M-H]-257.0840aC15H14O43.42243.0658b, 242.0568, 225.0651225.0473, 224.0497, 213.05687-Methoxy-9,1-dihydro-phenanthrene-2,4,5-triolPhenanthrene
DN-Cp.166.52[M-H]-259.0963C15H16O44.93244.0537, 137.0562, 123.0555, 121.0294b-TristinBibenzyl
DN-Cp.178.42[M+H]+273.0739C15H12O510.11153.0180, 147.0436137.8841, 125.0204NaringeninFlavonoids
8.36[M-H]-271.0606a2.19177.0222b 151.0056107.0167
DN-Cp.189.07[M-H]-419.1325aC21H24O95.37401.1343, 383.1510b, 318.0311, 283.0610, 217.0579365.1326, 283.06134-Methoxy-2,5,9R-trihydroxy-phenanthrene2-O-β-d-glucopyranoside APhenanthrene
DN-Cp.199.55[M+H]+305.1368C17H20O55.10--MoscatilinBibenzyl
9.60[M-H]-303.1229a2.95288.0998b, 271.0588, 189.0675, 137.0243153.0359, 137.0282
DN-Cp.209.61[M-H]-255.0669aC15H12O42.41240.0371, 239.0317b, 210.0706212.0455, 167.0440Fimbriol-BPhenanthrene
[M+H]+257.07887.95--
DN-Cp.219.78[M-H]-191.0714aC11H12O37.16163.0409, 145.0370, 119.0492-(E)-4-(4-hydroxy-3-methoxyphenyl)but-3-ene-2-oneOther
DN-Cp.229.97[M-H]-273.1106aC16H18O413.62258.0904, 137.0603, 122.0405, 121.03345b-GigantolBibenzyl
10.02[M+H]+275.1244a12.72257.1134, 243.0976, 151.0745, 137.0585b, 119.0446122.0348
DN-Cp.2311.33[M-H]-329.1357aC19H22O5-1.39179.0731b, 149.0640135.0830Dihydroconiferyl dihydro-p-coumarateOther
DN-Cp.2411.68[M-H]-241.0846aC15H14O39.99226.0626b, 154.0647198.0688, 169.0483Lusianthridin or CoeloninPhenanthrene
[M+H]+243.10285.08--
DN-Cp.2511.75[M-H]-241.0846aC15H14O32.14226.0627b, 154.0647198.0652Lusianthridin or CoeloninPhenanthrene
DN-Cp.2611.85[M-H]-253.0511aC15H10O41.84238.0266, 225.0582, 211.0385b, 210.0315182.0351Densiflorol BPhenanthrene
[M+H]+255.0643a3.48--
DN-Cp.2711.92[M]+240.0755aC15H12O39.95225.0526b, 197.0583197.0580, 169.0628MoscatinPhenanthrene
[M+H]+241.08591.70--
12.02[M-H]-239.0704a4.45224.0463b, 211.0389, 196.0497, 183.0564196.0533, 133.1477
DN-Cp.2812.09[M-H]-269.0799aC16H14O47.53255.0560b, 254.0573, 226.0706239.0292, 226.0617, 211.0399, 197.0679NudolPhenanthrene
[M+H]+271.09438.09--
DN-Cp.2912.60[M-H]-269.0816aC16H14O41.23255.0560, 254.0573b, 226.0701211.0410Ephemeranthol BPhenanthrene
[M+H]+271.09389.94--
DN-Cp.3015.89[M-H]-293.2137aC18H30O35.04275.2004b, 195.1381, 185.1190234.1787, 205.1613Unknown 1Other
DN-Cp.3117.06[M-H]-295.2260aC18H32O33.61277.2154b, 233.2251, 171.1028233.2224, 205.1281Unknown 2Other
DN-Cp.3218.56[M+Na]+332.1832C17H27NO46.83--6-HydroxynobilineAlkaloid
DN-Cp.3318.60[M+Na]+286.1178C16H25NO28.21--DendrobineAlkaloid
DN-Cp.3421.23[M-H]-277.2149aC18H30O28.64275.1946, 233.2220b-Unknown 3Other
DN-Cp.3522.30[M-H]-279.2346aC18H32O25.52261.2197, 206.0371-Unknown 4Other
Table 2.

Identified natural compounds of D. officinale by UPLC-IT TOF

No.tR (min)IonMS1 (m/z)FormulaDiff (ppm)MS2 (m/z)MS3 (m/z)Identification
DO-Cp.011.11[M-H]-341.1047aC12H22O115.66323.0924, 281.0875, 251.0779, 221.0663, 179.0571b, 161.0456, 143.0383, 131.0365161.0606, 143.0313Sucrose
[M+Na]+365.1054C12H22O114.20--
DO-Cp.021.23[M+H]+268.1018C10H13N5O48.32249.1201, 136.0603, 135.9986b119.0329, 112.0277Adenosine
DO-Cp.033.96[M-H]-579.2089aC28H36O131.01556.3611, 487.6456, 417.1536b402.1356, 181.0541, 166.0274Acanthoside B
DO-Cp.045.40[M-H]-341.1228aC16H22O84.07179.0717, 164.0499-Coniferin
DO-Cp.055.61[M-H]-225.0549aC14H10O33.62--7-Hydroxy-9,10-dihydro-1,4-phenanthrenedione
DO-Cp.065.70[M-H]-227.0731aC14H12O36.72209.0620, 185.0628b, 183.0828, 181.0653156.06289,10-Dihydro-2,4,7-phenanthrenetriol
DO-Cp.075.85[M+H]+284.1266C17H17NO37.35--Trans-N-coumaroyltyramine
[M-H]-282.1142aC17H17NO32.24162.0596, 145.0362, 134.0620, 119.0529b, 117.0388-
DO-Cp.086.06[M-H]-312.1238aC18H19NO46.49297.0967, 270.1120, 178.0536, 176.0395, 160.0493, 148.0568, 135.0461b-Trans-N-feruloyltyramine
6.12[M+H]+314.1386C17H17NO30.27177.0542b, 145.0273145.0273, 117.0339
DO-Cp.096.52[M-H]-259.1028C15H16O49.87244.0537, 137.0594, 121.0294-Tristin
DO-Cp.108.32[M-H]-271.0611aC15H12O50.36177.0187b, 151.0054, 125.0271133.0277, 107.0153Naringenin
8.38[M+H]+273.072213.05179.0353, 153.0181b, 147.0439125.0250, 111.0098, 109.0278
DO-Cp.119.26[M+H]+275.125C16H18O410.16--4,4′-dihydroxy-3,5-dimethoxybibenzyl
[M-H]-273.11579.00258.0912b, 242.0501, 167.0337, 152.0508, 137.0159152.0408, 137.0224, 109.0301
DO-Cp.129.55[M+H]+305.1384C17H20O55.41--Moscatilin
[M-H]-303.1238C17H20O58.90288.0998b, 271.0588, 189.0675, 137.0243153.0359, 137.0282
DO-Cp.139.77[M-H]-243.0997aC15H16O32.33183.0446, 137.0593,122.0417, 121.0384-Batatasin Ⅲ
DO-Cp.149.86[M-H]-289.1074aC16H18O52.23273.0745, 243.0987b, 138.0385137.0647, 122.04033, 4, α-Trihydroxy-5, 4′-dimethoxy-bibenzyl
DO-Cp.159.95[M-H]-273.1110C16H18O48.15258.0876, 214.0593, 137.0603, 122.0395, 121.0322b-Gigantol
10.01[M+H]+275.1256aC16H18O47.97257.1163, 243.0971, 151.0750, 137.0593, 123.0803, 108.0435136.0544, 119.0503, 108.0517
DO-Cp.1610.35[M+H]+469.1840C26H28O83.61--Dendrocandin U
[M-H]-467.1711C26H28O85.65449.1630, 437.1571, 424.0270, 257.0811b, 209.0815,194.0650242.0606, 151.0419
DO-Cp.1710.65[M+Na]+295.0937C16H16O41.29273.5656, 265.0157, 258.0071, 223.0908, 205.0851b, 149.0221176.0633, 149.0334Erianthridin or epemeranthol A
DO-Cp.1811.74[M+H]+275.1260C16H18O46.52--Moniliformine
[M-H]-273.1137a1.70258.0902b, 137.0815137.0277
DO-Cp.1911.86[M+H]+289.1406C17H20O49.84--3-Methylgigantol
[M-H]-287.1222a7.53272.1026, 257.0955, 137.0593b121.0319
DO-Cp.2012.64[M+H]+285.0737C16H12O57.22--Physcion
[M-H]-283.0624a4.23269.0380, 268.0284, 253.0172-
DO-Cp.2115.72[M-H]-293.2090aC18H30O35.84275.1941b, 231.2160, 185.1190234.1714, 231.2257Unknown 1
DO-Cp.2217.05[M-H]-295.2264aC18H32O34.96277.2179b, 183.1041, 171.1078137.1094Unknown 2
DO-Cp.2321.14[M-H]-277.2167aC18H30O22.17233.2290b, 205.1203,135.0830-Unknown 3
DO-Cp.2422.41[M-H]-279.2328aC18H32O20.55261.2284b-Unknown 4

Bibenzyls

Bibenzyls is an important phenolic compounds in the Dendrobium genus with a wide range of biological activities, there were nine bibenzyls identified from D. nobile extract and D. officinale extract. DN−Cp.22 appeared as a peak in t R 10.02 min, its positive and negative multistage fragment ion were collected. The positive protonated precursor ion +MS1 peak m/z 275.1243 [M+H]+ (calcd. C16H19O4 +, mass error 12.72 ppm) was fragmented into +MS2 fragment ion m/z 257.1134 [M+H−H2O]+, m/z 151.0745 [M+H−C7H7O2]+ and m/z 122.0348 [M+H−C9H13O2]+. +MS2 fragment ion m/z 257.1134 could drop −CH3, −C6H5O to produce m/z 243.0976 and m/z 151.0745 in turn, the latter further lost −CH2, −CH3 and obtained +MS2 fragment ion m/z 137.0586, +MS3 fragment ion m/z 122.0348 (Figure S3). The negative deprotonated precursor ion of DN−Cp.22 was 273.1095 [M−H] (calcd. C16H18O4 , mass error 13.62 ppm), it can generate −MS2 fragment ion m/z 258.0904 [M−H−CH3], m/z 151.0734 [M−H−C7H7O2] and m/z 122.0405[M−H−C9H12O2] by the loss of −CH3, −C7H7O2, −C9H12O2. −MS2 fragment ion m/z 258.0904 can cleave to −MS2 m/z 137.0602. DN−Cp.22 was finally identified as gigantol (C16H18O4). There is a chromatographic peak of the same compound named DO−Cp.15 in t R 10.01min of UPLC-ESI-IT-TOF chromatograms of D. officinale extract, the positive protonated precursor ion +MS1 peak m/z 275.1255 [M+H]+ (calcd. C16H19O4 +, mass error 8.34 ppm) was fragmented into +MS2 fragment ion m/z 257.1163 [M+H−H2O]+, m/z 243.0971 [M+H−H2O−CH3]+, m/z 151.0749 [M+H−C7H7O2]+ and m/z 137.0593 [M+H−C8H9O2]+. The negative deprotonated precursor ion of DO−Cp.15 was 273.1110 [M−H] (calcd. C16H18O4 , mass error 8.15 ppm), it can generate −MS2 fragment ion m/z 258.0876 [M−H−CH3], m/z 151.0758 [M−H−C7H7O2] and m/z 122.0395 [M−H−C9H12O2] by the loss of −CH3, −C7H7O2, −C9H12O2. −MS2 fragment ion m/z 258.0876 can cleave to −MS2 m/z 137.0603 (Figure S4). DO−Cp.15 was also identified as gigantol (C16H18O4). Other identified bibenzyl compounds in D. nobile extract and D. officinale extracts are shown in Tables 1 and 2.

Others

Other compounds include flavonoids, polysaccharides and so on. DO−Cp.10 in D. officinale was separated as a peak in t R 8.38 min, obtained its positive and negative ion mode data, the positive protonated precursor ion +MS1 peak m/z 273.0720 [M+H]+ (calcd. C15H13O5 +, mass error 13.78 ppm). The positive +MS2 fragment ion m/z 153.0181 [M−C8H9O]+ and m/z 147.0439 [M−C6H5O3]+ were derived from the loss of −C8H9O and −C6H5O3 by positive molecular ion peak of DO−Cp.10 respectively, both of they were discarded −CO to generate +MS3 fragment ion m/z 125.0250 and m/z 119.0490 (Fig. 3). The negative deprotonated precursor ion 271.0611 [M−H] (calcd. C15H11O5 , mass error 0.36 ppm) was fragmented into −MS2 fragment ion m/z 177.0187 [M−H−C6H6O] and m/z 151.0054 [M−H−C8H8O], the latter was further cleaved to produce −MS3 fragment ion m/z 133.0277 [M−H−C8H8O−H2O] and −MS2 fragment ion m/z 125.0267 [M−H−C8H8O−CO], −MS3 fragment ion m/z 109.0341 [M−H−C8H8O−CO-OH] can be deduced from the loss of -OH from m/z 125.0267 (Table 2), DO−Cp.10 was inferred as naringenin (C15H12O5). Meantime, DN−Cp.17 in D. nobile was separated as a peak in t R 8.42 min, the positive protonated precursor ion +MS1 peak m/z 273.0730 [M+H]+ (calcd. C15H13O5 +, mass error 10.11 ppm) can cleave to produce +MS2 fragment ion m/z 153.0181 [M+H−C8H9O]+ and +MS2 fragment ion m/z 147.0436, both of they were discarded −CO to generate +MS3 fragment ion m/z 125.0204 and m/z 119.0483. The negative deprotonated precursor ion 271.0606 [M−H] (calcd. C15H11O5 , mass error 2.19 ppm) was fragmented into −MS2 fragment ion m/z 177.0222 [M−H−C6H6O] and m/z 151.0054 [M−H−C8H8O] (Table 1). After the relevant research literature and reference substance comparison, DN−Cp.17 was also identified as naringenin (C15H12O5).

Fig. 3.
Fig. 3.

UPLC-ESI-IT-TOF extracted ion chromatogram (A), multigrade MS spectra (event 1 to event 6) (B) and proposed main fragmentation (C) of representative compound naringenin (DO-Cp.10) in D. officinale extract

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Network pharmacology analysis

PPI network analysis According to DL and GIA, 23 compounds and 18 compounds were screened from D. nobile and D. officinale respectively. A total of 591 targets for D. nobile and 676 targets for D. officinale were obtained as potential targets, of which 465 were the same. 4658 genes associated with cancer were retrieved in GeneCards database. We integrated the compound targets and cancer targets to obtain the common targets. 388 and 440 common targets were acquired from D. nobile and D. officinale respectively, there were 329 common targets from both of them after Venn analysis (Figure S5), the PPI network of D. nobile and D. officinale was constructed respectively using the medians of degree, betweenness centrality, closeness centrality as thresholds, containing 141 nodes and 1517 edges, 128 nodes and 1539 edges. Node size and color depth represented high degree value, the importance of targets was positively correlated with degree value. After topological analysis, ten core targets were screened from D. nobile and D. officinale, seven common targets including SRC, HSP90AA1, AKT1, EGFR, MAPK1, PIK3R1 and RHOA were derived from statistical calculations, STAT3, PIK3CA, EP300, MAPK3, HRAS and GRB2 were differential core proteins. The detailed information of 13 core target proteins were presented in Table 3.

Table 3.

The core target proteins for the antitumor effects of D. nobile extract and D. officinale extract

Gene IDGene namesSourceDegreeBetweenness CentralityCloseness Centrality
SRCProto-oncogene tyrosine-protein kinase SrcD. nobile1010.07820.5146
D. officinale1050.05710.4951
HSP90AA1Heat shock protein HSP 90-alphaD. nobile960.07540.5007
D. officinale1060.08260.4957
AKT1RAC-alpha serine/threonine-protein kinaseD. nobile840.05000.4913
D. officinale940.06400.5006
EGFREpidermal growth factor receptorD. nobile840.05270.4973
D. officinale880.04240.4892
MAPK1Mitogen-activated protein kinase 1D. nobile750.04360.4798
D. officinale860.03050.4749
PIK3R1Phosphatidylinositol 3-kinase regulatory subunit alphaD. nobile750.01770.4595
D. officinale790.01630.4553
RHOATransforming protein RhoAD. nobile610.03320.4590
D. officinale720.03860.4646
STAT3Signal transducer and activator of transcription 3D. nobile850.04140.4927
PIK3CAPhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoformD. nobile660.01130.4500
EP300Histone acetyltransferase p300D. nobile580.04340.4683
MAPK3Mitogen-activated protein kinase 3D. officinale900.04150.4904
HRASGTPase HRasD. officinale810.02720.4678
GRB2Growth factor receptor-bound protein 2D. officinale720.03860.4646

Herb−Compounds-Targets−Cancer network analysis The D. nobile-compounds-targets-cancer network and D. officinale-compounds-targets-cancer network were constructed successively to explore the relationship between compounds and intersection targets to find key compounds. The compounds-targets-cancer network of D. nobile and D. officinale contained 416 nodes and 2326 edges, 451 nodes and 2724 edges respectively (Fig. 4). 11 and 8 key compounds from D. nobile and D. officinale had been obtained respectively using the medians of degree, betweenness centrality and closeness centrality as thresholds, the detailed information of key compounds was shown in Table 4. Eight of them in D. officinale including tristin (DO−Cp.09), 4,4′-dihydroxy-3,5-dimethoxybibenzyl (DO−Cp.11), moscatilin (DO−Cp.12), batatasin III (DO−Cp.13), gigantol (DO−Cp.15), dendrocandin U (DO−Cp.16), moniliformine (DO−Cp.18) and 3-methylgigantol (DO−Cp.19) belong to bibenzyl. The key compounds of D. nobile were mainly composed of bibenzyls, phenanthrenes and others. D. nobile possess three identical bibenzyls with D. officinale, namely, tristin (DN−Cp.16 and DO−Cp.09), moscatilin (DN−Cp.19 and DO−Cp.12), gigantol (DN−Cp.22 and DO−Cp.15).

Fig. 4.
Fig. 4.

D. nobile-Compounds-Targets-Cancer network (A), D. officinale-compounds-targets-cancer network (B), bubble diagrams of the KEGG pathway enrichment analysis of D. nobile (C) and D. officinale (D)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Table 4.

The key antitumor compounds in D. nobile and D. officinale

CompoundsSourceDegreeBetweenness CentralityCloseness Centrality
GigantolD. nobile1870.05250.4673
D. officinale2360.02810.4491
MoscatilinD. nobile1540.03560.4415
D. officinale1390.01840.4269
TristinD. nobile1380.02750.4278
D. officinale1240.01530.4151
Dendrocandin UD. officinale1690.04120.4527
MoniliformineD. officinale1650.09010.5233
4,4′-Dihydroxy-3,5-DimethoxybibenzylD. officinale1640.02670.4482
3-MethylgigantolD. officinale1560.02720.4412
ConiferinD. officinale1410.02990.4286
Batatasin IIID. officinale1340.01800.4229
Dihydroconiferyl Dihydro-P-CoumarateD. nobile1330.03550.4350
Trans-N-CoumaroyltyramineD. nobile1240.02580.4209
Trans-N-FeruloyltyramineD. nobile1080.02190.4077
NudolD. nobile1040.01320.3990
NaringeninD. nobile990.01720.3983
Densiflrol BD. nobile930.01370.3945
6-HydroxynobilineD. nobile910.02080.3975
MoscatinD. nobile890.01130.3937
Ephemeranthol BD. nobile740.00760.3871

KEGG pathway enrichment analysis To further understand the antitumor mechanism of D. nobile and D. officinale, enrichment analysis of KEGG pathway was carried out, 128 related pathways (P < 0.05) were retrieved in D. nobile and 135 (P < 0.05) associated pathway were obtained in D. officinale. The top 20 related pathways were visualized in Fig. 4. Among the 20 pathways, D. nobile and D. officinale possessed 18 common pathways. The common pathways were categorized for four class, one of them belonged to the cellular processes (Focal adhesion), one of them belonged to the metabolism (Central carbon metabolism in cancer), eight of them belonged to human diseases or substance dependence (prostate cancer, non-small cell lung cancer, proteoglycans in cancer, glioma, Pancreatic cancer, hepatitis B, chronic myeloid leukemia, melanoma, acute myeloid leukemia), eight of them belonged to environmental information processing or signal pathway transduction (pathways in cancer, Thyroid hormone signaling pathway, HIF-1 signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, progesterone-mediated oocyte maturation, Rap1 signaling pathway, sphingolipid signaling pathway).

Experimental verification

Molecular docking Molecular docking was further applied to explore the interaction between core proteins and key compounds in D. nobile and D. officinale to confirm the reliability of network pharmacology. It is generally believed that the binding energy of the ligand and the receptor was lower, stability of conformation was stronger. The binding energy (≤−5.0 kJ mol−1) is regarded as a good binding activity [18]. As shown in Fig. 5, molecular docking results revealed that the key compounds had a good binding activity with core proteins, molecular docking results of the compounds and the target proteins in the lowest binding energy were displayed in 2D and 3D structures. The major binding interactions of them mainly contain the hydrogen bonding and hydrophobic interactions. Taking the gigantol-PI3K docking as an example, the binding energy of them was −8.5 kJ mol−1. Gigantol could form intermolecular hydrogen bonds with amino acid residues, including Glu880A, Val882A, Asp964A and Lys833A of the PI3K proteins. Moreover, we observed hydrophobic interactions with amino acid residues-ll963A, Met953A, Asp964A and lle879A.

Fig. 5.
Fig. 5.

Heatmap analysis of the molecular docking of key compounds of D. nobile (A) and D. officinale (B) with their core targets, the color represents the Vina score, the bluer the color, the lower the Vina score and the stronger the bind between the receptor and the ligand. Molecular docking models of PI3K and four key compounds (gigantol, moscatilin, densiflorol B and moscatin) (C), AKT and these four key compounds (D) and SRC and these four key compounds (E)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Western blotting D. nobile and D. officinale could inhibited PI3K-Akt and HIF-1 signaling pathways according to current study data, we carried out this mechanism verification through western blotting method. D. nobile extract and D. officinale extract showed that the strongest antitumor activity on A549 cells and non-small cell lung cancer pathway was in the forefront of all the enriched cancer pathways based on the KEGG pathway enrichment analysis. Therefore, we chose A549 cells for mechanism verification. p-PI3K and its downstream AKT and p-AKT expression levels were significantly reduced by D. nobile treatment (Fig. 6). In addition, cells treated with D. nobile extract also showed a tendency of downregulation of HIF-1α compared with the control group, similar results were obtained in D. officinale. Western blotting results confirmed that D. nobile extract and D. officinale extract could inhibited PI3K-Akt and HIF-1α signaling pathways, which was consistent with our prediction of network pharmacology.

Fig. 6.
Fig. 6.

The expression level of PI3K, p-PI3K, AKT, p-AKT and HIF-1α in human lung cancer A549 cells under the effect of different concentrations of D. nobile extract (A) and D. officinale extract (B). The data are presented as the mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 v.s. blank control group

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01012

Discussion

An analytical strategy combining UPLC-IT-TOF, network pharmacology, molecular docking and experimental verification as one of the most effective methods for exploring material basis and pharmacological mechanisms of complex natural drugs were established for explaining antitumor chemical constituents and mechanisms of D. nobile and D. officinale, the accurate-mass error of IT-TOF was slightly larger than that of other high-resolution tandem mass spectrometry, Q-TOF, orbitrap mass spectrometry through the experience of the use of several high-resolution mass spectrometry, accurate-mass error threshold of IT-TOF was generally set at <15 ppm. IT-TOF as a high-resolution tandem mass spectrometry can provide a wealth of multistage fragmentation information, which showed its unique characteristic qualitative advantage, the stronger qualitative analysis power of unknown compounds. Meanwhile, relatively-comprehensive natural compound data of D. nobile and D. officinale provided by several important natural product databases strongly supported the results of IT-TOF mass spectrometry analysis. UPLC-IT-TOF analytical technology was made full use of the advantage of the multistage fragment ion information of a single compound and also made up for the disadvantage of slightly larger accurate-mass error. Antitumor activity of D. nobile and D. officinale is associated with their own chemical constituents, phenanthrenes were detected to be mainly present in D. nobile, and bibenzyls were distributed in D. officinale, there were 16 and 35 bibenzyl compounds, 35 and 14 phenanthrene compounds separated and purified from D. nobile and D. officinale respectively [4, 5], TCMs rich in phenanthrenes and bibenzyls tended to show good antitumor effects. Bibenzyls were confirmed to have good antitumor activity in vitro and vivo [19, 20], moscatin and densiflorol B remarkably inhibited the proliferation of MCF-7 cells [15]. Different bibenzyls and phenanthrenes distributed in D. nobile and D. officinale may lead to variability in their antitumor activity, gigantol, moscatilin, tristin, moscatin and densiflorol B were screened out as key antitumor compounds in D. nobile, bibenzyls were the main antitumor compounds of D. officinale based on D. officinale-compounds-targets-cancer network. Moscatlin can block the metastasis of human breast cancer MDA-MB-231 cells via suppressing Akt and Twist-dependent pathways, significantly suppressed the metastasis of these cancer cells in a MDA-MB-231 metastatic animal tumor model [21]. Tristin had toxic activity on human leukemic promyelocytic cells and human acute monocytic leukemia cells with IC50 value 29.06 ± 1.13 μmol L−1 and 13.53 ± 1.10 μmol L−1 respectively [22]. Gigantol was applied to reduce the viability of human breast cancer cells and destabilize tumors via inhibiting PI3K/AKT, PI3K/Akt/mTOR and JAK/STAT signaling pathways [20, 23].

Data from this experiment told us that D. officinale extract had more antitumor targets than D. nobile extract, the antitumor effect of D. officinale extract was stronger than that of D. nobile extract. The result of molecular docking showed that antitumor core targets usually could be formed stable conformations with most of antitumor key compounds of D. nobile extract and D. officinale extract, and obtained good docking scores of compounds – targets while combining via hydrogen bonding and hydrophobic interaction. Phenanthrenes and bibenzyls are rich in phenolic hydroxyl groups and methoxy groups, are easy to form stable conformations with most core proteins, such as densiflorol B-PI3K (−7.9 kcal mol−1), densiflorol B -AKT (−8.2 kcal mol−1), densiflorol B-SRC (−8.8 kcal mol−1), gigantol-PI3K (−8.5 kcal mol−1), gigantol-AKT (−7.7 kcal mol−1) and gigantol-SRC (−7.4 kcal mol−1). Most antitumor core targets of D. nobile and D. officinale were associated with PI3K-Akt and HIF-1 signaling pathways, inhibition of PI3K-AKT signaling pathway can induce apoptosis of tumor cells [24], D. nobile extract and D. officinale extract can significantly down-regulate PI3K-AKT and HIF-1α signaling pathways. Hypoxia-inducible factor 1 (HIF-1) is a heterodimer consisting of the active subunits HIF-1α subunit and the constitutively HIF-1β subunit, HIF-1α can induce protein expression and regulate cell proliferation, cell apoptosis and tumor angiogenesis [25].

Conclusion

An efficient and sensitive UPLC-IT-TOF, network pharmacology and experimental verification was firstly established for quickly identifying and comparing the antitumor compounds of D. nobile and D. officinale in this study, a total of 40 potential antitumor compounds were characterized, the antitumor effect of D. officinale extract was stronger than that of D. nobile extract, gigantol, moscatilin, tristin, moscatin and densiflorol B were considered as key antitumor compounds of D. nobile and D. officinale. D. nobile and D. officinale had great similarities in terms of antitumor core targets and KEGG pathways, the antitumor mechanisms of D. nobile and D. officinale may be related to downregulation of PI3K-Akt and HIF-1α signaling pathway. These findings would show us more clear material basis of D. nobile and D. officinale, guiding further pharmacological mechanism research of D. nobile and D. officinale.

Conflict of interests

The authors declare no conflict of interest.

Supplementary material

Supplementary material to this article can be found online at https://doi.org/10.1556/1326.2022.01012.

Acknowledgments

This study was supported by grants from Guizhou Provincial Science and Technology Support Project (No. 2021-General 415), Guizhou Provincial Natural Science Foundation (2020-1Y404), Guizhou Provincial Innovation and Entrepreneurship Training Project for College Students (No. S202110660009, No. S202010660006) and Reform Project of Undergraduate Teaching Content and Course System of Guizhou Medical University (No. JG2021042, No. JG2019-60).

Abbreviations

DN−Cp.

compound in Dendrobium nobile

DO−Cp.

compound in Dendrobium officinale

EIC

extracted ion chromatogram

TIC

total ion current

TCM

traditional Chinese medicine

DDA

data dependent acquisition

MDF

mass defect filter

UPLC-ESI-IT-TOF

ultra performance liquid chromatography - electrospray ionization - ion trap - time of flight

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    Zhang, X. Y. ; Sun, W. B. ; Yang, Z. ; Liang, Y. ; Zhou, W. ; Tang, L. Hemostatic chemical constituents from natural medicine Toddalia asiatica root bark by LC-ESI Q-TOF MSE . Chem. Cent. J. 2017, 11, 55.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Xia, J. ; Yang, Z. ; Zeng, Q. F. ; Liang, Y. ; Hao, X. Y. ; Zhou, W. Analysis of chemical constituents in dendrobium nobile by UPLC-Q-TOF. J. Chin. Med. Mat. 2018, 41, 600607.

    • Search Google Scholar
    • Export Citation
  • 10.

    Tao, Y. ; Cai, H. ; Li, W. D. ; Cai, B. C. Ultrafiltration coupled with high-performance liquid chromatography and quadrupole-time-of-flight mass spectrometry for screening lipase binders from different extracts of Dendrobium officinale. Anal. Bioanal. Chem. 2015, 407, 60816093.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Wang, X. ; Wang, Z. Y. ; Zheng, J. H. ; Li, S. TCM network pharmacology: a new trend towards combining computational, experimental and clinical approaches. Chin. J. Nat. Medicines 2021, 19, 111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Li, S. Network pharmacology evaluation method guidance-Draft. World J. Tradit. Chin. Med. 2021, 7, 146154.

  • 13.

    Luo, C. R. ; Liu, J. ; Liang, Y. ; Sheng, Y. ; He, Y. Q. ; Zhou, W. Screening of anti-inflammatory alkaloids of Toddalia asiatica and the action mechanism based on molecular docking technology. J. Guizhou Med. Univ. 2021, 46, 639646.

    • Search Google Scholar
    • Export Citation
  • 14.

    Xia, J. Study on Material Basis of Antioxidant Activity of Ethyl Acetate Extract of Dendrobium Nobile, Dissertation; Guizhou Medical University: Guiyang, 2018; p 13.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zhou, W. ; Zeng, Q. F. ; Xia, J. ; Wang, L. ; Tao, L. ; Shen, X. C. Antitumor phenanthrene constituents of dendrobium nobile. Chin. Pharm. J. 2018, 53, 17221725.

    • Search Google Scholar
    • Export Citation
  • 16.

    He, L. L. ; Jiang, H. ; Lan, T. H. ; Qiu, Y. ; Yang, K. F. ; Chen, K. J. ; Yao, X. S. ; Yao, Z. H. ; Lu, W. H. Chemical profile and potential mechanisms of Huo-Tan−Chu-Shi decoction in the treatment of coronary heart disease by UHPLC-Q/TOF−MS in combination with network pharmacology analysis and experimental verification. J. Chromatogr. B 2021, 1175, 122729.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Daina, A. ; Michielin, O. ; Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Tong, H. J. ; Yu, M. T. ; Fei, C. H. ; Ji, D. ; Dong, J. J. ; Su, L. L. ; Gu, W. ; Mao, C. Q. ; Li, L ; Bian, Z. H. ; Lu, T. L. ; Hao, M. ; Zeng, B. L. Bioactive constituents and the molecular mechanism of Curcumae Rhizoma in the treatment of primary dysmenorrhea based on network pharmacology and molecular docking. Phytomedicine 2021, 86, 153558.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Tsai, A. C. ; Pan, S. L. ; Liao, C. H. ; Guh, J. H. ; Wang, S. W. ; Sun, H. L. ; Liu, Y. N. ; Chen, C. C. ; Shen, C. C. ; Chang, Y. L. ; Teng, C. M. Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo. Cancer Lett. 2010, 292, 163170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Losuwannarak, N. ; Maiuthed, A. ; Kitkumthorn, N ; Leelahavanichkul, A ; Roytrakul, S. ; Chanvorachote, P. Gigantol targets cancer stem cells and destabilizes tumors via the suppression of the PI3K/AKT and JAK/STAT pathways in ectopic lung cancer xenografts. Cancers 2019, 11, 2032.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Pai, H. C. ; Chang, L. H. ; Peng, C. Y ; Chang, Y. L ; Chen, C. C ; Shen, C. C. ; Teng, C. M. ; Pan, S. L. Moscatilin inhibits migration and metastasis of human breast cancer MDA-MB-231 cells through inhibition of Akt and Twist signaling pathway. J. Mol. Med. 2013, 91, 347356.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Zhao, G. Y. ; Deng, B. W. ; Zhang, C. Y ; Cui, Y. D. ; Bi, J. Y. ; Zhang, G. G. New phenanthrene and 9, 10-dihydrophenanthrene derivatives from the stems of Dendrobium officinale with their cytotoxic activities. J. Nat. Med. 2018, 72, 246251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Huang, J. H. ; Liu, C. X. ; Duan, S. N. ; Lin, J. ; Luo, Y. Y. ; Tao, S. C. ; Xing, S. P. ; Zhang, X. F. ; Du, H. Y. ; Wang, H. ; Huang, C. L. ; Wei, G. Gigantol inhibits proliferation and enhances DDP-induced apoptosis in breast-cancer cells by downregulating the PI3K/Akt/mTOR signaling pathway. Life Sci. 2021, 274, 119354.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Tewari, D. ; Patni, P. ; Bishayee, A. ; Sah, A. N ; Bishayee, A. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: a novel therapeutic strategy. Semin. Cancer Biol. 2021; https://doi.org/10.1016/j.semcancer.2019.12.008.

    • Search Google Scholar
    • Export Citation
  • 25.

    Tang, W. ; Zhao, G. Small molecules targeting HIF-1α pathway for cancer therapy in recent years. Bioorg. Med. Chem. 2020, 28, 115235.

Supplementary Materials

  • 1.

    Cakova, V. ; Bonte, F. ; Lobstein, A. Dendrobium: sources of active ingredients to treat age-related pathologies. Aging Dis. 2017, 8, 827849.

  • 2.

    Nie, X. Q. ; Chen, Y. ; Li, W. ; Lu, Y. L. Anti-aging properties of Dendrobium nobile Lindl.: from molecular mechanisms to potential treatments. J. Ethnopharmacol. 2020, 257, 112839.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Yan, M. Q. ; Yang, Z. Y. ; Shi, Q. Q. ; Wang, T. ; Chen, S. H. ; Lv, G. Y. Research progress on protective effects and mechanism of Dendrobii Caulis on metabolic disturbances. Chin. Trad. Herbal Drugs 2019, 50, 24912497.

    • Search Google Scholar
    • Export Citation
  • 4.

    Tang, H. X. ; Zhao, T. W. ; Sheng, Y. J. ; Zheng, T. ; Fu, L. Z. ; Zhang, Y. S. Dendrobium officinale Kimura et Migo: a Review on Its Ethnopharmacology, Phytochemistry, Pharmacology and Industrialization. Evid. Based Complement. Alternat. Med. 2017, 2017, 7436259.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Zhou, W. ; Xia, J. ; Sun, W. B. ; Liang, Y. ; Hao, X. Y. ; Tang, L. Current research status of chemical constituents and pharmacological effects of Dendrobium nobile. Chin. J. New Drugs 2017, 26, 26932700.

    • Search Google Scholar
    • Export Citation
  • 6.

    Pang, C. ; Zhang, X. L. ; Zhang, X. L. Research progress in inhibitory epithelium-derived malignant tumors by dendrobium officinale. Pract. Oncol. J. 2020, 34, 362367.

    • Search Google Scholar
    • Export Citation
  • 7.

    He, K. ; Zhang, D. ; Li, X. F. ; Liu, R. H. ; Huang, J. L. ; Liu, M. H. Inhibitory effect of dendrobium nobile lindl wall-broken powder on tumor growth of transplanted human liver cancer cell line HepG2 in nude mice. Chin. Pharm. J. 2019, 54, 18651870.

    • Search Google Scholar
    • Export Citation
  • 8.

    Zhang, X. Y. ; Sun, W. B. ; Yang, Z. ; Liang, Y. ; Zhou, W. ; Tang, L. Hemostatic chemical constituents from natural medicine Toddalia asiatica root bark by LC-ESI Q-TOF MSE . Chem. Cent. J. 2017, 11, 55.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Xia, J. ; Yang, Z. ; Zeng, Q. F. ; Liang, Y. ; Hao, X. Y. ; Zhou, W. Analysis of chemical constituents in dendrobium nobile by UPLC-Q-TOF. J. Chin. Med. Mat. 2018, 41, 600607.

    • Search Google Scholar
    • Export Citation
  • 10.

    Tao, Y. ; Cai, H. ; Li, W. D. ; Cai, B. C. Ultrafiltration coupled with high-performance liquid chromatography and quadrupole-time-of-flight mass spectrometry for screening lipase binders from different extracts of Dendrobium officinale. Anal. Bioanal. Chem. 2015, 407, 60816093.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Wang, X. ; Wang, Z. Y. ; Zheng, J. H. ; Li, S. TCM network pharmacology: a new trend towards combining computational, experimental and clinical approaches. Chin. J. Nat. Medicines 2021, 19, 111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Li, S. Network pharmacology evaluation method guidance-Draft. World J. Tradit. Chin. Med. 2021, 7, 146154.

  • 13.

    Luo, C. R. ; Liu, J. ; Liang, Y. ; Sheng, Y. ; He, Y. Q. ; Zhou, W. Screening of anti-inflammatory alkaloids of Toddalia asiatica and the action mechanism based on molecular docking technology. J. Guizhou Med. Univ. 2021, 46, 639646.

    • Search Google Scholar
    • Export Citation
  • 14.

    Xia, J. Study on Material Basis of Antioxidant Activity of Ethyl Acetate Extract of Dendrobium Nobile, Dissertation; Guizhou Medical University: Guiyang, 2018; p 13.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zhou, W. ; Zeng, Q. F. ; Xia, J. ; Wang, L. ; Tao, L. ; Shen, X. C. Antitumor phenanthrene constituents of dendrobium nobile. Chin. Pharm. J. 2018, 53, 17221725.

    • Search Google Scholar
    • Export Citation
  • 16.

    He, L. L. ; Jiang, H. ; Lan, T. H. ; Qiu, Y. ; Yang, K. F. ; Chen, K. J. ; Yao, X. S. ; Yao, Z. H. ; Lu, W. H. Chemical profile and potential mechanisms of Huo-Tan−Chu-Shi decoction in the treatment of coronary heart disease by UHPLC-Q/TOF−MS in combination with network pharmacology analysis and experimental verification. J. Chromatogr. B 2021, 1175, 122729.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Daina, A. ; Michielin, O. ; Zoete, V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Tong, H. J. ; Yu, M. T. ; Fei, C. H. ; Ji, D. ; Dong, J. J. ; Su, L. L. ; Gu, W. ; Mao, C. Q. ; Li, L ; Bian, Z. H. ; Lu, T. L. ; Hao, M. ; Zeng, B. L. Bioactive constituents and the molecular mechanism of Curcumae Rhizoma in the treatment of primary dysmenorrhea based on network pharmacology and molecular docking. Phytomedicine 2021, 86, 153558.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Tsai, A. C. ; Pan, S. L. ; Liao, C. H. ; Guh, J. H. ; Wang, S. W. ; Sun, H. L. ; Liu, Y. N. ; Chen, C. C. ; Shen, C. C. ; Chang, Y. L. ; Teng, C. M. Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo. Cancer Lett. 2010, 292, 163170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Losuwannarak, N. ; Maiuthed, A. ; Kitkumthorn, N ; Leelahavanichkul, A ; Roytrakul, S. ; Chanvorachote, P. Gigantol targets cancer stem cells and destabilizes tumors via the suppression of the PI3K/AKT and JAK/STAT pathways in ectopic lung cancer xenografts. Cancers 2019, 11, 2032.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Pai, H. C. ; Chang, L. H. ; Peng, C. Y ; Chang, Y. L ; Chen, C. C ; Shen, C. C. ; Teng, C. M. ; Pan, S. L. Moscatilin inhibits migration and metastasis of human breast cancer MDA-MB-231 cells through inhibition of Akt and Twist signaling pathway. J. Mol. Med. 2013, 91, 347356.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Zhao, G. Y. ; Deng, B. W. ; Zhang, C. Y ; Cui, Y. D. ; Bi, J. Y. ; Zhang, G. G. New phenanthrene and 9, 10-dihydrophenanthrene derivatives from the stems of Dendrobium officinale with their cytotoxic activities. J. Nat. Med. 2018, 72, 246251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Huang, J. H. ; Liu, C. X. ; Duan, S. N. ; Lin, J. ; Luo, Y. Y. ; Tao, S. C. ; Xing, S. P. ; Zhang, X. F. ; Du, H. Y. ; Wang, H. ; Huang, C. L. ; Wei, G. Gigantol inhibits proliferation and enhances DDP-induced apoptosis in breast-cancer cells by downregulating the PI3K/Akt/mTOR signaling pathway. Life Sci. 2021, 274, 119354.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Tewari, D. ; Patni, P. ; Bishayee, A. ; Sah, A. N ; Bishayee, A. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: a novel therapeutic strategy. Semin. Cancer Biol. 2021; https://doi.org/10.1016/j.semcancer.2019.12.008.

    • Search Google Scholar
    • Export Citation
  • 25.

    Tang, W. ; Zhao, G. Small molecules targeting HIF-1α pathway for cancer therapy in recent years. Bioorg. Med. Chem. 2020, 28, 115235.

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

Indexing and Abstracting Services:

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2021  
Web of Science  
Total Cites
WoS
652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
Impact Factor
without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
Journal Citation Indicator 0,40
Rank by Journal Citation Indicator Chemistry, Analytical 72/99
Scimago  
Scimago
H-index
29
Scimago
Journal Rank
0,27
Scimago Quartile Score Chemistry (miscellaneous) (Q3)
Scopus  
Scopus
Cite Score
2,8
Scopus
CIte Score Rank
General Chemistry 210/409 (Q3)
Scopus
SNIP
0,586

2020
 
Total Cites
650
WoS
Journal
Impact Factor
1,639
Rank by
Chemistry, Analytical 71/83 (Q4)
Impact Factor
 
Impact Factor
1,412
without
Journal Self Cites
5 Year
1,301
Impact Factor
Journal
0,34
Citation Indicator
 
Rank by Journal
Chemistry, Analytical 75/93 (Q4)
Citation Indicator
 
Citable
45
Items
Total
43
Articles
Total
2
Reviews
Scimago
28
H-index
Scimago
0,316
Journal Rank
Scimago
Chemistry (miscellaneous) Q3
Quartile Score
 
Scopus
393/181=2,2
Scite Score
 
Scopus
General Chemistry 215/398 (Q3)
Scite Score Rank
 
Scopus
0,560
SNIP
 
Days from
58
submission
 
to acceptance
 
Days from
68
acceptance
 
to publication
 
Acceptance
51%
Rate

2019  
Total Cites
WoS
495
Impact Factor 1,418
Impact Factor
without
Journal Self Cites
1,374
5 Year
Impact Factor
0,936
Immediacy
Index
0,460
Citable
Items
50
Total
Articles
50
Total
Reviews
0
Cited
Half-Life
6,2
Citing
Half-Life
8,3
Eigenfactor
Score
0,00048
Article Influence
Score
0,164
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,05895
Average
IF
Percentile
20,349
Scimago
H-index
26
Scimago
Journal Rank
0,255
Scopus
Scite Score
226/167=1,4
Scopus
Scite Score Rank
Chemistry (miscellaneous) 240/398 (Q3)
Scopus
SNIP
0,494
Acceptance
Rate
41%

 

Acta Chromatographica
Publication Model Online only
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Submission Fee none
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Acta Chromatographica
Language English
Size A4
Year of
Foundation
1992
Volumes
per Year
1
Issues
per Year
4
Founder Institute of Chemistry, University of Silesia
Founder's
Address
PL-40-007 Katowice, Poland, Bankowa 12
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 2083-5736 (Online)

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