NF-κB

The network pharmacology results indicated that Phellodendrine (PHE) had drug potential. Phellodendrine acted directly on 12 targets, including PTGS1, PTGS2, HTR1A, and PIK3CA, and then regulated cAMP, estrogen, TNF, serotonergic synapse, and other signaling pathways to exert anti-inflammatory effects. The experimental results showed that phellodendrine reduced the levels of IL-6 compared with the LPS group in 24 h and changed the mRNA expression of PTGS1, PTGS2, HSP90ab1, AKT1, HTR1A, PI3CA, and F10. Conclusion: Our research preliminarily uncovered the therapeutic mechanisms of phellodendrine on inflammation with multiple targets and pathways. Phellodendrine may be a potential treatment for inflammation-related diseases related to the cAMP and TNF signaling pathways. [2]

Phellodendrine (PHE) obviously improved the decreased survival rate and abnormally elevated heart-beating rate of zebrafish embryos caused by AAPH. Especially 200μg/mL of PHE make the survival rate increased to 90.26±1.40% at 72hfp and the heartbeat back to normal. Besides, AAPH caused a significant increase in the production of reactive oxygen species (ROS), lipid-peroxidation and cell death rate, all of which could be decreased after PHE treatment dose-dependently. And PHE exerted the protective activity against AAPH-induced oxidative stress through down-regulating AKT phosphorylation and NF-kB3 expression, which associate with modulation of IKK phosphorylation in zebrafish embryos. Significance: The PHE showed a good antioxidant effect in vivo, and the mechanism has been stated that the PHE can down-regulating AKT, IKK, NF-kB phosphorylation and COX-2 expression induced by AAPH. Moreover, the PHE also ameliorated the ROS-mediated inflammatory response. [1]

Drug-likeness and other characteristics of Phellodendrine (PHE) [2]
Lipinski’s rule of five (RO5) is used to evaluate the drug-likeness of oral drugs in humans. The parameters are mainly composed of molecular weight (MW, lower than 500 g/mol), topological polar surface area (TPSA, less than 140 A2), octanol–water partition coefficient (log P0/W (MLOGP), lower than 5), number of hydrogen-bond acceptors (nHAcc, less than 10), number of hydrogen-bond donors (nHDon, less than 5), and number of rotatable bonds (lower than 10). Other characteristics such as log S (ESOL), GI absorption, BBB permeant, log Kp (skin permeation), and bioavailability score were recorded. The chemical structure and canonical SMILES of phellodendrine were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). To estimate the drug-likeness properties and other characteristics of phellodendrine, we imported the Canonical SMILES C[N+]12CCC3=CC(=C(C=C3C1CC4=CC(=C(C=C4C2)OC)O)O)OC of Phellodendrine (PHE) into the SwissADME database (http://www.swissadme.ch/) (Daina, Michielin & Zoete, 2017).

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Collection of Phellodendrine (PHE)-related targets and inflammation-related targets [2]
TCMSP (https://old.tcmsp-e.com/tcmsp.php) and SwissTargetPrediction (http://www.swisstargetprediction.ch/) were required to collect the phellodendrine-related targets depending on chemical names (phellodendrine) and canonical SMILES (C[N+]12CCC3=CC(=C(C=C3C1CC4=CC(=C(C=C4C2)OC)O)O)OC) (probability > 0.09724). The union of targets from the two databases was the phellodendrine-related targets. Inflammation-related targets were selected from the OMIM (http://www.omim.org), Drugbank (https://go.drugbank.com/), and GeneCards (https://www.genecards.org/) databases by searching “inflammation” as a key word. The obtained protein target name was converted into gene name (office gene symbol) using the UniProt database (https://www.uniprot.org). Duplicate genes were deleted. Both Phellodendrine (PHE) targets and inflammation targets were imported into Venny 2.1 software (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to obtain the common targets of phellodendrine against inflammation.

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Network construction of Phellodendrine (PHE) inflammation common targets [2]
The PPI network was built using the STRING database (https://www.string-db.org/). Common targets were uploaded into the STRING database, Homo sapiens was selected in the term of organism, and only a medium confidence score of 0.400 was chosen. Common target interaction information obtained from the STRING database was integrated and visualized by Cytoscape (version 3.7.2) software.

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GO and KEGG pathway enrichment analyses [2]
In our study, GO and KEGG pathway enrichment analyses were carried out using the DAVID database (DAVID Bioinformatics Resources 6.8, https://david.ncifcrf.gov/), and statistical significance was set at P < 0.05. A heatmap was plotted by bioinformatics (http://www.bioinformatics.com.cn), which is an online platform for data analysis and visualization.

Cell viability [2]
MTT assay was used to measure cell viability. In brief, the RAW 264.7 cells were seeded into 96-well plates with a density of 5,000 per well and then incubated at 5% CO2 and 37 °C overnight. Various concentrations of Phellodendrine (PHE) (5, 10, 20, 40, 80, and 160 mg/L) were added to the cells in the presence or absence of LPS (1 mg/L) for 24, 48, and 72 h. The cells were subjected to MTT (20 μL, 5 mg/mL) and then incubated for 4 h. The supernatant was discarded completely, and the formed formazan was dissolved by adding 150 μL of DMSO. Finally, optical density (OD) was measured at 492 nm.

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Cytokine measurement [2]
To detect cytokine production, we seeded RAW 264.7 cells into 96-well plates (1 × 104 cells/well) overnight. The following groups and protocols were consistent with MTT assay. Cytokines in each group were detected by the IL-1β and IL-6 ELISA kits in accordance with the manufacturer’s instructions.

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Quantitative real-time PCR [2]
Total RNA was extracted from each group and then reverse-transcribed into cDNA using PrimeScript™ RT reagent Kit. The primers of the target genes and β-actin were used as shown in Table 1. Quantitative real-time PCR was performed with TB Green® Premix Ex Taq™. The relative expression levels were computed using the 2−ΔΔCT method.

Aims: This study is to investigate the effect of Phellodendrine (PHE) against AAPH-induced oxidative stress and find out the biological mechanism of PHE by using the zebrafish embryo model. [1]
Main methods: After treatments by AAPH or PHE, the mortality and heartbeat of zebrafish embryos were recorded and the production of reactive oxygen species (ROS), lipid-peroxidation and the rate of cell death were detected by fluorescence spectrophotometry respectively. Whereafter, the pathways of PHE against AAPH-induced oxidative stress were screened by inhibitors to explore its biological mechanism. The related genes and proteins expressions were analyzed by real-time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and western blotting.[1]
Waterborne exposure of embryos to Phellodendrine (PHE) and AAPH [1]
Embryos were transferred to individual wells of a 24-well plate (50 embryos/well) and incubated for 2 mL of embryo medium with PHE at 0, 50, 100, 200, 500, and 1000 μg/mL, respectively. Then the survival rates of embryos were recorded at 24, 48, 72, 96 and 120 hpf to determine the toxicity and select the optimal concentration of PHE. In addition, the embryos were treated with 12.5 mmol/L AAPH or co-treated with AAPH and PHE, and the survival rates of embryos were recorded at 24 hpf, 48 hpf and 72 hpf.

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Measurement of the heartbeat of zebrafish embryos [1]
Embryos were incubated in 1 mL of embryo medium with or without Phellodendrine (PHE) at 25, 50, 100 and 200 μg/mL respectively for 1 h, then treated with or without 12.5 mmol/L AAPH for 36 h. Subsequently, embryos were anaesthetized with 0.02% tricaine. The heartbeat rates of both atrium and ventricle were recorded for 3 min under the microscope, and the results were shown as the mean heartbeat rate per minute7].

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Estimation of ROS generation and image analysis in zebrafish embryos [1]
The fluorescent probe DCFH-DA is an oxidation-sensitive fluorescent probe dye, which can be used to analyze the generation of ROS in zebrafish embryos. Embryos were incubated in 1 mL of embryo medium with or without 50 μg/mL of Vc and 50, 100 and 200 μg/mL of Phellodendrine (PHE) for 1 h, then treated with 12.5 mmol/L AAPH for 12 h. Next, the medium was changed into normal embryo one to allow embryos developed to 2 dpf, and the embryos were transferred into 96-well plates and treated with 20 μg/mL DCFH-DA solution, then incubated in dark for 1 h at 28.5 °C and washed with fresh medium, which were anaesthetized with 0.02% tricaine and photographed by the fluorescence microscope. The fluorescent intensity of individual embryo was quantified using Nikon Elements-DR software for image analysis.

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Lipid peroxidation inhibitory activity and image analysis in zebrafish embryos [1]
The non-fluorescent DPPP became fluorescent after oxidized by lipid peroxide, and which could be used to measure lipid peroxidation in zebrafish embryos. The embryos were administrated according to the procedure of Section 2.6. The embryos were treated with 25 μg/mL fluorescent probe DPPP solution for 1 h in the dark at 28.5 °C and then rinsed with fresh medium containing 0.5% of Tween-80 solution and anaesthetized with 0.02% tricaine. The embryos were photographed by fluorescence microscope, and we analyzed their fluorescent intensity by Nikon Elements-DR software.

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Measurement of cell death caused by AAPH-induced oxidative stress in zebrafish embryos [1]
Acridine orange (AO) is a nucleic acid selective fluorescent cationic dye useful for apoptotic cells. The embryos were administrated according to the procedure of Section 2.6. The embryos were treated with 7 μg/mL fluorescent probe AO solution for 0.5 h in the dark [17] and washed with fresh medium. Then the embryos were anaesthetized and photographed by the fluorescence microscope and analyzed the fluorescent intensity.

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Treatment of inhibitors in zebrafish embryos [1]
To clarify the antioxidative mechanism of Phellodendrine (PHE), we chose five inhibitors including SB203580 (5 μg/mL), BAY 11-7082 (0.1 μg/mL), SP600125 (1 μg/mL), LY294002 (2 μg/mL) and U0126-EtOH (1 μg/mL) which correspondingly targeted on p38 MAPK, NF-κB, JNK, AKT and MAPK pathway. The concentrations of inhibitors were chose by LC50 and this experiment was carried out in advance. Embryos were placed into 12-well plates (50 embryos/well) and incubated in 2 mL of embryo medium, and then treated with Phellodendrine (PHE) (100 μg/mL) or inhibitors at 3–4 hpf for 1 h later, followed by treatment with or without AAPH for 12 h, then removed the AAPH and inhibitors developed up to 2 dpf. Finally, the production of ROS was detected by fluorescence microscope to screen the oxidative pathway induced by AAPH.

As mentioned above in the Methods section, we examined 11 properties. The MW of Phellodendrine (PHE) was 342.41 g/mol, nHAcc was 4, nHDon was 2, logP0/W(MLOGP) was −1.71, number of rotatable bonds was 2, TPSA was 58.92 A2, log S (ESOL) was −3.82, GI absorption was high, BBB permeant was “yes,” log Kp (skin permeation) was −6.54 cm/s, and bioavailability score was 0.55 (Table 2). The properties of Phellodendrine (PHE) were in accordance with the RO5, implying that it had drug potential. [2]

Toxicity of Phellodendrine (PHE) or AAPH in zebrafish embryo [1]
Researchers analyzed the survival rate of zebrafish embryos in 5 dpf shown in Table 1 and found the LD50 of PHE was about 500 μg/mL, therefore chosen 50, 100 and 200 μg/mL for the next experiments. When the embryos were exposed to AAPH, their survival rates (SR) were decreased to 73.33 ± 3.33%, 68.49 ± 2.90% and 65.12 ± 1.50% time-dependently and the heartbeat showed an obvious acceleration comparing with the NC group (P = 0.005 ). After co-treated with PHE, the SRs were obviously increased dose-dependently as shown in Fig. 3A. Especially PHE at a dose of 200 μg/mL caused a very significant increase. Besides, PHE could make the abnormally accelerating heartbeat decreased in a dose-dependent manner. From Fig. 3B, 200 μg/mL of PHE led heartbeat of embryos reduced to 97.80 ± 4.50% of NC group.

[1]. The defensive effect of phellodendrine against AAPH-induced oxidative stress through regulating the AKT/NF-κB pathway in zebrafish embryos. Life Sci. 2016 Jul 15;157:97-106.

[2]. Utilizing network pharmacology and experimental validation to investigate the underlying mechanism of phellodendrine on inflammation. PeerJ. 2022 Sep 23:10:e13852.

Phellodendrine (PHE) is an alkaloid.
Phellodendrine has been reported in Xylopia parviflora, Phellodendron chinense var. glabriusculum, and other organisms with data available.

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This study started from research on anti-oxidative mechanisms to find more about therapy of Phellodendrine (PHE), such as the anti-inflammatory effect. The PI3K/AKT signaling pathway plays an essential role in the development heart disease and doxorubicin-induced cardiomyocytes apoptosis, and the NF-κB signal pathway activation can promote the release of inflammatory factors as TNF-α, IL-6, and IL-8. Previous literatures prove the PHE has anti-nephritis effect, but has not studied the effect of the PHE on heart disease. So this study can provide an important theory basis for the research of PHE anti-heart disease. But this is only a preliminary research, and it needs to do more for further investigation. In conclusion, our study has demonstrated the PHE isolated from Phellodendri chinensis cortex has an ability of antioxidant through regulating the AKT/ NF-κB pathway in zebrafish embryo. And the PHE could reverse the expression of AKT and NF-κB3, IKK, COX-2 which were abnormally changed by AAPH-induced oxidative stress.[1]

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In summary, network pharmacological analyses and experimental validation were conducted to uncover the pharmacological mechanism of Phellodendrine (PHE) on inflammation. In this study, 12 common targets against inflammation were acquired through network pharmacology. Phellodendrine exerted anti-inflammation effects through the cAMP signaling pathway, TNF signaling pathway, serotonergic synapse, and so on. Further validation with experimental evidence demonstrated that phellodendrine inhibited inflammation in RAW264.7 cells by regulating the expression of IL-6, IL-1β, PTGS1, AKT1, HSP90ab1, HTR1A, and PI3CA. Our research might provide new treatment regimens for inflammation-related diseases. However, details about the anti-inflammation of phellodendrine need to be further investigated. [2]

C20H24NO4

342.4089

377.139

C, 70.15; H, 7.07; N, 4.09; O, 18.69

6873-13-8

104112-82-5

3081405

Off-white to light yellow solid powder

Xylopia parviflora, Phellodendron chinense var. glabriusculum

2.873

2

4

2

25

488

2

C[N@@+]12CCC3=CC(=C(C=C3[C@@H]1CC4=CC(=C(C=C4C2)OC)O)O)OC

RBBVPNQTBKHOEQ-KKSFZXQISA-O

InChI=1S/C20H23NO4/c1-21-5-4-12-8-19(24-2)18(23)10-15(12)16(21)6-13-7-17(22)20(25-3)9-14(13)11-21/h7-10,16H,4-6,11H2,1-3H3,(H-,22,23)/p+1/t16-,21-/m0/s1

(7S,13aS)-3,10-dimethoxy-7-methyl-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinolin-7-ium-2,11-diol

Phellodendrine; Phallodendrin; 6873-13-8; OB-5 Compound; AR68S526RB; (7S,13aS)-3,10-dimethoxy-7-methyl-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinolin-7-ium-2,11-diol; UNII-AR68S526RB; DTXSID50218855;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~5 mg/mL (~14.60 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)