Natural alkaloid; NF-κB

Despite the massive efforts to develop the treatment of pancreatic cancers, no effective application exhibits satisfactory clinical outcome. Macropinocytosis plays a critical role for continuous proliferation of pancreatic ductal adenocarcinoma (PDAC). In this study, we generated a screening method and identified Phellodendrine (PHE) chloride (PC) as a potential macropinocytosis inhibitor. PC significantly inhibited the viability of KRAS mutant pancreatic cancer cells (PANC-1 and MiaPaCa-2) in a dose-dependent manner; however, it did not affect the wild type KRAS pancreatic cancer cells (BxPC-3). Further experiments indicated that PC reduced the growth of PANC-1 cells through inhibition of macropinocytosis and diminishing the intracellular glutamine level. Disruption of glutamine metabolism led to enhance the reactive oxygen species level and induce mitochondrial membrane potential depolarization in PANC-1 cells. PC treatment caused increased Bax and decreased Bcl-2 expression, along with the activation of cleaved caspase-3, 7, 9 and cleaved-PARP, thus induced mitochondrial apoptosis. Moreover, PC inhibited macropinocytosis in vivo and effectively reduced the growth of PANC-1 xenograft tumors. All together, we demonstrated that inhibition of macropinocytosis might be an effective strategy to treat pancreatic cancers. Thus, PC could be a potential compound with improved therapeutic efficacy in patients with pancreatic cancers. [3]

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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]

To investigate the therapeutic effects of Phellodendrine (PHE) in ulcerative colitis (UC) through the AMPK/mTOR pathway. Volunteers were recruited to observe the therapeutic effects of Compound Cortex Phellodendri Liquid (Huangbai liniment). The main components of Compound Cortex Phellodendri Liquid were analysed via network pharmacology. The target of Phellodendrine (PHE) was further analysed. Caco-2 cells were cultured, and H2 O2 was used to stimulate in vitro cell model. Expression levels of LC3, AMPK, p-AMPK, mTOR and p-mTOR were detected via Western blotting and through immunofluorescence experiments. The therapeutic effects of phellodendrine were analysed via expression spectrum chip sequencing. The sequencing of intestinal flora further elucidated the therapeutic effects of phellodendrine. Compared with the control group, Compound Cortex Phellodendri Liquid could substantially improve the healing of intestinal mucosa. Network pharmacology analysis revealed that phellodendrine is the main component of Compound Cortex Phellodendri Liquid. Moreover, this alkaloid targets the AMPK signalling pathway. Results of animal experiments showed that phellodendrine could reduce the intestinal damage of UC compared with the model group. Findings of cell experiments indicated that phellodendrine treatment could activate the p-AMPK /mTOR signalling pathway, as well as autophagy. Expression spectrum chip sequencing showed that treatment with phellodendrine could promote mucosal healing and reduce inflammatory responses. Results of intestinal flora detection demonstrated that treatment with phellodendrine could increase the abundance of flora and the content of beneficial bacteria. Phellodendrine may promote autophagy by regulating the AMPK-mTOR signalling pathway, thereby reducing intestinal injury due to UC. [4]

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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.

Mice were allowed to acclimatize for one week. Then, PANC-1 cells (2 × 106 cells) in equal volume with matrigel, the total volume of 200 μl, were injected subcutaneously (s.c.) to the right flank of mice. When tumor volume reached 60–100 mm3, mice were randomly divided into 4 groups (n = 7/group): control (vehicle; 1:4 ratio of DMSO and PBS, i.p.), EIPA (2 mg/kg/day, i.p.), and Phellodendrine (PC) (30 mg/kg/day and 60 mg/kg/day, i.p.). Tumor volumes and body weights were measured and recorded every two days. Tumor volume was measured using digital caliper and calculated according to the formula: V = 0.52 × ab2, where a represents the large diameter and b is the small diameter of the tumor (Zhou et al., 2014). On the end of drug treatment, mice were killed and tumors were excised. Tumor weights were measured and tumor weight inhibition (%) was calculated based on the weight of excised tumors (W) using the equation: Tumor weight inhibition (%) = W (vehicle) – W (treatment)/ W (vehicle) × 100 (Wang et al., 2017). [3]

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In this study, SPF grade female C57BL/6 mice (18‐22 g) were selected as the animal model. According to weight, the mice were randomly divided into four groups with five mice in each group: normal control group, model group, positive control group and Phellodendrine (PC) group. Phellodendrine (PC) was given at a dosage of 30 mg/kg. The positive control group was treated with salazosulphapyridine (40 mg/mL) at a dose of 200 mg/kg. The normal control and the model control groups were treated with double steam water. The acute phase model of UC was induced by 5% dextran sulphate sodium (DSS). During the adaptation period, the mice were freely given food and water. The experiment was began by replacing the drinking water with a 5% DSS solution (except for the normal control group) and giving the mice a free drink. The daily water intake of each mouse was calculated as 6 mL, and a sufficient amount of the DSS solution was added the next day. The DSS solution was administered for 8 days. After the initial administration of DSS for 24 h, soft stool, diarrhoea or blood stool was found in the mice, that is the mice were given the drug by gavage. The mice in the normal control and the model control groups were given 0.1 mL of double‐steamed water every day. The mice in the positive control group were given 0.1 mL salazosulphapyridine (40 mg/mL) every day. The Phellodendrine (PC) group was administered with 30 mg/kg of Phellodendrine (PC). The overall performance of each mouse was assessed by disease activity index (DAI), including bodyweight loss, stool consistency and faecal blood, and the range of every item is from 0 to 4. The sum of all scores from these three parameters was calculated as the DAI. After 7 days of administration, all the mice were killed. The abdominal cavity of the mice was exposed, and their colon was dissected and its length was measured. The colon was cut lengthwise along the mesentery, and the stool was cleaned with normal saline and fixed with 10% neutral formalin solution. Gross injuries were observed and scored under a stereomicroscope, and severe ulcers were taken for pathological examination. [4]

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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.

[3]. Phellodendrine chloride suppresses proliferation of KRAS mutated pancreatic cancer cells through inhibition of nutrients uptake via macropinocytosis. Eur J Pharmacol. 2019 May 5;850:23-34.

[4]. Phellodendrine promotes autophagy by regulating the AMPK/mTOR pathway and treats ulcerative colitis. J Cell Mol Med. 2021;25(12):5707-5720.

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]

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Collectively, in this work, we demonstrated the anticancer effects of Phellodendrine (PC) might be via decreased macropinocytosis and altered glutamine metabolism. Moreover, PC treatment of cells with or without NAC also confirmed that reactive oxygen species generation was involved in PC induced mitochondrial apoptosis cell death. Our results revealed that inhibition of macropinocytosis could be a potent therapeutic strategy to discover and develop the compounds to treat pancreatic cancers.[3]

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This study investigated the effects and molecular mechanism of Phellodendrine (PC) in Compound Phellodendrine solution for UC treatment. Phellodendrine processing may promote autophagy by activating the AMPK‐mTOR signalling pathway and ultimately exert a protective effect on UC. However, whether phellodendrine regulates autophagy through other signalling pathways remains to be explored. Clarifying its specific mechanism of action in UC will provide a theoretical basis for the clinical applications of phellodendrine and new ideas for clinical treatment of UC. [4]

C20H24CLNO4

377.86

377.139

C, 63.57; H, 6.40; Cl, 9.38; N, 3.71; O, 16.94

104112-82-5

Phellodendrine;6873-13-8

59818

White to off-white solid powder

249-251℃ (methanol )

2

5

2

26

488

1

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

DGLDSNPMIYUWGN-OOJQBDKLSA-N

InChI=1S/C20H23NO4.ClH/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);1H/t16-,21?;/m0./s1

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

l-(-)-N-Methylcoreximine; Phellodendrine chloride; 104112-82-5; l-(-)-N-Methylcoreximine; Phellodendrine Hydrochloride; 13a-alpha-BERBINIUM, 2,11-DIHYDROXY-3,10-DIMETHOXY-7-METHYL-, CHLORIDE; (13aS)-2,11-dihydroxy-3,10-dimethoxy-7-methyl-5,6,7,8,13,13a-hexahydroisoquinolino[3,2-a]isoquinolin-7-ium chloride; (13aS)-3,10-dimethoxy-7-methyl-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinolin-7-ium-2,11-diol;chloride; Phellodendrine HCl; Phellodendrine chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~50 mg/mL (~132.32 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (13.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (13.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 5 mg/mL (13.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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