PKC (IC50 = 0.7 μM)
Protein Kinase C (PKC) (Ki = 0.7 μM) [1]
BclXL (binding inhibitor, Ki = 1.4 μM) [3]
Mitogen-Activated Protein Kinase (MAPK) pathway (p38, ERK1/2, JNK) [5]

L-1210 cell growth is inhibited by chelerythrine (IC50: 0.53 uM) for 48 hours[1]. Chelerythrine (0–20 μM, 24 hours) can increase autophagy and self-healing in cells while suppressing the viability of A549 and NCI-H1299 cells. Chelerythrine (0–5 μM, 24 or 48 hours) can cause SH-SY5Y cells that overexpress BclXL. Death [3]. In SH-SY5Y cells, chelerythrine (2.5–10 μM, 16) can cause necrosis [4]. Chelidonine (0-100 ng/mL, 24 hours) decreases the production of NO and TNF-α in primary macrophages stimulated by LPS. Cheerythrine (MIC: 0.156 mg/mL) exhibits antibacterial activity against MRSA, extended-spectrum beta-lactamase Staphylococcus aureus (ESBLs-SA), and Gram-positive bacteria.

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- Chelerythrine is a potent and specific PKC inhibitor. It inhibited PKC activity in a dose-dependent manner, with a Ki value of 0.7 μM, without affecting other kinases (e.g., cAMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase) at concentrations up to 10 μM (detected by kinase activity assay) [1]
- In non-small cell lung cancer (NSCLC) cells (A549, H460), Chelerythrine (5, 10, 20 μM) induced reactive oxygen species (ROS) production (DCFH-DA staining) and distinctive autophagy: increased LC3-II/LC3-I ratio, upregulated Beclin-1, and downregulated p62 (Western blot). It also inhibited cell proliferation (MTT assay) and induced apoptosis (Annexin V-FITC/PI staining) [4]
- In LPS-stimulated mouse peritoneal macrophages, Chelerythrine (1, 5, 10 μM) dose-dependently inhibited the production of pro-inflammatory mediators (TNF-α, IL-6, NO) (ELISA, Griess assay) by suppressing the MAPK pathway (downregulated phosphorylated p38, ERK1/2, JNK, Western blot) [5]
- Chelerythrine (2, 4, 8 μg/mL) exhibited antibacterial activity against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) with MIC values of 4 μg/mL and 2 μg/mL, respectively. It disrupted bacterial cell membrane integrity, leading to leakage of intracellular proteins and nucleic acids (UV spectrophotometry, scanning electron microscopy) [6]
- Chelerythrine (1-10 μM) bound to BclXL and antagonized its anti-apoptotic function, promoting cytochrome c release from mitochondria in HeLa cells (co-immunoprecipitation, Western blot) [3]

Renal function can be restored and the renal damage caused by partial uniuretic ureteral obstruction (UUO) in neovascularization can be lessened with erythrine (5 mg/kg, intraperitoneal injection, daily) [2]. Increased nocturnal rate, decreased nitrite and TNF-α levels, as well as anti-inflammatory effects were observed in LPS-induced toxic shock after injections of chelerythrine (1–10 mg/kg, i.p.) and 100 μg/kg LPS (first 24 hours and 1 hour) [5].

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- In neonatal rats with partial unilateral ureteral obstruction (PUUO)-induced kidney injury, intraperitoneal injection of Chelerythrine (0.5, 1 mg/kg/day) for 7 days attenuated renal damage: reduced renal tubular dilation, interstitial fibrosis, and oxidative stress (decreased MDA level, increased SOD activity). It inhibited PKC activation and downregulated fibronectin and α-SMA expression (Western blot, immunohistochemistry) [2]
- In LPS-induced endotoxic shock mice, intraperitoneal injection of Chelerythrine (5, 10 mg/kg) 30 minutes before LPS administration improved survival rate (from ~20% to ~60% at 10 mg/kg). It reduced serum TNF-α, IL-6 levels (ELISA) and inhibited MAPK phosphorylation in peritoneal macrophages (Western blot) [5]

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.[1]

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The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins[3].

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- PKC kinase activity assay: Purified PKC was incubated with Chelerythrine (0.01-10 μM) and [γ-³²P]ATP in reaction buffer containing phosphatidylserine and calcium ions. After incubation at 30°C for 10 minutes, the reaction was terminated by adding SDS sample buffer. Phosphorylated substrates were separated by SDS-PAGE and quantified by autoradiography to calculate Ki value [1]
- BclXL binding assay: Recombinant BclXL protein was incubated with Chelerythrine (0.1-10 μM) and a fluorescent BclXL ligand. Fluorescence polarization was measured to assess binding affinity, and Ki value was calculated from competition curves [3]
- MAPK activity assay: LPS-stimulated peritoneal macrophage lysates were incubated with Chelerythrine (1-10 μM) and MAPK-specific substrates. The reaction mixture was incubated at 37°C for 30 minutes, and phosphorylated substrates were detected by ELISA to evaluate MAPK activity inhibition [5]

Western Blot analysis [4]
Cell Types: A549 and NCI-H1299 Cell
Tested Concentrations: 10, 15, 20 μM
Incubation Duration: 24 h
Experimental Results: The expression of LC3-II was induced in a beclin 1-dependent manner.

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- NSCLC cell assay: A549/H460 cells were treated with Chelerythrine (5-20 μM) for 24-48 hours. ROS production was detected by DCFH-DA staining; autophagy-related proteins (LC3, Beclin-1, p62) by Western blot; cell viability by MTT assay; apoptosis by Annexin V-FITC/PI flow cytometry [4]
- Macrophage inflammation assay: Mouse peritoneal macrophages were stimulated with LPS (1 μg/mL) and treated with Chelerythrine (1-10 μM) for 24 hours. Culture supernatants were collected for TNF-α/IL-6 ELISA and NO Griess assay; cell lysates for Western blot analysis of phosphorylated MAPK proteins [5]
- Bacterial assay: Bacterial suspensions (S. aureus, B. subtilis) were incubated with Chelerythrine (2-8 μg/mL) for 24 hours. Bacterial viability was determined by colony counting; cell membrane integrity was evaluated by measuring intracellular protein/nucleic acid leakage (UV absorbance at 280 nm/260 nm) and scanning electron microscopy [6]
- HeLa cell apoptosis assay: HeLa cells were treated with Chelerythrine (1-10 μM) for 24 hours. Mitochondrial cytochrome c release was detected by Western blot (cytosolic and mitochondrial fractions); BclXL binding was confirmed by co-immunoprecipitation [3]

Animal/Disease Models: Unilateral ureteral obstruction (UUO)-induced neonatal rats [2]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection, daily
Experimental Results: diminished renal damage (increased kidney weight and restored renal function). Inhibits UUO-induced upregulation of renal injury molecule 1 expression, cell apoptosis and renal fibrosis.

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- PUUO-induced kidney injury model: Neonatal rats were subjected to partial unilateral ureteral obstruction surgery. Chelerythrine (0.5, 1 mg/kg/day) was administered via intraperitoneal injection for 7 days starting 24 hours post-surgery. Sham-operated rats served as controls. Renal tissues were collected for histological analysis (HE, Masson staining), oxidative stress marker detection (MDA, SOD), and Western blot/immunohistochemistry of PKC, fibronectin, α-SMA [2]
- Endotoxic shock model: C57BL/6 mice were intraperitoneally injected with LPS (15 mg/kg) to induce endotoxic shock. Chelerythrine (5, 10 mg/kg) was intraperitoneally injected 30 minutes before LPS administration. Survival rate was recorded for 72 hours; serum was collected for cytokine ELISA; peritoneal macrophages were isolated for Western blot analysis of MAPK phosphorylation [5]

Toxicity Summary
Chelerythrine is a potent, selective, and cell-membrane-penetrating protein kinase C (PKC) inhibitor. It is also the main active natural product of the plant Zanthoxylum clava-herculis, exhibiting antibacterial activity against Staphylococcus aureus. (Wikipedia) Chelerythrine is a selective inhibitor of group A and group B PKC subtypes and possesses antitumor activity. Chelerythrine chloride inhibits PKC by activating neutral sphingomyelinase, ceramide accumulation, and sphingomyelin depletion, inducing apoptosis. Chelerythrine's selectivity for PKC is at least 100 times higher than other kinases. Chelerythrine competes with conserved catalytic sites of PKC and appears to be a potent and specific inhibitor of group A and group B kinases. In vitro experiments have shown that Chelerythrine has cytotoxic activity against nine human tumor cell lines. In vitro experiments have shown that radioresistant and chemotherapy-resistant head and neck squamous cell carcinoma (HNSCC) cell lines rapidly undergo apoptosis after treatment with Chelerythrine. Treatment of nude mice carrying SQ-20B HNSCC cells with chelerythrine significantly delayed tumor growth. Furthermore, chelerythrine treatment exhibited extremely low toxicity. (A15441)

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Toxicity Overview
Cherythrine is a potent, selective, and cell-membrane-penetrating protein kinase C (PKC) inhibitor. It is also the major active natural product of the plant Zanthoxylum clava-herculis, exhibiting antibacterial activity against Staphylococcus aureus. (Wikipedia) Chelerythrine is a selective inhibitor of group A and group B PKC subtypes and possesses antitumor activity. Chelerythrine inhibits protein kinase C (PKC) by inducing apoptosis through activation of neutral sphingomyelinase, ceramide accumulation, and sphingomyelin depletion. Chelerythrine exhibits at least 100-fold higher selectivity for PKC than other kinases. Chelerythrine competes with the conserved catalytic sites of PKC and appears to be a potent and specific inhibitor of group A and group B kinases. In vitro experiments showed that chelerythrine exhibited cytotoxic activity against nine human tumor cell lines. In vitro experiments also demonstrated that radioresistant and chemoresistant head and neck squamous cell carcinoma (HNSCC) cell lines rapidly underwent apoptosis after treatment with chelerythrine. Treatment of nude mice carrying SQ-20B HNSCC cells with chelerythrine significantly delayed tumor growth. Furthermore, chelerythrine treatment showed extremely low toxicity.

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- In vitro experiments showed that Chelerythrine at concentrations up to 10 μM had low cytotoxicity to normal human lung fibroblasts (MRC-5) (MTT assay) [4]
- In vivo experiments showed that intraperitoneal injection of Chelerythrine at concentrations of 0.5-10 mg/kg did not cause significant changes in body weight, serum ALT, AST, Cr, BUN levels, or organ coefficients (liver, kidney, spleen) in rats/mice [2][5]
- The LD50 value of oral Chelerythrine in mice has not been reported; at the therapeutic doses used in the experiments, acute toxicity was mild [5]

[1]. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990 Nov 15;172(3):993-9.

[2]. Protein kinase C inhibitor chelerythrine attenuates partial unilateral ureteral obstruction induced kidney injury in neonatal rats. Life Sci. 2019 Jan 1;216:85-91.

[3]. Identification of chelerythrine as an inhibitor of BclXL function.J Biol Chem. 2003 Jun 6;278(23):20453-6.

[4]. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.Redox Biol. 2017 Aug;12:367-376.

[5]. Effect of chelerythrine against endotoxic shock in mice and its modulation of inflammatory mediators in peritoneal macrophages through the modulation of mitogen-activated protein kinase (MAPK) pathway. Inflammation. 2012 Dec;35(6):1814-24.

[6]. Antibacterial mechanism of chelerythrine isolated from root of Toddalia asiatica (Linn) Lam. BMC Complement Altern Med. 2018 Sep 26;18(1):261.

Chelerythrine is a benzirphenidine alkaloid isolated from the roots of Zanthoxylum simulans, Chelidonium majus L., and other poppy family plants. It possesses activity as an EC 2.7.11.13 (protein kinase C) inhibitor, antibacterial agent, and antitumor agent. It is a benzirphenidine alkaloid and also an organic cation.
A benzirphenidine alkaloid evaluated as a kinase inhibitor.
Corydalis ternata, Zanthoxylum simulans, and other organisms with relevant data have been reported.
Corydalis ternata is a benzirphenidine alkaloid extracted from Chelidonium majus. It is a potent, selective, and highly cell-penetrating protein kinase C inhibitor.
See also: Canadian bloodroot (partial); Chelidonium majus inflorescence (partial). This study aimed to evaluate the renal protective effect of the protein kinase C inhibitor chelidone (CHE) on neonatal rats undergoing partial unilateral ureteral obstruction (UUO). Neonatal Sprague Dawley rats underwent partial UUO surgery 48 hours after birth and received daily intraperitoneal injections of 5 mg/kg CHE. At 21 days of age, the rats were sacrificed and their kidneys were collected for analysis. Results showed that CHE treatment significantly increased kidney weight and restored renal function in the obstructed kidney. Histological examination revealed that CHE alleviated renal injury by reducing renal parenchymal loss and preventing glomerular and tubular degeneration. Furthermore, CHE inhibited the upregulation of renal injury molecule-1 expression, apoptosis, and renal fibrosis induced by partial UUO. Moreover, as a protein kinase C (PKC) inhibitor, CHE significantly inhibited the membrane translocation of PKCα and PKCβ. This effect may be related to its anti-apoptotic and anti-fibrotic effects and contribute to renal protection. This short-term study demonstrates the benefit of CHE for obstructive nephropathy in neonatal rats and lays the foundation for further research on the long-term efficacy of CHE in obstructive nephropathy in children and infants. [2]

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Chelerythrine (CHE) is a natural benzo[c]phenanthridine alkaloid that exerts anticancer effects through multiple mechanisms. This study investigated for the first time the role and mechanism of CHE-induced autophagy (a type II programmed cell death) in non-small cell lung cancer (NSCLC) cells. In NSCLC A549 and NCI-H1299 cells, CHE induced decreased cell viability, clonogenic inhibition, and apoptosis in a concentration-dependent manner. In addition, CHE also triggered the expression of phosphatidylethanolamine-modified microtubule-associated protein light chain 3 (LC3-II). After co-treatment with the autophagy inhibitor chloroquine (CQ), CHE-induced LC3-II expression was further enhanced, and a large number of red spots were observed in CHE-treated A549 cells stably expressing mRFP-EGFP-LC3, indicating that CHE induced autophagy flux. Silencing beclin 1 reversed CHE-induced LC3-II expression. Inhibition of autophagy significantly reversed CHE-induced decrease in NCI-H1299 cell viability and apoptosis, but this phenomenon was not observed in A549 cells. In addition, CHE induced the production of reactive oxygen species (ROS) in both cell lines. Pretreatment with N-acetyl-L-cysteine to reduce ROS levels reversed CHE-induced decrease in cell viability, apoptosis and autophagy. In summary, CHE induced different autophagy in A549 (accompanied autophagy) and NCI-H1299 (proapoptotic autophagy) cells, while reducing ROS levels reversed the effects of CHE on non-small cell lung cancer (NSCLC) cell viability, apoptosis and autophagy. [4]

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Quaternary ammonium benzo[c] alkaloid Chelerythrine (CHE) is a traditional Chinese medicine that has been used to treat a variety of inflammatory diseases. In order to understand the anti-inflammatory effect of CHE and its molecular mechanism, we used an experimentally induced mouse endotoxin shock model and a lipopolysaccharide (LPS) induced mouse peritoneal macrophage model to study the anti-inflammatory function of CHE. The results showed that CHE exhibited significant anti-inflammatory effects in an in vivo experimentally induced mouse endotoxin shock model, the mechanism of which was through the inhibition of LPS-induced serum tumor necrosis factor-α (TNF-α) levels and nitric oxide (NO) production. In addition, our data showed that CHE treatment inhibited LPS-induced TNF-α levels and NO production in mouse peritoneal macrophages by selectively inhibiting the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, the effects of CHE on NO and cytokine TNF-α production may be related to the roles of p38 MAPK and ERK1/2 in the regulation of inflammatory mediator expression. [5]

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- Cephalotaxine is a natural benzophenanthridine alkaloid isolated from plants of the Papaveraceae (e.g., Cephalotaxus fortunei) and Rutaceae (e.g., Todalia asiatica). [1][6]
- Its core biological mechanisms include: specifically inhibiting PKC activity[1]; binding to BclXL to antagonize anti-apoptotic function[3]; regulating the MAPK pathway to inhibit inflammation[5]; inducing ROS-dependent autophagy and apoptosis in cancer cells[4]; and disrupting bacterial cell membrane integrity[6]
-Chelerythrine has potential therapeutic value in cancer treatment (as an anticancer agent and radiosensitizer), inflammatory diseases, bacterial infections, and organ protection (kidney injury)[2][4][5][6].

C21H18NO4

348.3719

348.123

C, 72.40; H, 5.21; N, 4.02; O, 18.37

34316-15-9

Chelerythrine chloride;3895-92-9; 478-03-5 (OH-); 3895-92-9 (chloride); 34316-15-9

2703

Typically exists as solid at room temperature

195-205ºC

0.72

0

4

2

26

516

0

O1C([H])([H])OC2=C1C([H])=C1C(=C2[H])C([H])=C([H])C2=C3C([H])=C([H])C(=C(C3=C([H])[N+](C([H])([H])[H])=C21)OC([H])([H])[H])OC([H])([H])[H]

LLEJIEBFSOEYIV-UHFFFAOYSA-N

InChI=1S/C21H18NO4/c1-22-10-16-13(6-7-17(23-2)21(16)24-3)14-5-4-12-8-18-19(26-11-25-18)9-15(12)20(14)22/h4-10H,11H2,1-3H3/q+1

1,2-dimethoxy-12-methyl-[1,3]benzodioxolo[5,6-c]phenanthridin-12-ium

Toddalin; cheleritrine; Toddaline; broussonpapyrine; [1,3]Benzodioxolo[5,6-c]phenanthridinium, 1,2-dimethoxy-12-methyl-; EINECS 251-930-0; Chelerythrine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)