The research investigates its anti-inflammatory effects on LPS-activated human neutrophils, involving modulation of surface markers (CD16, CD62L), cytokine expression (TNF-α, IL-6, IL-8), and nitric oxide (NO) production. [1]

Mitraphylline treatment (25 μM) of LPS-activated human primary neutrophils significantly reduced nitrite production (by 54%, p < 0.05) compared to LPS-only treated cells, approaching levels achieved with the NOS inhibitor L-NAME. This was accompanied by a marked downregulation of iNOS mRNA expression (by 67%, p < 0.01). [1]
- Mitraphylline significantly downregulated the gene expression of pro-inflammatory cytokines in LPS-activated neutrophils: IL-6 by 83% (p < 0.05), IL-8 by 37% (p < 0.05), and TNF-α by 53% (p < 0.05). [1]
- Consistent with gene expression data, Mitraphylline significantly reduced the secretion of these cytokines from LPS-activated neutrophils: IL-6 by 62% (p < 0.05), IL-8 by 79% (p < 0.05), and TNF-α by 65% (p < 0.05). [1]
- Flow cytometry analysis of neutrophil surface markers showed that LPS activation nearly depleted the CD16⁺CD62L⁺ population (by 98%, p < 0.001), shifting cells to an activated phenotype. Co-incubation with Mitraphylline partially reversed this effect, increasing the CD16⁺CD62L⁺ population to 10-15% of total neutrophils (p < 0.05), indicating a shift towards a less inflammatory phenotype. [1]

Cytotoxicity Assay (MTT): Human neutrophils were seeded in 96-well plates (1×10⁵ cells/well) and incubated with various concentrations of Mitraphylline (0, 15, 25, 35, 45, 75, 100 μM) for 6 and 24 hours. Staurosporine (1 nM) was used as a reference control. Cell viability was measured by MTT colorimetric assay. The IC₅₀ (concentration for 50% reduction in viability) was calculated as 50 μM at 6 hours and 35 μM at 24 hours. A non-toxic concentration of 25 μM was selected for subsequent experiments. [1]
- Neutrophil Isolation and Culture: Human neutrophils were isolated from peripheral venous blood of healthy volunteers by dextran sedimentation and Ficoll-Histopaque gradient centrifugation, followed by hypotonic lysis of erythrocytes. Purity was >97% by trypan blue exclusion. Cells were suspended in RPMI 1640 medium with supplements and 1% heat-inactivated fetal bovine serum, and seeded at 3×10⁶ cells/mL. [1]
- Cell Stimulation and Treatment: Neutrophils were pre-treated with or without Mitraphylline (25 μM) for 30 minutes, then stimulated with LPS (0.1 μg/mL from E. coli 055:B5) for 6 hours. [1]
- Nitrite/Nitric Oxide Assay: Culture supernatants were mixed with Griess reagent and incubated for 15 minutes at room temperature. Nitrite concentration, as an indicator of NO production, was measured by absorbance at 540 nm using an ELISA reader. Results were extrapolated from a sodium nitrite standard curve and expressed as a percentage of the LPS-only treated control. [1]
- Flow Cytometry for Surface Markers: Neutrophils (10⁶ cells) were incubated with FITC-anti-human CD62L and PE-anti-human CD16 antibodies for 15 minutes at room temperature. Erythrocytes were lysed with FACS lysing solution. Fluorescence intensity was measured using a FACS Canto II flow cytometer. Neutrophils were gated as FSCʰⁱᵍʰSSCʰⁱᵍʰCD16ʰⁱᵍʰ cells, and mean fluorescence intensity (MFI) was measured for 10,000 cells per sample. [1]
- RNA Isolation and qRT-PCR: Total RNA was extracted using Trisure Reagent. RNA (1 μg) was reverse transcribed to cDNA. Real-time PCR was performed using Brilliant SYBR Green QPCR Supermix with specific primers for TNF-α, IL-6, IL-8, and housekeeping genes (GAPDH, β-actin). The 2⁻⁽ΔΔCt⁾ method was used to calculate relative mRNA expression. [1]
- Cytokine Quantification by ELISA: Levels of TNF-α, IL-6, and IL-8 in cell culture supernatants were measured using commercial ELISA kits, following the manufacturer's instructions. [1]

The cytotoxicity of Mitraphylline was assessed in human primary neutrophils using the MTT assay. The calculated IC₅₀ values were 50 μM at 6 hours and 35 μM at 24 hours of treatment. A concentration of 25 μM was determined to be non-toxic and was used for all subsequent functional experiments. [1]

[1]. Mitraphylline inhibits lipopolysaccharide-mediated activation of primary human neutrophils. Phytomedicine. 2016 Feb 15;23(2):141-8.

Mitraphylline belongs to the indoleazine class of compounds. It has been reported to be found in Uncaria tomentosa, Cephalanthus tetrandrus, and other organisms with relevant data. See also: Uncaria tomentosa (partial); Mitragyna speciosa leaves (partial).

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Mitraphylline is the major pentacyclic oxindole alkaloid found in the bark of Uncaria tomentosa (cat's claw), a plant traditionally used in folk medicine to treat inflammatory disorders, arthritis, and cancer. [1]
- This study demonstrates that Mitraphylline can modulate the activation of primary human neutrophils, steering them towards a less inflammatory phenotype. This is evidenced by reduced shedding of CD62L (resulting in increased CD16⁺CD62L⁺ population), decreased production of nitric oxide (via iNOS downregulation), and reduced expression and secretion of key pro-inflammatory cytokines (TNF-α, IL-6, IL-8). [1]
- The findings suggest that Mitraphylline contributes to the anti-inflammatory properties attributed to U. tomentosa extracts. By regulating neutrophil activation and promoting a less inflammatory state, it may have potential therapeutic value for chronic inflammatory diseases where neutrophils play a pivotal role. [1]

C21H24N2O4

368.4263

368.174

509-80-8

94160

White to off-white solid powder

1.33 g/cm3

555.2ºC

258-267 °C

289.6ºC

1.635

2.138

1

5

2

27

692

5

C[C@H]1[C@H]2CN3CC[C@]4([C@@H]3C[C@@H]2C(=CO1)C(=O)OC)C5=CC=CC=C5NC4=O

JMIAZDVHNCCPDM-DAFCLMLCSA-N

InChI=1S/C21H24N2O4/c1-12-14-10-23-8-7-21(16-5-3-4-6-17(16)22-20(21)25)18(23)9-13(14)15(11-27-12)19(24)26-2/h3-6,11-14,18H,7-10H2,1-2H3,(H,22,25)/t12-,13-,14+,18-,21+/m0/s1

methyl (1S,4aS,5aS,6R,10aR)-1-methyl-2'-oxospiro[1,4a,5,5a,7,8,10,10a-octahydropyrano[3,4-f]indolizine-6,3'-1H-indole]-4-carboxylate

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 : ~6.76 mg/mL (~18.35 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.)