Kalanchoe pinnata modulates production of TNF-α in neutrophils

Kalanchoe pinnata (Lam.) Pers. (syn. Bryophyllum pinnatum, Crassulaceae) is a popular medicinal plant used in Brazil and worldwide. It is popularly used to treat inflammatory disease, gastric ulcer [1]. We investigated the effects of a hydroethanolic extract (HE) and the major compound, quercetin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside (QAR) on TNF-α liberation, membrane integrity (necrosis), and DNA fragmentation (apoptosis) in human neutrophils. These cells play a significant role in the inflammatory response, by releasing inflammatory mediators such as tumor necrosis factor alpha (TNF-α)[2].

K. pinnata was collected at CPQBA-UNICAMP. A voucher specimen (337) is deposited at the CPMA. The dried leaves were macerated in ethanol 70%. Quercetin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside was isolated from the ethyl acetate fraction and identified by NMR. Human blood samples were collected from healthy volunteers. After collecting, the blood was processed to isolate neutrophils. Neutrophils (1 × 10⁶ cells/mL) were treated with HE and QAR at different concentrations for 4h at 37 °C in 5% CO₂ atmosphere. LPS (5 µg/mL) was used as positive stimulus for each sample tested. Level of TNF-α was determined by ELISA. Neutrophil viability and DNA fragmentation were analyzed by flow cytometry using propidium iodide [3]. Fluorescence was measured by FL2 channel 585/545nm. The procedure was approved by the Ethics Committee of Cruzeiro do Sul University (077/2015).

Levels of TNF-α were lowered in cells treated with HE at all concentrations tested (p < 0.05), but QAR was not potent as HE. Neutrophil viability dropped when cells were treated with doses higher than 0.25 mg/mL (EB) and 0.02 mg/mL (QAR). DNA fragmentation occurred when cells were treated with doses higher than 0.75 mg/mL (EB) and 0.04 mg/mL (QAR), respectively. HE has inhibitory activity on pro-inflammatory mediator.

Keywords: Kalanchoe pinnata, Crassulaceae, quercetin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside, TNF-α.

References:

[1] Kamboj A, Saluja AK. Bryophyllum pinnatum (Lam.) Kurz: phytochemical and pharmacological profile: A review. Pharmacogn Rev 2009; 3: 364 – 375

[2] Sugimoto MA, Vago JP, Teixeira MM, Sousa LP. Annexin A1 and the resolution of inflammation: Modulation of Neutrophil Recruitment, Apoptosis, and Clearance. J Immunol Res, 2016; 2016: 8239258

[3] Borges LdaS, Bortolon JR, Santos VC, De Moura NR, Dermargos A, Cury-Boaventura MF, Gorjão R, Pithon-Curi TC, Hatanaka E. Chronic inflammation and neutrophil activation as possible causes of joint diseases in ballet dancers. Mediators Inflamm, 2014; 2014: ID846021