RESUMEN
Piper longum L.(PL) is considered one of the most important species traditionally used for treating various ailments and has indicated the presence of alkaloids, flavonoids, and steroids. In this study, we isolated the chemical compounds of PLleaves,andmeasuredNO, IL-6, iNOS, as well as COX-2 protein levels. In addition, molecular docking analysis were used to further understand anti-inflammation effect of the compounds. We identified one new alkaloid named piperlongumine A (1) with ten known compounds (2-11). The new compound (1) and two other alkaloids 2E)-3-(4-hydroxy-3-methoxyphenyl)-1-(pyrrol-1-yl)propanone (7) and piperchabamide A (8) significantlyreduced NO production in LPS-stimulated RAW 264.7 cells with the IC50 values of 0.97 ± 0.05 mM, 0.91 ± 0.07mM, 1.63 ± 0.14 mM, respectively. Moreover, at concentration of 2 mM, compound 1 inhibited approximately 98 ± 0.64 % of IL-6 secretion, and decreased iNOS and COX-2 protein level by about 96 and 19 folds compared to LPS treatment alone, respectively. Furthermore, compounds 1, 7, and 8 were predicted to bind and inhibit IL-6, TNF-a, and iNOS, with compound 1 showing the highest binding energy of -7.09 kcal/mol. This study provides new insights for potential anti-inflammatory drug design and warrants further investigation.
RESUMEN
Context: The Piper species was studied several potential properties such as anti-tumor, anti-inflammatory and antioxidant activity. However, the specific anti-inflammatory activity of the extract from the fruits of P. longum L. has not been investigated. Objectives: Our study want to examine the anti-inflammatory effects of P. longum L. fruit methanolic extracts (PLE) on lipopolysachharide (LPS)-stimulated RAW 264.7 murine macrophages to understand the mechanism of this effect. Method: This study examined the chemical profiling of PLE by LC-HRMS analysis and measured the presence of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the supernatant using the Griess reagent assay and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA expression of IL-6, TNF-α, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were evaluated by using real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, the protein expression of COX-2, iNOS and the phosphorylation of MAPK family, c-Jun N-terminal kinase (JNK), p38 in protein level were observed by western blotting. Result: PLE have detected 66 compounds which belong to different classes such as alkaloids, flavonoids, terpenoids, phenolics, lactones, and organic acids inhibited nitric oxide products with the IC50 = 28.5 ± 0.91 µg/mL. Moreover, PLE at 10-100 µg/mL up-regulate HO-1 protein expression from 3 to 10 folds at 3 h. It also downregulated the mRNA and protein expression of iNOS, COX-2, decreased IL-6 and TNF-α secretion by modulating the mitogen-activated protein kinase (MAPK) signaling pathway, specifically by decreasing the phosphorylation of p38 and JNK. Conclusion: These results shown chemical profiling of PLE and demonstrated that PLE exhibits anti-inflammatory effects by regulating the MAPK family and could be a potential candidate for the treatment of inflammatory diseases.