Fatty acids in Plantago asiatica seeds are responsible for the production of the pro-inflammatory mediator nitric oxide

Background: Plantago asiatica L. (Plantaginaceae) commonly grows in East Asia, with its seeds (Shazenshi), having been used as diuretic and anti-inflammatory drugs in traditional Japanese medicine. It is not known which constituents of P. asiatica seeds elicit the anti-inflammatory effects, such as reduced expression of the inducible nitric oxide synthase (iNOS) in interleukin (IL)-1β-treated hepatocytes which leads to a reduction in the pro-inflammatory mediator nitric oxide (NO).  Objective: To identify the anti-inflammatory constituents of P. asiatica seeds, the anti-inflammatory activity of purified constituents was determined by assaying NO production in IL-1β-treated hepatocytes. Methods: P. asiatica seeds were extracted with 50% methanol and successively fractionated into three crude fractions with ethyl acetate (EtOAc) and n-butanol. The compounds were methylated and analyzed by gas chromatography–mass spectrometry (GC–MS). Primary cultured rat hepatocytes were prepared by collagenase perfusion, and P. asiatica seed extract (PASE), a fraction, or a compound was added to the culture medium with IL-1β and incubated at 37 °C. Potency of each fraction was determined by the Griess method for measuring the levels of nitrite in the medium. Results: PASE suppressed IL-1β-induced NO production without showing cytotoxicity, and an EtOAc-soluble fraction of PASE significantly inhibited NO production. GC–MS analysis detected 26 distinct fatty acids as their methyl esters in this fraction. Among them, three unsaturated fatty acids (linoleic, oleic, and α-linolenic acids) and palmitic acid were abundant. These unsaturated fatty acids are known to reduce NO levels. In contrast, acteoside and aucubin, which are thought to be present in the n-butanol-soluble fraction, showed only a low level of NO production suppression. Conclusion: The EtOAc-soluble fraction of PASE included many fatty acids, which may suppress the production of NO. The results imply that the anti-inflammatory activity of P. asiatica seeds may be produced by three unsaturated fatty acids. Because the fatty acids are abundant in the seeds of medicinal plants, they are likely to contribute to anti-inflammatory activity of the seeds. Keywords: Plantain seed, polyunsaturated fatty acid, Kampo medicine, hepatocyte, nitric oxide.