# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 1 Introduction  
The plant species _A. pichinchensis_ ([PERSON]) [PERSON]. [PERSON]. [PERSON]. (Asteraceae) is native to Mexico and grows in 28 of its 32 states. This species is also identified by the synonyms _Eupatorium aschembornianum_, _Eupatorium pichinchense_, and _Ageratina aschemborniana_[1, 2, 3]. Particularly, in the state of Morelos, it is popularly known as \"axihutil\", and it is used in traditional medicine for treating stomach pain, respiratory issues, gastrointestinal problems, and skin infections [4, 5].  
Scientific studies carried out using different biological evaluation models reveal that the aerial parts of _A. pichinchensis_ exhibit activity against onychomycosis and tinea pedis, gastroprotective and healing effects, and may even inhibit the in vitro proliferation of keratinocytes [6, 7, 8, 9, 10, 11]. The main compounds associated with this type of biological effect arechromenes, furans, terpenes, essential oils, and glycosylated flavonoids [8; 9; 10; 11; 12; 13]. However, the anti-inflammatory effect of this plant species has not been reported.  
Inflammation, a response of the immune system which stimulation can be caused by infections or stress, is associated with multiple diseases and triggers numerous biochemical, immunological, and cellular reactions [14; 15]. During the inflammatory process, mediating substances that generate pain are released into the nervous system. Among these are interleukins that induce the expression of genes that encode enzymes and other chemical species that contribute to the inflammatory response [16; 17; 18].  
In particular, the enzyme nitric oxide synthase (iNOS) catalyzes the formation of the free radical nitric oxide (NO) in a gaseous state that stimulates vasodilation and cannot be stored [19; 20; 21]. Notably, at low concentrations, NO plays a mediating and protective role, acting as an immunoregulatory and antimicrobial agent due to its ability to generate DNA damage, inhibit enzymes of energy metabolism and oxide proteins, and peroxididize the membrane lipids of pathogens [19; 20; 21]. However, high concentrations of NO can damage tissues by causing iNOS overexpression and cytotoxicity, which manifests itself in acute and chronic pathological responses mediated by cytokines and endotoxins of various cell types, mainly macrophages [22; 23; 24].  
The iNOS enzyme can be elicited in different cell types, such as macrophages, hepatocytes, neutrophils, etc., generating a large amount of NO that can be toxic, which is why NO functions as a proinflammatory molecule [25; 26; 24]. In the chronic phase of inflammation, the inflammatory signal can be magnified by the production of molecules, such as nuclear transcription factor kappa \(\beta\) (NF-k\(\beta\)), TNF-\(\alpha\), and interferon-gamma (IFN-\(\gamma\)), which promote the transcription of iNOS and increase vasodilation, edema, and plasma exudation [26; 27; 28; 29]. A high production of NO can be associated with the development of diseases, such as Alzheimer's, cardiovascular, rheumatoid arthritis, pulmonary fibrosis, diabetes, and cancer, among others [27; 28; 29]. This is why the iNOS enzyme is essential in the inflammatory process and allows us to understand the mechanism of action of active ingredients from medicinal plants [30; 31].  
This study reports the anti-inflammatory activity of chemical compounds isolated from the aerial parts (leaves and flowers) of _A. pichinensis_ with respect to the inhibition of NO in RAW 264.7 mouse macrophage cells induced with lipopolysaccharides.