Secondary metabolite profile in mature and old leaves of four piper species: Forest Betel (Piper aduncum L.), Red Betel (Piper crocatum Ruiz & Pav.), Javanese Chili Betel (Piper retrofractum Vahl.), and Green Betel (Piper betle L.)

Piper species is a potential medicinal plant, empirically known for its effec- tiveness in curing various diseases, particularly in Indonesia. Therefore, this research aimed to evaluate the similarities and differences in the profile of secondary metabolite compounds in mature and old leaves of four Piper species using Gas Chromatography-Mass Spectrometry (GC-MS). The analy- sis was also carried out to identify the specific compounds found in each species at different leaf development stages. Samples used were mature and old leaves from four species of Piper, namely forest betel (Piper aduncum L.) (PA), red betel (Piper crocatum Ruiz & Pav.) (PC), Javanese chili betel (Piper retrofractum Vahl.) (PR), and green betel (Piper betle L.) (PB). Subsequently, samples were extracted using ethanol solvent and secondary metabolite profile was detected through GC-MS. A total of 40 secondary metabolite compounds were found in mature and old leaves of four species. The results showed that alkaloid content contributed 25% of the total compounds detected, while fatty acids yielded the largest portion (27.5%). Based on PCA score plot analysis, a significant grouping of secondary metabolite compounds was observed in all species, where PC was catego- rized separately on the right, and the other species were on the left. Several specific compounds were also found only in one species and not in others. Similar to mature and old leaves, some compounds were discovered in one of the developmental phases. Dillapiole and hexadecane were only found in PA, both in mature and old leaves; germacrene A, eugenol, and chavicol were found in mature and old leaves of PB, but beta-selinene, delta- guaiene, and linolelaidic acid were only found in mature leaves. In PC, there were specific compounds namely benzofuran, myrcene, and 1,6,10- dodecatrien that were found in mature and old leaves. Besides that, trans- ocimene and alpha-bisabolol were only found in old leaves of PC. In PR, only tetracosane was found in mature leaves, while piperidine, octanodeca- noic acid, and benzylmalonic acid were found in old leaves