28Eurycoma longifolia is a well-known medicinal plant with pharmacological effects and 29 important bioactive compounds such as alkaloids and quassinoids. The suitable age of 30 harvesting E. longifolia root for commercial use is relatively unknown and could influence the 31 overall bioactive compounds present in the plant. In this study, non-targeted liquid 32 chromatography mass spectrometer (LC-MS) and multivariate analyses (MVA) were 33 performed to determine the chemical constituent of aqueous extract of 3-month-old E. 34 longifolia hairy root (HR) culture, 1-, 4-and 11-year-old harvested E. longifolia roots. 35 Unsupervised principle component analysis (PCA) and supervised partial least square 36 discriminant analysis (PLSDA) were applied to evaluate metabolic similarities and differences 37 in E. longifolia roots and hairy root in response to different harvesting age, locations and 38 propagation methods. A total of 34 significant buckets with variable importance in projection 39 (VIP) exceeding 1 were selected and identified. It was found that putatively identified 40 quassinoids were significantly higher in 1-, 4-and 11-year-old E. longifolia roots whereas 41 putative canthin-6-one alkaloids were abundantly present in 3-month-old E. longifolia hairy 42 root (HR). These findings may be applicable to improve the harvesting age and increase the 43 content of bioactive compounds in E. longifolia roots. 44 Introduction 54 Eurycoma longifolia Jack, locally known as Tongkat Ali, is an herbaceous tree of the family 55 Simaroubaceae, commonly found in Southeast Asian countries like Malaysia, Indonesia, 56 Thailand, Myanmar, Cambodia and Laos. E. longifolia, particularly its roots is traditionally 57 used for improvement of general health and libido. E. longifolia roots have been reported to 58 exhibit various pharmacological and biological activities such as antimicrobial [1-4], anti-59 diabetic [5], antimalarial [6, 7], aphrodisiac activities [8] and cytotoxicity against cancerous 60 cells [9-11]. The chemical profiling of E. longifolia root extract indicates alkaloids and 61 quassinoids derivatives as the major bioactive compounds found in this plant [12, 28]. 62 Quassinoids, particularly eurycomanone and their derivatives were reported to be abundant in 63 E. longifolia roots and responsible for various pharmacological properties. 64 The increasing demands of E. longifolia root as herbal medicinal products have been 65 impeded by the slow growth and late maturity of this plant. The suitable harvesting age of E. 66 longifolia roots is unclear, but for commercial use, E. longifolia roots are usually harvested at 67 the age of four years old [13]. Today, approximately 21,000 kg of E. longifolia were harvested 68 to meet a demand of more than 54,000 kg per year [12]. As a result, this practice has led to 69 excessive exploitation, extinction and adulterations in E. longifolia based products. Therefore, 70 there is a need to standardize harvesting age to ensure sufficient supply and sustainability of 71...