Background during growing stage when combined with chemometrics. potential evidences for the rational application and exploitation of Franch belonging to Gentianaceae is an important medicinal plant in China for treatment of hepatitis, jaundice and dysentery. The root and rhizome of this plant has been officially documented in Chinese Pharmacopoeia as one of raw materials of Gentianae Radix et Rhizoma (Longdan in Chinese), a hepatoprotective agent (State Pharmacopoeia Commission [2010]). According to ethnobotanical information, its aerial parts are also used as folk medicine for treatment of fever and rheumatic arthritis or as antiophidica, when prepared with vegetable oil. The phytochemistry and bioactivities of have been intensively studied. More than 100 secondary metabolites with different activities like hepatoprotective, anti-inflammatory, antioxidant and neuritogenic growth have been isolated from this plant (Gao et al. [2010a], [2010b]; Xu et al. [2006], [2007], [2009a]; Wang et al. [2012]). Among them, iridoid glycosides is the most abundant components especially gentiopicroside which content is more than 4.5?% and serve as major active ingredient and standard for quality control (Jiang et al. [2005]; Pan et al. [2015a]; Wang et al. [2012]). As one of well-known traditional Chinese medicine with remarkable medicinal functions, the wild resources have been under heavy threat owing to human activities and environmental pollution. Although have been extensively planted in Yunnan, some disadvantages on cultivation such as continuous cropping obstacle, time-consuming and laborintensive, etc. result in decline of production and quality. Fortunately, plant tissue 123562-20-9 culture is conducive to the accumulation of biomass and metabolites, in particular with individual metabolites, which amount is multifold higher than control group when treated with appropriate elicitors (Chuang et al. [2014]; Huang et al. [2014]; Kuzovkina et al. [2014]; Kumari et al. [2015]; Marsh et al. [2014]; Su et al. [2014]). However, the efficacy of medical plant, to a large extend, are attributed to synergistic effect of a number of metabolites. The amount of individual metabolites is significantly increased, which might lead to different pharmacological activities and therapeutic effects. On the other hand, previous studies (Jiang et al. [2005]; Pan et al. [2014], [2015a]; Wang et al. [2012]) on metabolites of were most focused on quality assessment, phytochemistry, pharmacology, etc. However, metabolites are not only the efficacious properties for maintaining human health, but also play an important role for resistance to abiotic and biotic threats during plant growth (Hall et al. [2008]; Wink [2003]). To our best 123562-20-9 knowledge, the accumulation and variation of metabolites in have not yet clear. Currently, analysis of metabolites based on separation technologies such as gas chromatographyCmass spectrometry (Hu et al. [2014]), liquid chromatography coupled with photodiode array detector (Yu et al. [2014]), mass spectrometry detector (Won et al. [2014]) and nuclear magnetic resonance spectroscopy (Hilbert et al. [2015]) can rapidly provide complex chemical information and clarify the similarities and differences of bio-samples when combined with chemometrics. However, comprehensive chemical information on metabolites cannot be analyzed in a single chromatogram. Fourier transform infrared spectroscopy (FT-IR) enables to rapid 123562-20-9 reflect holistic molecular structure-analyte relationships, which is considered as a well-established and non-destructive method for analysis of bio-sample, whereas it fails to recognise the variation of specific compound in sample due to the limited specificity and sensitivity (Karoui et al. [2010]; Lohumi et al. [2014]; Zhao et al. [2014]). In this study, the variation on distribution and accumulation of metabolites in are investigated based on plant tissue culture. Rabbit Polyclonal to TIGD3 Individual parts of sample during different growing stage are subjected to targeted and non-targeted analysis using FT-IR and liquid chromatography tandem mass spectrometry (LC-MS/MS). Moreover, the biosynthetic pathway of iridoid glycosides is also discussed. The combinative comparison approach can reflect the overall chemical difference during different growing stage, which may provide the useful information for reasonable utilization of resources. Methods Materials and chemicals Tissue culture materials provide by Dr. Heng-Yu Huang (College of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine) were established using leaves of The operations of tissue culture were completed on a super-clean bench. The plantlets were grown on Murashige-Skoog (MS) medium supplemented different concentration of indoleacetic acid (IAA), zeatin(ZT), dichlorphenoxyacetic acid (2,4-D), kinetin (KT) and 6-benzyladenine (BA) during different stage. The processed of tissue culture is present in Fig.?1. The culturing conditions were incubated at 23??2?C under cool white fluorescent light at 1500C2000?lx under 10?h per day. Fig. 1 Tissue culture flowchart of 100C1000. 123562-20-9 The capillary voltages were set at 3000?V (positive mode) and 2700?V (negative mode), respectively, and nozzle.