Citation: Mahar, R.; Manivel, N.; Kanojiya, S.; Mishra, D.K.; Shukla, S.K. Assessment of Tissue Specific Distribution and Seasonal Variation of Alkaloids in Alstonia scholaris. Metabolites 2022, 12, 607. https:// doi.org/10.3390/metabo12070607 Academic Editor: Miroslava Zhiponova Received: 5 June 2022 Accepted: 27 June 2022 Published: 30 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). metabolites H OH OH Article Assessment of Tissue Specific Distribution and Seasonal Variation of Alkaloids in Alstonia scholaris Rohit Mahar , Nagarajan Manivel, Sanjeev Kanojiya, Dipak K. Mishra and Sanjeev K. Shukla * Sophisticated Analytical Instrument Facility and Research, CSIR-Central Drug Research Institute, Lucknow 226031, India; rohitmahar4u@gmail.com (R.M.); nagambt@gmail.com (N.M.); sanjeev_kanojiya@cdri.res.in (S.K.); dk_mishra@cdri.res.in (D.K.M.) * Correspondence: skshukla@cdri.res.in or sanshukla@gmail.com Abstract: Alstonia scholaris is a well-known source of alkaloids and widely recognized for therapeutic purposes to treat the ailments in human and livestock. However, the composition and production of alkaloids vary due to tissue specific metabolism and seasonal variation. This study investigated alkaloids in leaves, stems, trunk barks, fruits, and flowers of A. scholaris. The impact of seasonal changes on the production of alkaloids in the leaves of A. scholaris was also investigated. One and two-dimensional Nuclear Magnetic Resonance (NMR) experiments were utilized for the characteri- zation of alkaloids and total eight alkaloids (picrinine, picralinal, akuammidine, 19 S scholaricine, 19,20 E vallesamine, Nb-demethylalstogustine N-Oxide, Nb-demethylalstogustine, and echitamine) were characterized and quantified. Quantitative and multivariate analysis suggested that the al- kaloids content is tissue specific, illustrating the effect of plant tissue organization on alkaloidal production in A. scholaris. The results suggest that the best part to obtain alkaloids is trunk barks, since it contains 7 alkaloids. However, the best part for isolating picrinine, picralinal, akuammidine, 19 S scholaricine, and 19,20 E vallesamine is fruit, since it shows highest amount of these alkaloids. Undoubtedly, NMR and statistical methods are very helpful to differentiate the profile of alkaloids in A. scholaris. Keywords: metabolic profiling; multivariate analysis; clustering analysis; quantitative analysis; chemical shift 1. Introduction Alstonia scholaris belongs to the family Apocynaceae and is well known as Devil’s tree or Saptparni. It is widely distributed in Asia especially in India, China and Malaysia [1]. It has been used as a traditional medicine for the treatment of various human and livestock diseases and several biologically active compounds have been isolated from this plant [2,3]. Alstonia scholaris (AS) is a very rich source of alkaloids, terpenoids and flavonoids etc. There is therefore great interest among scientific groups to explore this medicinal plant for therapeutic applications. Plants have been a great source of secondary metabolites. The presence and quantity of secondary metabolites depends upon the specific part or tissues of the plant, and seasonal variation of the particular region where the plant is grown. Different chemical composition and variable production of secondary metabolites among different geographical regions and seasons have been well documented for terrestrial plants [4]. Herbal medicine is an integral part of drug discovery and development, but due to the complexity of phytoconstituents, the methodology of choice for the identification of phytoconstituents in herbal medicine is mainly to get a characteristic fingerprint of a plant part, that represents the inherent chemical information of the plant species. It has been recognized that a slight variation in the metabolome of plants could be explained by the perturbations imposed on plants. These perturbations might include environmental, physi- cal, abiotic, nutritional stresses and several prolonged mutations. The term “metabolome” refers to the observable chemical profile of the metabolites in a sample [5]. Metabolic Metabolites 2022, 12, 607. https://doi.org/10.3390/metabo12070607 https://www.mdpi.com/journal/metabolites