Biosynthesis of Ginsenosides in Field-Grown Panax Ginseng

JSM Central Biotechnology & Biomedical Engineering Special Issue on Industrial Biotechnology-Made in Germany: The path from policies to sustainable energy, commodity and specialty products Edited by: Dr. Thomas Brück Professor of Industrial Biocatalysis, Dept. of Chemistry, Technische Universität München (TUM), Germany Research Article *Corresponding author Prof.Dr. Wolfgang Eisenreich, Lehrstuhl für Biochemie, Technische Universität München, Lichtenbergstr. 4, Biosynthesis of Ginsenosides in 85747 Garching, Germany, Tel: +49-89-289-13336; Fax: +49-89-289-13363; E-Mail: Field-Grown Panax Ginseng Submitted: 14 April 2013 Schramek, N.1, Huber, C.1, Schmidt, S.1, Dvorski, S.E.1, Kmispel, Accepted: 12 May 2014 Published: 14 May 2014 N.1, Ostrozhenkova, E.1, Pena-Rodriguez, L.M.1,2, Cusido, R.M.3, ISSN: 2333-7117 Wischmann, G.4 and Eisenreich, W.1* Copyright 1Lehrstuhl für Biochemie, Technische Universität München, Germany © 2014 Eisenreich et al. 2Laboratorio de Química Orgánica, Centro de Investigación Científica de Yucatán, México OPEN ACCESS 3Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Spain 4 FloraFarm GmbH, Walsrode-Bockhorn, Germany Keywords • Araliaceae • Triterpene Abstract • Mevalonate The biosynthesis of the triterpenoid ginsenosides Rg and Rb was studied by • Isotopologue Profiling 1 1 13 • 13 CO2 CO2 pulse-chase experiments in six-year-old Panax ginseng growing in the field. A pulse period of 7 hours followed by a chase period of 8 days was shown to generate 13 significant C-enrichments in both ginsenosides (Rg1> Rb1), as well as in free sugars 13 and amino acids. More specifically, CO2-labeled Rg1 and Rb1 were characterized 13 by specific NMR couplings due to C2-units indicating the mevalonate origin of the 13 triterpenes. C3-Labeled motifs in Rg1 or Rg1 that should be generated by the 13 alternative methylerythritol phosphate pathway from a C3-triose phosphate precursor 13 were apparently absent, whereas C3-isotopologues were detected in free sugars, amino acids and the sugar moieties of the ginsensosides from the same experiment. It can be concluded that ginsenosides are predominantly or entirely biosynthesized in P. ginseng via the mevalonate route, under the physiological conditions of the field experiment. The observed labeling patterns were also in perfect agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor entering the cyclization process with the dammarenyl intermediate. The higher 13 enrichments of C2-isotopologues in the protopanaxatriol-type Rg1 in comparison to the protopanaxadiol-type Rb1 indicated higher rates of Rg1 biosynthesis during the pulse/chase experiment with the six-year-old plant of P. ginseng. In summary, the study reveals the nature and dynamics of the ginsenoside biosynthetic pathway as a welcome basis for future biotechnological means. INTRODUCTION roots of four to seven year-old Panax e.g. in South Korea, China, Canada, and the US. These cultivations Medicinal plants are important sources of therapeutics aimed produce about 80,000 tons of fresh Ginsengplants rootsgrown per in year.the field, The at alleviating human disease. With increasing awareness of the world Ginseng market has been estimated to be around US$ health hazards and toxicity associated with the indiscriminate 2,000 million [7]. use of synthetic drugs and antibiotics, interest in plant-based The multiple effects of Ginseng on human health are drugs has revived throughout the world [1]. The World Health intensely studied but still not completely understood [5,11-25]. Organization has estimated that more than 75 % of the population Its bioactivity is mainly assigned to ginsenosides, a group of in developing countries depends primarily on traditional herbal triterpene saponins that are unique to Panax plants and especially medicine for basic healthcare needs and the worldwide annual abundant in the roots of P. ginseng. In the meantime, more than market for these products approached about US $ 60 billion [2- 150 different ginsenosides are known [13,24,26-28]. Most of 3]. Based on the current research, world-wide strategies and them belong to the dammarane or the oleanane-type, respectively (Figure 1). According to their glycone moieties, dammarane- will continue to play an important role in health care [4]. financial investments, it can be assumed that medicinal plants type ginsenosides (for example, Rb ) and protopanaxatriol-type Ginseng (Panax ginseng C.A. Meyer), belonging to the 1 ginsenosidestype ginsenosides (for example, are further Rg ) (Figureclassified 1). into Recently, protopanaxadiol- suppressive Araliaceae family, is one of the most important plants in traditional 1 effects of Rg1 on liver glucose production could be determined and modern East Asian Medicine. Since centuries, Ginseng in HepG2 human hepatoma cells via the LKB1-AMPK-FoxO1 extracts are used for anti-aging, anti-oxidative, adaptogenic and pathway [29]. Moreover, Rg1 and especially Rb1 cell-protective effects in models for ischemic disease [30-31], has now also been established in the phytotherapy of Western oxidative cellular stress [32], and hyper permeability-relatedshowed beneficial other health-benefitting effects [5,6]. Not surprisingly, Ginseng Medicine [7]. diseases induced by bacterial pathogens [33]. Although some advances in the production of ginsenosides Despite the enormous pharmacological importance of these by tissue culture and biotransformation have been made recently compounds, the biosynthesis of ginsenosides is not completely [8-10], Ginseng drugs are mostly based on extracts from the understood [9,24,34-38]. Generally, triterpenoid saponins are Cite this article: Schramek N, Huber C, Schmidt S, Dvorski SEM, Knispel N, et al. (2014) Biosynthesis of Ginsenosides in Field-Grown Panax Ginseng. JSM Biotechnol Bioeng 2(1): 1033. Eisenreich et al. (2014) Email: Central Figure 1 1 and Rg1. Carbon atoms derived from DMAPP and IPP are indicated in green and red, respectively. The used carbon numbering of Rb1 and Rg1 is also indicated. Formation and isoprenoid dissection of β-amyrin and ginsenosides Rb believed to be derived from the linear C30 compound, squalene, made by the head-to-head condensation of two molecules of be isomerized to DMAPP [49-52]. farnesyl diphosphate (FPP), each of them assembled from elimination, mevalonate is finally converted into IPP which can However, in the plastidic compartment of plant cells, also a dimethylallyl diphosphate (DMAPP) and two molecules of mevalonate-independent pathway (methylerythritol phosphate isopentenyl diphosphate (IPP) [8]. In an O -dependent process, 2 pathway, MEP pathway) exists for the biosynthesis of IPP and squalene is converted into (S)-2,3-oxidosqualene [39] which DMAPP (Figure 2B). This pathway starts with the condensation of glyceraldehyde 3-phosphate (GAP) and a C2-unit from pyruvate 45] triterpenes. These intermediates are then converted into affording 1-deoxy-D-xylulose 5-phosphate (DXP). DXP is then reacts to dammarane [40-43] or oleanane (β-amyrin) [44- ginsenosides by multiple oxidations (e.g. catalyzed by cytochrome rearranged and reduced to 2-C-methyl-D-erythritol 4-phosphate P450-dependent monooxygenases) [46-48] and glycosylations (MEP), which leads via four steps to (E)-4-hydroxy-3-methyl- [34,36] which are largely unknown (Figure 1). reduced to a mixture of IPP and DMAPP [53-56]. Also less understood is the generation of the early precursors but-2-enyl diphosphate (HMBPP). In the final reaction, HMBPP is IPP and DMAPP used in the biosynthesis of triterpenes including Due to the assignment of the mevalonate and MEP pathway ginsenosides. Generally, these early precursors are formed in the to the cytosolic or plastidic compartments, respectively, plant cytosolic compartment of plant cells by the classical mevalonate terpenes made in the cytosol (e.g. phytosterols) are thought to pathway (Figure 2A). Three molecules of acetyl-CoA produce be derived from the mevalonate pathway, whereas terpenes 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) which is reduced formed in the plastids (e.g. monoterpenes, diterpenes and to mevalonate. By phosphorylation and decarboxylation/ carotenoids) are believed to be made from the MEP route. JSM Biotechnol Bioeng 2(1): 1033 (2014) 3/17 Eisenreich et al. (2014) Email: Central Figure 2 Biosynthetic origin of IPP and DMAPP via the mevalonate pathway (A) and the alternative MEP pathway (B). [64]. The same conclusion was made from recent experiments for many terpenes, mainly on the basis of labeling experiments with hairy root cultures of P. ginseng exposed to mevinolin or withIndeed, plant this cell classification cultures (reviewed has now been in [56]). approved On the experimentally other hand, exchange of mevalonate or MEP-derived precursors between the pathway, respectively [65]. However, it became not clear from compartments has been observed affording plant terpenes with thefosmidomycin, later study specificwhether inhibitors this mixed of contribution the mevalonate is also or thevalid MEP for mixed biosynthetic origin [57-62]. ginsenoside formation in whole plants of Panax. The biosynthesis of ginsenosides occurs in the cytosolic In the present work, we have labeled full-grown plants of P. 13 compartment, and, on this basis, a cytoplasm-located mevalonate ginseng CO2 as a tracer. Indeed, origin of the precursors can be assumed. However, direct this method allows the maintenance of physiological conditions experimental evidence for the biosynthetic assignment

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