Ginsenosides — triterpene saponins are secondary metabolites that are found almost exclusively in the plant genus Panax. A long history of the use such species as Panax ginseng or P. quinquefolius in traditional medicine has led to numerous investigation of pharmacological effects of ginseng compounds conducted both in vitro and in vivo. These studies confirmed high therapeutic potential of ginsenosides proving their regulating action on the nervous, endocrine, cardiac and immune systems. Additionally many reports indicated multiple properties of ginseng saponins including antimicrobial, antioxidant, anti-inflammation, anticancer, radioprotective, and anti-aging (Liu et al., 2020). Most ginsenosides are classified as members of the dammarane family. Each ginsenoside has at least two (carbon-3 and -20) or three (carbon-3, -6, and -20) hydroxyl groups free or bound to monomeric, dimeric, or trimeric sugars. Ginsenosides also exist as stereoisomers, depending on the position of the hydroxyl group on carbon-20. These metabolites are divided into two groups based on their chemical structures: protopanaxadiol (PD) and protopanaxatriol (PT). The sugar moieties in the PD group attach to the 3-position of the dammarane-type triterpine, including Rb1, Rb2, Rc, Rd, Rb3, Rh2, and Rh3; the sugar moieties in the PT group attach to the 6-position of the dammarane-type triterpine, including Re, Rf, Rg1, Rg2, and Rh1 (Szczuka et al., 2019).
Initially, the raw material for the production of medicinal ginseng products was obtained from natural places, but due to the depletion of these resources, different attempts were made to cultivate ginseng, first under natural conditions, and then under field conditions. Currently, high demand and high prices (from 20 to 1105 dollars per kilogram) for ginseng root, as well as the inability to obtain this raw material from the natural places (since 1972 ginseng has been entered into the "Red Book of Endangered Species") have caused an increase in surface area for field ginseng cultivation. Soil cultivation of this plant is very labor-intensive, characterized by a long period of time (minimum 3-4 years) needed to obtain valuable raw material and high costs associated with agrotechny and the use of prophylactic plant protection treatments (ginseng is a plant extremely susceptible to fungal diseases, as well as eagerly attacked by pests) (Proctor et al. 2013: Jia et al. 2009). An opportunity to faster obtain in well-standardized conditions a raw material rich in biologically active ingredients, are in vitro cultures. In recent years the interest of hairy root culture cultivation increase. Numerous literature reports demonstrated that this type of roots can become an alternative way to obtain valuable secondary metabolites for field crops or cultures of cell suspensions. Hairy root cultures are characterized by the advantages that cell cultures do not have (Hussain et al. 2022). They are characterized by rapid growth without the need for additional phytohormones, which gives the opportunity to produce a large amount of biomass in a relatively short time, they are genetically stable and no drastic decline in metabolite accumulation was observed as the root line grows older. Their valuable advantage, in compering to susupension cultures, is tissue and structural differentiation which plays an important role in the normal course of metabolic processes, especially that some metabolites are synthesized only in specialized organs of plants. Important factors affecting the production of biomass and secondary metabolites, besides optimal concentration of sugars, nitrogen or phosphorus in the medium, are the technological methods. One of the most frequently used is the elicitation process. It consists in subjecting the in vitro culture to the activity of the elicitor. Elicitors in plant biology are extrinsic or foreign molecules often associated with plant pests, diseases, or synergistic organisms. Elicitor molecules can attach to special receptor proteins on plant cell membranes. These receptors can recognize the molecular pattern of elicitors and trigger intracellular defense signaling. This response results in metabolites’ enhanced synthesis, reducing damage, and increasing pest resistance, disease, or environmental stress. The effectiveness of the elicitation process depends primarily on the interaction between the plant cell and the elicitor. In addition literature data indicate that parameters such as a type and concentration of elicitor, duration of elicitation, age and line of the culture, the cell lines, addition of growth regulators, nutrient composition and culture conditions have an effect on the performance of the elicitor. The acting of elicitors is not specific thus their selection and the establishing of optimal condition for their acting have to definy experimentally for each cultures. (Halder et al. 2019)
Linalool (other names: β-linalool, linalyl alcohol, linaloyl oxide, allo-ocimenol, and 3,7-dimethyl-1,6-octadien-3-ol) is a monoterpene compound which exists in two enantiomeric forms. It has a stereogenic center at C3, and therefore there are two stereoisomers: (R)-(–)-linalool, known as licareol, and (S)-(+)-linalool, known as coriandrol. Licareol has lavender or lily-of-the-valley odor; it is a colorless liquid and practically insoluble in water, miscible with alcohol and ether, while coriandrol has a herbaceous and musty green smell and is often the major component in several essential oils. Coriandrol is also a colorless liquid soluble in alcohol (Ozek et al., 2010; Sarkic and Stappen, 2018). Linalool is recognized as safe (GRAS). Over 200 plants of the various genus produce it come from families, e.g., Lamiaceae (coriandrum, lavender, basil, mint, salvia, origanum, nepeta, thymus), Lauraceae (laurel, cinnamon), and Rutaceae (citrus fruits). In bergamot-mint and lavender essential oils got from plants of different geographical origin, linalool and linalyl acetate are the main constituents (Avetisyan et al., 2017; Kofidis et al., 2004). Linalool is used in the industrial production of various terpenes such as geraniol, nerol, citral, and its derivatives and the synthesis of vitamins A and E. (Kamatou and Viljoen, 2008). Because it is a safe compound, extensive research is carried out on its medical use. The linalool forms are shown to have different pharmacological effects: act on the brain neurotransmitters glutamic acid, y-aminobutyric acid (GASA), acetylcholine, and dopamine, exhibit local anesthetic properties. Other activities such as anti-inflammatory, anti-nociceptive, and anti-allergic activity in various animal models and antimicrobial, antioxidant, and cytostatic in cancer cells in vitro have also been described. Linalool show sedative effects on the central nervous system and causes the growth of levels of acetylcholine, which may be the great potential to use in Alzheimer’s disease (Kamatou and Viljoen, 2008; Peana and Moretti, 2008; Rodenak-Kladniew et al., 2018). Linalool is applied in aromatherapy and various industries: food, pharmaceutical or cometics (Kamatou and Viljoen, 2008).
Based on linalool’s various biological properties, our team used this compound to improve ginsenoside production in hairy root cultures. As a model culture, we applied the transformed roots of five-leaf ginseng (P. quinquefolium, American ginseng). The hairy root of P. quinquefolium may be an excellent source of ginsenosides, which levels are like those in ginseng roots cultivated in the field. The primary reasons that our research focuses on American ginseng cultures of hairy roots are unflagging interest in ginseng saponins and their therapeutic properties. Considering the payability (time and cost) of obtaining these biologically active compounds, our breeding using derived cultures in vitro allows getting in a much shorter time of ginseng biomass (only 28 days). It does not require agrotechnical work in contrast to getting valuable material derived from traditional cultivation (a minimum of three years) (Kochan et al., 2018b, 2018a, 2016). In previous reports, we described the influence of some elicitors to increase ginsenoside production in hairy roots of American ginseng (Kochan et al., 2018b, 2018a, 2017). Among them there are yeast extract (YE), methyl jasmonate (MeJa), abscisic acid (ABA) (Alcalde et al. 2022, Markowski et al. 2022), that were used for improving for secondary metabolite contents in many in vitro cultures, and essential oil compound - trans-anethole (t-A), that was succesfully applied a an factor intensifying of ginseg saponin accumulation for the first time (Kochan et al. 2018b). Obtained results encouraged us to search for other essential oil ingredients to get even higher saponin synthesis efficiency in the studied cultures. The present study’s primary object was examining whether linalool can be used as a potential new elicitor to enhance triterpene saponin production in P. quinquefolium hairy roots cultivated in shake flasks. This research estimated the optimum dose and elicitation time of linalool for effective ginsenoside biosynthesis in the studied cultures. The influence of linalool to accumulation secondary metabolites has not been documented in any plant in vitro cultures.