The influence of cultivation conditions on the formation of psychoactive salvinorin A, salvinorin B, rosmarinic acid and caffeic acid in Coleus scutellarioides

Coleus scutellarioides (L.) Benth. is a popular species in the world, known for its characteristic magnificent colourful leaves. The study has revealed that the contents of rosmarinic acid and caffeic acid are significantly higher in the plant tissues cultivated in vivo than when under in vitro conditions. The performed qualitative and quantitative analyses confirmed the presence (whose averaged content) of salvinorin A (6.65 µg/1 g of fresh plant) and salvinorin B (50.46 µg/1 g of fresh plant) in tissues of Coleus scutellarioides (L.) Benth. of ‘Electric lime’ variety. The greatest quantities of these compounds were recorded for plants cultivated in vitro on the MS medium enriched with NAA (naphthyl-1-acetic acid) at a concentration of 0.5 mg∙ dm–3. The research detected differences in the amounts of compounds between plants grown in vivo and those cultivated in vitro. Addition of plant growth regulators into the breeding medium under in vitro conditions was found affecting the amounts of compounds in plant tissues.

So far there has been no records in the literature about identified psychoactive substances occurring in Coleus scutellarioides.And that is why an attempt was undertaken to perform detailed analyses with a view to detect presence of psychoactive compounds in a strictly specified variety of Coleus scutellarioides.

Results
The qualitative analysis of the contents of all the four compounds, i.e. salvinorin A, salvinorin B, caffeic acid and rosmarinic acid, successfully confirmed their occurrence in the tissues of Coleus scutellarioides (L.) Benth.variability 'Electric lime' .Quantitative analysis was carried out for salvinorin A, salvinorin B, caffeic acid (Fig. 2) and rosmarinic acid (Fig. 3).
Plant growing conditions (in vivo or in vitro) as well as supplementing the breeding medium with growth regulators when under in vitro conditions significantly affect the contents of particular compounds in plant tissues (Diagram 1a-d).The highest content of salvinorin A (Diagram 1a), calculated in relation to the salvinorin B content, was found in plant tissues cultivated in vitro on the medium enriched with NAA -over three times exceeding the values obtained for plants bred on the medium without phytohormones.On the BA medium and under in vivo conditions the content of salvinorin A was over three times lower than in plants grown on the control medium.The greatest content of salvinorin B (Diagram 1b) was determined in the tissues of plants enriched with NAA -over three times higher than in plants cultivated without addition of phytohormones.The plants bred on the medium supplemented with BA were characterized by over three times lower content of salvinorin B as compared with the control medium.Similar levels of salvinorin B were found in plants cultivated in vivo and under in vitro conditions on the medium containing no phytohormones.The highest content of caffeic acid (Diagram 1c) was established to occur in plants coming from in vivo cultivation.The content of this compound exceeded 1,4 times the values obtained for the control medium in vitro.In plants bred in vitro on the medium enriched with BA the content of caffeic acid was over twice lower than in the case of plants growing on the medium without phytohormones.The highest amount of rosmarinic acid was revealed in plants cultivated in vivo -twenty times more as much in comparison with plants under in vitro conditions (Diagram 1d).Summing up, the highest mean values of the analyzed compounds were recorded for plants cultivated under in vivo conditions, i.e. 1871.39 µg • 1 g -1 of fresh tissue, while the lowest contents in plants bred in vitro and supplemented with BA -341.98 µg • 1 g -1 of fresh tissue.

Discussion
Rosmarinic acid exhibits an array of interesting biological effects.More comprehensive knowledge of this acid could contribute to its proper use in treatment and prevention of a wide range of health ailments [17][18][19][20][21] .
In plants rosmarinic acid should act as a pre-formed constitutively assembled defense compound.Rosmarinic acid is easily accumulated in undiffentiated plant cell cultures, in certain cases at concentrations highly exceeding those recorded for the plant itself.The first plant which was employed for production of rosmarinic acid in cell culture was Coleus scutellarioides 10,11 .
There is one publication which compares three varieties of Coleus scutellarioides with respect to the content of rosmarinic acid in plants grown in vivo.This research compares the following varieties: 'Defiance' , 'Shocking Pink' and 'Small Leaf ' .Based on thin-layer chromatography, a conclusion has been reached that variety 'Small Leaf ' may have the highest content of rosmarinic acid as related to the others 22 .
Plants representing the family Lamiaceae are characterized by a high content of rosmarinic acid.For instance, Mellisa officinalis contains 36.5 mg• 1 g -1 of rosmarinic acid, Mentha piperita -28.2 mg• 1 g -1 , Mentha spicata -58.5 mg• 1 g -1 whereas Rosmarinus officinalis only 7.2 mg• 1 g -123 .Increasing the biosynthesis of rosmarinic acid in Coleus scutellarioides has been an object of investigations undertaken by numerous research teams.Application of the combination IBA (indolylbutyric acid), BAP (6-benzylaminopurine) and GA 3 (gibberellic acid) (0.5 mg• dm -3 ) induced the highest efficiency of the rosmarinic acid production, amounting to 65.2% in in vitro cultures of whole plants 24 .Improvement in the biosynthesis of rosmarinic acid under in vitro plant cultures has been attained in the common sage Salvia officinalis L. via supplementation of the breeding medium with phytohormones: in these experiments IAA (indole-3-acetic acid) combined with BAP (6-benzylaminopurine) was added 25 .
Caffeic acid possesses a potential applicable in cosmetology and medicine thanks to its anti-oxidant, antimicrobial and also anti-cancer activity [26][27][28][29] .The content of caffeic acid in the roots of Asparagus officinalis amounts to 1.7-3.0mg• 1 g -1 whereas in Ocimum basilicum it reaches up to a level of 0.83 mg • 1 g -112,30 .Effective growth of the caffeic acid content has been achieved in tissue cultures of the Eastern purple coneflower Echinacea purpurea L. Moench.through application of the combination GA 3 (gibberellic acid), paclobutrazole and uniconazole 31 .
The content of salvinorin A in dry leaves of Salvia divinorum of different origin can reach from 55.75 to 163.97 μg • 100mg -1 dry leaves.Employment of the DART-HRMS method shows that normally the content of salvinorin A amounts to 1.54 mg • 1 g -1 raw leaves, whereas in improved extracts salvirnorin A is present at a level of 13.0-53.2mg • 1 g -1 raw leaves.In Mexico research on legal drugs determined the salvinorin A range from 8.32 to 56.52 mg • 1 g -1 dried leaf in the species mentioned.Investigators have established that 200-500 μg of salvinorin A induces strong hallucinogenic effects lasting for 20-30 min [32][33][34][35] .Salvinorins A and B represent diterpenes of neo clerodane.Salvinorin A is a hallucinogen occurring naturally in Salvia divinorum.With regard to strength it competes with a synthetic hallucinogen, namely diethylamide of lysergic acid.Salvinorin A is a typical non-nitrogenous selective antagonist of the opioid kappa receptor.Depending on the selectivity of salvinorin A for the opioid kappa receptor, this receptor constitutes a potential molecular target for the development of pharmaceuticals used for the study of characteristic features of different perception disorders, including schisophrenia, the Alzheimer's disease and bipolar affective disorder 36 .It possesses therapeutic potential in the treatment of pain, mood and personality disorders, substance abuse and gastro-intestinal disorders, which implies that non-alkaloids can be a potential scaffolding for the development of medicaments for the receptors coupled with aminergic proteins G 37 .Salvinorin B is a strong and selective analogue of the salvinorin A opioid kappa receptor.It is characterized by pro-depressant, anti-cocaine activity.However, in antinociceptive tests, this compound was not found acting as strongly as salvinorin A in reducing pain 38 .
Derivatives of salvinorin A and salvinorin B are salvianic acid A and salvianic acid B, a biosynthetic increse of which has been attained through supplementation of the breeding media with ABA (abscisic acid) in tissue cultivation of the red sage Salvia miltiorrhiza Bunge 39 .
Metabolic engineering is aimed at creation of new compounds in an organism, improvement in the production of the present compounds or mediation in the degradation of compounds 40 .Investigations carried out yield a conclusion that the metabolic pathways should be studied in the context of a whole plant and not only of a single cell.Even the simplest modifications can lead to induction of changes in the entire organism.At present the commonest strategy of targeted metabolic engineering is modification of a gene or genes, study of their expression and control enzymes involved in the metabolic pathways [41][42][43] .Yet, this is not the only means to modify the metabolic pathways in plants.So far numerous biosynthetic pathways in plant compounds have been successfully modified with the application of plant growth regulators 44,45 .Determination and understanding of the mechanism of plants' reaction to phytohormones and identification of specific genes undergoing expression www.nature.com/scientificreports/under supplementation will be of key importance in the future planning of improvement or modification of metabolic pathways in the biosynthesis of plant compounds.The precursor of salvinorins is geranylgeranyl diphosphate (GGPP), the biosynthesis of which occurs via the 2-C-methyl-erythritol 4-phosphate (MEP) pathway 46,47 .6-benzyloaminopurine reduce the amount of compounds witch are biosynthesized on the MEP pathway 48 , similar as in our case.The other studies indicate the influence of NAA on the MEP pathway 49,50 .Our results indicate that this plant growth regulator stimulated the biosynthesis of identificed secondary metabolites.Fluridone is commonly known to interfere with carotenoid and chlorophyll biosynthesis by modifying the MEP pathway 51,52 .Caffeic acid is one of the key intermediates of the plant phenylpropanoid pathway 53,54 .Rosmarinic acid biosynthesis is related to the phenylpropanoid pathway and the shikimic acid pathway 55 .It was proven that it was early steps of the phenylpropanoid pathway are regulated by auxins 56 .Moreover, the influence of cytokines on the discussed pathway was demonstrated 57 .Auxins are synthesized from L-tryptophan which is produced on the shikimate pathway 58 .Thus, the addition of auxins modifies this pathway, however, there are insufficient studies describing this effect.The effect of cytokinins has not been sufficiently studied so far.Generaly plant hormones have a comprehensive impact on the biosynthetic pathways of secondary metabolites in plants.Which is also confirmed by our results when using chemical compounds: BA, 2IP, fluridone, NAA and IAA.

Conclusions
The hitherto conducted investigations concentrated mainly on analysis of compounds contained in Coleus scutellarioides (L.) Benth.without specification of the variety studied.This research has revealed that specifically in Coleus scutellarioides variety 'Electric lime' the contents of rosmarinic and caffeic acids are significantly higher in tissues of plants cultivated under in vivo conditions.What is more, the performed qualitative and quantitative analyses confirm the presence of salvinorin A and salvinorin B in Coleus scutellarioides (L.) Benth.The presently discussed is the first successful attempt at such confirmation.And thus, this finding corroborates the reports indicating presence of psychoactive compounds in the species under analysis.
The present experiments detected differences in the amounts of the compounds concerned relative to the mode of cultivation, which is whether under in vivo or in vitro conditions.Supplementation of the breeding media with phytohormones under in vitro conditions was found affecting the percentage ratio and contents of particular compounds in plant tissues.Thereby, plant growth regulators have been proven to possess a potential for inducing modifications in the biosynthesis of compounds in plants.
The common occurrence of Coleus scutellaroides (L.) Benth., whose tissues contain salvinorins, poses a threat to humans since these psychoactive compounds can be dangerous to health, and that is why they should not be easily available.

Research material and conditions of in vitro culture
The initial material was composed of above-ground parts of Coleus scutellarioides (L.) Benth.plants representing the 'Electric lime' variety, coming from supermarket.For inducing in vitro cultures, fragments of stems with nodes were used.The prepared material was put onto the MS medium (Murashige & Skoog 59 ) at pH = 5.8.The plants were micropropagated on the MS medium without growth regulators (Fig. 4).

Quantification of compounds by the LC-MS/MS technique
The plant material constituted leaves and stems coming from plants bred on varied substrata under in vitro conditions and also from plants cultivated in vivo.The initial plant used for inducing in vitro cultures was homogenized by grinding in a mortar in liquid nitrogen, after which 1 g of the homogenate was macerated in 2 ml of 96% ethanol.The material was vortexed and subsequently transported into the ultrasonic bath for 20 min.The samples were kept in darkness for 18 h due to photolability of compounds and improved extraction efficiency.Then, the material was vortexed again and placed into the ultrasonic bath for 20 min.The samples were centrifuged for 10 min at 12,000 RPM and collection of the extract was carried out.To the remaining plant material 2 ml of pure methanol was added, after which the material was vortexed, transferred to the ultrasonic bath for 20 min, centrifuged for 10 min at 12,000 RPM and subsequently the extract was collected.The procedure was repeated until the moment when 6 ml of extract was obtained.The extract was divided into two samples.Through a filter (PTFE 0.22 µm), the extract was transferred to round bottom flasks, and evaporated using a vacuum evaporator at room temperature (SAVANT SPD121P SpeedVac Concentrator The dry remnant was dissolved in 1 ml of acetonitrile, then transferred for 20 min to the ultrasonic bath in order to make sure that the extract got entirely dissolved in ACN.This was followed by centrifugation for 5 min at 13,000 RPM.Then, 250 µl of the solution was poured into a vial of a volume of 2 ml, to which 500 µl of acetonitrile was added.The quantitative analysis of salvinorin B, salvinorin A (as related to the amount of salvinorin B), caffeic acid and rosmarinic acid was performed on an LCMS 8045 (Shimadzu, Kyoto, Japan) equipped with the C18 Kinetex (2.6u, 100A, 100 × 3.0 mm, Phenomenex, Torrance, CA, USA) column.The analysis was conducted in two replications.Chromatographic separation for quantitative determination of the contents of salvinorin B, rosmarinic acid and caffeic acid was performed with the use of two mobile phases: water with addition of 0.1% [v/v] formic acid (A and acetonitrile (B).The following gradient programme was used for analytes separation: initially 10% of B, then 20% B in 5th min, then 60% B in 10th min, then 10% B in 13th min and kept up to 20th min.The flow rate was set at 0.3 ml/min, column oven was set for 35 °C while the injection was set at 25.0 µl for salvinorin B and caffeic acid, and at 5.0 µl for rosmarinic acid quantification.Mass spectrometry was carried out with ionization through electrospraying and mode MRM (Table 1): At given parameters: the flow of the sprayed gas was 3 L/min, flow of the heating and drying gas -10 L/min, interphase temperature -300 °C, desolvation temperature -526 °C, temperature of the semiconductor laser -250 °C, of the heating block -400 °C.
Quantitative determination of salvinorin B, rosmarinic acid and caffeic acid in Coleus scutellarioides (L.) Benth.variability 'Electric lime' was performed in comparison with the pure model and the calibration curve whereas the quantification of salvinorin A was calculated as related to the content of salvinorin B. Qantitative analysis was performed based on the external standard method.

Statistical analysis
The results were expressed as mean measurements and recorded as a mean value.One-factor variance analysis (ANOVA) was carried out in order to verify lack of significant effect of plant growth regulators applied in in vitro cultures on the chemical compostion of Coleus scutellarioides (L.) Benth.'Electric lime' .The hypothesis assumed no influence of the used breeding medium on a plant's chemical compostion.Significant differences were estimated at a level of 0.05 and 0.01.In cases when variance analysis yielded a significant result, the Tukey honestly Table 1.Parameters of mass spectrometry.

Statement
All plant experiments were conducted in accordance with institutional, national and international guidelines and regulations.