Synergy and Antagonism of Active Constituents of ADAPT-232 on Transcriptional Level of Metabolic Regulation of Isolated Neuroglial Cells

CORE Metadata, citation and similar papers at core.ac.uk ORIGINAL RESEARCH ARTICLE Provided by Frontiers - Publisher Connector published: 20 February 2013 doi: 10.3389/fnins.2013.00016 Synergy and antagonism of active constituents of ADAPT-232 on transcriptional level of metabolic regulation of isolated neuroglial cells

Alexander Panossian1*, Rebecca Hamm2, Onat Kadioglu 2, Georg Wikman1 andThomas Efferth2

1 Swedish Herbal Institute Research and Development, Göteborg, Sweden 2 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany

Edited by: Gene expression profiling was performed on the human neuroglial cell line T98G after María M. Malagón, University of treatment with adaptogen ADAPT-232 and its constituents – extracts of Eleutherococcus Cordoba, Spain senticosus root, Schisandra chinensis berry, and Rhodiola rosea root as well as several Reviewed by: Jae Young Seong, Korea University, constituents individually, namely, eleutheroside E, schizandrin B, salidroside, triandrin, and South Korea tyrosol. A common feature for all tested adaptogens was their effect on G-protein-coupled JulieAnn Chowen, Hospital Infantil receptor signaling pathways, i.e., cAMP, phospholipase C (PLC), and phosphatidylinosi- Universitario Niño Jesús, Spain tol signal transduction pathways. Adaptogens may reduce the cAMP level in brain cells by *Correspondence: down-regulation of adenylate cyclase gene ADC2Y and up-regulation of phosphodiesterase Alexander Panossian, Swedish Herbal Institute Research and Development, gene PDE4D that is essential for energy homeostasis as well as for switching from cata- Gröndalsgatan 11 A, SE-412 62 bolic to anabolic states and vice versa. Down-regulation of cAMP by adaptogens may Göteborg, Sweden. decrease cAMP-dependent protein kinase A activity in various cells resulting in inhibition e-mail: [email protected] stress-induced catabolic transformations and saving of ATP for many ATP-dependant meta- bolic transformations. All tested adaptogens up-regulated the PLCB1 gene, which encodes phosphoinositide-specific PLC and phosphatidylinositol 3-kinases (PI3Ks), key players for the regulation of NF-κB-mediated defense responses. Other common targets of adap- togens included genes encoding ERα estrogen receptor (2.9–22.6 fold down-regulation), cholesterol ester transfer protein (5.1–10.6 fold down-regulation), heat shock protein Hsp70 (3.0–45.0 fold up-regulation), serpin peptidase inhibitor (neuroserpin), and 5-HT3 receptor of serotonin (2.2–6.6 fold down-regulation). These findings can be reconciled with the observed beneficial effects of adaptogens in behavioral, mental, and aging-associated dis- orders. Combining two or more active substances in one mixture significantly changes deregulated genes profiles: synergetic interactions result in activation of genes that none of the individual substances affected, while antagonistic interactions result in suppression some genes activated by individual substances. These interactions can have an influence on transcriptional control of metabolic regulation both on the cellular level and the level of the whole organism. Merging of deregulated genes array profiles and intracellular networks is specific to the new substance with unique pharmacological characteristics. Presumably, this phenomenon could be used to eliminate undesirable effects (e.g., toxic effects) and increase the selectivity of pharmacological intervention.

Keywords: pharmacogenomics, Rhodiola rosea, Schisandra chinensis, Eleutherococcus senticosus, ADAPT-232, salidroside, eleutheroside E, schizandrin B

INTRODUCTION functions irrespective of the nature of stressors (Brekhman II and The term adaptogen was introduced in scientific literature in Dardymov, 1969). On the verge of the new millennium, the def- the 1950s to refer to substances that increase the “state of non- inition of adaptogens was updated to “a new class of metabolic specific resistance”under stress conditions (Lazarev, 1958; Lazarev regulators which increase the ability of an organism to adapt to envi- et al., 1959). The adaptogen concept was based on the theory ronmental factors and to avoid damage from such factors”(Panoss- of stress (Selye, 1950), initially defined as a state of threatened ian et al., 1999a). The concept of adaptogens is now generally homeostasis, and a general adaptation syndrome characterized accepted by the scientific community (EMEA/HMPC/102655/, by a non-specific response of the organism to diverse stressors 2007; Samuelsson and Bohlin, 2009), although it has yet to gain (physical, emotional, environmental, etc.). It has been postulated prominence in mainstream pharmacology. In this context recent that adaptogens should be safe and are able to normalize body research progress has taken two main forms:

• Abbreviations: FOXO, forkhead box O; HPA, hypothalamic-pituitary-adrenal; Convincing clinical evidence has been generated on the efficacy JNK1, c-Jun N-terminal protein kinase; NO, nitric oxide; NPY, neuropeptide Y. of adaptogens, based on clinical trials performed in accordance

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with the International Conference in Harmonization Regu- than a single mechanism may account for their pharmacological lations (ICH) standards for good clinical practice (Davydov effects. and Krikorian, 2000; Goulet and Dionne, 2005; Sarris, 2007; Any pharmacological effect represents an integrated response Bleakney, 2008; Panossian and Wikman, 2009, 2010; Dwyer of an organism to the drug. The response can be associated with et al., 2011; Hung et al., 2011; Iovieno et al., 2011; Sarris et al., interactions between the drug and the cell at various levels of 2011; Chan, 2012; Ishaque et al., 2012), regulation: • Some key molecular mechanisms of adaptogen activity were defined (Panossian et al., 2007, 2009, 2012). • The level of a small physiologically active molecules, e.g., cAMP plays an important role in the integrative response of the organ- The stress-protective activity of adaptogens is associated with ism. This is the so-called metabolomic level, because ATP is a regulation of homeostasis both on: precursor of cAMP and AMP is a metabolite. • The level of proteins involved in the synthesis or degradation • the system level via several mechanisms of action which are of ligands or ligand receptors. This is the so-called proteomics linked to the hypothalamic-pituitary-adrenal (HPA) axis, and level of regulation. the cellular level via activation of molecular chaperones, mainly • Genes encoding proteins involved in synthesis, degradation, sig- hsp70 proteins, and the regulation of key mediators of the nal reception, and regulation. This is the so-called genomic and stress response, including neuropeptide Y (NPY), cortisol, nitric transcriptional level of regulation. oxide, stress-activated protein kinase JNK, and forkhead box O transcription factor (Panossian et al., 2007, 2009, 2012; Wiegant Activation or suppression of gene expression results in acti- et al., 2009). vation or inhibition of the biosynthesis of encoded proteins (enzymes, cofactors, or receptors), which in turn affects the pro- An important role of CNS system in stress is generally accepted, duction and function of active small molecules. Gene expression since the stress concept has been defined by Selye (Fink, 2000). profiles aid in tracing molecular interactions and signal transduc- Effect of adaptogens on CNS system, particularly neuroprotective tion pathways and to predict the pharmacological effect a drug will activity has been demonstrated in many animal and human stud- have on various cellular functions. ies (Panossian and Wikman, 2010; Panossian et al., 2011). Clinical Recently, the so-called “-omics” technologies have been estab- efficacy of adaptogens in behavioral and mental disorders such as lished for the analysis of pharmacological effects (Ulrich- depression, anxiety, bipolar disorder, chronic, and stress-induced Merzenich et al., 2007; Sarris et al., 2012). Since transcriptomics fatigue has been recently reviewed (Panossian and Wikman, 2009, and proteomics can monitor cellular changes in gene or pro- 2010). tein expression upon drug treatment in a comprehensive fashion, ADAPT-232 (Chisan®) is a traditional herbal medicinal prod- these techniques may be exquisitely suited to investigate the multi- uct consisting of a fixed combination of extracts from Rhodiola faceted mechanisms of herbal formulas. Therefore, we have ana- rosea root, Schisandra chinensis berry, and Eleutherococcus senti- lyzed the microarray-based transcriptome-wide mRNA expression cosus root. It is taken for decreased body performance such as profiles of the neuroglial cell line T98G after exposure to ADAPT- fatigue and weakness (Bogatova et al., 1997; Narimanian et al., 232 and its herbal components, R. rosea, S. chinensis, and E. 2005; Aslanyan et al., 2010). In general, herbal mixtures exert senticosus. their bioactivities through synergistic interactions of single com- The choice of neuroblastoma cell line T98G for our study ponents. This synergism can be attributed to the fact that med- is based on results obtained in many publications (Stein, 1979; icinal herbs contain many different phytochemicals, which may Guzhova et al., 2001; Su et al., 2012). In the central nervous sys- mutually influence each other’s activity. More than 140 com- tem, approximately 90% of the cells are glia. Glia has been shown pounds have been identified in R. rosea roots (Panossian and to have several functions, including serving as a transportation Wikman, 2010), 100 compounds in E. senticosus roots (Huang link between the bloodstream and neurons, uptake of neurotrans- et al., 2011), and about 200 compounds in S. chinensis berries mitters, synthesis and release of neurotrophic factors, immune (Panossian and Wikman, 2008). Many of them were shown to regulation, and modulation of synaptic activity (Henn and Ham- be active in pharmacological in vivo and in vitro experiments, berger, 1971; Haydon, 2001; Ullian et al., 2001). Glia contributes and likely contribute to the activity of the total extracts (Wag- to the defense of the brain through the expression of the innate ner et al., 1994; Panossian, 2003; Panossian and Wagner, 2005, immune response, promoting the clearance of neurotoxic pro- 2011; Panossian and Wikman, 2008, 2010; Panossian et al., 2008; teins and apoptotic cells from the CNS as well as by regulating Huang et al., 2011). It has been shown that the adaptogenic the entry of inflammatory systemic cells into the brain at the activity of ADAPT-232 is associated with key mediators of stress blood-brain barrier (Nguyen et al., 2002; Hauwel et al., 2005). response, e.g., heat-shock-proteins (Hsp70) and NPY, involved in This stimulates both tissue repair and the rapid restoration of the regulation of homeostasis, oxidative stress, energy metabo- tissue homeostasis. An important physiological function of neu- lism, cognitive function, and activation of the immune system roglial cells is metabolic supply of energy and other substances, during fatigue and exhaustion (Prodius et al., 1997; Panossian maintaining brain homeostasis – a function supposed to be the et al., 2009). However, the cellular and molecular modes of action characteristic for adaptogens by definition. Glial cell express a vari- are not well understood due to the fact that herbal remedies ety of hormonal receptors, which are critical during stress-induced in general have multiple targets and several mechanisms rather diseases. Glial cell express steroid receptors and generate many

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steroid hormones which elicit rapid non-genomic effects on neu- vitamin, calcium pantothenate). We also compared these two rons via both membrane-bound G-protein coupled receptors and fixed-composition mixtures to individual active compounds iso- nuclear genomic receptors known to activate RNA and protein lated from these plants, namely, salidroside, triandrin, tyrosol, synthesis (Bennett, 2000). Guzhova et al.(2001) show that glioma eleutheroside E, and schizandrin B (Figure 1). This pharma- cells export Hsp70 into the culture medium whether under nor- cogenomic analysis facilitates increased understanding of the mal conditions or subjected to heat shock. Hsp70 can be released molecular modes of action of adaptogens and the synergism of by glia and that exogenous Hsp70 can enhance neuronal stress ADAPT-232 forte. tolerance (Guzhova et al., 2001). Finally, an ability of astrocytes, but not neurons, to prevent macrophage and T-Cell inflammation MATERIALS AND METHODS in the CNS, to attenuate axonal loss and gliosis resulting in neu- DRUGS AND CHEMICALS roprotection in experimental autoimmune encephalomyelitis, the Eleutheroside E, schizandrin B, salidroside, and tyrosol, were pur- most widely used mouse model of multiple sclerosis (Spencea et al., chased from Chromadex (Irvine, CA, USA). Triandrin was isolated 2011) was the reason to select neuroglia cells in our studies devoted in Swedish Herbal Institute (SHI) Research and Development to understand molecular mechanisms of action of adaptogens. (Göteborg, Sweden), and identified by comparison (HPLC, TLC, The aim of the investigation was to compare similarities UV) with the authentic reference sample kindly provided by Prof. and differences in gene expression profiles upon treatment with Zapesoznaya (Institute of Officinal and Aromatic plants VILAR, these three adaptogenic plants. We compared the two plant Moscow,Russia). Pharmaceutical grade standardized extracts of R. extracts ADAPT-232 and ADAPT-232 forte (which is the same rosea L. roots, E. senticosus (Rupr. et Maxim) Harms roots, and S. composition as ADAPT-232 except also includes the anti-stress chinensis (Turcz.) Baill. berries, were manufactured in accordance

Eleutherococcus eleutheroside E Schisandra schizandrin B ADAPT-232 forte forte ADAPT-232 salidroside Rhodiola tyrosol Ca pantothenate triandrin

0,36 Salidroside Rt 8.38 0,34 OH 0,32 H O 0,30 O O Eleutheroside E Rt 21.63 0,28 OH Schizandrin Rt 51.17 HO 0,26 HO Tyrosol Rt 9.53 MeO O OH CH3 0,24 O OH H OH CH3O OH O OH CH3 0,22 OMe H H 0,20 CH3O HO MeO Triandrin R 10.43 CH3O t O AU 0,18 CH3O O CH3 HO 0,16 OH HO O O HO γ-Schizandrin Rt 75.21 O O OM 0,14 OH Rosavin Rt 23.56 HO CH3

0,12 OH H OH Eleutheroside B R HO CH3O CH3 0,10 t O O OH O 0,08 10.88 O OH CH3O CH3O 0,06 MeO HO HO CH3O O CH3

OH OH 0,04 Glc-O O CH3

0,02 0,00

250,00 300,00

350,00

5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00 55,00 60,00 65,00 70,000 Minutes

FIGURE 1 | Experimental setting for microarray-based transcriptome-wide mRNA profiling and 3D-HPLC fingerprint of ADAPT-232.

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to ICH Q7A and EMEA guidelines for Good Agricultural and calculated using specifications of their combinations (ADAPT- Collecting Practice (GACP) and Good Manufacturing Practice 232 and ADAPT-232 forte) to ensure that they corresponds to (GMP) of active pharmaceutical ingredients (API). We have pre- therapeutically effective doses. viously described the detailed composition and chemical charac- terization of ADAPT-232 and ADAPT-232 forte (Panossian et al., CELL CULTURE 2009, 2012; Aslanyan et al., 2010). The content of plant extracts The human neuroglial cell line T98G was purchased from the and their active markers was the same in both adaptogen prepara- American Type Culture Collection (ATCC, CRL-1690). Cells were tions; the only difference was the presence of calcium pantothenate grown in DMEM + GlutaMAX-I (Gibco, Darmstadt, Germany) in ADAPT-232 forte. Working samples used in experiments were with 10% fetal bovine serum (Gibco, Darmstadt, Germany) and prepared by dilution of stock solutions (5 mg/mL) of ADAPT-232, 1% penicillin/streptomycin (Gibco, Darmstadt, Germany). They ADAPT-232 forte, Radix Eleutherococci genuine extract, Radix were passaged twice a week and maintained in a 37˚C incubator Rhodiola genuine extract, Fructus Schisandrae genuine extract in a humidified atmosphere with 5% CO2. All experiments were or 10 mM solutions of purified analytical markers – salidroside conducted using cells in the logarithmic growth phase. (3 mg/mL), triandrin (3.1 mg/mL), eleutheroside E (7.4 mg/mL), schizandrin B (4 mg/mL), or tyrosol (1.4 mg/mL) with appropri- DRUG TREATMENT ate volumes of phosphate buffered saline solution (PBS). Working T98G cells were seeded 24 h before treatment on 6-well plates in solutions of 200 µL were added to 3 mL of cell culture to obtain the a density of 150,000 cells per well. The next day, old medium was same final concentrations of active markers and genuine extract removed and cells were treated in a final volume of 3 mL (Table 1). as in the incubation media of ADAPT-232 and ADAPT-232 forte The ethanol content was 0.8% for cells treated with isolated (Table 1). An exception was test sample B, where the concentration compounds and vehicle-treated control cells (test sample Z). Two was 100-fold lower than in test sample A. technical replicates were performed for each sample. Cells were The dose of 300 µg/mL (final concentration of ADAPT-232 incubated with the test substances for 24 h at 37˚C and then forte in incubation media) is based on the results of a recent phar- subjected to RNA isolation. macokinetic study of R. rosea-derived salidroside in human blood plasma, where we measured concentrations of ∼1 µg/mL = 3 µM mRNA ISOLATION AND QUALITY CONTROL (Panossian et al., 2010). However, in recent in vitro experiments, Cells were harvested after 24 h of treatment. TotalRNA was isolated an up-regulation of HSF1, Hsp72, and NPY was observed at 100- using InviTrap Spin Universal RNA Mini kit (Stratec Molecular, fold lower concentrations of ADAPT-232 forte (Panossian et al., Berlin, Germany) and dissolved in RNAse free-water. The RNA 2012). Therefore, in the present investigation ADAPT-232 forte of the two technical replicates was pooled (1:1) resulting in one was tested in two concentrations, 300 and 3 µg/mL (Table 1). sample for each treatment/control. The quality of total RNA was The concentrations of the total extracts of three herbal ingre- checked by gel analysis using the Total RNA Nano chip assay on dients and their active constituents are compatible is all test an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, samples: e.g., final concentration of salidroside is the same – Germany). All samples were of highest quality with RIN values 3 µM (900 µg/L) in all test samples containing salidroside, namely of 10. in Rhodiola extract, ADAPT-232, and ADAPT-232 forte. Sim- ilarly – schizandrin B, eleutheroside E triandrin, and tyrosol, GENE EXPRESSION PROFILING their concentrations were calculated based of the results of Microarray hybridizations were performed at the Institute of Mol- HPLC analysis of their content in genuine extracts and their ecular Biology (Mainz, Germany). Whole Human Genome RNA combinations. The concentrations of genuine extracts have been chips (860K Agilent) were used for gene expression profiling. Probe labeling and hybridization procedures were carried out following the One-Color Microarray-Based Gene Expression Analysis Proto- col1. Briefly, total RNA was labeled and converted to cDNA. Then, Table 1 | Concentrations used to treatT98G neuroglial cells for fluorescent cRNA (Cyanine 3-CTP) was synthesized and purified microarray experiments. using the QIAgen RNeasy Kit. After fragmentation of the cRNA,

Drug Concentration Designation samples were hybridized for 17 h at 65˚C. Microarray slides were washed and scanned with the Agilent Microarray Scanning system. ADAPT-232 forte 300 µg/mL Test sample A Images were analyzed and data was extracted. The background was ADAPT-232 forte 3 µg/mL Test sample B subtracted and data was normalized using the standard procedures ADAPT-232 172 µg/mL Test sample C of Agilent Feature Extraction Software. Eleutherococcus extract 30 µg/mL Test sample D Schisandra extract 100 µg/mL Test sample E MICROARRAY DATA ANALYSIS 2 Rhodiola extract 40 µg/mL Test sample F Expression data was further analyzed using Chipster software to Eleutheroside E 0.67 µM Test sample G filter genes by varying expression and significance. These steps Schisandrin B 5 µM Test sample H Salidroside 3 µM Test sample I 1http://www.chem.agilent.com/Library/usermanuals/Public/G414090040_ Triandrin 1.5 µM Test sample J GeneExpression_One-color_v6.5.pdf Tyrosol 3 µM Test sample K 2http://chipster.csc.fi/

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include filtering genes to isolate those that were up- or down- multiplying with the Ct value of RPS13 in the control. Fold changes regulated by one to three times the standard deviation (depend- were calculated with the ∆Ct (Standardized Ct of the gene in drug- ing on the total number of extremely up- or down-regulated treated sample – Ct of the gene in untreated sample) method where genes). A subsequent assessment of significance using empiri- the fold change is equal to 2−∆Ct for the up-regulated genes and cal Bayes t-test further narrowed the pool of genes. All genes −(2−∆Ct ) for the down-regulated genes. further considered showed a significant difference from the con- The correlation coefficient between mRNA expression values trol with p-value < 0.05, or otherwise are noted. Finally, filtered determined by microarray hybridization and real-time RT-PCR data was used in Ingenuity pathway analysis for Core analysis, in was R = 0.81 (Pearson Correlation Test), Table 3. order to determine networks and pathways influenced by the drug treatments3. RESULTS A microarray-based transcriptome-wide mRNA expression analy- REAL-TIME RT-PCR sis was performed to identify possible targets of the tested sub- Validation of the microarray data was done by real-time RT-PCR stances in T98G cells. T98G cells were treated with test substances for five selected genes which were tested for two drug-treated for 24 h in two technical replicates before total RNA was isolated samples (Tables 2 and 3). PCR primers were designed using and pooled for microarray hybridization. Significantly deregulated Roche Universal Probe Design4 and GenScript Real-Time PCR genes were identified compared to untreated controls (p < 0.05) by Primer Design5 tools. Amplification specificities were checked means of Chipster software analysis. Ingenuity Pathway analyses with Primer Blast6 using the sequence data from the NCBI RefSeq were performed with data sets of significantly up- or down- Human mRNA data base. Primer nucleotide sequences and used regulated genes: 536 genes for ADAPT-232 forte, 777 genes for primer concentrations are shown in Table 2. Oligonucleotides ADAPT-232 forte tested at the low concentration, 534 genes for were synthesized by MWG Eurofins. Extracted RNA was converted ADAPT-232 at the higher concentration, 591 genes for Eleuthe- to cDNA with RevertAid H Minus First Strand cDNA Synthesis Kit rococcus extract, 599 genes for Schisandra extract, 561 genes for (Thermo Scientific). Real-time RT-PCR experiments were per- substance Rhodiola extract, 567 genes for eleutheroside E, 620 formed with the Roche Bio-Rad CFX384 real-time PCR detection genes for schizandrin B, 640 genes for salidroside, 601 genes for system. Total reaction volume was 20 µL. About 4 µL 5x Hot Start triandrin,and 562 genes for tyrosol. TheVenn diagrams in Figure 2 TaqEvaGreen qPCR Mix (no ROX;Axon),75–250 nM final primer shows the number of unique deregulated genes for each treatment concentration, and about 500 ng RNA (converted to cDNA) were extract in comparison to the number of common deregulated used per reaction. The protocol was as follows: 50, 0˚C for 2 min, genes. Table 4 shows the main cellular functions and networks 95, 0˚C for 10 min, 45 cycle of 95, 0˚C for 15 s, Annealing Tem- most significantly affected by various adaptogens. Tables 5 and 6 perature for 1 min, 72, 0˚C for 1 min and 95, 0˚C for 1 min. The show genes up- or down-regulated by adaptogens. housekeeping gene RPS13 served as reference for standardization. To exemplarily validate the microarray-based mRNA expres- All measurements were done in duplicates to calculate mean values sions, real-time RT-PCR reactions were performed for five genes and standard deviations. Standardized Ct (cycle threshold) values using each two different pairs of treated and untreated sam- for the genes in samples were obtained by dividing the Ct values ples. The expression of these genes were quantified and the of genes in drug-treated samples by Ct values of RPS13 gene and ratios between treated and untreated samples were calculated and compared to the ratios obtained from microarray experiments

3 (Table 3). The correlation coefficient between mRNA expres- http://www.ingenuity.com/ sion values determined by microarray hybridization and real-time 4http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 5https://www.genscript.com/ssl-bin/app/primer RT-PCR was R = 0.81, indicating a high degree of concordance 6http://www.ncbi.nlm.nih.gov/tools/primer-blast between results obtained by both methods.

Table 2 | Primer nucleotide sequences and used primer concentrations.

Target gene Sequence Concentration (nM) Annealing temperature (˚C)

ADCY2 Fw: 50-CTGCTCGCCGTCTTCTTCGCG-30 125 57.9 Rev: 50-CGCCAGGGCAGTTGGAACTGTTAT-30 CETP Fw: 50-GAGACTGCCAAGGTGATCCAGA-30 125 58 Rev: 50-GTGGTGTAGCCATACTTCAGGG-30 ESR1 Fw: 50-CACCCAGGGAAGCTACTGTTTG-30 125 58 Rev: 50-ATCTCCACCATGCCCTCTACAC-30 HTR3D Fw: 50-TGACTGTTCTGCTGGGCTACA-30 125 58 Rev: 50-GCGAAGTAGACACCTCGCTT-30 PDE4D Fw: 50-GAATCAGAGAACATTCAACGACCAACCAG-30 75 63.4 Rev: 50-GCAGATGTGCCATTGTCCACATCAAAA-30 RPS13 Fw: 50-GGTTGAAGTTGACATCTGACGA-30 250 57.9–63.4 Rev: 50-CTTGTGCAACACATGTGAAT-30

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Table 3 | Validation of microarray-based mRNA expression by quantitative real-time RT-PCR.

Gene Sample Method Untreated cells Treated cells FC (log) FC

ADCY2 ADAPT-232 forte* Microarray 1,00 1,00 0 1,00 Real-time RT-PCR 1,08 Schisandra extract Microarray 1,00 1,00 0 1,00 Real-time RT-PCR 1,04 CETP ADAPT-232 forte* Microarray 1,00 1,00 0 1,00 Real-time RT-PCR 1,52 Eleutherococcus extract Microarray 8,75 6,13 −2,62 −6,15 Real-time RT-PCR −1,21 ESR1 ADAPT-232 forte** Microarray 8,23 4,63 −3,60 −12,13 Real-time RT-PCR −1,74 ADAPT-232 forte* Microarray 1,00 1,00 0 1,00 Real-time RT-PCR 1,42 HTR3D Eleutherococcus extract Microarray 6,93 5,65 −1,28 −2,43 Real-time RT-PCR −2,75 Salidroside Microarray 6,93 4,21 −2,72 −6,59 Real-time RT-PCR −2,81 PDE4D ADAPT-232 Microarray 1,00 1,00 0 1,00 Real-time RT-PCR 1,44 Rhodiola extract Microarray 4,84 6,85 2,01 4,03 Real-time RT-PCR 2,91

*Concentration 3 µg/mL, **Concentration 300 µg/mL.

FIGURE 2 | Venn diagrams of deregulated genes induced by the genes deregulated by each extract alone and the number of deregulated treatment of neuroglial cells with Rhodiola rosea root, Schisandra genes that overlapped multiple extracts. (B) Pool of all genes whose chinensis berries, and Eleutherococcus senticosus root extracts alone expression was affected by any of the three extracts alone in comparison and their fixed combination, ADAPT-232. (A) The number of unique to ADAPT-232.

DISCUSSION phospholipase C (PLC), and phosphatidylinositol mediated signal Based on the analysis of results obtained in this study, we can draw transduction (Table 6; Figure 3). several conclusions discussed in detail below. G-protein-coupled receptors (Gilman, 1987; Foord et al., 2005) are involved in a wide variety of physiological processes THE EFFECT OF ADAPTOGENS ON MEMBRANE-BOUND (Wettschureck and Offermanns, 2005; Hazell et al., 2011) G-PROTEIN-COUPLED RECEPTORS including: A common feature of all drugs tested is their effect on G- protein-coupled receptor (GPCR) signaling pathways. All tested adaptogens significantly deregulated the expression of a large • Regulation of behavior and mood including binding of number of genes encoding (a) GPCRs (Table 5) and (b) neurotransmitters such as serotonin, dopamine, GABA, and key proteins of G-protein downstream pathways, i.e., cAMP, glutamate.

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Table 4 | Cellular functions associated with genes and genetic of serotonin receptors modulate the release of many neuro- networks that showed significant differences in expression after transmitters including glutamate, GABA, dopamine, epineph- treatment with the test samples. rine/norepinephrine, and acetylcholine, as well as many hormones including oxytocin, prolactin, vasopressin, cortisol, corticotropin, Functions Test samples substance P, and others. Activation of serotonin receptors also influence various biological and neurological processes such as Antigen presentation Low concentration of ADAPT-322 forte aggression, anxiety, appetite, cognition, learning, memory, mood, Cancer Tyrosol nausea, sleep, and thermoregulation. A variety of pharmaceuti- Carbohydrate metabolism Eleutheroside E cal agents and illicit drugs, including many antidepressants and Cell cycle Salidroside antipsychotics, target serotonin receptors (Yakel, 2000; Kennett Cell death ADAPT-232 forte, schizandrin B, tyrosol et al., 1997). The binding of serotonin to the 5-HT receptor in Cell growth and proliferation ADAPT-232 3 neuronal cells of the central (e.g., brain-stem cells) and peripheral Cell–cell signaling and interaction ADAPT-232 forte, Schisandra extract, nervous systems opens the membrane channels, which in turn schizandrin B, ADAPT-232, Rhodiola leads to an excitatory response and anxiety (Kennett et al., 1997). extract, eleutheroside E Thus, the observed down-regulation of the HTR1A gene by tyrosol Cellular assembly and Low concentration of ADAPT-322 forte, (Table 6) is in line with publications that have demonstrated the organization schizandrin B anti-depressive effects of tyrosol in rats (Wikman and Panossian, Cellular compromise Schizandrin B 2004; Panossian et al., 2008) and recent publications showing that Cellular development ADAPT-232, Rhodiola extract, Rhodiola extracts affect serotonin receptors (Chen et al., 2009b; Schisandra extract Mannucci et al., 2012). Serotonin receptors are also known to reg- Cellular movement ADAPT-232, ADAPT-232 forte, ulate longevity (Murakami and Murakami, 2008) and behavioral salidroside aging (Murakami et al., 2008) in the nematode Caenorhabditis Inflammatory disease Triandrin elegans. This agrees with several publications demonstrating the Lipid metabolism All tested substances except beneficial effects of R. rosea on the lifespan of C. elegance and the schizandrin B fruit fly, Drosophila melanogaster (Jafari et al., 2007; Wiegant et al., Molecular transport Triandrin, tyrosol 2009). Nervous system development Tyrosol G-protein-coupled lysophospholipid receptors regulate cellu- and function lar Ca2+ homoeostasis, cytoskeletal architecture, proliferation Neurological disease Salidroside and survival, migration, and adhesion. They have been impli- Nucleic acid metabolism Eleutherococcus extract cated in development; regulation of the cardiovascular; immune Small molecule biochemistry Eleutherococcus extract, Schisandra and nervous systems; inflammation; arteriosclerosis; and cancer. extract, Rhodiola extract, eleutheroside Lysophospholipid receptor ligands bind to and activate their corre- E, triandrin, tyrosol sponding transmembrane receptors. The activated receptors have Vitamin and mineral metabolism Schisandra extract a wide range of cellular effects depending on which ligand, recep- tor, and cell-type is involved. Primary effects include inhibition • Sympathetic and parasympathetic nervous systems, which are of adenylyl cyclase and release of calcium from the endoplasmic regulated by GPCR pathways, and are responsible for control reticulum, while secondary effects include preventing apoptosis of many automatic body functions such as regulating blood and increasing cell proliferation (Meyer zu Heringdorf and Jakobs, pressure, heart rate, and digestive processes. 2007). Down-regulation of GPCRs by adaptogens (Table 6) indi- • Regulation of immune system activity and inflammation cates that the number of receptors is greatly reduced leading • Neuroendocrine homeostasis modulation. to strongly attenuated signal transduction via G-proteins and their effectors (Von Zastrow, 2002), which may contribute to the All of these physiological processes are associated with stress beneficial effect of adaptogens in stress-related disorders. and adaptogen activity, which increases stress tolerance. G-protein-coupled receptor expression and G-protein- The regulation of GPCRs is thought to play a fundamental role mediated signal transduction are affected by age and play an in maintaining physiologic homeostasis during stress (Carman important role in the development of the major human patholo- and Benovic, 1998). A number of pathologic states are associ- gies associated with aging, cancer, neurodegenerative disorders, ated with disturbances in the functional activity of certain GPCRs and cardiovascular diseases (Joseph et al., 1993; Alemany et al., (Lefkowitz, 1996). GPCRs are extremely important targets for 2007). This supports the results of in vivo studies in rats show- neuropsycho-pharmacology (Roth et al., 1998). Many neuropsy- ing that ADAPT-232 has potential value for the treatment of chiatric drugs either bind directly to specific GPCRs (e.g., antipsy- age-related disorders of the stress system and may be effective chotics) or affect GPCRs indirectly by influencing the amount of in maintaining the health in elderly individuals (Makarov et al., available native agonist (e.g., antidepressants; Von Zastrow, 2002). 2007). ADAPT-232 counters effects of aging associated with: Table 6 shows that adaptogens down-regulated the HTR1A gene encoding the 5-HT3 GPCR, which is known to activate • deregulation of programmed cell death (apoptosis), an intracellular second messenger cascade resulting in excitatory • suppression of the immune system and spontaneous tumors or inhibitory neurotransmission (Hoyer et al., 1994). Activation promotion,

www.frontiersin.org February 2013 | Volume 7 | Article 16| 7 Panossian et al. Gene expression profiles of ADAPT-232 2,657 5,657 2.9 2,676 − − − − 4,141 2,412 2,639 − − − 3,317 2,479 7,210 5,205 3,555 − − − − Salidroside Triandrin Tyrosol 3.4 8,754 4,563 2,770 11,959 3,411 2,990 − − − − − Schizandrin B − 2,282 2,639 4,170 − − − Eleutheroside E 3,434 2,888 3,972 3,434 2,990 3,294 5,464 3,294 − − − − − − Rhodiola extract − 2,329 2,928 2,990 6,589 2,676 − − − − Schisandra extract − 2,428 3,918 3,095 2,969 2,445 − − − − Eleutherococcus extract − 3,630 6,869 6,869 3,411 3,555 2,462 − − − − − − ADAPT-232 forte (3 mg/L) ADAPT-232 2,266 3,972 6,409 9,448 3,095 3,918 3,580 3,837 2,313 2,346 2,639 2,639 2,770 2,514 6,233 3,434 − − − − − forte (300 mg/L) Glutamate 2.3 2.5 3.1 3.0 2.7 2.5 3.5 Ligand ADAPT-232 Calcium sensor Orpahn A6 5-HTA Serotonin LNB7TMOrphanOrphanOrphan Orphan 3,681 5,540 3,9725-HTA 3,555 Serotonin 2,908 3,010 3,411 5,242 6,277 2,751 3,531 3,891 2,908 3,249 3,053 3,458 6,821 4,595 3,458 5,205 2,694 4,287 Orphan Orphan Metabotropic glutamate LNB7TM LNB7TMLNB7TM 6,543 2,282 2,395 3,053 Orphan LNB7TM Orphan Orphan SREB Orphan A3 Orphan Orphan A9 Orphan Orphan A2OrphanSPC/LPC 190,019 126,238 Orphan orphan A4 Orphan SPC/LPCorphan A1 lysoPLorphan A1 sphingosine 1P Orphan sphingosine 1P 86,823 51,268 4,084 125,366 4,287 3,482 3,340 family Orphan GPR179 GPR182 GPRC6A GPR37L1 HTR1F GPR110 GPR111 GPR114 GPR128 GPR141 GPR148 GPR150 GPR171 HTR1A GPR101 GPR112 GPR151 GPR160 GRM6 GPR88 GPR85 GPR78 GPR82 GPR83 GPR55 GPR52 GPR61 GPR65 GPR22 GPR37 GPR18 GPR4 GPR6 GPR12 GPR17 Genes Genes GPR1 The values show fold changes compared to the control. Table 5 | Genes up- and down-regulated by adaptogens inT98G cells.

Frontiers in Neuroscience | Neuroendocrine Science February 2013 | Volume 7 | Article 16| 8 Panossian et al. Gene expression profiles of ADAPT-232 − 6.9 21.7 440 FOXA1 + P4 − RBFOX1 FOX1 2.5 + 44.9 + 3.3 3.3 3.6 44.0 HSPA1B heat shock protein 72 3.0 + + + + 3 2.2 3.4 2.2 6.6 6.3 4.3 − − − − − HTR3D 5-HT receptor of serotonin − 3.3 3.3 3.1 2.4 3.3 4.5 3.4 4.2 2.7 4.3 3.0 2.4 2.6 − − − − − − − − − − − − − ** SERPIN B B2 B4 B2 B3 B2 B4 B9 B9 B2 B2 B4 B9 38.3 B8 9.3 B9 2.7 3.6 12.7 5.4 6.5 132.5 4.4 + − + + + + + + + ** SERPIN A1 A1 A1 A1 A1 A 6.6 − 12.0 A3 19.3 A1 22.6 5.8 7.5 7.1 3.4 3.0 2.9 ESR1 estrogen receptor 1 − − − − − − − − − 8.9 7.6 6.1 5.1 9.8 8.3 10.6 6.3 9.5 5.9 − CETP cholesterol ether trans − − − − − − − − − 6.1 13.2 9.6 5.1 10.1 10.1 5.1 9.9 12.7 − − PLCD4 phospholipase C* − − − − − − − 4.7 3.7 3.7 4.4 3.1 4.1 5.0 3.4 3.0 + PLCB1 phospholipase C beta 1 (phosphoinositide- specific) + + + + + + + + 67.2 A7 115.4 A10 + PRKCG and PRKCZ protein kinase C + 5.1 6.1 2.7 7.2 6.6 3.2 + + + + + + PI3KC2G phosphatidyl inositol-3 -kinase PIK3 44.5 4.3 5.0 2.7 4.0 3.2 4.2 4.6 96.3 + + + + + + + + + PDE4D phospho- diesterase 2.8 2.9 2.4 3.7 2.8 3.1 − adenylate cyclase − − − − − ADCY4 ADCY2 M) µ M) g/mL) g/mL) µ M) g/mL) g/mL) g/mL) µ M) µ µ M) µ µ µ g/mL) µ µ µ ) Indicates up-regulation. ) Indicates down-regulation. (1.5 Eleutherococcus (30 Schisandra (100 Rhodiola (40 Eleutheroside E (0.67 Schisandrin B (5 Salidroside (3 Triandrin Tyrosol (3 + − (300 (3 ( ( *PLCD4 expression is a marker for cancer.isoforms **Effects of of PKC the are SERPINs. cell-type specific: in CNS activation of PKC was associated with neuronal excitation (5-HT) and memory. **A1-A10 and B2-B9 represent the different Gene and corresponding protein (172 ADAPT-232 forte ADAPT-232 forte ADAPT-232 The values show fold changes compared to control. Table 6 | Common genes up- and down-regulated by adaptogens and corresponding proteins inT98G cells.

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FIGURE 3 | Hypothetic molecular mechanisms by which adaptogens (PKA), and phosphatidylinositol-3-kinase (PI3K). Effect of adaptogens on activate adaptive stress response pathways. Neurons normally receive G-protein-coupled receptors pathways: up-regulated genes are represented in

signals from multiple extracellular stressors that activate adaptive cellular red, down-regulated in blue color. The Gs alpha subunit (or Gs protein)

signaling pathways, e.g., many neurotransmitters activate GTP-binding protein activates the cAMP-dependent pathway by activating adenylate cyclase. Gi

coupled receptors (GPCR). The receptors in turn activate kinase cascades alpha subunit (or Gi/G0 or Gi protein) inhibits the production of cAMP from including those that activate protein kinase C (PKC), activate protein kinase A ATP.DAG, diacylglycerol; IP3, inositol triphosphate; PLC, phospholipase C.

• decrease in liver detoxification, The regulation of cAMP levels and PKA activity is a key • CNS malfunction (loss of memory and learning ability), and mechanism of energy homeostasis and represents a metabolic the development and progression of cardiac insufficiency, and switch between catabolism and anabolism (Walsh and Van Pat- hypercholesterolemia. ten, 1994; Conti, 2000; Abramovitch et al., 2004; Vandamme et al., 2012). Down-regulation of cAMP and PKA by adapto- THE EFFECT OF ADAPTOGENS ON cAMP-MEDIATED G-PROTEIN gens decreases stress-induced catabolic transformations. PKA is SIGNALING PATHWAYS presumably responsible for the stress-induced protective energy- Taking into account that adaptogens down-regulated the expres- saving effects of adaptogens. This energy-saving effect favors ATP- sion of genes encoding adenylate cyclase and up-regulated gene consuming anabolic transformations. Indeed, the inhibition of expression for phosphodiesterase, we suggest that adaptogens adenylate cyclase by adaptogens can increase intracellular ATP reduce the cAMP level in brain cells by (a) inhibiting adeny- levels and prevent ATP from being converted to cAMP (Taussig late cyclase (inhibiting synthesis of cAMP) and (b) stimulating and Gilman, 1995). This increased store of ATP might represent phosphodiesterase (stimulating degradation of cAMP; Figure 3). an energy source for other ATP-dependent enzymatic reactions Protein kinase A (PKA) acts in a cAMP-dependent manner required for metabolism. (Walsh and Van Patten, 1994). Low levels of cAMP down-regulate Mitochondrial decay is a major cause of aging, leading to PKA activity. PKA has several cellular functions including regula- the subsequent death of aerobic organisms including humans. tion of glycogen, sugar, and lipid metabolism. The effects of PKA Impairments in mitochondrial antioxidant status were invariably activation vary with the cell-type, e.g., in adipocytes PKA stimu- associated with decreased mitochondrial ATP-generation capacities late lipases while in myocytes (skeletal muscle) and hepatocytes as well as with increases in mitochondria-driven reactive oxygen PKA increases glucose formation (by stimulating glycogenolysis species (ROS) production in all tested tissues. and inhibiting glycogenesis) and its catabolic transformation Long-term supplementation of male and female C57BL/6J to pyruvate (glycolysis). This provides free energy in the form mice (starting from the age of 36 weeks) with schizandrin of ATP and NADH, which play an important role in stress B (administered at 0.012%, w/w) enhanced mitochondrial ATP- response. generation capacity in various tissues (brain, heart, liver, and

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kidney) during aging and mitigated age-dependent impairments other proteins (Nishizuka, 1995) and plays an important role in in mitochondrial antioxidant capacity and functional ability, tumor growth (Yamasaki et al., 2009). thereby retarding the aging process in mice, particularly during PI3K is a key upstream mediator of signal transduction and early aging,up to the age of 120 weeks (Ko et al.,2008). Schizandrin plays an important role in the regulation of NF-kB-mediated B was shown to increase the intracellular ATP levels and to protect defense responses as well as apoptosis. PI3K is required for long- against Gentamicin and Cyclosporine A-induced nephrotoxicity term enhancement of signal transmission between neurons, which by decreasing oxidative stress and cell death in vitro and in vivo is associated with potentiating memory and learning (Opazo et al., (Chiu et al., 2008; Zhu et al., 2012). R. rosea extract activated the 2003; Karpova et al., 2006; Yang et al., 2008). synthesis or resynthesis of ATP in skeletal muscles mitochondria and stimulated reparative energy processes after intense exercise in COMMON EFFECTS ON GENE EXPRESSION INVOLVED IN REGULATION rats. Treatment with R. rosea extract significantly (by 24.6%) pro- OF CYTOPLASMATIC AND NUCLEAR PROTEINS longed the duration of exhaustive swimming in comparison with ADAPT-232 up-regulated the SERPINI1 gene (serpin peptidase control rats (Abidov et al., 2003). Salidroside was shown to signif- inhibitor, neuroserpin), which is involved in the development and icantly increase the rate of ATP in isolated keratinocyte (Delepee function of the nervous system (Teesalu et al., 2004; Yepes and et al., 2004). These studies are in line with our hypothesis on the Lawrence, 2004). Neuroserpin controls axon growth and thereby mechanism of ATP-generation by adaptogens and their potential supports the transmission of nerve impulses. It plays an impor- benefits in fatigue and aging-related diseases. tant role in synapse development and regulates synaptic plasticity, The brain’s prefrontal cortex cells are known to contain suggesting that it may be important for learning and memory hyperpolarization-activated channels that can open when they (Hastings et al., 1997; Davis et al., 1999; Kinghorn et al., 2006). are exposed to cAMP in stress. Excessive opening of these chan- Moreover, neuroserpin inhibits the activity of tissue plasminogen nels impairs cognitive function. It has been suggested that cAMP activator (tPA), which plays a role in cell migration, blood clotting, inhibitors can inactivate the channels allowing for normal neu- and inflammation (Yepes and Lawrence, 2004). ron function. This reconnects the hyperpolarized cells to the A characteristic feature common to all tested adaptogens was neural network, and thus improves working memory, which plays down-regulation of the CETP gene, which encodes cholesteryl a key role in abstract thinking, planning, organizing, etc. (Wang ester transfer protein, a lipid plasma protein that facilitates the et al., 2007; Arnsten, 2011). Adaptogens may help in therapies transport of cholesterol esters and triglycerides between low- for cognitive deficits associated with normal aging, and for cog- density lipoproteins (LDL) and high-density lipoproteins (HDL; nitive changes associated with schizophrenia, bipolar disorder, Bruce et al., 1998). Pharmacological inhibition of CETP allevi- or attention deficit hyperactivity disorder (ADHD; Wang et al., ated atherosclerosis and other cardiovascular diseases, as well as 2011). metabolic syndrome (Barter et al., 2003). This hypothesis is in accordance with our findings and publica- Adaptogens down-regulated the ESR1 gene (Table 6). ESR1 tions,whereADAPT-232 and other adaptogens have demonstrated encodes estrogen receptor alpha (ERα), a nuclear receptor belong- beneficial effects on cognitive function in humans (Bogatova et al., ing to a large family of transcription factors,transducing hormonal 1997; Hartz et al., 2004; Narimanian et al., 2005; Darbinyan et al., signals, and regulating expression of target genes (Evans, 1988). 2007; Fintelmann and Gruenwald, 2007; Weng et al., 2007; Ols- ERα is primarily localized in the cytoplasm (Welshons et al., son et al., 2009; Aslanyan et al., 2010; Panossian and Wikman, 1984) in complexes with heat-shock-proteins (hsp90, hsp70, and 2010; Edwards et al., 2012). Two clinical pilot studies showed that hsp56) in a transcriptionally inactive form (Pratt, 1992). ERα are ADAPT-232 significantly improved attention and increased speed expressed in various tissues such as the breast, ovaries, testis, lung, and accuracy during stressful cognitive tasks in comparison to a uterus, bone, liver, and brain. In human forebrain, ERα are pre- placebo control (Aslanyan et al., 2010). ADAPT-232 significantly dominantly localized in amygdale (suggested to be involved in decreased the number of mistakes made by test subjects in com- mood and cognition), hypothalamus (involved in learning and plicated psychometric tests (Bogatova et al., 1997) and improved memory),and septum (Österlund and Hurd,2001;Yaghmaie et al., the quality-of-life and recovery period of patients suffering from 2005; Dahlman-Wright et al., 2006). Affective mood disorders, acute non-specific pneumonia (Narimanian et al., 2005). such as premenstrual syndrome, postnatal depression, and post- menopausal depression are associated with low serum-levels of ADAPTOGEN EFFECTS ON G-PROTEIN SIGNALING estrogens. Estradiol treatment has been shown to down-regulate PHOSPHATIDYLINOSITOL AND PHOSPHOLIPASE C PATHWAYS ERα in the uterus and in several brain areas (Shupnik et al., 1989; All tested adaptogens up-regulate the PLCB1 gene, which encodes Lauber et al., 1990,1991; Simerly andYoung, 1991; Österlund et al., phosphoinositide-specific PLC and the PI3KC2G gene, which 1998). encodes phosphatidylinositol 3-kinases (PI3Ks; Table 6; Figure 3). ESR1 expression is tissue-specific and differentially regulated When activated by a G-protein, PLC catalyses hydrolysis of in a region specific manner in the brain. It has been shown that phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol estrogen signaling via ERα can significantly attenuate an inflam- (DAG) and inositol-1,4,5-triphosphate (IP3; Figure 3; Gilman, matory neurodegenerative process by acting through astrocytes. 1987). IP3 is involved in a variety of cellular signaling pathways ERα expression is necessary in astrocytes, but not neurons for associated with phenomena as diverse as depression and tumor neuroprotection in experimental autoimmune encephalomyelitis, growth (Stutzmann, 2005; Sarkisov and Wang, 2008). DAG acti- the most widely used mouse model of multiple sclerosis – an vates protein kinase C (PKC), which phosphorylates numerous autoimmune disease characterized by demyelination and axonal

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degeneration (Spencea et al., 2011). Consequently, it can be ovarian cancer, colon cancer, prostate cancer, and endometrial expected that neuroprotective effect of adaptogens (Kim et al., cancer. (Fabian and Kimler, 2005; Ascenzi et al., 2006). 2004; Bu et al., 2005; Chen et al., 2009a; Qu et al., 2009; Bocharov Moreover, some of tested samples down-regulated the OLFM4 et al., 2010; Zhang et al., 2007, 2010; Li et al., 2011; Lee et al., gene, which was recently reported to inhibit apoptotic path- 2012a,b; Palumbo et al., 2012; Shi et al., 2012; Zeng et al., 2012) ways and to promote tumor cell proliferation. This suggests that must be associated with up-regulation ESR1 in glia cells. Surpris- OLFM4 could serve as a diagnostic marker for human can- ingly, the results of our study are not in line with this antici- cers (Koshida et al., 2007; Oh et al., 2011). Reduced expression pation because adaptogens down-regulate ERα gene expression. of the OLFM4 gene inhibits cell growth and increases sensiti- How to interpret these contradictory observations – neuroprotec- zation to hydrogen peroxide and tumor necrosis factor alpha- tion accompanied by down-regulation of ERα gene expression? induced apoptosis in gastric cancer cells (Liu et al., 2012). Down- This case is similar to another, when it was expected that stress- regulation of the PLCD4 gene (Table 6), which encodes cyto- protective adaptogens have to reduce cortisol, because normally plasmatic PLC, is apparently associated with catabolic activity of cortisol is increasing in stress. Indeed, adaptogens reduce corti- adaptogens. sol in stress (Panossian et al., 1999b). However, adaptogens can increase secretion of cortisol/corticosterone when added to iso- THE SYNERGY/ANTAGONISM CONCEPT AND CONSIDERATIONS ON lated adrenocortical cells (Panossian et al., 1999b; Panossian et al., DOSE-EFFECT DEPENDENCY 2007). Cell response (secretion of cortisol/corticosterone) to adap- The mechanism of action of adaptogens in T98G cells was togens and stress is similar – an activation, mild to adaptogens associated with deregulation of numerous genes. Even one and much stronger in stress. However, stress response (secretion single compound purified from the extracts, e.g., salidroside, of cortisol/corticosterone) was significantly low in adaptogen pre- triandrin, tyrosol from Rhodiola, schizandrin B from Schisan- treated cells than in control cells (Panossian et al.,1999b; Panossian dra, or eleutheroside E from Eleutherococcus affected the activ- et al., 2007). Pretreatment with adaptogen, so-called “adaptive ity of 500–700 genes belonging to several different functional stress response”or“preconditioning”or“hormesis”(Mattson et al., networks. 2007), acting as a mild stress or “stress vaccine,” adapts the cell The approximate number of genes affected by any single com- to stress (Panossian et al., 1999b; Wiegant et al., 2008; Boon- pound in ADAPT-232 was on the same order as the number of Niermeijer et al., 2012). Similarly, it can be speculated that down- genes affected by the entire mixture. Even if the gene expres- regulation of ERα gene expression by adaptogens is a signal for glia sion profile contained the same genes (Figure 2), their expression cell to initiate a feedback regulation of ERα. In a broader sense this remains of the same extent (the same fold range, Tables 5 and 6 concept is related to the inflammation, which is a defense response and Supplementary Material). The total number of interactions (“switch on” defense system) to cope the infection. In order to in the molecular networks did not increase proportionally with prevent an overreaction, a feedback mechanism of regulation of the number of active compounds in the mixture. Further, the inflammation is activated, e.g., cortisol and anti-inflammatory number of target genes affected by the complex mixture was cytokines are increasing (“switch off”defense system). Mild stress, decreased in some pathways as compared to single compounds. in general, is a defense response in order activate innate immunity. Figure 2 shows that 735 deregulated genes overlap between the In this context, adaptogens are natural substances initiating acti- single-component compounds. However, they do not account for vation of the innate defense systems, including ERα as one of the all 1056 genes affected by ADAPT-232, suggesting that deregula- component of stress system. tion of 321 genes by ADAPT-232 is due to synergic interactions. In addition to “classical” model of steroid receptor function, Thus, it is possible that some of ADAPT-232’s pharmacologi- when the ligand-bound estrogen receptor regulates transcription cal effects are associated with these 321 genes, supporting the of target genes in the nucleus by binding to estrogen response synergy concept for herbal mixtures. On the other hand, the element regulatory sequences in target genes, estrogen activated complete pool of all genes deregulated by three single extracts membrane-bound CRα initiates rapid signaling PI3K/PLC path- is 2188, while ADAPT-232 deregulates only 1056 genes. This indi- ways in astrocytes to indirectly modulate neuronal function and cates that antagonistic interactions of the three components in the survival (Deroo and Korach, 2006; Mhyre et al., 2006). Estro- mixture may result in suppression of some effects (possibly neg- gen treatment attenuates “non-classical” transcription at estrogen ative) of the mono-drugs in fixed combination in ADAPT-232. response elements sites in glioma cells (Mhyre et al., 2006). Adap- This is also in line with synergy/antagonism concept for herbal togens up-regulate PLCB1, PI3KC2G, and cAMP relate genes, mixtures. modulate NO and SAPK. These similarities of estrogens and Purified single substances revealed differential regulation of adaptogens allow to suggest that their mechanisms of actions more genes than did the extracts, e.g., Rhodiola extract affected someway interfere. Are they competing as mimetic or antago- more genes than salidroside, tyrosol, or triandrin, and ADAPT-232 nists is still unclear albeit both are neuroprotective. Further study affected more genes than Rhodiola, Schisandra, or Eleutherococcus on elucidation of this mechanism of action of adaptogens is extracts (Tables 5 and 6). required. For example, schizandrin B at a concentration of 3 µM sig- ERs are over-expressed in around 70% of breast cancers (Deroo nificantly down-regulated a number of genes (up to 12854 fold, and Korach, 2006). Pharmacological down-regulation of ESR1 Table 7) which were not affected in the total extract (in which may be effective in treatment and prevention of breast cancer, the concentration of schizandrin was comparable). Moreover,

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Table 7 |Top down-regulated genes in schisandrin B-treatedT98G cells In can be concluded that cellular sensitivity/reactivity is as investigated by Ingenuity Pathway analysis. decreasing at higher doses of adaptogens resulted in increased cel- lular tolerance to other stressors. Some effects of individual ingre- Symbol Description Fold change dients on gene expression were neutralized when the ingredients were combined in mixtures. In general, only the most common DOWN-REGULATED GENES effects attributed to the individual ingredients were observed in XRCC6 Ku70 protein* −12854.6 the complex mixtures. Interestingly, rather than potentiating gene RPS21 Ribosomal protein S21 −1618.0 expression levels, the combination of these components resulted YPEL5 Yippee-like 5 (Drosophila) −1082.4 in somewhat of a “smoothing effect” on gene expression level. KCTD20 Potassium channel tetramerization −252.5 The limitations of any in virto cell models experiment obvi- domain containing 20 ously should be considered when conclusions are generalized. The DSTN Destrin (actin depolymerizing factor) −126.2 changes in gene profile of any isolated cell line can not reflect all of *Ku70 is a protein that, in humans, is encoded by the XRCC6 gene, which is those in entire organism, because of numerous interactions with involved in DNA repair and chromatin remodeling. It is possible that XRCC6 is circulating hormones and mediators of immune system. However, involved in aging, although further results are necessary to determine whether these in vitro experiments can be helpful in finding of possible key it plays this role in humans. “Entrez Gene: XRCC6 X-ray repair complementing mechanisms of action of adaptogens, particularly when they are defective repair in Chinese hamster cells 6 (Ku autoantigen, 70 kDa).” considered in the context of huge body of evidences obtained in clinical and preclinical studies. It is just the first step in order Table 8 |Top up-regulated genes inT98G cells treated with ADAPT-232 to define a realistic hypothesis. Further studies can be focused forte (3 mg/L) as investigated by Ingenuity Pathway analysis. on evidences supporting this hypothesis. It is not excluded that plant extracts are metabolized in the liver thus, their metabo- Symbol Description Fold change lites may also compete with precursors. However, relevance of this in vitro model to the results of clinical studies is supported UP-REGULATED GENES by pharmacokinetic studies of salidroside, tyrosol, eleutheroside GCM2 Glial cells missing homolog 2 +948.8 E, and schizandrin. The half-life (t 1/2) was 4.662 h for eleuthero- (Drosophila) side E (Feng et al., 2006), 4.5 h for salidroside in SHR-5 extract EPHB1 EPH receptor B1 +903.9 (Panossian et al., 2010), 2 h for tyrosol (Panossian et al., 2009), ALX1 ALX homeobox 1 +867.1 and 4.6 h for schizandrin (Wang et al., 2008). Pharmacokinetic CHRNB4 Cholinergic receptor, nicotinic, beta 4 +670.9 curve of schizandrin has three maximums of concentration s in (neuronal) blood at 1, 3, and 8 h and it is still detectable in blood at least for SF3B2 Splicing factor 3b, subunit 2, 145 kDa +652.6 20 h (Wang et al., 2008; Liang et al., 2010). That is enough to reach WNT2B Wingless-type MMTV integration site +584.1 brain from the bloodstream, to pass the blood-brain barrier and to family, member 2B interact with membranes of glial cells, where G-protein receptors AICDA Activation-induced cytidine deaminase +552.6 are located. CD37 CD37 molecule +505.0 In conclusion, the effects of adaptogens on gene expression MDFI MyoD family inhibitor +487.8 profiles in neuroglial cells mainly affected energy metabolism TNNT2 TroponinT type 2 (cardiac) +461.4 (anabolic effects) and CNS functions. These results can be recon- HSPA12B Heat shock 70 kDa protein 12B +44.0 ciled with beneficial effects of adaptogens in behavioral, mental, HSPB8 Heat shock 22 kDa protein 8 +46.8 and aging-related disorders. HTR5A 5-Hydroxytryptamine (serotonin) +75.0 We conclude also, that blending two or more active substances receptor 5A, G-protein-coupled in one mixture significantly changes deregulated genes profiles: synergetic interactions result in activation of genes that none of the individual substances affected, while antagonistic interactions the gene expression profiles of schizandrin B and Schisandra result in suppression some genes activated by individual sub- extract were quite different (Table 7). The same is true for stances. Consequently, these changes can have an influence on other compounds as shown in Tables 6–8. This data may sup- transcriptional control of metabolic regulation both on the cel- port the reductionistic concept of drug discovery common in lular level and the level of the whole organism. As a result, the “western medicine” that is based on the isolation and applica- pharmacological profile of the combination may differ from the tion of active constituents rather than the use of complex plant profiles of the ingredients. Similarly, pharmacological profiles of extracts. purified compounds might differ from the pharmacological pro- The dose at which the compounds were administered strongly file the total plant extract. Presumably, this phenomenon could influenced both the intensity of the cellular response and the be used to eliminate undesirable effects (e.g., toxic effects) and profile of differentially expressed genes. For instance, ADAPT- increase the selectivity of pharmacological intervention. 232 forte taken at two different concentrations (3 and 300 mg/L) resulted in quite different gene expression profiles, including some ACKNOWLEDGMENTS instances of dose-dependent reversal effects on the same gene, e.g., This work was supported in part by the Swedish Herbal Insti- the adenylate cyclase-encoding gene (Table 8). tute (Alexander Panossian, Georg Wikman). We are indebted to

www.frontiersin.org February 2013 | Volume 7 | Article 16| 13 Panossian et al. Gene expression profiles of ADAPT-232

Dr. Tolga Eichhorn (Department of Pharmaceutical Biology, Insti- SUPPLEMENTARY MATERIAL tute of Pharmacy and Biochemistry, University of Mainz, Mainz, The Supplementary Material for this article can be found online Germany) for his scientific discussions and Karen Duffy (Cornell at http://www.frontiersin.org/Neuroendocrine_Science/10.3389/ University, Ithaka, NY, USA) for critically reading the manuscript. fnins.2013.00016/abstract

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