ARTICLE IN PRESS Phytomedicine 17 (2010) 494–499
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Double-blind, placebo-controlled, randomised study of single dose effects of ADAPT-232 on cognitive functions G. Aslanyan a, E. Amroyan b, E. Gabrielyan b, M. Nylander d, G. Wikman c, A. Panossian c,n a
Department of Clinical Pharmacology, National Institute of Health, Yerevan, Armenia Scientific Centre of Drug and Medical Technology Expertise, Ministry of Health, 375001 Yerevan, Armenia c Swedish Herbal Institute, Prinsgatan 12, SE-411 32, Gothenburg, Sweden d BioCare Research and Development, Stockholm, Sweden b
a r t i c l e in f o
Keywords: Adaptogens Rhodiola rosea Schisandra chinensis Eleutherococcus senticosus Mental performance Attention Accuracy Speed
a b s t r a c t The aim of this study was to assess the effect of a single dose of ADAPT-232 (a standardised fixed combination of Rhodiola rosea L., Schisandra chinensis (Turcz.) Baill., and Eleutherococcus senticosus Maxim) extracts on mental performance, such as attention, speed and accuracy, in tired individuals performing stressful cognitive tasks. The pilot study (phase IIa) clinical trial took the form of a double-blind, placebo-controlled, randomised, with two parallel groups. Forty healthy females aged between 20-68 years, who claimed to have felt stressed over a long period of time due to living under psychologically stressful conditions were selected to participate in the pilot study. In addition, a Stroop Colour-Word test (Stroop CW) was used to exhaust/prepare the volunteers prior to the d2 test used for assessment of cognitive function of patients. The participants were randomised into two groups, one (n = 20) of which received a single tablet of ADAPT-232 (270 mg), while a second (n =20) received a single tablet of placebo. The effects of the extract were measured prior to treatment and two hours after treatment using the d2 Test of Attention (d2). The results of the d2 test showed a significant difference (p o 0.05) in attention, speed, and accuracy (TN-E scores) between the two treatment groups. The subjects in the ADAPT-232 group quickly (two hours after verum was taken) gained improved attention and increased speed and accuracy during stressful cognitive tasks, in comparison to placebo. There was also a tendency of ADAPT-232 to reduce percentage of errors, which means better accuracy, quality of the work, and degree of care in the volunteers under stressful conditions. No serious side effects were reported, although a few minor adverse events, such as sleepiness and cold extremities, were observed in both treatment groups. & 2010 Elsevier GmbH. All rights reserved.
Introduction A group of herbal preparations known collectively as adaptogens (Brekhman and Dardymov, 1969; Panossian et al., 1999; Panossian, 2004) can increase tolerance to mental exhaustion and may enhance attention in cases of decreased performance, such as in fatigue and in the sensation of weakness (Olsson et al., 2009; Panossian and Wikman, 2009; Panossian and Wikman, 2010). The beneficial effects of multi-dose administration of adaptogens are mainly associated with the hypothalamic-pituitary-adrenal axis, a part of the stress-system that is believed to play a primary role in the reactions of the body to repeated stress and adaptation
n
Corresponding author: E-mail addresses: gayane@pharm.am (G. Aslanyan), elmira@pharm.am (E. Amroyan), egabri@pharm.am (E. Gabrielyan), ma.n@swipnet.se (M. Nylander), christina.holm@shi.se (G. Wikman), alexander.panossian@shi.se (A. Panossian). 0944-7113/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2010.02.005
(Panossian et al., 1999; Olsson et al., 2009; Panossian et al., 2007; Panossian and Wikman, 2008; Panossian and Wikman, 2010). A single dose application of adaptogens is important in situations, which requires a rapid response to tension or to a stressful situation (Panossian and Wagner, 2005). Additionally, a single administration of these adaptogens increases mental performance and physical working capacity in humans (Panossian and Wagner, 2005). The use of herbal preparations derived from Rhodiola rosea L, Schizandra chinensis (Turcz.) Baill. and Eleutherococcus senticosus Maxim. is not generally associated with deleterious side effects. In contrast, traditional stimulants may cause addiction, tolerance and abuse, and give rise to negative effects on sleep structure, thus cause rebound hypersomnolence or ’’come down’’ effects. ADAPT-232 is a standardised fixed combination of extracts of R. rosea, S. chinensis and E. senticosus. The aim of the present double-blind, parallel-group, randomised, pilot (phase IIa) study was to evaluate the effect of a single dose of ADAPT-232 on
ARTICLE IN PRESS G. Aslanyan et al. / Phytomedicine 17 (2010) 494–499
mental performance, such as attention, speed, and accuracy, on tired subjects performing stressful cognitive tasks.
three days was carefully explained to all volunteers. The selected participants, who also accepted the trial protocol, were included in the double-blind comparative study. Written informed consent was obtained from each participant in accordance with the revised declaration of Helsinki (World Medical Association Declaration of Helsinki, 2000).
Subjects and Methods The study protocols were reviewed and approved by the Ethics Committee of the Armenian Drug and Medical Technology Agency of the Ministry of Health of the Republic of Armenia. The pilot study involved 40 patients and was carried out at the Department of Clinical Pharmacology of the National Institute of Health (Yerevan, Armenia) between January and February 2008.
Study drugs The study drugs were manufactured according to Good Manufacturing Practice (GMP) by the Swedish Herbal Institute (Gothenburg, Sweden) and presented in the form of tablets. The ADAPT-232 (batch 1121) tablets comprised a fixed combination of dried extracts from roots of R. rosea (drug extract ratio 2.8:1; extraction solvent 70% ethanol), berries of S. chinensis (drug extract ratio 1.4:1; extraction solvent 95% ethanol) and roots of E. senticosus (drug extract ratio 10.5:1; extraction solvent 70% ethanol), and had been standardised with respect to rhodioloside (0.32%), rosavin, (0.5%), tyrosol (0.05%) schizandrin (0.37%), g-schizandrin (0.24%) and eleutherosides B and E (0.15%). The verum was presented as a single 420 mg tablet, which contains 270 mg of ADAPT-232. The amounts of the active ingredients were determined by analytical RP-HPLC using an acetonitrile-water gradient system as mobile phase. The peaks were detected by UV-PAD (Fig. 1) and the analytes were quantified at 221 nm (rhodioloside and tyrosol), 252 nm (rosavin), 220 nm (eleutheroside B), and 210 nm (eleutheroside E, schizandrin and g-schizandrin). All the analytical methods were validated for selectivity, peak purity, precision (RSD o5%) and accuracy in the range 50 - 150% of the target amounts of analytes in accordance with International Conference on Harmonisation (ICH) guidelines (International Conference on Harmonization, 1995) using Effi Validation 3 software (version 1.03) for testing and calibration laboratories subject to EN ISO/IEC 17 025:2001 (International Organisation for Standardisation, 2001).
Study population The study population included healthy female volunteers (nurses, doctors and teachers), which were recruited from a polyclinic, a school and a hospital close to the Department of Clinical Pharmacology. The included individuals, aged between 20-68 years, had experienced stress over a long period of time due to living under psychologically stressful conditions. Pregnant or breast-feeding subjects; individuals with identified medical conditions (e.g. HIV, cancer or cardiovascular, joint, liver, kidney or psychiatric diseases); those diagnosed with psychiatric problems; those presenting allergic reactions to herbal products; those who had used adaptogens within the previous two months or corticosteroids within the previous 6 months; those misusing tranquillisers, pain killing drugs or narcotics; those suffering from caffeine withdrawal syndrome or were heavy coffee drinkers; and those subjects who were deemed to be unwilling to cooperate or unable to participate in the study were excluded. The strict requirement to avoid coffee during the complete period of the trial and to abstain from alcohol and caffeine for a minimum of one day prior to the testing session on the morning of the first day of the trial and throughout the following 0.36
Salidroside Rt 8.38 OH
0.34 0.32
HO O
0.30 0.28 0.26
O
OH Eleutheroside E Rt 21.63
HO
O
Tyrosol Rt 9.53 OH
Schizandrin Rt 51.17
HO
MeO
OH
0.24
O
O
OH OH
CH3 OH
H CH3O
OH
CH3
OMe
0.22
H
H
CH3O
MeO
HO
0.20
AU
495
CH3O CH3O
O Triandrin Rt
0.18
10.43
HO HO
0.16 0.14
O
O OMe γ-Schizandrin Rt 75.21
OH
H
Rosavin Rt 23.56
0.12
HO Eleutheroside B
0.08
Rt 10.88 MeO
0.06
CH3 OH
CH3O
CH3
O O
O
OH HO OH
Glc-O
0.04
O OH
0.10
OCH3 OCH3
HO
CH3O CH3O CH3O
HO OH
OCH3 OCH3
MeO
0.02 0.00
λ.
250.00 300.00
nm
350.00 5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
Minutes Fig. 1. HPLC fingerprint of ADAPT-232. Rt is the retention time (min).
60.00
65.00
70.00
75.00
80.00
ARTICLE IN PRESS 496
G. Aslanyan et al. / Phytomedicine 17 (2010) 494–499
The 420 mg placebo tablets (batch 1116) contained dark brown muscovado sugar and the same inactive excipients (lactose monohydrate, microcrystalline cellulose, magnesium stearate) as in the verum tablets. The medication was provided in blister packs labelled ‘‘ADAPT-232/Placebo’’ followed by a code number (1 to 40), the identification number of the participant, dosage, batch number, and expiry date. The packaging, appearance and organoleptic characteristics of the verum and placebo tablets were similar, such that they could not be distinguished. The study drugs were stored separately at room temperature in a secure location to prevent their use in other ways than the described study.
Advertising for volunteers
Medical examination and evaluation for eligibility (inclusion and exclusion parameters)
Randomisation
Study design
Evaluation of primary and secondary endpoints, evaluation of the affect of learning and degree of tiredness
The selected participants were divided into two equal groups (n= 20) using a simple randomisation procedure. Group A received tablets containing ADAPT-232, whilst the negative control (Group B) received identical-looking tablets containing placebo. The identification number of each participant and the drug code number (this was encoded in a table of random numbers, which were generated at the production site under the control of quality assurance personnel and kept unbroken until the end of the study) were recorded in a protocol and in the Case Report Form (CRF), in order to allow subsequent identification. Information about the placebo and the verum became known to the statistician, investigators and volunteers only after completion of the study and final statistical analysis of the results. On Day 1, the subjects were examined medically and assessed for base-line primary and secondary stress-related endpoints by psychometric tests conducted in the morning (9 - 10 am) when patients were not tired (Tm1). On day 2, the tests were repeated in the morning (Tm2) and afternoon ( 4 pm when subjects were supposedly tired; Ta2), and the degree of tiredness was evaluated by comparing the results of Tm2 and Ta2, while the effects of learning were assessed by comparison of the results of Tm1 and Tm2. In the afternoon ( 4 pm) of day 3, a single dose of the study drug (ADAPT-232) or of placebo was administered and the effects were evaluated 2 h later by psychometric tests (Ta3). A schematic diagram of the trial is displayed in Chart 1.
ADAPT
Placebo
Administration of single dose
Two hours later, evaluation of primary endpoints and single dose effect
Monitoring of data. Analysis of results. Report of study. Chart 1. Schematic diagram of the design of the trial .
d2 test Methodology The efficacy of the treatment was evaluated by psychometric tests, which assessed the state of functional components of mental activity, such as short-term memory, attention (stability and intensity), speed and accuracy. Participants were set the task of completing forms, with a pencil, associated with the d2 Test of Attention (d2) and the Stroop Colour-Word (Stroop CW) tests. Stroop CW is a cognitive aspects of mental disorders such as brain damage, attention deficit, hyperactivity disorder, dementias. It is also used as a stressfull cognitive task for measuring stress response, such as arterial blood pressure, heart rate, etc. (Facchinetti et al., 2002). At the start of the test, subjects signed their forms and were given the appropriate instructions. A strict time limit (measured using a stop-watch) was allowed for the completion of the tests. Subsequently, the results were checked with the help of keys, which have scales in the upper and the lower lines that make it possible to read numbers of processed letters easily and quickly.
This test is a valid measure of selective attention and is sensitive to the speed and quality of performance (Brickenkamp and Zillmer, 1998) with following parameters:
(i) Total number of items processed (TN) – is a quantitative measure of performance in terms of all items processed (both relevant and irrelevant). TN is highly reliable and measures attention, processing speed, amount of work completed, and motivation. Keys 1 and 2 were used for scoring. (ii) Errors of omission (E1 - under-inclusion) - occur when relevant items (’’d’’ with two dashes) are not crossed out. Under-inclusion is a relatively common mistake and is sensitive to attention control, rule compliance, accuracy of visual scanning and quality of performance. Key 1 was used to determine the number of mistakes of omission. (iii) Errors of commission (E2 – over-inclusion) - occur when irrelevant letters are crossed out in contravention of the instructions. Over-inclusion is related to ability to focus, rule
ARTICLE IN PRESS G. Aslanyan et al. / Phytomedicine 17 (2010) 494–499
compliance, accuracy of visual scanning, care, and cognitive flexibility. Key 2 was used to score the errors of commission. (iv) Raw error score (E= E1+ E2) – represents the total number of errors committed and is a preliminary statistic for computing other parameters. P P (v) Percentage of errors (E%= 100 (E1+ E2)/ N) - is a variable measuring the qualitative aspects of performance and proportion of errors made, in terms of all items processed. The smaller the value of E%, the better is the accuracy, quality of the work, and degree of care. (vi) Total number of items processed minus raw error score (TN – E) – is a highly reliable parameter that measures the amount of work completed after a single correction for errors, in terms of ability to focus, and the relationship between speed and accuracy. Stroop CW test Stroop CW is used to measure stressful cognitive tasks and stress responses, such as arterial blood pressure and heart rate, etc. (Facchinetti et al., 2002). In the present study, a test or manual (30150M) for clinical and experimental application was employed (Golden and Freshwater, 2002). This test was based on three pages, which each contained five columns of 20 items, in total 100 items. The following parameters were employed: (i) Word score (W) - is the number of items (words) completed in a set time (45 s). The ‘‘word page’’ consists of the words ’’RED’’, ’’GREEN’’ and ’’BLUE’’, which were arranged randomly and printed in black ink on a white background. The objective was to choose the word that matched the colour printed at the top of the page. (ii) Colour score (C) - is the number of items (coloured symbols) completed in a set time (45 s). The ‘‘colour page’’ consists of 100 items, all written as XXXX, printed in either red, green, or blue ink on a white background. The objective was to choose the coloured symbols that matched the word at the top of the page. (iii) Colour-word score (CW) - is the number of items (coloured words) completed in a set time (45 s). The ‘‘colour-word page’’ consists of words from the ‘‘word page’’ printed in colours from the ‘‘colour page’’. The objective was to choose the colour in which the word at the top of the page was written, rather than the colour that the word names.
497
Clinical Practice (GCP) by an independent monitor that visited the study site on the first and third day of the trial. Thus, adherence to the study protocol, performance of the psychometric tests, completeness and accuracy of the CRFs, and the overall conduct of the trial was verified. Statistical methods Standard methods were used to calculate the mean, standard error of the mean and standard deviation values of the scores in the psychometric tests. The significance of differences between groups A and B were evaluated using Student t-tests. The significance of the differences in test scores (day 1 versus day 2; morning versus afternoon on day 2; day 2 versus day 3) within each group, and of the mean test scores (both before and after treatment) between groups, were determined using one-way ANOVA with Tukey multiple comparison post tests. Data management and calculations were performed with GraphPad (San Diego, CA, USA) Prism software (version 3.03 for Windows).
Results A total of 40 females aged between 20 and 68 (mean age ADAPT-232: 42.7; mean age placebo: 45.4) completed the trial and none reported adverse reactions to the medication administered. Although, one subject from the ADAPT group reported cold extremities after treatment and one subject from each of the groups claimed that treatment had caused severe sleepiness. In addition, one subject from the treatment group reported an improvement in sleep following medication. All participants were submitted to stressful cognitive tasks (Stroop CW and d2 tests), four times over three consecutive days (Day 1: in the morning when subjects were not tired; Day 2: in the morning when subjects were not tired and in the afternoon when subjects were tired; Day 3: in the afternoon, two hours after treatment with ADAPT or placebo, when subjects were tired). Repeated testing in the mornings of days 1 and 2 allowed the effect of learning (Table 1; Fig. 2), which were subsequently excluded from the overall results. The degree of tiredness in the afternoon of day 2 was assessed by comparing these results with those obtained in the morning of the same day (Table 2; Fig. 2). It was seen that there was a concomitant increase in the d2 test score due to learning and that tiredness decreased the score in the afternoon test. These two effects tended to neutralise each other,
Patient compliance and withdrawals All blister packs were collected at the end of the trial to ensure patient compliance. The lower limit for compliance was set to 100%. It was seen that all participants that were included in the study completed the observation period.
Table 1 Evaluation of the efficacy of learning in the 40 participants of the phase IIa trial. Test and parameter
Monitoring of the study The trial was monitored in accordance with the ICH (International Conference on Harmonisation, 2000) guidelines for Good
Tm1 versus Tm2p value (statistical significance)
1st day/morning (Tm1)
2nd day/morning (Tm2)
Stroop-CW test W score C score CW score
35.65 7 17.13 46.77 7 11.89 72.757 12.97
37.15 715.98 49.4 711.55 72.23 7 15.40
0.3310 (ns) 0.1702 (ns) 0.8293 (ns)
d2 test TN-E score E% score
485.4 7103.0 31.83 722.00
535.8 788.37 20.73 714.89
0.0216 (n) 0.0099 (nnn)
Data management procedures All information relevant to the study, including the results of the d2 and the Stroop CW tests, was entered carefully and accurately into the CRF for each participant, which were recognized by their number and trial identification. Data were subsequently entered on a participant-by-participant basis into a TM Microsoft Excels 2000 database, for further analysis.
Time of application of test
Values are means 7 SD. Significance of differences between means analysed by unpaired t-test is indicated by: n p o 0.05, nn p o0.01, nnn po 0.001. ns Indicates no significant difference (p 40.05) between the scores before and after treatment.
ARTICLE IN PRESS 498
G. Aslanyan et al. / Phytomedicine 17 (2010) 494–499
d2 test (TN-E score)
700
learning effect
effect of tiredness
effect of ADAPT-232
TN-E Score
*
600
500 ADAPT Placebo
400 Tm1
Tm2
Ta2
Ta3
test Fig. 2. Results of the stressful cognitive tasks applied during the phase IIa trial showing TN-E scores in the d2 test. n Indicates significant differences (p o0.05) between two groups.
Table 2 Evaluation of the degree of tiredness in the 40 participants of the phase IIa trial. Test and parameter
Time of application of test
Tm2 versus Ta2p value (statistical significance)
2nt day/morning (Tm2)
2rd day/afternoon (Ta2)
Stroop-CW test W score C score CW score
37.15 7 15.98 49.4 7 11.55 72.23 7 15.40
37.877 16.54 51.777 11.23 75.98710.9
0.5534 (ns) 0.0625 (ns) 0.0663 (ns)
d2 test TN-E score E% score
535.8 7 88.37 20.73 7 14.89
543.27 89.67 19.787 15.61
0.7111 (ns) 0.7822 (ns)
Values are means7 SD. Significance of differences between means was analysed by unpaired t-test. ns Indicates no significant difference (p 40.05) between the scores before and after treatment.
which resulted in the observed flattening of the (TN – E) curve markedly between Tm2 and Ta2. A significant difference (po0.05) between the two groups was observed in respect of TN-E score - the main parameter in the d2 test where ADAPT-232 increases speed and accuracy of mental performance improving attention, in comparison to placebo (Fig. 2). Thus, TN-E score increased for 43 units (from 552 to 593) in ADAPR-232 group, while in placebo group the increase was for 8 units (from 534 to 542).
Discussion The herbal drug ADAPT-232 comprises a fixed combination of extracts from the adaptogens R. rosea, S. chinensis and E. senticosus, which are known to increase non-specific resistance of an organism to stress (Panossian et al., 2008; Panossian et al., 2009). A single dose administration of ADAPT-232 has been shown to increase mental performance, short-term memory, the power of comprehension and oculomotor co-ordination in Russian cosmonauts undergoing training involving long periods of monotonous work during prolonged (90 days) isolation. In complicated psychometric tests carried out 4 h after administration of the drug, the number of mistakes made by the trainees was significantly reduced (Bogatova et al., 1997).
Furthermore, in an extended study of ADAPT-232 conducted at the Moscow Aviation Institute on 60 volunteers, it was (Bogatova et al., 1994) demonstrated that repeated administration of ADAPT-232 significantly increased the capacity for mental and physical (mainly cardiovascular) work carried out against a background of fatigue and/or stress (i.e. monotonous and longterm work that simulated the environment of the Saljut and Mir space stations). The results obtained in a recent study (Narimanian et al., 2005) indicated that adjuvant treatment of pneumonia patients with repeated administration of ADAPT-232 has a positive effect on the recovery of patients by decreasing the duration of the acute phase of the illness, by increasing mental performance of patients in the rehabilitation period, and by improving their quality-of-life. In the present study, we assessed the mental performance (attention, speed and accuracy) in stressed and tired subjects on a single dose of ADAPT-232 or placebo, whilst they performed stressful cognitive tasks (d2 test) under time pressure. The d2 test measures selective attention and is sensitive to the speed and quality of performance. The results obtained in this study clearly showed that ADAPT-232 significantly improved scores resulting from the psychometric test, in comparison to placebo. It was demonstrated that subjects taking ADAPT-232 increases the amount of precise/correct work completed on the background of mental fatigue. In other words, their ability to focus, processing speed and accuracy of performing specific tasks improved. These effects were seen already two hours after intake of ADAPT-232. There was also a tendency that ADAPT-232 reduced percentage of errors subjects made in the stressful psychometric test, in comparison to placebo. This suggests ADAPT-232 improves the quality of work and degree of care in subjects under stressful conditions. In conclusion, this pilot clinical trial suggests that ADAPT-232 efficiently and quickly improves attention, speed and accuray in situations of decreased performance caused by stress and tiredness. The results confirms previous findings and is of value to anyone who wants to improve their mental performance in work and everyday life. Further studies are required in order to find the optimal dose level and regimen providing contineously stable effect.
Notice of Conflict of Interest The study was conceived by GW, the Director and a stockholder of SHI Research and Development. The funding sponsor, however, had no role in any practical aspect of the study including experimental design, data collection, management, or analysis and interpretation of the results. The study protocol was formulated by AP and EA, and the ethical applications were prepared by GA and EG. GA enrolled the participants and performed the study interventions and, together with EA, conducted the data analysis. AP drafted the manuscript, and all authors were involved in its critical appraisal and final approval. AP is employed by the Sponsor on a permanent basis. GA receives an honorarium from the sponsor for contract research carried out on behalf of SHI Research and Development. EG and EA have no conflicts of interest of a commercial or financial nature in respect of this study.
Acknowledgments The study was sponsored by the Swedish Herbal Institute (SHI) Research and Development, Sweden, and materials were supplied by the sponsor.
ARTICLE IN PRESS G. Aslanyan et al. / Phytomedicine 17 (2010) 494–499
References Bogatova, R.I., Shlyakova, L.V., Malozemov, V.V., Narinskaja, A.L., Roslyakova, N.A., Shangin, A.L., 1994. Experimental trials of phytoadaptogen effect on the quality of operators’ activity, mental and physical working capacity. Final Report on ADAPT 232. Institute of Medical and Biological Problems (IMBP), Moscow, pp. 1–151. Bogatova, R.I., Shlyakova, L.V., Salnitsky, V.P., Wikman, G., 1997. Evaluation of the effect of a single dose of a phytoadaptogen on human subjects’ work ability during long isolation. Aerospace Environ. Med. 31, 51–54. Brekhman, I.I., Dardymov, I.V., 1969. New substances of plant origin which increase non-specific resistance. Annu. Rev. Pharmacol. 9, 419–430. Brickenkamp, R., Zillmer, E., 1998. The d2 Test of Attention 1st Edition Hogrefe and Huber Publishers, Go¨ttingen 1-72. Facchinetti, F., Neri, I., Tarabusi, M., 2002. Eleutherococcus senticosus reduces cardiovascular stress response in healthy subjects: a randomized, placebocontrolled trial. Stress Health 18, 11–17. Golden, C.J., Freshwater, S.M., 2002. The Stroop Color and Word Test. A Manual for Clinical and Experimental Uses. Stoelting Co., Chicago 1-68. International Conference on Harmonisation, 2000. ICH Harmonised Tripartite Guideline. Guideline for Good Clinical Practice E6(R1). Current Step 4 version dated 10 June 1996. Available at /http://www.ich.org/LOB/media/MEDIA482. pdfS. International Conference on Harmonization (ICH). Topic Q2 (R1), Validation of Analytical Procedures: Text and Methodology, Step 5, Note for Guidance for Validation of Analytical Procedure4 Text and Methodologies. CPMP/ICH/381/ 95, June 1995. Available at /http://www.emea.europa.eu/pdfs/human/ich/ 038195en.pdfS. International Organisation for Standardisation. EN ISO/IEC 17 025:2001 Standard Accreditation Requirements for QC Laboratories Operating under the GxP Compliance Requirements 2001. Available at /http://www.effichem.comS. Narimanian, M., Badalyan, M., Panosyan, V., Gabrielyan, E., Panossian, A., Wikman, G., Wagner, H., 2005. Impact of ChisanR (ADAPT 232) on the Quality – of - life and its Efficacy as an Adjuvant in the treatment of Acute Non-specific Pneumonia. Phytomedicine 12, 723–729.
499
Olsson E.M.G., von Sche´ele B., Panossian A.G., 2009. A randomized double-blind placebo controlled parallel group study of SHR-5 extract of Rhodiola rosea roots as treatment for patients with stress related fatigue. Planta Med. 75, 105–112. Panossian, A., Hambartsumyan, M., Hovanissian, A., Gabrielyan, E., Wikman, G., 2007. The adaptogens Rhodiola and Schizandra modify the response to immobilization stress in rabbits by suppressing the increase of phosphorylated stress-activated protein kinase, nitric oxide and cortisol. Drug Target Insights 1, 39–54. Available at /http://www.la-press.com/the-adaptogens-rhodiolaand-schizandra-modify-the-response-to-immobili-a260S. Panossian, A., Wagner, H., 2005. Stimulating effects of adaptogens: an overview of clinical trials of adaptogens with particular reference on their efficacy at single dose administration. Phytother Res. 19, 819–838. Panossian, A., Wikman, G., Andreeva, L., Boykova, A., Nikiforova, D., Timonina, N., 2008. Adaptogens exert a stress protective effect by modulation of expression of molecular chaperons. Planta Med. 74, 1018. Panossian, A., Wikman, G., Kaur, P., Asea, A., 2009. Adaptogens exert a stress protective effect by modulation of expression of molecular chaperons. Phytomedicine, 16, 617–622. Panossian, A., Wikman, G., Wagner, H., 1999. Plant adaptogens III: Earlier and more recent aspects and concepts on their modes of action. Phytomedicine 6, 287–300. Panossian, A., Wikman, G., 2008. Pharmacology of Schisandra chinensis Bail.: An overview of Russian research and uses in medicine. J. Ethnopharmacol. 118, 183–212. Panossian, A., Wikman, G., 2009. Evidence based efficacy of adaptogens in fatigue and molecular mechanisms related to their stress protective action. Curr. Clin. Pharmacol. 4, 198–219. Panossian, A.G., 2004. Adaptogens: tonic herbs for fatigue and stress. Altern. Complement. Ther. 9, 327–332. Panossian, A., Wikman, G., 2010. Effects of adaptogens on the central nervous system and the molecular mechanisms associated with their stress-protective activity. Pharmaceuticals 3, 188–224 http://www.mdpi.com/1424-8247/3/1/ 188/pdf. World Medical Association Declaration of Helsinki. Ethical principles for medical research involving human subjects 2000. 18th WMA General Assembly, Helsinki (Finland) June 1964 and 52nd WMA General Assembly, Edinburgh (Scotland), October 2000.