Effect of commercial Rhodiola rosea on

CYP enzyme activity in humans

European Journal of Clinical Pharmacology

Ole Kristian Thu*, Olav Spigset**, Odd Georg Nilsen* and Bent Hellum*

*Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway

** Department of Clinical Pharmacology, St. Olav University Hospital, Trondheim, Norway, and Department of Laboratory Medicine, Children’s and Women’s Health, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway

Short title:CYP interaction by Rhodiola rosea in humans

Corresponding Author:

Ole Kristian Forstrønen Thu

Norwegian University of Science and Technology (NTNU)

Faculty of Medicine,

Department of Cancer Research and Molecular Medicine,

Gastro Center,

St. Olav University Hospital

7006 Trondheim

Norway

E-mail:

Telephone: +47 90 56 56 72

Supplementary Table 1. Overview of parameters for the mass spectrometrysettings

Analyte / Q1 / Q3
[m/z] / Dwell time [ms] / DP [V] / EP [V] / CE [V] / CXP [V]
Caffeine / 195 / 138.3 / 100 / 40 / 10 / 27 / 12
Paraxanthine / 181 / 124.4 / 100 / 46 / 10 / 21 / 8
Losartan / 423.2 / 207.2 / 100 / 101 / 10 / 31 / 12
EXP-3174 / 437.2 / 235 / 100 / 111 / 10 / 25 / 14
Omeprazole / 346.2 / 198.1 / 100 / 126 / 10 / 33 / 16
5-OH-omeprazole / 362.2 / 214.1 / 100 / 46 / 10 / 17 / 12
Dextromethorphan / 272.3 / 147 / 100 / 81 / 10 / 41 / 14
Dextrorphan / 258.3 / 199 / 100 / 81 / 10 / 37 / 14
Midazolam / 326.2 / 291.1 / 100 / 140 / 10 / 40 / 8
1-OH-midazolam / 342.2 / 203.2 / 100 / 131 / 10 / 37 / 10
Propranolol (IS) / 260.3 / 116.2 / 100 / 111 / 10 / 25 / 12

IS = internal standard

Q1 / Q3 = quadrupole 1 / quadrupole 3

Dwell time = time system searches for each mass, each cycle

DP = declustering potential

EP = entrance potential

CE = collision energy

CXP = collision cell exit potential

Supplementary Table 2. Calibration curve linear ranges for each analyte, values for inter-day accuracy and precision (n = 6)

Analyte / LOQ (ng/ml) / Linear range
(ng/ml) / Inter-day precision
(%CV) / Inter-day accuracy
(%)
Caffeine / 5.0 / 5.0 – 500 / 4.6 – 9.6 / 93.0 – 97.3
Paraxanthine / 5.0 / 5.0 – 250 / 7.3 – 12.7 / 100.3 – 104.1
Losartan / 2.5 / 2.5 – 40 / 4.8 – 11.9 / 89.9 – 97.4
EXP-3174 / 2.5 / 2.5 – 40 / 5.3 – 13.6 / 91.0 – 102.6
Omeprazole / 2.5 / 2.5 – 60 / 4.7 – 5.8 / 96.8 – 106.8
5-OH-omeprazole / 2.5 / 2.5 – 300 / 9.4 – 12.9 / 99.3 – 103.9
Dextromethorphan / 0.1 / 0.1 – 1.5 / 6.6 – 10.7 / 95.4 – 98.4
Dextrorphan / 0.1 / 0.1 – 4.0 / 6.4 – 11.5 / 94.1 – 102.1
Midazolam / 0.05 / 0.05 – 2.0 / 8.1 – 11.2 / 87.1 – 108.6
1-OH-midazolam / 0.05 / 0.05 – 1.8 / 9.1 – 14.0 / 98.3 – 108.7

LOQ = limit of quantitation

CV = coefficient of variation

Supplementary information – analytical method and and concentrations of bioactive ingredients in the Rhodiola rosea product “Arctic root”

The bioactive constituents of R. rosea are suggested to be salidroside, tyrosol and rosavins (rosavin, rosin and rosarin). The rosavins areusually used in quality assurance as these constituents are specifically found in R. rosea [1-3]. The constituents in “Arctic root” were analyzed using a slightly modified methodology described previously[4].

Extraction

An “Arctic root” tablet of an identical batch as used in the study was weighed, crushed to powder using a mortar and extracted with 15 ml 50% ethanol in water at 37°C with constant stirring for one hour. The extraction solution was transferred to a 20 ml falcon tube and centrifuged at 3.000 rpm for 10 min and decanted into a new container. The product residue was added 5 ml of 50% ethanol and the extraction process was repeated. The first and second extract was pooled.The pooled herbal extract solution was evaporated to dryness at 40°C under a gentle stream of air overnight and weighed. Dried extract was stored in falcon tubes at 4°C and dissolved in 50% ethanol, not earlier than one day before analysis. A stock solution was made with a concentration of 25 mg crude extract / ml extraction solvent.

Analysis

An Agilent 1200 HPLC system was used and the constituents were separated with a Zorbax Eclipse XDB C18column (5 µm, 150 mm x 4.6 mm), connected after a Zorbax SB-C18 guard column (5 µm, 12.5 mm x 4.6 mm), at 30ºC. UV detection at 250 nm was used for rosavin, rosin and rosarin, whereas salidroside and tyrosol were detected at 210 nm. Flow was 1 ml/min with a sample injection volume of 10 µl Mobile phase was water (A) and MeOH (B). Gradient: initially 20%B, after 5 min B was increased to 60% over 9 min, hold for 1 min, and at 15 min B was ramped to 80%. After 18 min, B was decreased to 20% and the system was allowed two min to equilibrate. Total run time was 20 min, retention timeswere: salidroside 5 min, tyrosol 6.5 min, rosarin 13.2 min, rosavin 13.6 min and rosin 14 min.

Linear standard curves were constructed for each constituent for all analytical series. Standard curve range was 0.1 µg/ml – 10 µg/ml (7 points, r2 >0.998) with quality controls (QCs)as appropriate. LOQ was 0.1 µg/ml for tyrosol and 0.5 µg/ml for the other analytes. Intra- and inter-day CVs were less than 14% at the LOQ concentrations and less than 10.3% for the QCs. The overall inaccuracy was less than 11%.

The concentrations found are shown in the table below.

Constituent / Concentration [mg/g extract]
Rosavin / 7.99
Rosin / 2.32
Rosarin / 3.10
Salidroside / 8.42
Tyrosol / 1.27

References

1. Kucinskaite A, L P-O, M K-B, M S, A S, V B (2007) Evaluation of biologically active compounds in roots and rhizomes of Rhodiola rosea L. cultivated in Lithuania. Medicina (Kaunas) 43:487-494

2. Panossian A, Wikman G, Sarris J (2010) Rosenroot (Rhodiola rosea): traditional use, chemical composition, pharmacology and clinical efficacy. Phytomedicine 17 (7):481-493. doi:10.1016/j.phymed.2010.02.002

3. Panossian A, Wagner H (2005) Stimulating effect of adaptogens: an overview with particular reference to their efficacy following single dose administration. Phytother Res 19 (10):819-838. doi:10.1002/ptr.1751

4. Hellum BH, Tosse A, Hoybakk K, Thomsen M, Rohloff J, Georg Nilsen O (2010) Potent in vitro inhibition of CYP3A4 and P-glycoprotein by Rhodiola rosea. Planta Med 76 (4):331-338. doi:10.1055/s-0029-1186153

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