VoI 2
IIIicaGy, tualuafion I $afety&Glinical ry"-N
UKGupta
Comprehensiae
Bioactive Natural Products VoL ZzEfficasSrrsafety& Clinical Evaluation I @Copyright 2010
This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety ofreferences are listed. Reasonable efforts have been made to publish reliable data and information, but the editor and the publisher cannot assume responsibility forthe validity ofall materials or for the consequences oftheir use. All rights are reserved under International and Pan-American Copyright Conventions. Apart from any fair dealing for the purpose of private study, research, criticism or review, as permitted under the Copynght Act, 1956, no part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means-electronic, electrical, chemical, mechanical, optical, photocopying, recording or otherwise-without the prior permission of the copyright owner.
ISBN: 1-933699-52-3 SERIES ISBN : 1-933699-50-7
STI]DII.]M PRESS, LLC P.O. Box-722200,Houston, Texas-77072,USA Tel. 713-541-9400; Fax:713-541-9401 E -mail:
[email protected]
Printed at: Thomson hress (Indid Ltd.
16 Pharmacokinetics of Active Constituents of Rhodiola rosea SHR-5 Extract A. PaNossrax2,*,A. HolueNNrsyANl, H. AenanalrvaNl, E. GmnrnryeNlaNo G. Wrxuan2
ABSTRACT The actiueconstituentsof Rhodola roseaextract SHR-S of the popular plant adaptogenRhodiolq.roseaare tyrosol, rhodioloside q.ndrosauin. Validated capillary electrophoretic methods for the determination of thesecomponents in the blood was deuelopedand used for a pharmacokinetic study in rats and human (n=16) uolunteers.Rhodioloside was found to be quickly and completely absorbed into the blood of rats (bioauailability-75-90Eo), distributed within org&ns and tissues,and rapidly metabolised to tyrosol following oral administration of SHR-5 at dosesof 20 and 50 mg /hg. Many of the measured pharmacokinetic parameters of rhodioloside were significantly different when the pure conlpound was administered rather than the extrdct. The basal leuelof tyrosol in bloodplasma of rats increased following administration of SHR-S as a result both of absorption of free tyrosolpresent in the extract and ofbiotransformation of rhodioloside into tyrosol,which occurred within the first 2 h. The pharmacohinetics and the rate of biotransformation of rhodioloside were essentially the samefollowing single or multiple regimes of administration of SHR-S. Rosauin has low bioauailability (20-267o)and was quickly eliminated from the blood of rats that haue been administered SHR-\. The results of the study in humans showedthat 16 healthy uolunteers,after oral administration of two tablets of Rosenroot,reached a maximum concentration of rhodioloside qnd rosauin in bloodplasma after 2 h, where the absorption rate constant was equal in 1. "Exlab" Expert Analytical Laboratory of Armenian Scientific Centre of Drug and Medical Technology Expertise, Komitas Av e. 49/4, 3 75051 Yerevan, Armenia.
2. Swedish Herbal Institute, Prinsgatan 12, SE-413 05, Gothenburg, Sweden. Corresponding author : E-mail :
[email protected]
Cornp. Bio. Nat. Prod..,VoI.2 -Efficacy, Safety & Clinical Eualuation I
both components.Although, in contrast to rhodioloside, rosauin was not detectedin bloodplasma at 0.5 h and t h after oral administration of two tablets of Rosenroot.The maximum concentrationand elimination half-life of rhodiolosidewas 2-3 x higher than in rosauin.TheAUC, -C *,and AUC,-, ualues of rhodioloside were 3.5, 2.2 and 3.I-fold higher than in rosauin. Thus, the elimination of rhodioloside from the blood was 1.8 fold longer than the eliminationtime of rosauin. Key w ords : Capillary electrophoresis,pharmacokinetic, ft hodiola roseclL., rhodioloside,rosavin, tyrosol, SHR-5 INTRODUCTION Rhodiola roseaisone ofthe most popular plant adaptogensin Russia.Since 7969 Rhodiola rosea has been recommended by the Pharmacological Committee of the Ministry of Health of the USSR for the medicinal use as a stimulant of fatigue in patients experiencing aesthetic states or in healthy peopie with a tendency to asthenisation during their work, which requires high mental exertion or to recover their work capacity during and after long periods of intensive physical work. In addition, Rhodiola rosea is recommendedin casesof borderline nervous-mental diseases,neuroses, neurosis-Iike disorders, and psychopathies.In psychiatric practice, Rhodiola rosea extract is indicated for the correction ofneurological side effects of psychopharmacolo'gicaltherapy and for the intensification and stabilisation of remissions of aesthetic and apathico-abulictype schizophrenia patients (Saratikov et aL,1965; Krasiket aI.,1970; Saratikov, 1973;Mikhailova, 1983;Brichenko et al.,1986; National Pharmacopoeiaof the USSR, 1987; Saratikov & Krasnov 1987). A number of double-blind, placebo-controlledrandomised clinical trials of the standardised extract (SHR-5) of R. roseahave shown that use of the drug can reduce general fatigue (as determined by tests measuring complex perceptive and cognitive cerebral functions) under certain stress conditions by increasing total rnental performance (Darbinyan et a|.,2000), and can signifrcantly improve general well-being, physical fitness, mental fatigue, and coordination in healthy subjects when taken repeatedly for a short period of time (7-10 days) (Spasovet aL.,2000 a,b). A similar effect was observedafter single doseadministration of standardised SHR-5 extract on the capacity for mental work against a background of fatigue and stress on ahighly uniform population comprisingof 161cadets(Shevtsovet a|.,2003). The anti-fatigue effect of Rhodiola SHR-5 extract, as well as improved attention in fatigue and stressful conditions,were earlier observedin patients with chronic stress induced fatigue (Olsson et a|.,2008). Moreover, it has been beneficial in mild and moderate depression(Darbinyan et a|.,2007).
Pharmacohinetic s of Actiue Constituents of Rhodiola rosea
309
The active principles of Rhodiola extracts are tyrosol (syn. salidrosol) [2{4-hydroxyphenyl)ethanol], and the phenolic glycosides,rhodioloside (syz. rhodosin, salidroside) l2{4-hydroxyphenyl)ethyl-1-B-D-glucopyranoside]and rosavin (syz. cinnamyl alcoholB-vicianoside)[3-phenyl-2-propenyl-O{cr-Larabinopyranosyl-(1-6)-9-D-glucopyranosidel (Saratikov et al., 1^gGB; Saratikov, 1973;Barnaulov et al.,1986; Sokolovet a|.,1g90; Linh et al., 2000; Panossianef a|.,2007; Panossianet a1.,2008).In order to determine the contribution of individual componentswith respect to the overall curative effect of an herbal extract, it is necessary to study the pharmacokinetics of the active compounds in the particular preparation, since the presence of even inactive ingredients can influence the bioavailability, absorption, distribution, metabolism and excretion of the active compounds. The aim of the present study was to the investigate the pharmacokinetics of rhodiolosideand rosavin in rats (Panossianet a\.,2002) and healthy human volunteers after single and multiple oral administration of Rhodiola rosea SHR-5 extract-used as an active ingredient of 'Rosenroot @"tablets used in sc andinavia as an adaptogen to increase impaired performance in fatigue and weakness. MATERIALS
AND METIIODS
Chemicals HPLC-grade methanol (Rotisolv@;# 7342.I) and acetone (# 7328.2)were purchased from CarI Roth (Barcelona, Spain); HPCE-grade water @ EAG285780),50 mm borate buffer (pH 9.3;# 5062-8573)and 0.1M sodium hydroxide (# 5062-8575)were from Hewlett-Packard (PaloAlto, CA, USA); ethyl acetate (# 15485-7;1otKI 01058DI) and B-glucuronidase(# G-7770;loL 22K8942) were from Sigma-Aldrich (St Louis, MO, USA); and heparin lock flush solution (containing 500 USP heparin units/ml) was from Abbott Laboratories (Chicago, fL, USA). Unless stated otherwise, the water employed was deionised and distilled. Stud.y d.rug Rhodiola roseaL. roots soft extract SHR-5 (batch No EX 204G8,Swedish Herbal Institute, Sweden),quantified for rhodioloside content, (10 * 2mdg), rosavin (20 t 3 mg/g) and tyr.osol (1.5 r 0.3 mg/g). Rosenroottablets (400 mg, batch Nr L211) contain 144 mg SHR-5, 3.63 mg rhodioloside and 4.2 ng rosavin. Standard Solutions Reference standards stock solution. T A standard solution ofthe reference compounds tyrosol (Swedish Herbal Institute, Goteborg, Sweden; series 22.10.200I),rhodioloside(VILAR, Moscow,Russia;series510402)androsavin (VILAR; series 510402) was prepared by accurately weighing 5.0 mg of each
310
Cotnp. Bio. Nat. Prod., Vol. 2 - Efficacy, Safety & Clinical Euatuation I
compound into a 5 mL volumetric flask, dissolving and adjusting to volume with methanol Stud.y animals Three hundred time-mated male Wistar rats were obtained from the Institute of Fine organic chemistry of the National Academy of science, yerevan, Armenia. All animals were clinically examined upon arrival and any animal showing signs of abnormality or diseasewas replaced before the start of the study. No animals were replaced after the study had commenced.Animals were kept for 10-15days prior to the start of the study und.er al2L2h lighv dark cycle and had free accessto standard rat chow. During the study, a standardised diet for rats (combi/Yerevan, combi-corm plant; for composition, Zapadnyuk et aI., 1988) was provided, but feeding was discontinued before administration of a test substance. only tap water was offered adlib. Throughout the observation period, animals were kept separately in cages (55 x 35 x 25 cm), each consisting of a polystyren" ""se and a Iattice-framed steel lid, and supplied with wood-sawdustbedding. A 12/12 h lighudark cycle was maintained in the animal room throughout the study period, and the temperature and humidity were 22 + 4'c and 40 + Svo,respectively,with 1-2 changes of airlh. Each day, or after completion of work, the floor of the animal room was swept and mopped with an atmospheric biocide-cleanser.Excretions were collectedevery secondday on trays lined with absorbent paper which were suspendedbeneath each cage. D osag e and Administration Dosage,Sampling, Drug Safety Control. The concentrationsof rhodioloside,rosavin and tyrosol in the SHR-5 solutions were analysed by HPCE before their administration to the animals. The administered concentrations were 57.6, 92.5 and 6.5 pglmL,respectively. In addition, two dosesof SHR-5 were used in this study. The 20 mg/hg dose shows maximum antiarrhybhmic and antistressor effect in rats (saralikov, 1973;Sokolovet a|.,1990) and its rhodioloside content was equivalent to a human therapeutic dose (10 drops of Rhodiola extract), which is close to one tablet of "Rosenroot @". The b0 mg/kg dose contained rhodioloside equivalent to a human mean daily dose (2b drops of Rhodiola extract) (saratikov, 1973),which correspondsto two tablets of "Rosenroot@".Three sets of experiments aimed at determining the pharmacokinetics of tyrosol, rhodioloside and rosavin in the blood of animals were performed, whilst two further groups of animals were used to study the biotransformation of the active componentsof SHR-5 (Table 1).
Pharmacohinetics of Actiue Constituents of Rhodiola rosea
311
Table 1. Experimental design of the study of pharmacokinetics and biotransformations following administration of special extract SHR-5 of Rhodiola roseaL. to rats
ii : P f r BT e " b ' -3s!: E5 i*g
E
2
. E E E g ; !E - f El- : Hi $ $ g 3 E g g$ ;5 i 6 :
I E,
HE i ls$E ! ! E E E ! E g;E E;E 63 A F Hi E € E€E55iIi€H *fr.E 1
SHR-s 54
20
Single
I.V.
2
0 . 0 2 6 0 . 2 3 0 0 . 3 9 0 0; 0.06;0.33;0.5;
2
SHR-s 54
20
Single
Oral
10
0.026 0.230 0.390 0; 0.5; 1.0;1.25;1.5;
3
SHR-5 54
50
Single
Oral
10
0 . 0 6 5 0 . 5 7 6 0 . 9 7 5 0; 0.5; 1.0; 1,.25;1.5;
Oral
10
0 . 0 2 6 0 . 2 3 0 0 . 3 9 0 0; 0.5; 1.0; 1.25;
0.75; 1.0;1.5;2.0; 3.0 2 . 0 ;4 . 0 ; 5 . 0 ;6 . 0 2 . 0 ;4 . 0 ; 6 . 0 ;8 . 0 4
SHR-5 48 20/d,aiIy Multiple (5 days) 5 SHR-s 36 50/daily Multiple (5 days) 6 Rhodiolo-42 0.570 Single side 7 Water 42 Single
1 . 5 ; 2 . 0 ; 4 . 06;. 0 ; OraI 10 Oral
l-0
Oral
10
0 . 0 6 5 0 . 5 7 6 0 . 9 7 5 0; 1.0; 1.5;2.0; 4.0; -
0.576
5.0; 0; 0.5; 1.0; I.5; 2.0; 3.0; 5.0 0; 0.5; 1.0; I.5; 2.0; 3.0; 5.0
P harm.ac okinetic Studying Rat s In the frrst set of pharmacokinetic experiments, sHR-5 was administrated intravenously uia the tail vein at a dose of 20 mgkg to 48 animals in group 1(mean weight 112 + 10 g;range 100-120g). Blood samples(4-6 mL each) were collected in heparinised centrifuge tubes immediately before administration of drug and at various times between 0.06 and 3 h after injection (Table 1). Following collection,blood sampleswere centrifuged at 300 x g for 15 min to obtain blood plasma which was stored at -20'C until required for analysis. In the secondset of experiments, SHR-5 was administrated orally to two groups of 48 animals that had been deprived of food for 12 h: free accessto food was provided 2.5 h after the administration of the drug. Animals in group 2 (mean weight 167 t 10 g; range 150-180g) received SHR-5 solution at a dose of 20 mglkg, whilst those in group B (mean weight 120 + 8 g; range 110-130g) received a doseof 50 mg/kg. Blood samples(4-6 mL each) were collected in heparinised centrifuge tubes before administration ald at various times between 0.5 and 6 h after administration (Table 1). In the third set of experiments, oral administration of SHR-S was repeated for 5 days at daily dosesof 20 mg/kg (gfoup 4: mean weight 1b7
3r2
Comp. Bio. Nat. Prod.., VoI. 2 - Efficacy, Safety & Clinical Eualuation I
+ 8 g; range 150-1809) and 50 mg/kg (group 5: mean weight 145 * 10 g; range 130-160 g). Animals were deprived of food for 12 h prior to the administration (at 9.00 pm) of a single daily doseof SHR-5 solution: 2.5 h after drug administration, animals were allowed free accessto food until 9.00 am. Blood samples (4-6 mL each) were collected in heparinised centrifuge tubes before administration and at various times between 1.0 and 5 h after the fifth administration (Table 1). Study of Biotr ansformation
of Rhod.iolo sid.e
In order to study the biotransformation of active compounds, a group of 36 animals (group 6; mean weight 125tg g; range 110-140g) was fasted for l-6 h and rhodioloside was administrated orally at a dose of 0.570 mg/kg. After administration of the drug, only water was supplied during the whole experiment. Blood samples (4-6 mL each) were collected in heparinised centrifuge tubes before administration and at various times between 0.5 and 5 h after administration (Table 1). Hurnan Stud.y Design The study was performed as a blind randomized study, in compliance with the revised declaration of Helsinki (Hong Kong, 1989)and The Ministry of Health in Armenia. Volunteers The clinical part of the study was carried out at the Centre of TraumatologSz, Orthopedics, Burns and Radiology at the Ministry of Health of Armenia and written informed consent was obtained from each subject before the study was initiated. In total, 16 normal and healthy volunteers were scheduled to participate (two groups of 8-male and S-female).All volunteers had been donors at the Centre of Hematology for more than four years and a written anci signed agreement between the head of investigation and the volunteer were included in the study. Prior to the study, each subject underwenta brief physical examination and had blood drawn for serum chemistry and hematology analysis, according to the inclusion/ exclusion criteria. During the study each volunteer received a number and a special code,in compliancewith their sex. Stud.y Schedule The study included the following stages:selection of volunteers, receiving written signed agreements (information for volunteers), medical examination and blood biochemistry and hematology analysis, disposition in groups, drug administration andblood sampling, determination of drug in blood samples, and calculation of pharmacokinetic parameters. The study was provided according to the schemenotified in the investigation protocol.
- : I
Pharrnacokinetic s of Actiue Constitue nts of Rhodiola ro sea
313
Sele ct ion of Volunte er s The volunteer inclusion criteria focussed on healthy males and females aged 28-55, without allergenic anamnesis or illnesses, such as chronic diseases of the kidney, Iiver, cardiovascular and nervous system. In addition, the volunteers have to have signed €rnagreement written by the head of the investigation. Finally, they needed to be able to collaborate adequately during the entire length of the study. The volunteer exclusion criteria concerned volunteers suffering from chronic illnesses, such as cardiovascular diseases,diabetes(etc.), and arterial hypotension (etc.). In addition, volunteers addicted to, for example, medicines, narcotics and tobaccowere also excluded. Finally, they were not allowed to participate in any other trials. In addition, volunteers were removed from the investigation ifthey experienced an individual reaction, adverse reactions, serious adverse events or if their health and physical condition deteriorated. The Dates ofVolunteers
Enrolled
in the Stud.y
Bearing in mind the expected intra-individual variability, 16 healthy volunteers (caucasians), which had not received. any drugs in the past month prior to this study were enrolled. The volunteers, aged 28-55, were both female (mean age 4I.87 + 11.6) and male (mean age 45.50+ 6.9). Dosage, Blood Sampling
and Safety Control
D o sag e and Blo od, Samp ling The subjects were fasting during one night and then received two tablets of Rosenroot with 200 mL water two h before breakfast at 9 a.m. The standard breakfast time was at 9.80 a.m. - 10.00a.m., while the standard dinner time was 15.30-16.00.The period measured was 10 h. In addition, the subjects agreed to refrain from the use of other prescription or nonprescriptiondrugs (including vitamins), alcohol and coffeeduring the entire study period. At the end of the study, each subject underwent a brief physical examination and had blood drawn for serum chemistryand hematology analysis. The volunteers were all Armenian and live in the capital Yerevan. A 20-gauge heparinised catheter was inserted into a vein of the forearm to collect blood samples in a dilute heparin solution (10 to 15 u/ mL). TWo millilitres ofblood was drawn and discarded prior to collection of each blood sample (6-8 mL) into heparinised tubes. These tubes were centrifuged at 300 g for 15 min to prepare plasma and stored at -20oC until further analysis. For each volunteer, a set of blood samples (6-g mL each) were drawn before(0) administration and at 0.5, 1.0, 1.8,2.0,9.0,4.0, 6.0, 8.0 and 10.0h thereafter.
31,4
Comp. Bio. Na,t. Prod.., VoL 2 - Efficacy, Safety & Clinical Eualuation I
Safety Control Drug safety was rated from physical examination, measurement of blood pressure, heart rate, and laboratory examination, such as hematology (leukocytes, erythrocytes, and hemoglobin), biochemistry and documentation of adverse events (AE), which were asked and recorded by the clinical staff every h after drug intake. The classification of the AE's was in accordancewith the EC-guideline Ill/3445/91-EN and termed as mild, moderate and severe. In addition, the causes were classifred as definite, probable, possible,unlikely, not related and not possibleto judge. HPCEAnalyses In order to analyse rhodioloside and rosavin in blood, 1.5 mL of acetone was added to 1.5 mL of the plasma sample, the mixture was vortex-mixed, a further 5 mL of acetone added, the whole vortex-mixed again and the proteins precipitated at 4"C by centrifugation at 2000 x g for 15 min. The supernatant was removed, evaporated to dryness al 40'C using a vacuum rotary evaporator and the residue dissolved in 100 mL of methanol, transferred into HPCE vials and subjected to analysis. For the analysis of tyrosol in blood, 1.0 mL of methanol was added to 1.0 mL of the plasma sample, the mixture was vortex-mixed, a further 3.0 mL of methanol added, the whole vortex-mixed and the proteins precipitated at 4"C by centrifugation at 2000 x g for l-5 min. The supernatant was removed and treated exactly as describedabove. For the analyse of tyrosol in urine, ca. 50 pl of B-glucuronidase solution was added to 1.0 mL of urine sample, the whole mixed vigorously for 30 s and then incubated at 55'C for 3 h. After incubation, the mixture was purified uio solid phase extraction by applying the sample to Supelclean LC-18 SPE tubes (Supelco,Belefonte,PA, USA; 3 mL; lot SP1662A),which had been pre-washed with 1 mL of methanol and 1 mL ofwater, and eluting with 3 mL each of water: methanol mixtures of compositions 95:5, 85:15 and 50:50. The second and third eluents were evaporated to dryness at 50'C using a vacuum rotary evaporator: the residue was dissolved in 1.0 mL of acetate buffer (pH 5.2), 5.0 mL of ethyl acetate added, the whole vortex-mixed for 60 s, and the two layers separated. The organic phase was evaporatedto dryness at 50'C using a vacuum rotary evaporator, the residue was dissolvedin 100 ml of methanol and subjectedimmediately to HPCE analysis. Analyses were carried out using a Hewlett Packard HPCE system comprising a model HPBDCE apparatus interfaced to a HP Kayak XA workstation and a HP Laser Jet 4000 printer, and equipped with a HP fused silica capillary (# G 1600-61232; tohal length 56 cm; effective length 50 cm; i.d. 50 mm; optical path length 150 mm) maintained at 10"C. The column was preconditioned by flushing for 1.0 min. with 0.1 M sodium hydroxide, rinsingwith water, and then flushingwith mobile phase [50 mM
Pharmacokinetics of Actiue Constituents of Rhodiola rosea
*
U
f
\
315
u
g
h'r
X
{ }
a *
\ a , r ' & q #
o F > = o q
6 = E I P-b t- E i p
AD
tr a€
--1'
?:'r = t
| A
t F I lrg-, : g
d(E o * r o 9 6 9 H d
H ' -
i i j tr d . = =
6
k h
;
E r.;i ^ ooq
s c - , E - X tr O.n Iot.! 6t - ^ i =i
. ! a> - cq , >
xr
8 rr I I
BS r; I
t
r
t
y
dro
o o 9 @ : 0 ) F{ O':
i- oq) -ia
a
8E +r Z,6 c\' o l E H a 5 *
E * c o = F
iiO*o'
$€dF l E c o o H tr@ rl
H9; < b R b.o * F F F 6"a cd
L ri
E.r-' o ?v
U; fi P
t r a . gE j b0
316
Comp. Bio. No;t. Prod.,Vol.
2 -Efficacy, Safety & Clinical Eualuation I
borate buffer (pH 9.3) : methanol in the ratio 85:15 (v/v) for rhodioloside and rosavin, and 60:40 (v/v) for tyrosoll for 2 min. Injection was at b0 mbar for 4 s, the running voltage was 25 kV, detection was at20L nm (reference 300 nm), 25Lnm (reference450 nm) and275 nm (reference400 nm), and the run time was 15 min. The capillary was washed for b min. after very frfth run and the mobile phase replaced. Fig 1 shows a capillary electropherogram of rhodioloside and rosavin (migration times 11.86 t 0.06 and 12.39 x.0.06 min, respectively), whilst Fig 2 depicts the electropherogram for tyr.osol (migration Lime L2.7g t 0.10 min). Calibration curves for all analytes were linear in the range 2.80 125.00 mg/l with correlation coefficients of 0.9g86, 0.gg\2 and 0.9984, respectively, for tyrosol, rhodioloside and rosavin. The limit of detection for aII analytes was 1.0 mg/l (at a signav noise ratio of 3) and the limit of quantifrcation was ca. 2.5 mgA. The accuracy w as g8.5g7ofor rhodioloside, 97.20vofor rosavin and97.34vofor tyrosol. The recoverieswere 90.38 and 93.82Vo,respectively, for rhodioloside and rosavin from blood samples, and 86.5 and 99.\Vo,respectively, for tyr.osolfrom blood and urine samples. The repeatability for all ana\rtes was ca. 947o,and the relative standard deviation (RSD) value was established at
