Comparative phytochemical characterization of three Rhodiola species

Article abstract of DOI:10.1016/j.phytochem.2006.07.026

In comparison to the well-recognized adaptogenic herb Rhodiola rosea, phytochemical constituents of two other Rhodiola species (R. heterodonta and R. semenovii) were elucidated and characterized. Two major phytochemical groups; phenolic and/or cyanogenic glycosides and proanthocyanidins, were isolated and identified in the three species. Chemical similarities among the three species were observed; however, each species displayed differences in phytochemical constituents. R. heterodonta contained a newly detected phenylethanoid glycoside, heterodontoside, in addition to the known compounds tyrosol, viridoside, salidroside, and rhodiocyanoside A. Both R. heterodonta and R. rosea contained phenylethanoid/propanoid compounds that were not detected in R. semenovii. For R. semenovii, the cyanogenic glucosides rhodiocyanoside A and lotaustralin were detected. Although the three species have proanthocyanidins composed of (-)-epigallocatechin and its 3-O-gallate esters in common, the degree of polymerization greatly differed between them. In contrast to R. heterodonta and R. semenovii, R. rosea has higher molecular weight polymeric proanthocyanidins. This study resulted in the identification and isolation of phytochemical constituents for direct cross-comparison between three Rhodiola species of medicinal and pharmacological value.

Full text of DOI:10.1016/j.phytochem.2006.07.026

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 2380–2391
www.elsevier.com/locate/phytochem
Comparative phytochemical characterization of three Rhodiola species
a
a
a
b
Gad G. Yousef , Mary H. Grace , Diana M. Cheng , Igor V. Belolipov ,
c
a,
*
Ilya Raskin , Mary Ann Lila
a
Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801, USA
b
Tashkent State Agrarian University, Tashkent, Uzbekistan
Biotech Center, Cook College, Rutgers University, New Brunswick, NJ 08901, USA
c
Received 30 March 2006; received in revised form 1 June 2006
Available online 7 September 2006
Abstract
In comparison to the well-recognized adaptogenic herb Rhodiola rosea, phytochemical constituents of two other Rhodiola species (R.
heterodonta and R. semenovii) were elucidated and characterized. Two major phytochemical groups; phenolic and/or cyanogenic glyco-
sides and proanthocyanidins, were isolated and identified in the three species. Chemical similarities among the three species were
observed; however, each species displayed differences in phytochemical constituents. R. heterodonta contained a newly detected phenyl-
ethanoid glycoside, heterodontoside, in addition to the known compounds tyrosol, viridoside, salidroside, and rhodiocyanoside A. Both
R. heterodonta and R. rosea contained phenylethanoid/propanoid compounds that were not detected in R. semenovii. For R. semenovii,
the cyanogenic glucosides rhodiocyanoside A and lotaustralin were detected. Although the three species have proanthocyanidins com-
posed of (ꢀ)-epigallocatechin and its 3-O-gallate esters in common, the degree of polymerization greatly differed between them. In con-
trast to R. heterodonta and R. semenovii, R. rosea has higher molecular weight polymeric proanthocyanidins. This study resulted in the
identification and isolation of phytochemical constituents for direct cross-comparison between three Rhodiola species of medicinal and
pharmacological value.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Rhodiola heterodonta; Rhodiola semenovii; Rhodiola rosea; Crassulaceae; Heterodontoside; Phenylethanoid glycosides; Cyanogenic glucosides;
Proanthocyanidins; Prodelphinidins; EGCG; EGC; HPLC-ESI-MS
1
. Introduction
(Tolonen et al., 2003a). While phenylethanoid derivatives
including salidroside (3) (rhodioloside) (Fig. 1) were previ-
ously used exclusively to standardize medicinal prepara-
tions of R. rosea extracts, it is now believed that a variety
of co-occurring phytochemical constituents in the plant
(including the phenylpropanoids [e.g. rosavins] and possi-
bly terpenoids and flavonoids) may be responsible for its
unique pharmacological activity (Brown et al., 2002). The
phytochemical constituents in Rhodiola are species-depen-
dent, and the predominant species screened in efficacy stud-
ies has been R. rosea, although salidroside (3) production
in other species including R. sachalinensis, R. kirilowii,
and R. crenulata has also been reported (Kurkin and
Zapesochnaya, 1986; Wu et al., 2003).
Rhodiola rosea L. (golden root), historically used as an
adaptogen in Russia, northern Europe, and in China as a
traditional herbal medicine, is valued for its ability to
enhance human resistance to stress or fatigue, and promote
longevity (Spasov et al., 2000; Kurkin and Zapesochnaya,
1
986; Tolonen et al., 2003a). Golden root phytochemical
extracts are the source of important commercial prepara-
tions widely used throughout Europe, Asia, and more
recently in the USA, with biological activities including
antiallergenic and anti-inflammatory effects, enhanced
mental alertness, and a variety of therapeutic applications
*
Two central Asian Rhodiola genotypes (R. heterodonta
and R. semenovii), Crassulaceae family, have similar rich
Corresponding author. Tel.: +1 217 333 5154; fax: +1 217 244 3469.
E-mail address: imagemal@uiuc.edu (M.A. Lila).
0
031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.07.026

G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
2381
O
O
HO
HO
2
3
OH
O
7
R O
OR1
HO
HO
O
OMe
2
1
4
OH
8
6
5
R =R =H, Tyrosol (1)
1
2
R =CH , R = Glucose, Viridoside (2)
Heterodontoside (5)
1
3
2
R =H, R = Glucose Salidroside (3)
1
2
H
CH3
C
N
OH
OH
CH3
O
O
O
O
N
HO
HO
HO
HO
CH CH
2
3
OH
OH
Rhodiocyanoside A (4)
Lotaustralin (6)
OH
OH
OH
OH
OH
OH
HO
O
HO
O
OH
OH
OH
OH
OH
S
O
O
OH
OH
OH
(
-)-Epigallocatechin-3-O-gallate
(-)-EGCG-4b-benzylthioether (7a)
(
EGCG, 7)
Fig. 1. Isolated and characterized phenolic and cyanogenic glycosides 1–6 and proanthocyanidin basic unit 7 and its benzylthioether adduct (7a) from the
three Rhodiola species; R. heterodonta, R. semenovii, and R. rosea.
ethnobotanical histories and are locally valued as adapto-
gens, but have not been well investigated outside of the
former Soviet Union. R. heterodonta, indigenous to Uzbe-
kistan, has been found to contain salidroside (3) (rhodiol-
oside) in common with golden root, and its extract is
included, along with extracts of other Rhodiola species, in
a medicinal preparation (carpediol) used to treat depres-
sion (Krasnov et al., 1976; Wikman and Panossian,
processors. The objectives of this study were to compare
the phytochemical constituents of the two central Asian
Rhodiola species (R. heterodonta and R. semenovii) to the
well-characterized golden root (R. rosea), using freshly
harvested live rhizomes and standardized reproducible
extraction and separation methods, in order to examine
the inherent constituents potentially responsible for the
above mentioned metabolic-enhancing properties. All
three species were characterized in terms of phenylethanoid
and/or cyanogenic glycosides as well as proanthocyanidin
content.
2
003). R. semenovii, indigenous to Kyrgyzstan and locally
valued for endurance-enhancing and anti-hypoxic proper-
ties, contains dimeric and oligomeric proanthocyanidins
(
Kuliev et al., 2004; Matamarova et al., 1999) which have
been linked to the hypolipidemic, hypocholesteremic, and
anti-inflammatory properties of extracts from this plant.
R. semenovii was reclassified by some Russian authors as
a separate genus (Clementsia semenovii), because it did
not contain detectable levels of tyrosol (1), salidroside
2. Results and discussion
2.1. Identification of phytochemicals in Rhodiola species
(
2), and herbacetin characteristic of other members of Rho-
Two distinct groups of compounds, which have recog-
nized pharmacological activities were isolated and identi-
fied from the three Rhodiola species. The first group
included phenolic and/or cyanogenic glycosides with anti-
allergy, anti-inflammatory, anti-anoxia, anti-fatigue, hepa-
toprotective, and cognitive-enhancing properties (Brown
et al., 2002; Darbinyan et al., 2000; Diaz-Lanza et al.,
2001). The second group was proanthocyanidins, which
were noted for significant bioactivities including anti-
oxidant, anti-cancer, anti-inflammatory, anti-allergic,
anti-mutation, anti-aging and improving liver function
diola (Kurkin et al., 1986).
In previous studies, complete phytochemical character-
ization of, and cross-comparison between, various species
has been complicated by the intermittent use of dried,
powdered and/or pulverized plant preparations for analy-
sis. Although the natural isomeric forms, functional
groups, and bioactivity of many phytochemical compo-
nents may be altered during extraction and processing, in
many cases the methods used to prepare herbal extracts/
dried preparations are unknown or vary widely between

2382
G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
(
Demeule et al., 2002; Gross, 2004; Kris-Etherton and
and spectroscopic data (NMR and HPLC-ESI-MS), litera-
ture, and comparison to spectra for commercial standards
of compounds 1 and 3.
Keen, 2002; Marchand, 2002; Takahata et al., 2001).
When chromatographically fractionating the 70% aq.
acetone crude extract using either silica gel or Sephadex
LH-20 columns, phenolic and cyanogenic glycosides along
with the lipophylic materials were eluted in the early frac-
tions. The proanthocyanidins with higher polarity and
molecular weights were detected in the later fractions,
Heterodontoside (5), purified as a white powder, had a
molecular formula of C H O according to its HRESI/
2
0
31 11
TOFMS molecular ion peak observed at m/z 447.1875,
1
13
1
and its H and C NMR spectroscopic data. The
H
NMR spectra (Table 1) exhibited two signals in the aro-
matic region at d 7.16 (d, J = 8.5 Hz, H-2, 6) and 6.82 (d,
J = 8.5 Hz, H-3, 5) indicating a para-substituted phenyl
ring. A singlet observed at d 3.74 integrated for three pro-
tons is characteristic for an O-methyl group located at posi-
tion 4 of the phenyl ring. Two anomeric protons were
which eluted with a mixture of MeOH–acetone–H O.
2
Applying vacuum liquid chromatography to the 70% aq.
acetone crude extract using a silica gel column resulted in
a wide range of separated fractions (22 fractions) that facil-
itated characterization of the compounds using TLC and
HPLC-ESI-MS, whereas using a Sephadex LH-20 column
separated this crude extract into two distinct chemical
groups; the phenolic and/or cyanogenic glycosides, and
proanthocyanidins.
0
overlapped at d 4.29 (d, J = 7.8 Hz, H-1 and d, J =
00
6.8 Hz, H-1 ). Chemical shifts and coupling constants of
sugar residues showed that they were b-glucopyranose
and b-xylopyranose. The complexity of the upfield signals
1
1
was resolved by H– H COSY spectrum of 5 (Table 1).
1
3
2
.2. Phenolic and cyanogenic glycosides
The C-chemical shifts of the sugar moieties demon-
0
strated that xylose is attached at the 6 position of glucose.
Subsequent separation of the phenolic glycoside mixture
This was shown from the downfield shift of this carbon (dc
collected from a Sephadex LH-20 column for R. hetero-
donta (Fr. 3–11) by column chromatography on silica gel
afforded 2-(4-hydroxyphenyl)ethanol (tyrosol, 1), 2-(4-
methoxyphenyl)ethyl-b-D-glucopyranoside (viridoside, 2),
68.2) compared to viridoside (2) (d 61.5). The proposed
c
structure for 5 was further confirmed by comparing its
1
3
C NMR spectrum with that of the monosaccharide (2)
(Table 1) and other related and reported compounds (Bis-
set et al., 1989). Acid hydrolysis (HCl) afforded p-meth-
oxyphenylethanol, D-glucose and D-xylose. The sugars
were identified by co-chromatography with standard
samples. The optical rotation of the hydrolyzed sugars
indicated that both are in the D-configuration. Acetylation
2
-(4-hydroxyphenyl)ethyl-b-D-glucopyranoside (salidroside
or rhodioloside 3), and rhodiocyanoside A (4) (Fig. 1). The
identification of these compounds was consistent with pre-
vious reports (Golovina and Nikonov, 1988; Nishimura
et al., 1990; LaLonde et al., 1976; Yoshikawa et al.,
1
996). In addition to the above compounds, a new phenyl-
ethanoid glycoside designated ‘‘heterodontoside’’ (5)
Fig. 1) was identified. The isolated, previously reported
of 5 (Ac O, pyr, 25 ꢁC) gave the hexaacetate which was
2
1
13
identified by its H and C NMR spectra and by ESIMS,
(
in which exhibited molecular ion peaks at m/z 721 [M +
+
+
compounds were identified according to their chemical
Na] and 716 [M + NH4] corresponding to a molecular
Table 1
1
13
a
H and C NMR spectroscopic data of two phenolic glycosides; viridoside (2) and heterodontoside (5) isolated from R. heterodonta
Position
Viridoside (2)
Heterodontoside (5)
1
13
1
13
H NMR
C NMR
H NMR
H-H-COSY
C NMR
1
–
130.7
129.7
113.5
158.4
35.1
70.8
54.4
103.1
73.9
76.7
70.4
76.7
61.5
–
–
–
130.8
129.8
113.6
158.4
35.1
70.9
54.4
103.2
73.8
75.7
70.3
76.7
68.2
103.9
71.1
72.9
68.3
65.5
2
3
4
7
8
,6
,5
7.17 (d, J = 8.7)
6.82 (d, J = 8.5)
–
2.86 (t, J = 7.4)
4.04 (dd, J = 15.0, 7.5)
3.75 (s)
4.29 (d, J = 7.8)
3.18 (d, J = 7.8)
3.25–3.35 (m)
3.25–3.35 (m)
3.25–3.35 (m)
7.16 (d, J = 8.7)
6.82 (d, J = 8.5)
–
2.85 (t, J = 7.4)
4.02 (dd, J = 14.8, 7.4), 3.72 (m)
3.74 (s)
4.29 (d, J = 7.8)
3.18 (d, J = 7.8)
3.34 (t, J = 7.8)
3.30 (m)
3,5
2,6
–
8
7
OMe
1
2
3
4
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1 ,3
2
0
3.43 (m)
0
6
3.86 (dd, J = 11.8, 1.8)
4.08 (dd, J = 11.3, 1.8), 3.72 (m)
4.29 (d, J = 6.7)
3.57 (d, J = 6.9)
3.48 (m)
3.78 (m)
3.84 (dd, J = 12.4, 3.1), 3.42 (m)
6
2
0
0
0
0
0
00
1
2
3
4
5
–
–
–
–
–
00 00
–
–
–
–
1 ,3
00
00
2 , 4
00
3
00
5
a
1
13
H and C NMR spectra were acquired in CD
3
OD at 500 and 125 MHz, respectively; TMS was used as internal standard; chemical shifts are shown in
the d scale with J values (Hz) in parentheses.

G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
2383
formula of C H O . From these structural elements we
ESI positive-ion mode spectra for the three species (Fr.
14, Sephadex LH-20) are shown in Fig. 2. A series of singly
3
2
42 17
conclude that heterodontoside (5) is a phenylethanoid
disaccharide and defined as 2-(4-methoxyphenyl)-ethyl-b-
O-D-glucopyranosyl-6-b-O-D-xylopyranoside (Fig. 1).
Re-combined early fractions of R. semenovii (Fr. 3–11),
fractionated nearly the same way as for R. heterodonta,
afforded the two cyanogenic glucosides; rhodiocyanoside
A (4) and lotaustralin (6) (Fig. 1). They were identified
from their physical, chemical and spectroscopic data
+
charged ions [M + 1] , m/z 459, 611, 763, 915, 1067, 1219,
+
1371, 1524 [M + 2] , 1675, 1827, and 1979 were detected;
all of these were consistent with molecular masses of single
carbon-carbon linkages. The most abundant ion at m/z 763
was consistent with a molecular mass of heterogenic
dimeric prodelphinidin, suggesting one unit of epigallocate-
chin linked to one unit of epigallocatechin gallate by a sin-
gle carbon–carbon bond (B-type). These results were
consistent with previous reports on R. semenovii (Mata-
marova et al., 1999; Kuliev et al., 2004). Data also showed
similar proanthocyanidin contents for all three species;
dimers, trimers, tetramers, and pentamers of heterogenic
constitution as well as homogenic oligomers, but with dif-
ferent proportional compositions. The R. rosea proantho-
cyanidin fraction showed different spectra compared to
the other two species, where the homogenic oligomeric pro-
delphinidin gallates were the predominant forms of pro-
anthocyanidins. This was clear from the relative
(
NMR and HPLC-ESI-MS). Lotaustralin (6) was previ-
ously reported in R. rosea (Akgul et al., 2004).
The phenolic glycoside mixture of R. rosea obtained
from the early fractions of the Sephadex LH-20 column
(
Fr. 1–11) was characterized by HPLC-ESI-MS. Salidro-
side (3), rosin (8), rosarin (9), and rosavin (10) were identi-
fied according to their mass units and by comparing their
retention times with commercial standards and reported lit-
erature (Tolonen et al., 2003a,b).
2
.3. Proanthocyanidins
+
abundance of the peaks at m/z 915 [M + 1] for EGCG
1
13
H and C NMR spectra (acetone-d ) of the early pro-
+
dimer, m/z 1371 [M + 1] for EGCG trimer and m/z
6
+
anthocyanidin fraction (Fr. 12) of R. heterodonta showed a
simple spectrum for epigallocatechin-3-O-gallate (EGCG).
1827 [M + 1] for EGCG tetramer (Fig. 2). HPLC-ESI-
MS data provided no evidence for the absolute and relative
orientation of the many chiral centers or the location of the
interflavanyl bond.
1
H NMR showed two sharp singlets at d 7.03 and 6.63,
each integrated for two protons, characteristic for gallate
moiety and pyrogallol B-ring substitution, respectively.
Also signals at d 3.04 (1H, dd, J = 17.1, 4.5 Hz, H-4b)
and 2.91 (1H, dd, J = 17.1, 2.0 Hz, H-4a), in addition to
the small coupling constant (J2,3 < 2.0 Hz) confirmed a rel-
ative 2,3-cis configuration. The downfield shift of H-3 at d
1
The H NMR spectra of the proanthocyanidin fractions
12, 13, 14, and 15 from R. rosea displayed a complex pat-
tern with broad signals, indicating highly polymerized pro-
anthocyanidins.
A
subsequent fractionation of the
combined fractions (Fr. 12–15) on a Sephadex LH-20 col-
umn afforded a purified polymeric proanthocyanidin frac-
tion, obtained after freeze-drying as a light-brown
5
.56 indicated the attachment of the galloyl moiety at this
1
3
position. The C NMR spectrum was consistent with the
1
25
D
13
H NMR spectrum, where it showed peaks at d 106.8
powder, ½aꢁ +86.4 (c 2.14 MeOH–H O 1:1). The
C
c
2
0
0
00
00
(
C-2 , 6 ) of ring B and 110.0 (C-2 , 6 ) of the gallate ester
moiety. Also, the upfield chemical shift of C-2 and C-3 (dc
8.0 and 69.3), and the downfield shift of C-4 (d 26.6) con-
NMR spectrum (acetone-d -D O 1:1 v/v) (Fig. 3) showed
6 2
a sharp peak at d 110.0 indicating a gallate functionality,
c
0
0
7
and at d 106.3 characteristic for 2 , 6 nonoxygenated car-
c
c
firmed the 2,3-cis configuration. The 2R, 3R configuration
bons of the pyrogallol B-rings. These two prominent peaks
indicated the presence of prodelphinidin gallate esters
(Porter et al., 1982). The presence of weak signals around
115.5 ppm indicated also the presence of some units in
the polymer with a dihydroxy B-ring substitution (epicate-
chin-gallate esters, ECG). The upfield resonances at dc
33.0–36.0 and 26.0 ppm were attributable to the C-4 of
the extender and terminal flavanoid units, respectively.
The flavonoid units were predominantly in the 2,3-cis
configuration as indicated by the upfield resonance of the
heterocyclic ring carbons around dc 76 (C-2) and the
absence of the corresponding downfield signal for any trans
ꢂ
5
2
was based on the negative ½aꢁ value (ꢀ122, acetone, c
D
1
13
5
.0). Both H and C NMR spectra were identical to the
corresponding spectra obtained from the commercial stan-
dard and consistent with the reported data for (ꢀ)-EGCG
1
(
(
Davis et al., 1996). The H NMR spectra of later fractions
Fr. 13–15) were difficult to interpret where they showed
broadening and complexity of signals; however, the prom-
inent peaks of the gallate moiety and trihydroxy substitu-
tion of the B-ring were clearly recognized in the spectra.
The composition of the proanthocyanidin fractions (Fr.
1
2–15) was also investigated by HPLC-ESI-MS in the posi-
tive ion mode. This soft ionization technique usually gives
ion peaks making it useful for molecular mass determina-
tion (Guyot et al., 1997). Fraction 12 of both R. hetero-
donta and R. semenovii showed the presence of only one
isomer (d 84) (Porter et al., 1982; Haslam, 1989). The
c
results also suggest that the terminal unit was predomi-
nantly 3-O-galloylepigallocatechin as indicated by the
absence of resonances in the 27–29 ppm region and the
presence of the characteristic upfield C-4 chemical shift at
about 26.0 ppm. Also the C-3 resonance of the terminal
unit was clearly at 69.5 ppm indicating a flavan-3-O-galloyl
terminal unit (Foo et al., 1996). This was further confirmed
+
peak with m/z 459 [M + 1] corresponding to EGCG, in
agreement with its NMR spectroscopic data. Later frac-
tions from both plant extracts showed mixtures of oligo-
meric and polymeric proanthocyanidins. Representative

2384
G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
Fig. 2. ESI positive-ion mode mass spectra of proanthocyanidin fraction 14, Sephadex LH-20, from the three Rhodiola species. epigallocatechin (EGC),
epigallocatechin gallate (EGCG), and degree of polymerization (DP).
by thiolysis degradation (see below) of proanthocyanidin
polymers using toluene-a-thiol followed by identification
using HPLC-ESI-MS and NMR analysis of the purified
thioadducts. Although we had no evidence for the location
of the interflavanyl bond, the C-4 to C-8 linkages were
favored in these compounds since the alternative C-4 to
C-6 linkages were less likely due to their more restricted
occurrence (Fletcher et al., 1977; Hemingway et al., 1982).
reaction was catalyzed by acid under mild conditions; there-
fore the flavan-3-O-gallate units were preserved (Gu et al.,
2002). To settle the absolute configuration of the extension
units, the benzyl thioether derivative was isolated and puri-
fied on Sephadex column using ethanol as eluting solvent to
1
give compound 7a (Fig. 1). Its H NMR spectra (see Section
3) showed the appearance of downfield signals due to the
benzene ring. The small coupling constants of the heterocy-
clic proton resonances are in accord with those of the 2,3-cis
and 3,4-trans configuration in the C-ring (Haslam, 1989).
The negative optical rotation (ꢀ108) and comparison with
reported data (Tanaka et al., 1994; Hashimoto et al.,
1989) indicated a 2R, 2R, 3S configuration, and it is identi-
fied as (ꢀ)-epigallocatechin-3-O-gallate-(4b-S)-benzylthioe-
ther (7a). The identities of the individual peaks for the three
plants were determined by HPLC-ESI-MS (LCQ) using
the same method run conditions as done with the HPLC
(Agilent 1100 instrument). The HPLC chromatogram for
2
.4. Thiolytic degradation of proanthocyanidins
Thiolytic degradation coupled with the reversed-phase
HPLC was applied to elucidate the average degree of poly-
merization (DPn) of the polymeric proanthocyanidins. In
this reaction, the flavonoid extension units of the proantho-
cyanidins are captured by toluene-a-thiol to form the ben-
zylthioether derivatives and only the terminal units are
released as the free flavan-ol or flavan-3-O-gallates. The

G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
2385
1
3
6 2
Fig. 3. C NMR spectrum of the proanthocyanidin polymer fraction from R. rosea (750 MHz, acetone-d -D O 1:1 v/v).
the three Rhodiola species after thiolysis (Fig. 4) showed two
2.5.1. Phenyl and cyanogenic glycosides
+
main peaks corresponding to m/z 459 [M + 1] EGCG,
The R. heterodonta genotype contained a newly discov-
ered phenolic glycoside designated ‘‘heterodontoside’’ (5)
that has not been previously reported and was not detected
in either of the other two species. R. heterodonta and R.
rosea contained phenylethanoid and phenylpropanoid
compounds that were not detected in R. semenovii. R.
semenovii contained mainly the two major cyanogenic glu-
cosides; rhodiocyanoside A (4) and lotaustralin (6). Major
phenolic glycosides detected in R. heterodonta included, in
addition to heterodontoside (5), tyrosol (1), viridoside (2),
salidroside (3), and rhodiocyanoside A (4). The most com-
mon phenyl glycosides isolated in this study from R. rosea
were salidroside (3), rosarin, rosavin, and rosiridin. None
of these ethanoid or propanoid glycosides were detected
in R. semenovii. These findings indicated that phytochemi-
cal constituents among the three Rhodiola species are spe-
cies-dependent and may account for different
phytochemical activities and benefits from each.
+
terminal unit, R 9.1 min, and m/z 581 [M + 1] EGCG-
t
benzylthioether, extension units, R 27.4 min, in addition
t
to other minor peaks and the peak corresponding to the
residual reagent at R 31.9 min. The estimated degree of
t
polymerization showed that R. heterodonta, R. semenovii,
and R. rosea had divergent DPn values of 5 ± 1, 6 ± 1,
and 13 ± 3 (n = 4), respectively. These results were also in
agreement with the HPLC-ESI-MS and NMR spectro-
scopic data for the polymeric proanthocyanidin fractions
from the three species.
2.5. Comparison of phytochemical constituents in Rhodiola
species
Characterization of phytochemical compounds present
in the three Rhodiola species was accomplished using frac-
tions obtained from a 70% acetone plant crude extract and
HPLC-ESI-MS and NMR spectroscopic analysis. In gen-
eral, the two central Asian Rhodiola genotypes (R. hetero-
donta and R. semenovii) had several phenolic glycosides
and proanthocyanidin compounds in common with R.
rosea. However, an in-depth characterization of Rhodiola
extracts revealed some unique differences in both the phe-
nyl glycoside and proanthocyanidin constituents in the
three Rhodiola genotypes as detailed below.
2.5.2. Proanthocyanidins
Aqueous acetone (70%) was reported as the most effi-
cient solvent to extract proanthocyanidins from plant tis-
sues (Gu et al., 2003a, 2003b; Yousef et al., 2004). Of the
dry 70% acetone extract, pure proanthocyanidin oligomeric
and polymeric fractions from Sephadex LH-20 separation
collectively constituted ca. 28%, 33%, and 35% dry weight

2386
G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
Fig. 4. RP-HPLC chromatogram for R. heterodonta, R. semenovii, and R. rosea, proanthocyanidin polymers detected at 280 nm after thiolysis. Terminal
units (ꢀ)-EGCG, R : 9.1 min (A), (ꢀ)-EGCG-benzylthioether, R : 27.4 min (B), and reaction reagent, toluene-a-thiol, R : 31.9 min (C).
t
t
t
for R. heterodonta, R. semenovii, and R. rosea respectively.
This high proportion of proanthocyanidin content is
expected to have a significant role in the overall phyto-
chemical activities of the three Rhodiola species studied
to the well-known R. rosea. Using fresh rhizomes with the
same techniques of extraction, isolation, and identification
of phytochemical components offered data which eluci-
dated direct cross-comparison between the three Rhodiola
species. Phytochemical constituents and compounds
unique to these species are most likely to be responsible
for their pharmaceutical interest in the central Asian
region, Europe, and more recently in the USA. A new phe-
nylethanoid glycoside; heterodontoside (5), was identified
in the R. heterodonta species. Proanthocyanidins known
to have pharmacological activities, constituting a fairly
large portion of the Rhodiola extracts (ca. 30% of the
70% acetone dry crude extract), were also characterized
in all three species. This is the first report to study in detail
the chemistry of R. heterodonta and to isolate cyanogenic
compounds in R. semenovii. To the best of our knowledge,
this is also the first report on the isolation and characteriza-
tion of oligomeric and polymeric proanthocyanidins from
R. rosea.
(
2
Brown et al., 2002; Prior and Gu, 2005; Xie and Dixon,
005). The three Rhodiola species contained similar pro-
anthocyanidin constituents including monomers, dimers,
trimers, tetramers, and pentamers of epigallocatechin and
its gallate esters. However, the relative abundance of these
polymers differed among the three species. While the dimer
EGC-EGCG (m/z 763) was the predominant proanthocy-
anidin in R. heterodonta and R. semenovii, more abundant
homogenic prodelphinidin gallate oligomers were found in
R. rosea, in particular the EGCG dimer (m/z 915), EGCG
trimer (m/z 1371), and EGC tetramer (m/z 1827). The esti-
mated degree of polymerization (DP) of proanthocyanidin
was ꢃ5 and 6 for R. heterodonta and R. semenovii, respec-
tively, but ꢃ13 for R. rosea as stated above. In the three
species, dimeric and oligomeric proanthocyanidins were
only reported before in R. semenovii (Matamarova et al.,
1
999; Kuliev et al., 2004). While proanthocyanidin compo-
sition was nearly similar in the three species, R. rosea con-
tained a more highly polymerized ratio and this was
associated with more difficulties in the isolation and purifi-
cation during both silica gel and Sephadex LH-20 column
chromatography separations.
3. Experimental
3.1. General
HPLC-ESI-MS data were obtained, unless otherwise
stated, using a LCQ Deca XP mass spectrometer (150–
2000 m/z) attached to photodiode array (PDA) detector
(200–600 nm) (Thermo Finnigan Corp., San Jose, CA).
Analyses were performed according to procedures devel-
oped by Yousef et al. (2004) with minor modifications.
2
.5.3. Conclusion remarks
In summary, this study presented a comprehensive phy-
tochemical characterization of two limitedly-known Rhodi-
ola species (R. heterodonta, and R. semenovii) as compared

G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
2387
Briefly, the HPLC separations were carried out on a C -
& Herder, Boriss) (I. Sodombekov s.n., 28 July 2004, ICBG
Central Asia voucher KPL_00142, ILLS, MO) were har-
vested in the mountainous regions of Uzbekistan and
Kyrgyzstan, respectively, in 2004. Fresh rhizomes of
golden root (R. rosea) harvested in May 2005 from coastal
islands in central Norway were generously donated by
Rosenrot Norge Ltd., Oslo, Norway. Rhizomes were
immediately upon arrival cleaned thoroughly with water,
cut into segments (2–5 g), and stored at ꢀ80 ꢁC until
extraction.
1
8
reversed-phase column (2.1 · 150 mm, VYDAC, Cat. #:
01 SPS215). The mobile phase solvent consisted of 95%
DD H O and 5% CH CN with 0.1% formic acid (A) and
2
2
3
9
5% CH CN and 5% DD H O with 0.1% formic acid
3 2
(
B). A step gradient of 0%, 30%, 60%, 90%, and 0% of sol-
vent B was used at 3, 30, 45, 50, and 60 min, respectively,
with 200 ll/min flow rate and 10-ll injection volumes.
The column was equilibrated with solvent A for 10 min
between samples at the same flow rate. Low resolution
and high resolution electronic spray ionization mass spec-
tra (LR-ESI-MS and HR-ESI-MS) were recorded on a
Micromass ZAB-SE Spectrometer (Waters Corporation,
Beverly, MA, USA) in the Mass Spectrometry Laboratory
of the School of Chemical Sciences, University of Illinois,
Urbana-Champaign.
3.3. Extraction and isolation
Frozen rhizome tissues (150 g) were exhaustively
extracted with 70% aq-acetone (7 · 500 ml, 2 min each)
at room temperature using a Turbo-twister blender (Ham-
ilton Beach/Proctor-Silex Inc., Southern Pines, NC). The
rhizome tissue was blended for 2 min at high speed and
filtered first with cheesecloth, then through Whatman #
4 paper. The acetone and some of the water were
removed under reduced pressure at 40 ꢁC. The concen-
trate was then frozen at ꢀ20 ꢁC and lyophilized. The
average 70% acetone crude-extract dry mass was 17%,
10%, and 11% for R. heterodonta, R. semenovii, and R.
rosea, respectively. This crude extract (10 g) was then frac-
tionated via vacuum liquid chromatography (VLC) over
silica gel (Type 60, 10–40 lm, CaSO4 binder S-6503,
Sigma Chemical Co., St. Louis, MO). Twenty-two frac-
tions (100 ml each) of increasing polarity were obtained
(Yousef et al., 2004). The elution system started with
petroleum ether (100%), petroleum ether–EtOAc (1:1),
EtOAc (100%), followed by 2%, 5%, 8%, 12%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%,
1
13
The H NMR, 1D and 2D (500 MHz) and C-NMR
(
125 MHz) spectra (CD OD for phenolic glycosides, ace-
3
tone-d6 or acetone-d -D O 1:1 for proanthocyanidins)
6
2
were recorded on a Varian Inova 500 spectrometer (Var-
ian Instruments, Palo Alto, CA), at a proton frequency
of 500.078 MHz, using TMS as internal standard. The
1
3
C NMR spectra of the highly polymerized PA fraction
was obtained on the Varian Unity Nova 750 spectrome-
ter with a 10 mm broadband probe at 20ꢁC, at an oper-
ating frequency of 188.55 MHz over a sweep width of
4
5 kHz, using a 90ꢁ pulse width of 18.00 ls and an
acquisition time of 2.89 s. Optical rotations were mea-
sured in CH OH solution using a JASCO DIP-370 digi-
3
tal polarimeter (Jasco Limited, UK) at 25 ꢁC. UV spectra
were obtained on a Shimadzu UV-2401 PC UV–Vis
Recording Spectrophotometer (Shimadzo, North Amer-
ica). Chromatographic separations were achieved by cc
using lipophilic Sephadex LH-20 (Sigma, St. Louis,
MO) and silica gel 60 230–400 mesh ASTM (Merck,
Whitehouse Station, NJ). Fractions were analyzed by
Thin Layer Chromatography (TLC) using silica gel 60
100% of MeOH–H O (1:1) in EtOAc. Solvents were
2
removed using reduced pressure at 40 ꢁC, and the dried
fractions were stored at ꢀ20 ꢁC. From each powdered
dry fraction, 5 mg were dissolved in 1 ml 50% aq-MeOH,
filtered though 0.45 nm nylon filters and 10 ll were
injected for analysis by HPLC-ESI-MS.
F254 pre-coated plates with
a solvent system of
EtOAc–MeOH–H O (77:13:19). The TLC plates were
2
visualized by spraying with reagents for primary separa-
tion and identification compounds. The spraying reagents
used were vanillin–HCl (vanillin–HCl–MeOH, 1:1:20, v/
v/v) and cleaning reagent (sodium dichromate
Na Cr O Æ 2H O–H O–H SO , 1:5:8, w/v/v) at 110 ꢁC
3.4. Purification and characterization of phenolic and/or
cyanogenic glycosides and proanthocyanidins
To separate phenolic and/or cyanogenic glycosides from
proanthocyanidins, a portion of the 70% acetone crude
extract (2.8 g) of each plant was applied to a Sephadex
LH-20 column (2.5 · 30 cm) which had been equilibrated
with EtOH, collecting a total of 15 50-ml fractions. Elution
started with EtOH (Fr. 1–11) to collect phenolic and
cyanogenic glycosides. Oligomeric and polymeric proanth-
ocyanidins were recovered from the column using MeOH–
2
2
7
2
2
2
4
for 10 min.
Commercial standards of (+)-catechin, (ꢀ)-epicatechin,
(
3
ꢀ)-gallocatechin, (ꢀ)-epigallocatechin, (ꢀ)-gallocatechin-
-O-gallate, (ꢀ)-epigallocatechin-3-O-gallate, tyrosol (1),
salidroside (3), rosin, rosarin and rosavin (Chromadex,
Laguna Hills, CA) were used to compare with compounds
isolated from Rhodiola extracts.
acetone–H O (1:1:1) as the elution solvent (Fr. 12–15),
2
3
.2. Plant material
Schemes 1–3.
Fresh rhizomes of R. heterodonta (Hooker & Thomson,
Boriss) (K. Tojboev s.n., 12 June 2004, ICBG Central Asia
voucher UPL_00085, ILLS, MO) and R. semenovii (Regel
3.4.1. R. heterodonta phytochemical constituents
Fractions 3–11 from the Sephadex LH-20 column were
combined (1.23 g) and applied to a silica gel column

2388
G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
Rhodiola heterodonta
7
0% acetone extract of fresh rhizomes
2
.8 g
Sephadex LH-20
EtOH
MeOH-Acetone- H2O 1:1:1
Fr.1-2
.46 g
Lipophylic fraction
Fr. 14 Fr.15
0.18g
0.33g
Fr.3-11
1.23 g
Fr.12
.22g
EGCG (7)
Fr 13
0.24 g
0
0
Oligomeric and polymeric
prodelphinidins (HPLC-MS)
Silica gel
EtOAc
50% MeOH in EtOAc
Fr.19-21
Fr. 22-25
Fr.11-14
Fr.1-2
.034 g
Lipophylic Tyrosol (1)
material
Fr.3-4 Fr.5-10
0.033 g 0.010 g
Fr.15-18
0.027 g
0
.136 g
0.337 g
0
0.504 g
Carbohydrate
fraction
Rhodiocyanoside A (4)
Silica gel
Silica gel
Viridoside (2) Salidroside (3) Heterodontoside (5) Carbohydrate
0.052 g
0.0973 g
.064 g fraction
.114 g
0
0
Scheme 1. Sequential fractionation of the 70% aq. acetone crude extract from R. heterodonta.
Rhodiola semenovii
7
0% acetone extract of fresh rhizomes
2
.8 g
Sephadex LH-20
EtOH
MeOH-Acetone- H2O 1:1:1
Fr.1-2
.35 g
Lipophylic fraction
Fr.12
Fr 13
Fr. 14 Fr.15
0.31 g 0.20 g
Fr.3-11
.40 g
0
0.07 g
0.37 g
1
EGCG (7)
Oligomeric and polymeric
prodelphinidins (HPLC-MS)
Silica gel
EtOAc
50% MeOH in EtOAc
Fr.1-4
.055 g
Lipophylic fraction
Fr. 5-11 Fr.26-31
Fr.32-40
0.040 g
Fr.41-60
1.053 g
0
0.010 g
0.035 g
Lotaustralin (6) Rhodiocyanoside A (4) Carbohydrate
fraction
Scheme 2. Sequential fractionation of the 70% aq. acetone crude extract from R. semenovii.
(
2.5 cm · 35 cm). Elution started with EtOAc followed by
allowed the characterization of proanthocyanidins in the
fractions 12–15.
an EtOAc–MeOH gradient (2–50%), collecting 50-ml frac-
tions (Scheme 1). Compound 1 (tyrosol, 33 mg) was eluted
in the early fractions (Fr. 3–4) with 100% EtOAc. Com-
pounds 2 and 3 were obtained in a mixture in fractions
3.4.2. R. semenovii phytochemical constituents
Fractions 3–11 from Sephadex LH-20 column were
combined (1.4 g) and fractionated over a silica gel column
similar to the above procedure with R. heterodonta where
compounds 6 (lotaustralin, 35 mg) and 4 (rhodiocyanoside
A, 40 mg) were isolated (Scheme 2). Proanthocyanidin
fractions (12–15), containing monomeric and polymeric
proanthocyanidins, were similarly characterized by TLC,
NMR, and HPLC-MS.
1
1–14 eluted with 10% MeOH in EtOAc. This mixture
was further separated on a second silica gel column using
EtOAc–MeOH (9:1, v/v) solvent where 64 mg and 52 mg
of viridoside (2) and salidroside (3), respectively, were iso-
lated. Compound 4 (rhodiocyanoside A, 27 mg) was
obtained in the fractions 15–18 eluted with 16% MeOH
in EtOAc. Fractions 19–20 eluted with 20% MeOH in
EtOAc, were combined and further chromatographed on
a silica gel column (EtOAc–MeOH–H O, 79:11:10) to give
compound 5 (heterodontoside) (97.3 mg). Preliminary TLC
for the fractions followed by NMR and HPLC-ESI-MS
3.4.3. R. rosea phytochemical constituents
Fractions 1–11 containing the ethanoid and propa-
noid glycosides were combined (1.6 g), and subjected to
2

G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
2389
Rhodiola rosea
7
0% acetone extract of fresh rhizomes
2
.8 g
Sephadex LH-20
EtOH
MeOH-Acetone- H2O 1:1:1
Fr.1-11
.6 g
Phenyl glycoside mixture
HPLC-MS)
Fr.12
Fr.13
Fr.14
Fr.15
1
0.01 g 0.34 g
0.38 g
0.12 g
Polymeric prodelphinidins (HPLC-MS)
(
Salidroside (3)
Rosin (8)
Rosarin (9)
Rosavin (10)
Sephadex LH-20
5
0% aq. EtOH 60% aq. acetone
0
.35 g
0.39 g
Purified
prodelphinidin polymer
Unidentified
complex mixture
Scheme 3. Sequential fractionation of the 70% aq. acetone crude extract from R. rosea.
HPLC-MS analysis. In a similar way to the above two spe-
cies, fractions 12–15, containing oligomeric and polymeric
proanthocyanidins, were further analyzed by HPLC-ESI-
MS. For NMR analysis, the fractions from 12 to 15
Rhodiola samples both before and after thiolysis, were
directly injected into the HPLC. Degree of polymerization
was calculated by dividing the peak area of the extension
units (benzylthioethers) by that of the terminal units for
each sample (Guyot et al., 2001).
(
0.85 g) were combined and further purified on Sephadex
LH-20 column (Scheme 3). The column was first washed
with an excess of EtOH–H O (1:1, v/v) before eluting the
3.5.1. Isolation and purification of the thiolysis products
To separate the individual components from the thiolysis
mixture, a larger concentration of the same fraction in
MeOH (ꢃ100 mg/ml) was subjected to thiolysis the same
way as above. The thiolysis mixture was freeze-dried and
the obtained dry powder was chromatographed over Sepha-
dex LH-20 column (EtOH) to afford two main compounds;
2
proanthocyanidin polymer with 60% aq. acetone. The puri-
fied polymeric proanthocyanidin fraction was freeze-dried
1
13
(
0.394 g) before HPLC-ESI-MS, and H and C NMR
analyses.
3
.5. Thiolytic degradation of proanthocyanidin polymers
(
ꢀ)-epigallocatechin-3-O-gallate-(4b-S)
benzylthioether
To estimate the degree of polymerization and identify
(extension units, 7a) and (ꢀ)-epigallocatechin-3-O-gallate
the stereochemistry of the extension units of the proantho-
cyanidin polymeric fractions for the three Rhodiola
genotypes, a thiolysis degradation method was used (Gu
et al., 2002; Guyot et al., 1998, 2001). Based on the
HPLC-ESI-MS profile for Sephadex LH-20 fractions, frac-
tion 14 (highly polymerized) from each plant was used for
this analysis. The thiolysis analysis was performed using an
Agilent 1100 HPLC system (Agilent Technologies Inc.,
Wilmington, DE) with autosampler, diode array detector
(terminal units, 7).
(ꢀ)-Epigallocatechin-3-O-gallate-(4b-S) benzylthioether
2
D
5
(7a): pale brown amorphous powder, ½aꢁ – 108 (c 5.5, ace-
+
1
tone), ESI-MS [M + 1] m/z 581, H NMR (500 MHz, ace-
0
0
00
tone-d ): d 7.21–7.52 (5H, m, benzene), 7.01 (1H, s, 2 , 6
6
0
0
gallate), 6.6 (1H, s, 2 ,6 ), 6.03, 6.05 (1H each, d,
J = 2.2 Hz, H-6 and 8), 5.50 (1H, s, H-2), 5.46 (1H, m,
H-3), 4.25 (1H, d, J = 2.2 Hz, H-4), 4.11 and 4.20 (1H
1
3
each, d, J = 13.2 Hz, Ph-CH -S); C NMR (125 MHz) dc
2
(
DAD, 280 nm) and 24 mm · 4 mm · 5 lm Supelcosil
36.3 (–CH S–), 40.0 (C4), 72.1 (C3), 73.6 (C2), 95.0 (C6),
2
0 0 00 00
LC-18 reversed-phase column (25 ꢁC) (Sigma-Aldrich,
Bellefonte, PA). The elution solvents consisted of 2% acetic
acid in H O (A) and 100% MeOH (B). The linear gradient
started at 15% B and increased to 80% B in 45 min and
continued for 5 min at 80% B followed by 15 min of 15%
B to equilibrate the column with a constant flow rate of
96.4 (C8), 98.2 (C10), 106.0 (C2 ,6 ), 109 (C ,6 ), 120.5
00
(C1 ), 127.1 (C4 benzene), 128.6, 129.3 (C2,3,4,5 benzene),
0 0 00 0 0
132.6 (C1 ), 138.4, 139.0 (C4 ,C4 ), 145.3, 145.7 (C3 ,5 and
2
00
00
3 ,5 ), 156.3, 157.5, 158.8 (C5,7,9), 165.6 (C@O).
3.6. Heterodontoside (5)
1
ml/min.
2
D
5
Duplicates of commercial standards (1.25 mg/ml 100%
White powder; ½aꢁ – 28.5 (c 1.0, MeOH); UV (MeOH)
1
MeOH) including (+)-catechin, (ꢀ)-epicatechin, (ꢀ)-gallo-
catechin and (ꢀ)-epigallochatechin, (ꢀ)-gallocatechin-3-O-
gallate, and (ꢀ)-epigallocatechin-3-O-gallate, and the
k
(loge) 225 (1.01), 277 (0.25), 280 (0.19) nm; H NMR
and C NMR: see Table 1; HRESIMS m/z 447.1875 (Calc.
for C H O 477.1866 [M + 1] ).
max
1
3
+
2
0
31 11

2390
G.G. Yousef et al. / Phytochemistry 67 (2006) 2380–2391
Acknowledgements
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3
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(Malus domestica var. Kermerrien). J. Agric. Food Chem. 46,
We gratefully acknowledge the support of Fogarty
International Center of the NIH under U01 TW006674
for the International Cooperative Biodiversity Groups.
The authors thank Drs. David Seigler and Robert Coates,
University of Illinois, for helpful advice on some spectro-
scopic interpretations. We thank Mr. Carl Ivar Storøy of
Rosenrot Norge Ltd. (www.rosenrot.no) for donating fresh
rhizomes of R. rosea for this comparative analysis.
1
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