Phytochemistry Letters 15 (2016) 81–87

Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Mini review

Structure-antioxidant and anti-tumor activity of Teucrium polium

phytochemicals
Wael A. Elmasria , Mohamed-Elamir F. Hegazyb , Yehia Mechrefa , Paul W. Paréa,*
a
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
b
Department of Medicinal Plants, National Research Centre, Giza, Egypt

A R T I C L E I N F O A B S T R A C T

Article history: Chemical characterization as well as antioxidant and anti-tumor activity are reported for isolated
Received 4 September 2015 metabolites from Teucrium polium L. (Lamiaceae). Structures were identified using standard MS and NMR
Received in revised form 3 November 2015 spectroscopic methods. Sesquiterpene absolute stereochemistry was determined based on a modified
Accepted 9 November 2015
Mosher’s reaction. Biological activity was evaluated by a cupric reducing antioxidant capacity (CUPRAC)
Available online xxx
assay and select compounds screened for anti-tumor activity. (1R,4S,10R) 10,11-dimethyl-dicyclohex-5
(6)-en-1,4-diol-7-one and (R)-mandelonitrile-b-laminaribioside, together with ten previously reported
Keywords:
compounds were identified. Antioxidant versus tumor-inhibition relationships was examined.
Teucrium polium
Lamiacae
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Antioxidant activity
Mosher’ reaction
Sesquiterpenes
Anti-tumor activity

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.1. General procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.2. Reagent and chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.3. Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.4. Extraction and isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.5. Acid hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.6. Gas chromatography analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.7. Preparation of (R) and (S)-MTPA esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.8. Cu+2 reduction assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.9. Cytotoxicity screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.10. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.1. (1R,4S,10R) 10,11-dimethyl-dicyclohex-5(6)-en-1,4-diol-7-one (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.2. (R)-MTPA ester of 1 (1a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.3. (S)-MTPA ester of 1 (1b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.4. (R)-mandelonitrile-b-laminaribioside (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.5. (R)-MTPA ester of 3 (3a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.6. (S)-MTPA ester of 3 (3b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.7. (R)-MTPA ester of 4 (4a) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.8. (S)-MTPA ester of 4 (4b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

* Corresponding author. Fax: +1 806 742 1289.

E-mail address: Paul.Pare@ttu.edu (P.W. Paré).

http://dx.doi.org/10.1016/j.phytol.2015.11.007
1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
82 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87
1. Introduction
Oxidants are ubiquitous in biological systems and can cause
significant damage ranging from reversible biochemical mod-
ifications to permanent DNA alterations. In fact, ailments such as
cardiovascular diseases, cancer and aging can result from such
oxidative damage (Rahal et al., 2013). While biological systems
have antioxidants to minimize such cellular oxidative damage,
including enzymes (e.g. catalase) and organic compounds (metal-
lothionein and ascorbic acid) (Klimp et al., 2002), excessive
generation of oxidants can overload protective mechanisms of an
organism (Rahal et al., 2013). Exogenous antioxidants can assist in
reducing such oxidative damage and indeed a plethora of
antioxidants exist ranging from water-soluble flavonoid glycosides
to water-insoluble alpha-tocopherol.
The array of structurally different exogenous antioxidants
points to a complexity of structural motifs responsible for
antioxidant activity. In fact, simple biochemical modifications
such as aglycone methylation and/or glycosylation structure–
activity relationships have not been fully resolved. For example,
with polyphenol glycosides, the number of sugars (Fiol et al.,
2012), and/or phenolic groups (Siquet et al., 2006) can have a
bearing on antioxidant activity. It has been suggested that free
phenolic hydroxyl pairs form stabilized phenoxy radical
intermediates increasing activity (Christodouleas et al., 2009).
Indeed O-modifications of flavonoid hydroxyl substitutions can
directly reduce flavonoid antioxidant activity (Cao et al., 1997).
For phenylpropanoid glycosides the degree of glycosylation has
been shown to influence antioxidant activity (Rahman et al.,
2006). Antioxidant activity can be additionally affected by the
cellular environment with some antioxidants functioning both as
pro-oxidants and antioxidants. For example, in tumor cells
vitamin C acts as pro-oxidants producing hydrogen peroxide that
can toxify the cell; whereas, in healthy cells the metabolite
functions as a protective antioxidant (Casciari et al., 2001).
Additional empirical-based structure–reactivity relationships are
necessary to predict antioxidant activity based on characteristic
chemical motifs.
The medicinal plant Tecrium polium, rich in antioxidants, was
selected as a promising source of polyphenolic glycosides to
examine structure–reactivity relationships of aglycone and sugar
substitutions with antioxidant activity. Indeed, several glycosides
(e.g. phenylpropanoid and iridoid) and aglycones (e.g. sesquiter-
penes) have already been isolated from T. polium (Cozzani et al.,
2005; De Marino et al., 2012; Elmasri et al., 2015a; Elmasri et al.,
2015b). Building on such antioxidant chemical studies, structure–
activity relationships were examined for phenylpropenoid metab-
olites isolated from T. polium. The compounds isolated including
newly identified sesquiterpene and mandelonitrile glycoside
(Fig. 1) allowed for anti-oxidation activity to be examined for
comparable structures in methylation and glycosylation substitu-
tions. In addition to investigating structure–antioxidant relation-
ships, metabolite cytotoxicity against tumor cell lines was
examined to provide insight into possible links between antioxi-
dant and anti-tumor activity. Compound cytotoxicity was evaluat-
ed using a human tumor screen including cell lines for leukemia,
melanoma, ovarian, breast, colon, lung, CNS, renal and prostate
cancers (Shoemaker, 2006).
2. Materials and methods
2.1. General procedures
Optical rotations were measured in MeOH on an Autopal IV
automatic polarimeter (Rudolph Research Analytical) equipped
with a 10 cm microcell and a sodium lamp (lmax = 589 nm). UV data
were obtained on a Genesys 20 spectrophotometer. IR (KBr)
spectra were recorded on a ThermoNicolet model IR 100 spectro-
photometer. NMR spectra were obtained on a Joel NMR spectrom-
eter equipped with Delta software; chemical shifts were reported
in d (ppm) and J coupling in Hz. The 13C NMR multiplicities were
determined by DEPT experiments. NOE measurements were
obtained from 2D NOESY experiments. One-bond heteronuclear
1
H-13C connectivities were determined by HMQC, and two- and
three-bond 1H-13C connectivities were determined by HMBC
experimentation. HRESIMS was performed on a Dionex Ultimate
3000 UHPLC system interfaced with an LTQ Orbitrap Velos
(Thermo Scientific, Pittsburgh, PA, USA) mass spectrometer. Data
were processed using Xcalibur Qual browser software (Thermo
Scientific, Pittsburgh, PA, USA). GCMS analysis was performed on
an ISQ QD Single Quadrupole GC–MS system and data were
processed using Xcalibur software (Thermo Scientific, Pittsburgh,
PA, USA). HPLC was performed using a prep-C18 column
(21.2
250 mm, 10 mm) on an Agilent 1100 apparatus equipped
with a Rheodyne injector and with UV detectors. Column
chromatography was carried out using EMD silica gel 60 (70–
230 mesh). Analytical TLC was performed on EMD silica gel 60 F254
sheets, 0.25 mm thick.
2.2. Reagent and chemicals
Trolox (purity 97%), a-tocopherol (purity >97%), butylated
hydroxyanisole (BHA) (purity >98.5%), neocuproine (purity 99%),
and ammonium acetate (purity 97%) were purchased from Fisher
Scientific Inc.
2.3. Plant material
Aerial parts of T. polium were collected in June 2010, from North
Sinai, Egypt and authenticated by ME Hegazy (National Research
Centre, Egypt). A voucher specimen has deposited in the herbarium
of St. Katherine protectorate, Egypt (voucher ID: SK-105).
2.4. Extraction and isolation
Air-dried aerial plant tissue (2 kg) was crushed. The resulted
powder was extracted at room temperature with CH2Cl2–MeOH
(1:1). The solvent was evaporated and the residue (210 g) subjected
to silica gel CC eluting with n-hexanes, CH2Cl2, and MeOH in
increasing order of polarity up to 15% MeOH in CH2Cl2 to afford
398 fractions. Based on metabolite complexity of chromatographic
analysis, fractions A (225–232), B (250–265), and C (284–293)
were combined and further purified for spectroscopic analysis and
biological testing.
Fraction A (13 g) was concentrated in vacuo and subjected to
silica gel CC eluted with a CH2Cl2–MeOH gradient (75:25) up to
100% MeOH; fractions were monitored by TLC with
CH2Cl2–MeOH–H2O (6:2:0.5) to afford 42 fractions. Fractions
25–40 (3.2 g) were pooled and purified by reversed phase (RP)
HPLC eluting with MeOH–H2O (0.1% HCHO) (25:75) to afford 1
(4.8 mg), 2 (4.8 mg), 3 (14 mg), 10 (12.2 mg), 11 (3.5 mg), and 12
(21 mg).
Fraction B (9 g) was concentrated in vacuo and subjected to
silica gel CC eluting with a gradient of CH2Cl2–MeOH starting with
(9:1) up to (0.5:9.5); fractions were monitored by TLC with
CH2Cl2–MeOH–H2O (3:5:0.5) to afford 73 fractions. Sub-fractions
18–30 (1.5 g) were pooled and purified by RP HPLC eluting with an
isocratic MeOH–H2O system (35:65) to afford 5 (21 mg).
Fraction C (6 g) was concentrated in vacuo and subjected to
silica gel CC eluted with a CH2Cl2–MeOH (7:2) gradient up to
CH2Cl2–MeOH (1:9); fractions were monitored by TLC with
CH2Cl2–MeOH–H2O (6:2:0.5) to afford 46 fractions. Fractions
 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87
3–4 (250 mg) were pooled and purified by reversed phase (RP)
HPLC eluting with gradient MeOH–H2O system (41:59) to (50:50)
to afford 6 (7.5 mg), 7 (2.8 mg), 8 (1.5 mg), and 9 (6.5 mg).
2.5. Acid hydrolysis
A solution of 2 (1 mg in H2O: dioxane (1:1) with 1 N HCl (1 mL
total volume)) was heated to 80  C for 2 h. After cooling, the
reaction mixture was neutralized using Amberlite IRA-68, the resin
removed by filtration and the filtrate extracted with EtOAc. The
aqueous layer was concentrated and the sugars were identified by
TLC eluting with n-hexane:EtOAc:MeOH:AcOH:H2O
(1:4:2:0.5:0.5) by comparison with authentic standards (Wang
et al., 2004).
2.6. Gas chromatography analysis
For sugar identification, an aqueous aliquot was silylated with
N-trimethylsilylimidazole (TMSI) for 2 h at room temperature.
After reaction termination with dist. H2O, the mixture was
partitioned with n-hexane and the organic layer was analyzed
by GCMS. For sugar configuration identification, L-cysteine methyl
ester hydrochloride in pyridine (0.2 mL, 0.06 M) was added to the
aqueous layer, stirred at 60  C for 1 h and incubated at RT for 2 h
with TMSI (0.2 mL). The reaction mixture was partitioned with n-
hexane and dist. H2O, and the organic layer was analyzed by GCMS
(Elmasri et al., 2015a). D-Glucose was confirmed by retention-time
comparison (26.2 min) with derivatized authentic standards.
2.7. Preparation of (R) and (S)-MTPA esters
An aliquot of 1, 3 and 4 (2 mg each) were dissolved in CHCl3
(2 mL) with dry pyridine under N2 (g). (S)- or (R)-a-methoxy-
a-(trifluoromethyl)phenylacetyl (MTPA) chloride (0.1 mL) was
added to each aliquot. The reaction was stirred overnight,
quenched with a saturated NaHCO3 solution and the CHCl3 layer
was aqueous rinsed and dried under reduced pressure to afford the
(R) and (S)-MTPA esters (Elmasri et al., 2015a).
2.8. Cu+2 reduction assay
The cupric ion (Cu+2) reducing method was used with slight
modifications (Ak and Gulcin, 2008). To an aqueous CuCl2 solution
(0.01 M, 0.25 mL), ethanolic neocuprine (7.5
103 M, 0.25 mL) and
CH3COONH4 (1 M, 0.25 mL, pH 6.5) along with different concen-
trations of the test compound (15–60 mM) or a synthetic positive
control were combined. Absorbance was recorded at 450 nm, after
30 min. TEAC coefficients were calculated by dividing the
extinction coefficient of the test compound (e) by that of trolox
(Gungor et al., 2011; Nechifor et al., 2012).
2.9. Cytotoxicity screening
Anti-tumor screening was performed on 4, 5, 6, 10, and 12 at a
single concentration (10 mM) at the National Cancer Institute
according to standard procedures (Alley et al., 1988; Boyd and
Paull, 1995; Grever et al., 1992; Shoemaker, 2006) with a NCI-
60 DTP human tumor cell line screen (http://dtp.nci.nih.gov/
branches/btb/ivclsp.html).
2.10. Statistical analysis
Data were expressed as the mean  standard deviation (SD) of
three measurements. A Student t test was used to determine
significant differences between means with a significant difference
defined at p  0.05.
83
3. Results and discussion
The CH2Cl2–MeOH extract of T. polium aerial plant material was
partitioned with a gradient of n-hexane, dichloromethane, and
methanol. Compounds from the eluted fractions were purified
using a combination of Sephadex, silica gel CC and RP-HLPC. The
absolute stereochemistry was assigned for 1, 3 and 4 for the first
time.
Compound 1 was isolated as a yellow amorphous powder, with
a specific rotation of ½a25
D ¼ 7:20 (c 0.25, MeOH). The HR-ESI–MS
exhibited a molecular ion peak [M + H]+ at m/z 211.1330 (calc.
211.1329), and [M + Na]+ at m/z 233.1149 (calc. 233.1148) suggesting
a molecular formula of C12H18O3. 13C NMR and DEPT spectra
exhibited 12 signals corresponding to two methyls, four methyl-
enes, a carbonyl, sp2 hybridized carbon, an oxygenated methine,
and two quaternary carbons (one oxygenated and one aliphatic).
The 1H NMR spectrum displayed signals corresponding to two
tertiary methyl groups at dH 1.33, 1.38 (both s, 3H), an olefinic
proton at dH 5.99 (s, 1H), and an oxygenated methine proton at dH
3.32 (dd, J = 4.12 Hz, 1H). An alpha–beta unsaturated carbonyl was
identified based on characteristic NMR signals and assigned to
C-5-C-7 (Table 1). HMBC correlations between H-6 (dH 5.99) and
34.4 allowed for the assignment of C-8 (Fig. 2) ; H2-8 were
identified by HMQC (Table 1). 1H-1H COSY correlations between
Ha-8 at 2.55 and Hb-8 at dH 2.31 correlated with dH 2.18 and 1.76
allowing for the assignment of H2-9 (Fig. 2). This was supported by
HMBC correlations between Ha-9 and C-8/C-7 and between H2-
8 and C-9/C-10 confirming the presence of an a,b-unsaturated
cyclohexanone substructure. HMBC correlations from H-8 to dC
42.6 and 37.5 and from H-9 to dC 42.6, 17.8, 79.1, and 171.7 allowed
for assignment of C-10, C-11, and C-1. This was supported by HMBC
from dH 3.32 (H-1) to C-5, C9, C-10, and C-11. COSY correlation
between H-1 and dH 1.59 (H2-2) and in turn H2-2 and dH 1.51 (H-3a)
and 1.87 (H-3b) allowed for H-1–H2-3 assignments. HMBC
confirmed these assignments with H-1 correlating with dC 26.7
(C-2) and 38.3 (C-3). HMBC correlations were also observed
between H2-3 and dC 71.2 and 28.5 allowed for assignment of C-4C-
12, respectively.
Relative stereochemistry was resolved by NOESY with corre-
lations between Me-12 and H-3a (a), H-3a and H-1, and Me-11 and
H-9a (a) and H-2 (Fig. 3); this implied that OH-1 and Me-12 are on
the same molecular face. A modified Mosher’s reaction (Ohtani
et al., 1991) was performed to determine the absolute configura-
tion of the secondary alcohol at C-1. Treatment of two aliquots of 2
with (S)- and (R)-MTPA chloride afforded the corresponding esters
1a and 1b, with molecular ion peaks at m/z 427.1702 and 427.1701,
respectively, consistent with successful derivatization. The pattern
of dH (S–R) values (Fig. 4) allowed for determining the absolute
configuration at C-1 to be R and the stereochemistry based on NMR
and Mosher data was assigned to 1R, 4S, and 10R as shown in Fig. 1.
The structure of 1 is (1R, 4S, 10R) 10,11-dimethyl-dicyclohex-5(6)-
en-1,4-diol-7-one, a new natural product.
Compound 2 was isolated as a yellow amorphous powder with
a specific rotation of ½a25 D ¼ 13:84 (c 0.13, MeOH). HR-ESI–MS
exhibited a molecular ion peak [M + Na]+ at m/z 480.1486 (calc.
480.1481), suggesting a molecular formula C20H27NO11. The 1H
NMR spectrum displayed an AA0 BB0 ring system assigned to a
mono-substituted aromatic ring at dH 7.59 (m, J = 1.83 Hz, 1H) and
7.43 (m, J = 2.29, 1.83 Hz, 4H). In addition, a downfield proton at dH
6.02 (s, 1H), was consistent with a nitrogenous substitution. Two
anomeric signals at dH 4.57 (d, J = 7.79 Hz, 1H), 4.79 (d, J = 7.33 Hz,
1H) were consistent with a b-D-glucose group also observed after
acid hydrolysis via GC comparison with an authentic standard.
HMBC correlations from H-10 to C-7 at dC 68.1 and from H-100 to
C-30 supported the linkage of both glucose units to C-7 and C-30 ,
84 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87

Table 1
1
H and 13C NMR data of 1 and 2 (d in ppm, J in Hz) (400 MHz, methanol d4). Signals OH OH OH
were assigned on the basis of DEPT, 1H-1H COSY, HMQC, and HMBC experiments. HO O O CN
1 2
O O
Position 1
H 13
C 1
H 13
C O HO HO
1 3.32, d (4.12) 79.1 135.1 OH
2 1.59, m 26.7 7.59, m (1.83) 128.5 HO HO
3 1.51, m (3.66) 38.3 7.43, m (2.29) 129.9
1.87, m
4 71.2 7.43, m (2.29) 130.7
1 2
5 171.7 7.43, m (2.29) 129.9
Fig. 2. HMBC and COSY observed with compounds 1–2. HMBC (solid arrow) and
6 5.99, s 125.0 7.43, m (1.83) 128.5
COSY (solid, black line) correlations observed with compounds 1-2.
7 203.4 6.02, s 68.1
8 2.31, m 34.4
2.55, m(5.04) 118.4 data, the structure was identified as (R)-mandelonitrile-b-lami-
9 1.76, m 37.5 naribioside, a new natural product.
2.18, m (2.75)
Compound 3 was isolated as a yellow amorphous powder, with
10 42.6
11 17.8 a specific rotation of ½a25
D ¼ þ6:66 (c 0.45, MeOH). The HR-ESI–MS
12 28.5 exhibited a molecular ion peak [M + Na]+ at m/z 289.1406 (calc.
289.1410) suggesting a molecular formula of C15H22O4. Based on 1H
Glucose0
10 4.57, d (7.79) 100.3
and 13C (Table 1), 2D NMR data (Fig. 2), similarity to a previously
20 3.63, m 77.7 reported germacranolide is established (Gordon et al., 1981).
30 3.54, t (7.79) 83.0 Because the absolute stereochemistry is inexorably linked to
40 3.35, ma 71.1 biological activity and the enantiomers of a given chiral molecules
50 3.18, ma 78.0
should be considered two different drugs (McConathy and Owens,
60 3.67, ma 62.4
3.84, ma 2003), a modified Mosher’s reaction (Ohtani et al., 1991) was
performed to establish the absolute configuration of 3. Treatment
Glucose00 of two aliquots of 1 with (S)- and (R)-MTPA chloride afforded the
100 4.79, d (7.33) 105.5
corresponding diesters 3a and 3b, with molecular ion peaks at m/z
200 3.25, ma 76.0
300 3.36, ma 77.7
721.2189 and 721.2191 (calc. 721.2206), respectively, consistent
400 3.35, ma 71.1 with derivatized products. The pattern of dH (S–R) values (Fig. 4)
500 3.34, ma 78.4 allowed for the determination of R at C-1 and C-6. Therefore 3 was
600 3.67, ma 62.5 assigned as (1R,6R,7R,8S,11R)-1,6-dihydroxy-4,11-dimethyl-ger-
3.84, ma
macran-4(5), 10(14)-dien-8,12-olide.
a
Signal patterns are unclear due to overlapping. The relative stereochemistry of 4 was previously reported (Sanz
and Marco, 1991) while the absolute stereochemistry was
respectively (Fig. 2). These data indicate a mandelonitrile established here again based on a modified Mosher’s method.
disaccharide similar to what has been previously reported (Neilson Treatment of two aliquots of 4 with (S)- and (R)-MTPA chloride
et al., 2011) except for the configuration at C-7. Prediction of the afforded the corresponding esters 4a and 4b, respectively. The
stereochemistry of cyanogens aglycone moiety was suggested by pattern of dH (S–R) values (Fig. 4) allowed for determining the
Nahrstedt based on chemical shift data (Huebel et al., 1981) and absolute configuration at C-6 to be R and other position were
complexity of the anomeric proton signals (Seigler et al., 2002). assigned to 1R, 4S, 5S, 6R, 7S, and 10R as shown in Fig. 1. Thus, the
This signal complexity is attributable to anisotropism of the structure of 4 was established as (10R,1R,4S,5S,6R,7S)-4,10-die-
neighboring aromatic ring. The dC of the (S)-configuration appears poxygermacran-6-ol.
up-field relative to the (R)-configuration (Huebel et al., 1981). In addition, eight previously reported compounds: 8-acetyl-
Examination of both 1H and 13C NMR spectral data and comparison harpagide (5) (Takeda et al., 1987) polumoside (6) (Elmasri et al.,
with published mandelonitrile glycosides (Neilson et al., 2011) as 2014), 2-(3-hydroxy-4-methoxyphenyl)-ethyl-O-(aL-rhamnosyl)-
well as 6 indicated an (R)-configuration at C-6. This was confirmed
by an absence of higher-order spin multiplicities which gives rise
to an obscured doublet (observed as a multiplet) in the case of
(S)-configurations (Huebel et al., 1981). On the basis of this spectral

OR OH OH
1 HO O O 7 CN
8
O O 1
3 5 O HO HO
12 OH HO HO 4

R
1 H 2
1a (R) MTPA
1b (S) MTPA
Fig. 1. Chemically identified metabolites. Consensus structures of newly identified
phytochemicals. Fig. 3. NOESY correlations observed with compounds 1.
 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87 85

OR -0.04 O - 0.07
O
+0.01 -0.15 OR
+0.12 -0.09 - 0.09 - 0.05
+ 0.014
- 0.19 - 0.01 -0.09 +0.04 +0.03
+0.03 + 0.048 + 0.104
O - 0.028 -0.02 +0.06
-0.02 O
OR
+0.01 OH
+ 0.12 -0.04
+ 0.004 OR +0.07

1 3 4
Fig. 4. Absolute configuration for compounds 1,3, and 4. Modified Mosher’s method Dd values are calculated based on the difference of 1H NMR (S-R) in ppm.

(1!3)-O-(a-L-rhamnosyl)-(1!6)-4-O-E-feruloyl-b-D-glucopyra-
noside (7) (Zhou et al., 1998), 2-(3,4-dihydroxyphenyl)-ethyl-O-
a-L-rhamnopyranosyl-(1!3)-O-a-L-rhamnopyranosyl-(1!6)-4-
[O-E-3-(4-hydroxy-3-methoxyphenyl)]-2-propenoate-b-D-gluco-
pyranoside (8) (Abdalla and Abu, 1987), acteoside (9) (Zhou et al.,
1998), prunasin (10) (Huebel et al., 1981), la-hydroxyisoondetm-
none (11) (Zdero and Bohlmann, 1989), and teucardoside (12)
(Elmasri et al., 2014).
All isolated compounds were assayed for anti-oxidant activity
based on a well-established CUPRAC assay with 2 exhibiting low
activity with a TEAC value of 0.11  0.03 mmol TE/100 mmol
compound in comparison with the positive control trolox. Since
a series of the isolated phenylpropanoid metabolites varied in their
glycosylation and methylation functionality, structure–antioxidant
relationships for compounds 6–9 were examined. Increasing sugar
units with accompanying free-phenolic-hydroxyl pairs increased
anti-oxidant activity. Compound 6 exhibited higher antioxidant
capacity than any of the other tri-saccharide phenylpropanoids
assayed. Specifically activities descended in order from 6 (three
sugars, no methylation) >7 (three sugars, single methylation) >8 Fig. 5. TEAC values for tested compounds. Determination of TEAC (Trolox
(three sugars, double methylation) and >9 (two sugars, no equivalent antioxidant capacity) values with CUPRAC assay to study the structure
methylation). Consistent with this trend, polumoside B (De Marino antioxidant activity relationships for glycosylated and methylated phenylpropa-
et al., 2012) (4 sugars, no methylation) has an antioxidant capacity noids including: two sugar moiety with no methylation (9), three sugar moiety with
no methylation (6), three sugar moiety with one methylation (7), and three sugar
greater than 6 suggesting that increasing the number of sugar moiety with two methylation (8) in comparison with trolox, a-tocopherol, and BHA
moieties enhances antioxidant activity. Interestingly, NaOH (median  SD, n = 3).
disruption of polysaccharide spatial structure as observed by
atomic force microscopy has been shown to be correlated with LOX IMVI, and UACC-257, respectively. Compound 5 has induced
reduced antioxidant activity (Liu et al., 2011). Moreover, 6 and 9 the growth of cancer cell lines HOP-92 and HCC-2998 by 29 and
with free ortho-dihydroxyl groups showed higher activity than the 16%, respectively, and decreased the growth of SNB-75 cell line by
positive controls, trolox and a-tocopherol with non-adjacent 17%. Notably, polumoside and teucardoside, which have significant
hydroxyl groups (Fig. 5). These results are consistent with previous antioxidant properties, resulted in varied effects on several cancer
reports that increasing free phenolic hydroxyl pairs enhances cell lines. This inconsistent connection between anti-tumor and
antioxidant activity (Siquet et al., 2006; Krishnamachari et al., antioxidant activity observed with the compounds isolated here
2004). Such phenolic hydroxyl pairs are thought to form hydrogen from T. polium is consistent with in vivo studies (Patterson et al.,
bond between adjacent groups that can stabilize phenoxy radical 1997). In one case, antioxidant supplements had decreased the risk
intermediates (Christodouleas et al., 2009). Hydroxyl methylation of death from gastric cancer but not from esophageal cancer.
decreases antioxidant activity as it destabilizes the intermediate by However, risk of developing gastric cancer and/or esophageal
disrupting hydrogen bonding. cancer were not affected by antioxidant supplementation (Blot
Compounds 4–6, 10 and 12 were also assayed for tumor et al., 1993). In another study, an increase in the incidence of lung
cytotoxicity; growth inhibition percent (GIP) was compared to no- cancer occurred with beta-carotene supplements; in contrast,
drug control and relative to time zero number of cells (Table 2). alpha-tocopherol had no effect on lung cancer incidence (Heino-
This assay allows for detection of both growth inhibition percent nen et al., 1994). Antioxidants supplements in combination with
(GIP) (values between 0 and 100) and lethality (values less than 0). cancer therapy can alter the effectiveness or reduces the toxicity of
Polumoside (6) showed GIP of 10, 9, 10, 12, 12 against NCI- specific drugs (Lawenda et al., 2008). Clearly, additional studies are
H322M, SF-539, OVCAR-4, OVCAR-8, and ACHN cell lines, needed to clarify possible connections between antioxidant
respectively. On the other hand, 6 showed a GIP of 12, 10, and supplements and tumor progression.
10 against NCI-H460, SNB-19, and SK-OV-3 cell lines, respectively.
Teucardoside (12) showed GIP of 12, 9, 10, 16 and 25 3.1. (1R,4S,10R) 10,11-dimethyl-dicyclohex-5(6)-en-1,4-diol-7-one (1)
against NCI/ADR-RES, NCI-H322M, SK-MEL-28, HOP-92 and RXF
393, respectively. However, 12 also exhibited a GIP of 10, 11, 13, 14, Yellow amorphous powder. ½a25 D = 7.20 (c 0.25, MeOH). UVmax
12, 19, and 10 against SR, SK-OV-3, NCI-H23, NCI-H460, SNB-19,
286. IR (KBr) cm1: 3405, 2933, 1724, 1685, 1594, 1374, 1240, 1042,
86 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87

Table 2 3.3. (S)-MTPA ester of 1 (1b)

Growth inhibition percent (GIP) for 4–6, 10 and 12 against human cancer cell line at
the concentration of 10 mM.a 1
H NMR (CD3OD, 400 MHz): 4.80 (1H, dd, H-1), 2.25 (2H, m*,H-
Cancer panel name Cell line name GIP 2), 1.88 (2H, m*,H-2), 1.69 (2H, m, H-3a), 6.02 (1H, s, H-6), 2.39 (1H,
4 5 6 10 12 m*, H-8a), 2.32 (1H, m*, H-8b), 1.75 (1H, m,H-9a), 1.38 (3H, brs, H-
Breast MCF7 7 6 1 2 2
11), 1.37 (3H, s, H-12); an astericks (*) indicates overlaping signals.
MDA-MB-231/ATCC 4 1 3 3 8 HRESIMS m/z: [M + H]+ 427.1701 (calc. 427.1727).
HS 578T 8 12 6 7 8
T-47D 3 8 14 7 17 3.4. (R)-mandelonitrile-b-laminaribioside (2)
Colon COLO 205 11 8 3 6 1
HCC-2998 10 16 2 7 3 Yellow amorphous powder. ½a25 D = 13.84 (c 0.13, MeOH).
HT29 5 3 8 5 1 UVmax 283. IR (KBr) cm1: 3288, 2932, 1713, 1598, 1077, 609.
KM12 4 5 4 0 8
HRESIMS m/z: [M + Na]+ 480.1486 (calc.480.1481) for C20H27NO11.
SW-620 11 13 3 8 3
Elemental analysis C, 52.51; H, 5.95; N, 3.06; O, 38.47.
CNS SF-268 5 6 6 4 -3
SF-295 2 9 4 0 6 3.5. (R)-MTPA ester of 3 (3a)
SF-539 7 2 9 4 7
SNB-19 12 0 10 10 14 1
SNB-75 21 17 2 20 1
H NMR (CDCl3, 400 MHz): 4.83(1H, d, H-1), 1.96 (2H, m*,H-2),
2.18 (2H, m*, H-3), 5.10 (1H, m, H-5), 5.34 (1H, d, H-6), 2.25 (1H, m*,
Leukemia CCRF-CEM 10 9 1 13 3 H-7), 3.84 (1H, m*, H-8), 3.12 (1H, m,H-9a), 5.37 (1H, brs, H-14a),
HL-60(TB) 3 8 3 1 5 5.27 (1H, brd, H-14b), 1.72 (3H, s, H-15); an astericks (*) indicates
RPMI-8226 0 4 0 11 5
overlaping signals. HRESIMS m/z: [M + Na]+ 721.2189 (calc.
SR 3 8 2 3 10
721.2206).
Melanoma LOX IMVI 1 5 7 0 12
M14 1 1 9 1 3 3.6. (S)-MTPA ester of 3 (3b)
MDA-MB-435 10 2 5 9 0
SK-MEL-28 0 12 8 4 10 1
H NMR (CDCl3, 400 MHz): 4.80 (1H, d, H-1), 2.08 (2H, m*,H-2),
SK-MEL-5 17 3 1 15 3
UACC-257 6 9 7 8 19 2.22 (2H, m*, H-3), 5.20 (1H, m, H-5), 5.36 (1H, d, H-6), 2.18 (1H, m*,
UACC-62 9 0 2 3 1 H-7), 3.79 (1H, m*, H-8), 3.11 (1H, m,H-9a), 5.18 (1H, brs, H-14a),
5.17 (1H, brd, H-14b), 1.72 (3H, s, H-15); an astericks (*) indicates
Non-Small Cell Lung A549/ATCC 6 5 4 0 9
overlaping signals. HRESIMS m/z: [M + Na]+ 721.2191 (calc.
EKVX 3 7 6 6 3
HOP-62 6 5 4 15 2 721.2206).
HOP-92 5 29 3 8 12
NCI-H226 9 3 4 10 1 3.7. (R)-MTPA ester of 4 (4a)
NCI-H23 7 1 1 6 11
NCI-H322M 7 1 10 14 9 1
H NMR (CDCl3, 400 MHz): 2.98 (1H, m, H-1), 2.28 (1H, m*,H-2),
NCI-H460 11 10 12 10 13
NCI-H522 16 5 6 19 4 1.14 (1H, m*, H-3a), 2.09 (1H, m, H-3b), 2.94 (1H, d, H-5), 5.22 (1H,
dd, H-6), 1.06 (1H, m, H-7), 1.81 (1H, m*, H-8a), 2.20 (1H, m,H-9a),
Ovarian OVCAR-3 9 8 8 3 8 0.85 (3H, d, H-12), 0.92 (3H, d, H-13), 1.45 (1H, s, H-14), 1.29 (3H, s,
OVCAR-4 7 0 10 4 4
H-15); an astericks (*) indicates overlaping signals.
Prostate PC-3 7 3 5 9 0
DU-145 11 2 5 7 6 3.8. (S)-MTPA ester of 4 (4b)

Renal 786-0 0 7 1 6 1 1
H NMR (CDCl3, 400 MHz): 2.98 (1H, m, H-1), 2.30 (1H, m*,H-2),
A498 1 10 2 4 7
1.18 (1H, m*, H-3a), 2.12 (1H, m, H-3b), 2.85 (1H, d, H-5), 5.24 (1H,
ACHN 11 8 12 12 3
CAKI-1 15 2 4 8 3 dd, H-6), 1.02 (1H, m, H-7), 1.75 (1H, m*, H-8a), 2.19 (1H, m,H-9a),
RXF 393 3 9 16 1 25 0.92 (3H, d*, H-12), 0.95 (3H, d*, H-13), 1.46 (1H, s, H-14), 1.25 (3H, s,
SN12C 16 1 3 17 6 H-15); an astericks (*) indicates overlaping signals.
TK-10 6 4 1 5 0
UO-31 3 9 10 4 10
Conflict of interest
a
Data obtained from NCI-60 DTP human tumor cell line screening. Negative
values expressing growth promotion. The authors declare no conflict of interest.

602. HRESIMS m/z: [M + Na]+ 233.1149 (calc. 233.1148), [M + H]+
211.1330 (calc. 211.1329) for C15H22O10. Elemental analysis C,
68.54; H, 8.63; O, 22.83.
3.2. (R)-MTPA ester of 1 (1a)
1
H NMR (CD3OD, 400 MHz): 4.79 (1H, dd, H-1), 2.13 (2H, m, H-
2), 1.82 (2H, m*,H-2), 1.66 (2H, m*, H-3a), 6.04 (1H, s, H-6), 2.48 (1H,
m*, H-8a), 2.36 (1H, m*,H-8b), 1.90 (1H, m,H-9a), 1.42 (3H, brs, H-
11), 1.36 (3H, s, H-12); an astericks (*) indicates overlaping signals.
HRESIMS m/z: [M + H]+ 427.1702 (calc. 427.1727).
Acknowledgments
The authors are grateful for in vitro screening by the National
Cancer Institute (Bethesda, MD) (http://dtp.cancer.gov). Research
was supported in part by the Robert Welch Foundation (D-1478),
NSF equipment grant CHE-1048553 and NSF CRIF program.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2015.
11.007.
 W.A. Elmasri et al. / Phytochemistry Letters 15 (2016) 81–87
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