Food Chemistry 136 (2013) 120–129

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Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v ie r . c o m / l o c a te / f o o d c h e m

Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and

correlation to their antioxidant and anti-proliferative activity
a,⇑ b b a a
Vassiliki G. Kontogianni , Goran Tomic , Ivana Nikolic , Alexandra A. Nerantzaki , Nisar Sayyad , Stanislava
⇑ ⇑
Stosic-Grujicic b, Ivana Stojanovic b, , Ioannis P. Gerothanassis a, Andreas G. Tzakos a,
aDepartment of Chemistry, University of Ioannina, Ioannina GR-45110, Greece
b Department of Immunology, Institute for Biological Research ‘‘Sinisa Stankovic’’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade, Serbia

article info abstract

Article history: The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia offi-cinalis extracts
Received 29 February 2012 against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array
Received in revised form 18 July 2012 n
detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ ESI-MS ). For the quantitative estimation of triterpenic
Accepted 19 July 2012 Available online 31
July 2012 acids in the crude extracts an NMR based meth-odology was used and compared with the HPLC measurements, both applied
for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of
viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by
Keywords:
Cancer cell apoptosis
nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-a release from macrophages
Nitric oxide therefore enhancing their cytocidal action. The rosemary extract devel-oped more pronounced antioxidant, cytotoxic and
RINm5F immunomodifying activities, probably due to the pres-ence of betulinic acid and a higher concentration of carnosic acid in its
Rosmarinus officinalis extract phytochemical profile.
Salvia officinalis extract LC–
MS/MS 2012 Elsevier Ltd. All rights reserved.
1 13
H– C HSQC and HMBC NMR

1. Introduction Rosemary extract has shown anti-proliferative effects on vari-ous tumor

Medicinal plants have served as rich sources of pharmacologi-cally active
substances. Herbs have been used in a diverse array of purposes including
medicine, nutrition, flavorings, beverages, dyeing, repellents, fragrances,
cosmetics, charms, smoking and industrial uses. Today, herbs are still found in
40% of prescription drugs (Newman & Cragg, 2007). Lamiaceae plants are
now culti-vated worldwide, mainly for use as culinary and medicinal herbs,
and are widely studied as natural antioxidant sources since they are enriched
in polyphenols. Their potent bioactivity and relatively low toxicity have
rendered them useful ingredients in complemen-tary alternative medicine and
as nutritional supplements. Rose-mary (Rosmarinus officinalis L.) and sage
(Salvia officinalis) leaf are popular herbal teas and essential-oil containing
drugs. Rosemary and sage are rich sources of di- and triterpenoids, phenolic
acids, and flavonoids. Carnosic acid, carnosol and rosmarinic acid are the
main antioxidant compounds present in them (Cuvelier, Berset,
cell lines (Cheung & Tai, 2007). Carnosol and carnosic acid isolated from
rosemary leaves have also presented anticancer properties, as determined in
HL-60 cells (Bai et al., 2010). The cyto-toxic activity of sage has not been
studied in detail, but it has been shown that rosmarinic acid and sage extracts
induce apoptosis in colorectal cancer cell lines (Xavier, Lima, Fernandes-
Ferreira, & Pereira-Wilson, 2009). These findings suggest that Lamiaceae
herbs contain several compounds with anti-proliferative activity against
different cancers. As for their immunomodifying properties, the rosemary
extract was found to be mainly anti-inflammatory (Che-ung & Tai, 2007),
while sage extract exhibited pro-inflammatory effects during its anti-
leishmanial activity (Radtke, Yeap Foo, Lu, Kiderlen, & Kolodziej, 2003).
There are still no studies looking into the anti-proliferative activity of sage
and rosemary extracts and also at the effects of their major constituents on rat
insullinoma RINm5F cells. There is also no general investigation of their
phyto-chemical composition and there has been no comparative evalua-tion of
their antioxidant and cytotoxic activities.

& Richard, 1994).

⇑ Corresponding authors. Tel.: +381 11 2078 390; fax: +381 11 2761 433 (I. Stojanovic), tel.:
+30 26510 08397; fax: +30 26510 08799 (V. G. Kontogianni), tel.: +30 26510 08387; fax: +30
26510 08799 (A. G. Tzakos).
E-mail addresses: me01375@cc.uoi.gr (V.G. Kontogianni), ivana@ibiss.bg.ac.rs (I.
Stojanovic), atzakos@cc.uoi.gr (A.G. Tzakos).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.091
The aim of the present study was to characterize the composi-tion of plant
extracts rich in biophenols belonging to the Lamiacea family (ethyl acetate
extracts of R. officinalis and S. officinalis) using LC/DAD/ESI-MS n.
The
main compounds of the extracts were quan-tified using HPLC
and NMR methods. Furthermore, extracts were studied
colorimetrically for their total phenolic and flavonoid con-
tents. Their antioxidant activities were evaluated using the
DPPH
 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
(1,1-diphenyl-2-picrylhydrazyl) method. In addition, the effect of these plant
extracts on viability, apoptosis and NO production was investigated in rat
insulinoma RINm5F cells and their potential immunomodifying properties
were monitored.
2. Materials and methods
2.1. Plant material, reagents and standards
R. officinalis and S. officinalis were commercial samples. All sol-vents
were of appropriate purity and were purchased from various suppliers. Acetic
acid (glacial) was of analytical grade from Merck (Darmstadt, Germany).
Folin–Ciocalteu, phenol reagent and alu-minium chloride were obtained from
Fluka (Switzerland), DPPH ( 90%) was obtained from Sigma–Aldrich
(Steinheim, Germany). Standard compounds were: ursolic acid (90%), caffeic
acid (98%) and betulinic acid (90%) from Aldrich (Steinheim, Germany),
olean-olic acid (97%), carnosic acid, carnosol and rutin (94%) from Sigma
(Steinheim, Germany), rosmarinic acid (95%) and apigenin-7-O-glucoside
(97%) from Fluka (Steinheim, Germany), genkwanin, luteolin (99%) and
apigenin (99%) from Extrasynthese (Genay, France).
2.2. Sample preparation
The plant material was dried with liquid nitrogen and pulver-ized into a
fine powder. The ground leaves were subsequently ex-tracted with two
solvents of increasing polarity, hexane (extraction volume 200 mL), and ethyl
acetate (extraction volume 200 mL), in a Soxhlet apparatus for 6 h for each
solvent. The ethyl acetate ex-tracts were concentrated in a rotary evaporator
and kept into sealed flasks.
2.3. LC–MS analysis
2.3.1. Instrumentation
n
All LC-MS experiments were performed on a quadrupole ion trap mass
analyzer (Agilent Technologies, model MSD trap SL) ret-rofitted to a 1100
binary HPLC system equipped with an degasser, autosampler, diode array
detector and electrospray ionization source (Agilent Technologies, Karlsruhe,
Germany). All hardware components were controlled by Agilent Chemstation
Software.
2.3.2. Analysis
Rosemary and sage extracts were dissolved in ethanol to the de-sired
concentration of 1 mg of dry extract/mL. A 10 ll aliquot was filtered (0.45 lm)
and injected into the LC–MS instrument. Separa-tion was achieved on a 25
cm 4.6 mm i.d., 5 lm Altima C18 ana-lytical column (Alltech, Deerfield,
USA), at a flow rate of 0.6 mL/ min, using as solvent A (water/acetic acid,
99.9: 0.1 v/v) and sol-vent B (acetonitrile). The gradient used for the analysis
of rosemary and sage extracts was: 0–15 min, 80–40% A; 15–20 min 40–25%
A; 20–45 min 25–15% A; 45–50 min 15–10% A; 50–65 min 10% A; 65– 70
min 10–80% A. The UV/vis spectra were recorded in the range 200–400 nm
and chromatograms were acquired at 210, 280 and 330 nm.
2 3
Both precursor and product (MS and MS ) ions scanning of the phenolic
compounds were monitored between m/z 50–m/z 1,000 in negative polarity.
The ionization source conditions were as fol-lows: capillary voltage, 3.5 kV;
drying gas temperature, 350 LC; nitrogen flow and pressure, 12 L/min and 12
psi, respectively. Max-imum accumulation time of ion trap and the number of
MS repeti-tions to obtain the MS average spectra were set at 30 and 3 ms,
respectively.
121
2.4. NMR analysis for the presence of triterpenic acids
NMR experiments were performed on a Bruker AV-500 spec-trometer
equipped with a cryoprobe. Spectra were obtained from ethyl acetate extracts
dissolved in pyridine-d5 (20 mg of material in 0.5 mL of solution in pyridine-
d5) to investigate triterpenoids. The procedure followed was according to the
method employed by Kontogianni, Exarchou, Troganis, and Gerothanassis,
(2009) and described in detail in Supplementary material (Section S1). All
measurements were performed in triplicate (on three different extracts of the
same sample).
2.5. HPLC analysis for the presence of triterpenic acids
Instrumentation and detection mode are described in Supple-mentary
material (Section S2). For the analysis of triterpenoids the extracts were
dissolved in methanol (1 mg/mL). Identification of triterpenoids was based on
retention time, UV spectra and spik-ing. All measurements were performed in
triplicate (on three dif-ferent extracts of the same sample).
2.6. Determination of the total phenolic content
The total phenolic content of the extracts was measured using the Folin–
Ciocalteu method according to the procedure of Gutfin-ger (1981), described
in Supplementary material (Section S3). The results were expressed as mg of
caffeic acid per g of dry ex-tract. All measurements were performed in
duplicate.
2.7. Determination of the total flavonoid content
The content of flavonoids was determined according to Miliaus-kas,
Venskutonis, and Van Beek (2004), described in Supplemen-tary material
(Section S4) using rutin as a reference compound. All determinations were
carried out in duplicate.
2.8. Antioxidant activity: DPPH- radical scavenging assay
The DPPH radical-scavenging effect was evaluated according to the
method employed by Choi et al. (2002), described in Supple-mentary material
(Section S5). All measurements were performed in duplicate.
2.9. Tests performed on rat insulinoma cells (RINm5F) and immune cells
2.9.1. Animals
C57BL/6 mice were bred and housed conventionally at the Insti-tute for
Biological Research ‘‘Sinisa Stankovic’’ (Belgrade, Serbia) under standard
laboratory conditions (non-specific pathogen free) and provided with standard
rodent chow and water ad libitum. The handling of animals and the study
protocol were in accordance with international guidelines and approved by
the local Institu-tional Animal Care and Use Committee.
2.9.2. Cell line
Rat insulinoma RINm5F cells were kindly donated by Dr. Kar-sten
Buschard (Bartholin Instituttet, Copenhagen, Denmark).
2.9.3. Isolation of peritoneal macrophages
Resident peritoneal macrophages were collected by peritoneal lavage with
3 mL cold PBS, centrifuged at 500g and their viability was assessed by
conventional trypan-blue staining (Sigma–Aldrich). Afterwards macrophages
were suspended in culture medium (RPMI-1640 medium (Sigma–Aldrich, St.
Louis, MO, USA) supple-mented with 2 mM L-glutamine, 20 lg/mL
gentamycine (Galenika
122 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
5
a.d., Serbia), 0.01% sodium pyruvate, 5 10 M 2-mercap-toethanol,
antibiotics (all from Sigma–Aldrich) and 5% (v/v) heat-inactivated FCS
(foetal calf serum) (PAA Chemicals, Pasching, Aus-tria)) and seeded into 96-
5
well flat-bottom plates (Sartstedt, Numbr-echt, Germany) (1 10 /well) for the
6
Griess reaction and into 24-well plates (Sartstedt) (1 10 /well) for ELISA.
2.9.4. Determination of cell viability and apoptosis
RINm5F cells were grown in culture medium containing 10% FCS in a
4
humidified (5% CO2, 95% air) atmosphere at 37 LC. RINm5F cells (1 10
cells/well) were seeded in 96-well plates (Sardstedt, Numbrecht, Germany)
and treated with specific extracts dissolved in DMSO (Sigma–Aldrich) (12.5–
100 lg/mL) or matching concen-trations of the mentioned solvent. After 24 h
incubation, the pro-portion of attached cell (cell viability) was assessed by
crystal violet staining. Briefly, after fixation with methanol for 10 min, crystal
violet solution (MOL, Belgrade, Serbia) was added to the culture wells and
incubated at 25 LC for the next 15 min. After thorough washing in tap water,
incorporated dye was solubilized in 33% acetic acid (Zorka Farma, Sabac,
Serbia) and the developed colour was recorded in an automated microplate
reader (LKB 5060–006, LKB, Vienna, Austria) at 570 nm, with a correction at
670 nm. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide
(MTT) (Sigma–Aldrich) assay, cell respiration was as-sessed by the
mitochondrial-dependent reduction of MTT to coloured formazan product
(Mosmann, 1983), which reflected cell viability. Briefly, following incubation
with the extracts, RINm5F cells were pulsed for an additional hour with 0.5
mg/mL MTT, cul-ture media were aspirated and the cells lysed in DMSO. The
con-version of MTT to formazan was monitored in an automated microplate
reader at 570 nm, with a correction at 670 nm. Cell via-bility was expressed
as a percentage of the control value (un-treated cells) which was arbitrarily set
to 100%. The concentration of the extracts that induced 50% reduction in cell
viability (IC50) was calculated according to the formula:
((50% lower inhibition)/(higher inhibition lower inhibi-tion) (higher dose
lower dose)) + lower dose, where lower inhi-bition represented % of non-
viable cells below 50%, higher inhibition represented % of non-viable cells
above 50%, higher and lower dose were corresponding concentrations of
extracts.
For detection of DNA–histone complexes present in the cytoplas-mic
4
fraction of apoptotic RINm5F cells (1 10 cells/well), Cell Death Detection
ELISA (Roche, Basel, Switzerland) was used. Briefly, after 24 h treatment
with the specific extracts, cells were disrupted in the supplied lysis buffer for
30 min at room temperature and then centrifuged at 20,000g for 10 min. After
centrifugation, supernatants containing the cytoplasmic fraction were
removed carefully and as-sayed following manufacturer’s instructions.
Results were displayed as absorbances detected at 450 nm, where higher
absorbance repre-sents more DNA–histone complexes present.
2.9.5. Determination of nitric oxide (NO) production
Nitrite accumulation, an indicator of NO production, was mea-sured in
cell culture supernatants using the Griess reagent. The supernatants (50 lL)
were mixed with an equal volume of Griess reagent (a mixture at 1:1 of 0.1%
naphthylethylenediamine dihy-drochloride and 1% sulfanilamide in 5%
H3PO4–all from Sigma–Al-drich) and the absorbance at 570 nm was
measured on a microplate reader. The nitrite concentration was calculated
from a NaNO2 standard curve. The data obtained from triplicates are
presented as lM of nitrite.
2.9.6. Determination of tumor necrosis factor a (TNF-a) secretion The content
of TNF-a (tumor necrosis factor alpha) in cell-free
culture supernatants was determined by sandwich ELISA using MaxiSorp
plates (Nunck, Rochild, Denmark). Samples were ana-
lysed in duplicate for murine TNF- a (BD Pharmingen, San Diego, CA, USA)
according to the manufacturer’s instructions. The absor-bance at 450 nm was
determined in a microplate reader. The re-sults were calculated using standard
curves made on the basis of known concentrations of the recombinant TNF- a.
To allow statisti-cal evaluation of the data, the samples in which the value of
the cytokine was below the limit of sensitivity of the assay was as-signed
values equivalent to this limit.
2.9.7. Statistical analysis
The results are presented as means ± SD of triplicate observa-tions from
one representative of at least three experiments with similar results. The
significance of the differences between various treatments was analysed by
analysis of variance (ANOVA), fol-lowed by Student-Newman–Keul’s test. A
p value less than 0.05 was considered to be statistically significant.
3. Results and discussion
3.1. The phytochemical composition of extracts – LC–MS analysis
n
The LC/DAD/ESI-MS analysis of the rosemary extract led to the
separation and identification of the majority of the constituents; overall, 17
compounds were identified belonging to three repre-sentative classes of
constituents: diterpenes, flavonoids and trite-rpenic acids. Rosmarinic acid
was the only hydroxycinnamic derivative that was identified. Because
polyphenols contain one or more hydroxyl and/or carboxylic acid groups, MS
data were ac-quired in negative ionization mode. Identification of the com-
pounds was carried out by comparing retention times and masses with those
of the 7 authentic standards. For the remaining 10 compounds for which no
standards were available, identifica-tion was based on accurate mass
measurements of the pseudomo-lecular [M–H] ions and their fragmentation
pattern, as has been documented in the literature (Cuvelier et al., 1994). In
Fig. 1A, the total ion current (TIC) chromatogram (i) and UV chromatogram
(ii) at 280 nm of the investigated extract are presented. Data ob-tained from
n
the ESI-MS analysis of the extract are summarised in Table 1.
Compound 1 gave a [M–H] ion at m/z 477 attributed to isorh-amnetin-3-
O-hexoside. Its MS/MS data showed the loss of a hexose moiety (162 amu)
resulting in the fragment ions at m/z 315 corre-sponding to a deprotonated
molecular ion of isorhamnetin and at m/z 300, whereas further fragmentation
of the ion at m/z 315 pro-duced ions at m/z 300 and m/z 271 corresponding to
subsequent
loss of methyl groups, in agreement with literature data (Hossain, Rai,
2 3
Brunton, Martin-Diana, & Barry-Ryan, 2010). Likewise, the MS and MS
experiments for compound 2 with [M–H] at m/z 461 showed the loss of a
hexose moiety and of a methyl group, respec-tively, confirming the presence
of homoplantaginin, which has been previously reported in rosemary and sage
extracts (Borrás Linares et al., 2011; Cuvelier, Richard, & Berset, 1996). The
last fla-vonoid identified, compound 4, was the aglycon hispidulin (at m/z
299) eluting at 18.8 min.
Three peaks with [M–H] at m/z 345, were found, i.e., isomers of rosmanol
(compounds 5, 6 and 7; retention times 21.1, 22.0, and 22.6 min,
2
respectively). MS analysis at m/z 345 gave the m/z 301 and m/z 283 peaks,
corresponding to the [M–H–CO2] and [M– H–CO2–H2O] fragments,
respectively, in agreement with litera-ture data (Cuvelier et al., 1996;
Zimmermann, Walch, Tinzoh, Stüh-
linger, & Lachenmeier, 2011). Compound 9 could be attributed to
methoxycarnosol (m/z 359), as its MS2 analysis gave one main product ion at
m/z 329 corresponding to loss of methoxy group and the MS 3 spectra of the
ion at m/z 329 yielded the ion at m/z 285 attributable to the
subsequent loss of a carbon dioxide mole-
 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
Fig. 1. (A) Total ion chromatogram (i) and UV chromatogram (ii) at 280 nm of rosemary extract. (B) Total ion chromatogram (i) and UV chromatogram (ii) at 280 nm of sage extract.
cule. The deprotonated molecular ion [M–H] of compound 10 was detected at
2
m/z 343, and its MS spectra gave the m/z 315 and m/z
300 peaks, consistent with [M–H–CH2CH3] and [M–H– CH2CH2CH3]
fragment ions, respectively. Such a fragmentation pathway for rosmadial has
been described in the literature (Hoss-ain et al., 2010). The precursor MS
analysis of compound 12 re-sulted in the deprotonated ion [M–H] at m/z 315
that could be
tentatively characterized as rosmaridiphenol (Borrás Linares et al., 2011).
2
Finally, compound 14 (m/z 345) corresponded to methyl carnosate as its MS
spectra gave a base peak at m/z 301 due to loss of a carbon dioxide molecule,
with further loss of a methyl group producing m/z 286 ions. This
fragmentation pattern was in agreement with that reported by Herrero, Plaza,
Cifuentes, and Ibáñez (2010) and Hossain et al. (2010).
n
Respectively, the LC/DAD/ESI-MS analysis led to the separation and
identification of the majority of the constituents; overall, 18 compounds were
identified in the sage extract (Table 1), most of them being common with
those of rosemary extract. In Fig. 1B the total ion current (TIC)
chromatogram (i) and UV chromatogram
(ii) at 280 nm of the investigated extract are presented. Compound 1 and 2
(apigenin-7-O-glucoside and homoplantaginin), having very close retention
times, co-eluted giving one peak in the UV chromatogram and their
discrimination was achieved using their
123
MS spectra. Compound 4 gave a [M–H] ion at m/z 474 attributed to apigenin-
acetyl-glucoside. Its MS/MS data showed the loss of an acetylhexose moiety
(204 amu) resulting in the fragment ion at m/ z 269 corresponding to a
deprotonated molecular ion of apigenin. Compound 5 (m/z 315) eluting at
16.2 min was identified as isorh-amnetin since the loss of methyl groups,
resulting in fragment ions m/z 300 and 271, was observed in its fragmentation
pathway. Lute-olin was co-eluted with isorhamnetin, identified by comparison
of its retention time and mass with those of the authentic standard.
3.2. Quantitative analysis
3.2.1. Content of triterpenic acids
The triterpenic acids (oleanolic, ursolic and betulinic acid) have
previously been identified to possess anticancer properties in both rat and
human cell lines (Janakiram et al., 2008; Kassi et al., 2007; Pandey, Sung, &
Aggarwal, 2010). Additionaly, oleanolic acid and its analogues prevented
colon carcinogenesis in male F344 rats (Janakiram et al., 2008).
Since the
major purpose of our current study was to investigate the
effects of rosemary and sage extracts on the apoptosis of
cancer cell lines, it was essential to determine the
composition of these extracts regarding to the exact quantity
of the above triterpenic acids.
124 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
Table 1

Peak assignments of rosemary and sage extracts. (Rt: retention time).

[M–H] (m/z) 2 3 Compounds b
Rt (min) MS [M–H] (m/z) (%) MS [base peak] (m/z) (%) Detected in
10.9 477 315 (100), 300 (27), 462 (4) 300 (100), 271 (41) Isorhamnetin-3-O-hexoside R1
11.9 431 269 (100) 269 (100) a S1
Apigenin-7-O-glucoside
12.1 461 299 (100), 284 (70), 446 (44) 284 (100), 297 (97), 266 (10) Homoplantaginin R2, S2
12.4 359 161 (100), 179 (30), 197 (24) 133 (100) a R3, S3
Rosmarinic acid
14.2 474 269 (100) 269 (100) Apigenin-acetylglucoside S4
a
16.2 315 300 (100), 271 (40) 300 (100) Isorhamnetin-luteolin S5
18.3 269 269 (100) 269 (100) a S6
Apigenin
18.8 299 283 (100), 217 (21) 226 (100), 137 (51) Hispidulin R4, S7
21.1 345, 691 301 (100), 283 (4) 283 (100), 258 (19) Rosmanol isomer R5, S8
22.0 345 283 (100), 301 (85) 227 (100), 267 (47), 289 (60) Rosmanol isomer R6,S9
22.6 345 283 (100) 227 (100), 267 (50) Rosmanol isomer/ Epirosmanol R7, S10
23.5 388 344 (100), 284 (15) 284 (100) Unknown 0
S1
0
25.0 390 346 (100) 346 (100) Unknown S2
0
26.5 687 299 (100) 299 (100) Unknown S3
24.0 283 268 (100) 268 (100) a R8
Genkwanin
26.9 359 329 (100) 285 (100) Methoxycarnosol R9, S11
27.7 329 285 (100) 269 (100), 299 (27), 201 (17) a R10, S12
Carnosol
28.8 343 315 (100) 300 (100) Rosmadial R11, S13
30.9 315 300 (100) 300 (100) Rosmaridiphenol R12
32.4 331, 685 287 (100) 243 (100) a R13, S14
Carnosic acid
36.2 345 301 (100), 286 (21) 286 (100) Methyl carnosate R14, S15
37.4 317 317 (100), 179 (88) 314 (100) Unknown 0 0
R1 , S4
0 0
40.9 317 315 (100), 287 (41) 287 (100) Unknown R2 ,S5
0 0
49.0 453 405 (100) 405 (100) Unknown R3 , S6
a
52.2 455, 565 452 (100) 452 (100) Betulinic acid R15, S16
a
54.5 455, 565 407 (100) 377 (100) Oleanolic acid R16, S17
55.1 455, 565 453 (100) 453 (100) a R17, S18
Ursolic acid
a
Identification confirmed using commercial standards.
b 0
R: rosemary, S: sage, number indicates the peak in TIC chromatogram, is used for nonidentified compounds.

For the quantitative estimation of triterpenic acids a methodol-ogy
introduced by our group for the discrimination and quantifica-tion of
oleanolic and ursolic acid (Kontogianni et al., 2009) was expanded and
applied to the identification of betulinic acid in the rosemary extract. Since
these analytes have weak chromoph-ores and low UV absorption, their
resolution by LC is rather diffi-cult. Therefore, we employed a strategy based
1 13 1 13
on 2D H– C HSQC ( H– C Heteronuclear Single-Quantum Coherence)
1 13 1 13
and H– C HMBC ( H– C Heteronuclear Multiple-Bond Correlation) NMR
1 13 1 13
experiments. H– C HSQC and H– C HMBC spectra of a mixture of
oleanolic, ursolic and betulinic acid were first recorded in order to investigate
whether their identification in the 2D NMR map is feasible. Most of the one-
bond proton and carbon nuclei interactions of these triterpenic acids overlap;
however, there are significant differences in the cross peaks of C12–H12,
C18–H18, and C22–H22 for oleanolic and ursolic acid and in the cross peaks
of C13–H13, C16–H16, C19–H19, C22–H22 and C29–H29 of betuli-nic acid.
1 13
The H and C chemical shifts of the NMR signals that were utilized for the
identification and quantification of these mol-ecules (the selected cross peaks
that were plotted as a function of concentration), are reported in Table S1
(Supplementary data). The peak intensities were calculated as the mean value
of the absolute intensities of the cross peaks of C13–H13 and C19–H19 of
betulinic acid and C12–H12 and C18–H18 for oleanolic and ursolic acid (Fig.
2A).
1 13
Focusing on the long range connectivities in the 2D H– C HMBC map,
of diagnostic importance for the presence of betulinic acid could be the cross-
peaks of the allylic H29, showing long-range connectivities with C19 and
C30 (Fig. 2B). Furthermore, of diagnostic importance could be the cross-
peaks of H16 to C13, C14, C18, C17 and C28. For oleanolic and ursolic acid
1 13
the diagnostic long range connectivities in the 2D H– C HMBC map are
given in Table S1 (Supplementary data).
1 13
Quantitative results could be obtained from 2-D H– C data acquisitions
of 14 min. The concentration limit was found to be 3 mM. To further compare
our NMR data, detailed HPLC measure-
ments were also carried out. For the HPLC analysis of triterpenic acids,
addition of derivatized cyclodextrins (HP-c-CD) to the aceto-nitrile–
phosphate buffer mobile phase according to Claude, Morin, Lafosse, and
Andre (2004) was used to improve their resolution. The use of cyclodextrins
for the determination of betulinic acid in plant extracts is reported for the first
time herein. Using a flow rate of 1.0 mL/min, the retention time of betulinic
acid was 15.8 min, of oleanolic acid 30.2 min and of ursolic acid 33.5 min
(Figure S1 Supplementary data).
In the sage extract, betulinic acid was found in trace amounts so it was not
possible to quantify it either with HPLC or with NMR based methods. The
relevant results are presented in Table 2. In sage ex-tract, as in the case of the
rosemary extract, the dominant com-pounds were ursolic acid, having a
significantly high concen-tration, followed by oleanolic acid. Baricevic et al.,
(2001), reported ursolic acid as the main component of a chloroform extract
of sage, detected at a concentration of 480 mg/g dry extract, which was re-
ported to have anti-infammatory activity, along with oleanolic acid.
3.2.2. Quantitative measurement of diterpenes, flavonoids and
rosmarinic acid
Quantitative analysis was performed by means of HPLC methods using a
diode array detector. Since reference compounds were not available for most
of the detected compounds, flavonoid glucosides were quantitated as
apigenin-7-O-glucoside (at 330 nm), flavonoid aglycones as apigenin (at 330
nm) and diterpenes as carnosic acid (at 280 nm). The calibration curves were
2
proved to be linear in the ranges of 0.5–10 mg/L with R 0.9984 (apigenin-7-
2
O-glucoside), 0.5–10 mg/L with R 0.9909 (apigenin) and 2–250 mg/L with
2
R
0.9952 (carnosic acid). The calibration curves for genkwanin and rosmarinic
acid were also found to be linear in the ranges 1– 20 mg/L with R 2 0.9947 and
5–20 mg/L with R2 0.9947, respec-tively. In total, 7 flavonoids, 8 diterpenes,
3 triterpenic acids and rosmarinic acid were quantified. In S. officinalis
extract the content of luteolin was determined together with isorhamnetin and
apige-nin-7-O-glucoside together with homoplantaginin.
 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129 125

1 13 1 13
Fig. 2. (A) 500 MHz 2D H– C HSQC spectrum of the rosemary extract (40 mg/ ml) (ns = 2, experimental time: 14 min); (B) 500 MHz 2D H– C HMBC spectrum of the same
solution as in (A) (ns = 64, mixing time 50 ms, experimental time: 4 h & 16 min). (OA: oleanolic acid, UA: ursolic acid, BA: betulinic acid).

The quantitative results are summarised in Table 2. The quanti-tatively
dominating compounds in rosemary extract were ursolic acid followed by
carnosic acid, while in the sage extract they were ursolic acid followed by
oleanolic acid. In the rosemary extract, rosmanol and carnosol had similar
concentrations, being eight times smaller in comparison with carnosic acid. In
the sage extract, rosmanol had the same concentration as carnosic acid,
followed by carnosol. The content of rosmarinic acid in both extracts was the
same, while the flavonoids identified were minor components in both
extracts. The most frequently used extraction solvents re-ported in the
literature for rosemary and sage were water, metha-nol, ethanol and acetone,
resulting in extracts with main compounds carnosic acid and rosmanol
(Cuvelier et al., 1996), car-nosol and carnosic acid (Herrero, Plaza, Cifuentes,
& Ibáñez, 2010), rosmarinic acid and luteolin-7-O-glucoside (Zimmermann et
al., 2011).
126 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
Table 2

Concentrations (± standard deviation) of compounds in rosemary and sage extracts (mg/g dry extract).

Compound Measured as Rosemary extract Sage extract

Isorhamnetin-3-O-hexoside Ag 1.0 ± 0.21 –
a – 3.6 ± 0.75
Apigenin-7-O-glucoside
Homoplantaginin Ag 1.5 ± 0.09
a
Rosmarinic acid 11.6 ± 1.20 10.0 ± 0.92
a
Isorhamnetin-luteolin A – 2.9 ± 0.11
a – 2.5 ± 0.38
Apigenin
Hispidulin A 1.5 ± 0.29 6.3 ± 0.58
Rosmanol isomer (Rt 21.1 min) CA 11.0 ± 0.18 6.1 ± 1.16
Rosmanol isomer (Rt 22.0 min) CA 23.5 ± 2.35 42.8 ± 3.05
Rosmanol isomer/ Epirosmanol (Rt 22.6 min) CA 1.7 ± 0.33 4.0 ± 0.24
Genkwanin 2.0 ± 0.52 –
Carnosol CA 21.5 ± 2.11 31.1 ± 1.00
Rosmadial CA 8.7 ± 0.22 6.8 ± 0.42
Rosmaridiphenol CA 18.2 ± 0.94 –
a * 42.9 ± 3.05
Carnosic acid 177.3 ± 9.71
Methyl carnosate CA 7.9 ± 0.13 8.6 ± 0.22
HSQC HPLC HSQC HPLC
a
Betulinic acid 46.9 ± 5.9* *
34.8 ± 6.7 – –
a
Oleanolic acid 89.7 ± 4.6 97.1 ± 12.7 171.9 ± 10.6 178.7 ± 13.0
a * * 358.8 ± 14.2 349.7 ± 17.6
Ursolic acid 190.1 ± 8.3 186.7 ± 19.2
Total phenolic content mg CfA/g d. e. 54.6 ± 2.2 73.7 ± 2.6

Total flavonoids content mg R/g d. e. 24.6 ± 5.0 39.3 ± 5.3

Abbreviations: Ag: apigenin-7-O-glucoside; A: apigenin; CA: carnosic acid; CfA: caffeic acid; R: rutin; d.e.: dry extract; R t: retention time.
(The triterpenic concentrations of sage extract are already published (Kontogianni et al., 2009), but are given for comparison reasons).
a
Identification confirmed using commercial standards.
*
Denotes the difference in the concentration of these compounds that are responsible for the cytotoxic activity of the extracts.

3.3. Antioxidant activity and total phenolic and flavonoid content
The total phenolic and total flavonoid content are given in Table 2. Sage
extract showed higher phenolic and flavonoid con-tents in comparison to
rosemary extract, as was expected from the higher concentration of
flavonoids. The antioxidant activity evaluated by the use of the DPPH radical-
scavenging assay was found SC50 = 40.6 ± 2.6 lg/mL for the rosemary extract
and SC50 = 78.0 ± 1.7 lg/mL for the sage extract. The concentration of the
sample (standard compound or plant extract) ( lg/mL) that is sufficient to
obtain 50% of a maximum scavenging capacity of the stable DPPH radical is
defined as SC50. Thus, the SC50 value is neg-atively related to the antioxidant
activity. The antioxidant activity of these extracts is due to the content of
phenolic abietane diter-penes such as carnosic acid and its derivatives,
carnosol and rosm-anol isomers. Moreover, the presence of rosmarinic acid
additionally contributes to the activity detected. Cuvelier et al. (1996)
provided a correlation between antioxidant efficiency and the composition of
sage, indicating that carnosol, rosmarinic acid and carnosic acid had the
greatest antioxidant activities among its constituents. Although some
flavonoids are potent antioxidants, the identified flavonoids made a rather
small contribution to the total antioxidant capacity of the extracts due to their
low abun-dance. This could provide a reasonable explanation for the obser-
vation that the sage extract, although containing increased levels of phenolics
and flavonoids, exhibited lower antioxidant capacity in comparison to the
rosemary extract. Furthermore, the enhanced antioxidant effect that rosemary
extract possessed, can probably be attributed to the significantly higher
concentration of carnosic acid, or to the synergistic effects of its different
constituents.
3.4. The influence of plant extracts rich in biophenols on viability of rat
insulinoma RINm5F cells
In order to evaluate potential anticancer properties of the stud-ied plant
extracts, RINm5F cells were treated with increasing con-centrations of the
extracts. The viability of treated cells was
determined by the crystal violet assay, where the intensity of the dye
correlates to the number of attached viable cells. Results ob-tained indicate
that the rosemary extract was more potent in reduc-ing RINm5F cell viability
with an IC50 of 35.6 lg/mL compared to the sage extract. S. officinalis extract
was operative at concentrations of
50 to 100 lg/mL (IC50 101.5 lg/mL). When tested at 100 lg/mL, both
rosemary and sage extracts induced a decrease in RINm5F cell viability by
91% and 40%, respectively. Importantly, the solvent of both extracts (DMSO)
was ineffective regarding its impact on cell viability (Fig 3A). Although these
extracts have never been com-pared for their efficacy, similar results were
obtained with both ex-tracts in vitro on human melanoma M14, A375 cells,
human colon carcinoma cells Caco-2, hepatoma cells HepG2 and HCT-116
colon cancer cells. Rosemary and sage extracts reduced the growth and in-
duced cell death in the respective cancer cell lines (Russo, Lom-bardo,
Troncoso, Garbarino, & Cardile, 2009). These in vitro data are in accordance
with the observed effects of these extracts on the reduction of mammary
tumor growth and a number of skin tu-mors in mice (Visanji, Thompson, &
Padfield, 2006).
Next, we wanted to determine the effect of rosemary and sage extracts on
the function of mitochondria, key energy producers and controllers of cell
death. Mitochondrial function was measured by the colorimetric MTT assay
which detects cell respiration. Both rosemary and sage extracts down-
regulated cell respiration of RINm5F. Seemingly, their effects on
mitochondrial function strongly correlated with the previously observed
influence on cell viability (Fig 3B).
The observed difference in cytotoxic activity of these extracts probably
stems from the difference in their phytochemical compo-sitions. In order to
determine the primary cytotoxic components of sage and rosemary extracts,
the cytotoxic activity of the major con-stituents was evaluated. The major
differences in the phytochem-ical profile of rosemary extract relative to the
sage extract were located in the composition of carnosic acid, ursolic acid and
shows that the rosemary extract
betu-linic acid (Fig 3C). Table 2
con-tains a fourfold higher concentration of carnosic acid and
a twofold lower concentration of ursolic acid (concentrations
are de-
 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129 127

Fig. 3. The effect of sage and rosemary extracts on RINm5F cell viability. Viability of RINm5F cells cultured for 24 h with increasing concentrations of extracts or left untreated was determined by
crystal violet assay (A) or MTT assay (B). Data are presented as mean ± SD. indicates p < 0.05 of extract-treated vs. untreated cells, while # indicates p < 0.05 of Salvia-treated vs. Rosmarinus-treated
RINm5F cells. (C) Structures of phytochemicals that were altered in the phytochemical profile of the sage and rosemary extract: carnosic acid, ursolic acid and betulinic acid (D). Cytotoxic effects of
carnosic acid, ursolic acid and betulinic acid. Viability of RINm5F cells cultured for 48 h with carnosic, ursolic or betulinic acid (each at a concentration of 50 g/ml) or left untreated was determined
by crystal violet assay. Results are presented as % of remaining viable cells compared to untreated cells (100% viable). Data are presented as mean ± SD. indicates p < 0.05 of acid-treated vs.
untreated cells, while # indicates p < 0.05 of ursolic or betulinic-treated vs. carnosic acid-treated RINm5F cells. (E) Rosemary and sage extracts induce apoptotic cell death in RINm5F. Apoptosis of
RINm5F cells was determined after 24 h of incubation with extracts. Data are presented as mean ± SD. indicates p < 0.05 of extract-treated vs. untreated cells, while # indicates p < 0.05 of Salvia-
treated vs. Rosmarinus-treated RINm5F cells. (F) The effect of extracts on NO production in RINm5F cells. Nitrite accumulation, as a measure of NO production was determined after 24 h incubation
with the relevant extracts. Data are presented as mean ± SD. indicates p < 0.05 of extract-treated vs. untreated cells, while # indicates p < 0.05 of Salvia-treated vs. Rosmarinus-treated RINm5F cells.
128 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129

⁄
noted with ), compared to sage. Furthermore, the sage extract has only traces
of betulinic acid. Our results indicate that carnosic acid was the most potent in
inducing cell death of RINm5F cells com-pared to ursolic and betulinic acid
(Fig 3D). After 48 h of cultivation with carnosic acid only 23% of RINm5F
cells were viable, 33% in the presence of ursolic, while 73% cells survived
betulinic acid treat-ment. Therefore, the observed strong cytotoxic effect of
rosemary extract could be attributed to the higher concentration of carnosic
acid and probably to its additive effect with betulinic acid, the lat-ter of which
is detected only in trace amounts in sage extract. In-deed, many reports
indicate that carnosic acid and betulinic acid act in a cytotoxic and anti-
proliferative manner in tumor cells (Bai et al., 2010). Carnosic acid also
promotes generation of reac-tive oxygen species and mitochondria
dysfunction in human neu-roblastoma IMR-32 cells (Tsai, Lin, Lin, & Chen,
2011). Similarly, we showed here that rosemary extract (containing a high
concen-tration of carnosic acid) strongly impaired mitochondrial function in
RINm5F cells, probably inducing apoptosis.

3.5. Rosemary and sage extracts induce apoptosis of RINm5F cells

In order to determine whether the observed cytotoxic effect ex-erted by

the extracts was mediated by induction of apoptotic death, internucleosomal
DNA fragmentation was monitored. The frag-mented DNA is released into
the cytoplasm as a DNA–histone com-plex. Therefore, we detected DNA–
histone complexes that are present in apoptotic cells. Interestingly, we found
that rosemary ex-tract induced apoptotic cell death in a dose-dependent
manner, whereas sage extract was significantly less pro-apoptotic (Fig 3E).
This is in accordance with findings by other groups suggesting that the mode
of action of rosemary and sage cytotoxic effects is medi-ated via apoptosis
(Xavier et al., 2009). Furthermore, apoptosis induction in RINm5F cells by
the extracts could be related to the pre-viously observed potent inhibition of
mitochondrial function. Fig. 4. Salvia and Rosmarinus extracts exert an immunomodifying effect. Peritoneal
macrophages were incubated with increasing concentrations of extracts and NO production (A)
or TNF-a secretion (B) were determined after 24 h. Data are presented as mean ± SD. indicates
p < 0.05 of extract-treated vs. untreated macrophages, while # indicates p < 0.05 of Salvia-
3.6. Rosemary and sage extracts induce RINm5F cell apoptosis via NO treated vs. Rosmarinus-treated macrophages.
production

Several clinical and experimental studies indicate that the pres-ence of NO anti-cancer properties of the studied plant extracts could be acti-vated
in tumor microenvironments is detrimental to tumor cell survival and indirectly through activation of macrophages. Again, rose-mary extract is
metastasis (Singh & Gupta, 2011). Therefore, we tested NO production after significantly more potent in inducing NO in macrophages, as compared to the
24 h of RINm5F cell cultivation in the presence of the extracts. The results sage extract. However, up-regu-lation of TNF- a secretion correlates with the
illustrated that both extracts significantly increased NO production in
IC50 concentrations of extracts: rosemary extract was more effective in a dose
RINm5F cells. As was also identified in the case of apoptosis induction,
of 50 lg/ mL, while 100 lg/mL of sage extract elevated TNF- a level to a high-
rosemary extract was again a stronger activator of NO production compared
er extent in comparison to the level elevated by the rosemary ex-tract. In
to matching concentrations of sage extract (Fig 3F). The specific role of NO
addition to boosting immune cell function, sage and rosemary extracts (i.e.
in the regulation of apoptosis/survival-related genes expression seems to tilt rosmarinic acid) effectively inhibit angio-genesis (Keshavarz et al., 2010) and
the balance toward the promotion of pro-apoptotic genes and the suppression (Huang & Zheng, 2006) there-fore create a potent anti-tumor environment.
of anti-apoptotic genes (Olson & Gar-bán, 2008). Therefore, the observed up-
regulation of NO in cancer cells could, at least partly, account for the
induction of apoptosis by the investigated extracts.

4. Conclusions

3.7. The effect of rosemary and sage extracts on the function of
macrophages
Macrophages are cells of innate immunity and have the ability to destroy
cancer cells by secreting various pro-inflammatory mediators (Klimp, De
Vries, Scherphof, & Daemen, 2002). One of these mediators, NO, was
enhanced in a dose-dependent manner after the treatment of macrophages
with the extracts (Fig 4A). In addition, cytotoxic cytokine TNF-a was
significantly up-regulated in macrophages treated with either rosemary or
sage extracts (Fig 4B). Since macrophage cytocidal function is largely
mediated by the production of NO and TNF-a, it can be concluded that
The phytochemical analysis of R. officinalis and S. officinalis ex-tracts
revealed the presence of several constituents, classified in the diterpene,
triterpenoid and flavonoid natural product classes, most of them being
common for both extracts. The quantitatively dominant compounds in these
extracts, quantified by HPLC and NMR methods, were ursolic acid followed
by carnosic acid in the rosemary extract, while in the sage extract, they were
ursolic acid followed by oleanolic acid. Also, another difference between the
two extracts was the presence of betulinic acid, in significant amounts, in the
rosemary extract.
This study also illustrated that rosemary and sage extracts
pos-sess strong anticancer properties. However, rosemary
extract pre-
 V.G. Kontogianni et al. / Food Chemistry 136 (2013) 120–129
sented higher cytotoxic effects in comparison to the sage extract. Importantly,
a positive linear correlation between antioxidant activity and cytotoxicity was
determined between the two ex-tracts, with the rosemary extract presenting
higher antioxidant and cytotoxic activities. The antioxidant activity of
rosemary ex-tract is attributed to carnosol, rosmarinic acid and carnosic acid,
while its cytotoxic activity could be attributed to carnosic acid, and the
triterpenoids betulinic and ursolic acid. When combined, these two features of
the investigated extracts could provide both direct destruction of cancer cells
and protection of healthy cells during cancer treatment (antioxidant activity).
To gain a better understanding of the enhanced cytotoxic activity
illustrated by the rosemary extract relative to the sage extract, direct mapping
of the network of interactions developed between individ-ual constituents and
cellular components needs to be performed (Janga & Tzakos, 2009). The
observed differentiation in the cytotoxic activity between the two extracts
could be primarily attributed to their altered phytochemical profiles. The
major differences in the phytochemical profiles of the two extracts were
identified as the fourfold higher concentration of carnosic acid in the
rosemary ex-tract as also the presence of a significantly higher amount of
betuli-nic acid in it with respect to the sage extract. Among the major
constituents presented in the two extracts carnosic acid was found to present
the strongest cytotoxic activity and a potential synergistic effect with betulinic
acid could be responsible for the differential cytotoxic activity determined for
the two extracts.
Furthermore, it was found that both extracts exerted direct cyto-cydal
effects via up-regulation of nitric oxide (NO) in cancer cells, which in turn
acts in a pro-apoptotic manner and induces cell apop-tosis. On the other hand,
through activation of macrophages and their secretion of pro-inflammatory
mediators, extracts could prob-ably indirectly induce cancer cell death.
Preferentially, for the elim-ination of cancer cells, anticancer compounds
should both directly induce cancer cell death and also boost the immune
system to exert their anti-tumor properties. Therefore, the recorded
potentiation of immune cell function by these extracts is of great importance
for the future development of potential anticancer therapy.
Acknowledgements
This work was co-funded by Esthir Gkani Foundation, (Ioannina, Greece)
and the Project of Ministry of Education and Science, Republic of Serbia
(No.: 173013). Special thanks are given to the Mass Spectrometry Unit and
NMR center of University of Ioannina for providing access to LC-MS/MS,
HPLC and NMR facilities. The authors have declared no conflict of interest.
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.foodchem.2012. 07.091.
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