PHYTOTHERAPY RESEARCH

Phytother. Res. 27: 1206–1213 (2013)

Published online 2 October 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.4853

Chemical Composition and Biological Activities of

Polar Extracts and Essential Oil of Rose-scented
Geranium, Pelargonium graveolens

Maher Boukhris,1 Monique S. J. Simmonds,2 Sami Sayadi1 and Mohamed Bouaziz1,2,3*

1
Laboratoire des Bioprocédés Environnementaux, Centre de Biotechnologie de Sfax, BP: «1177» 3018, Sfax, Tunisie
2
Royal Botanic Gardens Kew, Richmond Surrey TW9 3AB, UK
3
Laboratoire d’Electrochimie et Environnement, Ecole National d’Ingénieur de Sfax, Université de Sfax, BP « 1173» 3038, Tunisie

Pelargonium graveolens (Geraniaceae) was characterized with respect to its chemical composition, antioxidant
potential and antimicrobial activities. This is the first investigation focusing on the comparison of both essential oil
and polar extracts from this species. The chemical composition of the essential oil of the aerial parts of P. graveolens
was analyzed by gas chromatography/mass spectrometry. The main constituents of the oil were found to be
b-citronellol (21.9%), citronellyl formate (13.2%), geraniol (11.1%), 10-epi-g-eudesmol (7.9%), geranyl formate
(6.2%) and (l)-linalool (5.6%). Nine flavonoids were identified by high-performance liquid chromatography–MS
in leaf and flower extracts. Kaempferol 3-O-rhamnoside-glucoside, isorhamnetin aglycone, quercetin 3-O-glucoside,
kaempferol 3,7-di-O-glucoside, quercetin 3-O-pentose and kaempferol 3-O-glucoside, quercetin 3-O-rhamnoside-
glucoside, quercetin 3-O-pentoside-glucoside, myrisetin 3-O-glucoside-rhamnoside flavonoids were detected in
methanolic and aqueous extracts, respectively. The total flavonoids ranged between 29.9 and 78.2 mg QE/g in flower
water and methanol extracts, respectively, and 22.5 and 71.2 mg QE/g dry weight in leaf water and methanol extracts,
respectively. The highest antioxidant activities using two methods of free radical scavenging capacities were obtained
with the essential oil (9.16 mM of Trolox and 2.68 mg/ml). All P. graveolens essential oil and polar extracts were
active against at least one bacterium. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: Pelargonium graveolens; polar extracts; essential oil; flavonoids; antimicrobial activity; antioxidant activity.

The rose-scented geranium Pelargonium graveolens

INTRODUCTION
L’Hér. (Geraniaceae) is an erect, much-branched shrub,
which can reach up to 1.3 m in height and 1 m in spread.
It is recognized that a diversity of plant species contain The hairy stems are herbaceous when young, becoming
compounds that exhibit antioxidant properties. Although woody with age (Van der walt and Volster, 1988). The
numerous studies on species such as rosemary, sage and
deeply incised leaves are velvety and soft to the touch
oregano have resulted in the development of natural
due to the presence of numerous glandular hairs. The
antioxidant formulations for food, cosmetic and other
applications, scientific information on the antioxidant plant is cultivated for its high value essential oil, which
properties of various plants, particularly those which are is used in aromatherapy and in the production of high-
less widely used in culinary and medicine is still scarce grade perfumes and cosmetic products (Eiasu et al.,
(Miliauskasa et al., 2004). The implication of oxidative 2009). Its oil has been used for many years in traditional
and nitrosative stress in the etiology and progression of medicine as antiasthmatic, antiallergic, antioxidant, anti-
several severe and chronic clinical disorders has led to diarrhoeic, antihepatotoxic, diuretic, tonic, haemostatic,
the suggestion that antioxidants can have health benefits stomachic and antidiabetic (Lis-Balchin, 2002; Abe
as prophylactic agents (Soobrattee et al., 2005). The et al., 2004; Maruyama et al., 2006).
number of individual phytochemicals identified in fruits Studies on Pelargonium species have focused on the
and vegetables is estimated to be greater than 5000, but chemical composition of the essential oils and a number
the health benefits of these and other phytochemicals in of flavonoid surveys (Mitchell et al., 1998; Williams and
foods are poorly studied (Liu, 2004). Phytochemicals Harborne, 2002). Rana et al., (2002) determined the
can include not only secondary metabolites such as presence of 30 compounds in the essential oil from
phenolic compounds but also vitamins, sugars and fatty P. graveolens, accounting for 99.1% of the oil. The
acids, and these compounds can be used as nutraceuticals main components identified were citronellol (33.6%),
(Guimarães et al., 2009). Therefore, research on biologic- geraniol (26.8%), linalool (10.5%), citronellyl formate
ally active extracts and compounds from natural sources (9.7%) and p-menthone (6.0%).
has been of great interest to scientists involved in different The present study evaluates for the first time the
aspects of health care. in vitro antimicrobial and antioxidant activities of
both the essential oil and polar extracts of P. graveolens
growing in Tunisia and the chemical compositions of
* Correspondence to: Dr. Mohamed Bouaziz, Institut Supérieur de
Biotechnologie de Sfax, Route Soukra Km:3.5, BP’1175’ Sfax 3038-
these extracts using gas chromatography/mass spec-
Université de Sfax, Tunisie. trometry (GC/MS) and liquid chromatography/mass
E-mail: mohamed.bouaziz@fsg.rnu.tn spectrometry (LC/MS), respectively.
Received 17 April 2012
Revised 17 August 2012
Copyright © 2012 John Wiley & Sons, Ltd. Accepted 31 August 2012
 10991573, 2013, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ptr.4853 by Universidade Federal Do ABC, Wiley Online Library on [12/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF GERANIUM 1207

LC-MS analysis. The composition of flavonoids in the

MATERIALS AND METHODS
methanol and water extracts of the flowers and leaves
were analysed using an HPLC-MS/MS system (Survey
Chemicals. Methanol, ethyl acetate, formic acid, acetic or autosampler and pumps and LCQ Classic ion trap
acid, and acetonitrile HPLC-grade solvents were purchased mass spectrometer, Thermo-Finnigan, San Jose, CA,
from Riedel-de Haen (Seelze, Germany). The solvents USA) fitted with a 150 mm  4.6 mm, 5 mm, C-18
were of appropriate purity. 2,2-Diphenyl-1-picrylhydrazyl chromatography column (Luna C-18; Phenomenex UK,
(DPPH), 2,6-di-tert-butyl-4-hydroxy-boxylic acid (BHT), Macclesfield, UK). The acidified aqueous acetonitrile and
2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) acidified aqueous methanol HPLC mobile phase gradients
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid used were, 90:0:10 (t = 0 min), 0:90:10 (t = 20 min), 0:90:10
(Trolox) were purchased from Sigma-Aldrich (Chemie (t = 25 min), 90:0:10 (t = 27 min) and 90:0:10 (t = 37 min),
Gmbh, Steinhein, Germany). Besides, malt extract broth water/methanol/acetonitrile + 1% acetic acid, with a flow
(MEB) and Agar (MEA) were purchased from Fluka. rate of 1 mL/min. The column was equilibrated in
Other chemicals, such as hydrogen peroxide, sodium the start conditions for 8 min prior to the injection of
hydroxide, were of analytical grade. Folin–Ciocalteu 10 mL of the sample. The HPLC was interfaced to the
reagent was obtained from Fluka (Switzerland). Sodium MS via an atmospheric pressure chemical ionisation
acetate (CH3COONa.3H2O) were purchased from Merck (APCI) source operated in the positive mode under the
(Darmstadt, Germany). Double distilled water was used general conditions recommended by the manufacturer:
in the HPLC mobile phase. APCI needle voltage  5 kV; heated capillary temperature
150  C; sheath and auxiliary nitrogen gas flows 20 units;
Plant material. Samples of P. graveolens were collected APCI vaporiser temperature 450  C; tube lens offset
in March 2008 from Sfax, South of Tunisia (Latitude voltage  10 V.
34 43´ N, Longitude 10 41´ N, Altitude 21 m). This
region is characterized by its arid climate having a mean Total phenolics determination. The total phenol content
rainfall of 200 mm per year. Pelargonium graveolens was of extracts was determined using the phenol reagent
identified by Professor Makki Boukhris, Science Faculty (Bouaziz et al., 2010). Briefly, 50 mL aliquot of different
of Sfax, Tunisia. Voucher specimen were deposited at extracts was assayed with 250 mL of phenol reagent
the Laboratory of Environmental Bioprocesses, Biotech- and 500 mL of aqueous sodium carbonate (20%, w/v).
nology Centre of Sfax, Tunisia (Voucher N Pg 22). The mixture was vortexed and diluted with water to a
final volume of 5 mL. After incubation for 30 min at
Extraction of the essential oil. The aerial parts of P. room temperature, the absorbance was measured at
graveolens (Geraniaceae) (600 g) were subjected to 765 nm. The total phenols were expressed as gallic
hydrodistillation for 3 h, using a modified Clevenger- acid equivalents (GAE/g of dry weight), using a
type apparatus. The oil was dried over anhydrous calibration curve of a freshly prepared gallic acid
Na2SO4 and preserved in a sealed vial at +4  C prior to solution. For the gallic acid, the curve absorbance
further analysis with a yield of 0.16% (w/w). versus concentration is described by the equation
y = 0.0012 x - 0.0345 (r2 = 0.9997).
Extraction procedures. Methanol and water were used Total flavonoids determination. The total flavonoids
sequentially to fractionate the soluble compounds of P. were measured by a colorimetric assay developed by
graveolens. The dried flowers (100 g) and leaves Zhishen et al. (1999). One mL aliquot of the appropri-
(100 g) of P. graveolens were subjected to successive ately diluted sample or standard solution of quercetin
solvent extraction methanol and water for 24 h under a (20, 40, 60, 80 and 100 mg/L) was added to a 10 mL
continuous reflux setup in a Soxhlet extractor. The volumetric flask containing 4 mL dd H2O. At zero time,
extracts were concentrated under reduced pressure (rotary 0.3 mL of NaNO2 (5%, w/w) was added to the flask.
evaporator) then preserved at +4  C in glass in the dark After 5 min, 0.3 mL AlCl3 (10% w/w) was added. At
until its use. 6 min, 2 mL of NaOH (1 M) was added to the mixture.
Immediately, the reaction flask was diluted to volume
GC/MS analysis. The analyses were performed using an with the addition of 2.4 mL of dd H2O and thoroughly
Agilent 5975B mass spectrometer coupled to an Agilent mixed. The absorbance of the mixture, characterized
6890 N gas chromatograph. An aliquot of 1 ml extract by a pink colour, was determined at 510 nm compared
was then injected splitlessly, into the GC/MS. Next, the to a water control. The total flavonoid were expressed
data were displayed on a DB-5MS column, 30 m in as fresh weight mg/g quercetin equivalents (QE). For
length, 0.25 mm i.d. and 0.25 mm in thickness (Agilent quercetin, the curve absorbance against concentration
Technologies, J&W Scientific Products, USA). The was described by the equation y = 0.0049x (r2 = 0.9984).
carrier gas was helium. GC oven temperature started
at 100  C and was held for 1 min at 260  C and then Total flavonols determination. The content of flavonols
for 10 min with program rate 4  C/min. The injector and was determined by the method of Yermakov et al.
detector temperatures were set at 250 and 230  C, (1987). A standard curve of rutin was obtained by
respectively. The mass range was scanned from 50 mixing 2 mL of different concentrations of methanolic
to 550 amu. The identification of components was solutions of rutin with 2 mL of AlCl3 (20 mg/mL) and
assigned by matching their mass spectra with Wiley 6 mL of sodium acetate (50 mg/mL). The absorbance
and NIST library data, standards of the main compo- was measured after 2.5 h at 440 nm. The same procedure
nents and comparing their Kovats retention indices is followed for 2 mL of plant extract (3.5 mg/mL). The
with reference libraries (Davies, 1990; Adams, 2001) total flavonols were expressed as fresh weight mg/g rutin
and from the literature. equivalents (RE). For rutin, the curve absorbance
Copyright © 2012 John Wiley & Sons, Ltd. Phytother. Res. 27: 1206–1213 (2013)
 10991573, 2013, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ptr.4853 by Universidade Federal Do ABC, Wiley Online Library on [12/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1208 M. BOUKHRIS ET AL.

against concentration was described by the equation • Standard bacterial strains: Pseudomonas aeruginosa
y = 0.0407x- 0.2091 (r2 = 0.9034). ATCC 15442, Escherichia coli ATCC 10536,
Staphylococcus aureus ATCC 6538 and Bacillus
Mineral analyses. Each mineral composition was deter- subtilis ATCC 6633.
mined according to the specificities of each element. • Yeast and fungi: Candida albicans ATCC 10231 and
Sodium and potassium were determined by flame Aspergillus niger ATCC 16404.
spectrophotometer, magnesium by atomic absorption The bacteria were cultivated in tryptic soy broth (TSB)
spectrophotometer, calcium by titration, phosphorus by (Sigma) at the appropriate temperature (30–37  C) for
colorimetric phosphomolybdate, ammonium nitrogen the strain. Fungi and yeasts were cultured on MEB or
by distillation of ammonia, sulphur and chloride by agar (MEA) (Fluka) at 28  C. The cultures of bacteria
ion chromatography. and fungi were maintained in their appropriate agar slants
at 4  C throughout the study and used as stock cultures.
Antioxidant potential of P. graveolens extracts. Inocula were prepared by adjusting the turbidity of each
DPPH assay. The DPPH radical scavenging effect was bacterial and yeast cultures or fungal spore’ suspension,
evaluated following the procedure described in a to reach an optical comparison to that of a 0.5 McFarland
previous study (Bouaziz et al., 2010). Aliquots (50 mL) standard, resulting in a suspension containing approxi-
of various concentrations (5, 10, 15, 20 and 25 mg/mL) mately 1 to 5 108 CFU/ml.
of the test extracts in methanol were added to 5 mL of
methanolic solution containing DPPH radicals (6 10 6 M). Minimum inhibitory concentration and minimum bacteri-
After a 30 min incubation period at room temperature cidal concentration. Minimum inhibitory concentration
and in the dark, the absorbance was read in opposition (MIC) and minimum bactericidal concentration (MBC)
to the control at 517 nm. The inhibition (IC50) of free were determined by the broth dilution method as recom-
radical DPPH (IC50 %) was calculated in percentage: mended by the National Committee for Clinical Labora-
IC50 % = [(Ablank – Asample)/Ablank] 100, where Ablank tory Standards (NCCLS, 2000). The test was performed
is the absorbance of the control reaction (containing all in sterile 96-well microplates. As regards the inhibitory
reagents except the test extract), and Asample is the activity of the essential oils, they were properly prepared
absorbance of the test extract. The concentration of and transferred to each microplate well in order to obtain
the test extract providing 50% inhibition (IC50, a twofold serial dilution of the original sample. In order to
expressed in mg/mL) was calculated from the graph obtain stable diffusion, stock solutions of the essential oils
plotted with inhibition percentage against the extract were prepared in 0.1% ethanol. The inocula (100 mL)
concentration. The synthetic antioxidant reagent butylated containing 104 (exponent) CFU of bacteria or fungi
hydroxytoluene (BHT) was used as positive control and were added to each well. While the negative control
all tests were carried out in triplicate. wells contained bacteria or fungi only in their adequate
ABTS assay. The Trolox equivalent antioxidant medium, the positive ones contained 10 mg/L of Chlor-
capacity (TEAC) assay, which measures the reduction amphenicol and Amphotericin B antibiotics for bacteria
of the radical cation of ABTS by antioxidants, was per- and fungi, respectively. Subsequently, 30 mL of 0.02%
formed as previously described by Laporta et al. (2007). resazurin and 12.5 mL of 20% Tween 80 were added.
Briefly, 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) Plates were aerobically incubated at 30  C for 16–20 h.
radical cation (ABTS +) was produced by reacting an
•
After incubation, the wells were observed for a colour
ABTS stock solution (7 mM) with 2.45 mM potassium change from blue to pink. MIC was defined as the
persulfate (final concentration) and conserving the mix- lowest concentration of test samples that inhibited the
ture in the dark at room temperature for 12–16 h before bacterial growth. To determine MBC, broth was taken
use. With regard to the study of phenolic compounds, from each tube and sub-cultured for 24 h in TSB for
the ABTS + solution was diluted with ethanol to an
•
bacteria and MEB for fungi.
absorbance of 0.7  0.02 at 734 nm. Briefly, 1 mL of
the ABTS + solution and 100 mL antioxidant solution
•
Statistical analyses. The values were expressed as mean
were mixed for 45 s and measured immediately after  standard deviation of three parallel measurements.
5 min at 734 nm. The active extracts were assessed at five The data were evaluated by a one-way analysis of
different concentrations determined within the linear variance using SPSS (Chicago, IL) software, and differ-
range of the dose–response curve. A calibration curve ences between the means were determined using
was prepared with different concentrations of Trolox Student’s t-test. Values were considered statistically sig-
(0.2–0.8 mM), and the results were expressed in mM of nificant when p < 0.05.
Trolox, (y = 0.6792 x). The results were expressed in
TEAC (mM).

Bactericidal and fungicidal activities. To evaluate the RESULTS AND DISCUSSION

bactericidal and the fungicidal activities of the essential
oil and polar extracts of P. graveolens, pure cultures Chemical composition of the essential oil of the aerial
of authenticated bacteria and fungi were obtained parts
from the international culture collections (The American
Type Culture Collection (ATCC) and ‘La Collection The essential oil extracted by hydrodistillation from the
de l’Institut Pasteur’). The locally isolated and identi- dried aerial part of P. graveolens was quantitatively
fied strains were provided by CTM: Tunisian culture analyzed by GC-MS. Thirty two components were iden-
collection of microorganisms, Biotechnology Centre tified in the analyzed oil sample amounting to 97.4% of
of Sfax, Tunisia. the total oil (Table 1). The yield of essential oils from
Copyright © 2012 John Wiley & Sons, Ltd. Phytother. Res. 27: 1206–1213 (2013)
 10991573, 2013, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ptr.4853 by Universidade Federal Do ABC, Wiley Online Library on [12/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF GERANIUM 1209

Table 1. Chemical composition of essential oil of aerial part of P. the aerial parts of P. graveolens was 0.16% (w/w).
graveolens The main constituents of P. graveolens essential oil were
b-citronellol (21.9%), citronellyl formate (13.2%),
Compound TRa KIb % geraniol (11.1%), 10-epi-g-eudesmol (7.9%), geranyl
formate (6.2%) and (l)-linalool (5.6%). Although the
1 a-pinene 8.58 930 0.76
oxygenated monoterpenes (67.8%) and sesquiterpene
2 b -phellandrene 11.45 1023 0.12
hydrocarbons (20.0%) dominated the essential oil,
3 b- ocimene 11.78 1034 0.31
monoterpene hydrocarbons were present in small
4 (l)-linalool 13.65 1074 5.6
amounts (1.2%). The essential oil of P. graveolens
5 rose oxide-trans 13.95 1079 2.01
6 l-menthone 15.20 1140 0.31
contained high level of monoterpenes and oxygenated
7 iso menthone 15.52 1177 4.42
terpenes such as b-citronellol, citronellyl formate,
8 b-citronellol 17.45 1238 21.93
geraniol and menthone. These results were in agree-
9 geraniol 18.16 1261 11.07
ment with those reported by Southwell et al. (1995),
10 (E)-citral 18.56 1277 0.38
who claimed that geranium oil from Algeria and China
11 citronellyl formate 18.7 1280 13.24
contained high amounts of citronellol and low amounts
12 geranyl formate 19.42 1307 6.22
of geraniol.
13 a-cubebene 20.69 1357 0.11
14 a-copaene 21.39 1369 0.99
15 b-bourbonene 21.63 1388 3.14
Chemical composition of P. graveolens extracts
16 b-cubebene 21.74 1396 0.18
17 trans-caryophyllene 22.52 1403 1.02 The HPLC analysis of the methanol and aqueous
18 isoledene 23.09 1435 0.47 extracts of P. graveolens showed the presence of peaks
19 isolongifolene 23.25 1450 0.54 with flavonoid-type UV spectra (two bands, l max of
20 a-caryophyllene 23.39 1454 0.37 band 1 between 320 and 350 nm and l max of band 2
21 aromadendrene 23.56 1467 0.68 between 250 and 270 nm) and interfering peaks of other
22 germacrene D 24.07 1474 4.33 phenolics. Table 2 lists the identified compounds in
23 viridiflore 24.41 1486 2.35 order of elution. The structure assignment of flavonoids
24 a-muurolene 24.52 1495 0.44 for which no standards were available was based on a
25 g-cadinene 24.86 1499 2.38 systematic search for molecular ions using extracted
26 d-cadinene 25.07 1511 1.33 ion mass chromatograms and comparing them with the
27 epizonaren 25.13 1517 0.42 data in the literature (Heller and Forkmann, 1988;
28 a-agarofuran 25.64 1530 1.28 Grayer et al., 2000; Bouaziz et al., 2005; Heneidak
29 geranyl propionate 26.88 1643 0.22 et al., 2006). For example, flavonoid 4 had a protonated
30 10-epi-g-eudesmol 27.39 1703 7.92 molecule [M + H]+ at m/z 317 and a strong fragment
31 b-eudesmol 28.04 1728 0.51 at m/z 302. This suggests that this compound was the
32 geranyl tiglate 29.12 1785 2.39
isorhamnetin aglycone.
Total 97.44
Compound 6 exhibited a base peak [M + H]+ at m/z
Chemical classes
611 and an intermediate ion at m/z 465 as well as an
Monoterpenes 68.98
aglycon ion at m/z 303. The loss of 146 amu from the
Monoterpenes hydrocarbons 1.19
pseudomolecular ion represents the sugar rhamnose
Oxygenated monoterpenes 67.79
and the loss of 162 amu from the intermediate ion is
Sesquiterpenes 28.46
due to the loss of glucose. The obtained MS spectra
Sesquiterpenes hydrocarbons 20.03
suggest that compound 6 was rutin. Compound 7, whose
Oxygenated sesquiterpenes 8.43
UV spectra suggest that it was the flavonoid quercetin
3-O-pentosides (l max 257, 362 nm), showed an [M + H]+
a
TR: retention time; ion at m/z 435 with significant fragments at m/z 303.
b
KI: retention index (Kovalts) The identification of the compound was confirmed by

Table 2. List of the detected compounds from water and methanol extracts in P. graveolens with their HPLC retention times, UV
maximum and mass spectral data.

Comp. no. Rt (min) Solvant UV l max (nm) [M + H]+ MSn Compounds

1 8.87 b 264, 356 627 627, 481, 319 Myrisetin 3-O-glu-rha

2 9.06 b 255, 356 597 597, 465, 434, 303 Quercetin 3-O-pent-glu
3 9.21 a 266, 303 sh, 350 611 611, 449, 287 Kaempferol 3,7-di-O-glu
4 9.79 a 253, 370 317 317, 302 Isorhamnetin aglycone
5 10.06 a 256, 350 465 465, 303 Quercetin 3-O-glu
6 10.24 b 256, 269, 372 611 611, 465, 303, 285, 257, 247, 229, 165 Quercetin 3-O-rha-glu (Rutin)
7 10.95 a 257, 362 435 435, 303 Quercetin 3-O-pent
8 11.24 b 265, 348 449 449, 287, 241, 213, 165 Kaempferol 3-O-glu
9 11.33 a 264, 350 595 595, 449, 287 Kaempferol 3-O- rha-glu

a: water, b: methanol, [M + H]+ = pseudomolecular ion obtained by HPLC-APCI-MS, MS = mass fragmentation,

glu: gluciside; rha: rhamnoside; pent: pentoside.

Copyright © 2012 John Wiley & Sons, Ltd. Phytother. Res. 27: 1206–1213 (2013)
 10991573, 2013, 8, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ptr.4853 by Universidade Federal Do ABC, Wiley Online Library on [12/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1210 M. BOUKHRIS ET AL.

comparing its HPLC retention time, UV spectra and mass Mineral composition
spectra with the data obtained from standard in-house
libraries. The APCI mass spectrum in the positive mode
of compound 9 exhibited a base peak [M + H]+ at m/z The elemental analysis in mg/100 g (DW) indicated that
595 (Table 2), an intermediate ion at m/z 449 and an the flowers, petiole and blade contained, respectively,
aglycone ion at m/z 287. While the loss of 146 amu from calcium (2.51, 2.31 and 2.6), magnesium (0.32, 0.3 and
the pseudomolecular ion indicates the sugar rhamnose, 0.42), sodium (1.32, 1.43 and 1.27), potassium (1.9, 1.82
the loss of 162 amu from the intermediate ion is due and 0.66), phosphorus (0.22, 0.17 and 0.15), nitrogen
to the loss of glucose. The l max of the UV spectrum at (0.64, 0.81 and 1.73) chlorides (1.53, 1.88 and 0.6) and
264 and 350 nm suggests that compound 9 is a kaempferol sulphur (0.14, 0.046 and 0.056) (Fig. 1). The calcium
3-O-sugar, and the combined results of the MS and UV level in the studied leaves compares favourably with
spectra suggest that compound 9 could be kaempferol the value reported by Lokhande et al. (2010) who has
3-O-rhamnoside-glucoside. The latter was confirmed by also found that the medicinal species Bryophyllum
cochromatography with authentic standards as kaempferol pinnatum has high levels of calcium (0.41%). The accu-
3-O-rutinoside.
mulation of calcium is related to the phenological stage
In the same way, compounds 1–3, 5 and 8 were iden-
tified as flavonoids, myristin 3-O-glucoside-rhamnoside of the plant (reproductive organs), and the soil which
(1), quercetin 3-O-pentose-glucoside (2), kaempferol was rich in limestone (Osowski and Fahlenkamp, 2006;
3,7-di-O-glucoside (3), quercetin 3-O-glucoside (5) and Monti et al., 2008). It is well-known that calcium and
kaempferol 3-O-glucoside (8). To the best of our know- phosphorus are important for growth and maintenance
ledge, these compounds were identified for the first time of bones, teeth and muscles (Dosunmu, 1997; Turan
from P. graveolens. et al., 2003). Plants are capable of absorbing a wide
range of mineral ions with relevance to human nutrition
and health. Mineral concentrations in many plant foods
Total phenolic, flavonoids and flavonol content are low, relative to human requirements; this has elicited
efforts to enhance the mineral content in food plants
The total phenolic, flavonoid and flavonol content of
methanol and water extracts of flowers and leaves of (Grusak, 2002).
P. graveolens are shown in Table 3. The highest amounts Sodium is essential for regulation of osmotic pressure
of total phenolic content were obtained in the methanol of the body and helps to maintain acid–base and water
extracts of flowers and leaves (109.8 and 84.1 mg GAE/g
of dry weight extracts, respectively) compared to the
amounts present in water extracts of flowers and leaves
(60.76 and 54.71 mg GAE/g of dry weight extracts,
respectively). This result was in agreement with the
reports of Hertog et al. (1993) and Yen et al. (1996),
which proved that the methanol is the most suitable
solvent for the extraction of phenolic compounds. The
total flavonoid content in extracts varied considerably
according to solvent used (Table 3). Flavonoid content
in the methanol extracts of the flowers and leaves
showed significant differences (78.4 and 71.2 mg QE/g
(dry wt of extract) and higher than those in the water
extracts (Table 3). Such results are in agreement with
those of Hajji et al. (2010) who showed that flavonoid
content increases significantly with solvent polarity.
When the concentration of flavonols was expressed in
mg rutin/g (dry wt) of plant extract the methanol
extracted was again shown, to contain more flavonols than Figure 1. Diagrams representing the polygonal mineral contents
water extracts (Table 3). This analysis shows that methanol of flower, petiole and blade of Pelargonium graveolens. (g/100 g
of D.W). (S: sodium, K: potassium, Mg: magnesium, Ca: calcium,
extracted a higher amount of flavonols from the leaves P: phosphorus, N: ammonium nitrogen, S: sulphur, Cl: chloride).
(23.8 total flavonols mg rutin/g (dry wt)) than from the This figure is available in colour online at wileyonlinelibrary.com/
flowers (23.8 total flavonols mg rutin/g of dry weight). journal/ptr.

Table 3. Total phenolic, flavonoid and flavonol content of P. graveolens polar extracts.

Total phenolic content mg GAE/g Total flavonoid content mg QE/g Total flavonols mg rutin/g
Extracts (dry weight) (dry weight) (dry weight)

Methanol flowers 109.76  16.06a 78.49  6.19a 23.87  3.02a

Aqueous flowers 60.76  3.70b 29.87  3.12b 8.44  0.81b
Methanol leaves 84.18  9.41c 71.21  3.54c 43.59  3.46c
Aqueous leaves 54.71  3.53d 22.45  1.87d 10.28  2.24b

Results are expressed as mean  standard deviation of three determinations.

Means with different letters were significantly different at p < 0.05.

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CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF GERANIUM 1211

Table 5. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of P. graveolens essential oil and extracts on standard bacterial and fungal strains (mg/ml). na, not

2.5–5
2.5–5
MCC
balance of the body. Its deficiency causes loss of body

Chloramphenicol Amphotericin B
weight and nerve disorder. Potassium is accumulated

–
–
–
–
within human cells by the action of the Na+, K+ -

1.25–2.5
1.25–2.5
ATPase (sodium pump), and it is an activator of some

MIC
enzymes; in particular, co-enzyme for normal growth

Controls
and muscle function (Birch and Padgham, 1994). Potas-

–
–
–
–
sium deficiency causes nervous disorder, diabetes, and

MCC
poor muscular control resulting in paralysis (Lokhande

na
na
na
na
–
–
et al., 2010). The Na/K ratio in the body is of great
concern for the prevention of high blood pressure

3.12–6.25
3.12–6.25
3.12–6.25
3.12–6.25
(FND, 2002). Actually, it is recommended that Na/K

MIC
ratio be less than one. Therefore, the consumption of
flowers and petiole of P. graveolens would probably

–
–
reduce high blood pressure diseases because their Na/K
is less than one (0.69 and 0.78, respectively).

10–20
10–20
20–40
20–40
10–20
Leaves methanol

MCC

2.5–5
extract

1.25–2.5
5–10
5–10
10–20
10–20
5–10
MIC
Antioxidant activity

In the ABTS assay, the methanol and water extracts

10–20
5–10
10–20
20–40
20–40
10–20
Leaves methanol

MCC
of the flowers and leaves of P. graveolens all exhibited

extract
moderate antioxidant activities ( 1 mM TEAC) that
were comparable with BHT (1.32 mM TEAC) (Table 4).

5–10

5–10
10–20
10–20
5–10
In the DPPH assay, a comparison of IC50 of extracts

MIC

2.5–5
and essential oil shows significant differences (Table 4).
Indeed, essential oil possesses the greatest DPPH
radical scavenging activities (2.62 mg/mL) and is more

5–10
10–20

5–10
MCC
Flowers aqueous

2.5–5

2.5–5
2.5–5
active than BHT which is used as an antioxidant in the
food industry. These studies have shown that the bioas- extract
says used to evaluate the antioxidant potential of the

1.25–2.5

1.25–2.5
1.25–2.5
5–10
extracts of P. graveolens highlight the fact that different

2.5–5

2.5–5
MIC
groups of compounds in a plant have antioxidant
activity. For example, the essential oil of the aerial parts
of P. graveolens has revealed the highest antioxidant
5–10

5–10
2.5–5
2.5–5

2.5–5
2.5–5
Flowers methanol

activity in the DPPH assay. These results were similar

MCC

to those obtained by ABTS assay. The antioxidant

extract

activity of the compounds in both the essential oil and

the extracts of P. graveolens could contribute to the
1.25–2.5
1.25–2.5
1.25–2.5

1.25–2.5

medicinal uses of this species. The activity could be

MIC

2.5–5

2.5–5

attributed in part to the presence of compounds such

as b-citronellol and geraniol in the essential oil as well
as the phenolics in the methanol and water extracts.
3.52–7.04
3.52–7.04
3.52–7.04
1.76–3.52
1.76–3.52
3.52–7.04

Lu and Foo (2001) reported that most natural antioxi-

MCC

dant compounds often work with each other to produce

Essential oil

1.76–3.52
1.76–3.52
1.76–3.52
0.88–1.76
0.88–1.76
1.76–3.52
MIC

Table 4. Free radical scavenging capacities of P. graveolens

essential oil and polar extracts measured by DPPH and ABTS
assays.
Pseudomonas aeruginosa ATCC 15442

ABTS (mM of Trolox)DPPH (IC50 mg/mL)

Staphylococcus aureus ATCC 9144

Extracts

1.32  0.21a 8.32  0.21a

Candida albicans ATCC 10231
Aspergillus niger ATCC 16404

BHT
Escherichia coli ATCC 10536

9.16  0.11b 2.68  0.21b

Bacillus subtilis ATCC 6633
Microorganisms

Essential oil
Methanol flowers extract 1.13  0.11c 10.30  0.45c
Aqueous flowers extract 0.98  0.02c 12.85  0.83d
Methanol leaves extract 0.86  0.18c 20.71  0.72e
Aqueous leaves extract 0.93  0.15c 16.59  0.40f

Results are expressed as mean  standard deviation of three

determinations.
active.

Means with different letters were significantly different at

p < 0.05.

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1212 M. BOUKHRIS ET AL.

a broad spectrum of antioxidant properties that create phenolic compounds in these extracts (Table 2) that can
an effective defence system against free radicals. effect cellular membranes altering their permeability and
The antioxidant activity of phenolic compounds is release of intracellular constituents (e.g. ribose and Na
mainly due to their redox properties and chemical struc- glutamate), but also interfere with membrane functions
ture, which contribute to their ability in chelating transi- (electron transport, nutrient uptake, protein, nucleic acid
tional metals, inhibiting lipoxygenase and scavenging synthesis and enzyme activity) (Bajpai et al., 2009).
free radicals (Decker, 1997; Kubola and Siriamornpun,
2008). Detailed examination of the composition of
phenolic compounds in plant extracts as well as those
absorbed through the gut is required for the comprehen- CONCLUSIONS
sive assessment of the importance of individual com-
pounds to the antioxidant properties of a plant extract. The present study has shown that the essential oil and/or
polar extracts of P. graveolens may be a potential source
of natural antioxidants. The essential oils from many
Antimicrobial plants have recently been of increased interest in
research as well as the food industry that are looking
The MICs and MBCs of the essential oil and polar for natural additives to replace synthetic antioxidants
extracts of P. graveolens evaluated against the tested in food products. The biological activities of the essential
microorganisms are shown in Table 5. The results show oil and polar extract of Pelargonium graveolens suggest
that the essential oil and polar extracts exhibit bacteri- that this species justifies further study. The results of
cide and fungicide effects, and in most cases, the concen- this study also provide some scientific support as to why
trations required to kill the bacteria and fungi are two the species is used medicinally in North Africa. The anti-
times higher than the concentration required to inhibit oxidant and antimicrobial activities could be accredited to
the bacteria and fungi as represented by the respective the high content of flavonoids and volatile components
MBC and MIC values. Overall, the essential oil was identified for the first time by HPLC/MS and GC/MS,
more potent than the methanol and water extracts. respectively. This study demonstrates that P. graveolens
Previous research would suggest that the bactericidal essential oil and polar extracts could be considered as an
and fungicidal activities of the essential oil could be alternative to ‘synthetic food preservatives’. In fact, it
explained by the presence of high concentrations of helps in reducing radical scavenging activities, eliminating
oxygenated monoterpenes (Sousa et al., 2006; Singh or reducing the growth of important pathogens and
et al., 2008; Yangui et al., 2009). b-Citronellol and contributing to enhance food safety and shelf life. This
geraniol, the two major monoterpenes component of plant could therefore be used to improve the treatment
geranium oil, have reported antimicrobial activity (Inouye of candidose or as a food-conserving additive.
et al., 2001; Lorenzi et al., 2009). Moreover, Zore et al.
(2009) have stated that the anti-Candida activity of
geraniol, geranyl acetate and citronellol appears to be
associated with their ability to damage membrane Acknowledgements
integrity. They inhibit germ tube induction at very low This research was supported by Contrats Programmes Ministry of
concentrations and arrest C. albicans cell cycle. These Higher Education, Scientific Research and Technology in Tunisia.
authors showed that geraniol and geranyl acetate have Our thanks go to Prof. Monem Kallel, from ENIS (Sfax) for mineral
fungicidal activity at 0.064% v⁄v concentrations, i.e. MICs composition analyses, Lobna JLAIL for her help with GC-MS analysis
(561 mg/mL and 584 mg/mL, respectively) and killed 99.9% and Dr. Elaine Porter (Kew-UK) for help with LC-MS analyses.
of inoculum within 15 and 30 min of exposures, respect-
ively. On the other hand, the methanol and water extracts
of flowers and leaves did show antimicrobial activity, with Conflict of Interest
the flower extracts being overall more active than the leaf
extracts. This activity is most likely associated with the The authors have declared that there is no conflict of interest.

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