Digest Journal of Nanomaterials and Biostructures Vol. 9, No. 3, July - September 2014, p.

1085 - 1094

COMPARATIVE STUDY OF POLYPHENOLIC CONTENT, ANTIOXIDANT

AND ANTIMICROBIAL ACTIVITY OF FOUR GALIUM SPECIES (RUBIACEAE)

L. VLASEa,b, A. MOCANa*1, D. HANGANUc, D. BENEDECc, A. GHELDIUb, G.

CRIȘANa
a
Department of Pharmaceutical Botany, Faculty of Pharmacy, "Iuliu Hatieganu"
University of Medicine and Pharmacy, 12 Ion Creanga Street, 400010 Cluj-
Napoca, Romania
b
Department of Pharmaceutical Technology and Biopharmaceutics, 12 Ion
Creanga Street, Cluj-Napoca, 400010, Romania
c
Department of Pharmacognosy “Iuliu Hatieganu” University of Medicine and
Pharmacy, 12 Ion Creanga Street, Cluj-Napoca, 400010, Romania

Four Galium species (G. verum L., G. mollugo L., G. aparine L. and G. odoratum L.) were
analyzed in order to evaluate their polyphenolic content, antioxidant and antimicrobial
activities. The identification and quantification of major phenolic compounds content was
performed using a HPLC-MS/MS method. The total polyphenols, caffeic acid derivatives
and flavonoids content was spectrophotometrically determined. The antioxidant activity
was evaluated using the DPPH bleaching method and all data indicates that G. verum was
the best source of antioxidants amongst the four studied species. Rutin was the compound
found in largest amount in all analysed extracts. Luteolin was found only in the extracts of
G. mollugo and G. aparine and kaempferol only in the extracts of G. verum and G.
odoratum, but in different amounts. Chlorogenic acid, p-coumaric acid and ferulic acid
were identified in all extracts in different amounts and caftaric acid only in G. aparine
extract. The antimicrobial activity was determined using the diffusimetric method. Galium
extracts showed inhibitory activity on various pathogenic bacteria and fungi tested. The
quantitative and qualitative differences between the four species of Galium concerning the
polyphenolic content, could serve as a taxonomic marker in order to distinguish species
and to avoid adulterations.

(Received June 30, 2014; Accepted September 1, 2014)

Keywords: Galium species; Polyphenols; Antioxidant activity; Antimicrobial activity;

DPPH.

1. Introduction

Plant-origin polyphenols are antioxidant agents that have been suggested to exert
beneficial pharmacological effects on neurodegenerative disorders. Plant polyphenols might also
display anticarcinogenic, antimutagenic and cardioprotective effects assumed by their free radical
scavenging properties. Polyphenols have been also reported as chemopreventive agents by
lowering the cholesterol level and roughly limiting cell damage. Their ability to delay lipid
oxidation in foodstuffs and biological membranes, in addition to their propensity to act as a
prophylactic agent has motivated their research in food science and biomedicine [1,2].
The cosmopolitan genus Galium, belonging to Rubiaceae, consists of about 300
herbaceous plant species [3]. The species are used in food manufacturing and folk medicine all
over the world. Galium species are herbaceous, annual or perennial, with thin rhizome and

*
Corresponding authors: [email protected]
1086

cylinder-form stem, that presents four prominent longitudinal lines. The leaves are in verticil but
just two of these are genuine leaves, the others being stipules. The flowers are of four types in
ended panicles and the fruits are twin nucules [4,5]. The European flora gathers more than 145
species [6]; in the Romanian flora, there are between 28 species quoted, most with white flowers
and six with yellow flowers [7]. The aerial parts are gathered for medicinal purposes in the
blossoming period. Because the identification of these plants is frequently vague or imprecise
owing to their highly similar morphological characteristics, the results of chemotaxonomic
investigations could be valuable for the systematic evaluation of this genus.
Galium verum L. – yellow “Lady’s bedstraw”, with golden yellow flowers, contains
polyphenols, flavonoids, phenyl-propanoids compounds and iridoids. They are used as diuretic,
depurative, light sedative, spasmolytic in kidney stones, and externally for injuries and skin
damages as wound healing, psoriasis treatment and rheumatism [8]. In some countries, the drug is
still used as a dye including the milk industries. In the same places with G. verum, vegetates G.
mollugo L., with white flowers, which is considered an adulteration for “Lady’s bedstraw”
[9,10,11,12].
Galium aparine L. is a typical scrambling climbing plant. Decoction of whole plant is
locally used as a tea. Aerial parts of G. aparine contain anthraquinones, iridoids, alkanes,
flavonoids, tannins, polyphenolic acids, and vitamin C. Some scientists have reported that the
whole plant of G. aparine is used for the treatment of lymph swellings, tonsillitis, jaundice,
wounds, cancer, fever, scurvy, hypertension and leukemia [13].
Galium odoratum L. – “sweet woodruff” (formerly known as Asperula odorata L.) is a
mat-forming perennial plant that is most often seen as a ground cover in shady areas. Plants emit a
strong odor of freshly mown hay when foliage is crushed or cut. Aromatic intensity of the foliage
increases when dried, thus dried leaves are popularly used in sachets or potpourris. The plant has
also been used commercially, in perfumes. Leaves are sometimes used to flavor teas and cold fruit
drinks. “Sweet woodruff” was widely used in herbal medicine during the Middle Ages, gaining a
reputation as an external application to wounds and cuts and also taken internally in the treatment
of digestive and liver problems. In current day phytotherapy it is valued mainly for its tonic,
diuretic and anti-inflammatory effect. The leaves are antispasmodic, cardiac, diaphoretic, diuretic,
sedative and are used in the treatment of insomnia and nervous tension, varicose veins, biliary
obstruction, hepatitis and jaundice. The plant contains coumarins, polyphenols, iridoids and
flavonoids. It is commercially grown as a source of coumarin and it is not recommended to be
used alongside conventional medicine for circulatory problems or in pregnancy state [14].
The aim of this study was to determine the polyphenolic content, and to evaluate
antioxidant and antimicrobial activities of G. verum L., G. mollugo L., G. aparine L. and G.
odoratum L. (Rubiaceae). None of these plants have subjected before to detailed studies in order to
reveal their polyphenolic content or antioxidant and antimicrobial activities.

2. Experimental

2.1 Plant materials

Plant materials (aerial parts) from the four species were collected in 2013, during the
blooming period (May-June) from the Province of Transylvania (NW of Romania) and identified
by Professor Gianina Crișan from the Pharmaceutical Botany Department, one of the coauthors.
Authenticated voucher specimens were deposited in the Herbarium of the Department of
Pharmaceutical Botany, Faculty of Pharmacy, “Iuliu Hațieganu” University of Medicine and
Pharmacy. The vegetal material was air dried at room temperature in shade, separated and grinded
to fine powder (300 µm).

2.2 Preparation of the extracts

2.0 g of plant material (aerial parts – herba) from each species, after being grinded to a
proper degree of fineness (300 µm), were ultrasonicated with 20 ml of 70% ethanol (Merck,
 1087

Darmstadt, Germany), for 30 min at 60°C. The samples were then cooled down and centrifuged at
4500 rpm for 15 min, and the supernatant was recovered [2,15]. All the samples were filtered
using Whatman filter paper and preserved at 4 ºC.

2.3 Chemicals and bacterial strains

Ferulic acid, sinapic acid, gentisic acid, gallic acid, patuletin, luteolin were products from
Roth (Karlsruhe, Germany), cichoric acid, caftaric acid were products from Dalton (Toronto, ON,
Canada), chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin,
quercitrin, hyperoside, kaempferol, myricetol, fisetin were products from Sigma Aldrich (St.
Louis, MO, USA). HPLC grade methanol, analytical grade orthophosphoric acid, hydrochloric
acid and Folin-Ciocâlteu reagent were purchased from Merck (Darmstadt, Germany), aluminum
chloride, sodium acetate, sodium carbonate, ethanol (Merck, Darmstadt, Germany), DPPH (2,2-
diphenyl-1-picrylhydrazyl) and BHT (butylated hydroxytoluene) were obtained from Alfa-Aesar
(Germany). For the antibacterial potential assaying of the plant extracts, all microorganism strains
were distributed by MicroBioLogics®: Staphylococcus aureus ATCC 49444 (Gram+), Listeria
monocytogenes ATCC 13076 (Gram+), Escherichia coli ATCC 25922 (Gram-), and Salmonella
typhimurium ATCC 14028 (Gram-) and one fungal strain, Candida albicans ATCC10231.

2.4 LC/MS analysis

2.4.1 Apparatus and Chromatographic Conditions

The experiment was carried out using an Agilent Technologies 1100 HPLC Series system
(Agilent, Santa Clara, CA, USA) equipped with G1322A degasser, G13311A binary gradient
pump, column thermostat, G1313A auto sampler and G1316A UV detector. The HPLC system
was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). For the separation,
a reverse-phase analytical column was employed (Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 μm
particle); the work temperature was 48°C. The detection of the compounds was performed on both
UV and MS mode. The UV detector was set at 330 nm until 17.5 min, then at 370 nm. The MS
system functioned using an electrospray ion source in negative mode. The chromatographic data
were processed using ChemStation and DataAnalysis software from Agilent. The mobile phase
was a binary gradient: methanol and acetic acid 0.1% (v/v). The elution started with a linear
gradient, beginning with 5% methanol and ending at 42% methanol, for 35 minutes; then 42%
methanol for the next 3 minutes [15,16]. The flow rate was 1 mLmin-1 and the injection volume
was 5 µL. The MS signal was used only for qualitative analysis based on specific mass spectra of
each polyphenol. The MS spectra obtained from a standard solution of polyphenols were
integrated in a mass spectra library. Later, the MS traces/spectra of the analyzed samples were
compared to spectra from library, which allowed positive identification of compounds, based on
spectral mach. The UV trace was used for quantification of identified compounds from MS
detection. Four polyphenols cannot be quantified in current chromatographic conditions due
overlapping (caftaric acid with gentisic acid and caffeic acid with chlorogenic acid). However, all
four compounds can be selectively identified in MS detection (qualitative analysis) based on
differences between their molecular mass and MS spectra. For all compounds, the limit of
quantification was 0.5 μg/mL, and the limit of detection was 0.1 μg/mL. The detection limits were
calculated as minimal concentration producing a reproductive peak with a signal-to-noise ratio
greater than three. Quantitative determinations were performed using an external standard method.
Calibration curves in the 0.5–50 μg/mL range with good linearity (R2 > 0.999) for a five point plot
were used to determine the concentration of polyphenols in plant samples [2,16,17].

2.5 Determination of total polyphenols, caffeic acid derivatives and flavonoids

content

The total phenolic content (TPC) of the extracts was determined using the Folin-Ciocalteu
method with some modifications [18,19]. 2 mL of each ethanolic extract wich was diluted 25 times
1088

were mixed with 1.0 mL of Folin-Ciocalteu reagent, 10.0 mL of distilled water and diluted to 25.0
mL with a 290 g/L solution of sodium carbonate [1,2]. The samples were incubated in the dark for
30 min. The absorbance was measured at 760 nm. Gallic acid was used as standard for the
calibration curve and was plotted at 0.02, 0.04, 0.06, 0.08, and 0.10 mg/mL, prepared in
methanol:water (50:50, v/v). TPC values were determined using an equation obtained from the
calibration curve of gallic acid graph (R2 = 0.999).
The phenolic acids content in the vegetal materials was determined using the
spectrophotometric method with Arnow’s reagent [20]. The percentage of phenolic acids,
expressed as caffeic acid equivalent on dry vegetal material (mg CAE/g vegetal material), was
determined using an equation that was obtained from calibration curve of caffeic acid (R2 = 0.994).
The spectrophotometric aluminum chloride method was used for flavonoids
determination. 5 mL of each extract were mixed with 5.0 mL of sodium acetate 100 gL -1, 3.0 mL
of aluminum chloride 25 gL-1, and filled up to 25 mL by methanol in a calibrated flask. The
absorbance was measured at 430 nm [20]. Total flavonoids content values was determined using
an equation obtained from calibration curve of the rutin graph (R2 = 0.999). All analyses were
done in triplicate.

2.6 DPPH• Radical Scavenging Assay

The free radical scavenging activity of the ethanolic extracts from the herba of four
species of Galium was quantified in terms of hydrogen donating or radical scavenging ability
using the stable DPPH radical method. The DPPH solution (0.1 gL-1) in methanol was prepared
and 4.0 ml of this solution were added to 4.0 ml of extract solution (or standard) in methanol at
different concentrations (10-50 μg/ml). After 30 minutes of incubation at 40°C in a thermostatic
bath, the decrease in the absorbance (n = 3) was measured at 517 nm. The percent of DPPH
scavenging ability was calculated as: DPPH scavenging ability = (Acontrol – A sample/Acontrol) × 100,
where Abscontrol is the absorbance of DPPH radical + methanol (containing all reagents except the
sample) and Abssample is the absorbance of DPPH radical + sample extract. Afterwards, a curve of
% DPPH scavenging capacity versus concentration was plotted and IC50 values were calculated.
IC50 denotes the concentration of sample required to scavenge 50% of DPPH free radicals
[17,19,21,22,23]. The positive controls were those using the standard solution quercetin and
butylated hydroxytoluene (BHT). IC50 value is correlated with the antioxidant capacity. So, IC50 ≤
50 µg/mL value means a high antioxidant capacity; 50 µg/mL < IC50 ≤ 100 µg/mL value means a
moderate antioxidant capacity and IC50 > 200 µg/mL value means no relevant antioxidant capacity
[24].
2.7 Antimicrobial activity test

The ethanolic extracts of Gallium species were tested for the antimicrobial activity against
two Gram-positive bacterial strains: Staphylococcus aureus (ATCC 49444), Listeria
monocytogenes (ATCC 13076), against two Gram-negative bacterial strains: Escherichia coli
(ATCC 25922), Salmonella typhimurium (ATCC 14028) and one fungal strain: Candida albicans
(ATCC10231) by a previously described disc diffusion method, in Petri dishes. Each
microorganism was suspended in Mueller Hinton (MH) broth and diluted approximately to 10E6
colony forming unit (cfu)/mL. They were “flood-inoculated” onto the surface of MH agar and MH
Dextroxe Agar (MDA) and then dried. Six-millimeter diameter wells were cut from the agar using
a sterile cork-borer, and 60 μL of each extract were added into the wells. The plates were
incubated at 37°C and the diameters of the growth inhibition zones were measured after 24 h.
Gentamicin (10 µg/well) and Fluconazole (25 µg/well) were used as standard drugs. The controls
were performed only with sterile broth and overnight culture and 10 μL of 70% ethanol [25]. All
tests were performed in triplicate, and clear halos greater than 10 mm were considered as positive
results.
 1089

3. Results and discussions

3.1 LC – MS results

In this paper, 19 phenolic compounds have been investigated by HPLC-MS, in four 70%
ethanolic extracts of G. verum L., G. mollugo L., G. aparine L. and G. odoratum L. herba. HPLC
coupled with MS is a very powerful analytical technique, due to its high sensitivity and the
structural information that can be obtained about the analytes. A high-performance liquid
chromatographic (HPLC) method has been developed for the determination of 19 phenolic
compounds (eight phenolic acids, four quercetin glycosides, and seven flavonol and flavone
aglycones) from plant material. The applicability of the proposed analytical method and the
qualitative and quantitative determination of the standard phenolic compounds have already been
verified [2,15,16,]. This method allows a simultaneous analysis of different classes of polyphenols
by a single pass column (the separation of all examined compounds was carried out in 35 min).
The concentrations of identified polyphenolic compounds in the analyzed samples are presented in
Table 1.
Table 1: The polyphenolic compounds in the extracts of Galium species
(mg polyphenolic compounds/100 g dried vegetal material)

Polyphenolic Rt±SD (min) G. verum L. G. mollugo L. G. aparine L. G. odoratum L.

compounds
Caftaric acid 3.54 ± 0.05 NF NF <0,2 NF
Gentisic acid 6.52 ± 0.04 NF NF <0,2 <0,2
Caffeic acid 5.60 ± 0.04 NF <0,2 <0,2 <0,2
Chlorogenic acid 5.62 ± 0.05 <0.2 <0,2 <0,2 <0,2
p-Coumaric acid 9.48 ± 0.08 0.983 ± 0.10 0.682 ± 0.06 1.404 ± 0.28 0.802 ± 0.08
Ferulic acid 12.8 ± 0.10 <0.2 <0,2 3.793 ± 0.31 0.658 ± 0.05
Sinapic acid 15.00 ± 0.10 NF NF NF NF
Cichoric acid 15.96 ± 0.13 NF NF NF NF
Hyperoside 18.60 ± 0.12 NF NF 0.300 ± 0.03 NF
Isoquercitrin 19.60 ± 0.10 78.021 ± 0.95 <0,2 0.967 ± 0.13 NF
Rutin 20.20 ± 0.15 804.262 ± 1.89 24.312 ± 0.93 7.983 ± 0.30 11.248 ± 0.87
Myricetin 21.13 ± 0.12 NF NF NF NF
Fisetin 22.91 ± 0.15 NF NF NF NF
Quercitrin 23.64 ± 0.13 4.291 ± 0.21 4.478 ± 0.20 NF 2.048 ± 0.45
Quercetin 26.80 ± 0.15 2.651 ± 0.13 0.339 ± 0.04 5.679 ± 0.26 0.339 ± 0.37
Patuletin 29.41 ± 0.12 NF NF NF NF
Luteolin 29.10 ± 0.19 NF 0.606 ± 0.08 0.467 ± 0.07 NF
Kaempferol 32.48 ± 0.17 3.069 ± 0.17 NF NF 1.345 ± 0.09
Apigenin 33.10 ± 0.15 NF NF NF NF
Notes: NF- not found, below limit detection. Values are the mean ± SD (n = 3).

The compounds were shown in Table 1 in the order of their retention time. The HPLC
chromatogram of G. verum sample is presented in Figure 1, for G. mollugo sample in Figure 2, for
G. aparine sample in Figure 3, and for G. odoratum in Figure 4, respectively. All quantitative
determinations were performed using the external standard method. In the ethanolic extract of G.
verum, three flavonoid glycosides were identified as main components. Rutin (quercetin 3-O-
rutinoside) was the compound found in the largest amount (804.262 ± 1.89 mg/100 g) followed by
isoquercitrin - quercetin 3-O-glucoside (78.021 ± 0.95 mg/100 g). Quercitrin (quercetin 3-O-
rhamnoside), quercetin and kaempferol were detected at lower levels than major flavonoids (4.291
± 0.21 mg/100 g, 2.651 ± 0.13 mg/100 g, 3.069 ± 0.17 mg/100 g,). p-coumaric acid was also
detected but in a lower amount (0.983 ± 0.10 mg/100g). Ferulic acid and chlorogenic acid were
also identified in this extract, but they were in too low concentration to be quantified (Table 1).
1090

In the ethanolic extract of G. mollugo only two flavonoid glycosides were identified in
large amount. Rutin was the compound found in the largest amount in the extract (24.312 ± 0.93
mg/100 g) followed by quercitrin (4.478 ± 0.20 mg/100 g). Luteolin and quercetin were detected at
lower levels (0.606 ± 0.08 mg/100 g, 0.339 ± 0.04 mg/100g respectively) and isoquercitrin was
also identified in the extract, but in too low concentration in order to be quantified (< 0.2 mg/100
g). Regarding the phenolic acids, p-coumaric acid was found in the largest amount (0.682 ± 0.06
mg/100g). Caffeic acid, chlorogenic acid, ferulic acid were identified, but they were in too low
concentration (< 0.2 mg/100 g) to be quantified (Table 1). The presence of luteoline in the G.
mollugo extract could serve as an important chemotaxonomic marker that could avoid the
adulteration of G. verum with these taxa.
In the ethanolic extract of G. aparine, rutin was present in the largest amount (7.983 ±
0.30 mg/100 g) followed by quercetin (5.679 ± 0.26 mg/100 g). Isoquercitrin, luteoline and
hyperoside were detected in lower amounts (0.967 ± 0.13 mg/100 g, 0.467 ± 0.07 mg/100 g and
0.300 ± 0.03 mg/100 g, respectively). In this extract were also identified three quercetin
glycosides: rutin isoquercitrin and hyperoside and two free aglycones, quercetin (5.679 ± 0.26
mg/100 g) and luteolin (0.467 ± 0.07 mg/100 g). Regarding the phenolic acids, ferulic acid was
found in the largest amount (3.793 ± 0.31 mg/100 g) followed by p-coumaric acid (1.404 ± 0.28
mg/100 g). Caftaric acid, gentisic acid, caffeic acid and chlorogenic acid were identified, but they
were in too low concentration to be quantified (<0.2 mg/100 g) (Table 1). The presence of
hyperoside and caftaric acid only in the G. aparine extract could constitute an important source of
taxonomical differentiation.
In the ethanolic extract of G. odoratum, rutin was also the compound present in the largest
amount (11.248 ± 0.87 mg/100 g) followed by quercitrin (2.048 ± 0.45 mg/100g). Two flavonoid
glycosides were identified, e.g,. rutin and quercitrin and two free aglycones – kaempferol (1.345 ±
0.09 mg/100 g) and quercetin (0.339 ± 0.37 mg/100 g) in lower amounts than the flavonoid
glycosides (Table 1). Regarding the phenolic acids composition, p-coumaric acid was detected in
the largest amount (0.802 ± 0.08 mg/100 g) followed by ferulic acid (0.658 ± 0.05 mg/100 g).
Other phenolic acids, like gentisic acid, caffeic acid, chlorogenic acid were only identified but not
quantified due to their low concentration (<0.2 mg/100 g). Considering the 19 standard
compounds used in this study (Table 1), some other peaks were not identified.
Thus, the comparative study showed large differences, especially quantitative, between the
four Galium species. The major compound, found in all the analyzed extracts, was rutin. The
richest species in rutin was G. verum (804.262 ± 1.89 mg/100 g) followed by G. mollugo (24.312
± 0.93 mg/100 g). The aglycon, luteolin was detected only in G.mollugo and G. aparine.
Kaempferol was detected in a larger amount in G. verum (3.069 ± 0.17 mg/100 g) than in G.
odoratum (1.345 ± 0.09 mg/100 g). Quercetin was the aglycon present in all analysed extracts.
Intens.
mAU 3

400

300

200

100
2

1 4 5 6
0
0 5 10 15 20 25 30 Time [min]

Fig. 1: HPLC chromatogram of G. verum sample

(1-p-coumaric acid, 2-isoquercitrin, 3-rutin, 4-quercitrin, 5-quercetin, 6-kaempferol)
 1091
Intens.
mAU

400

300

200

100

2
1 3 4 5
0
0 5 10 15 20 25 30 Time [min]

Fig. 2: HPLC chromatogram of G. mollugo sample

(1-p-coumaric acid, 2-rutin, 3-quercitrin, 4-quercetin, 5-luteolin)

Intens.
mAU

80

60

40

20

2 5 6
1
0 3 4 7

0 5 10 15 20 25 30 Time [min]

Fig. 3: HPLC chromatogram of G. aparine sample

(1-p-coumaric acid, 2-ferulic acid, 3-hyperoside, 4-isoquercitrin, 5-rutin, 6-quercetin, 7-luteolin)

Intens.
mAU
300

250

200

150

100

50

1 2 3 4 5 6
0
0 5 10 15 20 25 30 Time [min]

Fig. 4: HPLC chromatogram of G. odoratum sample

(1-p-coumaric acid, 2-ferulic acid, 3-rutin, 4-quercitrin, 5-quercetin, 6-kaempferol)

3.2 Determination of polyphenolic compounds content: total polyphenols, flavonoids

and caffeic acid derivatives

Table 2: Quantitative determination of polyphenolic compounds content

(g compounds/100 g dried vegetal material)
Plant species G. verum L. G. mollugo L. G. aparine L. G. odoratum L.
Total polyphenolic 2.6 ± 0.12 3.46 ± 0.13 2.40 ± 0.24 1.4 ± 0.35
compounds – TPC
(g GAE/100 g dry mass)
Flavonoids 5.21 ± 0.24 6.93 ± 0.44 1.60 ± 0.53 2.81 ± 0.54
(g RE/100 g dry mass)
Caffeic acid derivatives 1.329 ± 0.10 2.152 ± 0,19 0.348 ± 0.09 0.419 ± 0.17
(g CAE/100 g dry mass)
Each value is the mean ± SD of three independent measurements. GAE: Gallic acid equivalents;
RE: rutin equivalents; CAE: caffeic acid equivalents.
1092

There are also quantitative differences in the total polyphenolic compounds, flavonoids
and caffeic acid derivatives between these four Galium species. The highest amount of the total
polyphenols was determined for G. mollugo (3.46 ± 0.13 g/100 g) followed by G. verum (2.6 ±
0.12 g/100 g), G. aparine (2.4 ± 0.24 g/100 g) and G. odoratum (1.4 ± 0.35 g/100 g) – Table 2.
There is no literature data regarding the total polyphenolic compounds of these four species.
Concerning the content of flavonoids, the highest amount was determined also for the extract of G.
mollugo (6.93 ± 0.44g/100 g), followed by the extract of G. verum (5.21 ± 0.24 g/100 g), G.
odoratum (2.81 ± 0.54g/100 g) and G. aparine (1.60 ± 0.53g/100 g). Tămaș et al. report that the
total content of flavonoids was over three times higher for G.verum (2.24%) compared to G.
molugo (0.72%). The difference in the extraction yield could be the result of using different
extraction method in that work and ultrasonication in ours [4]. The phenolic acids values were
expressed as caffeic acid equivalent (g CAE/100 g dried product). The highest amount of phenolic
acids was determined for the ethanolic extract of G. mollugo (2.152 ± 0.19 g/100 g) followed by
G. verum (1.329 ± 0.10 g/100 g). These results were in compliance with the determinations of the
polyphenolic content and suggest that both G. verum and G. mollugo can be used as valuable
sources of antioxidants.

3.3 In-vitro antioxidant activity

Determination of the free radical scavenging activity

Table 3: DPPH free radical scavenging activity

Samples IC50 (µgmL-1)

BHT (standard control) 16 ± 0.54
Quercetin (standard control) 5.60 ± 0.35
G. verum 105.43 ± 0.15
G. mollugo 107.45 ± 0.53
G. aparine 116.43 ± 0.46
G. odoratum 264.42 ± 0.74
Each value is the mean ± SD of three independent measurements.

The antioxidant activity of these ethanol extracts was assessed by the DPPH radical
bleaching method. BHT and quercetin (0.025 mg mL-1) were used as the positive control (Table 3).
The highest radical scavenging activity was showed by the extract of G. verum with IC50 = 105.43
± 0.15 µgmL-1, followed by the extract of G. mollugo with IC50 = 107.45 ± 0.53µgmL-1 and the
extract of G. aparine with IC50 = 116.43 ± 0.46 µgmL-1. The lowest radical scavenging activity
was showed by the extract of G. odoratum with IC50 = 264.42±0.74 µgmL-1. The IC50(DPPH•) values
of the extracts increased in the following order: G. verum < G.mollugo < G. aparine < G.
odoratum. This was in good agreement with the total polyphenolic compounds values listed in
Table 3. The lowest IC50 value means the most powerful antioxidant potential. According to this
method, G. verum extract exhibited a moderate antioxidant capacity. Compared to the reference
compounds, quercetin (IC50 = 5.60 ± 0.35 µgmL-1) and BHT (IC50 = 16 ± 0.54 µgmL-1), the ethanol
extracts of G.verum showed lower antioxidant capacity. The results presented here constitute the
first information on the antioxidant activities of Romanian Galium species ethanolic extracts.
There is no literature data regarding the comparison of the antioxidant activities of these four
Galium species.

3.4 Antibacterial activity

The disc-diffusion assay was used to determine the antimicrobial activity of the
investigated ethanolic extracts of Galium against a panel of microorganisms including two gram-
positive bacteria Staphylococcus aureus (S. aureus), and Listeria monocytogenes (L.
monocytogenes), two gram-negative bacteria, Salmonella typhimurium (S. typhimurium) and
Escherichia coli (E. coli), and the fungus Candida albicans (C. albicans). The results of the
 1093

antimicrobial activity of these extracts are shown in Table 4. The antibacterial activity is ranked
from no activity (inhibition diameter < 10 mm), low (inhibition diameter between 10 and 15 mm),
moderate (inhibition diameter between 15 and 20 mm) and high activity (diameter inhibition
≥ 20 mm) [26].

Table 4. Results of the antimicrobial activity of Galium extracts in agar diffusion method.

Samples Inhibition zone in diameter (mm)

Staphylococcus Listeria Escherichia Salmonella Candida
aureus monocytogenes coli typhimurium albicans
Galium verum 11 ± 0.05 16 ± 0.05 10 ± 0.05 12 ± 0.05 15 ± 0.05
Galium - 10 ± 0.05 - - 8 ± 0.05
mollugo
Galium - - - - -
aparine
Galium 16 ± 0.05 16 ± 0.05 14 ± 0.05 10 ± 0.05 10 ± 0.05
odoratum
Gentamicin 19 ± 0.05 18 ± 0.1 22 ± 0.00 18 ± 0.05 -
Fluconazole - - - - 25 ± 0.00
The values represent the average of three determinations ± standard deviations.
Gentamicin (10µg/disk) and Fluconazole (10µg/disk) were used as a positive control.

The results showed variation in the antimicrobial properties of these four extracts of
Galium. As it can be seen from the Table 4, all investigated ethanolic extracts were active against
all the bacteria tested. The antimicrobial activity of these extracts was exhibited mainly against the
Gram-positive bacteria (S. aureus, L. monocytogenes). All four samples showed a low activity
against Gram-negative bacteria (S. typhimurium, E. coli) with diameters of inhibition zones
between 10 and 15 mm. The extract of G. verum showed a moderate antibacterial activity against
L. monocytogenes (inhibition diameter - 16 mm), and limited activity against the other bacterial
pathogens tested. The most pronounced activity with inhibition zones more than 14 mm was
shown by the ethanolic extract of G. odoratum. Gentamicin was used as a reference antibiotic. The
majority of these ethanolic extracts showed a weak antifungal activity against C. albicans. The
extract of G. mollugo was inactive on this fungal strain with the diameter of inhibition zone from 8
mm. Fluconazole was used as reference antifungal agent.
The results of the present investigation suggest that various extracts of Galium exhibited an
important activity against Gram-positive bacteria, especially.

3.4 Statistical analysis

All samples were analyzed in triplicate; average and relative SD were calculated using
Excel software package.

4. Conclusions

We have determined the phenolic profile, the antioxidant and antimicrobial activities for
four indigenous species of Galium genus (Rubiaceae) and we have completed the lack of literature
data with new information concerning the polyphenolic compounds and their bioactivity. The
simultaneous determination of a wide range of polyphenolic compounds was performed using a
rapid, highly accurate and sensitive HPLC method assisted by mass spectrometry detection. The
antioxidant activity evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) bleaching method
indicate that G. verum was the best source of antioxidants amongst the four studied species, related
with the total polyphenolic content. The antimicrobial tests underlined an important activity
against Gram-positive bacteria for the extracts of G. odoratum and G. verum. The comparative
1094

study showed significant differences, both qualitative and especially quantitative, among the four
species of Galium. This study suggests that the aerial parts (herba) of G. verum and G. mollugo
may be considered a source of important polyphenols with bioactive properties, a source that could
be pharmaceutically exploited.

Acknowledgements

This paper was published under the frame of European Social Found, Human Resources
Development Operational Programme 2007-2013, project no. POSDRU/159/1.5/S/136893.

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