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Campanula portenschlagiana Roem. et Schult.: Chemical and Antimicrobial Activities by Olivera Politeo* a ), Mirjana Skocibusic b ), Franko Burcul a ), Ana Maravic b ), Ivana Carev a ), Mirko Ruscic b ), and Mladen Milos a ) a
) Department of Biochemistry, Faculty of Chemistry and Technology, University of Split, N. Tesle 10/V, HR-21000 Split (phone: þ 385-21329437; fax: þ 385-21329461; e-mail:
[email protected]) b ) Department of Biology, Faculty of Science, University of Split, N. Tesle 12, HR-21000 Split
The phytochemical profile and the antimicrobial effects of the volatile oil and the aqueous extract of Campanula portenschlagiana, a wild growing plant endemic to Croatia, were described. In the volatile oil, 53 compounds were identified by GC-FID and GC/MS analyses. Diterpene alcohols constituted the major compound class with labda-13(16),14-dien-8-ol as the main compound. The aqueous extract was characterized by the total phenolic content. The antimicrobial potential of the volatile oil and the aqueous extract was evaluated against a diverse range of microorganisms comprising food-spoilage and food-borne pathogens. The volatile oil exhibited interesting and promising antimicrobial effects against the tested species, which were generally more pronounced against Gram-negative bacteria. In addition, the inhibitory effect of this volatile oil was also evaluated against eleven extended-spectrum b-lactamase (ESBL)-producing isolates. The results suggest that the C. portenschlagiana volatile oil might be used as antimicrobial agent against ESBL-producing isolates and Gram-negative bacteria.
Introduction. – Campanula portenschlagiana Roem. et Schult., commonly known as Dalmatian bellflower, is an endemic plant which grows wild, at low altitudes, on rocky slopes and cliffs of the Dalmatian mountains, along the Adriatic coast and islands [1]. It is an evergreen perennial with basal leaves that are kidney like, circled heartshaped, with serrated edges and without or uniformly covered with hairs. The light blue to violet-blue, bell-shaped flowers grow in panicles. Flowering commonly occurs between June and September, resulting in pouch-shaped fruits. The genus Campanula is represented in the Croatian flora by more than 50 species, of which more than ten Croatian native campanula species are endemic [2]. The portenschlagiana taxon has been classified as near threatened (NT) on the Croatian, rare (R) on the Bosnian and Herzegovinian, rare (R) on the European, and rare (R) on the World Red List [3]. Some campanulas have been used for centuries in traditional oriental medicines and therefore represent a source of potentially bioactive compounds that have been shown to promote health and reduce risks against a wide spectrum of respiratory diseases including asthma, bronchitis, and pulmonary tuberculosis as well as inflammatory diseases. Previous phytochemical studies on the Campanulaceae family indicated the presence of a great diversity of metabolites. Most of these identified compounds were triterpenoids [4 – 7] and alkaloids [8]. Indeed, triterpenoid saponins and their 2013 Verlag Helvetica Chimica Acta AG, Zrich
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glycosides have widely been found in Campanula species and are known to have antimicrobial, antifungal, insecticidal, anticancer, and antioxidant potential [6] [9]. The Campanula species were also reported to be a source of high diversity and variable levels of anthocyanins, phenylpropanoids, coumarins, and catechins [10] [11]. Pharmacological studies and clinical practice have demonstrated that several species possess many biological functions, including antitumor [12], antioxidant [11] [13] [14], antiinflammatory [6], neuroprotective [7], and hepatoprotective activities [15]. The volatile oils and extracts of various plants have been investigated for their biological activities, notably the antibacterial and antifungal properties. However, little attention has been given to the study of the phytochemical constituents of C. portenschlagiana volatile oil as well as of any other plant of the Campanula genus. Hence, efforts have been made to investigate the potential role of the volatile oil and the aqueous extract of C. portenschlagiana as natural antimicrobial agents. In the present study, the volatile constituents of C. portenschlagiana were examined by GC-FID and GC/MS analyses, and the efficacy of the volatile oil and the aqueous extract against Gram-positive and Gram-negative bacteria, including important multidrug-resistant strains, was evaluated. This study represents the first report of the phytochemical profile and biological screening of C. portenschlagiana.
Results and Discussion. – Chemical Composition of the C. portenschlagiana Volatile Oil. The C. portenschlagiana volatile oil was isolated by hydrodistillation and characterized by GC-FID and GC/MS analyses. Fifty-three compounds were identified, representing 81.3% of the total volatile oil (Table 1). Diterpene alcohols constituted the major compound class (Table 2). Among them, labda-13(16),14-dien-8-ol was the main compound (29.6%), followed by phytol (5.1%), abienol (2.6%), and feruginol (1.3%). Sesquiterpenes also composed a large portion of this oil (9.2%) with bcaryophyllene being the major sesquiterpene (3.1%), followed by bicyclogermacrene (1.7%). The nonoxygenated diterpene cembrene A (6.2%) was also identified in significant quantitiy, followed by the alkane tricosane (3.2%), the diterpene aldehyde pimarinal (2.7%), and oxygenated nonterpene compounds such as hexahydro farnesyl acetone (2.7%), nonanal (2.4%), and methyl stearate (2.1%). Labda-13(16),14-dien-8-ol was, to this date, identified with quantitatively significant amounts in Anthemis werneri L. ssp werneri volatile oil [16]. It was also reported to be present at low content in the essential oils of Stachys alopecuros L. from Greece [17] and Pinus canariensis from Tenerife [18]. Antimicrobial Activity. The antimicrobial activities of the C. portenschlagiana volatile oil and aqueous extract were determined against a panel of eleven microorganisms. The inhibitory potential was assessed qualitatively, by measuring inhibitionzone diameters in the disc-diffusion (DD) assay, and quantitatively, by determining the minimum inhibitory concentrations (MICs) in the broth microdilution bioassay. The results showed interesting and promising antimicrobial effects (Table 3). The volatile oil exhibited moderate to strong in vitro antimicrobial effects on the Grampositive bacteria tested, with inhibition-zone diameters (DD) of 19.6 – 24.8 mm at a concentration of 250 mg/disc and MIC values ranging from 62.5 to 125.0 mg/ml. Concerning the Gram-positive bacteria, the volatile oil was equally effective against
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Table 1. Chemical Composition of the Campanula portenschlagiana Volatile Oil Compound
RI a )
Content [% (w/w)]
Identification b )
( E )-Hex-2-enal c ) ( Z )-Hex-3-en-1-ol c ) m-Xylene Heptanal a-Pinene ( E )-Hept-2-enal c ) Benzaldehyde b-Pinene 2-Pentylfuran Octanal ( Z,E )-Hepta-2,4-dienal c ) Limonene Phenylacetaldehyde Linalool Nonanal a-Terpineol Decanal ( E,E )-Deca-2,4-dienal c ) b-Damascenone b-Elemene b-Caryophyllene a-Humulene ( Z )-b-Farnesene c ) Pentadecane b-Selinene Germacrene D Bicyclogermacrene a-Muurolene ( E,E )-a-Farnesene c ) d-Cadinene Caryophyllene oxide Isospathulenol t-Cadinol t-Muurolol Heptadecane Pentadecanal Hexahydrofarnesyl acetone Butyl isobutyl phthalate Nonadecane Dibutyl phthalate Cembrene A Manoyl oxide Octadecanal Heneicosane Labda-13(16),14-dien-8-ol c ) e ) Phytol Methyl stearate Abienol Pimarinal
< 900 < 900 < 900 905 941 962 969 980 996 1007 1015 1036 1052 1106 1109 1192 1212 1325 1387 1397 1425 1460 1465 1500 1483 1487 1497 1506 1514 1531 1589 1648 1652 1657 1700 1719 1843 1888 1900 1973 1992 2003 2017 2100 2112 2129 2142 2152 2178
0.8 1.3 tr d ) 0.1 1.7 0.2 0.4 0.4 0.4 0.4 tr 0.1 1.3 tr 2.4 0.4 0.3 0.3 tr tr 3.0 0.4 0.3 0.9 tr 0.4 1.7 0.2 0.2 0.2 0.9 0.4 0.5 1.0 tr 0.4 2.7 0.9 0.3 0.7 6.2 tr 0.4 0.9 29.6 5.1 2.1 2.6 2.7
RI, MS RI, MS RI, MS RI, MS RI, MS, RI, MS RI, MS RI, MS, RI, MS RI, MS RI, MS RI, MS, RI, MS RI, MS, RI, MS RI, MS, RI, MS RI, MS RI, MS RI, MS RI, MS, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, MS RI, MS RI, MS RI, MS MS
Co-GC
Co-GC
Co-GC Co-GC Co-GC
Co-GC
Co-GC
Co-GC
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Table 1 (cont.) Compound
RI a )
Docosane Tricosane Feruginol Methyl heneicosanoate
2200 2300 2305 2434
Identification b )
Content [% (w/w)] 0.6 3.2 1.3 1.0
Total
RI, RI, RI, RI,
MS, Co-GC MS, Co-GC MS MS
81.3
a
) RI: Retention index determined relative to n-alkanes (C9C40 ) on a HP-5MS capillary column ) Identification: RI, comparison of RI with literature data [28] and/or homemade library; MS, comparison of mass spectra with NIST02 and Wiley 7 mass spectral databases; Co-GC, coinjection with reference compound. c ) Correct isomer not identified. d ) tr: Trace ( < 0.1%). e ) Identification confirmed by comparison of the RI with published data [18 – 20]. b
Table 2. Compound Classes Represented in the C. portenschlagiana Volatile Oil Compound class Nonterpene compounds Nonoxygenated Compounds Oxygenated Compounds Alcohols Aldehydes Esters Terpene compounds Monoterpenes Sesquiterpenes Diterpenes Nonoxygenated Diterpenes Oxygenated Diterpenes Diterpene oxides Diterpene alcohols Diterpene aldehydes Ketones Other compounds Total
Content [% (w/w)]
No. of compounds
5.9
7
1.3 7.0 4.7
1 12 4
2.6 9.2
5 15
6.2
1
tr 38.6 2.7 2.7
1 4 1 1
0.4
1
81.3
Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Listeria monocytogenes (MIC values of 125.0 mg/ml), whereas Bacillus cereus was the most sensitive Gram-positive bacterium with a MIC value of 62.5 mg/ml. Against the Gram-negative bacteria tested, stronger inhibitory effects of the volatile oil were observed, with DD values at a concentration of 250 mg/disc of 24.7 0.6 mm for Escherichia coli, 27.9 1.7 mm for Klebsiella pneumoniae, and 28.3 2.4 mm for Pseudomonas aeruginosa. These DD values showed to be higher than those obtained with 15 mg/disc of gentamicin. The MIC values of the volatile oil against the Gram-negative bacteria tested were found to be in the range from 7.8 to 62.5 mg/ml. A particularly high potential of the volatile oil was observed against P. aeruginosa (MIC
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Table 3. In vitro Antimicrobial Activity of the C. portenschlagiana Volatile Oil and Aqueous Extract in Comparison with the Standard Antibiotics Cefotaxime (CTX ) and Gentamicin (GEN) and the Antifungal Drug Amphotericin B ( AM B ) Microorganisms
Volatile oil a
Aqueous extract b
c
DD )
MIC ) DD )
Bacteria Bacillus cereus ATCC11778 Enterococcus faecalis ATCC 29212 Staphylococcus aureus ATCC 25923 Clostridium perfringens FNSST 4999 Listeria monocytogenes ATCC15313 Escherichia coli FNSST 982 Klebsiella pneumoniae FNSST 011 Pseudomonas aeruginosa FNSST 014
24.8 1.5 19.6 0.9 21.4 2.3 21.8 3.2 22.8 0.9 24.7 0.6 27.9 1.7 28.3 2.4
62.5 125.0 125.0 125.0 125.0 62.5 15.6 7. 8
Fungi Candida albicans ATCC 6275 Penicillium sp. FNSST 3724 Rhizopus stolonifer FNSST 3833
29.5 1.8 24.7 0.6 27.3 2.2
3.9 62.5 7.8
13.6 1.5 18.4 0.7 21.5 0.6 10.8 2.3 12.4 0.8 13.4 1.2 14.4 0.9 11.2 0.7
b
CTX
GEN/AM B
MIC ) DD ) MIC ) DD e ) f ) MIC b ) 250.0 125.0 125.0 500.0 250.0 250.0 125.0 125.0
10.4 0.2 250.0 12.6 1.4 250.0 16.2 1.7 62.5
d
26.8 23.5 21.7 28.5 23.2 22.8 21.4 10.7
b
0.25 0.5 0.5 0.1 0.5 0.5 0.5 16.0
18.2 14.6 23.9 21.7 19.4 11.5 18.2 9.5
4.0 4.0 1.2 0.5 2.0 32.0 8.0 64.0
21.6 17.3 19.2
1.0 4.0 2.0
a
) DD: Inhibition-zone diameter around the disc [mm] obtained by the disc-diffusion method at a concentration of 250 mg oil/disc. b ) MIC: Minimum inhibitory concentration [mg/ml]. c ) DD at 750 mg extract/disc. d ) DD at 30 mg cefotaxime/disc. e ) DD at 15 mg gentamicin/disc. f ) DD at 10 mg amphotericin B/disc.
of 7.8 mg/ml), an activity which was even higher than that observed for the positive controls cefotaxime and gentamicin. Moreover, the volatile oil of C. portenschlagiana was also found to inhibit the growth of the fungi Candida albicans, Penicillium sp., and Rhizopus stolonifer, with DD values of 29.5 1.8, 24.7 0.6 and 27.3 2.2 mm, respectively, and MIC values of 3.9, 62.5, and 7.8 mg/ml, respectively. When compared to the volatile oil, the aqueous extract showed a significantly lower antimicrobial potential against both the Gram-positive and Gram-negative bacteria as well as the fungal strains tested. The MIC values of the aqueous extract against Grampositive and Gram-negative bacteria were found to be in the range of 125 – 500 and 125 – 250 mg/ml, respectively. Hence, the aqueous extract showed no interesting antimicrobial activity when compared to the standard antibiotics (Table 3). Given the promising MIC values of the C. portenschlagiana volatile oil against Gram-negative bacteria, we extended our study to multidrug resistant (MDR) Gramnegative strains including extended-spectrum b-lactamase (ESBL)-producing isolates of P. aeruginosa, P. fluorescens, Stenotrophomonas maltophilia, and Burkholderia cepacia as well as the Enterobacteriaceae E. coli and K. pneumoniae. Infections caused by ESBL-producing bacteria have become a clinical and therapeutic problem because these organisms are resistant not only to b-lactams but also to many other structurally unrelated antimicrobial agents. Multidrug resistance is especially common in the setting of cystic fibrosis, where such species commonly infect the lungs causing extensive morbidity and premature death. Treatment of nonfermentative Gram-negative bacteria such as P. aeruginosa, P. fluorescens, S. maltophilia, and the B. cepacia complex (BCC) can be particularly problematic [19].
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C. portenschlagiana volatile oil showed promising activity against the eleven ESBLproducing isolates tested, when compared to the activity of eight broad-spectrum antibiotics, with MIC values ranging from 3.9 to 62.5 mg/ml (Table 4). The most significant activity was obtained against the two S. maltophilia strains FNSST138 and FNSST166 as well as against K. pneumoniae FNSST271 (equal MIC values of 3.9 mg/ ml), which is one of the most prevalent nosocomial enterobacterial pathogens, causing infections that range from mild urinary infections to severe bacteremia and pneumonia with a high rate of morbidity and mortality [20]. Table 4. Minimum Inhibitory Concentrations (MICs in mg/ml) of the C. portenschlagiana Volatile Oil and Standard Antibiotics against Gram-Negative Extended-Spectrum b-Lactamase ( ESBL )-Producing Isolates ESBL-Producing isolates
Volatile Standard antibiotics a ) oil CAZ CTX CIP GEN
TET TOB IMP
MEM
Burkholderia cepacia FNSST154 Burkholderia cepacia FNSST206 Pseudomonas aeruginosa FNSST008 Pseudomonas fluorescens FNSST216 Stenotrophomonas maltophilia FNSST124 Stenotrophomonas maltophilia FNSST138 Stenotrophomonas maltophilia FNSST166 Klebsiella pneumoniae FNSST271 Klebsiella pneumoniae FNSST272 Escherichia coli FNSST235 Escherichia coli FNSST236
15.6 62.5 15.6 31.1 62.5 3.9 3.9 3.9 15.6 31.1 62.5
128 256 16 8 64 8 8 64 128 128 128
128 256 128 16 64 64 128 0.125 1 4 4
128 32 128 64 128 64 > 256 128 16 128 128 256 128 256 16 32 64 64 32 256 32 > 256
0.25 2 16 0.125 16 0.5 2 0.5 1 0.25 4
2 4 32 0.125 128 2 4 128 32 64 8
8 2 128 0.5 > 32 > 32 16 32 32 32 64
0.125 256 64 0.25 32 8 32 0.25 2 0.25 0.125
a ) MIC Values highlighted by shading represent resistance according to the breakpoints established by the Clinical and Laboratory Standards Institute (CLSI; M100-S17, 2007); abbreviations of the standard antibiotics: CAZ, ceftazidime; CTX, cefotaxime; CIP, ciprofloxacin; GEN, gentamicin; TET tetracycline; TOB, tobramycin; IMP, imipenem; MEM, meropenem.
Notable reduction of bacterial growth was also observed for B. cepacia FNSST154, P. aeruginosa FNSST008, and K. pneumoniae FNSST272 with equal MIC values of 15.6 mg/ml. However, the B. cepacia FNSST206, E. coli FNSST236, and S. maltophilia FNSST124 isolates showed less susceptibility (MICs of 62.5mg/ml), when compared with the other ESBL-producing bacteria tested. Phenolic diterpenes and their derivates are an important class of plant metabolites, which were reported to be responsible for the antimicrobial activity of several volatile oils [21] [22]. Consequently, the high antibacterial efficacy of C. portenschlagiana volatile oil could be attributed to the high percentage of diterpenes such as cembrene A, phytol, primarinal, and abienol. Moreover, the predominance of labda-13(16),14dien-8-ol could also be responsible for the strong antimicrobial activity observed in this study. Possible synergistic effects of the compounds in the oil should also be taken into consideration. The results described in this study clearly indicated that the volatile oil isolated form C. portenschlagiana possessed the antimicrobial potential to control the growth of
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several pathogens, including ESBL-producing isolates, and these findings are in strong agreement with previous reports [23] [24]. Total Phenolic Content of the Aqueous Extract. The total phenolic content (TPC) was determined by the FolinCiocalteu method and expressed as gallic acid equivalents (mg GAE/g extract). The TPC of the aqueous extract of C. portenschlagiana was found to be 40.6 5.1 mg GAE/g extract. Conclusions. – This investigation is part of our ongoing research on the biological activity of volatile and nonvolatile compounds isolated from wild growing plants in Croatia. This is the first report on the phytochemical analysis and bioactivity of endemic C. portenschlagiana. The results showed a very interesting chemical and antimicrobial profile of the plant volatile oil. Diterpene alcohols were identified to constitute the major compound class. The results of antimicrobial activity showed that the volatile oil was more effective against some microorganisms than standard antibiotics, especially against Gram-negative ESBL-producing isolates. The determination of the in vivo toxicity and the antimicrobial properties should be included in our further research. The findings of this study may be used as foundation for further chemotaxonomic studies on the complex Campanula genus as well as on other species of the Campanulaceae family. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (Project Antioxidative Constituents and Cholinesterase Inhibitors from Aromatic Herbs, Grant 0112160547-1330, and Project Faecal Indicator and Potential Pathogens in Coastal and Marine Waters, Grant 177-0000000-3182).
Experimental Part General. The FolinCiocalteu phenol reagent as well as all the solvents employed were purchased from Kemika, Zagreb, Croatia. Plant Material. The plant material (whole plants without roots) was collected from its natural habitat, i.e., Mt. Sveti Ilija, Biokovo Mountain, at the Makarska coast (Central Dalmatia, Croatia), during flowering, in June, 2010, at 1494 m.a.s.l. (GaussKrger coordinates: X ¼ 5663090, Y ¼ 4804135). Typical habitats are rock fissures within the stenoendemic community of rock-fissure vegetation of Portenschlagia and Campanula portenschlagiana associations (As. PortenschlagielloCampanuletum portenschlagianae) Trinajstic¤ (1980) ex Zi.Pavletic¤ 1986 [25]. The botanical identity of the plant material was confirmed by a botanist and voucher specimens have been deposited with the Department of Biology, Faculty of Science, University of Split, Croatia. Volatile Oil Isolation. Aliquots of 100 g of fresh plant material and 500 ml of H2O were placed in a Clevenger-type apparatus. The volatile oil was isolated by hydrodistillation for 3 h. The obtained oil was separated, dried (anh. Na2SO4 ), and stored under Ar in a sealed vial at 208 before use. Preparation of Aqueous Extract. Aliquots of 100 g of fresh plant material and 500 ml of H2O were placed in an Erlenmeyer flask and refluxed in an ultrasound bath for 2 h. The mixture was then filtered through a filter paper, and the extract was centrifuged, evaporated to dryness in a rotary evaporator under reduced pressure, and stored at 208 before use. GC-FID and GC/MS Analyses. The GC-FID and GC/MS analyses were carried out with an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A equipped with a flame ionization detector (FID), an Agilent Technologies mass-selective detector model 5975C, and an Agilent J&W HP5MS ((5%-phenyl) methylpolysiloxane) cap. column (30 m 0.25 mm i.d., film thickness 0.25 mm). The oven temp. was programmed isothermal at 708 for 2 min, then rising from 70 to 2008 at 38/min, and finally
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held isothermal at 2008 for 18 min; injector temp., 2508; carrier gas, He (1 ml/min); injection vol., 1 ml; split ratio, 1 : 50. The FID temp. was 3008 and the MS detector conditions were as follows: ionization voltage, 70 eV; ion source temp., 2308; mass scan range, 45 – 350 mass units. The analyses were carried out in duplicate. Identification and Quantification of Components. The individual peaks of the volatile oil were identified by comparing i) their retention indices (RIs), determined relative to n-alkanes (C9 – C40) on the HP-5MS column, with those of a homemade library, literature data, and/or data of authentic samples [26] as well as ii) their mass spectra with literature data, the Wiley 7 (Wiley, NY, USA) and the NIST02 (Gaithersburg, MD, USA) mass spectral databases [16 – 18]. The homemade library was created from data of authentic compounds obtained commercially and from the main components of many essential oils obtained during our previous studies. The component percentages were calculated as mean values form the GC-FID and GC/MS peak areas using the normalization method without correction factors (Table 1). Microbial Strains and Culture Conditions. The volatile oil and the aqueous extract were tested against 22 or 11 microbial strains, resp. Most of these strains are pathogenic for humans and show multidrug resistance to antibiotics. The microbial strains were obtained from various sources including the American Type Culture Collection (ATCC, Manassas, VA) and the Faculty of Natural Science, University of Split (FNSST). The strains included Bacillus cereus (ATTC 11778), Enterococcus faecalis (ATCC 29212), Listeria monocytogenes (ATCC 15313), Staphylococcus aureus (ATCC 25923), as well as the yeast Candida albicans (ATCC 6275) and the filamentous fungal strains Rhizopus stolonifer (FNSST 3833) and Penicillium sp. (FNSST 3724). Additionally, the following food spoilage and food-borne pathogens were tested: Clostridium perfringens (FNSST 4999), Escherichia coli (FNSST 982), Klebsiella pneumoniae (FNSST 011), and Pseudomonas aeruginosa (FNSST 014). The multidrug resistant (MDR) extended-spectrum b-lactamase (ESBL)-producing isolates tested included Burkholderia cepacia (FNSST 154 and FNSST 206), Pseudomonas aeruginosa (FNSST 008), P. fluorescens (FNSST 216), Stenotrophomonas maltophilia (FNSST 124, FNSST 138, and FNSST 166), Klebsiella pneumoniae (FNSST 271 and FNSST 272), and Escherichia coli (FNSST 235 and FNSST 236). The microbial strains were stored as 30% glycerol stocks at 808 and subcultured on tryptic soy-agar plates (TSA, Becton Dickinson) at 378 before testing. Determination of the Antimicrobial Activity. The antimicrobial screening of the C. portenschlagiana volatile oil and aqueous extract was carried out with the agar-disc diffusion and the broth-microdilution bioassays, according to the procedures described previously [27]. Determination of the Total Phenol Content. The total phenol content (TPC) was determined spectrophotometrically, according to the FolinCiocalteu colorimetric method [28] [29]. The TPC of all samples was measured in triplicate. Gallic acid was used as standard for the calibration curve. The TPC was expressed as gallic acid equivalents (mg GAE/g extract).
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