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Natural Product Communications
Chemical Composition of Fatty Acid and Unsaponifiable Fractions of Leaves, Stems and Roots of Arbutus unedo and in vitro Antimicrobial Activity of Unsaponifiable Extracts
2010 Vol. 5 No. 7 1085 - 1090
Mohamed Amine Diba, Julien Paolinib,*, Mourad Bendahoua, Laurent Varesib, Hocine Allalia, Jean-Marie Desjobertb, Boufeldja Tabtia and Jean Costab a
Université Aboubekr Belkaïd, Laboratoire de Chimie Organique Substances Naturelles et Analyse, BP 119, 13000 Tlemcen, Algérie b
Université de Corse, Equipe Chimie des Produits Naturels, UMR-CNRS 6134, BP 52, 20250 Corte, France
[email protected] Received: November 23rd, 2009; Accepted: April 20th, 2010
The chemical composition of the fatty acid and unsaponifiable fractions of the leaves, stems and roots of Arbutus unedo L. were determined using gas chromatography and gas chromatography-mass spectrometry. The fatty acid fractions of the leaves, stems and roots contained 38.5%, 31.3% and 14.1% palmitic acid, respectively, along with other long-chain fatty acids (up to C22). The chemical composition of the unsaponifiable fractions differed: the leaf and stem fractions contained high levels of aliphatic (32.1% and 62.6%, respectively) and terpenic compounds (49.6% and 25.7%, respectively), and the root fraction mainly contained esters, of which the most abundant was benzyl cinnamate (36.6%). The antimicrobial activities of the unsaponifiable fractions against nine species of microorganisms were assessed. The unsaponifiable leaf and stem extracts inhibited the growth of Klebsiella pneumoniae, Enterococcus faecalis and Candida albicans. Keywords: Arbutus unedo L., strawberry tree, fatty acid, unsaponifiable fraction, GC/MS, antimicrobial activity.
The strawberry tree (Arbutus unedo L., Ericaceae) is a 5–15 m tall evergreen shrub that flowers from September to October [1]. The 2–3 cm diameter spherical fruits are red, and are tasty only when fully ripe. They are used mainly for the production of jams, jellies and alcoholic beverages [2,3]. In the Mediterranean region and North Africa, A. unedo is traditionally used as an alternative medicine for its biological properties. The fruit has antiseptic, diuretic and laxative effects, and the leaves have astringent, urinary tract antiseptic, anti-diarrheal and depurative properties [4–8]. Ziyyat et al. [9,10] showed that an aqueous extract of A. unedo exhibited antihypertensive [9] and vasorelaxant properties [10]. Furthermore, an in vitro study indicated that diethylether and ethyl acetate extracts of A. unedo leaves have an anti-aggregating effect on human platelets [11]. This effect is likely mediated by its antioxidant activity, which may inhibit protein tyrosine phosphorylation and Ca2+ influx into platelets [12–15].
Several compounds have been isolated from A. unedo, including aromatic acids, iridoids, monoterpenoids, phenylpropanoids, sterols and triterpenoids [16]. For example, Ayaz et al. [17] detected lactic, malic, suberic and fumaric acids. Phytochemical studies have shown that the leaf extract contains phenolic antioxidant compounds, such as flavonoids (quercitin, isoquercetin, kaempferol, hyperoside and rutin) [18,19], tannins, phenolic glycosides, anthocyanins and gallic acid derivatives [19,20]. Vitamin C, vitamin E, α-terpineol, (E)-2-α-tocopherol and carotenoids are also present in A. unedo [3,5,17,19,21,22]. To our knowledge, only one study has investigated the essential oil content of the leaves (sample origin: Turkey) [23]; the essential oils consisted mainly of terpenic and aliphatic compounds, of which (E)-2-decenal (12.0%), α-terpineol (8.8%), hexadecanoic acid (5.1%), and (E)-2-undecenal (4.8%) were the major components. In Algeria, many patients use medicinal plants as well as conventional medical regimens to treat a variety of diseases. Interest in the use of bioactive compounds
1086 Natural Product Communications Vol. 5 (7) 2010
extracted from plants for developing new drugs has increased in recent years [5]. The aim of this study was to determine the chemical composition of the fatty acid and unsaponifiable fractions of solvent extracts of the stems, leaves and roots of A. unedo, to quantify the antibacterial and antifungal activities of the unsaponifiable fractions, and to assess the potential of the species as a source of natural compounds for pharmaceutical applications. Gas chromatography (GC) and GC-mass spectrometry (GC-MS) were used to determine the chemical composition of the extracts. Antibacterial and antifungal activity was assessed using nine species of microorganisms and a microdilution method. The light petroleum extracts of A. unedo were first separated into two fractions, namely the fatty acid and unsaponifiable fractions. Fatty acids in the leaf, stem and root extracts were tentatively identified on the basis of their fatty acid methyl ester (FAME) derivatives after derivatization with BF3/MeOH (Table 1). The 14 fatty acids identified in the leaf, stem and root extracts accounted for 83.6%, 74.3% and 85.7% of the total fatty acid fractions, respectively (Table 1). The fatty acid compositions of the three fractions were qualitatively similar. However, oleic acid was present only in the leaf extract (10.6%), nonadecanoic acid in the stem extract (0.6%), and enanthic acid in the root extract (5.2%). The leaf, stem and root extracts had a high saturated fatty acid content, of which palmitic acid was the predominant compound (38.5%, 31.3% and 14.1%, respectively), followed by lauric acid (5.2%, 9.1% and 13.2%, respectively). The main unsaturated fatty acids were, for the leaf extract, oleic acid (10.6%), linolenic acid (9.3%) and linoleic acid (5.5%); for the stem extract, linolenic acid (8.5%) and linoleic acid (7.8%); and for the root extract, linolenic acid (8.6%) and linoleic acid (13.6%). Combined GC and GC-MS analysis of the unsaponifiable fractions detected 32 compounds, of which 18 were present in the leaf fraction, 24 in the stem fraction and seven in the root fraction, accounting for 88.2%, 90.4% and 80.2% of the total unsaponifiable content of these fractions, respectively (Table 2). All three fractions contained high levels of aliphatic compounds, but they differed qualitatively and quantitatively in chemical composition. The leaf fraction was dominated by aliphatic compounds (32.1%), monoterpene hydrocarbons (30.7%) and oxygenated sesquiterpenes (18.5%), whereas the stem fraction contained a higher level of aliphatic compounds (62.6%), a similar level of monoterpene hydrocarbons (21.9%) and a much smaller amount of oxygenated sesquiterpenes (3.8%) than the leaf fraction. The fractions also differed qualitatively and quantitatively in
Dib et al. Table 1: Fatty acids of leaf, stem and root extracts of Arbutus unedo (proportion w/w expressed with regard to the total of fatty acids). N°a 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fatty Acidb Enanthic Lauric Tridecylic Myristic Pentadecanoic Palmitic Margaric Linoleic Linolenic Oleic Stearic Nonadecanoic Arachidic Behenic
Il c 1005 1521 1608 1708 1808 1908 2008 2083 2081 2106 2109 2210 2311 2511
Ia d Leavese Stemse Rootse 1016 5.2 1529 5.2 9.1 13.2 1592 2.3 2.8 1.5 1707 2.6 4.5 3.2 1796 2.4 1.4 5.2 1908 38.5 31.3 14.1 2015 1.6 5.1 2071 5.5 7.8 13.6 2080 9.3 8.5 8.6 2098 10.6 2108 2.5 2.1 4.6 2199 0.6 2318 1.2 1.1 4.9 2510 1.9 5.1 6.5
Total identified 83.6 74.3 85.7 Orders of elution are given on apolar column (Rtx-1). b Fatty acids were tentatively identified on the basis of their fatty acid methyl ester (FAME) derivatives. c Retention indices in literature of corresponding esters on the apolar column (I l) (Jennings and Shibamoto [34]; König et al.. [36]) d Retention indices of corresponding esters on the apolar Rtx-1 column (I a). e Relative percentages (%) on apolar column (Rtx-1); tr = trace (< 0.1%). a
respect of individual monoterpene hydrocarbons. (Z)-βocimene was the major component of the leaf fraction (22.4%), which also contained significant amounts of limonene (3.2%), α-pinene (2.6%) and sabinene (2.5%), whereas the major component of the stem fraction was myrcene (8.5%), followed by sabinene (4.6%), limonene (3.2%), terpinene (2.7%), α-pinene (2.4%) and (Z)-β-ocimene (0.5%). The leaf and stem fractions contained low levels of aromatic ester compounds; (E)anyl 2-methylbutyrate constituted only 6.5% and 2.1%, respectively. Conversely, the root fraction was characterized by a high level of aromatic ester compounds (37.1%) and no terpenic compounds. The major components of the root fraction were benzyl cinnamate (36.6%), undecane (14.9%) and 2.4-dimethyl nonane (12.4%). The root fraction also contained carbonyl and carboxylic compounds (52.9%) in the form of decanal, (E)-2-nonenal, decyl acetate, (Z)-3hexenyl benzoate and benzyl cinnamate. We evaluated the antibacterial activity of the unsaponifiable extracts against pathogenic strains of Gram-positive (Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter frendii and Enterococcus faecalis). Table 3 shows the mean percentage bacterial growth inhibition of the fractions according to the agar dilution method. The unsaponifiable extracts of leaves were effective against K. pneumoniae and E. faecalis (minimum inhibitory concentration [MIC] = 108 and 112 μg/mL,
Chemical composition of Arbutus unedo extracts
Natural Product Communications Vol. 5 (7) 2010 1087
Table 2: Chemical composition of unsaponifiable fractions of leaves, stems and roots from Arbutus unedo. N°a Components 1 2-Methyl heptane 2 Hexan-2-one 3 Octane 4 Ethyl cyclohexane 5 2,6-Dimethyl heptane 6 Nonane 7 α-pinene 8 Sabinene 9 Myrcene 10 2,4-Dimethyl nonane 11 Limonene 12 (Z)-β-ocimene 13 Terpinene 14 Nonanal 15 Undecane 16 (E)-2-Nonenal 17 Decanal 18 Decanol 19 Tridecane 20 Dodecanal 21 Tetradecane 22 Decyl acetate 23 Pentadecane 24 Cadina-3,9-diene 25 Elemol 26 (Z)-3-hexenyl benzoate 27 Dodecyl acetate 28 (E)-Anyl 2-methyl butyrate 29 Tetradecanol 30 Heptadecane 31 Hexadecanal 32 Benzyl cinnamate
Il b 766 791 800 827 830 900 936 973 987 1012 1020 1029 1051 1087 1100 1163 1188 1263 1300 1391 1400 1406 1500 1519 1550 1570 1606 1651 1676 1700 1814 2042
Ia c Leavesd Stemsd Rootsd 765 5.5 794 3.2 800 0.4 822 2.4 827 1.3 900 5.1 938 2.6 2.4 972 2.5 4.6 994 8.5 1009 12.4 1013 3.2 3.2 1026 22.4 0.5 1048 2.7 1089 tr 1100 2.1 9.9 14.9 1168 4.5 1178 3.8 5.7 1261 tr 1300 tr 1389 3.8 10 .7 1400 tr 1410 tr tr 5.6 1500 1.9 3.8 1520 0.4 1542 18.5 3.8 1579 0.5 1591 10.5 1.7 1651 6.5 2.1 1664 7.9 8.2 1700 2.4 5.7 1826 3.5 0.9 2050 36.6
Total identified
88.2
90.4
80.2
Aliphatic compounds Phenolic compounds Hydrocarbon monoterpenes Hydrocarbon sesquiterpenes Oxygenated sesquiterpenes
32.1 6.5 30.7 0.4 18.5
62.6 2.1 21.9 3.8
43.1 37.1 -
a
Order of elution are given on apolar column (Rtx-1). Retention indices of literature on the apolar column (I l) (Jennings and Shibamoto [34]; König et al. [36]) c Retention indices on the apolar Rtx-1 column (I a). d Relative percentages (%) on apolar column (Rtx-1); tr = trace (2200
>2200 >2200
4
nd
Staphylococcus aureus
>2200
1820
>2200
2
nd
Listeria monocytogenes
>2200
2200
>2200
2
nd
Escherichia coli
>2200
>2200 >2200
4
nd
Klebsiella pneumoniae
108
>2200
4
nd
Pseudomonas aeruginosa Citrobacter frendii
>2200
>2200 >2200
4
nd
>2200
2000
>2200
4
nd
Enteroccocus faecalis
112
>2200 >2200
4
nd
nd
15
Gram-negative bacteria
110
Yeasts Candida albicans a
>2200
110
>2200
GEN: Gentamicin; bAmB: Amphotericin B; nd: not determined
The leaves, stems and roots of A. unedo were characterized in terms of fatty acids. All parts of the plant were rich in saturated fatty acids, of which palmitic acid was the main component (leaves, 38.5%; stems, 31.3% and roots, 14.1%). All extracts contained long-chain fatty acids (C12 to C22). The unsaponifiable fractions of the leaves and stems were characterized by aliphatic compounds and monoterpene hydrocarbons. Sesquiterpenoids were also present, but in smaller amounts. Conversely, the unsaponifiable root fraction was dominated by benzyl cinnamate. The antimicrobial properties of the unsaponifiable extracts were tested using nine species of microorganisms. The leaf extract exhibited activity against K. pneumoniae and E. faecalis, and the stem extract against K. pneumoniae and C. albicans, the latter a fungal strain deemed dangerous and difficult to eliminate. The monoterpene hydrocarbon contents of stems and leaves (21.9% and 30.7%, respectively) may be partly responsible fo r their antibacterial activity [29] as the root extract contained no monoterpene compounds and had no antibacterial activity. To the best of our knowledge, this is the first report on the fatty acid composition of A. unedo and its antimicrobial activity. Experimental Plant material: Samples of A. unedo L. stems, leaves and roots were collected in Terni Forest (about 20 km south of Tlemcen, Algeria; altitude, 1190 m; 34° 49′ N, 1° 19′ E) in September 2008. Voucher specimens were deposited in the herbarium of the Tlemcen University Botanical Laboratory (voucher no. Er 09.08). A portion of each sample was stored at 4°C for future studies.
1088 Natural Product Communications Vol. 5 (7) 2010
Sample preparation: Leaf and stem samples were airdried and crushed. The resulting stem and leaf portions were sieved through 0.355 mm and 0.600 mm sieves, respectively. The roots were cleaned and cut into small pieces. Thirty g leaf, stem and root samples were extracted twice using 200 mL of light petroleum under reflux using a Soxhlet apparatus. After evaporation of the solvent under reduced pressure, the extracts were dried over MgSO4. The mean yields of triplicate extractions were stems, 0.86%; leaves, 3.8% and roots, 0.36%. Extraction of unsaponifiable compounds: One g aliquots of the light petroleum extract were saponified using 50 mL methanolic potassium hydroxide solution (2 mol/L) for 1 h under reflux. The unsaponifiable components were then extracted 3 times with 100 mL diethylether. The pooled extracts were washed 3 times with 50 mL deionized water. The solvent was removed at 35°C under reduced pressure using a rotary evaporator. The unsaponifiable fractions of leaves, stems and roots constituted 55.2%, 50.3% and 25.1% (w/w) of the light petroleum extract, respectively. Extraction of fatty acids: After extraction of the unsaponifiable components, the remainder of the methanolic potassium hydroxide solution was acidified to pH 5–6 with 1 N HCl to convert the fatty acid salts to free fatty acids, which were then extracted 3 times with 50 mL diethylether, dried over MgSO4 and weighed. A methanolic solution containing 10% BF3 was added to the free fatty acids to transform them into methyl ester derivatives [30]. The fatty acid derivatives were then extracted 3 times with 50 mL of n-hexane at room temperature. The organic layer was evaporated and dried over Na2SO4. The fatty acid fractions of the leaves, stems and roots constituted 23.5%, 31.4% and 10.4% (w/w) of the diethylether extract, respectively. Gas chromatographic analysis: GC analyses were carried out using a Perkin Elmer (Waltham, MA, USA) Autosystem XL GC apparatus equipped with a dual flame ionization detection system and fused Rtx-1silica capillary columns (60 m × 0.22 mm i.d., 0.25 μm film thickness; polydimethylsiloxane). The oven temperature was programmed to increase from 60–230°C at 2°C/min and was then held isothermally at 230°C for 35 min. Injector and detector temperatures were maintained at 280°C. Samples were injected in the split mode (1/50) using helium as carrier gas (1 mL/min); the injection volume was 0.2 μL. Retention indices of the compounds were determined relative to the retention times of a series of n-alkanes (C5–C30) using linear interpolation, the Van den Dool and Kratz equation [31], and software from
Dib et al.
PerkinElmer. Relative concentrations were calculated based on GC peak areas without using correction factors. Gas chromatography–mass spectrometry analysis: Samples were analyzed using a Perkin Elmer Turbo mass detector (quadrupole) coupled to a Perkin Elmer Autosystem XL equipped with Rtx-1 fused silica capillary columns and Rtx-Wax (ion source temperature, 150°C; ionization energy, 70 eV). Ionization energy MS were acquired over a mass range of 35–350 Da (scan time, 1 s). Other GC conditions were the same as described for GC, except the split was 1/80. Component identification: As previously reported [32,33], the method used for identification of individual components was based on (a) comparison of calculated retention indices on an apolar column with those of either authentic compounds or literature data [34–37] and (b) computer-matching using commercial MS libraries [36,38–41] and comparison of MS with those in either our own library of authentic compounds or in the literature [34,35]. Bacteria and yeast: The bacterial strains used in this study were obtained from the medical reanimation department of the Hospital University Center of Tlemcen and consisted of five Gram-negative (E. coli, P. aeruginosa, C. frendii, E. faecalis and K. pneumoniae) and three Gram-positive bacteria (B. cereus, S. aureus and L. monocytogenes). The C. albicans fungal isolate was obtained from the dermatology department of the Hospital University Center of Tlemcen. Preparation of inocula: Strains preserved in nutrient agar at 4°C were placed in nutrient solution and incubated at 37°C for 24 h. Culture aliquots (0.1 mL) were added to 10 mL of brain heart infusion broth (Pronadisa Hispanalab, S.A.). The fungus preserved at 4°C in Sabouraud agar supplemented with chloramphenicol was reconstituted in nutrient solution and incubated at 30°C for 24 h. Culture aliquots (0.1 mL) were added to 10 mL sterile physiological water. Muller– Hinton agar for the bacteria (Pronadisa Hispanalab) and Sabouraud dextrose agar plus chloramphenicol for the yeast (Merck) were utilized. The inocula were incubated until a microbial density of 106–107 colony-forming units per mL was attained. The agar dilution method was used to assess antimicrobial activity. The MIC was determined using the latter method. Dilution agar method: A dilution agar method was used to determine MIC. Extracts were dissolved in 1% DMSO and 0.2 mL aliquots were added in twofold increments from 0.1 mg/L 2.2 mg/L to either Mueller– Hinton agar (bacteria) or Sabouraud dextrose agar
Chemical composition of Arbutus unedo extracts
containing chloramphenicol (C. albicans). The media were cooled to 45–50°C prior to being poured into the plates. Gentamicin and amphotericin B were used as the standard antibiotics for bacteria and fungus, respectively [42,43]. Sterile solutions of DMSO and
Natural Product Communications Vol. 5 (7) 2010 1089
culture medium were used as controls. The experiments were performed in triplicate. The bacterial test was conducted at 37°C for 24 h and the fungal test at 30°C for 48 h.
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Coumarins from Seseli hartvigii Lin Zhang, Alev Tosun, Masaki Baba, Yoshihito Okada, Lijun Wu and Toru Okuyama
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Spartinoxide, a New Enantiomer of A82775C with Inhibitory Activity Toward HLE from the Marine-derived Fungus Phaeosphaeria spartinae Mahmoud Fahmi Elsebai, Stefan Kehraus, Michael Gütschow and Gabriele M. König
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Natural Product Communications 2010 Volume 5, Number 7 Contents Original Paper
Page
A DFT Analysis of Thermal Decomposition Reactions Important to Natural Products William N. Setzer
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Novel Terpenoids from the New Zealand Liverworts Jamesoniella colorata and Bazzania novae-zelandiae Masao Toyota, Ikuko Omatsu, Fumi Sakata, John Braggins and Yoshinori Asakawa
999
Two New C20-Diterpenoid Alkaloids from Delphinium anthriscifolium var. savatieri Xiao-Yu Liu, Lei Song, Qiao-Hong Chen, and Feng-Peng Wang
1005
Cytotoxic Activity of Quassinoids from Eurycoma longifolia Katsunori Miyake, Feng Li, Yasuhiro Tezuka, Suresh Awale and Shigetoshi Kadota
1009
Antifungal Activity of Saponin-rich Extracts of Phytolacca dioica and of the Sapogenins Obtained through Hydrolysis Melina Di Liberto, Laura Svetaz, Ricardo L. E. Furlán, Susana A. Zacchino, Carla Delporte, Marco A. Novoa, Marcelo Asencio and Bruce K. Cassels
1013
New Lupane-type Triterpenoid Saponins from Leaves of Oplopanax horridus (Devil’s Club) Pei-Pei Liu, Mo Li, Ting-Guo Kang, De-Qiang Dou and David C Smith
1019
Triterpene Saponins from Cyclamen persicum Ghezala Mihci-Gaidi, David Pertuit, Tomofumi Miyamoto, Jean-François Mirjolet, Olivier Duchamp, Anne-Claire Mitaine-Offer and Marie-Aleth Lacaille-Dubois
1023
Cytotoxic Pentacyclic Triterpenoids from Combretum oliviforme Xiao-Peng Wu, Chang-Ri Han, Guang-Ying Chen, Yuan Yuan and Jian-Ying Xie
1027
Preparative Separation of Four Major Bufadienolides from the Chinese Traditional Medicine, Chansu, Using High-Speed Counter-Current Chromatography Xiu Lan Xin, Junying Liu, Xiao Chi Ma, Qing Wei, Li Lv, Chang Yuan Wang, Ji Hong Yao and Jian Cui
1031
Acetylcholinesterase and Butyrylcholinesterase Inhibitory Compounds from Eschscholzia californica (Papaveraceae) Lucie Cahlíková, Kateřina Macáková, Jiří Kuneš, Milan Kurfürst, Lubomír Opletal, Josef Cvačka, Jakub Chlebek and Gerald Blunden
1035
In Vitro Testing for Genotoxicity of Indigo Naturalis Assessed by Micronucleus Test Luca Dominici, Barbara Cerbone, Milena Villarini, Cristina Fatigoni and Massimo Moretti
1039
Metabolites from Withania aristata with Potential Phytotoxic Activity Gabriel G. Llanos, Rosa M. Varela, Ignacio A. Jiménez, José M. G. Molinillo, Francisco A. Macías and Isabel L. Bazzocchi
1043
Bioassay-guided Isolation and Quantification of the α-Glucosidase Inhibitory Compound, Glycyrrhisoflavone, from Glycyrrhiza uralensis Wei Li, Songpei Li , Lin Lin, Hong Bai, YingHua Wang, Hiroyoshi Kato, Yoshihisa Asada, Qingbo Zhang and Kazuo Koike
1049
Xanthones, Biflavanones and Triterpenes from Pentadesma grandifolia (Clusiaceae): Structural Determination and Bioactivity Grace Leontine Nwabouloun Djoufack, Karin M. Valant-Vetschera, Johann Schinnerl, Lothar Brecker, Eberhard Lorbeer and Wolfgang Robien
1055
Gmelinoside I, a New Flavonol Glycoside from Limonium gmelinii Zhanar A. Kozhamkulova, Mohamed M. Radwan, Galiya E. Zhusupova, Zharilkasin Zh. Abilov, Saniya N. Rahadilova and Samir A. Ross
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2-Arylbenzofuran Neolignans from the Bark of Nectandra purpurascens (Lauraceae) Jaime Rios-Motta and Eliseo Avella Continued inside backcover
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