Anti-inflammatory, antioxidant and antifungal furanosesquiterpenoids isolated from Commiphora erythraea (Ehrenb.) Engl. resin

Fitoterapia 82 (2011) 654–661

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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Anti-inflammatory, antioxidant and antifungal furanosesquiterpenoids isolated from Commiphora erythraea (Ehrenb.) Engl. resin Daniele Fraternale a, Silvio Sosa b, Donata Ricci a, Salvatore Genovese c, Federica Messina d, Sabrina Tomasini d, Francesca Montanari d, Maria Carla Marcotullio d,⁎ a b c d

Dipartimento di Scienze dell'Uomo, dell'Ambiente e della Natura, Sez. Biologia Vegetale, Università degli Studi di Urbino “Carlo Bo”, Via Bramante, 28-61029 Urbino, Italy Dipartimento dei Materiali e delle Risorse Naturali, Università degli Studi di Trieste, Via A. Valerio, 6-34127 Trieste, Italy Dipartimento di Scienza del Farmaco, Università degli Studi di Chieti, Via de' Vestini, 31-66013 Chieti, Italy Dipartimento di Chimica e Tecnologia del Farmaco, Sez. Chimica Organica, Università degli Studi di Perugia, Via del Liceo, 1-06123 Perugia, Italy

a r t i c l e

i n f o

Article history: Received 9 December 2010 Accepted in revised form 3 February 2011 Available online 21 February 2011 Keywords: Antifungal Anti-inflammatory Antioxidant Commiphora erythraea DPPH Croton oil test

a b s t r a c t The topical anti-inflammatory, free radical scavenging and antifungal activities of essential oils and extracts of Commiphora erythraea (Ehrenb.) Engl. resin were investigated. The hexane extract significantly inhibited oedema when applied topically in Croton oil-induced ear oedema assay in mice. The same extract showed antioxidant activity in DPPH radical scavenging assay. A bioguided separation of the hexane extract led to the isolation of furanosesquiterpenoids 1 and 2 that showed a weak antifungal activity, while compounds 3–5 resulted to be antioxidant (EC50 4.28, 2.56 and 1.08 mg/mL, respectively) and antiinflammatory (30, 26 and 32% oedema reduction, respectively). © 2011 Elsevier B.V. All rights reserved.

1. Introduction The genus Commiphora (Burseraceae), native to the Arabian Peninsula, Africa and most of the Middle East and India, comprises more than 150 species that produce fragrant resins commonly known as myrrh [1]. The chemical constituents of several Commiphora plant extracts have been reviewed; characteristic compounds of myrrh are mainly terpenoids, including furanosesquiterpenoids with eudesmane, germacrane, elemane, or guaiane skeletons, that are responsible for the resin's odour [2]. Traditionally, Commiphora oleo-gum resins are largely used for their biological activities to treat colds, fever, malaria, in wound healing, as antiseptic and in skin infections [3]. The resins obtained from Commiphora spp play an important role in the economy of rural families in Ethiopia [4].

⁎ Corresponding author. Tel.: +39 075 5855107; fax: +39 075 5855116. E-mail address: [email protected] (M.C. Marcotullio). 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.02.002

Among various resins collected by local people, “agarsu” (Commiphora erythraea (Ehrenb.) Engl resin) is commonly used to protect livestock from ticks [5] and the larvicidal effect of a hexane extract of C. erythraea has been proved [6,7]. Furthermore, C. erythraea bark, sap and gum are used for the treatment of foetal membrane retention, of worm infestations, and as anti-inflammatory to treat eye problems [5]. C. erythraea is a taxonomically complex species and in the past has been considered a subspecies of C. kataf (Forssk.) Engl. [8]. The composition of the steam distilled oil from C. erythraea resin has been recently reported [9], and it showed to be different from that of C. kataf essential oil [10]. IPO (Increasing People Opportunities) is a no profit association whose work in Ethiopia is aimed in “increasing the value of incenses and enhancing gum and resins collection and processing, with the double aim of promoting aromatic materials' fair trade from Horn of Africa and, at the same time, protecting and preserving cultural heritage and natural landscape” [11]. C. erythraea resin is commercialized

D. Fraternale et al. / Fitoterapia 82 (2011) 654–661

by IPO in western countries mostly as “ethical incense” looking forward to better define the biological properties of agarsu. On demand of IPO association we decided to study C. erythraea resin and in the present paper, we report the evaluation of the antifungal, antioxidant and anti-inflammatory properties of differently prepared essential oils and extracts obtained from the commercial agarsu and the bioguided isolation of three anti-inflammatory and antioxidant furanosesquiterpenoids from the bioactive hexane extract.

2. Experimental 2.1. Plant material The resin of C. erythraea (Agarsu grade I), commercialized by Agarsu Liben Cooperative and imported into Italy by IPO (Increasing People Opportunities) association [11], was studied. A voucher specimen (# MCM-1) of the resin (Agarsu grade I) is deposited at the Dipartimento di Chimica e Tecnologia del Farmaco - Sez. Chimica Organica, University of Perugia (Italy).

2.2. Extraction of the resin 2.2.1. Steam distillation Steam distillation was performed in a glass laboratory apparatus assembled as described by Pourmortazavi et al. [12]. The steam generator flask was filled with 1000 mL of H2O and the ground resin (10 g) was distilled for 3 h. The oil (D) was dried over anhydrous sodium sulphate, filtered and stored at −20 °C. The yield (1.92 ± 0.52%, w/w) is the mean of three distillations ± standard deviation (SD).

2.2.2. Hydrodistillation The ground resin (22.3 g) was hydrodistilled in triplicate with a Clevenger-type apparatus for 3 h with 500 mL of H2O. The oil (HD) was dried over anhydrous sodium sulphate, filtered and stored at −20 °C. The yield (1.86 ± 0.13%, w/w) is the mean of three distillations ± SD.

2.2.3. CO2 extraction The SFE system (Jasco, Cremella, LC, Italy) consisted of a model PU-2080-CO2 plus and detector UV/Vis Jasco mod. 875-UV. Finely triturated resin (5 g) was placed in a 10-mL disposable SFE extraction cell. The resin was held in dynamic extraction at 20 °C and 20 MPa for 1 h (SF1) and at 40 °C and 100 MPa for 1 h (SF2) at a CO2 flow rate of 1 mL/min. The effluent was collected in a sample vial and stored at − 20 °C. The yields (SF1: 8.40 ± 0.15%, w/w, SF2: 10.00 ± 0.12%, w/w) are the mean of three extractions ± SD.

2.2.4. Hexane extraction The ground resin (5 g) was extracted at room temperature for 4 h with 250 mL of n-hexane. Then, the suspension was filtered under vacuum and the solvent removed under N2. Reported yield (24.27 ± 0.20%, w/w) is the mean of three extractions ± SD.

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2.3. GC–MS analyses Oils and extracts were analysed by GC and GC–MS. Analyses were performed on a Hewlett Packard HP 6890 gas chromatograph. The GC instrument was equipped with a Hewlett Packard MS 5973 mass selective detector and a fused silica capillary column (HP-5MS; 30 m × 0.25 mm i.d., 0.25 μm film thickness). The oven temperature was programmed from 60 °C for 5 min, then ramped at 4 °C/min to 270 °C, and held for 20 min. Injector and detector temperatures were 250 and 270 °C, respectively. Samples were dissolved in methylene chloride to give 1% w/v solutions and were injected in the splitless mode using helium as carrier gas (1 mL/min); the injection volume was 1 μL. The ionization energy was 70 eV. Percentage compositions of the components were obtained from electronic integration dividing the area of each component by the total area of all components. The percentage values are the mean ± SD of three injections of the sample. All compounds were identified by comparison of their linear retention indices (RI) relative to retention times on HP-5MS column of a homologous series of C5–C20 alkanes with those reported in the literature [13] and by comparison of mass spectra from the NIST98 Mass Spectral Database. Moreover, whenever possible, identification has been confirmed by injection of authentic sample of the compounds and by comparison of 1H and 13C NMR spectra with those already reported.

2.4. Isolation of tested furanosesquiterpenoids The hexane extract (H) (3.80 g) was fractionated by silica gel column chromatography (Davisil LC60A 60–200 μm). Three fractions were collected by sequentially eluting the column with 1 L each of hexane (H-1 fraction, 0.18 g), CH2Cl2 (H-2 fraction, 2.48 g), and CH2Cl2–EtOAc 95:5 (H-3 fraction, 0.78 mg). The biologically active fractions (H-2 and H-3) were further fractionated. Fraction H-2 was purified by SiO2 column chromatography and eluted using a step gradient of hexane/ethyl acetate, increasing the solvent system from 0% to 5% ethyl acetate. 10-mL fractions were collected throughout and pooled into seven groups (H2-1–H2-7) according to composition, as visualized by TLC (Thin Layer Chromatography, Merck TLC silica gel 60 F254) after spraying with panisaldehyde–H2SO4–MeOH (1:1:98) and using 5% hexane: ethyl acetate as eluent. Fraction H2-2 was constituted by furanogermacradienone 1 (42 mg), fraction H2-4 resulted to be furanodienone 2 (7 mg), fraction H2-5 (54 mg) was a mixture of compounds 2 and 3, and fraction H2-6 contained pure 3-methoxy-furanogermacradien-6-one 4 (62 mg). Fraction H-3 was purified on SiO2 column chromatography and eluted using a step gradient of hexane–Et2O from 0 to 10%. 10-mL fractions were collected throughout and combined by single spot compounds or by groups showing the same Rf values giving three fractions (H3-1–H3-3). H3-1 contained pure 4 (60 mg) and H3-3 pure 2-methoxy-furanogermacren-6one 5 (50 mg). Fraction H2-3 (0.10 g) was purified by preparative TLC and elution with hexane–Et2O 10% gave pure H2-31 (1, 70 mg) and pure H2-32 (2, 10 mg).

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Table 1 Chemical composition of Commiphora erythraea essential oils, extracts and fractions a. Dc

HD d

He

SF1 f

SF2 g

H-1

H-2

H-3

Identification h

α-Thujene α-Pinene Camphene Sabinene β-Pinene 3-Carene p-Cymene Limonene 4-Terpineol δ-Elemene α-Cubebene α-Copaene β-Bourbonene β-Elemene α-Gurjunene β-Caryophyllene α-Guaiene γ-Elemene Aromadendrene α-Humulene Alloaromadendrene Germacrene D β-Selinene α-Selinene Curzerene γ-Cadinene δ-Cadinene α-Cadinene Germacrene B 1(10),4-Furanodien-6-one (2) β-Elemenone Curzerenone (6) Alismol β-Eudesmol α-Eudesmol

0.20 ± 0.08 3.42 ± 0.09 = 0.10 ± 0.04 0.73 ± 0.06 0.40 ± 0.07 0.20 ± 0.10 0.50 ± 0.07 0.32 ± 0.04 1.60 ± 0.15 0.97 ± 0.10 4.35 ± 0.05 1.56 ± 0.07 5.39 ± 0.10 4.29 ± 0.05 1.48 ± 0.04 0.96 ± 0.04 = 3.51 ± 0.10 1.09 ± 0.05 2.04 ± 0.04 1.99 ± 0.03 3.25 ± 0.05 3.18 ± 0.03 = 1.16 ± 0.14 2.15 ± 0.15 = = 20.60 ± 0.11 = 2.85 ± 0.07 1.92 ± 0.06 2.56 ± 0.08 1.02 ± 0.06

1.52 ± 0.07 4.52 ± 0.13 8.16 ± 0.18 2.24 ± 0.16 = 3.00 ± 0.05 2.57 ± 0.08 = 0.98 ± 0.03 2.55 ± 0.07 1.31 ± 0.10 6.57 ± 0.07 2.01 ± 0.03 8.18 ± 0.12 6.02 ± 0.15 2.11 ± 0.04 = = 4.43 ± 0.14 1.78 ± 0.07 3.64 ± 0.10 1.72 ± 0.05 3.63 ± 0.08 3.82 ± 0.05 0.70 ± 0.13 1.28 ± 0.04 2.49 ± 0.12 0.43 ± 0.08 1.77 ± 0.15 9.00 ± 0.04 = 0.92 ± 0.06 1.05 ± 0.02 1.23 ± 0.12 0.52 ± 0.05

= = = = = = = = = 0.76 ± 0.13 2.00 ± 0.04 3.18 ± 0.15 1.31 ± 0.08 3.58 ± 0.06 2.43 ± 0.12 0.78 ± 0.02 = 2.03 ± 0.03 0.52 ± 0.04 0.54 ± 0.05 = 1.24 ± 0.03 1.82 ± 0.07 0.59 ± 0.03 = 0.70 ± 0.02 2.38 ± 0.03 = = 24.65 ± 0.05 = 3.59 ± 0.13 1.06 ± 0.03 1.36 ± 0.03 1.25 ± 0.04

= = = = = = = = = 0.18 ± 0.02 0.26 ± 0.08 1.40 ± 0.10 0.76 ± 0.09 2.79 ± 0.11 1.61 ± 0.04 = 0.40 ± 0.11 = 0.97 ± 0.10 0.21 ± 0.03 0.32 ± 0.04 = 1.40 ± 0.10 0.86 ± 0.11 = 1.01 ± 0.02 0.44 ± 0.07 = 0.96 ± 0.06 14.00 ± 0.10 2.940.08 3.16 ± 0.09 0.87 ± 0.09 = =

= = = = = = = = = = 0.67 ± 0.08 2.00 ± 0.13 1.00 ± 0.04 3.42 ± 0.07 1.81 ± 0.04 = 0.52 ± 0.06 1.36 ± 0.07 0.39 ± 0.11 = 0.37 ± 0.12 = 1.53 ± 0.09 0.56 ± 0.08 = = 0.57 ± 0.09 = 1.21 ± 0.09 15.16 ± 0.05 2.46 ± 0.16 3.49 ± 0.13 0.39 ± 0.05 = =

= = = = = = = = = 2.24 ± 0.04 1.75 ± 0.06 17.94 ± 0.12 4.89 ± 0.21 13.02 ± 0.05 13.48 ± 0.14 1.39 ± 0.08 = 4.83 ± 0.11 = = = = 14.21 ± 0.09 8.99 ± 0.20 = 4.07 ± 0.14 3.89 ± 0.05 0.94 ± 0.06 0.19 ± 0.14 = = = = = =

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = 40.20 ± 0.05 = 7.11 ± 0.06 = = =

= = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = 4.60 ± 0.02 0.35 ± 0.11 0.58 ± 0.06

MS, RI i MS, RI, Inj MS, RI, MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Inj MS, RI, Inj MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Inj MS, RI MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI, Ref MS, RI MS, RI, Ref MS, RI, Ref MS, RI MS, RI, Ref NMR MS, RI, Ref MS, Ref, Inj NMR MS, RI, Ref MS, RI, Ref

D. Fraternale et al. / Fitoterapia 82 (2011) 654–661

Component b

Table 1 (continued) Component b

Dc

HD d

He

SF1 f

SF2 g

H-1

H-2

H-3

Identification h

Germacrone 1, 10(15)-Furanogermacra-dien-6-one (1) NI j Dihydropyrocurzerenone rel-3R-Methoxy-4S-furanogermacra-1E, 10(15)-dien-6-one (4) NI rel-2R-Methoxy-4R-furanogermacra-1(10)E-en-6-one (5) Mirrhone (3) NI NI NI NI

1.26 ± 0.08 10.41 ± 0.05 = 4.21 ± 0.05 3.30 ± 0.06

0.37 ± 0.06 4.30 ± 0.08 = 1.11 ± 0.09 0.90 ± 0.07

= 10.36 ± 0.07 0.81 ± 0.10 3.38 ± 0.08 12.45 ± 0.14

0.68 ± 0.05 17.25 ± 0.13 = 2.20 ± 0.08 20.49 ± 0.11

0.55 ± 0.11 18.62 ± 0.14 = 3.27 ± 0.07 14.02 ± 0.07

= = = = =

= 36.05 ± 0.10 = 4.22 ± 0.14 =

= = = = 41.80 ± 0.08

MS, RI, Ref NMR, Inj

2.45 ± 0.06 3.09 ± 0.11 = 1.20 ± 0.17 = = =

0.78 ± 0.09 0.92 ± 0.05 = 1.22 ± 0.03 = = =

2.11 ± 0.05 9.91 ± 0.11 0.61 ± 0.10 3.13 ± 0.06 0.36 ± 0.14 tr k =

= 12.76 ± 0.07 4.69 ± 0.14 5.35 ± 0.09 1.68 ± 0.09 = =

6.07 ± 0.08 10.52 ± 0.09 1.99 ± 0.02 5.11 ± 0.08 1.83 ± 0.13 = tr

= = = 2.10 ± 0.20 5.20 ± 0.16 = =

= = = 6.58 ± 0.18 5.80 ± 0.09 = =

1.92 ± 0.14 34.68 ± 0.05 6.01 ± 0.10 6.02 ± 0.14 1.98 ± 0.12 = =

Total (%) Total identified Monoterpenes Monoterpenoids Sesquiterpenes Sesquiterpene alcohols Sesquiterpene ketones Furanosesquiterpenoids

99.71 96.06 5.55 0.32 38.97 5.50 1.26 44.45

99.74 97.74 22.00 0.98 53.74 2.80 0.37 17.86

98.82 92.42 = = 23.80 3.67 = 64.95

99.64 92.60 = = 13.57 0.87 3.62 74.54

98.89 85.88 = = 15.41 0.39 3.01 67.08

99.22 91.92 = = 91.92 = = =

99.96 87.58 = = = = = 87.58

97.94 88.02 = = = 5.53 = 82.49

NMR, Inj NMR, Inj

Percentage obtained by FID peak-area normalization. Values are presented as the mean ± SD (n = 3). Components are listed in order of their elution from a HP-5MS column. c D: Steam distilled oil. d HD: Hydrodistilled oil. e H: Hexane extract. f SF1: Obtained by extraction at 20 MPa and 20 °C for 1 h. g SF2: Obtained by extraction at 100 MPa and 40 °C for 1 h. h Identification: MS, by comparison of the mass spectrum with those of the computer NIST98 library (99% matching); RI, by comparison of RI with those reported from NIST databank; Ref, by comparison with literature data; Inj: positive identification from mass spectrum and retention time which agree with authentic compound; NMR, isolated compounds identified by NMR spectra. i RI, linear retention indices were determined relative to the retention times on HP-5MS column of homologous series of C5\C20 alkanes, using the equation of Van den Dool and Krantz. [44]. j NI: Not identified. k tr: taces, b0.10%. b

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a

NMR, Inj NMR, Inj

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Fraction H2-5 was purified by elution with hexane–EtOAc 30% giving H2-51 (2, 30 mg) and H2-52 (3, 12 mg). Purification of H3-2 on preparative TLC plates and elution with CH2Cl2 gave H3-21 (4, 10 mg) and H3-22 (5, 12 mg). The structures of all isolated compounds were determined spectroscopically by mono- and bidimensional NMR techniques (1H-, 13C, JMOD, COSY, HMQC, HMBC, NOESY experiments) and confirmed by comparison with literature data [6,10,14–17].

2.5. Antifungal activity 2.5.1. Fungal strains Fungal plant pathogens used in the tests were: Fusarium culmorum (Smith) Saccardo (ATCC 12656), Phytophtora cryptogea Pethyb. et Laff. (ATCC 33913), Alternaria solani Ell. et Mart. (ATCC 11078). All the organisms were maintained on potato-dextrose-agar (PDA, Difco) in the dark at 20 °C and subcultured in the same medium every three weeks.

2.6. Antioxidant activity Radical scavenging activity was determined by a spectrophotometric method based on the reduction of an ethanol solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) [19,22]. The antioxidant activity was expressed as EC50. The EC50 is defined as the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%. The results are the mean ± standard deviation (SD) of three experiments. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma), ascorbic acid (Sigma) and BHT (butylated hydroxytoluene, Sigma) were used as positive control. 2.7. Topical anti-inflammatory activity The topical anti-inflammatory activity was evaluated as inhibition of the Croton oil-induced ear oedema in mice. Ten animals were used for each group of treatment [23,24]. 2.8. Statistical analysis

2.5.2. Determination of antifungal activity The phytopathogenic fungi were tested according to a reported procedure [18,19]. The oils were dissolved in 20% dimethylsulphoxide (DMSO) plus 5% Tween 20 (Fluka). Concentrations of 3000, 6000 and 12,000 ppm were tested. Controls consisted of 3000, 6000 and 12,000 ppm of 20% DMSO + 5% Tween 20 mixed with PDA. All experiments were carried out in triplicate. The MIC values (ppm) were determined by the dilution method in solid medium. Dilutions of the emulsions of the oil were made in the culture medium over the concentration range of 3000 ppm and 6000 ppm, for all strains tested. MICs were determined as the concentration with no visible growth. The experiments that showed different MIC results were triplicated and the higher value obtained was noted. MFCs were obtained using a membrane filtration method [20] and were calculated from 6000 to 12,000 ppm for all strains tested. After homogenization of each mycelium, each fungal inoculum (1 mL; 108 spores/mL) was added to 10 mL of oil emulsion (oil + DMSO + 5% Tween 20); the mixture was kept at room temperature for a contact time of 15 min. After the contact time, 1 mL of the mixture was transferred into a sterile filtration apparatus (MILLIPORE SWINNEX — 25), washed twice with 100 mL of the neutralizing solution consisting of distilled water containing 10% Tween 20 sterilized at 121 °C for 20 min, filtered and rinsed with sterile distilled water. The membranes were separately laid out on the middle of the Petri dishes containing the corresponding agar medium (PDA). After incubation the colonies were counted on the membranes. Previously, we checked that the mixture DMSO plus 5% Tween 20 did not inhibit the germination of spores. MFCs were calculated as the lowest concentrations of oil which inhibited the recovery of fungus. In order to obtain the development of conidia, 7 day old mycelia were exposed for 10 days to 12 h of NUV light (near ultraviolet) and 12 h dark. Nystatin dihydrate (Fluka) (100 ppm) was used as positive control for fungi [21].

Oedema values, expressed as means ± standard error (SE) of the mean, were analysed by one way analysis of variance, followed by Dunnett's test for multiple comparison of unpaired data. A probability level lower than 0.05 was considered as significant. 3. Results and discussion The compositions of different oils and extracts are reported in Table 1 and the structures of the isolated pure compounds are reported in Fig. 1. Oils were obtained with lower yields (HD: 1.86%, D: 1.92%) with respect to the extracts (H: 24.27%, SF2: 10.00%, SF1: 8.40%). The steam distilled oil prepared for this study showed a composition similar to one already reported [9]. Sesquiterpenoids in all oils and extracts represented the most abundant components, except in HD oil that was richer in sesquiterpenes. In all cases furanosesquiterpenoids were the most representatives oxygenated sesquiterpene derivatives and they were particularly present in the extracts (H: 64.95%, SF1: 74.54%, SF2: 67.08%). Surprisingly, hexane extract was devoid of monoterpenes and monoterpenoids. In our opinion this could be due to the volatilization of these very volatile compounds during the removal of the solvent. The GC–MS spectrum of the hexane extract showed the presence of a compound (traces, b0.10%) with a M+ 410, but we were unable to identify it and it was undetectable in any of the three fractions we prepared. The same compound was not present in SF1 and SF2. On the other hand, in SF2 (40 °C, 100 MPa) there were traces of a different compound (M+ 492). 1(10), 4-furanodien-6-one 2 is the major constituent of the D oil (20.60%) and of H extract (24.65%). All the other furanoderivatives percentages differ notably, depending on the extraction procedure. All the oils and extracts showed a yellow-brownish colour and a semisolid consistence, the odour is bitter and woody and it is completely different from the “warm-balsamic, sweet and spicy-aromatic” odour reported for C. myrrha [16],

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659

O O O O

O

O

1

2

3

MeO MeO

O

O

O O

O 4

O

5

6

Fig. 1. Isolated compounds from Commiphora erythraea hexane extract.

as we had the opportunity to test oils and extract of this latter specie. The sensory observations of our preparations agree with the statement that the odour derives from furanosesquiterpenes [2], as oils and extracts do not differ very much in the qualitative composition, being furanosesquiterpenoids always the most characteristic components. Column chromatography and preparative TLC separation allowed us to obtain fractions containing pure furanosesquiterpenoids (1–5). Compounds 1, 2, 4 and 5 had been already isolated from the steam distilled essential oil of C. erythraea previously analysed [9]. Compound 3 was reported in the previous paper as NI (not identified) as we were not able to isolate it by column chromatography at that time. The chemical structure of 3 was elucidated as myrrhone by comparison of the spectral data with those previously reported [17]. Curzerenone (6) was isolated and identified in the previous study [9], while this time we could find it only as a GC–MS product, but we could not isolate it in the hexane extract. Ab initio calculations found furanodienone 2 more

stable than its rearranged product curzerenone 6. This could explain why in the GC–MS analysis of essential oils furanodienone predominates over curzerenone [25]. Nevertheless it is well known that furanodienone 2 is thermally sensitive and its decomposition into curzerenone (6) through Cope rearrangement is well documented [26–28]. In our opinion, distillation procedures are able to transform furanodienone 2 into curzerenone 6, while non-thermal procedures, such as hexane and CO2 extractions, prevent furanodienone decomposition. Antifungal activity against A. solani, F. culmorum and P. cryptogea of oils, extracts, fractions and pure compounds are reported in Table 2. Results obtained from the agar dilution method, followed by the determinations of MIC indicate that the values are 3000 ppm for A. solani, 5500 ppm for F. culmorum and P. cryptogea, for all tested oils and extracts (Table 3). The most active extract resulted to be H (Table 2), that was subfractioned in H-1, H-2 and H-3. Among the fractions obtained, fraction H-2 was the most active. From

Table 2 Free radical scavenging activity (expressed as EC50) and antifungal activity of oils, extracts, fractions and pure compounds.

Oils and extracts

Fractions

Pure compounds

a

Fungal growth inhibition (%) a

Sample

DPPH EC50 (mg/mL)

A. solani

F. culmorum

P. cryptogea

HD D SF1 SF2 H H-1 H-2 H-3 1 2 3 4 5 Ascorbic acid BHT Trolox Nystatin (100 ppm)

9.077 ± 0.66 14.286 ± 2.09 11.042 ± 0.99 8.515 ± 0.59 4.126 ± 0.17 169.549 ± 10.75 12.872 ± 1.75 3.487 ± 0.30 = = 1.080 ± 0.08 4.287 ± 0.33 2.563 ± 0.14 0.110 ± 0.07 0.086 ± 0.007 0.011 ± 0.001

100° 100° 63.2 ± 5.8 100° 100° 60.1 ± 5.6 86.2 ± 7.9 70.7 ± 6.8 24.7 ± 1.8 80.7 ± 7.5 = = = = = =

90.3 ± 8.3 98.5 ± 8.5 94.6 ± 8.9 92.2 ± 8.7 90.3 ± 8.6 50.5 ± 4.8 90.1 ± 8.7 76.7 ± 6.9 49.3 ± 4.2 82.4 ± 7.8 = = = = = =

55.5 ± 4.6 62.9 ± 5.9 63.2 ± 5.8 66.6 ± 6.0 66.0 ± 6.0 33.5 ± 2.7 67.3 ± 6.1 55.4 ± 4.9 23.4 ± 1.9 76.5 ± 7.1 = = = = = =

=

100*

100*

100*

Tested dose 3000 ppm; °fungistatic; *fungicidal.

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Table 3 Screening of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of C. erythraea oils and extracts. Phytopathogenic fungi

MIC ppm a

MFC ppm a

F. culmorum P. cryptogea A. solani

5500 5500 3000

N 12,000 12,000 N 12,000

a

Same values for all tested oil and extracts.

this fraction the most abundant compounds (1 and 2) were isolated and tested. Furanodienone 2 resulted to be the most active compound and the most sensitive strain was F. culmorum. The DPPH radical scavenging activity decreased in order H N SF2 N HD N SF1 N D (Table 2). After fractionation of H, DPPH assay indicated that best scavenging activity was exerted by fraction H-3 that contained compounds 3–5. These compounds were isolated and tested. Compound 3 resulted to be the most active with an EC50 of 1.080 mg/mL. As a reference, the EC50 of Trolox was 0.011 mg/mL. Hexane extract (H), possessing the highest antioxidant activity, was evaluated for its topical anti-inflammatory activity at the dose of 1000 μg/cm2, in comparison to its fractions (H-1, H-2 and H-3) which were administered at the respective doses of 10, 73 and 708 μg/cm2, calculated on the basis of the fractionation yields and corresponding to 1000 μg of the parent extract. H reduced the oedematous response by 84% and its activity passed completely into H-3 which induced 90% oedema reduction. On the other hand, H-1 and H-2 were inactive. The non-steroidal anti-inflammatory drug indomethacin (100 μg/cm2), used as a reference, provoked 62% oedema reduction (Table 4). Also the furanosesquiterpenoids 3–5 isolated from H-3 exerted a significant anti-inflammatory effect, reducing the oedematous response by 26–32% at the dose of 0.3 μmol/cm2. The equimolar dose of the reference drug indomethacin determined 58% oedema reduction, hence being only two times more active (Table 4). Fungal contamination and oxidation reactions cause a reduction in food quality and they may take place during processing and storage. Synthetic antioxidant and antifungal Table 4 Topical anti-inflammatory activity of hexane extract, its fractions and pure compounds from Commiphora erythraea resin. Substance

Dose

Oedema (mg) mean ± S.E.

% Oedema reduction

Controls H H-1 H-2 H-3 Indomethacin Controls 3 4 5 Indomethacin

– 1000 μg 10 μg a 73 μg a 708 μg a 100 μg – 0.3 μmol 0.3 μmol 0.3 μmol 0.3 μmol

6.8 ± 0.3 1.1 ± 0.1 ⁎ 6.0 ± 0.4 6.3 ± 0.4 0.7 ± 0.1 ⁎ 2.6 ± 0.3 ⁎

– 84 12 7 90 62 – 32 30 26 58

6.9 ± 0.3 4.7 ± 0.3 ⁎ 4.8 ± 0.3 ⁎ 5.1 ± 0.3 ⁎ 2.9 ± 0.3 ⁎

⁎ p b 0.001 at the analysis of variance, as compared to controls. a Dose equivalent to 1000 μg of the parent extract.

compounds, although they are important in crops and foods spoilage control, are often toxic to living organisms and increase environmental pollution and may affect human health [29,30]. Recently, there has been an increasing interest toward the use of plant extracts and essential oils as natural biodegradable pesticides [31–33] that could overcome the problem of the resistance that fungi usually develop toward conventional pesticides. Alternaria and Fusarium are phytopathogen fungi of plants (e.g. cereals, potatoes, tomatoes) known to produce several mycotoxins, such as alternariol and zearalenone, that represent a worldwide problem [34,35]. One of the aims of this study was to determine the biological activity of oils and extracts of C. erythraea against some phytopathogenic fungi to determine whether they could be used as natural preservatives by local population. The in vitro antifungal activity against A. solani, F. culmorum and P. cryptogea was studied and our experiments showed that none of the oils and extracts showed significant inhibition against tested phytopathogenic fungi. Compound 2 resulted to be the most active against all the tested strains. The radical scavenging activity of oils and extracts in the DPPH test, showed that the hexane extract had the best antioxidant activity that passed in fraction H-3. Compounds 3–5 that were the most abundant compounds in this fraction were purified and evaluated. In the reduction of DPPH radical (Table 2), the highest activity was observed for myrrhone 3 that was ten times less active than ascorbic acid and that may be regarded as natural antioxidant for food and storage products. The antioxidant activity of the hexane extract encouraged us to evaluate its topical anti-inflammatory activity. The bioguided fractionation of the hexane extract concentrated its activity in fraction H-3, which accounted for the total antiinflammatory effect of the parent extract. Three of the major components of these fractions (3–5) were tested and each of them showed a mild but significant activity that could contribute to the antiphlogistic effect of the parent fraction H-3 in a synergistic way. Furane derivatives could, in principle, be regarded as toxic compounds, due to the formation of toxic phase I metabolites [36]. The introduction of a benzene ring fused to the furan, or the conjugation with an electron withdrawing group (e.g. a carbonyl) can reduce the formation of adduct with DNA and then the toxicity of furane derivatives [37]. On the other hand, furanosesquiterpenoid are known to posses different interesting biological activities [38], indeed several biological actions have been recognised for furanodienone 2, which was previously shown to possess anti-inflammatory properties and to inhibit cyclooxygenases-1 and -2 as a possible mechanism involved in this effect [39–42]. The good radical scavenging activity against singlet oxygen, responsible of lipid peroxidation, of furanosesquiterpenoids has been proved [43]. As far as we know this is the first report about the anti-inflammatory and antioxidant activity of compounds 3 and 4 that can be regarded as good topic anti-inflammatory compounds. Acknowledgements The authors wish to thank Comune di Perugia for financial support, Ipoassociazione (http://www.ipoassociazione.org)

D. Fraternale et al. / Fitoterapia 82 (2011) 654–661

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