Original Papers
Effects of 7-Hydroxycalamenene Isolated from Croton cajucara Essential Oil on Growth, Lipid Content and Ultrastructural Aspects of Rhizopus oryzae
Authors
Mariana M. B. Azevedo 1, 2*, Catia A. Almeida 2*, Francisco C. M. Chaves 3, Galba M. Campos-Takaki 4, Sonia Rozental 5, Humberto R. Bizzo 6, Celuta S. Alviano 2, Daniela S. Alviano 2
Affiliations
The affiliations are listed at the end of the article
Key words " 7‑Hydroxycalamenene l " Rhizopus oryzae l " zygomycetes l " Croton cajucara l " Euphorbiaceae l " essential oil l
Abstract
received revised accepted
January 22, 2014 March 14, 2014 March 22, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368441 Planta Med 2014; 80: 550–556 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Mariana M. B. Azevedo Instituto de Química Universidade Federal do Rio de Janeiro CT, Bloco A, Ilha do Fundão Rio de Janeiro, RJ, 21941–909 Brazil Phone: + 55 21 25 62 67 11 [email protected]
!
The leaves and bark of Croton cajucara, a shrub from the Amazon region, have been used in folk medicine to treat diabetes, malaria, and gastrointestinal and liver disorders. The essential oil from the leaves, rich in linalool, presented antileishmanial and antimicrobial activities. A chemotype of this species was found with an essential oil rich in 7-hydroxycalamenene. During our studies of the C. cajucara essential oil, we isolated 7-hydroxycalamenene at > 98% purity. The minimum inhibitory concentration of 7-hydroxycalamenene against Absidia cylindrospora, Cunninghamella elegans, Mucor circinelloides, Mucor circinelloides f. circinelloides, Mucor mucedo, Mucor plumbeus, Mucor ramosissimus, Rhizopus microsporus, Rhizopus oryzae, and Syncephalastrum racemosum ranged from 19.53 to 2500 µg/mL. The reference drug used, amphotericin B, presented a minimum inhibitory concentration ranging from 0.085 µg/ mL to 43.87 µg/mL. 7-Hydroxycalamenene also altered spore differentiation and total lipid con-
Introduction !
Essential oils and products of plant secondary metabolism have a wide range of applications in folk medicine, fragrance industries, and food flavoring and preservation, but only in recent years have they started to be recognized for their potential antimicrobial properties [1]. In the search for new antifungal drugs, medicinal plants must not be ignored. Many plant extracts and essential oils possess antimicrobial activity and have been proposed for use in complementary medicine and are, therefore, important sources of new sub-
* Mariana M. B. Azevedo and Catia A. Almeida contributed equally to this study.
Azevedo MMB et al. Effects of 7-Hydroxycalamenene …
Planta Med 2014; 80: 550–556
tent. Ultrastructural analysis by transmission electron microscopy showed significant alterations in the cellular structure of R. oryzae.
Abbreviations !
AMB: CLSI:
amphotericin B Clinical and Laboratory Standards Institute FUN-1: 2-[(E)-(2-chloro-1-phenyl-4-quinolylidene)methyl]-3-methyl-1,3benzothiazol-3-ium iodide MRSA: methicillin-resistant Staphylococcus aureus RPMI-MOPS: Roswell Park Memorial Institute medium-3-(N-morpholino)propanesulfonic acid TEM: transmission electron microscopy WFCC: World Federation for Culture Collections
stances with good antimicrobial activity and minor toxicity [2]. Croton cajucara Benth. (Euphorbiaceae) is a very important traditional medicinal plant in Brazil. It occurs widely in the Amazon rainforest region (Brazil), where it is popularly known as “sacaca”. Infusions of the stem bark have been used for the treatment of liver and kidney disorders, diabetes, diarrhea, stomachaches, fever, jaundice, hepatitis, and malaria and to lower blood cholesterol [3, 4]. Two morphotypes of C. cajucara are known, white “sacaca” and red “sacaca”, which are mainly identified by the young leaf color and stems. In general, essential oils from the white morphotype are rich in linalool, while those from the red morphotype are rich in 7-hydroxycalamenene. However, some exceptions have been found, so this sub-
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Fig. 1 Structure of 7-hydroxycalamenene.
Table 1
Results and Discussion !
GC‑MS of the essential oil from C. cajucara SV6 showed that it is " Table 1), so it was chosen for rich in 7-hydroxycalamenene (l isolation according to the method described by Azevedo and coworkers [21]. Analysis by GC indicated a purity over 98 %. The results of the assessment of the antimicrobial activity of 7" Table 2. All species tested hydroxycalamenene are presented in l were sensitive to 7-hydroxycalamenene at concentrations ranging from 19.53 µg/mL to 2500 µg/mL. These results corroborate those of our previous study in which 7hydroxycalamenene was not only effective against R. oryzae and M. circinelloides but also against other species of zygomycetes [9]. MIC values between 50 and 500 µg/mL are usually considered to be strong activity, while moderate activity is between 600 and 1500 µg/mL, and weak activity is above 1500 µg/mL [22]. According to this classification, 7-hydroxycalamenene presents high activity against Absidia cylindrospora, Cunninghamella elegans, M. circinelloides, Mucor plumbeus, M. circinelloides f. circinelloides, R. oryzae, and Rhizopus microsporus, showing that it is a promising substance against zygomycetes. Furthermore, although 7-hydroxycalamenene presented fungistatic activity against R. oryzae at the MIC, this substance had fungicidal activity when it was concentrated fourfold. Thus, 7-hydroxycalamenene activity against R. oryzae should be considered strong and fungicidal based on the references mentioned above [22, 23].
a
Main components of C. cajucara essential oil.
LRIa
Name
SV6 %
1101 1375 1383 1417 1428 1452 1459 1476 1480 1494 1498 1501 1513 1522 1541 1554 1575 1580 1626 1640 1644 1652 1803
linalool α-copaene β-bourbonene β-caryophyllene β-copaene α-humulene allo-aromadendrene α-amorphene germacrene D bicyclogermacrene α-muurolene germacrene A γ-cadinene δ-cadinene α-calacorene germacrene B spathulenol caryophyllene oxide dillapiole T-cadinol T-muurolol α-cadinol 7-hydroxycalamenene
11.8 1.4 0.8 2.4 0.3 1.2 1.8 0.8 4.2 1.7 0.5 0.3 2.0 4.8 0.3 0.6 2.4 1.2 1.3 1.4 1.1 2.1 35.4
Linear retention index
The zygomycetes spores lose their outer wall during the germination process [24]. The assay with germinated spores of R. oryzae showed that the outer wall is not likely to alter the activity of 7hydroxycalamenene because the MIC for these spores is the same as that for ungerminated spores. The cytotoxic effect of 7-hydroxycalamenene was previously evaluated against peritoneal macrophages by Rodrigues et al. [25]. The cytotoxic concentration of 7-hydroxycalamenene for those cells was higher than 500 µg/mL. These results suggest that 7-hydroxycalamenene can prevent the evolution of zygomycosis with low cytotoxicity. According to Kontoyiannis and Lewis [26], Rhizopus is one of most common genera that cause zygomycosis, so R. oryzae was " Fig. 2 shows that 7-hydroxycalamenene at chosen as a model. l the MIC inhibited spore differentiation at the beginning of the process. This result suggests that the evolution of zygomycosis could be inhibited, increasing patient survival.
Azevedo MMB et al. Effects of 7-Hydroxycalamenene …
Planta Med 2014; 80: 550–556
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stance cannot be used as a chemical marker [5, 6]. The linaloolrich essential oil from the leaves of C. cajucara has shown good activity against Leishmania amazonensis and Candida albicans [7, 8]. In addition, our group demonstrated that the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara was effective against Mycobacterium tuberculosis, Mycobacterium smegmatis, MRSA, Rhizopus oryzae, and Mucor circinelloides [9]. Mucormycosis, also referred to as phycomycosis or zygomycosis, is an aggressive and rapidly progressive infection that primarily occurs in immunocompromised patients. Members of the genera Rhizopus, Mucor, and Absidia are the organisms most commonly isolated from patients with zygomycosis. Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea are other zygomycetes that have been implicated in human diseases. AMB, as well as its lipid formulation, has been essential for treatment for several decades. However, the drug posaconazole has exhibited promising activity and is currently undergoing extensive clinical investigations [10–12]. " Fig. 1) is a hydroxylated sesquiterpene 7-Hydroxycalamenene (l whose molecular weight is 218. It has been identified in Eremophila drummondii (Scrophulariaceae), Ulmus glabra (Ulmaceae), Ganoderma applanatum (Ganodermataceae), cotton leaves inoculated with Xanthomonas campestris pv. malvacearum (Malvaceae; Xanthomonadaceae), Syzygium cumini (Myrtaceae), Bazzania trilobata (Lepidoziaceae), and Tilia europaea (Tiliaceae). It shows good activity against Botrytis cinerea, Cladosporium cucumerinum, Phythophthora infestans, Pyricularia oryzae, and Septoria tritici [13–19]. Our group also demonstrated the promising activity of 7-hydroxycalamenene against some zygomycetes, such as R. oryzae [20]. In this context, the aim of this work was to evaluate the possible mechanism of action of 7-hydroxycalamenene against the main agent of zygomycosis.
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Microorganism
MIC (µg/mL) Amphotericin B
A. cylindrospora C. elegans M. circinelloides M. plumbeus M. circinelloides f. circinelloides M. mucedo M. ramosissimus R. oryzae R. microsporus S. racemosum
7-OH
19.53 19.53 19.53 19.53 19.53 2500 2500 39.06 19.53 2500
Table 2 MIC values of amphotericin B and 7-hydroxycalamenene.
staticb static static static static static static static static static
7-hydroxycalamenene; b fungistatic
Fig. 2 Growth inhibition curve of R. oryzae: absence of the growth of spores treated with 7-hydroxycalamenene (at 39.06 µg/mL) when compared with control for 24 h at 30 °C. The experiment was done three times.
Fig. 3 Differentiation kinetics of R. oryzae (at 39.06 µg/mL). A Control 0 h, B control 0 h stained with FUN-1, C treated 0 h, D treated 0 h stained with FUN-1, E control 6 h, F control 6 h stained with FUN-1, G treated 6 h, and H treated 6 h stained with FUN-1. (Color figure available online only.)
Azevedo MMB et al. Effects of 7-Hydroxycalamenene …
Planta Med 2014; 80: 550–556
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a
0.34 43.87 0.17 0.17 > 43.87 0.34 0.17 0.68 0.34 0.085
Effect a
Original Papers
553
Fig. 5 Total lipids yield of R. oryzae when treated with 7-hydroxycalamenene at the MIC concentration (39.06 µg/mL).
The use of FUN-1 showed that despite the absence of differentiation, all spores were viable after 7-hydroxycalamenene treat" Fig. 3 ment due to the presence of red fluorescence structures. l shows that 7-hydroxycalamenene inhibits differentiation. After 6 h, hyphae formation was observed in the fungal control, while the spores treated with 7-hydroxycalamenene were only swollen. The inhibition of spore germination in vitro could cause a decrease in the invasion of host tissue. Transmission electron microscope images of R. oryzae control " Fig. 4 A and C) show a round morphology with a homogecells (l nous cytoplasm, cylindrical mitochondria, nuclei with well-con" Fig. 4 C), and a densed chromatin, a linear plasma membrane (l cell wall with two distinct layers, an inner electron lucent layer and an outer electron dense layer. Spores treated for 18 h with " Fig. 4 B, D, and E) present an 7-hydroxycalamenene at the MIC (l increase in the cell wall thickness and electron density. Three distinct layers could be clearly visualized; the innermost and outermost layers are electron dense, whereas the intermediary layer is electron lucent. In addition, the nucleus presents more dispersed chromatin and the mitochondria appear rounded. According to
Hess and Weber [27], dormant spores present spherical mitochondria, membrane invagination, and the absence of an inner " Fig. 4 B. These characteristics were also wall [24], as shown in l shown by Ekundayo [28] when the spores were treated with sodium azide, a respiratory inhibitor. Sterols, mainly ergosterol, are important substances present in yeasts and filamentous fungi and are necessary for the growth and normal function of the fungal cell membrane. In addition to controlling the fluidity, asymmetry, and integrity of the membrane, ergosterol contributes to the proper functioning of enzymes bound to the membrane [29]. Ergosterol is the major sterol in Rhizopus arrhizus (currently R. oryzae) and Mucor species, with the exception of Mucor hiemalis, in which 22-dihydroergosterol is the principal sterol [30]. Eukaryotic membranes have low membrane potentials and high levels of sterols and neutral lipids [31], and the sterol is the major mechanism of action of most antifungals. However, no changes in the amount of ergosterol (evaluated by the Arthington-Skaggs and coworkers method [32]) or neutral lipids (determined by the Greenspan and cow-
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Planta Med 2014; 80: 550–556
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Fig. 4 Transmission electron microscopy images of R. oryzae. Control cells (A and C) present a round shape morphology with an homogenous cytoplasm, circular mitochondria (m), nuclei (nu) with a well-condensed chromatin, linear plasma membrane (pm in C), and a cell wall (cw) with two distinct layers: an inner electron lucent layer and outer electron dense layer. Treated spores at 39.06 µg/mL (B, D, and E) present an increase of cell wall thickness and electron density. Three distinct layers could be clearly visualized; the innermost and outermost layers are electron dense whereas the intermediary layer is electron lucent. The nucleus presents a more dispersed chromatin (nu in B). Bars A and B = 1 µm; C, D, and E = 0.5 µm.
Original Papers
orkers method [33]) were detected in spores treated with 7-hydroxycalamenene compared with untreated control spores. The role of lipids in the germination of spores has been associated with the utilization of lipids as carbon and energy sources by means of the glyoxylate cycle in the germination process [34]. A significant decrease in mycelial total lipids in R. oryzae is common, and this decrease may be associated with a high rate of respiration, which appears concurrently with reproductive pro" Fig. 5 showed that decesses in many fungi [35]. The results in l spite the low mycelial mass due to the inhibition of spore differentiation, the total lipids yield of R. oryzae spores treated with 7hydroxycalamenene was 120% greater than the control. It may be supposed that there was inhibition in lipid consumption, as the lipid anabolic process can still be active. Based on this fact, the results are very promising and suggest that 7-hydroxycalamenene affects the respiratory chain of R. oryzae and, hence, interferes in its development. In conclusion, 7-hydroxycalamenene is a promising substance to inhibit R. oryzae, the main agent of zygomycosis, potentially by affecting the respiratory chain and inhibiting fungal growth and germination.
Materials and Methods !
Plant material and essential oil extraction Plant material from C. cajucara was obtained from EMBRAPA Experimental Farm, Amazonas, Brazil. A voucher specimen was deposited at EMBRAPA Amazonia Oriental Herbarium (registry IAN 165 013) by J. M. Albuquerque. Leaves of C. cajucara were collected between 8 and 9 a. m., dried at room temperature, and coarsely ground into powder just before distillation. The oil was obtained by hydrodistillation in a modified Clevenger apparatus for 5 h [36].
Essential oil analyses Sample SV6 of C. cajucara essential oil was analyzed in an Agilent 6890 N gas chromatograph fitted with a 5 %-diphenyl-95%-dimethylpolysiloxane capillary column (HP-5, 25 m × 0.32 mm × 0.25 µm). Mass spectra were obtained with an Agilent 5973 N system according to Pereira et al. [6]. The results were compared to data from the literature [6, 37].
Isolation of 7-hydroxycalamenene from Croton cajucara essential oil The isolation of 7-hydroxycalamenene was performed by preparative column chromatography with silica gel (Merck, 70–230 mesh) according to Azevedo and coworkers [21]. The material was analyzed by gas chromatography to verify its purity.
Antifungal activity assay The antifungal activity of 7-hydroxycalamenene was evaluated against A. cylindrospora (URM4476), C. elegans (URM2084), M. circinelloides (LIKA0066), M. circinelloides f. circinelloides (URM480), Mucor mucedo (LIKA0072), M. plumbeus (URM3232), Mucor ramosissimus (URM3087), R. microsporus (LMC123), R. oryzae (UCP1506), and Syncephalastrum racemosum (UCP1550). All strains used belong to the culture collection of the “Universidade Católica de Pernambuco”, located in the Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Brazil – NPCIAMB/UNICAP. The culture collection is registered in the WFCC.
Azevedo MMB et al. Effects of 7-Hydroxycalamenene …
The microdilution broth method was used according to CLSI reference document M38-A [38] for filamentous fungi. Positive and negative growth controls were made. Amphotericin B (Sigma) was used to control for the sensitivity of tested microorganisms. All experiments were performed in duplicate and repeated twice. To evaluate the fungicide/fungistatic properties of 7-hydroxycalamenene, a 10-µl aliquot of serial dilutions from 2500 µg/mL to 39.06 µg/mL was dropped on the surface of potato dextrose agar and incubated at 30 °C for 48 h. The activity of 7-hydroxycalamenene was also evaluated with germinated spores of R. oryzae. Briefly, 5 × 104 spores/mL were placed in microtubes to differentiate for 8 h. In parallel, a serial dilution of 7-hydroxycalamenene (2500 to 1.22 µg/mL) was made and transferred to microtubes containing differentiated spores. This system was incubated for 18 h at 30 °C, and then an ELISA was performed (Stat Fax 2100) and the absorbance was read at 630 nm. The experiment was performed in duplicate and repeated twice.
Growth curve of Rhizopus oryzae To evaluate the extent to which 7-hydroxycalamenene inhibits the growth of R. oryzae, a growth inhibition curve was developed with 39.06 µg/mL of this substance. The microdilution broth method was repeated, and spores were incubated at 30 °C and read with a SpectraMax M5 (Molecular Devices) for 24 h at 530 nm. The experiment was performed in quintuplicate, with positive and negative growth controls.
Influence of 7-hydroxycalamenene on the cellular differentiation of Rhizopus oryzae The influence of 7-hydroxycalamenene on the differentiation kinetics of R. oryzae was determined as follows: 200 µl of content of each well at 2, 4, and 6 h was collected, centrifuged at 5000 rpm for 3 min, and washed with PBS, pH 7.2. The pellet was resuspended in 50 µl of 10 µM FUN-1 stain (Invitrogen) and incubated for 30 min at 30 °C in the dark. Then, the pellet was washed with 500 µl of PBS, pH 7.2, and visualized with an Axioplan II fluorescence microscope (Carl Zeiss) using a × 40 objective lens.
Transmission electron microscopy Fungal spores were treated with 7-hydroxycalamenene for 18 h at 39.06 µg/mL. Afterwards, cells were washed in PBS, pH 7.2, and fixed in a solution of 2.5 % glutaraldehyde and 4% freshly prepared formaldehyde in 0.1 M cacodylate buffer (Sigma), pH 7.2, for 2 h at room temperature. After fixation, cells were post-fixed for 1 h in 1 % osmium tetroxide containing 1.25 % potassium ferrocyanide and 5 mM CaCl2 in cacodylate buffer, pH 7.2, washed in the same buffer, dehydrated in ethanol, and embedded in Spurrʼs resin. Ultrathin sections were stained with uranyl acetate (Sigma) and lead citrate (Sigma), and images were obtained with a Zeiss 900 electron microscope equipped with a CCD Camera (Mega View III model, Soft Image System). Images were processed with iTEM software (Soft Image System).
Sterol quantitation method Total intracellular sterols were extracted as described by Arthington-Skaggs and coworkers [32] with slight modifications. Briefly, 106 spores of R. oryzae from a 5-day potato dextrose agar plate culture were used to inoculate 50 mL of RPMI-MOPS broth containing 39.06 µg/mL 7-hydroxycalamenene. The cultures were incubated for 18 h with shaking at 30 °C. The stationary phase cells were harvested by centrifugation at 4000 rpm for 5 min
Planta Med 2014; 80: 550–556
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Neutral lipids To evaluate the intracellular content of neutral lipids in R. oryzae, 500 µL of a solution of Nile red (Invitrogen) (200 nM in PBS, pH 7.2) was used to stain 5 × 105 spores. The spores were grown for 18 h in 39.06 µg/mL 7-hydroxycalamenene, centrifuged at 5000 rpm for 5 min, resuspended in 500 µL of Nile red solution, and incubated at room temperature for 10 min. After the cells were rinsed with PBS, pH 7.2, the fluorescence was measured by the flow cytometer Accuri with Cflow Plus software. We analyzed yellow-gold fluorescence (emission at 560 nm and excitation at 515 nm) [33].
Extraction and quantitation of total lipids The total lipid content was extracted as described by Weet and coworkers [39]. After growing in RPMI-MOPS (pH 7.2) for 18 h with 39.06 µg/mL 7-hydroxycalamenene, mycelial and the supernatant material were extracted with 100 mL of a chloroform : methanol (3 : 1) solution followed by 100 mL of hexane. All extractions were carried out overnight at room temperature with continuous agitation using a magnetic stirrer. Suspended material was removed by filtration, and the supernatants were evaporated under prepurified nitrogen. The lipid yields were measured and compared to the control (not treated).
Acknowledgments !
The authors thank Beatriz Bastos for technical assistance and sample preparation for electron microscopy. The present work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
Conflict of Interest !
The authors declare no conflict of interest.
Affiliations 1
2
3 4
5
6
Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil EMBRAPA Amazônia Ocidental, Manaus, AM, Brazil Núcleo de Pesquisas em Ciências Ambientais, Departamento de Química, Universidade Católica de Pernambuco, Recife, PE, Brazil Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil EMBRAPA Agroindústria de Alimentos, Rio de Janeiro, RJ, Brazil
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and washed once with sterile distilled water. The net wet weight of the cell pellet was determined. Three milliliters of 25 % alcoholic potassium hydroxide solution (25 g of KOH and 35 mL of sterile distilled water brought to 100 mL with 100 % ethanol) was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and incubated in an 85 °C water bath for 1 h. Following incubation, the tubes were allowed to cool at room temperature. Sterols were then extracted by the addition of a mixture of 1 mL of sterile distilled water and 3 mL of cyclohexane (Merck) followed by vigorous vortex mixing for 3 min. The upper layer was transferred to a clean borosilicate glass screw-cap tube and stored at − 20 °C for as long as 24 h. Prior to analysis, a 20-µL aliquot of the sterol extract was diluted fivefold in 100 % ethanol and scanned spectrophotometrically between 240 and 300 nm with a SpectraMax M5 (Molecular Devices).
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Effects of 7-Hydroxycalamenene Isolated from Croton cajucara Essential Oil on Growth, Lipid Content and Ultrastructural Aspects of Rhizopus oryzae
Authors
Mariana M. B. Azevedo 1, 2*, Catia A. Almeida 2*, Francisco C. M. Chaves 3, Galba M. Campos-Takaki 4, Sonia Rozental 5, Humberto R. Bizzo 6, Celuta S. Alviano 2, Daniela S. Alviano 2
Affiliations
The affiliations are listed at the end of the article
Key words " 7‑Hydroxycalamenene l " Rhizopus oryzae l " zygomycetes l " Croton cajucara l " Euphorbiaceae l " essential oil l
Abstract
received revised accepted
January 22, 2014 March 14, 2014 March 22, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368441 Planta Med 2014; 80: 550–556 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Mariana M. B. Azevedo Instituto de Química Universidade Federal do Rio de Janeiro CT, Bloco A, Ilha do Fundão Rio de Janeiro, RJ, 21941–909 Brazil Phone: + 55 21 25 62 67 11 [email protected]
!
The leaves and bark of Croton cajucara, a shrub from the Amazon region, have been used in folk medicine to treat diabetes, malaria, and gastrointestinal and liver disorders. The essential oil from the leaves, rich in linalool, presented antileishmanial and antimicrobial activities. A chemotype of this species was found with an essential oil rich in 7-hydroxycalamenene. During our studies of the C. cajucara essential oil, we isolated 7-hydroxycalamenene at > 98% purity. The minimum inhibitory concentration of 7-hydroxycalamenene against Absidia cylindrospora, Cunninghamella elegans, Mucor circinelloides, Mucor circinelloides f. circinelloides, Mucor mucedo, Mucor plumbeus, Mucor ramosissimus, Rhizopus microsporus, Rhizopus oryzae, and Syncephalastrum racemosum ranged from 19.53 to 2500 µg/mL. The reference drug used, amphotericin B, presented a minimum inhibitory concentration ranging from 0.085 µg/ mL to 43.87 µg/mL. 7-Hydroxycalamenene also altered spore differentiation and total lipid con-
Introduction !
Essential oils and products of plant secondary metabolism have a wide range of applications in folk medicine, fragrance industries, and food flavoring and preservation, but only in recent years have they started to be recognized for their potential antimicrobial properties [1]. In the search for new antifungal drugs, medicinal plants must not be ignored. Many plant extracts and essential oils possess antimicrobial activity and have been proposed for use in complementary medicine and are, therefore, important sources of new sub-
* Mariana M. B. Azevedo and Catia A. Almeida contributed equally to this study.
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tent. Ultrastructural analysis by transmission electron microscopy showed significant alterations in the cellular structure of R. oryzae.
Abbreviations !
AMB: CLSI:
amphotericin B Clinical and Laboratory Standards Institute FUN-1: 2-[(E)-(2-chloro-1-phenyl-4-quinolylidene)methyl]-3-methyl-1,3benzothiazol-3-ium iodide MRSA: methicillin-resistant Staphylococcus aureus RPMI-MOPS: Roswell Park Memorial Institute medium-3-(N-morpholino)propanesulfonic acid TEM: transmission electron microscopy WFCC: World Federation for Culture Collections
stances with good antimicrobial activity and minor toxicity [2]. Croton cajucara Benth. (Euphorbiaceae) is a very important traditional medicinal plant in Brazil. It occurs widely in the Amazon rainforest region (Brazil), where it is popularly known as “sacaca”. Infusions of the stem bark have been used for the treatment of liver and kidney disorders, diabetes, diarrhea, stomachaches, fever, jaundice, hepatitis, and malaria and to lower blood cholesterol [3, 4]. Two morphotypes of C. cajucara are known, white “sacaca” and red “sacaca”, which are mainly identified by the young leaf color and stems. In general, essential oils from the white morphotype are rich in linalool, while those from the red morphotype are rich in 7-hydroxycalamenene. However, some exceptions have been found, so this sub-
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Fig. 1 Structure of 7-hydroxycalamenene.
Table 1
Results and Discussion !
GC‑MS of the essential oil from C. cajucara SV6 showed that it is " Table 1), so it was chosen for rich in 7-hydroxycalamenene (l isolation according to the method described by Azevedo and coworkers [21]. Analysis by GC indicated a purity over 98 %. The results of the assessment of the antimicrobial activity of 7" Table 2. All species tested hydroxycalamenene are presented in l were sensitive to 7-hydroxycalamenene at concentrations ranging from 19.53 µg/mL to 2500 µg/mL. These results corroborate those of our previous study in which 7hydroxycalamenene was not only effective against R. oryzae and M. circinelloides but also against other species of zygomycetes [9]. MIC values between 50 and 500 µg/mL are usually considered to be strong activity, while moderate activity is between 600 and 1500 µg/mL, and weak activity is above 1500 µg/mL [22]. According to this classification, 7-hydroxycalamenene presents high activity against Absidia cylindrospora, Cunninghamella elegans, M. circinelloides, Mucor plumbeus, M. circinelloides f. circinelloides, R. oryzae, and Rhizopus microsporus, showing that it is a promising substance against zygomycetes. Furthermore, although 7-hydroxycalamenene presented fungistatic activity against R. oryzae at the MIC, this substance had fungicidal activity when it was concentrated fourfold. Thus, 7-hydroxycalamenene activity against R. oryzae should be considered strong and fungicidal based on the references mentioned above [22, 23].
a
Main components of C. cajucara essential oil.
LRIa
Name
SV6 %
1101 1375 1383 1417 1428 1452 1459 1476 1480 1494 1498 1501 1513 1522 1541 1554 1575 1580 1626 1640 1644 1652 1803
linalool α-copaene β-bourbonene β-caryophyllene β-copaene α-humulene allo-aromadendrene α-amorphene germacrene D bicyclogermacrene α-muurolene germacrene A γ-cadinene δ-cadinene α-calacorene germacrene B spathulenol caryophyllene oxide dillapiole T-cadinol T-muurolol α-cadinol 7-hydroxycalamenene
11.8 1.4 0.8 2.4 0.3 1.2 1.8 0.8 4.2 1.7 0.5 0.3 2.0 4.8 0.3 0.6 2.4 1.2 1.3 1.4 1.1 2.1 35.4
Linear retention index
The zygomycetes spores lose their outer wall during the germination process [24]. The assay with germinated spores of R. oryzae showed that the outer wall is not likely to alter the activity of 7hydroxycalamenene because the MIC for these spores is the same as that for ungerminated spores. The cytotoxic effect of 7-hydroxycalamenene was previously evaluated against peritoneal macrophages by Rodrigues et al. [25]. The cytotoxic concentration of 7-hydroxycalamenene for those cells was higher than 500 µg/mL. These results suggest that 7-hydroxycalamenene can prevent the evolution of zygomycosis with low cytotoxicity. According to Kontoyiannis and Lewis [26], Rhizopus is one of most common genera that cause zygomycosis, so R. oryzae was " Fig. 2 shows that 7-hydroxycalamenene at chosen as a model. l the MIC inhibited spore differentiation at the beginning of the process. This result suggests that the evolution of zygomycosis could be inhibited, increasing patient survival.
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stance cannot be used as a chemical marker [5, 6]. The linaloolrich essential oil from the leaves of C. cajucara has shown good activity against Leishmania amazonensis and Candida albicans [7, 8]. In addition, our group demonstrated that the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara was effective against Mycobacterium tuberculosis, Mycobacterium smegmatis, MRSA, Rhizopus oryzae, and Mucor circinelloides [9]. Mucormycosis, also referred to as phycomycosis or zygomycosis, is an aggressive and rapidly progressive infection that primarily occurs in immunocompromised patients. Members of the genera Rhizopus, Mucor, and Absidia are the organisms most commonly isolated from patients with zygomycosis. Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea are other zygomycetes that have been implicated in human diseases. AMB, as well as its lipid formulation, has been essential for treatment for several decades. However, the drug posaconazole has exhibited promising activity and is currently undergoing extensive clinical investigations [10–12]. " Fig. 1) is a hydroxylated sesquiterpene 7-Hydroxycalamenene (l whose molecular weight is 218. It has been identified in Eremophila drummondii (Scrophulariaceae), Ulmus glabra (Ulmaceae), Ganoderma applanatum (Ganodermataceae), cotton leaves inoculated with Xanthomonas campestris pv. malvacearum (Malvaceae; Xanthomonadaceae), Syzygium cumini (Myrtaceae), Bazzania trilobata (Lepidoziaceae), and Tilia europaea (Tiliaceae). It shows good activity against Botrytis cinerea, Cladosporium cucumerinum, Phythophthora infestans, Pyricularia oryzae, and Septoria tritici [13–19]. Our group also demonstrated the promising activity of 7-hydroxycalamenene against some zygomycetes, such as R. oryzae [20]. In this context, the aim of this work was to evaluate the possible mechanism of action of 7-hydroxycalamenene against the main agent of zygomycosis.
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Microorganism
MIC (µg/mL) Amphotericin B
A. cylindrospora C. elegans M. circinelloides M. plumbeus M. circinelloides f. circinelloides M. mucedo M. ramosissimus R. oryzae R. microsporus S. racemosum
7-OH
19.53 19.53 19.53 19.53 19.53 2500 2500 39.06 19.53 2500
Table 2 MIC values of amphotericin B and 7-hydroxycalamenene.
staticb static static static static static static static static static
7-hydroxycalamenene; b fungistatic
Fig. 2 Growth inhibition curve of R. oryzae: absence of the growth of spores treated with 7-hydroxycalamenene (at 39.06 µg/mL) when compared with control for 24 h at 30 °C. The experiment was done three times.
Fig. 3 Differentiation kinetics of R. oryzae (at 39.06 µg/mL). A Control 0 h, B control 0 h stained with FUN-1, C treated 0 h, D treated 0 h stained with FUN-1, E control 6 h, F control 6 h stained with FUN-1, G treated 6 h, and H treated 6 h stained with FUN-1. (Color figure available online only.)
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a
0.34 43.87 0.17 0.17 > 43.87 0.34 0.17 0.68 0.34 0.085
Effect a
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Fig. 5 Total lipids yield of R. oryzae when treated with 7-hydroxycalamenene at the MIC concentration (39.06 µg/mL).
The use of FUN-1 showed that despite the absence of differentiation, all spores were viable after 7-hydroxycalamenene treat" Fig. 3 ment due to the presence of red fluorescence structures. l shows that 7-hydroxycalamenene inhibits differentiation. After 6 h, hyphae formation was observed in the fungal control, while the spores treated with 7-hydroxycalamenene were only swollen. The inhibition of spore germination in vitro could cause a decrease in the invasion of host tissue. Transmission electron microscope images of R. oryzae control " Fig. 4 A and C) show a round morphology with a homogecells (l nous cytoplasm, cylindrical mitochondria, nuclei with well-con" Fig. 4 C), and a densed chromatin, a linear plasma membrane (l cell wall with two distinct layers, an inner electron lucent layer and an outer electron dense layer. Spores treated for 18 h with " Fig. 4 B, D, and E) present an 7-hydroxycalamenene at the MIC (l increase in the cell wall thickness and electron density. Three distinct layers could be clearly visualized; the innermost and outermost layers are electron dense, whereas the intermediary layer is electron lucent. In addition, the nucleus presents more dispersed chromatin and the mitochondria appear rounded. According to
Hess and Weber [27], dormant spores present spherical mitochondria, membrane invagination, and the absence of an inner " Fig. 4 B. These characteristics were also wall [24], as shown in l shown by Ekundayo [28] when the spores were treated with sodium azide, a respiratory inhibitor. Sterols, mainly ergosterol, are important substances present in yeasts and filamentous fungi and are necessary for the growth and normal function of the fungal cell membrane. In addition to controlling the fluidity, asymmetry, and integrity of the membrane, ergosterol contributes to the proper functioning of enzymes bound to the membrane [29]. Ergosterol is the major sterol in Rhizopus arrhizus (currently R. oryzae) and Mucor species, with the exception of Mucor hiemalis, in which 22-dihydroergosterol is the principal sterol [30]. Eukaryotic membranes have low membrane potentials and high levels of sterols and neutral lipids [31], and the sterol is the major mechanism of action of most antifungals. However, no changes in the amount of ergosterol (evaluated by the Arthington-Skaggs and coworkers method [32]) or neutral lipids (determined by the Greenspan and cow-
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Fig. 4 Transmission electron microscopy images of R. oryzae. Control cells (A and C) present a round shape morphology with an homogenous cytoplasm, circular mitochondria (m), nuclei (nu) with a well-condensed chromatin, linear plasma membrane (pm in C), and a cell wall (cw) with two distinct layers: an inner electron lucent layer and outer electron dense layer. Treated spores at 39.06 µg/mL (B, D, and E) present an increase of cell wall thickness and electron density. Three distinct layers could be clearly visualized; the innermost and outermost layers are electron dense whereas the intermediary layer is electron lucent. The nucleus presents a more dispersed chromatin (nu in B). Bars A and B = 1 µm; C, D, and E = 0.5 µm.
Original Papers
orkers method [33]) were detected in spores treated with 7-hydroxycalamenene compared with untreated control spores. The role of lipids in the germination of spores has been associated with the utilization of lipids as carbon and energy sources by means of the glyoxylate cycle in the germination process [34]. A significant decrease in mycelial total lipids in R. oryzae is common, and this decrease may be associated with a high rate of respiration, which appears concurrently with reproductive pro" Fig. 5 showed that decesses in many fungi [35]. The results in l spite the low mycelial mass due to the inhibition of spore differentiation, the total lipids yield of R. oryzae spores treated with 7hydroxycalamenene was 120% greater than the control. It may be supposed that there was inhibition in lipid consumption, as the lipid anabolic process can still be active. Based on this fact, the results are very promising and suggest that 7-hydroxycalamenene affects the respiratory chain of R. oryzae and, hence, interferes in its development. In conclusion, 7-hydroxycalamenene is a promising substance to inhibit R. oryzae, the main agent of zygomycosis, potentially by affecting the respiratory chain and inhibiting fungal growth and germination.
Materials and Methods !
Plant material and essential oil extraction Plant material from C. cajucara was obtained from EMBRAPA Experimental Farm, Amazonas, Brazil. A voucher specimen was deposited at EMBRAPA Amazonia Oriental Herbarium (registry IAN 165 013) by J. M. Albuquerque. Leaves of C. cajucara were collected between 8 and 9 a. m., dried at room temperature, and coarsely ground into powder just before distillation. The oil was obtained by hydrodistillation in a modified Clevenger apparatus for 5 h [36].
Essential oil analyses Sample SV6 of C. cajucara essential oil was analyzed in an Agilent 6890 N gas chromatograph fitted with a 5 %-diphenyl-95%-dimethylpolysiloxane capillary column (HP-5, 25 m × 0.32 mm × 0.25 µm). Mass spectra were obtained with an Agilent 5973 N system according to Pereira et al. [6]. The results were compared to data from the literature [6, 37].
Isolation of 7-hydroxycalamenene from Croton cajucara essential oil The isolation of 7-hydroxycalamenene was performed by preparative column chromatography with silica gel (Merck, 70–230 mesh) according to Azevedo and coworkers [21]. The material was analyzed by gas chromatography to verify its purity.
Antifungal activity assay The antifungal activity of 7-hydroxycalamenene was evaluated against A. cylindrospora (URM4476), C. elegans (URM2084), M. circinelloides (LIKA0066), M. circinelloides f. circinelloides (URM480), Mucor mucedo (LIKA0072), M. plumbeus (URM3232), Mucor ramosissimus (URM3087), R. microsporus (LMC123), R. oryzae (UCP1506), and Syncephalastrum racemosum (UCP1550). All strains used belong to the culture collection of the “Universidade Católica de Pernambuco”, located in the Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Brazil – NPCIAMB/UNICAP. The culture collection is registered in the WFCC.
Azevedo MMB et al. Effects of 7-Hydroxycalamenene …
The microdilution broth method was used according to CLSI reference document M38-A [38] for filamentous fungi. Positive and negative growth controls were made. Amphotericin B (Sigma) was used to control for the sensitivity of tested microorganisms. All experiments were performed in duplicate and repeated twice. To evaluate the fungicide/fungistatic properties of 7-hydroxycalamenene, a 10-µl aliquot of serial dilutions from 2500 µg/mL to 39.06 µg/mL was dropped on the surface of potato dextrose agar and incubated at 30 °C for 48 h. The activity of 7-hydroxycalamenene was also evaluated with germinated spores of R. oryzae. Briefly, 5 × 104 spores/mL were placed in microtubes to differentiate for 8 h. In parallel, a serial dilution of 7-hydroxycalamenene (2500 to 1.22 µg/mL) was made and transferred to microtubes containing differentiated spores. This system was incubated for 18 h at 30 °C, and then an ELISA was performed (Stat Fax 2100) and the absorbance was read at 630 nm. The experiment was performed in duplicate and repeated twice.
Growth curve of Rhizopus oryzae To evaluate the extent to which 7-hydroxycalamenene inhibits the growth of R. oryzae, a growth inhibition curve was developed with 39.06 µg/mL of this substance. The microdilution broth method was repeated, and spores were incubated at 30 °C and read with a SpectraMax M5 (Molecular Devices) for 24 h at 530 nm. The experiment was performed in quintuplicate, with positive and negative growth controls.
Influence of 7-hydroxycalamenene on the cellular differentiation of Rhizopus oryzae The influence of 7-hydroxycalamenene on the differentiation kinetics of R. oryzae was determined as follows: 200 µl of content of each well at 2, 4, and 6 h was collected, centrifuged at 5000 rpm for 3 min, and washed with PBS, pH 7.2. The pellet was resuspended in 50 µl of 10 µM FUN-1 stain (Invitrogen) and incubated for 30 min at 30 °C in the dark. Then, the pellet was washed with 500 µl of PBS, pH 7.2, and visualized with an Axioplan II fluorescence microscope (Carl Zeiss) using a × 40 objective lens.
Transmission electron microscopy Fungal spores were treated with 7-hydroxycalamenene for 18 h at 39.06 µg/mL. Afterwards, cells were washed in PBS, pH 7.2, and fixed in a solution of 2.5 % glutaraldehyde and 4% freshly prepared formaldehyde in 0.1 M cacodylate buffer (Sigma), pH 7.2, for 2 h at room temperature. After fixation, cells were post-fixed for 1 h in 1 % osmium tetroxide containing 1.25 % potassium ferrocyanide and 5 mM CaCl2 in cacodylate buffer, pH 7.2, washed in the same buffer, dehydrated in ethanol, and embedded in Spurrʼs resin. Ultrathin sections were stained with uranyl acetate (Sigma) and lead citrate (Sigma), and images were obtained with a Zeiss 900 electron microscope equipped with a CCD Camera (Mega View III model, Soft Image System). Images were processed with iTEM software (Soft Image System).
Sterol quantitation method Total intracellular sterols were extracted as described by Arthington-Skaggs and coworkers [32] with slight modifications. Briefly, 106 spores of R. oryzae from a 5-day potato dextrose agar plate culture were used to inoculate 50 mL of RPMI-MOPS broth containing 39.06 µg/mL 7-hydroxycalamenene. The cultures were incubated for 18 h with shaking at 30 °C. The stationary phase cells were harvested by centrifugation at 4000 rpm for 5 min
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Neutral lipids To evaluate the intracellular content of neutral lipids in R. oryzae, 500 µL of a solution of Nile red (Invitrogen) (200 nM in PBS, pH 7.2) was used to stain 5 × 105 spores. The spores were grown for 18 h in 39.06 µg/mL 7-hydroxycalamenene, centrifuged at 5000 rpm for 5 min, resuspended in 500 µL of Nile red solution, and incubated at room temperature for 10 min. After the cells were rinsed with PBS, pH 7.2, the fluorescence was measured by the flow cytometer Accuri with Cflow Plus software. We analyzed yellow-gold fluorescence (emission at 560 nm and excitation at 515 nm) [33].
Extraction and quantitation of total lipids The total lipid content was extracted as described by Weet and coworkers [39]. After growing in RPMI-MOPS (pH 7.2) for 18 h with 39.06 µg/mL 7-hydroxycalamenene, mycelial and the supernatant material were extracted with 100 mL of a chloroform : methanol (3 : 1) solution followed by 100 mL of hexane. All extractions were carried out overnight at room temperature with continuous agitation using a magnetic stirrer. Suspended material was removed by filtration, and the supernatants were evaporated under prepurified nitrogen. The lipid yields were measured and compared to the control (not treated).
Acknowledgments !
The authors thank Beatriz Bastos for technical assistance and sample preparation for electron microscopy. The present work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
Conflict of Interest !
The authors declare no conflict of interest.
Affiliations 1
2
3 4
5
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Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil EMBRAPA Amazônia Ocidental, Manaus, AM, Brazil Núcleo de Pesquisas em Ciências Ambientais, Departamento de Química, Universidade Católica de Pernambuco, Recife, PE, Brazil Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil EMBRAPA Agroindústria de Alimentos, Rio de Janeiro, RJ, Brazil
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and washed once with sterile distilled water. The net wet weight of the cell pellet was determined. Three milliliters of 25 % alcoholic potassium hydroxide solution (25 g of KOH and 35 mL of sterile distilled water brought to 100 mL with 100 % ethanol) was added to each pellet and vortex mixed for 1 min. Cell suspensions were transferred to sterile borosilicate glass screw-cap tubes and incubated in an 85 °C water bath for 1 h. Following incubation, the tubes were allowed to cool at room temperature. Sterols were then extracted by the addition of a mixture of 1 mL of sterile distilled water and 3 mL of cyclohexane (Merck) followed by vigorous vortex mixing for 3 min. The upper layer was transferred to a clean borosilicate glass screw-cap tube and stored at − 20 °C for as long as 24 h. Prior to analysis, a 20-µL aliquot of the sterol extract was diluted fivefold in 100 % ethanol and scanned spectrophotometrically between 240 and 300 nm with a SpectraMax M5 (Molecular Devices).
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