Piper Essential Oils Inhibit Rhizopus oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity.

Piper is the largest genus of the Piperaceae family. The species of this genus have diverse biological activities and are used in pharmacopeia throughout the world. They are also used in folk medicine for treatment of many diseases in several countries including Brazil, China, India, Jamaica, and Mexico. In Brazil, Piper species are distributed throughout the national territory, making this genus a good candidate for biological activity screening. During our studies with Piper essential oils, we evaluated its activity against Rhizopus oryzae, the main agent of mucormycosis. The main compounds of seven Piper essential oils analyzed were Piper callosum-safrole (53.8%), P. aduncum-dillapiole (76.0%), P. hispidinervum-safrole (91.4%), P. marginatum-propiopiperone (13.2%), P. hispidum-γ-terpinene (30.9%), P. tuberculatum-(E)-caryophyllene (30.1%), and Piper sp.-linalool (14.6%). The minimum inhibitory concentration of Piper essential oils against R. oryzae ranged from 78.12 to >1250 µg/mL. The best result of total inhibition of biofilm formation was obtained with Piper sp. starting from 4.88 µg/mL. Considering the bioactive potential of EOs against planktonic cells and biofilm formation of R. oryzae could be of great interest for development of antimicrobials for therapeutic use in treatment of fungal infection.

7-hydroxycalamenene Effects on Secreted Aspartic Proteases Activity and Biofilm Formation of Candida spp.

BACKGROUND: The 7-hydroxycalamenenene-rich essential oil (EO) obtained from the leaves of Croton cajucara (red morphotype) have been described as active against bacteria, protozoa, and fungi species. In this work, we aimed to evaluate the effectiveness of 7-hydroxycalamenenene against Candida albicans and nonalbicans species. MATERIALS AND METHODS: C. cajucara EO was obtained by hydrodistillation and its major compound, 7-hydroxycalamenene, was purified using preparative column chromatography. The anti-candidal activity was investigated by minimum inhibitory concentration (MIC) and secreted aspartic proteases (SAP) and biofilm inhibition assays. RESULTS: 7-hydroxycalamenene (98% purity) displayed anti-candidal activity against all Candida species tested. Higher activity was observed against Candida dubliniensis, Candida parapsilosis and Candida albicans, showing MIC values ranging from 39.06 µg/ml to 78.12 µg/ml. The purified 7-hydroxycalamenene was able to inhibit 58% of C. albicans ATCC 36801 SAP activity at MIC concentration (pH 7.0). However, 7-hydroxycalamenene demonstrated poor inhibitory activity on C. albicans ATCC 10231 biofilm formation even at the highest concentration tested (2500 µg/ml). CONCLUSION: The bioactive potential of 7-hydroxycalamenene against planktonic Candida spp. further supports its use for the development of antimicrobials with anti-candidal activity. SUMMARY: Croton cajucara Benth. essential oil provides high amounts of 7-hydroxycalamenene7-Hydroxycalameneneisolated from C. cajucarais active against Candida spp7-Hydroxycalameneneinhibits C. albicans aspartic protease activity7-Hydroxycalamenene was not active against C. albicans biofilm formation. Figure.

Effects of 7-hydroxycalamenene isolated from Croton cajucara essential oil on growth, lipid content and ultrastructural aspects of Rhizopus oryzae.

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 content. Ultrastructural analysis by transmission electron microscopy showed significant alterations in the cellular structure of R. oryzae.

Antioxidant and antimicrobial activities of 7-hydroxy-calamenene-rich essential oils from Croton cajucara Benth.

Croton cajucara is a shrub native to the Amazon region locally known as "sacaca". Two morphotypes are known: white and red "sacaca". The essential oils (EO) obtained by hydrodistillation from leaves of the red morphotype were, in general, rich in 7-hydroxycalamenene (28.4%-37.5%). The effectiveness of these EO regarding the antimicrobial activity against pathogenic microorganisms was initially investigated by the drop test method, showing significant inhibition zones. Among the microorganisms tested, the essential oils rich in 7-hydroxycalamenene were more effective against methicillin resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Mycobacterium tuberculosis, M. smegmatis, Mucor circinelloides and Rhizopus oryzae. The minimum inhibitory concentrations (MIC) of the oils were determined using the broth dilution assay. It was possible to observe that 7-hydroxycalamenene-rich oils presented high antimicrobial activity, with MIC of 4.76 × 10⁻³ µg/mL for MRSA, 4.88 µg/mL for M. tuberculosis, 39.06 µg/mL for M. smegmatis, and 0.152 µg/mL for R. oryzae and 3.63 × 10⁻8 µg/mL for M. circinelloides. The antioxidant activity of this EO suggests that 7-hydroxycalamenene provides more antioxidant activity according with EC(50) less than 63.59 µg/mL. Considering the bioactive potential of EOs and 7-hydroxycalamenene could be of great interest for development of antimicrobials for therapeutic use in treatment of bacterial and fungal infections in humans and/or veterinary practice.

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of Fonsecaea pedrosoi.

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing alpha 2,3- and alpha 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.