4.4 Ensaios da atividade antioxidante
4.4.2 Avaliação do potencial oxidante e antioxidante dos monoterpenos em eritrócitos
Para investigar o potencial oxidante dos monoterpenos foi preparada uma suspensão de eritrócitos a 30% em PBS (11,35g NaH2PO4.2H2O; 24,36g Na2HPO4 e 7,18g NaCl para 1
L; pH 7,4) suplementado com glicose (200 mg/dL), pH 7,6. Em seguida, as amostras dos monoterpenos em diferentes concentrações foram adicionadas a 2 mL da suspensão de eritrócitos e incubados por um período de 1 hora sob agitação lenta e constante (100 rpm) a 22±2 °C. Em seguida as amostras foram centrifugadas a 2500 rpm durante 5 minutos e a porcentagem de metahemoglobina (metHb) em relação a hemoglobina (Hb) total foi quantificada por espectrofotometria em comprimento de onda de 630 nm e 540 nm, respectivamente. A porcentagem de metHb formada foi comparada com os valores obtidos
para a fenilhidrazina (PH), um comprovado agente oxidante (WEFFORT-SANTOS et al., 2008).
Para investigar o potencial antioxidante, após o período de incubação de 1h referente a etapa descrita anteriormente, foi adicionado um 1 mmol/L do agente oxidante fenilhidrazina. As suspensões foram aeradas e mantidas sob agitação lenta e constante (100 rpm) por mais 20 minutos a 22±2 °C. Decorrido este período, as amostras foram centrifugadas a 2500 rpm durante 5 minutos, diluídas em tampão fosfato (9g Na2HPO4.12H2O, 5,7g KH2PO4 para 1 L) e
a porcentagem de metHb em relação a Hb total foi quantificada por espectrofotometria a 630 nm e 540 nm. Todos os experimentos foram realizados em triplicata.
4.4.3 Avaliação do potencial antioxidante dos monoterpenos frente ao peróxido de hidrogênio
Distribuiu-se 2 mL de tampão fosfato (9 g Na2HPO4.12H2O, 5,7 g KH2PO4 para 1 L)
em tubos de falcon, em seguida adicionou-se a solução de cada monoterpeno em diferentes concentrações e, por último, foi acrescentado 0,6 mL de solução de peróxido de hidrogênio (H2O2) 40 mM. Um grupo controle negativo foi feito apenas com a solução de tampão fosfato
e o controle positivo com o tampão fosfato, a solução de H2O2 40 mM e solução de ácido
ascórbico (vitamina C). Os experimentos foram realizados em triplicata e o resultado expresso em % de atividade antioxidante em comparação ao grupo controle negativo (tampão + H2O2)
(GULÇIN, et al. 2010).
4.5 Ensaios de citotoxicidade
4.5.1 Avaliação do potencial hemolítico dos monoterpenos em eritrócitos humanos
Amostras de sangue humano A, B e O foram misturadas com NaCl 0,9 % na proporção de 1:30 e centrifugadas a 2500 rpm durante 5 minutos para obtenção dos eritrócitos. Este procedimento foi repetido por mais duas vezes e, o sedimento da última centrifugação ressuspenso em NaCl 0,9% para obter uma suspensão a 0,5%. As amostras dos
monoterpenos em diferentes concentrações foram adicionadas à 2 mL da suspensão de eritrócitos para um volume final de 2,5 mL. Uma suspensão de eritrócitos foi utilizada como controle negativo (0 % de hemólise) e uma suspensão de eritrócitos acrescida de Triton X-100 a 1% como controle positivo (100 % de hemólise). Após isso, as amostras foram incubadas por 1 hora à 22 ± 2 ºC sob agitação lenta e constante (100 rpm). Decorrido este tempo as amostras foram centrifugadas a 2500 rpm durante 5 minutos e a hemólise quantificada por espectrofotometria em comprimento de onda de 540 nm (RANGEL et al.,1997).
Todos os experimentos foram realizados em triplicata e expressos como a média mais ou menos o erro padrão da média.
4.6 Análise Estatística
Os resultados obtidos nos experimentos tiveram seus valores expressos em média ± erro padrão da média (e.p.m.) e analisados empregando-se o teste t de Student não pareado, para análise de duas colunas. Os resultados foram considerados significativos quando p < 0,05. Para a análise dos dados utilizou-se o programa estatístico GraphPad Prisma versão 5.0®.
5.1 Antimicrobial analysis of three monoterpenes derived from citronellal:
an
in silicoapproach
Será submetido na Revista Cubana de Farmácia
Antimicrobial analysis of three monoterpenes derived from citronellal: an
in silico
approach
Heloísa M. B. F. de Oliveira1, Abrahão A. O. Filho2, José Pinto de Siqueira Júnior1, Edeltrudes O. Lima1 1 Program in Natural Products and Synthetic Bioactive, Federal University of Paraiba, Paraiba-Brazil
2 Academic Unit Biological Sciences, Federal University of Campina Grande, Paraiba – Brazil
*Corresponding author: heloisambf@gmail.com
ABSTRACT
Monoterpenes are known to exhibit a variety of effects in different biological systems. In the study, the (R)-(+)-citronellal, (S)-(-)-citronellal and 7-hydroxycitronellal were evaluated for its antimicrobial effects. The PASS online program was used in the study for activities in
silico. In silico models are being applied for the evaluation of pharmacological and toxicity
of compound in metabolic environment of mammals. The analysis of possible antimicrobial activity in silico citronellal derivatives of monoterpenes revealed that all the studied compounds are more likely to have a significant antifungal effect.
1. INTRODUCTION
Representative of a class of secondary metabolites, the monoterpenes are constituents of essencial oils present in species of aromatics herbs. It’s biosynthetic origin derived from isoprênica units, which are composed by ten atoms of carbons (1). Despite having a simple structure, these compounds have some biological activities (2,3,4,5).
Among the various monoterpenes studied can highlight the citronellal, which occurs naturally in essential oils of various herbs of the caatinga, such as gender Eucalyptus (ex.:
Eucalyptus citriodora). Other genders also produce citronellal, although in percentage
variables, such as, gender Melissa, Mentha, Allium, Cinnamomum, Cymbopogon (6,7,8,9). Several plants that produce citronellal are used worldwide, mainly in South America, in treating various health conditions, including primarily the treatment of pain (10). Also this monoterpene already revealed antimicrobial activity (7,11), allelopathic activity (6,9), antioxidante activity (12,13) and herbicide activity (6).
Based on this information , aimed to analyze in silico the antimicrobial properties of three monoterpenes derived from citronellal, (R)-(+)-citronellal (RC), (S)-(-)-citronellal (SC) and 7-hydroxycitronellal (7-OH).
2. MATERIALS AND METHODS
2.1 PASS online
Prediction of Activity Spectra for Substances (PASS) online (http://www.way2drug. com/passonline) is designed to evaluate the general biological potential of an organic drug- like molecule. It provides simultaneous predictions of many types of biological activities
based on the structure of organic compounds. The biological activity spectrum of a chemical compound is the set of different types of biological activity that reflect the results of the
compound’s interaction with various biological entities. PASS online gives various facets
of the biological action of a compound. Pa (probability "to be active") and Pi (probability "to be inactive") estimates the categorization of potential compound is belonging to the sub-class of active or inactive compounds respectively (14).
PASS gives hits based on the probability of new effects and mechanism of action with required activity spectra among the compounds from in-house, old and commercial databases. PASS online predicts the biological activity spectrum for the modified imprints on the basis of its structural formula, along with different descriptors like antifungal, antiviral, antihelmintic, antiprotozoal, etc., so it is possible to estimate if new compounds have a particular effect (14).
3. RESULTS AND DISCUSSION
In silico models are being applied for the evaluation of pharmacology and toxicity of
compound in metabolic environment of mammals. Hence, several efficient statistical machine learning methods have been used to develop in silico tools for the prediction of pharmacological and toxicological hazards of molecular structure (15).
Computer-assisted prediction models, so-called predictive tools, play an essential role in the proposed repertoire of alternative methods besides in vitro models. Hence, these tools
are used to study both existing and hypothetical compounds, which are fast, reproducible and are typically based on human bio- regulators (14,16).
The analysis of percentual value of “Pa” for antimicrobial activity in silico for citronellal derivatives revealed that all the studied monoterpenes are more likely to have a significant antifungal effect ( Pa 7-OH: 42,4%; Pa RC: 58%; Pa SC: 58%) (Figures 1,2,3).
Figure 1- Predicted antimicrobial activities of the 7-hydroxycitronellal depicted through Pass online tool.
Figure 2- Predicted antimicrobial activities of the ( R ) - ( + ) - citronellal depicted through Pass online tool.
Figure 3- Predicted antimicrobial activities of the ( S ) - ( - ) - citronellal depicted through Pass online tool.
Knowing that because there is a close relationship enters the pharmacological activity and the enantiomeric of active substances due to stereo selectivity of biological receptors and enzymes (17). We sought to compare the results between the isomers (R)-(+)- citronellal and (S)-(-)-citronellal, analyzing the data can be seen that these compounds have the same percentage of probability in relation to its possible pharmacological effects, suggesting that in such cases the isomerism does not interfere with potential antimicrobial effects presented by these monoterpenes.
4. CONCLUSION
In silico study of three monoterpenes derived from citronellal, (R)-(+)-citronellal,
(S)-(-)-citronellal and 7-hydroxycitronellal, demonstrated that these compounds have several possible biological effects on the pathogenics microorganisms, in special fungi species, besides the isomerism does not interfere in these activities.
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5.2 Antimicrobial effect of (R)-(+)-citronellal against
Candidastrains
Será submetido na Latin American Journal of Pharmacy.
Antimicrobial effect of (R)-(+)-citronellal against
Candidastrains
Heloísa Mara Batista Fernandes de Oliveira*1, Abrahão Alves de Oliveira Filho2, José Pinto de Siqueira Júnior1, Edeltrudes de Oliveira Lima1 1 Program in Natural Products and Synthetic Bioactive, Federal University of Paraiba, Paraiba-Brazil
2 Academic Unit Biological Sciences, Federal University of Campina Grande, Paraiba – Brazil
*Corresponding author: heloisambf@gmail.com
ABSTRACT
Fungi of Candida genus are responsible for most oral infections in immunocompromised patients, especially of people with HIV / AIDS. In the study, the monoterpene (R)-(+)- citronellal was evaluated for its antifungal effects. In the methodology five Candida albicans strains and five Candida tropicalis strains were used in the study for activities. All the microorganism strains were obtained from the Laboratory of Mycology collection. The Microdilution method was used for antifungal assay of the monoterpene. The Nistatin (100 UI/mL) was the standard drug. The obtained results showed activity fungicid against both fungistrains.
1. INTRODUCTION
In immunocompromised individuals, especially those affected by HIV / AIDS, about 74% have lesions in the oral mucosa caused by infections caused by Candida spp. (1,2).
Candida albicans is cited as the most pathogenic species, predominantly found in
candidiasis lesions of the buccal mucosa, however, the increase is observed in the number of
non-albicans species in the course of infection, such as: C. tropicalis, C. krusei, C.
parapsilosis e C. guilliermondii (3).
The use of medicinal plants due to its antiseptic properties is observed since ancient times. However, clinical and laboratory evidence of the effectiveness of these elements are still scarce in literatura (4,5). Thus, research on natural products from plants, such as monoterpenes it is a valid alternative.
Several studies demonstrate the antifungal potential of monoterpenes such as citral, geraniol and citronellal (racemic mixture) (6,7). In this context this work aimed to evaluate the antifungal potential of monoterpene (R)-(+)-citronellal.
2. MATERIALS AND METHODS
2.1 Obtained of monoterpene
The monoterpene (R)-(+)-citronellal was purchased from Sigma-Aldrich® Industry (São Paulo-SP). To achieve the pharmacological tests, the substance was solubilized in DMSO and diluted in distilled water. The concentration of DMSO was less than 0.1% v/v.
2.2 Determination of the minimum inhibitory concentration (MIC)
For methodology by antifungal activity, were selected five strains of Candida albicans strains (ATCC 76845, LM62, LM106, LM 108, LM 122) and five strains of Candida
tropicalis strains (ATCC 13803, LM 06, LM 14, LM 31, LM 36). All the microorganism strains were obtained from the Laboratory of Mycology collection.
The antifungal activity assays were carried out according to the protocols Cleeland and Squires (1991) (8), Hadacek and Greger (2000) (9) and CSLI (2008) (10).
The MICs of the monoterpene was determinated of the against Candida strains by broth microdilution technique. Initially was distributed 100 µL of Sabouraud dextrose broth doubly concentrated in the holes of microdilution plates. Then, 100 µL of the emulsion products also doubly concentrated, were dispensed in the wells of the first row of the plate. And by means of a serial dilution at a ratio of two concentrations were obtained 1024 µg/mL to 2 µg/mL, so that the first line of the plate was meet the highest concentration and last, the lowest concentration. Finally, it was added 10 µL of the inoculum of the species in the cavities, where each plate column refered to a fungal strain, in particular.
In parallel, it was carried out feasibility control of the tested strains. Also, sensitivity control these forward strains to antifungal action considered standards in clinical use (nystatin 100 UI/mL). To verify the absence of interference in the results for the solvent used in the preparation of the substance in the event the DMSO, in which a control was placed in the cavities 100 µL of the double-concentrated broth , 100 µL of DMSO and 10 µL of the suspension was made.
The plates were sealed asseptically and incubated at 35 ° C for 24 - 48 hours to the reading performed. MIC was defined for the products tested as the lowest concentration able to produce inhibition of visible fungal growth recorded in the holes, compared with the control growth. Testing was performed in duplicate and the result expressed by the arithmetic mean of the MIC's obtained in the two tests.
2.3 Determination of the minimum fungicide concentration (MFC)
An aliquot (20 μL) of each pit growth fungal (MIC, MIC x 2, MIC x 4) was grown in a plate with Sabouraud Dextrose Agar. It was then incubated at 35-37 ° C for 24 hours. The MFC was considered the lower concentration in Sabouraud Dextrose Agar planted where there was 3 lower growth units forming colonies (11).
3. RESULTS
The results of antifungal activity to determine the MIC (Minimum Inhibitory Concentration) and MFC (minimum fungicidal concentration) of monoterpene front of the
Candida albicans strains are shown in Table 1 and 2. The MIC and MFC of monoterpene
front of the Candida tropicalis strains are shown in Table 3 and 4. Observing these results can be seen that the monoterpene presented MIC50 and MFC50 the values of 256 µg/mL, and 512
Table 1- Antifungal activity for determination of the MIC of the (R)-(+)-citronellal (RC) from
Candida albicans strains. Fungal strains/ Substance Candida albicans ATCC 76845 Candida albicans LM 62 Candida albicans LM 106 Candida albicans LM 108 Candida albicans LM 122 RC (1024 g/mL) + + + + + RC (512 µg/mL) + + + + - RC (256 µg/mL) + + + + - RC (128 µg/mL) - - - - - RC (64 µg/mL) - - - - - RC (32 µg/mL) - - - - - RC (16 µg/mL) - - - - - Negative control - - - - - Positive control + + + + + (-) No inhibition (+) inhibition
Table 2- Antifungal activity for determination of the MFC of the (R)-(+)-citronellal (RC) from
Candida albicans strains. Fungal strains/ Substance Candida albicans ATCC 76845 Candida albicans LM 62 Candida albicans LM 106 Candida albicans LM 108 Candida albicans LM 122 RC (1024 g/mL) + + + + - RC (512 µg/mL) + + - + - RC (256 µg/mL) - - - - - RC (128 µg/mL) - - - - - RC (64 µg/mL) - - - - - RC (32 µg/mL) - - - - - RC (16 µg/mL) - - - - - Negative control - - - - - Positive control + + + + + (-) No inhibition (+) inhibition
Table 3- Antifungal activity for determination of the MIC of the (R)-(+)-citronellal (RC) from
Candida tropicalis strains. Fungal strains/ Substance Candida tropicalis ATCC 13803 Candida tropicalis LM 06 Candida tropicalis LM 14 Candida tropicalis LM 31 Candida tropicalis LM 36 RC (1024 µg/mL) + + + + + RC (512 µg/mL) + + + + - RC (256 µg/mL) + + + - - RC (128 µg/mL) - - - - - RC (64 µg/mL) - - - - - RC (32 µg/mL) - - - - - RC (16 µg/mL) - - - - - Negative control - - - - - Positive control + + + + + (-) No inhibition (+) inhibition
Table 4- Antifungal activity for determination of the MFC of the (R)-(+)-citronellal (RC) from
Candida tropicalis strains. Fungal strains/ Substance Candida tropicalis ATCC 13803 Candida tropicalis LM 06 Candida tropicalis LM 14 Candida tropicalis LM 31 Candida tropicalis LM 36 RC (1024 µg/mL) + + + + + RC (512 µg/mL) + + + + - RC (256 µg/mL) + - - - - RC (128 µg/mL) - - - - - RC (64 µg/mL) - - - - - RC (32 µg/mL) - - - - - RC (16 µg/mL) - - - - - Negative control - - - - - Positive control + + + + + (-) No inhibition (+) inhibition
4. DISCUSSION
Human mycoses are caused by primary pathogenic fungi that invade the tissues of a normal host, or by opportunistic fungi that invade the tissues of individuals with severe alterations in the immune system (6).
There is an increasing interest to obtain alternative antimicrobial agents from natural sources because the widespread use of antifungal synthetic preservatives has led to the appearance of resistant microorganisms, in this context include the monoterpenes (12).
The results showed that monoterpene (R)-(+)-citronellal present de strong effect against C. albicans and C. tropicalis strains with MIC50 for monoterpene is 256 µg/mL.
According with Sartoratto et al (2004) (13) results strong activity is for MIC values between 0.05 – 0.50 mg/mL, moderate activity MIC values between 0.6 – 1.50 mg/mL and weak activity above 1.50 mg/mL. These results are in agreement with the data obtained by Mirona et al. (2014) (6) in their study using several monoterpenes against various strains of Candida.
Analyzing the results of the MFC can be seen that the monoterpene does have fungicide activity against both species of Candida, because according Hafidh et al. (2011) (14) when the ratios of MFC/MIC were 1 or 2, indicating that the effect of the compound was fungicide in nature (and not fungistatic).
5. CONCLUSION
Based on the results obtained it can be concluded that the monoterpene tested features as a promising drug against infections from fungi of the genus Candida.
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