Spermostatic activity of Eugenia brejoensis and Myroxylon peruiferum essential oils toward human spermatozoa

Spermostatic activity of Eugenia brejoensis and Myroxylon peruiferum essential oils toward human spermatozoa José Adelson Alves do Nascimento Junior, Clovis Macêdo Bezerra Filho, Thiago Henrique Florencio de Oliveira, Alexandre Gomes da Silva, Patricia Cristina Bezerra-Silva Patricia Maria Guedes Paiva, Marcia Vanusa da Silva, Daniela Maria do Amaral Ferraz Navarro, Luis Claudio Nascimento da Silva and Maria Tereza dos Santos Correia


INTRODUCTION
Several contraceptive methods are described in the medical literature as alternatives to avoid unplanned pregnancy.These approaches include different compounds from various chemical classes, of various action mechanisms and with different levels of effectiveness.Barrier methods, for example, consist of the use of substances that prevent fertilization of the ovum by sperm by establishing physical or chemical barriers (Kumar et al., 2012).Vaginal spermicides, including creams, gels and foaming aerosols are examples of chemical barriers (Batár, 2010).Spermicidal substances are designed to prevent pregnancy through death or immobilization of spermatozoa so that they are unable to reach the oocyte and fertilize it (Schreiber et al., 2006).Despite their advantages, they are based mostly on nonoxynol-9, a compound that is capable of causing side-effects such as cervical and vaginal irritability and potentially favoring the emergence of infectious microorganisms and/or altering the growth of lactobacilli (Ojha et al., 2003).There is urgent need for replacement of such agents with safer and more effective alternatives such as natural contraceptives.
In this context, it is important to search for new spermicidal compounds from plants, especially those endemic to different and under-exploited ecosystems, such as the Caatinga (a biome exclusive to Brazil).This area is marked by extreme environmental conditions typical of a semi-arid tropical climate, such as high temperatures (annual average temperature above 25°C) and scarce and erratic rainfall with long periods of drought.It is believed that these climatic features play an essential role in the production of bioactive metabolites by plants of the Caatinga (de Albuquerque et al., 2012).Such metabolites provide a range of proven biomedical applications (de Oliveira et al., 2012;da Silva et al., 2013), thereby encouraging the search for new compounds from plants of the Caatinga.
This study aimed to evaluate the spermostatic activity of essential oils from two plants collected in the Caatinga: Eugenia brejoensis Mazin and Myroxylon peruiferum L. The first plant, E. brejoensis (Myrtaceae), is a recently described species first recorded from humid, highland forests, called brejos, in the Caatinga of Pernambuco State, Brazil.Later collections documented an expanded distribution now known to include other states of the Brazilian Northeast (Paraiba, Alagoas and Sergipe) and Southeast (Espírito Santo) and showed that the species occurs in both the Caatinga and Atlantic forest domains (Mazine and Souza, 2008;Giaretta and Peixoto, 2014).On the other hand, M. peruiferum (Fabaceae) is a wellknown species with a wide distribution in Brazil and

Collection of plant
Samples of leaves from E. brejoensis and M. peruiferum were collected from the "Parque Nacional do Catimbau" (Catimbau National Park, Buíque, Pernambuco, Brazil) in September, 2012.Botanical identification was done at the Herbarium of the Instituto Agronômico de Pernambuco (Agronomic Institute of Pernambuco; IPA-PE, Brazil) and specimen vouchers were deposited in the same herbarium (IPA 84.033 and IPA 84.113,respectively).

Extraction of the essential oils
Approximately, 200 g of powdered leaves of each plant (pre-dried at room temperature and ground in a mill) were subjected to hydrodistillation by heating for 3 h in a Clevenger-type apparatus.Each essential oil layer was separated, dried over anhydrous sodium sulfate and stored in a hermetically sealed glass vial.The essential oils were named from E. brejoensis as EbEO and from M. Peruiferum as MpEO.They were stored at 4°C prior to each assay.This protocol was repeated 3 times at least .
The GC oven temperature was set at 40°C, held for 2 min, increased to 230°C at 4°C/min, then held for 5 min.Helium (He) carrier gas flow (1 mL/min) was maintained at a constant pressure of 7.0 psi.MS Source and quadruple temperatures were set at 230 and 150°C, respectively.Mass spectra were taken at 70 eV (in EI mode) with a scanning speed of 1.0 scan from 35 to 350 m/z.The same conditions were used in the GC analysis performed in a Thermo Fisher Scientific (Waltham, MA, USA) Trace GC Ultra gas chromatograph equipped with a flame ionization detector (FID), a split/splitless injector and a Hamilton Bonaduz (Bonaduz Switzerland) HB-5 fused silica capillary column (30 m × 0.25 mm; film thickness 0.25 μm).The composition of essential oil was expressed in the form of percentages of total peak area as recorded by the GC-FID.The GC analyses were performed in triplicate.The medium percentage of peaks were processed.
Initial identification of the individual components of the essential oils was carried out by comparison with previously reported values of retention indexes (RI), obtained by co-injection of oil samples and C9-C30 linear hydrocarbons and calculated according to the Van den Dool and Kratz equation (Van Den Dool and Dec Kratz, 1963;Adams, 2009).Subsequently, the MS acquired for each component was matched with those stored in the mass spectral library of the GC-MS system (MassFinder 4, NIST08 and Wiley Registry™ 9th Edition) and with other published mass spectral data.
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Analysis of immobilizing activity
Semen samples were collected by masturbation from 12 fertile volunteers who had been pre-tested, were non-smoking, healthy, aged 18 to 45 years, and whose sperm showed normal morphology (> 60%), motility and viability more than 40 and 70%, respectively.Semen samples were collected after sexual abstinence of 48 to 120 h, as recommended by the WHO standard protocol (World Health Organization, 2010).A routine semen analysis was performed immediately after coagulation, followed by reliquefaction at room temperature.A free and informed consent form was signed by each volunteer before participation in the study.All experiments were performed according to ethical standards and were approved by the Ethics Committee of ASCES College (Caruaru, Brazil), under license number 02604312.6.0000.5203.
Different concentrations (25, 50, 100, 200 and 400 µg/mL) of each oil (prewarmed to 37°C) were mixed with human ejaculate at a 1:1 volume ratio.A solution of warmed DMSO (5%), diluted in phosphate-buffered saline (PBS; pH 7.4), was used as a control.Motility assessment was performed by putting 10 μL of each experimental mixture (Semen + EO or Semen + Control) on prewarmed slides, and at least 10 fields were viewed for a total count of 200 spermatozoa for each slide (Paul and Kang, 2011).The tests were carried out in triplicate for each sample after incubation for 5, 15 and 30 min.Sperm motility was assessed following the WHO protocol for seminal processing (World Health Organization, 2010).Tests were performed independently and six patients were tested for each essential oil.

Hemolytic assay
Blood (5 to 10 ml) was obtained from healthy volunteers by venipuncture and placed in heparinized tubes, after written informed consent was obtained.Human erythrocytes were isolated by centrifugation (1,500 rpm, 10 min at 4°C).The erythrocytes were washed three times with phosphate-buffered saline (PBS; pH 7.4).Each tube received 1.1 mL of erythrocyte suspension (1%) and 0.4 mL of various extract concentrations (0.15625 to 2.5 mg/mL).The controls were only solvent (negative) and Quillaja saponin (0.0025%, positive).After 60 min incubation, cells were centrifuged and the absorbance of supernatant was recorded at 540 nm.The hemolytic activity was expressed by the following formula (de Oliveira et al., 2012): Hemolytic activity (%) = (As -Ab) × 100 / (Ac -Ab) where Ab = solvent absorbance, As = sample absorbance, and Ac = saponin absorbance.

Statistical analysis
The results are expressed as mean ± standard deviation (SD).Statistical significance was determined by Student t and analysis of variance (ANOVA) tests comparing treated groups with untreated control.A p-value of <0.05 was considered to be statistically significant.In all graphs, the bars represent the mean value ± SD.Determinations of IC50 (concentration of 50% immobilized spermatozoa) and HC50 (the concentration needed for 50% of hemolysis) were performed by linear regression.
The effects of EbEO and MpEO on human spermatozoa motility is as shown in Figure 1.The essential oil from E. brejoensis showed the best spermicide activity.When treated with EbEO, significant immobilizing action was observed with all tested concentrations and at all times as compared to controls (p<0.05),except at a concentration of 25 µg/mL.The spermostatic activity of EbEO was time-and dose-dependent and ranged from 8.53 to 42.70% at 5 min, from 9.38 to 44.10% at 15 min, and from 9.60 to 47.56% at 30 min.
Regarding the action of MpEO, significant inhibition was found at 50 µg/mL after 15 and 30 min of incubation, and at higher concentrations (100, 200 and 400 µg/mL) in all treatment periods (p< 0.05).The immobilizing values were also dose-dependent and ranged from 6.2 to 33.33% at 5 min, 7.6 to 32.73% at 15 min, and from 9.66 to 36.66% at 30 min.The IC 50 values at 5, 15 and 30 min were 620.39,652.70 and 579.37 µg/mL, respectively.It is worth emphasizing that there are no reports in the literature regarding contraceptive potential for the genre Myroxylon.As part of its phytochemical composition, M. peruiferum possesses, volatile oils, esters, alcohols, triterpene alkaloids, phenols, proteins and glycosides (Schwarcz et al., 2014).
Chemical components identified in M. peruiferum   essential oil such as α-pinene, γ-muuroleno, spathulenol and caryophyllene oxide were not reported as contraceptive actives in previous studies.On the other hand, some major components (such as α-pinene, γmuurolene, caryophyllene oxide) have been detected in essential oils antimicrobial, antioxidant, and gastroprotective activities (Almeida et al., 2015;Saraswathy et al., 2010;Policegoudra et al., 2012).The cellular toxicity were evaluated using human erythrocytes as test system, both essential oils showed low toxicity with HC 50 of 8.66 and 6.85 mg/mL, for MPEO and EBEO, respectivelly.These concentrations are higher than IC 50 values found for both oils.

Conclusion
This study demonstrated that essential oils from E. brejoensis and M. peruiferum show a potential spermostatic effect on human sperm with low cytotoxicity.Future studies, including the identification of active compounds in each essential oil, their action mechanisms and in vivo activity, are necessary to support the use of these essential oils to develop new pharmaceutical preparations.

a
Constituents listed in order of elution on a non-polar DB-5 column; b Retention indices (RI) calculated from retention times in relation to those of a series of C9-C30.n-alkanes on a 30 m DB-5 capillary column; c Values taken fromAdams (2009).

Table 1 .
Identification of constituents of the essential oil obtained from Eugenia brejoensis and Myroxylon peruiferum.