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Chemical composition and toxicological evaluation of Hyptis suaveolens (L.) Poiteau (LAMIACEAE) in Drosophila melanogaster and Artemia salina
South African Journal of Botany 113 (2017) 437–442
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Chemical composition and toxicological evaluation of Hyptis suaveolens (L.) Poiteau (LAMIACEAE) in Drosophila melanogaster and Artemia salina J.W.A. Bezerra a, A.R. Costa a, M.A.P. da Silva b, M.I. Rocha b, A.A. Boligon c, J.B.T. da Rocha d, L.M. Barros b, J.P. Kamdem b,⁎ a
Course of Biological Sciences, Regional University of Cariri (URCA), Pimenta, Crato, CE CEP 63.105-000, Brazil Department of Biological Sciences, Regional University of Cariri (URCA), Pimenta, Crato, CE CEP 63.105-000, Brazil Phytochemical Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Santa Maria, RS CEP 97105-900, Brazil d Postgraduate Program in Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil b c
a r t i c l e
i n f o
Article history: Received 19 March 2017 Received in revised form 29 August 2017 Accepted 3 October 2017 Available online xxxx Edited by L Verschaeve Keywords: Drosophila melanogaster Artemia salina Essential oil Infusion Bamburral
a b s t r a c t Hyptis suaveolens (L.) Poiteau, known as “Bamburral” is a plant widely used in Brazilian folk medicine, however, there are no toxicological studies to ascertain its safety. Thus, we aimed to investigate the phytochemical and toxic effects of essential oil and infusion of the dry leaves of H. suaveolens in Drosophila melanogaster and Artermia salina. The chemical composition of the essential oil was performed by CG-MS, while that of the infused extract was analyzed by HPLC-DAD. The toxicity of the essential oil-OE (3.03–30.3 μg/mL air) to D. melanogaster was performed by means of volatilization; and for infusion (2.5–20 mg/mL), they were submitted to ingestion and then submitted to locomotive activity by means of negative geotaxis. However, for acute toxicity, A. salina cysts were exposed for 24 h to the tested samples (10–1000 μg/mL). Our results demonstrated that OE had three major components: β-Caryophyllene (18.57%), sabinene (15.99%) and spathulenol (11.09%) while the leaf infusion showed caffeic acid as the major component (12.76 mg/g). H. suaveolens leaves infusion was not toxic to the organisms at all the concentrations tested (p N 0.05). However, OE behaved as quite toxic with LC50 of 15.5 and 49.72 μg/mL in D. melanogaster and A. salina, respectively. In addition, OE caused impairment of the locomotor behavior of flies. Therefore, our study indicates that more caution should be taken regarding the dosage and frequent use of H. suaveolens leaf essential oil. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Medicinal and aromatic plants have always been part of human life since they are used for food and/or for the treatment of various illnesses. The use of plant products as therapeutic and pharmacological alternatives is affordable and widely accepted culturally. However, despite the widespread popular use, there is evidence that they can be potentially toxic. In recent years, there has been a growing scientific interest in essential oils and botanical products; however, there are few reports of toxicological studies of plant oils used by populations (Mishra and Tiwari, 2011; Zouari, 2013; Cunha et al., 2015). The Lamiaceae family consists of approximately 252 genus and 6800 species, being one of the important economical sources of essential oils. But, the biological activities of some plants of this family are unknown. The Hyptis genus has approximately 400 species (Judd et al., 2009), in which Hyptis suaveolens (L), commonly known in the Northeastern ⁎ Corresponding author at: Regional University of Cariri, Department of Biological Sciences, CEP: 63105-000 Crato, Ceará, Brazil. E-mail address: [email protected] (J.P. Kamdem).
https://doi.org/10.1016/j.sajb.2017.10.003 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
Brazil as “Bamburral”, is of great interest for medical applications. Its leaves are commonly used in the treatment of stomach pain (Shirwaikar et al., 2003; Shenoy et al., 2009). Numerous species including the fruit fly, Drosopila melanogaster and Artemia salina are widely used as model organism to investigate the pharmacological and/or toxicological effects of chemical compounds and natural products (Siddique et al., 2005; Zemolin et al., 2014). Of particular genetical importance, it has been suggested that D. melanogaster has about 75% human disease-causing genes (Jimenez-Del-Rio et al., 2010; Pandey and Nichols, 2011; Sobral-Souza et al., 2014). Therefore, its genetical importance associated with its low cost and its high sensitivity to toxic substances makes it a suitable model. Artemia salina Leach. (Artemiidae) is a crustacean from salt water environments. This organism plays an important role in the energy flow of the food chain in this environment. Due to the fact that it is cheap and does not require approval of the ethical committee, it is extensively used in toxicological studies to determine lethal concentration (LC) (Meyer et al., 1982; Parra et al., 2001; Gouveia et al., 2014). Substantial evidence from the literature indicates that H. suaveolens oil exerts a variety of pharmacological and biological activities such as
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antioxidant, antibacterial, with the major constituents of the oil being sabinene, α-Terpinolene and 1,8-cineole (Nantitanon et al., 2007) and nematicide with D-limonene and menthol as main constituents (Falcão and Menezes, 2003). In addition, there are studies with H. suaveolens oil showing that it has toxicity in some organisms such as gnat that transmits dengue Aedes aegypti (L.) (Diptera: Culicidae) (Tennyson et al., 2012), the beetle weevil-the-beans, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) (Ilboudo et al., 2010), the maize weevil, Sitophilus zeamais (Iloba and Ekrakene, 2006), the rice weevil, Sitophilus oryzae (Olotuah, 2011), one diptera host and transmitter of malaria, Anopheles gambiae (Abagli et al., 2012), and star tick Amblyomma cajennense (Soares et al., 2010). However, there is little information in the literature regarding the toxicity of H. suaveolens, particularly using D. melanogaster and A. salina as model. Thus, the objective of this study was (i) to chemically characterize the essential oil and infusion of the leaves of H. suaveolens and (ii) evaluate their potential toxic effects in D. melanogaster and Artemia salina. 2. Material and methods 2.1. Plant material The leaves of H. suaveolens were collected in March 2015 in the municipality of Quixelô - Ceará, Brazil, at 09:00 h, ±30 min in the following coordinates: Latitude − 6° 15 ′43.0056″, longitude − 39° 16 ′2.5926′ and 193.265 m sea level. The leaves were dried in an oven at 30 °C. The plant material was identified and a voucher specimen was deposited in the Herbarium Dárdano Andrade Lima - URCA under # 12.104. 2.2. Extraction of essential oil The essential oil of H. suaveolens was extracted from dried leaves, submitted to hydrodistillation in Clevenger apparatus. After collection, the leaves were crushed into small pieces (150 g) and filled into a 1 L volumetric flask, where 2 L of distilled water was added. The flask was coupled to the Clevenger apparatus under the heating mantle and the temperature adjustment was carried out until the water boiled. After boiling, the 2 h time of the extraction cycle was started. At the end of each extractive cycle, the oil contained in the apparatus was collected with the aid of a pipette and stored in amber and refrigerated bottles. After extraction, sodium sulphate was used to remove the aqueous phase present in the essential oil (Matos, 2009). 2.3. Preparation of the infusion of dried leaves of Hyptis suaveolens (ILHS) The leaves were collected, dried in the shade and then crushed. The infusion was prepared by soaking 80 g of dried leaves in 0.5 L of distilled water at 100 °C and put in repose for 1 h and then filtered. 2.4. Stock and creation of Drosophila melanogaster The creation of flies, D. melanogaster (Harwich strain) was made in Laboratory of Microbiology and Molecular Biology (LMBM), Regional University of Cariri - URCA. They were obtained from the Federal University of Santa Maria - UFSM, from strains originated from the National Species Stock Center, Bowling Green, OH. The flies were reared in a glass bottles 5.5 × 12 cm containing approximately 5 mL of standard medium (1% w/v yeast, 2% w/v sucrose, 1% w/v milk powder, 1% w/v agar, 0.08% w/v nipagin). They were maintained at 12:12 h (light/dark cycle), and constant temperature and humidity (25 ± 1 °C, 60% relative humidity, respectively). 2.5. Treatment, mortality test and negative geotaxis of D. melanogaster Twenty adult flies with 6 days of age (males and females) were placed in flasks of 330 mL containing the bottom filter paper
impregnated with 1 ml of 1% sucrose in distilled water. In the cover, it was attached a filter paper for the application of the essential oil so that the oil could volatilize from the top, in order to achieve the respiratory system of the flies. The experimental groups received different dosages of the essential oil of Hyptis suaveolens: 3.03, 7.57, 15.5, 22.72 and 30.3 μg/mL of air, while the control group received only the 1% sucrose. The total time of exposure to essential oil was 48 h. Regarding the treatment of flies with the infusion of the plant extract, the filter paper was impregnated with 2.5, 5, 10 and 20 mg/mL of sucrose solution and the flies were added in the flasks. Then, the mortality was taken every 3, 6, 12, 24 and 48 h and the results were expressed in percentage. The locomotor activity of the exposed flies was determined by the negative geotaxis test. 2.6. Toxicity against larvae of Artemia salina Leach In artificial seawater, it was added cysts of Artemia salina Leach and subjected to constant aeration for 24 h with light, the time required for hatching the larvae. Then, the concentrations of 10, 25, 50, 100, 250, 500 and 1000 μg/mL were prepared for H. suaveolens essential oil and for infusion of dry leaves of the plant (Meyer et al., 1982). Potassium dichromate (K2Cr2O7) was used as positive control at the same concentrations as essential oil and infusion of dry leaves, while the negative control was seawater. The reading was performed after 24 h, and the calculation of LC50 was obtained by linear regression using GraphPad Prism 6. 2.7. Phytochemical analysis of essential oil by Gas chromatography (GC-FID) The gas chromatography (GC) analysis was performed with Agilent Technologies 6890N GC-FID system, equipped with DB-5 capillary column (30 m × 0.32 mm; 0.50 mm) and connected to a FID detector. The thermal programmer was 60 °C (1 min) to 180 °C at 3 °C/min; injector temperature 220 °C; detector temperature 220 °C; split ratio 1:10; carrier gas Helium; flow rate: 1.0 mL/min. The injected volume of H. suaveolens essential oil was 1 μL diluted in chloroform (1:10). Two replicates of samples were processed in the same way. Component relative concentrations were calculated based on GC peak areas without using correction factors (Boligon et al., 2013). 2.8. Identification of the components Identification of the constituents was performed on the basis of retention index (RI), determined with reference to the homologous series of n-alkanes, C7–C30, under identical experimental conditions, compared with the mass spectra library search (NIST and Wiley), and with the mass spectra literature date Adams (2007). The relative amounts of individual components were calculated based on the CG peak area (FID response). 2.9. Phytochemical analysis of Hyptis suaveolens infusion by HPLC 2.9.1. Chemical, apparatus and general procedures All chemicals were of analytical grade. Acetonitrile, formic acid, caffeic acid, chlorogenic acid, caffeic acid and ellagic acid were purchased from Merck (Darmstadt, Germany). Apigenin, catechin, rutin and quercetin were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High-performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.
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2.9.2. Quantification of compounds by HPLC-DAD For analysis of the Hyptis suaveolens infusion of dried leaves, 15 mL of the extract was injected into a Phenomenex C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles and eluted at 0.6 mL/min with a 70-min linear gradient from 5 to 30% (v/v) acetonitrile in 0.1% (v/v) aqueous formic acid followed by a 5-min linear gradient to 100% (v/v) acetonitrile, following the method described by Reis et al. (2014) with some modifications. The wavelengths used were 254 nm for gallic acid; 280 nm for catechin; 327 for ellagic acid, chlorogenic acid and caffeic acid; and 356 nm rutin, quercetin and apigenin. The extract and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.020–0.250 mg/mL. Chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 500 nm). All chromatography operations were carried out at ambient temperature and in triplicate. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve (Kamdem et al., 2013; Boligon et al., 2014). 2.10. Statistical analysis Differences between groups of HPLC were assessed by an analysis of variance model and Tukey's test. The level of significance for the analyses was set at p b 0.05. These analyses were performed by using the free software R version 3.1.1. (R Core Team, 2014). Statistical analyses of the means in triplicate (n = 3) ± standard deviation were performed using the software program GraphPad Prism 6, using Twoway Variance Analysis (ANOVA), followed by the Tukey test at 5% reliability. 3. Results 3.1. Chemical composition of essential oil of H. suaveolens The essential oil had a yield of 0.153%. It was identified a total of 44 components in the essential oil of H. suaveolens representing 99.97% of the whole composition (Table 1). The major identified components of the oil were the β-Caryophyllene (18.57%), the sabinene (15.99%) and spathulenol (11.09%). However, the minor components were (Z)-β-Ocimene (0.07%), α-Copaene (0.09%) and δ-cadidene (0.09%).
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Table 1 Composition of Hyptis suaveolens essential oil. Compounds
RIa
RIb
α-Thujene α-Pinene Sabinene β-Pinene Myrcene δ-2-Carene α-Phellandrene α-Terpinene p-Cymene Limonene 1-8-Cineole (Z)-β-Ocimene (E)-β-Ocimene γ-Terpinene cis-Sabinene hydrate Linalool cis-p-Menth-2-en-1-ol t-Sabinol 4-Tepineol p-Cymen-8-ol α-Terpineol δ-Elemene α-Copaene β-Elemene β-Cedrene β-Caryophyllene β-Gurjunene γ-Elemene Aromadendrene α-Humelene Alloaromadendrene γ-Muurolene Germacreno D β-Selinene Bicyclogermacrene γ-Cadinene δ-Cadidene Germacrene B Spathulenol Caryophyllene oxide Globulol Cubenol β-Eudesmol α-Cadinol Total identified (%)
989 940 976 980 994 999 1006 1019 1030 1031 1037 1041 1055 1060 1068 1095 1123 1139 1178 1183 1191 1335 1377 1390 1416 1421 1433 1435 1439 1453 1461 1477 1481 1486 1501 1512 1525 1559 1576 1580 1582 1641 1649 1656
931 939 976 980 991 1001 1005 1018 1029 1031 1033 1040 1050 1061 1068 1098 1121 1140 1177 1183 1189 1338 1376 1391 1417 1418 1432 1433 1439 1454 1462 1477 1480 1485 1488 1513 1520 1556 1576 1581 1583 1642 1649 1653
Oil % 1.09 0.85 15.94 2.11 0.26 0.49 1.38 1.05 0.76 5.19 3.04 0.07 0.12 2.97 0.61 0.43 0.28 0.15 6.82 0.23 0.94 1.17 0.09 0.78 0.14 18.57 0.23 1.44 0.32 1.17 0.40 0.28 5.21 0.89 7.52 0.36 0.09 0.27 11.09 3.18 0.62 1.07 0.13 0.45 99.97
Relative proportions of the essential oil constituents were expressed as percentages. a Retention indices experimental (based on homologous series of n-alkane C7–C30). b Retention indices from literature (Adams, 2007).
3.2. HPLC analysis of ILHS HPLC fingerprinting composition of Hyptis suaveolens infusion of leaves revealed the presence of the gallic acid (retention time - tR = 8.95 min; peak a), catechin (tR = 15.93 min; peak b), chlorogenic acid (tR = 21.87 min; peak c), caffeic acid (tR = 24.31 min; peak d), ellagic acid (tR = 30.56 min; peak e), rutin (tR = 37.84 min; peak f), quercetin (tR = 48.05 min; peak g) and apigenin (tR = 56.41 min; peak h) (Fig. 1 and Table 2). 3.3. Test of mortality of D. melanogaster In fumigation test with the essential oil, significant effects of concentrations and their interaction were detected. It appears that the number of dead flies increased with the time and concentration-dependent manner (Fig. 2). All concentrations of the oil above of 15.5 μg/mL of air caused at least 50% mortality of the flies. In contrast, there was no significant dead of flies treated with infusion of H. suaveolens in comparison with the control (Fig. 3).
Fig. 1. Representative reverse-phase HPLC analysis of Hyptis suaveolens leaves infusion. Using standard and spectral analysis, peaks a, b, c, d, e, f, g and h were identified as gallic acid, catechin, chlorogenic acid, caffeic acid, ellagic acid, rutin, quercetin and apigenin respectively.
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Table 2 Composition of Hyptis suaveolens infusion. Compounds
Gallic acid Catechin Chlorogenic acid Caffeic acid Ellagic acid Rutin Quercetin Apigenin
LOD
LOQ
mg/g
H. suaveolens %
μg/mL
μg/mL
3.85 ± 0.01 a 5.94 ± 0.01 b 0.61 ± 0.02 c 12.76 ± 0.03 d 3.49 ± 0.01 e 3.84 ± 0.01 a 3.45 ± 0.02 e 5.90 ± 0.01 b
0.38 0.59 0.06 1.27 0.34 0.38 0.34 0.59
0.013 0.008 0.021 0.015 0.030 0.019 0.024 0.011
0.042 0.025 0.070 0.053 0.098 0.062 0.079 0.037
Results are expressed as mean ± standard deviations (SD) of three determinations. Averages followed by different letters differ by Tukey test at p b 0.05. LOD: limit of detection and LOQ: limit of quantification.
3.4. Test of geotaxia negative against D. melanogaster As depicted in Fig. 4, no significant change was detected on the locomotor activity of flies treated with the H. suaveolens essential oil in comparison to the control (p N 0.05). However, the percentage of flies that reached the top of the glass column (6 cm of in 5 s) significantly decreased with time and concentration dependent manner compared with the control (p b 0.05), suggesting deficit in their locomotor activity (Fig. 4), possibly as results of decreased acetylcholine esterase activity. Similar to what observed in the mortality of flies treated with H. suaveolens infusion (Fig. 3), exposure of flies to different times and concentrations of H. suaveolens infusion did not significantly alter locomotor activity evaluated as negative geotaxis (Fig. 5, p N 0.05). 3.5. Test for toxicity against larvae of Artemia salina Figs. 6 and 7 represent respectively the effect of H. suaveolens essential oil and H. suaveolens leaves infusion on the mortality of Artemia salina. It can be observed that both, the positive control (K2Cr2O7) and the essential oil start their toxicity at 25 μg/mL, above which it increased the mortality rate till 75 μg/mL where it reached its maximum toxic effect (Fig. 6). The essential oil was shown to have comparable toxic effect with that of the positive control by presenting LC50 of 49.72 μg/mL vs. LC50 of 53.05 μg/mL for the positive control (K2Cr2O7). In contrast, no toxic effect was noted when Artemia salina was treated with H. suaveolens leaves infusion (LC50 N 1000 μg/mL) in comparison to the control (Fig. 7). 4. Discussion H. suaveolens is widely used by rural and urban communities for medicinal purposes, and it is well known by farmers because it is rejected in animal feed. This may be justified because the essential oil of the species is toxic. The objective of this work was to evaluate the chemical
Fig. 2. Toxic effect of the essential oil of H. suaveolens OEHS (μg/mL) against D. melanogaster. **** Value statistically significant with p b 0.0001.
Fig. 3. Toxic effect of infusion of the leaves of H. suaveolens ILHS (μg/mL) against D. melanogaster. ns = no statistical significance.
composition of the essential oil and an infusion of the leaves of the species, as well as a prospection of toxicological tests with the model's organisms D. melanogaster and A. salina. Our results demonstrated that H. suaveolens presents/displays high lethality to the organisms tested, indicating that it can potentially be toxic to humans. Benelli et al. (2012) reported some changes in chemical composition of essential oil (EO) of H. suaveolens, highlighting the presence of sabinene (34.0%), β-caryophyllene (11.2%) and terpinolene (10.7%) as the main constituent. However, these differences in OE composition may be due to differences in cultivation, collection periods, climatical stress and, especially, the geographic origin of the plant (Miguel et al., 2005; Noudjou et al., 2007). An alternative to prevent a wide variation in the essential oil composition is that it must be extracted under the same conditions, resulting in a more constant composition (Kerbauy, 2004). In the flask assay, the LC50 of the essential oil was 15.5 μg/mL obtained after 12 h. If compared with the results obtained by Sobral-Souza et al. (2014) using essential oil of Eugenia jambolana under same conditions, it can be concluded that essential oil of H. suaveolens is more toxic than that of E. jambolana (LC 50 of 2.5 mg/mL vs. 15.5 μg/mL). Similarly, in the report of Benelli et al. (2012), the essential oil of H. suaveolens showed LC50 of 18.37 μL/mL with the Mediterranean fly Ceratitis capitata, which is comparable to what obtained with H. suaveolens essential oil used in this study. This toxic effect can be attributed to the synergism of chemical constituents present in the essential oil since they presented moderate toxicity when tested alone and higher toxicity when combined with two or more constituents (Benelli et al., 2012). As reported by Di Pasqua et al. (2006), the essential oils are typical lipophilic, and they can cross the plasma membrane and affect their structures such as polysaccharides, fatty acids and phospholipids, damaging the cytoplasmic components. Such damage may lead to leakage of macromolecules and cellular lysis hence, the death of biological organisms. This may explain the higher toxicity of essential oils when compared with the extracts.
Fig. 4. Effect of H. suaveolens essential oil in the locomotor activity (negative geotaxis behavior) of D. melanogaster. At the end of the exposure, the flies were submitted to negative geotaxis behavior test as described in Section 2. Y-axis represents the number of flies able to climb at least 6 cm of a glass column after 5 s (number of flies on top). Results are expressed as mean ± SD. * p b 0.05 compared to control at each exposure time.
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Fig. 5. Effect of H. suaveolens leaves infusion on the locomotor activity (negative geotaxis behavior) of D. melanogaster. At the end of the treatment, the flies were submitted to negative geotaxis behavior test as described in Section 2. Y-axis represents the number of flies able to climb at least 6 cm of a glass column after 5 s (number of flies on top). Results are expressed as mean ± SD. ns = no significance statistical compared to control at each exposure time.
Although several studies have described the ability of essential oils as antioxidants to inhibit lipid peroxidation, other recent studies have shown that they may behave as pro-oxidants affecting internal cell membranes and organelles, such as mitochondria (Azmi et al., 2006; Bakkali et al., 2008). Based on the different concentrations tested, the EOs presented cytotoxic effects on living cells, as observed with the A. salina and D. melanogaster. In the A. salina test, the components of essential oil (EO) may have caused depolarization of the mitochondrial membranes, decrease in the membrane potential or the EO may have caused the permeability of outer and inner mitochondrial membranes leading to death cell apoptosis and necrosis (Bakkali et al., 2008). However, this did not occur with the infusion of H. suaveolens. In the analysis of our results, it was observed that both botanical materials (essential oil and infusion) presented results of significant importance for studies to evaluate the toxicity of medicinal plants, where these toxicological studies are essential for their safe use, once it becomes essential to achieve their usefulness in pharmacological research.
5. Conclusion In general, the present study investigated first the toxicity of the compound and secondary infusion of H. suaveolens leaves in model organisms (Artemia salina and Drosophila melanogaster). The study demonstrated that the essential oil of H. suaveolens was toxic at the concentrations tested and this can be at least in part, attributed to the synergism of its chemical components. This indicates that more precautions should be exercised regarding the dosages and frequently use of H. suaveolens essential oil. In contrast, infusion of leaves of H. suaveolens did not show any toxicity to the model's organisms used.
Fig. 6. Percent mortality of Artemia salina exposed to Hyptis suaveolens essential oil (OEHS). Values were calculated as relative percentages in the live control, N = 3.
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Fig. 7. Percent mortality of A. salina exposed to H. suaveolens leaves infusion. Values were calculated the relative percentages in the live control, N = 3.
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Kerbauy, G.B., 2004. Fisiologia vegetal. first ed. Guanabara Koogan, Rio de Janeiro. Matos, F.J.A., 2009. Introdução à Fitoquímica Experimental. first ed. UFC, Fortaleza. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.J., McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica 45, 31–34. Miguel, M.G., Duarte, F., Venâncio, F., Tavares, R., 2005. Variation in the main components of the essential oils isolated from Thymbra capitata L. (Cav.) and Origanum vulgare L. Journal of Agricultural and Food Chemistry 53, 8162–8168. Mishra, B.B., Tiwari, V.K., 2011. Natural products: an evolving role in future drug discovery. European Journal of Medicinal Chemistry 46, 4769–4807. Nantitanon, W., Chowwanapoonpohn, S., Okonogi, S., 2007. Antioxidant and antimicrobial activities of Hyptis suaveolens essential oil. Scientia Pharmaceutica 75, 35–54. Noudjou, F., Kouninki, H., Ngamo, L.S., Maponmestsem, P.M., Ngassoum, M., Hance, T., Lognay, G.C., 2007. Effect of site location and collecting period on the chemical composition of Hyptis spicigera Lam. An insecticidal essential oil from North-Cameroon. Journal of Essential Oil Research 19, 597–601. Olotuah, O.O., 2011. Pesticidal activities of Hyptis suaveolens in pest management. Phytopathology 101, 132. Pandey, U.B., Nichols, C.D., 2011. Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacological Reviews 63, 411–436. Parra, A.L., Yhebra, R.S., Sardiñas, I.G., Buela, L.I., 2001. Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine 8, 395–400. R Core Team, 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Reis, E.M., Neto, F.W.S., Cattani, V.B., Peroza, L.R., Busanello, A., Leal, C.Q., Boligon, A.A., Lehmen, T.F., Libardoni, M., Athayde, M.L., Fachinetto, R., 2014. Antidepressant-like effect of Ilex paraguariensis in rats. BioMed Research International 2014, 1–9.
Shenoy, C., Patil, M.B., Kumar, R., 2009. Wound healing activity of Hyptis suaveolens (L.) Poit (Lamiaceae). International Journal of PharmTech Research 1, 737–744. Shirwaikar, A., Shenoy, R., Udupa, A.L., Shetty, S., 2003. Wound healing property of ethanolic extract of leaves of Hyptis suaveolens with supportive role of antioxidant enzymes. Indian Journal of Experimental Biology 41, 238–241. Siddique, H.R., Gupta, S.C., Dhawan, A., Murthy, R.C., Saxena, D.K., Chowdhuri, D.K., 2005. Genotoxicity of industrial solid waste leachates in Drosophila melanogaster. Environmental and Molecular Mutagenesis 46, 189–197. Soares, S.F., Borges, L.M.F., Sousa Braga, R., Ferreira, L.L., Louly, C.C.B., Tresvenzol, L.M.F., Ferri, P.H., 2010. Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs. Veterinary Parasitology 167, 67–73. Sobral-Souza, C.E., Leite, N.F., Cunha, F.A., Pinho, A.I., Albuquerque, R.S., Carneiro, J.N., Coutinho, H.D., 2014. Cytoprotective effect against mercury chloride and bioinsecticidal activity of Eugenia jambolana Lam. Arabian Journal of Chemistry 7, 165–170. Tennyson, S., Ravindran, K.J., Arivoli, S., 2012. Bioefficacy of botanical insecticides against the dengue and chikungunya vector Aedes aegypti (L.) (Diptera: Culicidae). Asian Pacific Journal of Tropical Biomedicine 2, 1842–1844. Zemolin, A.P.P., Cruz, L.C., Paula, M.T., Pereira, B.K., Albuquerque, M.P., Victoria, F.C., Franco, J.L., 2014. Toxicity induced by Prasiola crispa to fruit fly Drosophila melanogaster and cockroach Nauphoeta cinerea: evidence for bioinsecticide action. Journal of Toxicology and Environmental Health 77, 115–124. Zouari, N., 2013. Essential oils chemotypes: a less known side. Medicinal Aromatic Plants 2, 145–154.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Chemical composition and toxicological evaluation of Hyptis suaveolens (L.) Poiteau (LAMIACEAE) in Drosophila melanogaster and Artemia salina J.W.A. Bezerra a, A.R. Costa a, M.A.P. da Silva b, M.I. Rocha b, A.A. Boligon c, J.B.T. da Rocha d, L.M. Barros b, J.P. Kamdem b,⁎ a
Course of Biological Sciences, Regional University of Cariri (URCA), Pimenta, Crato, CE CEP 63.105-000, Brazil Department of Biological Sciences, Regional University of Cariri (URCA), Pimenta, Crato, CE CEP 63.105-000, Brazil Phytochemical Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Santa Maria, RS CEP 97105-900, Brazil d Postgraduate Program in Toxicological Biochemistry, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil b c
a r t i c l e
i n f o
Article history: Received 19 March 2017 Received in revised form 29 August 2017 Accepted 3 October 2017 Available online xxxx Edited by L Verschaeve Keywords: Drosophila melanogaster Artemia salina Essential oil Infusion Bamburral
a b s t r a c t Hyptis suaveolens (L.) Poiteau, known as “Bamburral” is a plant widely used in Brazilian folk medicine, however, there are no toxicological studies to ascertain its safety. Thus, we aimed to investigate the phytochemical and toxic effects of essential oil and infusion of the dry leaves of H. suaveolens in Drosophila melanogaster and Artermia salina. The chemical composition of the essential oil was performed by CG-MS, while that of the infused extract was analyzed by HPLC-DAD. The toxicity of the essential oil-OE (3.03–30.3 μg/mL air) to D. melanogaster was performed by means of volatilization; and for infusion (2.5–20 mg/mL), they were submitted to ingestion and then submitted to locomotive activity by means of negative geotaxis. However, for acute toxicity, A. salina cysts were exposed for 24 h to the tested samples (10–1000 μg/mL). Our results demonstrated that OE had three major components: β-Caryophyllene (18.57%), sabinene (15.99%) and spathulenol (11.09%) while the leaf infusion showed caffeic acid as the major component (12.76 mg/g). H. suaveolens leaves infusion was not toxic to the organisms at all the concentrations tested (p N 0.05). However, OE behaved as quite toxic with LC50 of 15.5 and 49.72 μg/mL in D. melanogaster and A. salina, respectively. In addition, OE caused impairment of the locomotor behavior of flies. Therefore, our study indicates that more caution should be taken regarding the dosage and frequent use of H. suaveolens leaf essential oil. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Medicinal and aromatic plants have always been part of human life since they are used for food and/or for the treatment of various illnesses. The use of plant products as therapeutic and pharmacological alternatives is affordable and widely accepted culturally. However, despite the widespread popular use, there is evidence that they can be potentially toxic. In recent years, there has been a growing scientific interest in essential oils and botanical products; however, there are few reports of toxicological studies of plant oils used by populations (Mishra and Tiwari, 2011; Zouari, 2013; Cunha et al., 2015). The Lamiaceae family consists of approximately 252 genus and 6800 species, being one of the important economical sources of essential oils. But, the biological activities of some plants of this family are unknown. The Hyptis genus has approximately 400 species (Judd et al., 2009), in which Hyptis suaveolens (L), commonly known in the Northeastern ⁎ Corresponding author at: Regional University of Cariri, Department of Biological Sciences, CEP: 63105-000 Crato, Ceará, Brazil. E-mail address: [email protected] (J.P. Kamdem).
https://doi.org/10.1016/j.sajb.2017.10.003 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
Brazil as “Bamburral”, is of great interest for medical applications. Its leaves are commonly used in the treatment of stomach pain (Shirwaikar et al., 2003; Shenoy et al., 2009). Numerous species including the fruit fly, Drosopila melanogaster and Artemia salina are widely used as model organism to investigate the pharmacological and/or toxicological effects of chemical compounds and natural products (Siddique et al., 2005; Zemolin et al., 2014). Of particular genetical importance, it has been suggested that D. melanogaster has about 75% human disease-causing genes (Jimenez-Del-Rio et al., 2010; Pandey and Nichols, 2011; Sobral-Souza et al., 2014). Therefore, its genetical importance associated with its low cost and its high sensitivity to toxic substances makes it a suitable model. Artemia salina Leach. (Artemiidae) is a crustacean from salt water environments. This organism plays an important role in the energy flow of the food chain in this environment. Due to the fact that it is cheap and does not require approval of the ethical committee, it is extensively used in toxicological studies to determine lethal concentration (LC) (Meyer et al., 1982; Parra et al., 2001; Gouveia et al., 2014). Substantial evidence from the literature indicates that H. suaveolens oil exerts a variety of pharmacological and biological activities such as
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antioxidant, antibacterial, with the major constituents of the oil being sabinene, α-Terpinolene and 1,8-cineole (Nantitanon et al., 2007) and nematicide with D-limonene and menthol as main constituents (Falcão and Menezes, 2003). In addition, there are studies with H. suaveolens oil showing that it has toxicity in some organisms such as gnat that transmits dengue Aedes aegypti (L.) (Diptera: Culicidae) (Tennyson et al., 2012), the beetle weevil-the-beans, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) (Ilboudo et al., 2010), the maize weevil, Sitophilus zeamais (Iloba and Ekrakene, 2006), the rice weevil, Sitophilus oryzae (Olotuah, 2011), one diptera host and transmitter of malaria, Anopheles gambiae (Abagli et al., 2012), and star tick Amblyomma cajennense (Soares et al., 2010). However, there is little information in the literature regarding the toxicity of H. suaveolens, particularly using D. melanogaster and A. salina as model. Thus, the objective of this study was (i) to chemically characterize the essential oil and infusion of the leaves of H. suaveolens and (ii) evaluate their potential toxic effects in D. melanogaster and Artemia salina. 2. Material and methods 2.1. Plant material The leaves of H. suaveolens were collected in March 2015 in the municipality of Quixelô - Ceará, Brazil, at 09:00 h, ±30 min in the following coordinates: Latitude − 6° 15 ′43.0056″, longitude − 39° 16 ′2.5926′ and 193.265 m sea level. The leaves were dried in an oven at 30 °C. The plant material was identified and a voucher specimen was deposited in the Herbarium Dárdano Andrade Lima - URCA under # 12.104. 2.2. Extraction of essential oil The essential oil of H. suaveolens was extracted from dried leaves, submitted to hydrodistillation in Clevenger apparatus. After collection, the leaves were crushed into small pieces (150 g) and filled into a 1 L volumetric flask, where 2 L of distilled water was added. The flask was coupled to the Clevenger apparatus under the heating mantle and the temperature adjustment was carried out until the water boiled. After boiling, the 2 h time of the extraction cycle was started. At the end of each extractive cycle, the oil contained in the apparatus was collected with the aid of a pipette and stored in amber and refrigerated bottles. After extraction, sodium sulphate was used to remove the aqueous phase present in the essential oil (Matos, 2009). 2.3. Preparation of the infusion of dried leaves of Hyptis suaveolens (ILHS) The leaves were collected, dried in the shade and then crushed. The infusion was prepared by soaking 80 g of dried leaves in 0.5 L of distilled water at 100 °C and put in repose for 1 h and then filtered. 2.4. Stock and creation of Drosophila melanogaster The creation of flies, D. melanogaster (Harwich strain) was made in Laboratory of Microbiology and Molecular Biology (LMBM), Regional University of Cariri - URCA. They were obtained from the Federal University of Santa Maria - UFSM, from strains originated from the National Species Stock Center, Bowling Green, OH. The flies were reared in a glass bottles 5.5 × 12 cm containing approximately 5 mL of standard medium (1% w/v yeast, 2% w/v sucrose, 1% w/v milk powder, 1% w/v agar, 0.08% w/v nipagin). They were maintained at 12:12 h (light/dark cycle), and constant temperature and humidity (25 ± 1 °C, 60% relative humidity, respectively). 2.5. Treatment, mortality test and negative geotaxis of D. melanogaster Twenty adult flies with 6 days of age (males and females) were placed in flasks of 330 mL containing the bottom filter paper
impregnated with 1 ml of 1% sucrose in distilled water. In the cover, it was attached a filter paper for the application of the essential oil so that the oil could volatilize from the top, in order to achieve the respiratory system of the flies. The experimental groups received different dosages of the essential oil of Hyptis suaveolens: 3.03, 7.57, 15.5, 22.72 and 30.3 μg/mL of air, while the control group received only the 1% sucrose. The total time of exposure to essential oil was 48 h. Regarding the treatment of flies with the infusion of the plant extract, the filter paper was impregnated with 2.5, 5, 10 and 20 mg/mL of sucrose solution and the flies were added in the flasks. Then, the mortality was taken every 3, 6, 12, 24 and 48 h and the results were expressed in percentage. The locomotor activity of the exposed flies was determined by the negative geotaxis test. 2.6. Toxicity against larvae of Artemia salina Leach In artificial seawater, it was added cysts of Artemia salina Leach and subjected to constant aeration for 24 h with light, the time required for hatching the larvae. Then, the concentrations of 10, 25, 50, 100, 250, 500 and 1000 μg/mL were prepared for H. suaveolens essential oil and for infusion of dry leaves of the plant (Meyer et al., 1982). Potassium dichromate (K2Cr2O7) was used as positive control at the same concentrations as essential oil and infusion of dry leaves, while the negative control was seawater. The reading was performed after 24 h, and the calculation of LC50 was obtained by linear regression using GraphPad Prism 6. 2.7. Phytochemical analysis of essential oil by Gas chromatography (GC-FID) The gas chromatography (GC) analysis was performed with Agilent Technologies 6890N GC-FID system, equipped with DB-5 capillary column (30 m × 0.32 mm; 0.50 mm) and connected to a FID detector. The thermal programmer was 60 °C (1 min) to 180 °C at 3 °C/min; injector temperature 220 °C; detector temperature 220 °C; split ratio 1:10; carrier gas Helium; flow rate: 1.0 mL/min. The injected volume of H. suaveolens essential oil was 1 μL diluted in chloroform (1:10). Two replicates of samples were processed in the same way. Component relative concentrations were calculated based on GC peak areas without using correction factors (Boligon et al., 2013). 2.8. Identification of the components Identification of the constituents was performed on the basis of retention index (RI), determined with reference to the homologous series of n-alkanes, C7–C30, under identical experimental conditions, compared with the mass spectra library search (NIST and Wiley), and with the mass spectra literature date Adams (2007). The relative amounts of individual components were calculated based on the CG peak area (FID response). 2.9. Phytochemical analysis of Hyptis suaveolens infusion by HPLC 2.9.1. Chemical, apparatus and general procedures All chemicals were of analytical grade. Acetonitrile, formic acid, caffeic acid, chlorogenic acid, caffeic acid and ellagic acid were purchased from Merck (Darmstadt, Germany). Apigenin, catechin, rutin and quercetin were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High-performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.
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2.9.2. Quantification of compounds by HPLC-DAD For analysis of the Hyptis suaveolens infusion of dried leaves, 15 mL of the extract was injected into a Phenomenex C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles and eluted at 0.6 mL/min with a 70-min linear gradient from 5 to 30% (v/v) acetonitrile in 0.1% (v/v) aqueous formic acid followed by a 5-min linear gradient to 100% (v/v) acetonitrile, following the method described by Reis et al. (2014) with some modifications. The wavelengths used were 254 nm for gallic acid; 280 nm for catechin; 327 for ellagic acid, chlorogenic acid and caffeic acid; and 356 nm rutin, quercetin and apigenin. The extract and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.020–0.250 mg/mL. Chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 500 nm). All chromatography operations were carried out at ambient temperature and in triplicate. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve (Kamdem et al., 2013; Boligon et al., 2014). 2.10. Statistical analysis Differences between groups of HPLC were assessed by an analysis of variance model and Tukey's test. The level of significance for the analyses was set at p b 0.05. These analyses were performed by using the free software R version 3.1.1. (R Core Team, 2014). Statistical analyses of the means in triplicate (n = 3) ± standard deviation were performed using the software program GraphPad Prism 6, using Twoway Variance Analysis (ANOVA), followed by the Tukey test at 5% reliability. 3. Results 3.1. Chemical composition of essential oil of H. suaveolens The essential oil had a yield of 0.153%. It was identified a total of 44 components in the essential oil of H. suaveolens representing 99.97% of the whole composition (Table 1). The major identified components of the oil were the β-Caryophyllene (18.57%), the sabinene (15.99%) and spathulenol (11.09%). However, the minor components were (Z)-β-Ocimene (0.07%), α-Copaene (0.09%) and δ-cadidene (0.09%).
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Table 1 Composition of Hyptis suaveolens essential oil. Compounds
RIa
RIb
α-Thujene α-Pinene Sabinene β-Pinene Myrcene δ-2-Carene α-Phellandrene α-Terpinene p-Cymene Limonene 1-8-Cineole (Z)-β-Ocimene (E)-β-Ocimene γ-Terpinene cis-Sabinene hydrate Linalool cis-p-Menth-2-en-1-ol t-Sabinol 4-Tepineol p-Cymen-8-ol α-Terpineol δ-Elemene α-Copaene β-Elemene β-Cedrene β-Caryophyllene β-Gurjunene γ-Elemene Aromadendrene α-Humelene Alloaromadendrene γ-Muurolene Germacreno D β-Selinene Bicyclogermacrene γ-Cadinene δ-Cadidene Germacrene B Spathulenol Caryophyllene oxide Globulol Cubenol β-Eudesmol α-Cadinol Total identified (%)
989 940 976 980 994 999 1006 1019 1030 1031 1037 1041 1055 1060 1068 1095 1123 1139 1178 1183 1191 1335 1377 1390 1416 1421 1433 1435 1439 1453 1461 1477 1481 1486 1501 1512 1525 1559 1576 1580 1582 1641 1649 1656
931 939 976 980 991 1001 1005 1018 1029 1031 1033 1040 1050 1061 1068 1098 1121 1140 1177 1183 1189 1338 1376 1391 1417 1418 1432 1433 1439 1454 1462 1477 1480 1485 1488 1513 1520 1556 1576 1581 1583 1642 1649 1653
Oil % 1.09 0.85 15.94 2.11 0.26 0.49 1.38 1.05 0.76 5.19 3.04 0.07 0.12 2.97 0.61 0.43 0.28 0.15 6.82 0.23 0.94 1.17 0.09 0.78 0.14 18.57 0.23 1.44 0.32 1.17 0.40 0.28 5.21 0.89 7.52 0.36 0.09 0.27 11.09 3.18 0.62 1.07 0.13 0.45 99.97
Relative proportions of the essential oil constituents were expressed as percentages. a Retention indices experimental (based on homologous series of n-alkane C7–C30). b Retention indices from literature (Adams, 2007).
3.2. HPLC analysis of ILHS HPLC fingerprinting composition of Hyptis suaveolens infusion of leaves revealed the presence of the gallic acid (retention time - tR = 8.95 min; peak a), catechin (tR = 15.93 min; peak b), chlorogenic acid (tR = 21.87 min; peak c), caffeic acid (tR = 24.31 min; peak d), ellagic acid (tR = 30.56 min; peak e), rutin (tR = 37.84 min; peak f), quercetin (tR = 48.05 min; peak g) and apigenin (tR = 56.41 min; peak h) (Fig. 1 and Table 2). 3.3. Test of mortality of D. melanogaster In fumigation test with the essential oil, significant effects of concentrations and their interaction were detected. It appears that the number of dead flies increased with the time and concentration-dependent manner (Fig. 2). All concentrations of the oil above of 15.5 μg/mL of air caused at least 50% mortality of the flies. In contrast, there was no significant dead of flies treated with infusion of H. suaveolens in comparison with the control (Fig. 3).
Fig. 1. Representative reverse-phase HPLC analysis of Hyptis suaveolens leaves infusion. Using standard and spectral analysis, peaks a, b, c, d, e, f, g and h were identified as gallic acid, catechin, chlorogenic acid, caffeic acid, ellagic acid, rutin, quercetin and apigenin respectively.
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Table 2 Composition of Hyptis suaveolens infusion. Compounds
Gallic acid Catechin Chlorogenic acid Caffeic acid Ellagic acid Rutin Quercetin Apigenin
LOD
LOQ
mg/g
H. suaveolens %
μg/mL
μg/mL
3.85 ± 0.01 a 5.94 ± 0.01 b 0.61 ± 0.02 c 12.76 ± 0.03 d 3.49 ± 0.01 e 3.84 ± 0.01 a 3.45 ± 0.02 e 5.90 ± 0.01 b
0.38 0.59 0.06 1.27 0.34 0.38 0.34 0.59
0.013 0.008 0.021 0.015 0.030 0.019 0.024 0.011
0.042 0.025 0.070 0.053 0.098 0.062 0.079 0.037
Results are expressed as mean ± standard deviations (SD) of three determinations. Averages followed by different letters differ by Tukey test at p b 0.05. LOD: limit of detection and LOQ: limit of quantification.
3.4. Test of geotaxia negative against D. melanogaster As depicted in Fig. 4, no significant change was detected on the locomotor activity of flies treated with the H. suaveolens essential oil in comparison to the control (p N 0.05). However, the percentage of flies that reached the top of the glass column (6 cm of in 5 s) significantly decreased with time and concentration dependent manner compared with the control (p b 0.05), suggesting deficit in their locomotor activity (Fig. 4), possibly as results of decreased acetylcholine esterase activity. Similar to what observed in the mortality of flies treated with H. suaveolens infusion (Fig. 3), exposure of flies to different times and concentrations of H. suaveolens infusion did not significantly alter locomotor activity evaluated as negative geotaxis (Fig. 5, p N 0.05). 3.5. Test for toxicity against larvae of Artemia salina Figs. 6 and 7 represent respectively the effect of H. suaveolens essential oil and H. suaveolens leaves infusion on the mortality of Artemia salina. It can be observed that both, the positive control (K2Cr2O7) and the essential oil start their toxicity at 25 μg/mL, above which it increased the mortality rate till 75 μg/mL where it reached its maximum toxic effect (Fig. 6). The essential oil was shown to have comparable toxic effect with that of the positive control by presenting LC50 of 49.72 μg/mL vs. LC50 of 53.05 μg/mL for the positive control (K2Cr2O7). In contrast, no toxic effect was noted when Artemia salina was treated with H. suaveolens leaves infusion (LC50 N 1000 μg/mL) in comparison to the control (Fig. 7). 4. Discussion H. suaveolens is widely used by rural and urban communities for medicinal purposes, and it is well known by farmers because it is rejected in animal feed. This may be justified because the essential oil of the species is toxic. The objective of this work was to evaluate the chemical
Fig. 2. Toxic effect of the essential oil of H. suaveolens OEHS (μg/mL) against D. melanogaster. **** Value statistically significant with p b 0.0001.
Fig. 3. Toxic effect of infusion of the leaves of H. suaveolens ILHS (μg/mL) against D. melanogaster. ns = no statistical significance.
composition of the essential oil and an infusion of the leaves of the species, as well as a prospection of toxicological tests with the model's organisms D. melanogaster and A. salina. Our results demonstrated that H. suaveolens presents/displays high lethality to the organisms tested, indicating that it can potentially be toxic to humans. Benelli et al. (2012) reported some changes in chemical composition of essential oil (EO) of H. suaveolens, highlighting the presence of sabinene (34.0%), β-caryophyllene (11.2%) and terpinolene (10.7%) as the main constituent. However, these differences in OE composition may be due to differences in cultivation, collection periods, climatical stress and, especially, the geographic origin of the plant (Miguel et al., 2005; Noudjou et al., 2007). An alternative to prevent a wide variation in the essential oil composition is that it must be extracted under the same conditions, resulting in a more constant composition (Kerbauy, 2004). In the flask assay, the LC50 of the essential oil was 15.5 μg/mL obtained after 12 h. If compared with the results obtained by Sobral-Souza et al. (2014) using essential oil of Eugenia jambolana under same conditions, it can be concluded that essential oil of H. suaveolens is more toxic than that of E. jambolana (LC 50 of 2.5 mg/mL vs. 15.5 μg/mL). Similarly, in the report of Benelli et al. (2012), the essential oil of H. suaveolens showed LC50 of 18.37 μL/mL with the Mediterranean fly Ceratitis capitata, which is comparable to what obtained with H. suaveolens essential oil used in this study. This toxic effect can be attributed to the synergism of chemical constituents present in the essential oil since they presented moderate toxicity when tested alone and higher toxicity when combined with two or more constituents (Benelli et al., 2012). As reported by Di Pasqua et al. (2006), the essential oils are typical lipophilic, and they can cross the plasma membrane and affect their structures such as polysaccharides, fatty acids and phospholipids, damaging the cytoplasmic components. Such damage may lead to leakage of macromolecules and cellular lysis hence, the death of biological organisms. This may explain the higher toxicity of essential oils when compared with the extracts.
Fig. 4. Effect of H. suaveolens essential oil in the locomotor activity (negative geotaxis behavior) of D. melanogaster. At the end of the exposure, the flies were submitted to negative geotaxis behavior test as described in Section 2. Y-axis represents the number of flies able to climb at least 6 cm of a glass column after 5 s (number of flies on top). Results are expressed as mean ± SD. * p b 0.05 compared to control at each exposure time.
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Fig. 5. Effect of H. suaveolens leaves infusion on the locomotor activity (negative geotaxis behavior) of D. melanogaster. At the end of the treatment, the flies were submitted to negative geotaxis behavior test as described in Section 2. Y-axis represents the number of flies able to climb at least 6 cm of a glass column after 5 s (number of flies on top). Results are expressed as mean ± SD. ns = no significance statistical compared to control at each exposure time.
Although several studies have described the ability of essential oils as antioxidants to inhibit lipid peroxidation, other recent studies have shown that they may behave as pro-oxidants affecting internal cell membranes and organelles, such as mitochondria (Azmi et al., 2006; Bakkali et al., 2008). Based on the different concentrations tested, the EOs presented cytotoxic effects on living cells, as observed with the A. salina and D. melanogaster. In the A. salina test, the components of essential oil (EO) may have caused depolarization of the mitochondrial membranes, decrease in the membrane potential or the EO may have caused the permeability of outer and inner mitochondrial membranes leading to death cell apoptosis and necrosis (Bakkali et al., 2008). However, this did not occur with the infusion of H. suaveolens. In the analysis of our results, it was observed that both botanical materials (essential oil and infusion) presented results of significant importance for studies to evaluate the toxicity of medicinal plants, where these toxicological studies are essential for their safe use, once it becomes essential to achieve their usefulness in pharmacological research.
5. Conclusion In general, the present study investigated first the toxicity of the compound and secondary infusion of H. suaveolens leaves in model organisms (Artemia salina and Drosophila melanogaster). The study demonstrated that the essential oil of H. suaveolens was toxic at the concentrations tested and this can be at least in part, attributed to the synergism of its chemical components. This indicates that more precautions should be exercised regarding the dosages and frequently use of H. suaveolens essential oil. In contrast, infusion of leaves of H. suaveolens did not show any toxicity to the model's organisms used.
Fig. 6. Percent mortality of Artemia salina exposed to Hyptis suaveolens essential oil (OEHS). Values were calculated as relative percentages in the live control, N = 3.
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Fig. 7. Percent mortality of A. salina exposed to H. suaveolens leaves infusion. Values were calculated the relative percentages in the live control, N = 3.
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